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CHEMOPREVENTATIVE PROPERTIES OF CRUCIFEROUS VEGETABLE EXTRACTS AND PURIFIED COMPONENTS FOR HUMAN PROSTATE CANCER
DISSERTAHON
Presented m Partial Fulfillment of the Requirements for
the Degree Doctor of Philosophy in the Graduate
School of the Ohio State University
By
Corey Edison Scott, M.S. *****
The Ohio State University 2001
Dissertation Committee:
Professor Steven Schwartz, Adviser Approved by
Professor Grady Chism
Professor David Min iviser Dr. Steven Clinton Food Scienc^and Nutrition Profa'am
Dr. Mark Morse UMl Number: 3031262
UMl*
UMl Microform 3031262 Copyright 2002 b/Bell & Howell Information and Learning Company. All rights reserved. This microform edition is protected against unauthori^ copying under Title 17, United States Code.
Bell & Howell Information and Learning Company 300 North Zeeb Road P.O. Box 1346 Ann Arbor, Ml 48106-1346 ABSTRACT
A recent case-control epidemiological study has shown a significant reduction (41%) in the risks of developing prostate cancer among men who consume at least three servings of cruciferous vegetables (e.g. broccoli, cabbage, watercress, brussel sprouts) per week versus those who consume less than one. Cruciferous vegetables are known to contain large amounts of isothiocyanates, which are potent PhaseH enzyme inducers. We are investigating new mechanisms of chemoprevention by cruciferous vegetables aside
&om Phase H enzyme manifestation and carcinogen metabolism. This research addresses the effects of extracts and purified components (glucosinolates, isothiocyanates, indoles, etc) from cruciferous vegetables on malignant (PC-3, DU145, and LNCaP(androgen sensitive)) and prostatic epithelial cell growth and isolation and identification of chemopreventative compounds from the cruciferous vegetables. The ultimate goal of this research is to illustrate a means of lowering prostate cancer risks by consumption of cruciferous vegetables and to identify the components that have biological activity that can be linked to chemoprevention for human and animal studies. Both extracts and purified isothiocyanates from cruciferous vegetables inhibit cell proliferation, arrest cell cycle progression, and induce apoptosis in each of the three studied malignant cell lines in a dose dependent manner. The most potent extracts in growth inhibition were the broccoli sprout extract (S phase arrest) and watercress extract (GzM phase arrest), as we
ti observed a greater concentratioa of isothiocyanates in these extracts. The most potent isothiocyanates were benzyl isothiocyanate, sulforaphane, and phenethyl isothiocyanate with IC50 values between 2.3 and 9.8 [xM. Purified glucosinolates and indoles were less effective in the growth inhibitory effects with IC 5Q values over 100 pM. Sulforaphane, benzyl isothiocyanate, and phenethyl isothiocyanate were able to induce apoptosis in each line by 12%, 45%, and 54% respectively after 72 hours. Human prostatic epithelial cells (non-malignant) were much more resistant to the growth inhibitory effects and pro- apoptotic effects of isothiocyanates up to 50 pM concentrations. Insulin like growth factor (IGF-l) was added to AT6.3 (rat prostate) cells and their proliferation increased
3.4-fold. Isothiocyanates sulforaphane, phenethyl, and benzyl isothiocyanate inhibited
IGF-l stimulated cell proliferation and induced apoptosis in the cells at concentrations under 5 pf/I. Ultimately, this research shows that compounds from cruciferous vegetables can lower cancer risks by a variety of mechanisms such as proliferation inhibition, induction of apoptosis, and through influencing signal transduction pathways in malignant prostate cells.
ui \
Dedicated to my family
IV ACKNOWLEDGMENTS
I wish to sincerely thank my advisor and mentor Professor Steven Schwartz for introducing me to the study of Food Science. His conscientious attitude, kind spirit, and vast expertise in analytical methods helped to guide me in this task of obtaining a PhJ) in Food Science.
E wish to acknowledge Dr. Steven Clinton for his efibrts in establishing the collaboration of Food Science and Medical Oncology. This was sometimes a difficult endeavor, but he was available and critical with all parts of this research.
I wish to thank Professor Grady Chism for his technical advice in the area of plant enzymes and Food Science. 1 truly appreciated the kind words of encouragement during my tenure at The Ohio State LTniversity.
I thank Professor David Min for his expert advice in essentially all areas of food chemistry. I will forever remember his active participation in my work and helping me become acclimated to food science.
I acknowledge Dr. Mark Morse for his expertise in dealing with isothiocyanates chemistry and isothiocyanate metabolism. He served a crucial role in the success of this collaboration of food science and medical oncology. VITA
October 14,1973 ...... Æom-Thomasville, NC
199 5...... BA Chemistry, The University ofNorth Carolina at Chapel Hill
199 6...... Researcher, UNC School of Medicine Cystic Fibrosis Center
1998...... MS Chemistry, North Carolina Agricultural and Technical State University
1998-present ...... USDA Fellow The Ohio State University
PUBLICATIONS
Research Publications
1. Adebodun, F., Scott, CH., Cunningham, C., Bustamante, PM., Bradshaw, A., Ping, L., Williams, K. Elevated levels of Ca(II) modulate the activity and inhibition of serine proteases: Implication in the mechanism of apoptosis. Cell Biochem, Funct, 18: 59-66.2000.
2. Scott, CH., Adebodun. F. ^^C-NMR investigation of protein synthesis during apoptosis in human leukemic cell lines./. Cell Physiol. 181:147-152.1999.
3. Scott, CH., L JT. Abdullah, Davis, C.W. Ca^^ and protein kinase C activation of mucin granule exocytosis in. permeabilized SPOCl cells. Am. J. Physiol. 275 {Cell Physiol. 44):C285-C292.1998
vr FIELDS OF STUDY
Major Field: Food Science and Nutrition
m TABLE OF CONTENTS
Page Abstract...... ü
Dedication ...... iv
Acknowledgements ...... v
Vita.______vi
List of Tables ...... xii
List of Figures ...... xii
Chapters:
L Review of the literature ...... I
LI Etiology of prostate cancer ...... I L.LI Ensulin-like growth factor and prostate cancer...... 3 1.1.2 Dietary influences on prostate cancer ...... 6 1.2 Chemopreventative properties of cruciferous vegetables ...... 9 1.3 Cruciferous vegetables and malignant cell culture studies ...... 23 1.4 Cruciferous vegetables and cancer in animal models ...... 25 1.5 Human and rodent metabolism of glucosinolates and isothiocyanates ...... 37 1.6 Implications for food processing of cruciferous vegetables and. anticancer Properties ...... 42 1.7 Analysis and detection of phytochemicals from cruciferous vegetab les ...... 50 1.8 Research objectives ...... 57 References ...... 58
2. Cruciferous vegetable extracts inhibit malignant prostate epithelial cell growth and lower the apoptotic threshold ...... 70
2.1 Abstract------71 22 Introduction ______72
viii 2.3 Materials and methods ...... 74 2.3.1 Cruciferous vegetable preparation for extraction ...... 74 2.3.2 Aqueous extraction of cruciferous vegetables ...... 75 2.3.3 Analytical HPLC analysis of cruciferous vegetable extracts ...... 75 2.3.4 Preparatory HPLC fractionation and isolation of bioactive components ...... 75 2.3.5 Cell culture...... 75 2.3.6 In vitro antiproliferation assay...... 76 23.7 Cell cycle and programmed cell death (apoptosis) analysis ...... 76 2.3.8 Statistical analyses ...... 77 2.4 Results...... 77 2.4.1 Components profiles from cruciferous vegetables ...... 77 2.4.2 Effects of cruciferous vegetable on malignant cell growth ...... 78 2.4.3 Effects of cruciferous vegetable extracts on cell cycle progression 79 2.4.4 Effects of cruciferous vegetable extracts on induction of apoptosis 80 2.5 Discussion ...... 81 2.6 Conclusions ...... 85 2.7 References ...... 86
3. The effects of natural isothiocyanates, sinigrin, and indole on prostatic and malignant prostate epithelial cell proliferation, cell cycle, and viability _____ 101
3.1 Abstract...... 102 33 Introduction ...... 103 3.3 Materials and methods ...... 105 3.3.1 Analytical HPLC analysis of glucosinolates, isothiocyanates, and indoles ...... 105 3.3.2 Cell cidture...... 106 3.3.3 In vitro antiproliferation assay ...... 106 3.3.4 Cell cycle and programmed cell death (apoptosis) analysis ...... 107 3.3.5 Statistical analyses...... 107 3.4 Results...... 108 3.4.1 Effects of glucosinolates, isothiocyanates, and indoles on prostatic and malignant prostate cell growth ...... 108 3.43 Effects of isothiocyanates, and indoles on cell cycle progression ...... 109 3.4.3 Effects of isothiocyanates and indoles on programmed cell death (apoptosis) ...... 110 3.5 Discussion ...... Ill 3.6 Conclusions ...... 118 3.7 References ...... 119
IX 4. The effects of glucosinolates, isothiocyanates, indoles, and cruciferous vegetable extracts on IGF-l mediated ATd.3 cell growth ...... 134
4.1 Abstract...... 135 4.2 Introduction ...... 136 43 Materials and methods ...... 138 4.3.1 Glucosinolate, isothiocyanate, indole, and IGF-l preparation ...... 138 4.3.2 HPLC analysis of glucosinolates, indoles, isothiocyanates, and broccoli sprouts ...... 139 4.3.3 Cell culture...... 139 4.3.4 In vitro IGF-1 growth inhibition assay ...... 140 4.3.5 Akt and MAP kinase western blot ...... 140 4.3.6 Statistical analyses...... 141 4.4 Results...... 141 4.4.1 Effects of sinigrin, indole-3-carbinol, isothiocyanates, and broccoli sprout extract on AT6.3 cell proliferation ...... 141 4.43 Effects of IGF-l on AT6.3 cell proliferation ...... 142 4.4.3 Effects of sinigrin, indole-3-carbinoI, isothiocyanates, and broccoli sprout extract on IGF-l stimulated AT6.3 cell growth—142 4.4.4 Effects of isothiocyanates and broccoli sprout extract on Akt activation ...... 143 4.4.5 Effects of phenethyl isothiocyanate on induction of apoptosis ...... 144 4.5 Discussion ...... 144 4.6 Conclusions ...... 148 4.7 References ...... 149
Appendix A ...... 167
Appendix B ...... 169
Appendix C ...... 176
Bibliography...... 179 LIST OF TABLES
LI Mean values of IGF-I, IGFD, IGF-l/PSA ratio and PSA in patients with no evidence of cancer and those with prostate cancer ...... 5
12 Major anticarcinogenic compounds found in cruciferous vegetables ...... 10
1.3 Glucosinolate levels in Brassica vegetables ...... 16
1.4 Isothiocyanate water/oil partition coefBcient and their respective IC 50 values 31
1.5 Characteristics of human prostate cancer cell lines ...... 34
1.6 Loss of isothiocyanates and mtrües during storage and cooking ______.43
2.1 Percentage of cells in each phase of cell cycle ...... 88
22 Percentage of apoptotic cells in treated samples ...... 89
3.1 ICso values for glucosinolates^ isothiocyanates, and indoles ...... 121
3 2 Percentage of cells in cell cycle ...... 122
4.1 ICso values for Glucosinolates, Indoles, and Isothiocyanates ...... 153
4.2 Percent Reduction in IGF-l Stimulated Cell Growth ...... 154
4.3 Percent Activation of Akt Activity by Isothiocyanates ...... 155
4.4 Percent Apoptotic AT6.3 cells treated with IGF-l phenethyl isothiocyanate or Both ...... 155
XI LISTOFnGURES
Page
LI Cell survival process by IGF-I receptor mediated binding ...... 4
12 Typical a), alkyl and b). indole glucosinolate structures ...... 11
1.3 Glucosino late derived from elongated amino acids ...... 12
1.4 Glucosinolate biosynthesis ...... 14
1.5 Hydrolysis of glucosinolates to isothiocyanates by myrosinase ...... 17
1.6 Hydrolysis products of non-indole glucosinolates ...... 18
1.7 Hydrolysis products of hydroxyl containing glucosinolates ...... 19
1.8 Hydrolysis products of indole glucosinolates ...... 20
1.9 Phase I and Phase H metabolism of benzo[a]pyrene ...... 22
1.10 Effects of isothiocyanates and nitriles on K562 human leukemic growth ...... 24
1.11 Percentage of cell populations in each phase of cell cycle treated with 15 pM sulforaphane for up to 72 hours, b.) Cell cycle histogram showing accumulation of HT29 cells in G%M phase of cell cycle after 15 pM sulforaphane treatment ...... 27
1.12 Percentage of apoptotic cells in HT29 control cells and HT29 cells treated with 15 pM sulforaphane ...... 28
1.13 Proposed, major reactive sites on isothiocyanates ...... 30
1.14 Correlation of initial rate constants with non-enzymatic glutathione conjugation rates ------33
1.15 Glutathione conjugation of isothiocyanates ...... 40 1.16 Heat induced isothiocyanate-thiocyanate isomerization and cleavage
xii pathway for allyl isothiocyanate ...... 46
L17 Heat induced formation of allylamine and dimerization of allyl isothiocyanate ...... 47
1.18 Major non-volatile thermal degradation products of allyl isothiocyanate ...... 48
1.19 Major non volatile thermal degradation products o f Sulforaphane...... 49
1.20 Gas chromatograms of sulforaphane and sulforaphane nitrile from broccoli ...... 51
1.21 Mass spectra o f sulforaphane, sulforaphane nitile, 3-butenyl isothiocyanate ....52
1.22 High performance liquid chromatography for various isothiocyanates and Oxazoladines ...... 53
1.23 High performance liquid chromatography separation of several naturally occurring glucosinolates ...... 54
1.24 Cyclocondensation reaction ...... 56
2.1 Glucosinolate hydrolysis ...... 90
2.2 HPLC chromatograms of cruciferous vegetables ...... 91
2.3 Effects of added myrosinase on extracts ...... 92
2.4 Effects of cruciferous extracts on DU145 cell growth ...... 93
2.5 Effects of cruciferous extracts on LNCaP cell growth ______.94
2.6 Effects of cruciferous extracts on PC-3 cell growth ...... 95
2.7 HPLC fractionatiott of broccoli sprout extract. ______96
2.8 Effects of broccoli sprout extract on cell cycle progression ...... 97
2.9 Effects of watercress extract on cell cycle progression ______98
2.10 Effects of broccoli extract on cell cycle progression ...... 99
2.11 Apoptotic effects of watercress extract on PC-3 cells ______100
xiii 3.1 Enzymatic hydrolysis of glucosinolates to isothiocyanates ...... 123
3.2 Effects of isothiocyanates and indoles on PC-3 cell growth ...... 124
3.3 Effects of isothiocyanates and indoles on DuI45 cell growth ...... 125
3.4 Effects of isothiocyanates and indoles on LNCaP cell growth ...... 126
3.5 Effects of sulforaphane on prostatic and malignant cell growth...... 127
3.6 Effects on added glutathione on PC-3 cell proliferation ...... 128
3.7 Effects of sulforaphane on PC-3 cell cycle ...... 129
3.8 Effects of benzyl isothiocyanate on LNCaP and PC-3 cells ...... 130
3.9 Effects of sulforaphane on prostatic cell cycle progression ...... 131
3.10 Annexin V staining of apoptotic PC-3 cells exposed to isothiocyanates ...... 132
3.11 Effects of low (SpM) and high(30pM) levels of sulforaphane and benzyl isothiocyanate on apoptosis in PC-3 cells ...... 133
4.1 lGF-1 cell signaling events leading to decreased apoptosis ...... 156
4.2 Effects of isothiocyanates and indole-3-carbinol on AT6.3 cell proliferation.. 157
4.3 Effects of broccoli sprout extract on AT63 cell growth ...... 158
4.4 ÎÎPLC chromatogram of broccoli sprout extract ...... 159
4.5 Effects of IGF-l on AT6.3 proliferation ...... 160
4.6 Effects of IGF-l on AT6.3 cell proliferation ...... 161
4.7 Effects of isothiocyanates on IGF-l stimulated AT6.3 cell proliferation ____ 162
4.8 Reduced apoptosis in IGF-l treated samples (24 hours) ______163
4.9 Morpholigical changes in IGF-l treated AT6.3 cells during mduction of apoptosis by benzyl isothiocyanate (48 hours) ...... 164
4.10 Effects of isothiocyanates on IGF-l Akt phosphorylation ______165
XIV 4.11 Annexin V Staining of Apoptotic cells induced by Phenethyl isothiocyanate. 166
XV CHAPTER 1
REVIEW OF THE LITERATURE
1.1 Etiology of Prostate Cancer
Cancer of the prostate is an unusually common malignancy relative to other organs in males. Prostate cancer usually accounts for 40% of new cancers diagnosed and results in
40,000 deaths annually, ranking it second in cancer deaths to lung cancerL With early detection, the overall five-year survival rate for localized prostate cancer is approximately
100%. However, after 5 years the survival rate gradually drops to 52% at ten years’.
Cancer of the prostate typically affects older men, with 75% of cases being diagnosed in men over the age of 65. The etiology of this type of cancer remains poorly understood but there are several factors that can lead to prostate cancer development. The risks of developing prostate cancer are largely influenced by dietary intake, tobacco use, and certain environmental exposures. Androgens also play a key role in prostate carcinogenesis since it has been suggested that elevated levels of testosterone can be linked to prostate cancer’. Testosterone is converted to an active metabolite dihydroxytestosterone (DHT) by an enzyme known as 5-alpha-reductase. DHT is the major hormonal regulator of prostate growth and function. DHT production is significant in prostatic neoplasia since Eunuchs and men with abnormal androgen metabolism do not develop prostate cancer^.
1 There is a tremendous ethnic variation in prostate cancer incidences as it e^cts racial groups to diSèring degrees. African American males have the highest incidences of prostate cancer (by as much as 40%) followed by Caucasian males L Asian males have the lowest incidence rates of prostate cancer^ Accumulating epidemiological data now suggests that the greater risks of prostate cancer among African American men cannot be attributed solely to diet alone^’^’'*^. Studies have suggested that African American males have higher circulating levels of testosterone than Caucasians, and it has been suggested that an elevated level of testosterone can be linked to developing prostate cancer via oxidative stress'^. Strikingly, ethnic groups who migrate from low risk areas to high-risk areas have significantly higher mortality rates from prostate cancer. This is evident in at least two separate ethnic groups, Japanese men and African American men. For example, between 1979 and 1986, there was a 13% rise in prostate cancer incidences reported among Japanese men who migrated to the United States and consumed a more western- type diet^. Genetics may play a key role in prostate cancer as men with a close family history of prostate cancer (from either father or grandfather) have a 2-3 fold greater likelihood of developing prostate cancer^.
Early detection of prostate cancer is generally performed by evaluating an individuals prostate specific antigen (PSA) blood levels^"^. PSA levels can now help difierentiate between prostate cancer and prostate hyperplasia. PSA is a naturally produced chemical that helps liquefy semen. A small amount can be found in the blood stream and elevated levels may be an indication of prostate infiammation, prostate enlargement, or prostate cancer. For these reasons PSA counts may not always be a precise estimate for prostate cancer. However, when cancer of the prostate is present, the PSA. test are capable of detection greater than 80% of the thne^.
1.1.1 Insulin-like Growth Factors and Prostate Cancer
Recently, insulin like growth factors (IGFs), have been implicated in prostate cell proliferation and cancer®’^*®. IGFs are single-chain peptides of about 70 amino acids which are 40%-50% identical to insulin and bind to extracellular receptors. IGFs have been shown to have mitogenic and anti-apoptotic effects on prostate epithelial cells in
IGFs are a family of growth factors largely produced in the liver, the most prevalent of which in serum is IGF-l. Biological activity of IGFs is mediated through a heterotetrameric tyrosine kinase receptor which is similar to an insulin receptor (Figure
I). IGFs influence cell survival and proliferation by binding to receptors causing autophosphorylation and activation of intrinsic tyrosine kinase which then goes on to phosphorylate intracellular substrates such as IRS-l and She. These events lead to activation of mitogen-activated protein kinases (MAPK), extracellular signal related kinases (ERK), and phospholidylinositol 3-kinase (P13K) pathways. An important target for P13BC is a serine/threonine kinase called Akt. Akt is involved in cell survival and is mediated by several growth factors. Akt can phosphorylate a regulatory member of the
Bcl-2 family, BAD, which can remove BAD from a death suppressor Bcl-xL. Bcl-xL can then remain associated with the caspace activator Apaf-L, thus preventing activation of caspase enzymes and preventing onset of programmed cell death (apoptosis). IGF-l receptor binding and cell proliferation effects can be inhibited by insulin-like growth factor binding proteins (IGFBPs), specifrcally IGFBP-3". Furthermore, PSA can cleave the IGFBP/IGF-lcompIex and release IGF-I, thereby increasing IGF-l serum levels^\
3 OrewttifKtora,
> î ^ X ïT'V ^lîw/ 1^ ^a—I a /
\ l i / Apoptosis
Figure 1.1: Cell survival and anti-apoptotic process by IGF receptor mediated binding (from Cell Signal) Thus IGF-l, IGFBP-3, and PSA levels collectively are beginning to be evaluated as biomarkers for prostate cancer. However, many epidemiological studies yield conflicting results regarding lGF-1 levels and prostate cancer risks. Mantzoro et a P report that an increase of one standard deviation of lGF-1 serum levels (60ng/ml in tbeir study) can be associated with a two-fold increase in prostate cancer risks. Other studies have shown that increased levels oflGF-l and decreased levels of IGFBPs can increase prostate cancer risks^^^t A study of245 men found 71 men (29%) to have prostate cancer and
174 men (71%) not to have any malignancies of the prostate'^ The mean ages were 67 +
9 years and 65+6 years respectively. This study found lGF-1 levels, IGF density (IGFD),
IGF-l/PSA ratio and PSA counts to be higher in patients with prostate cancer (Table l.l).
However, it was indicated firom this study that IGF-l and IGF-l density cannot be
IGF-l IGFD IGF-l/PSA PSA (ng/ml) (ng/ml/cc) Ratio (ng/ml)
No Cancer (n=174) 136 4.17 20.7 6.57
Prostate Cancer (n=7l) 176 9.41 28.6 7.49
P value 0.030 0.045 0.001 0.018
Table 1.1: Mean Values of IGF-l, IGFD, IGF-l/PSA ratio and PSA in patients with no evidence of cancer and those with prostate cancer‘s used alone to detennine prostate cancer risks. When IGF-l is used along with PSA, prostate cancer risks determinations can be more accurate. A study performed to ascertain racial difierences in IGF-I levels in 63 Affican American men and 42 Caucasian men aged 35-69 (no prostate malignancies, but a family history) found no significant difference in plasma levels of IGF-l (162.3 ng/ml vs 172.1 ng/ml p=0.4l5)^^ In contrast, Afiican American men had significantly lower lGFBP-3 serum levels than the
Caucasian men, 2798ng/ml vs 3216 ng/ml, p=0.0045. Also observed was an inverse relationship between IGF-l levels and age, but not with IGFBPs and age. Finally, a case- control study of 210 men with newly diagnosed prostate cancer and 224 control men found higher IGF-l serum levels in case patients (158 ng/ml) than in control (147.4 ng/ml)'**. However, there was no significant change in IGFBPs serum levels. A strong association was shown between increasing age and prostate cancer risks (0R=1.51) between men under the age of 70. Serum levels of IGF-l were not related with the disease stage. In this study, IGFBP-3 was not significantly associated with increased risk. Thus, firom these studies, the roles of IGF-l and IGFBP-3 on prostate carcinogenesis are conflicting- While each study’s results may be valid, there are certainly several extraneous factors such as physical stature, weight, environment, and energy intake which may have lead to the inconsistent results. Nonetheless, IGF-l, IGF-l density, and
IGF-l/PSA ratio may be valuable biomarkers for cancer of the prostate.
1.1.2 Dietary Influences on Prostate Cancer
Prostate cancer exhibits a long latency period relative to several other malignancies.
This suggests that diet and nutrient intake may influence the onset and progression of prostate cancer. Diet is a major cause of cancer, yet data is accumulating fiom both
6 epidemiological and experimental studies that suggest as a reduction in cancer at various organs, including the prostate, by consuming a healthy diet that includes a high intake of
Suits and vegetables and. a low intake of saturated fat and red meat Many case- control and cohort studies yield inconsistent results regarding dietary intake of speciffc
Suits and vegetables and prostate cancer risks. However, it is widely accepted that a diet high in saturated fat can contribute to an increased risk in developing prostate cancer'^.
Throughout the world, there is a strong correlation between fat intakes with mortality from prostate cancer^®. These findings have also been illustrated in laboratory studies where energy restrictions from saturated fats reduced the incidence and growth of experimental cancers in rodents. As far as red meat consumption, cohort and case-control studies alike have detected an increase in prostate cancer risks in men whose overall red meat or animal product intake is high. The overall risk was approximately two-fold greater than diets low in these products*®’^^
Based on several epidemiological studies, the evidence of an association between fruit and vegetable consumption and cancer prevention is now strong and consistent^’*®’^^’**’'®.
However, studies dealing with specific fruit and vegetable intakes and prostate cancer risks are inconsistent and contradictory®. Among the groups of vegetables that have shown an association as far as consumption with reduced prostate cancer risks are cruciferous vegetables®.
To date, the diets of many people across the world include large amounts of cruciferous vegetable intake, both raw and processed. Cruciferous vegetables (Brassica spp.) which include broccoli, kale, cabbage, brussel sprouts, turnips, kohlrabi, radishes, cauliflower and other vegetables, are known to be rich in nutrients such as carotenoids,
7 minerals, fiber, and various vitamins. The cultivation of cruciferous vegetables has been noted as far back as 4000 B.C. and the medicinal and nutritional aspects have been noted as far back as 530 B.C. as evidenced by carbonized seeds^"^**. Cruciferous vegetables are unique in that they have a characteristic pungent or biting taste, due to high concentrations of glucosinolates and their aglycone hydrolysis products (isothiocyanates, thiocyanates, nitriles, indoles etc.) versus other vegetables. Across the world, cabbage is the most widely consumed cruciferous vegetable. However, in the United States, broccoli is the most widely consumed cruciferous vegetable with its per capita use quadrupling since 1980. The average broccoli intake for Americans is 4 1/2 pounds per year, representing a 940% increase in intake over the past 20 years. The average American
aged 12 to 70 consumes approximately 40 to 90 grams per day of cruciferous
vegetables^.
There have been approximately 80 combined cohort and case-control studies on the association between cruciferous vegetable consumption and cancer risks. Of the case control studies, 64% showed an inverse relationship between consumption of one or more cruciferous vegetable and risk of cancer^. Fewer studies have shown a nonsignificant decreased risk, no effect, or increased risk in prostate cancer associated with consumption of cruciferous vegetables'"^*. However, in general there is a protective effect against cancer by consumption of cruciferous vegetables. Many of the inconsistencies in these
studies have arisen fi:om small sample size, non-comprehensive measures of fruit and
vegetable intake, and extraneous factors such as age, fat intake, and lifes^le. Also, many
epidemiological studies on fimits and vegetables neglect to separate the effects of total
fruit and vegetable intake from the efiects of specific fiuit and vegetables^. A recent case-control study performed on relatively young men age 40-65 (which are generally at a low risk of prostate cancer) who were diagnosed with, prostate cancer showed a protective effect against prostate cancer for total vegetable consumption and specifically for cruciferous vegetable consumption^. The study was unique in that after controlling for total vegetable consumption, there was found a 41% decrease in prostate cancer risks among men who consumed at least three or more servings of cruciferous vegetables per week versus those who consumed less than one. There was no association found between feuit intake and prostate cancer risks observed in this study. This suggests that consumption of cruciferous vegetables versus consumption of many other fruits and vegetables can significantly reduce prostate cancer risks.
1.2 Cbemopreventative Properties of Cruciferous Vegetables
Cruciferous vegetables are composed of many nutrient and non-nutrient phytochemicals that can protect against carcinogens and other toxic electrophiles (Table
1.2)^'^. The overall mechanisms of protection are not fidly understood, however much of the experimental cbemopreventative data is linked to glucosinolates, which are compounds found in large quantities in cruciferous vegetables, and their various hydrolysis products. All glucosinolates consist of a P-D-thioglucose group, a sulphated oxfrne moiety, and a variable side chain derived from methionine, tryptophan, phenylalanine, and branched-chain amino acids (Figures 1.2-1.3). Over 120 glucosinolates have been identified and eacli fall into three principal categories, (1) alkyl or alkenyl side chain, (2) aromatic group, and (3) indolyP'^'^’^’^®*^. Glucosinolates can essentially be regarded as the plant defenses or as a natural pesticide. Plants with high Compound Anti-Cancer Property
Glucosinolates Precursor to isothiocyanate and other phytochemicals
Isothiocyanates Phase I Enzyme Inhibition, Phase H enzyme Induction
Indoles Tumor growth and estrogen metabolism suppressor
Carotenoids Antioxidant/ Free radical quencher
Vitamins Antioxidants
Minerals Regulators of glutathione peroxidase (selenium)
Fiber Decrease carcinogen resident time
Table 1.2: Major anticarcinogenic compounds found in cruciferous vegetables.
glucosinolate content have been shown to be less preyed upon by insects, slugs, or pigeons^’^'*’^^. Humans are also sensitive to the pungent and bitter taste associated with glucosinolates which can effect overall sale and consumption^®’^’’^*'
Glucosinolate biosynthesis in the plant can be described by three steps, (I) ammo acid chain elongation, (2) synthesis of glucosinolate from amino acid, and (3) chain modification (Figure Very few enzymes from these steps have been isolated so the overall biochemical pathway remains elusive but the following pathways have been represented in several studies"'’’'^^*^®. The first proposed step in glucosinolate
biosynthesis is conversion of the amino acid to an oxime. The next step is the oxidation
of the oxime to an aci-nitro complex which then conjugates with cysteine and acts as a
10 thiol donor. This molecule is then cleaved by CS-lyase to yield thiohydroximate. The thiohydroximate is the S-glucosylatedby UDPGithiohydroximate glucosyltransferase (S-
HO
HO
OH
Figure 1.2 Typical a.) AUq^l and b.) Indole glucosinolate structures
GT) to produce a desulphoglucosinolate. This species is sulfated by phosphadenosine 5’* phosphosulfate (PAPS): desulphoglucosinolate sulphotransferase. Next the side-chain can undergo several modifications^'’^ . These modifications have received little attention and are poorly understood. Mostly, what is suggested to occur in methionine derived species is an oxidation to methylsulphinyl or methylsulphonyl followed by removal of the methylsulphinyl group and desaturation to result in an alkenyl
II M eth io n in e MethyMiioaUcyi C H j-S -C H j-C H ^ — [CH 2 l„—GSL n=*l-8
Methyhulphmytatkvl CHa-SO-CHj-CHj—ICHiln-GSL n » 1-9
MethybulphonYlalkyl CHj—SOi—CHj—CHj— {CH 2 l„ — 4-MethyhulphihyJ-3-butenyl CHa-SO-CHsCH—[CH^lj-GSL Alkenyl CH2=CH-[CH2l „-GSL n = 1, 2, 3 2-Hydraxy-3-butenyl CH2=CH-CH-CHi-GSL OH 2-Hydroxv-4-pemenyt CHiSCH-CHj-CH-CHa-GSL &H Hydroxalkyl CHa-CHa-lCHzIn-GSL n = 1,2,3 OH C-O-CHi-CHi—[CHatn-OSL ti “ 1 - A Banzoyloxyalkyt c r Valine CH/ n « 0 - 2 CHj-CHi-CH-lCHjl „-GSL n “ 0 .1 isoleucine CH, CHa—ICHjl^-GSL Phenylalanine Phanylalkyl a Figure 1J: Glucosinolate derived from elongated amino acids( n denotes number of methylene groups) 12 C H j-Q S L T yrosine p-Hydroxybenzyl T ryprophan 3-Indoiylmethyt OH H 4-Hydroxy'3*îndolylmethyl C H j-G S L 1 -Methoxy-3-indolylmethyl OCH, CHz-GSL Figure U Continued: Glucosinolates derived from amino acids(fi’om Ref. 23) 13 COOH COON COf / 0 5H" «-CH — R-CH R-CR=NOH \ 0 0 0 NH% WHOM # « ♦ ' aidoxIiM «•«minoMid SH M SN / R-CN R-CH \'Ô H NO NON M-OH K-osoj ttilohydroxafnlcacld Figure 1.4: Glucosinolate biosynthesis (from Ref. 24) glucosinolate or subsequent hydro^tylation to yield an hydroxyalkenyl. Typical glucosinolates found in brassica species are aliphatic, (a-methylthioalkyl, aromatic, and indole(heterocyclic) glucosinolates(TabIe most common glucosinolates contain either a straight or branched carbon chain‘‘*’^°. Many of these chains are unique containing double bonds, hydroxyl groups, carbonyl groups, or sulfiir atoms with, various oxidation states. Other interesting glucosinolates not in the Brassica family contain an additional rhamnose or arabinose sugar moiety^"^°. Glucosinolates are degraded by an enzyme called myrosinase (thioglucohydrolase) and undergo Lossen rearrangement to yield a variety of reactive products when the plant is damaged (Figure 1.5)^“^. Myrosinase is a P-thioglucosidase which is similar in structure and function to glycosylhydrolases. Immunological evidence has indicated that myrosinase is located in the cytoplasm of myrosin cells throughout the plant tissue and is chemically segregated from glucosinolates^^’^®’^. Multiple forms of myrosinase can exist in the same plant and the enzyme kinetics difièr widely^®*^. Myrosinase can also be activated by ascorbic acid, with some enzymes being inactive in the absence of ascorbic acid. Ascorbic acid acts as a nucleophilic catalyst and raises both the Km and Vmax for glucosinolate substrates^®. Myrosinase possesses a glutamate residue (Glu-426) which is required for nucleophilic catalytic activity and its substrates must contain an hydroxyl group at the C-2 position on the glucose moiety for enzyme binding. The P-glucosyl moiety is cleaved and the sulfate moiety is released. The unstable thiohydroxamate-0- sulfonate rearranges to form a variety of compounds. The primary products are isothiocyanates, thiocyanates, nitriles, and indole compotmds (Figures 1.6,1.7,1.8). 15 AUplialic Glucosinolates Indolyl glucosinolates Vegetable glucoiberin progoitrin sinigrin glucoraphanin glucobrassicin 4-meihoxyglucobrassicin Broccoli 0,5 6,2 0,0 5,9 2.1 0,2 Brussel sprouts 3,1 7.6 8,2 2,1 4,5 0,9 Cabbage (green) 7,3 0,2 10,2 0,3 6,8 1.3 Cabbage (red) 1.6 1.2 2,7 0,1 3,8 0,3 Cauliflower 0,2 0,0 0,2 0,3 0,7 0,2 o\ Kohlrabi 0,2 0,0 0,0 0,1 1.3 0,1 Radish 0,0 0.2 0.0 0,2 0,3 0,5 Table 1.3: Glucosinolate levels in brassica vegetables (pmol/g dry weight) (From Ref, 24) HO OHqh myrosinase N=C=S O-SO3 Figure 1.5: Hydrolysis of giucosinolates to isothiocyanates by myrosinase. Product formation depends on several factors such as glucosinolate structure, pH, temperature, Fe^% and epithiospecifierprotein^’^'*’^®’^®. The biological activity of glucosinolates and hydrolysis products depends on the glucosinolate chemical structure. Glucosinolates and their hydrolysis products from cruciferous vegetables have been shown to block toxic and neoplastic effects of a wide variety of chemical carcinogens (Phase I enzyme inhibition) and to induce eirqmies of xenobiotic metabolism, such as Phase H enzymes, which thereby accelerate xenobiotic metabolism and detoxifies certam. carcinogens^^^’®°’®'*®^ Procarcinogens can be activated by cytochrome P450 enzymes which renders a foreign molecule more polar and 17 / Sp-D-Glucose R-C % NOSOj- (NoQ-indole glucosinolate) Myrosinase HzO / +D-Glucose R-C NOSO^- -HSO pH 7 Low pH Unclear V 1r \ r R-N=C=S R-CN R-S=C=N Isothiocyanate Nitrile Thiocyanate Figure 1.6: Hydrolysis products of non-indole glucosinolates 18 S'P-D-Glucose CH2=CH-ÇH-CH2--^./ OH NOSO3 - myrosinase ,SH CH2 =CH-CH-CH2 -( r\u [ OH NOSO3 ] CH2 =CH-(pi 1 CH2 -CH-CH-CH2 CN CH2 =CH-ÇH-CH2 CN OH OH I epithionitrile nitrile OxazoIidine-2-thione Figure U : Hydrolysis products of hydroxyl containing glucosinolates. 19 ^/S-p-D-Glucose NOSO] R Myrosinase HiO pH T >^ pH 3-4 (Indole-3-acetoaitriie) (unstable isothiocyanate) CH 2 OH (indole alcohol) R -t-S=C=N- Figure 1.8: Hydrolysis products of indole glucosinolate 20 facilitates in its excretion. This is known as Phase I metabolism depicted in Figure 1.9 using benzo[a]pyrene for example. A. negative consequence of Phase 1 metabolism may be the generation of electrophiles which can react with and form adducts with DMA, RNA, proteins,, and lipids. If DNA adducts go unrepaired, mutations can occur. A second class of enzymes known as Phase II enzymes add polar groups to the Phase I metabolite making the molecule hydrophilic and more readily excretable. Phase H enzymes can deactivate carcinogens by destroying the reactive centers of the carcinogens or by conjugating with ligands and facilitating in excretion before they can damage DNA. Thus, induction of Phase E enzymes (e.g. glutathione S-transferase, NAD(P)H:quinone oxidoreductase, glucuronosyltransferase, and epoxide hydrolase) can play a major role in chemoprevention and is a. very relevant topic that is under continuing investigation’®”’. The major inducers of Phase E enzymes found in cruciferous vegetables are a class of compounds caUed isothiocyanates^®*^®. These compounds are mostly monofonctional inducers, in that they have low cytotoxicity and have been shown to induce Phase E enzymes and inhibit Phase I (sometimes toxifying or carcinogenic) enzymes. Glucosinolates, are relatively non-reactive towards carcinogens and have been shown not to induce Phase E enzymes.^®*^ However, their enzymatic breakdown products, isothiocyanates, are very potent in the manifestation of Phase E enzyme induction and have recently been shown to act as indirect antioxidants thereby increasing tolerance to oxidative stress^’. Isothiocyanates are not the only degradation products of glucosinolates. Depending on the type of glucosinolate (indole or non-indole), temperature, pH, and availability of ions there are several aglycones which can be formed^®. These include indole-carbinols, nitriles, thiocyanates, oxazolidine-2-thiones, 21 2JAKœeOxi Glucuranide Cajugaics SulEue Conjugates 3,6-Qumone Gluauonide Cojugates l,6^QuiBone SulÊue Conjugates 7,8-Arene Oxide 6,12^Quinone Cpoxute Hydrolase uomensanon Cytochrome P-450 Covalent Bintiing 7-Hydroxy BjtjP ofRNA. DNA.& Protein. OH Gtucufoniile Cojugates 7,8-OibydrodîoI 7,8-Dfol 9,10-Epoxide Sulfite Conjugates \ Gluemonide Cojugates CANCER Sulfite Conjugates OH Diot Phenol Figure 1.9: Phase I and Phase D Metabolism of Beazo[a]pyrene 22 hydroxynitriles and epithionitriles. Each, of these compounds has the potential ofbeing active in chemoprevention through other mechanisms aside from Phase H en^mie induction. Isothiocyanates have been shown to be potent inducers of Phase H enzymes, but the mechanism of Phase H gene regulation remains unclear. Several studies of Phase H genes have revealed many cis-acting regulatory elements^^. These include antioxidant/electrophiiic response element, xenobiotic/aromatic hydrocarbon response element, activator protein-1 response element, and nuclear factor kappa-B response element binding sites. Recently it has been indicated that the response element that plays the most important role in regulation of Phase H genes such as NAD(P)H:quinone oxidoreductase, glutathione-S-transferase, and UDP gluconosyltransferases is the antioxidant/electrophiiic response element. This illustrates that Phase H genes can be expressed by means of an electrophilic signal and independent of receptors. 1.3 Cruciferous Vegetables and Malignant Cell Culture Studies A combination of both epidemiological and experimental data supports the protective effects of cruciferous vegetables against various types of cancers. Recently, a small number of studies have been initiated to address the efiects of glucosinolates, isothiocyanates, indole-3-carbinol, and nitriles on malignant cell growth in vitroP"^ Isothiocyanates and corresponding nitriles generated from three classes of glucosinolates (aliphatic, thioaliphatic, and aromatic) epiprogoitrin, glucocheirolin, glucoraphenin, glucotropeolin, and sinalbin respectively have been shown to inhibit proliferation in human erythroleukemic K562 cells with isothiocyanates halting proliferation to a greater extent versus nitriles except for sinalbin(Figure LIO)®. Similar and almost identical 23 lOQl 0.1 100 1000 ICO glueachairaftn (iiM) Î 3 60 3 t I 3 I to ICO 1000 O.l 1 10 1000 9hican^nin (pM) glucokopMlin |wM| 3 e ! 3 O .l 100 1000 Figure 1.10: Effects of isothiocyanates * and nitriles^ on K562 hitman leukemia cell growth (from Ref. 63) 24 antiproliferative effects were shown on five other cell lines such as murine erythroleukemic FL cells, human T-lymphoid Jurkat cells, human cervix carcinoma HeLa cells, H9-38 cells, andH3T-l-l cells. Two sulfiir-containing side chain isothiocyanates 3-methylthiopropyl and 5- methylthiopentyl isothiocyanate were incubated with. B16-F10 melanoma cells in a similar study. Each isothiocyanate caused growth inhibition with IC 5 0 values of 48 nM and 170 nM respectively after 72 hours^. After 16 hours a significant buildup of cells in the GiM phase of cell cycle was observed with a loss of cell population in the Gi phase. These effects were cytostatic, rather than cytotoxic, as greater than 98% of these cells were still viable at the IC5 0 concentrations. 3-methylthiopropyl and 5-methylthiopentyl isothiocyanates were shown to modify the state of kinases and suppress signals for cell growth. Indole-3-carbinol (ICO ^iM), a glucosinolate hydrolysis product of glucobrassinin has been shown to arrest cell cycle progression along with tamoxifen at Gt phase and decrease DNA synthesis in MCF-7 human breast cancer cells independent of estrogen receptor binding®^. The CDBC2 dependent pathway and retinoblastoma phosphorylation, which are essential in Gt cell cycle regulation, were both decreased. Endole-3-carbinol and tamoxifen were shown to work by separate mechanisms. Tamoxifen competed for estrogen binding receptors, whereas indole-3-carbinol decreased CDK 6 expression and was shown not to bind estrogen receptors. A similar study addressed the effects of cruciferous vegetable extracts ficom cabbage, fermented cabbage, and brussel sprouts as estrogen receptor agonists or antagonists®®. Each, extract was observed to possess very little if any estrogen receptor binding. A recent study addressed the effects of an 25 isothiocyanate conjugate (a common metabolite iu humans and rodents), phenethyL isothiocyanate-iVacetylcysteine (PEITC-iVAC) on androgen dependent LNCaP and androgen independent DU145 human prostate cancer cells.®^ The researchers found that at high PEITC-iVAC concentrations cell lysis occurred and at low concentrations, inhibition of growth (50-65%), DNA synthesis, and clonogenecity occurred along with induction of apoptosis. However it was noted that these effects took several days. Other studies have shown that isothiocyanates iberin, sulforaphene (alkene isomer of sulforaphane), erucin, and pbenethyl isothiocyanates induce apoptosis via activation of caspase and p53 genes Surprisingly, the parent molecules, glucosinolates, have not been observed to inhibit proliferation, affect cell cycle, or induce apoptosis in malignant cell lines at concentrations up to 500 |xM in some studies®®. Sulforaphane has also been shown to be selectively cytotoxic to HT29 colon cancer cells causing antiproliferation and apoptosis (>25% at 72 hours), but has a much weaker effect on differentiated CaCo2 cells™. Sulforaphane was able to inhibit DNA, RNA, protein, and phospholipid synthesis in the malignant colon cells. thymidine incorporation, indicative of DNA synthesis, decreased by 30% within 30 minutes in the presence of 15 pM sulforaphane. A 30% decrease in RNA and protein synthesis was also observed after a I hour incubation in 15 pM sul&raphane. Phospholipid metabolism decreased more than 50% after a three hour incubation. HT29 cells treated with sulforaphane accumulated in the GzM phase of cell cycle accompanied by a decrease in cells in the S phase (Figure 1.11). There was also a high level of cyclin A and cyclin BI expression in the presence of sulforaphane, which regulate cdc2 kinase activity at the GzM phase of cell cycle™. Sulforaphane induced cell death via apoptosis(Figure 1.12 ). 26 50- 3 * 0 a 72 - 5 0 I * 25 b . 0 3* «0 72 m 75 12* a so- 25- 0 24 40 72 Maun 4b Figure L ll r a. Percentage of cell populatioas in each phase of cell cycle treated with 15 sulforaphane up to 72 hours (circles are treated, squares are control) b. Cell cycle histogram showing accumulation of HT29 cells in GzM phase of cell cycle after 15 {iM sulforaphane treatment (from Ref. 70) 27 12 o sa 2 0 - ao 9 % 10 - 0 24 Hours Figure 1.12: Percentage of Apoptotic Cells in HT29 Control Cells (squares) and HT29 Cells Treated with 15pM Sulforaphane (circles) (6om Ref. 70) 28 After a 24 hour sulforaphane incubation, bax, an apoptotic gene was expressed, but p53 expression remained unchanged. An anti-apototic gene, bcl-2 was not expressed. Apoptosis in this study was carried out via caspace enzyme cascade due to the proteolytic cleavage of its substrate PARP^. This study was significant because it illustrated that sulforaphane can induce programmed cell in a manner independent of p53. This information is valuable since approximately 70% of all cancers have either deleted or mutated p53 genes^'^. The cytotoxic effects of isothiocyanates are not completely understood but it has been shown that isothiocyanates can act under suppression mechanisms weakening genetic changes that can occur during neoplasia. The major antiproliferative eSects shown for isothiocyanates are structurally related and can be summarized into two chemical characteristics, reactivity and lipophilicity of the isothiocyanate. Each isothiocyanate contains essentially three functional groups that can participate in reactivity, primarily interfering with en^rme systems that control cell proliferation or viability®^ (Figure 1.13). The groups are the isothiocyanate functional group (N=C=S), the adjacent methylene group, and a functional group on the side chain such as an alkene, sulfur atom, ben^l group, or others. Since the N=C=S group is common to all isothiocyanates, other functional groups are significant in the degree of antiproliferation in malignant cell lines. Alkyl isothiocyanates with, longer chams lengths (n=4,5,6 vs. n=3) have a greater efiect on antiproliferation®. However, it has been shown that alkenyl isothiocyanates with increasing chain lengths decreases the effects of antiproliferation®'®. For sulfur containing isothiocyanates, an increasing oxidation state on the sulfur atom has an 29 o Various functional groups in side chain ■ N = C = S C H i Methylene hydrogens \ adjacent to N=C=S group Isothiocyanate group Figure 1.13: Proposed major reactive sites on isothiocyanates increasing effect on antiproliferation®^. Stereochemistry of isothiocyanates has little effect on antiproliferation^’®®. It has been suggested that the most important antiproliferative property of an isothiocyanate is its lipohilicity, or ability to pass through a ceil membrane as measured by water/oil partition coefficient®^’®^’™. The major bioactive isothiocyanates (in terms of antiproliferation) have low water/oil partition coefficients (Table 1.4). For example benzyl isothiocyanates and p-hydroxy benzyl isothiocyanate differ substantially in their antiproliferative potency. The water/oil partition coefficient for benzyl isothiocyanate is much less than p-hydroxy benzyl isothiocyanate suggesting benzyl isothiocyanate can pass through the cell membrane more readily and effect cell proliferation. Furthermore, isothiocyanates with similar methylene chemical shifts (an indication of reactivity) but with significantly lower water/oil partition coefficients show a mucli greater effect on 30 antiprollfération, again suggesting that lipophilicity is a governing factor in antiproliferation moreso than reactivity (Table L4)®. Native Glucosinolate water/oil Partition Coefficient ICsoCplVI) Sinigrin 0.015 <0.1 Gluconapin 0.628 24.0 Progoitrin ND 55.0 e-progoitrin 0.568 42.0 Sinalbin 0.468 320.0 Glucotropaeolin 0.006 <0.1 Glucoerucin 0.012 2.5 Glucocheirolin 0.580 6.0 Glucoraphenin 0.405 15.0 Table 1.4: Isothiocyanate water/oil partition coefficients and their respective ICso values, (IC 5o=The concentration, required to cause 50% growth inhibition) (Grom Ref. 69) Cellular uptake of isothiocyanates has recently been elucidated by studies using natural isothiocyanates and MCF-7 cells^\ Surprisingly, isothiocyanates have been shown to accumulate in cells to very high degrees, up to a millimolar level. This accumulation is essential in anticancer properties of isothiocyanates such as induction of 3L Phase H enzymes and malignant cell cytotoxicity. Cells gather isothiocyanates in the form of glutathione conjugates, but isothiocyanates pass through the cell membrane in the fiee form^^ Thus levels of glutathione and glutathione-S-transferase are critical in cellular uptake of isothiocyanates. Furthermore, isothiocyanate accumulation varies betweens cells due to varying levels of intracellular glutathione and varying activities of glutathione-S-transferases. Studies on several malignant cell lines (MCF-7, COS-7, and Hepa lclc7) showed that depleting levels of glutathione decreased isothiocyanate uptake and that increasing glutathione and glutathione-S-transferase increased isothiocyanate uptake^'. Initial rate constants for isothiocyanate uptake correlate strongly with Z"** order non-enzymatic glutathione conjugation rate constants, indicating that glutathione plays a central role in accrual (Figure 1.14). Isothiocyanates are taken in by simple diffiision and surprisingly independent of their respective lipophilicity. For example, sulforaphane, allyl isothiocyanate, benzyl isothiocyanate, and phenethyl isothiocyanate diSer approximately 700-fbld in range of oil/water partition coefGcients but differ only 4-fbld in initial uptake rates^L Furthermore, sulforaphane and phenethyl isothiocyanate have comparable initial uptake rates but differ in lipophilicity by 8-fbld. Thus, in regards to cellular accumulation, lipophilicity of isothiocyanates appears to be negligible, whereas glutathione conjugation is an essential feature. DU145, LNCaP, and PC-3 cell lines are commonly used malignant prostate cell culture models. Each of these cell lines is highly tumorigenic, malignant, and rapidly proliferating. These cell lines will be used principally m this research. The major characteristics of each cell line are listed in Table 15. 32 10 5 - ADyl-ITC ï 0 0 10 20 30 40 SO 60 Ratio of Specific QST activi^ CMCF-7/hOCTPl venusMCF-7Awt> 300 Benzyl-rrC m 250 200 I I 150 ADyi-rrC s|•a 2 100 SF 0t 2 3 4 Log P Values of rrCs Figure 1.14: a.) Initial uptake rates vs GST activity for isothiocyanates b.) effects of iipophilicity on initial uptake rates (ôom Ref. 71) 33 LNCaP PC-3 DU145 Source Lymph node Bone Brain lesion 50 yr old male 62 yr old male male with leukemia Androgen yes no no sensitivity Morphology mod-poor poor poor Acid yes no no Phosphatase PSA yes no no VitD receptor yes yes yes TGPp receptor no yes yes p53 normal deleted mutant Rb yes yes no Table 1.5: Characteristics of human prostate cancer cell lines. 34 1,4 Cruciferous Vegetables and Cancer in Animal Models There have also been many animal studies initiated to address the eSects of cruciferous vegetable feeds on malignant cell growth in vivo. The protective effects of cruciferous vegetables associated with a reduction in cancer risks can be grouped into two main mechanisms, which are acting as blocking agents or suppressing agents^®’^^. Blocking agents refer to compounds that can inhibit carcinogens from damaging DNA. This has been best described by induction of Phase H enzymes and inhibition of Phase I enzymes by giucosinolate hydrolysis products. Suppressing agents refer to compounds that interact with tumor cells and can slow or inhibit the progression of cancer. These studies have shown protective eSects against chemically induced tumorigenesis at various target organs by both blocking mechanisms (i.e. feeding protective components or diet followed by carcinogen administration) and by suppression (i.e. administering carcinogen followed by feeding compound or diet). A feeding study performed on male rats consisting of 10-40% Lyophilized cruciferous vegetable diet (from either broccoli or cabbage) was shown to increase the levels of antioxidant and anticarcinogenic glutathione in colon mucosal cells, but not liver cells^®. A non-cruciferous diet consisting of 10-40% lyophilized potato showed no such increase in glutathione levels. Male F334 rats were treated with azoxymethane (AOM) once per week for two weeks. Sulforaphane and phenethyl isothiocyanate and their MAC conjugates were administered by gavage after AOM insult ^ost initiation) and before AOM insult (initiation)^. After 10 weeks aberrant crypt foci were enumerated. Both sulforaphane and phenethyl isothiocyanate and their conjugates reduced the total number of aberrant crypt foci from 153 to 100-116 (p<0.01) and multicrypt fbci from 52 to 27-38 (p<0.05) 35 during post mitiatioa treatment However, only sulforaphane and phenethyl isothiocyanate were effective during the initiation stage. Studies on isothiocyanate effects on tumorigenesis indicate a specificity in activity. Benzyl isothiocyanate (BITC) and phenethyl isothiocyanate ^EITC) show good activity in inhibiting lung tumorigenesis by NNK with little toxicity’*’^®. The effects were shown given before or during carcinogen insult, but not after*°. Pure phenethyl isothiocyanate (25pM) was administered to A/J mice for four days. The mice were then challenged with a single dose of 10 [iM 4-(methylnitrosoamino)-l-(3-pyridyl)-l-butanone, a lung carcinogen. The investigators observed that prior administration of PEITC was able to inhibit tumor multiplicity in the lung by 90% and reduce the number of mice that developed tumors by 70%. However, in the case of benzyl isothiocyanate, DMB A tumorigenesis was only inhibited by a subsequent dose. Phenyl hexylisothiocyanate (PHITC) actually was shown to enhance colon tumorigenesis induced by azoxymethane both during and post carcinogen insult®’. BITC was shown to inhibit benzo[a]pyrene lung tumorigenesis in A/J mice whereas PEITC has no effect®”'®^. Yet PEITC is a potent inhibitor of lung tumorigenesis in A/J mice by NNK and esophageal tumorigenesis in rats by NBMA. whereas BITC was shown to be ineffective®®'*^. Furthermore, PHITC inhibits lung tumorigenesis in NNK treated A/J mice but enhances esophageal tumorigenesis in NBMA treated rats®*'®®. These studies indicate that the ability of these studied isothiocyanates to inhibit various tumorigenesis at certain organs can be attributed to the structure of the isothiocyanate and the specific P450 enzymes that they may be able to induce or inhibit. 36 Other studies have addressed the in vivo induction, of specific P-450 enzymes or glutathione-S-transferase isofbrms (GST) by cruciferous vegetable feeds or purified chemicals. One study showed the eSects of intact indoLyl glucosinolates (gIucobrassicin(GB) and neoglucobrassicin (NeoGB)) purified fiom broccoli on induction of hepatic CYP I A, 2B1/2 and 2E1 GST isoforms in male Wistar rats.^^ These researchers discovered that GB and NeoGB were able to induce several of the protective enzymes more so than their corresponding myrosinase breakdown products. Smith et al showed that sinigrin was able to suppress proliferation and induce apoptosis in colorectal crypts in rats previously treated with dimethylhydrazine®". Several studies have also been initiated to adt^ss the protective effects of cruciferous vegetable diets or purified compounds from cruciferous vegetable diets against administered carcinogens. For example, a diet consisting of aqueous broccoli sprout extracts and purified sulforaphane were shown to reduce incidence, multiplicity, and weight in mammary tumors in female Sprague-Dawley rats challenged with 7,12 dimethylbenz(a)anthracene (DMBA)‘*. Other isothiocyanates (BITC and PEITC) have been shown to inhibit metastasis and reduce lung colonization in syngemc mice. Isotopically labeled.indole-3-carbinol (13C) has been shown to decrease the effects of afiatoxin (AFBI) DNA binding in a dose dependent manner (0-400 ppm) when fed to rainbow trou^^. At this point there has been very little experimental data on the in vitro and in vivo effects of other giucosinolate hydrolysis products fiom cruciferous vegetables on animal models and malignant prostate cell lines. 1.5 Human and Rodent Metabolism of Glucosinolates and Isothiocyanates The bioavailability of glucosinolates and their hydrolysis products must be discernible before their chemopreventative aspects can truly be appreciated. The chemical diversity 37 of glucosinolates and the large variation, in hydrolysis products complicates the quantitative measure of bioavailabüity and uptake in humans^’^'*’^”. A small amount of giucosinolate degradation takes place in the upper intestinal tract, most likely the result of spontaneous chemical degradation. An in vitro study simulating peptic and small intestine digestion in pig found that giucosinolate losses ranged from 3 to 28% with indolyl glucosinolates being the most labile®*. A major portion of giucosinolate metabolism is carried out in the lower gastrointestinal tract. The degree of giucosinolate degradation in the gut depends on the amount of myrosinase present in the diet^*'®^’®®’®’. Mammalian cells do not possess myrosinase activity. However, the ability to degrade glucosinolates in largely influenced by intestinal bacteria such as Bacteroides, Peptostreptococcits, Enterococcus, Proteus, and Esherichia spp®^*®®. Little is known about the final structure of microbial giucosinolate degradation products due to possible rapid degradation or metabolism of the products. However, an in vivo study using 50 pmol sinigrin administered by gavage showed that as much as 100 nmol allyl isothiocyanate could be found in the cecal and colon of rats*®°. Glucosinolates may be partly absorbed because small amounts of glucosinolates have been isolated in the blood and urine^*. Also glucosinolates glucotropaeotin and sinigrin have been shown to be passively transported from mucosal to serosal side of everted sacs made from small intestine and colon ofhamsters^°\ Other minor pathways of metabolism have also been shown. Rats dosed with radiolabeled allyl isothiocyanate excreted approximately 15% as COz in exhaled afr and several unknown metabolites were found in the frces^®\ As far as tissue distribution, radiolabels were found to a large extent in liver, kidneys, and intestinal mucosal, while the heart and brain had very low levels 38 Glucosinolates and isothiocyanates are rapidly absorbed from the upper intestinal tract. The major metabolic pathway for isothiocyanates in conversion to iV-acetylcysteine derivatives(or mercapturic acids)Isothiocyanates are not only inducers of Phase H enzymes but are also substrates for glutathione-S-tranferases. The first step is initial conjugation with glutathione promoted by glutathione-S-transferase (Figure 1.14). The sulfhydryl group of the glutathione attacks the central carbon of the isothiocyanate. This results in the formation of glutathione dithiocarbamate. Through a series of enzymatic conversions by y-glutamyltranspeptidase, cysteineglycinase, and N- acetyltranferase, the final product is a i\T-acetylcysteine dithiocarbamate. iV-acetyl- cysteine derivatives are mostly excreted in the urine. The amount of firee isothiocyanates depends on the amount of myrosinase in the diet and the amount of isothiocyanates in the diet is directly proportional to dithiocarbamate excretion®^’®^'®^’*®^. Cooked cruciferous vegetables (with inactive myrosinase) and reduction in gut microflora by antibiotics or mechanical cleansing have both shown a reduction in dithiocarbamate excretion'®^. Several other vegetables such as tomatoes, com, green beans, and carrots yield no detectable dithiocarbamate excretion. Dithiocarbamate excretion is rapid, reaching a peak in approximately eight hours. Twenty four hours after cruciferous vegetable consumption, approximately 80% of dithiocarbamates are excreted^°^’^“’^°^. Dithiocarbamate levels return to baseline approximately 72 hours after vegetable consumption. Dithiocarbamate excretion in humans parallels isothiocyanate intake rather than giucosinolate intake. For isothiocyanates, the excretion is linear with intake and metabolism can account for as much as 40-50% of dose whereas giucosinolate 39 (glutathione) GST Y-GLU-CYS-GLY- y-g lu -c y s -g ly H - â s + R-NH-C=S R-N=C=S GGTP (isothiocyanate) CYS-GLY S R-NH-&S CG S-CYS-iVac S-CYS R-NH-C=S ^ -^^^R-NH-C=S (dithiocarbamate) Figure 1.15: Glutathione Conjugation of Isothiocyanates GST= Glutathione-S-transferase GCTP= Gammaglutamyl transpeptidase CG= cystein glycinase NAT=N acetyl transferase Nac= iV-acetylcysteine 40 metabolism accounts for only 10-20% of dose^‘^’^°^. Other minor metabolic pathways are oxidative metabolism and conjugation in the liver or by binary excretion. A recent feeding study in humans was performed to address the fate of glucosinolates and isothiocyanates consumption from cruciferous vegetables'®^. This study observed that a normal serving of broccoli containing approximately 0.8 pmol isothiocyanates/ g fresh weight resulted in 47% of the isothiocyanates metabolized. The major excreted metabolites of isothiocyanates are glutathione conjugates (excreted in bile) or M- acetylcysteine conjugates, dithiocarbamates and mercapturic acid (excreted in urine) in humans and rodents*®*. Isothiocyanates differ in their half-lives within the body, most likely due to Phase H enzyme affinity. However, essentially all metabolites from isothiocyanates are cleared from the body within 72 hours. Giucosinolate metabolism is much slower than isothiocyanate metabolism in humans (K 1/2 greater than 20 hours). It has been indicated that one of the first steps in giucosinolate metabolism is conversion to its corresponding isothiocyanate by gut microflora. However, a serving of broccoli containing equal amounts of glucosinolates and isothiocyanates resulted in only 10-20% of the glucosinolates metabolized vs. 44-56% of the isothiocyanates metabolized^"*'*®^. Male Sprague-Dawley rats were given a 50 mg/kg dose of sulforaphane and erucin (sulfide analog of sulforaphane) and their urine and bile was assayed for metabolites*®®. Five major metabolites were detected. The first metabolites found in bile were glutathione conj'ugates of sulforaphane and erucin. Another major metabolite was an aOcene analog of sulforaphane with a double bond at the C-3 position in the carbon chain, suggesting that sulforaphane can undergo oxidative metabolism. The final metabolites, identified in urme, were Aac conjugates of sulforaphane and erucin. Finally, erucin 41 conjugates found in rodents fed sulforaphane also suggest that removal of the sulfone group is a mechanism in sulforaphane metabolism(oxidation/reduction). Approximately 72% of the dose was excreted as iVac conjugates over 24 hours. The alkene metabolite of sulforaphane found can possibly be a desaturation/oxidation metabolite of cytochrome P450 (Phase I) enemies, since sulforaphane has been shown to inhibit Phase I enemies. The glutathione and cysteine adducts of sulforaphane are stable and after 4 hours <2% dissociate to give free sulforaphane. However an added thiol group (GSH or glutathione) can catalyze the dissociation and displace the isothiocyanate^^. After a glutathione incubation, 85% of the cysteine conjugate dissociates to give free sulforaphane after 4 hours. This phenomenon was also observed when the glutathione conjugate was incubated with cysteine, albeit to a lesser degree. Tins suggests that both conjugates can act as carbamoylating agents toward thiol groups. This release mechanism is important to re-circulate isothiocyanates in vivo to enhance their chemopreventative properties. 1.6 Implicatioas for Food Processing of Cruciferous Vegetables and Anticancer Properties Cruciferous vegetables are consumed as raw, cooked, and processed forms. The amount of phytochemicals lost during harvesting, processing, and cooking are paramount in terms of chemoprevention. It has been shown that as much as 40% of the phytochemicals (isothiocyanates such as sulforaphane, iberin, and erysolin) in broccoli can be lost just seven days after harvesting and 20%-90% of these isothiocyanates can be lost during normal processing and cooking (Table 1.6).“°’“^ Physical losses during cooking and food processing are significant since most glucosinolates and some isothiocyanates and other hydrolysis products are water-soluble 42 Processing DAH CHB Siilforapliane Sulforaphane nitrile Iberin i Fresh 1 127,2 36.7 51.2 51.1 Blanched 1 6.2 19.3 16.6 ND Cooked 1 33.6 24.9 13.6 9.4 Fresh 5 25.4 24.7 34.4 22.6 Cooked 5 8.9 22.6 6.9 4.2 Fresh 16 14.6 20.4 6.5 7,9 Cooked 16 7.1 16.9 2.7 3,3 w^ UAH=Days alter harvest CHB=CyanohydroxybiHene Table 1.6; Loss of isotliiocyaiiates and nilrilcs in broccoli dne to storage and cooking (from Ref, 110) and can leach, out mto cooking water. Glucosinolates also can degrade during normal cooking by as much as 40%, whereas indolyl glucosinolates have been shown to be more heat labile than non-indolyl glucosinolates^^ As such, glucosinolates are relatively more heat stable while isothiocyanates are more heat labile^Sulforaphane for example, has been shown to degrade almost completely at 60°C in aqueous solution^ Cruciferous vegetables are often thermally processed to inactivate myrosinase, the enzyme which degrades glucosinolates. Myrosinase, which is distributed throughout the plant tissue in myrosin cells, and glucosinolates are chemically segregated in plants. Myrosinase is relatively thermolabile when compared with other food related enemies and has been shown to lose 99.5% of its activity after heat treatment (100 “C for 15-60 min)23^4 Inactivation of myrosinase is performed to reduce the pungent, sulfury, and bitter flavors associated with giucosinolate hydrolysis products, but also greatly reduces the concentration of isothiocyanates and other phytochemicals. A recent method to inactivate myrosinase is by high pressure processing”'*’' Myrosinase was inactivated at 20°C at pressure between 300 and 500 MPa in broccoli. At 35‘’C and pressures <350 MPa, an inactivating temperature at atmospheric pressure, there was actually a retardation in thermal inactivation indicating an antagonistic or protective effect at low pressures. Furthermore, depending on the processing conditions, several aglycones can be formed aside from isothiocyanates including nitriles, thiocyanates, oxazolidine-2-thiones, hydroxynitriles and epithionitriles^’^'*. Thus, methods of food processing are potentially very important to help create and maintain the level of chemopreventative compounds in broccoli. Several studies have been performed to identify the thermal and acidic degradation products of isothiocyanates. The very pungent allyl isothiocyanate was 44 stored at 80“C. ia buffers at pH 4,6, and 8. Several degradation products were formed"^. In basic solution the major products were allylamine, allyl dithiocarbamate, diallylthiourea, and carbon disulfide. At neutral and acidic conditions the major products were carbon disulfide and allylamine. Previously unidentified products were also found such as diallylurea and diallyl sulfide. Allyl isothiocyanate can also rearrange to form allyl thiocyanates, however isomerization is favored more at neutral conditions rather than acidic or basic (Figure 1.16). Allyl isothiocyanate is much more stable under acidic conditions as degradation products were shown to increase with increasing pH. At 20“C, the same products were formed in each buffer but at much smaller amounts. Amounts of isothiocyanates lost during cooking are substantial. A study performed to simulate allyl isothiocyanate degradation during cooking (100°C for 1 hour) found that the major volatile degradation product was allylamine (Figure 1.17)“^. The major non volatile product was N,N diallylthiourea. Also produced were diallyl sulfide, diallyl disulfide, diallyl trisulfide, diallyl tetrasulfide, and allyl thiocyanates (Figure 1.18). This change from allyl isothiocyanate to the various degradation products was characterized by an odor change from pungent mustard-like odor to a garlic-like note. Sulforaphane degradation products were identified in essentially the same manner. The major non-volatile decomposition product was a dimer, N,N di(4- methyl(suifinyl)butyl thiourea(Figure 1.19)"^. The major volatile products were dimethyl disulfide, 1,2,4 trithiolane, 4-isothiocyanate-l-(methylthio)-I-butene, and 3- butenyl isothiocyante. The degree of temperature had a great effict on volatile concentrations, suggesting that higher temperatures accelerate the formation of volatile products. 45 Scheme 1. Major Path of AITC and ATC Isomerization CHîïîCH CHz=CH-CHz-N=C=S ^ N .CHj ^ C% =CH-C% -S-CsN allyl isothiocyanate pseudo-six Scheme 2. Heterolytic Cleavage of ITCs R-N=C=S R^SCN* R-S-CsN I K* + SCN* Scheme 3. Homolytic Cleavage of ITCs R—S—C=N ■ R* +■ SCN • R* + R—S—CsN ' R—N=C=S + R* Scheme 4. Decomposition of ITCs R-N=C=S ♦ R -N % ------R-NH-Ç-NH-R T HD* H amine dialkyl thiouna R-N=C=S — R-NH-C-0* — HiO isothiocyanate monothiocarbamate COS — ► HjS + COj Figure 1.16: Heat induced isothiocyanate-thiocyanate isomerhation and cleavage pathways for aiiyl isothiocyanate (firom Ref. 116) 46 ^j^^>V^N=C=S + H jO 0=C=( + ^j^v^N=C=S C x 2 - H Figure 1.17: Heat induced formation of allyiamine and dimeiization of allyl isothiocyanate (from Ref. 117) 47 i. 6 .ÿ^S^S^g-'SsgXV^ 1 10 S'®'S C r 2 i 0 Ô 8 s Figure 1.18: Major uoa-volatUe thermal degradation products of allyl isothiocyanate (from Ref. 117) 48 o II CH3— S—CHz— CH%—CH;—CH2— N = C = S + H2O o CH]— S—CH;—CH;—CHi—CH;—NH; O CHj— S—CH;—CH;—CH;—CH;—N =C =S ? CHj— S—CH;—CH;—CH;—CH;—N > = * CH]—S—CH;—CH;—CH;—CH;—N o " Figure 1.19: Major non-volatile thermal degradation product of sulforaphane (from Ref. 112) 49 1.7 Analysis and Detection of Phytochemicals from Cruciferous Vegetables There have been numerous comprehensive studies that have been performed to isolate and identify chemopreventative compounds from cruciferous vegetables’’*’^^®. The isolation, quantification, and detection methods are often complicated due to the reactivity and volatility of giucosinolate hydrolysis products. The earliest methods for isolating intact glucosinolates were gas chromatography and high performance liquid chromatography. The original gas chromatography method by Underwood and Kirkland converted parent glucosinolates to per-trimethylsilydesulfo glucosinolates before separation’ ’*. Others have expanded upon this method by using enzymes to desulfate the glucosinolates before analysis”®’’"’. A major limitation of this method was the large production of sulfuric acid. Many of the aglucone breakdown products began to be extracted with organic solvents. This was an important method as it stopped the need for steam distillation and large amount of water which had to be removed. MuUin and Sahasrabudhe began to lyophilize bulk vegetables and heat-inactivated myrosinase by the addition of hot buffer’^'’. UV detection began to be used to detect glucosinolates and hydrolysis products but excluded the detection of thiocyanates and nitriles. Kjaer et al’^ and Spencer and Daxenbieher’^® published the mass spectra of several isothiocyanates, nitriles, and oxazodinethiones. These methods lead to further advancements in both GC/MS detection and preparatory HPLC purification of isothiocyanates, indoles, nitriles, and oxazolidinethiones figures IJ20-123)’^^’’^ ’’^. Perhaps the most important separation and detection of intact glucosinolates, isothiocyanates and other hydrolysis products came in 1996 by Prestera et al and Zhang 50 1 1I I 8 a 3 < •• 1 1 w w i l L 1MI laji 14M wji MJi »m am mm Titoit(aua.) Figure 1.20; Gas Chromatogram of Isothiocyanates Sulforaphane and Sulforaphane Nitrile from Broccoli (ôomRef. 127) 51 a . ) I ? I, i . Im.T. m.1 m/z b.) £I ? I t I. f ) % — <— «I» JlilM mfz c.) 1 1 2 iw «1* m m/z Figure 1.21: Mass Spectra of a.) Siilforaphane, b.) Sulforaphane nitrile, and c.) 3- Butenyl Isothiocyanate (&om Ref. 127) 52 * 1 1 I g f 4- 15 20 25 30 40 Retention time [ntin] It c Q. 0 10 20 30 40 Retention time [irin] Figure 1.22: High Performance Liquid Chromatography for Various Isothiocyanates and Oxazoladines (firom Ref. 126) 53 l.Glucoiberin. 8. Gluconapin 0.3 « 2. Glucoiaphanin 9 .4-Hydroxyglucobrassicin 3 .2-hydroxyethyl 10. Glucobrassicanapin 3 4. Glucocheirolii 1L Glucoerucin 5. Progoitrin 12. Glucotropaeolia 1 .2 6. Smigrin 13. Glucobrassicia I 7. Glucosmalbia 14. Neoglucobrassicin 7 - 1 W 1 9 M s 9 Â a 1 2 a 1 i i 14 § V T-«—r- 10 1 5 20 25 MIN Figure 1.23: High Performance Liquid Chromatography Separation of Several Naturally Occurring Glucosinolates (&om Ref 129) 54 et al The first method facilitated in. analysis giucosinolate content by a series of 5 methods. In summary, reverse phase paired-ion chromatography (salt was tetradecylammonium bromide) was used on plant extracts. Endogenous glucosinolates were hydrolyzed to isothiocyanates by added purified myrosinase, which were quantified. Paired-ion chromatography counter ions were replaced with ammonium ions. This allowed for bioassays, negative-ion fast atom bombardment mass spectrometry, and high resolution NMR. These methods lead to the conclusive identification of the major glucosinolates in SAGA broccoli as 4-methylsulfinylbutyl (glucoraphanin) and 4- methylthiobutyl (glucoerucin) without derivatization or destruction of the glucosinolates. Quantification of isothiocyanates in plants is difficult due to the high reactivity and volatility of isothiocyanates relative to their parent glucosinolates. Traditionally, isothiocyanates are measured via UV/vis spectrometry due to the strong absorbance at 238 nm from the characteristic N=C=S group. Zhang et al dramatically improved the sensitivity of detection of isothiocyanates, dithiocarbamates, and carbon disulfide to the picomolar level This was performed by quantitatively converting isothiocyanates to their non-volatile NAC conj'ugates (dithiocarbamates) and then reacting them with 1,2 benzendithiol in a cyclocondensation reaction. The reaction product was 1,3 benzedithiole-2-thione can be measured spectroscopically at 365 nm (Figure 1.24). The reaction rates of isothiocyanates vary considerably, but thiocyanates, cyanates, isocyanates, cyanides, and other related compounds do not interfere with the cyclocondensation reaction. Tertiary isothiocyanates did not react with 1,2 benzendithiol probably due to steric hinderance of the central carbon atom or a reduced electrophilicity of the carbon atom due to the electron releasing 55 a. "5 R -N =C =S + R-NHz Rt R2 b. R-N=C=S + % S S f R-NHz HS SH (2) (1) c. S II ? Ri—NH—C—S—R2 R-f-NH—C—(S)n—R2 Dithiocarbamates Polythiocarbamates f S f Ri—NH'-C—NH—R2 Ri*—0 —C—S—R2 C *S Thioureas Xanthates Carbon disulfide Figure 1.24: Cyclocondensatioa reactiou a. general reaction scheme, b. Actual reaction scheme with isothiocyanates, c. Compounds that can react with benzene dithiol in a cyclocondensation reaction (Srotn Ref. 130) 56 methyl groups. Nonetheless, tertiary isothiocyanates have not been isolated from edible plants^°''^\ The exact reaction mechanisms are not known for dithiocarbamates and carbon disulfide but they are probably analogous to isothiocyanates. This method is useful because it can quantitatively determine the amoimts of isothiocyanates, dithiocarbamates, and carbon disulfide down to the picomolar level. This method’s limitation is that it fails to distinguish which types of reactants are present, as all isothiocyanates, dithiocarbamates, and carbon disulfides yield the same reaction product. 1.8 Research Objectives The objective of this research is to explore the biological properties of cruciferous vegetables in cancer chemoprevention by understanding the mechanisms of giucosinolate hydrolysis products aside from the well established Phase tt enzyme manifestation mechanism. This research is unique in that we are evaluating the ability of several cruciferous vegetable extracts and natural glucosinolates and hydrolysis products to inhibit malignant prostate cell proliferation, arrest cell cycle progression, and induce apoptosis (programmed cell death). Furthermore we are examining the ability of cruciferous extracts and purified compounds to influence cell-signaling pathways by inhibiting IGF-1 stimulated cell proliferation and anti-apoptosis. Finally, we are studying a novel method of food processing which can actually increase the concentration of chemopreventative isothiocyanates and indoles in situ. Overall, these findings will help illustrate a means of lowering prostate cancer risks by diet. 57 REFERENCES 1. Cancer Facts and Figures, American Cancer Society 200L 2. Clinton, S, Giovanucci, E. Diet, nutrition, and prostate cancer. Annu. Rev. Nutr. 1998; 18:413-40. 3. Powell I, Gelfand D, Parzuchowski J, Heilbrun L, Franklin A. 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Thermal degradation of allyl isothiocyanate in aqueous solution. / Agr, Food CAem. 1998;46:220-223. 118. Mcgregor D, MuUin W, Fenwick G. Analytical methodology for determining glucosinolate composition and content. J Amer OffAnal CAem. 1983;66:825-849. 119. Verberk R, Dekker M, Jongen W. Glucosinolates, in. Natural toxicants in foods, Ed by Watson DH. ShefiBeld Academic Press, Sheffield, pp 29-53.1998. 120. Underhill E, Kirkland D. Gas chromatography of trimethylsilyl derivatives of glucosinolates. J Chromatogr. 1971;57:47-53. 121. Persson S. A method fbr determination of glucosinolates in rapeseed as TMS derivatives. Proc 4'* int Rapeseed Cong, 1947.Giessen.381-392. 122. Thies W. Quantitative gas liquid chromatography of glucosinolates on a microliter scale. Fette, Seifen, XnstncA/n. 1976;78:231-245. 123. Hiltunen R, Huntikangas A, Drufra V. Determination if glucosides from Brassica species by gas capillary gas chromatography. Acta Pharm Fenn.l980;89:31-40. 124. Mullin W, Sahasrabudhe M. Effect of cooking of glucosinolates in cruciferous vegetables. Can Inst Food Sci TechnolJ,l97S;ll:50-55. 125. Kjaer A, Ohashi M, Wilson J, Djerassi C. Mass spectra of isothiocyanates. Acta ChemScand, 1963;17:2143-2141. 126. Spencer G, Daxenbichler M. Gas chromatography-mass spectrometry o f nitriles, isothiocyanates and oxazolidinethiones derived from cruciferous glucosinolates./ Sci Food Agric. 1980;31:359-367. 127 Chaing W, Donald, P., Leits, R. Gas chromatography/Mass spectrometry method for the determination of sulforaphane and sulforaphane nitrile in broccoli. /. Agr, Food Chem, 1998;46:1018-1021. 128. Matthaus, B., Luftman, H Glucosinolates in the family Brassicacea: Separation and identification by LC/ESI-MS-MS methods. J, Agr. Food Chem. 2000; 48, 2234-2239. 129. Prestera, T., Fahey, J., Holtzclaw, W., Abeygunawardana, C, Kachinski, J., Talalay, P. Comprehensive chromatographic and spectroscopic methods for the 68 separation and identification of intact glucosmolates. Anal. Biochem. 1996; 239, 168-179. 130. Zhang, Y., Wade, K., Prestera, T., Talalay, P. quantitative determination of isothiocyanates, dithiocarbamates, carbon disulfide, and related thiocarbonyl compounds by cyclocondensation with 1,2-benzenedithioL Xnnf Biochem. 1996; 239,160-167. 69 CHAPTER! CRUCIFEROUS VEGETABLE EXTRACTS INHIBIT MALIGNANT PROSTATE CELL GROWTH AND LOWER THE APOPTOTIC THRESHOLD Corey E. Scott^ Steven. K. Clmton% and Steven L Schwartz^ ^Department of Food Science and Technology Parker Hall, 2015 Fyffe Court Columbus OH, 43210 The OMo State University ^Department of Internal Medicine College of Medicine and Public Health Division of Hematology and Oncology Starling-Loving Hall Columbus OH 43210 The Ohio State University Keywords: Cruc^erous vegetables, broccoli, isothiocyanates, glucosinolates, HPLC, Prostate cancer 70 2.1 ABSTRACT A recent case-control study has shown a reduction in prostate cancer incidences in men who consume at least three servings of cruciferous vegetables (i.e. broccoli, cabbage, brussel sprouts, etc.) per week. Our current research eSbrts involve the characterization of potent extracts and individual components via high performance liquid chromatography (HPLC) from broccoli, broccoli sprouts, watercress, and brussel sprouts which possess bioactivity in malignant prostate cancer cell lines. HPLC analyses monitored at 235 nm yielded unique profiles from each vegetable. Each aqueous extract of similar concentration was serially diluted and added to cell cultures fbr 48 hours. Each extract inhibited proliferation in the studied malignant cell lines, but to differing degrees. The broccoli sprout extract inhibited cell proliferation to the greatest degree followed by watercress, brussel sprouts, then broccoli. Cell cycle analyses via flow cytometry indicated that the broccoli sprout extract arrests cell cycle progression at the S phase and watercress in the GiM phase of cell cycle. An HPLC preparatory fractionation was utilized to isolate the bioactive components from the broccoli sprout and watercress extract A total of 13 components were separately collected and evaluated in cell culture. We observed four components that caused at least a 70% infubitiott of proliferation in each of the malignant cell lines and three components that caused at least 50% inhibition of proliferation in the malignant cell lines. These findings will help lead to the 71 development of products for laboratory animal and human studies in prostate cancer prevention. 2.2 INTRODUCTION Prostate cancer is the second leading cause of cancer death among menL The risks of developing prostate cancer are largely influenced by dietary intake, environmental exposures, and genetic factors. Prostate cancer exhibits a relatively long latency period suggesting that diet and nutrient intake m ^ influence the onset and progression of this disease'. Evidence supporting the protective eSects of vegetable intake against cancers is strong and consistent. Of a total of 74 case-control studies, 46 studies (62%) found lower cancer risks associated with consumption of at least one type of cruciferous vegetable i.e. (broccoli, cabbage, kale, brussel sprouts, cauliflower)^ A recently published case-control study of adult males aged 40-64 found a 41% decrease in prostate cancer incidence in men who consumed three or more servings of cruciferous vegetables per week as opposed to many other fluits and vegetables^. This finding suggest that consumption of cruciferous vegetables may have a specific effect on reducing prostate cancer versus many other vegetables. Cruciferous vegetables (Brassica spp. ) i.e. broccoli, cabbage, brussel sprouts, watercress, kale, cauliflower, etc., are consumed fi’equently in a western diet and contain higli concentrations of a class of compounds called glucosmolates (GLs), which are hydrolyzed enzymatically to produce isothiocyanates (ITCs) and various other 72 hydrolysis products when the plant is damaged by insect infestation, mechanical processing or eating2.3,4,5 (Figure 2.1). Much of the chemopreventative data associated with cruciferous vegetable consumption deals with induction of detoxification enzymes (i.e. Phase H enzymes) and xenobiotic metabolism by glucosinolates and isothiocyanates found in cruciferous vegetables.'*’^’®’’^ Induction of phase H enemies (i.e. glutathione S-transferase, NAD(P)H:quinone oxidoreductase, giucuronosyltransferase, and epoxide hydrolase) can detoxify carcinogens or toxic electrophiles by destroying their reactive centers and by facilitating their excretion via conjugation before they can damage DNA. Isothiocyanates are monofimctional inducers in that they can actually inhibit Phase I enzymes, or enzymes that can activate procarcinogens^'^. Glucosinolates are relatively non-reactive towards carcinogens and have been shown not to induce Phase II enzymes. However, their enzymatic breakdown products, isothiocyanates, are very potent in the manifestation of Phase H enzyme induction®’^. The overall mechanisms for chemoprevention fi’om cruciferous vegetable consumption are incomplete and may involve several complex interactions. Thus, induction of Phase H enzymes is probably not the sole means of chemoprotection shown by cruciferous vegetable components. We hypothesize that known components and components yet to be identified in cruciferous vegetables can be involved in separate mechanisms that can lower prostate cancer risks such as inhibition of proliferation, disruption of the cell cycle, and induction of apoptosis in malignant prostate cells. Isothiocyanates have been shown to inhibit proliferation and induce apoptosis in malignant colorectal and erythroleukemic cell lines but the effects of cruciferous vegetable extracts and purified components on malignant prostate cells have not been 73 well documented*’®’^®’^^ The recent epidemiological data^ suggesting a protective role against prostate cancer by the consumption of cruciferous vegetables illustrates the significance of studying the above mentioned effects, fit this paper we employ high performance liquid chromatography to identify and isolate components from various cruciferous vegetables and examine the effects of individual components on cell viability, proliferation, and induction of apoptosis in three malignant prostate epithelial cell lines DU145, LNCaP (androgen sensitive), and PC-3. 2.3 MATERIALS AND METHODS 2 J.l Cruciferous Vegetable Preparation for Extraction Fresh cruciferous vegetables (broccoli, watercress, and brussel sprouts) were purchased from local Ohio produce companies. Broccosprouts® {Brassica oleracea italica, SAGA), a commercially available sprout with high levels of sulforaphane, was purchased from local grocery stores and used in this study. Each vegetable was thoroughly washed with 3 volumes of 70% ethanol and rinsed with 10 volumes of distilled water. A 300 gram portion of each vegetable (50 gram portion of Broccosprouts®) was separately placed in a blender (Osterizer Galaxy, I liter capacity) along with 300 ml of distilled water (100 ml distilled water with broccoli sprouts) and homogenized at maximum speed for 20 minutes at room temperature. Each solution was then lyophilized using a LabConco Lyph Con 4.5 fireeze dryer overnight The resulting powders were vacuum-sealed in aplastic pouch and stored at-20“C. For a rapid analysis, 74 vegetables were homogenized as previously stated and centrifuged at 7000 rpm fbr 5 min. Each supernatant was collected and normalized to similar UV/vis absorbance at 235 nm. Extracts were then sterile filtered and added to cell culture or made ready for HPLC analysis. 2.3.2 Aqueous Extractîou of Cruciferous Vegetables A 3-gram sample of each lyophilized powder was extracted with 20 ml of distilled water on a stir plate for I hour at room temperature. A 3 ml aliquot was taken from this solution through a 0J2 pm filter (Whatman) and immediately subjected to analytical HPLC analysis to profile components from each vegetable. 2 J.3 Analytical HPLC Analysis of Cruciferous Vegetable Extracts Aqueous cruciferous vegetable extracts were subjected to analytical HPLC analysis on a Waters 2690 system equipped with a 996 photodiode array (PDA) monitoring at 235 nm using Millennium software. A reverse phase Ctg column (Supelco, 24 cm x 4.6mm, 5um) was eluted isocratically with 90:10 waternnethanol mobile phase for 30 minutes with a flow rate of I ml/min at room temperature. Crude aqueous extracts were dissolved in 3 mis of mobile phase (methanolnvater) and injected as 50 pL aliquots. To obtain aqueous extracts of similar concentrations, each extract was measured at 235 nm in a I cm quartz cuvette using a Hewlett-Packard spectrophotometer with HP Chemstation software. The extracts were diluted to similar concentrations before added to cell culture. 2.3.4 Preparatory HPLC Fractionation and Isolation of Bioactive Components Compounds from crude extracts were collected using preparatory HPLC from cruciferous vegetables usmgaSP 8800 Spectra Physics Pump with a Waters 996 Photo Diode Array (PDA) monitoring at 235 am and a Cig preparatory column (Waters Bondak 75 ‘ 19 mm X 300 mm). Mobile phase was 90:10 water: methanol. A concentrated 1 ml aliquot from the crude extract was injected unto the HPLC system at a isocratic flow rate of 7 ml/min and components collected in glass vials at each respective elution time. Components were then separately sterile filtered and evaluated in cell culture for bioactivity. 2.3.5 Cell Culture Human prostate cancer cell lines (LNCaP, DU145, and PC-3) were cultured in RPMI media containing 10% heat inactivated fetal bovine serum, L-glutamine, and antibiotics streptomycin and penicillin in an atmosphere of 5% CO%l 95% 0% with fi-esh media supplied every 48 hours. For antiproliferation experiments, cells were seeded at 4x10^ cells per well in a 96 well plate and incubated as above overnight. One day after seeding, the cells were challenged with media containing various concentrations of crude cruciferous vegetable extracts and incubated fbr 48 hours. Crude extracts were added as 500 pL aliquots into 10 mis (1:20) of cell culture medium. This stock solution was serial diluted 3-fold and added to cell culture. 2.3.6 I n v itr o Antiproiiferation Assay Viable cell numbers were microscopically determined by counting cells using trypan blue exclusion and a hemocytometer. Cell proliferation was judged using an MTS- tetrazolium colorimetric assay. In this method, mitochondria of living cells metabolize the tétrazolium salt which yields a color change in the media. The intensity of the final color is proportional to the number of live and growing cells. Dead cells do not metabolize the salt. 4x10^ cells/well were placed into a 96-well micro titer plate fbr 24 hours. Fresh media containing cruciferous vegetable extracts were then added and 76 incubated, for 48 hours. Extracts of similar concentratioa were serially diluted 3 fold. After 48 hours, the MTS reagent was added to each well and cultures were incubated an additional 2 hoiu-s at 27°C before measuring absorbance at A 490 nm. 2.3.7 Cell Cycle and Programmed Cell Death (Apoptosis) Analysis Cruciferous extracts efiects on cell cycle progression and programmed cell death were analyzed by using a TUNEL Assay with an Intergen ApoTag Kit (Purchase, NY). Cell cycle determinations were based on the measurement of DNA content labeled with propridium iodide. Human prostate cancer cells (approx 3x10® ) were growth in 125 cm" flasks and incubated as above and exposed to control media and cruciferous vegetable extracts for 0-72 hours. The cells were then trypsinized and fixed with 1% paraformaldehyde and stored in 70% ethanol at -40°C. For cell cycle and apoptosis analyses, the cells were resuspended in 150 pL buffer containing RNase, andpropidium iodide. Cytometric analysis was performed using a Coulter Elite Flow cytometer through a 630 nm LP filter. We evaluated apoptosis via the TUNEL assay which uses a TdT enzyme and fiuorescien isothiocyanate (FITC) to flourescently label DNA stand breaks at the 3’-hydroxyl group. 2J.8 Statistical Analyses Data represent means ± SEM. Data were analyzed by ANOVA using Statview or Sigmastat software. 77 2.4 RESULTS 2.4.1 Component Profiles from Aqueous Cruciferous Vegetable Extracts HPLC chromatograms from cruciferous vegetables broccoli, Broccosprouts®, brussel sprouts and watercress are shown m Figure 2 2 . The HPLC chromatograms from the cruciferous vegetable extracts reveal several peaks absorbing in the region of235 nm, consistent with published reports of glucosinolates, isothiocyanates, and other hydrolysis products^'t We have identified peaks from the chromatograms of several cruciferous vegetables by online spectra generated with PDA detection and co-elution with authentic standards. The identified peaks represent indole-3-carbinol and four isothiocyanates, sulfbraphane, iberin, erucin, allyl isothiocyanate, and phenethyl isothiocyanate. Some of the unidentified peaks that elute early (between the two and six minutes) on the chromatograms are most likely glucosinolates since a decrease in area of these peaks occurs when purified myrosinase is added (Figure 2.3). None of the other separated components observed were myrosmase sensitive. 2.4.2 Effects of Cruciferous Vegetable Extracts on Malignant Prostate Cell Gro^vth Human prostate cancer cells were grown under conditions stated in materials and methods and treated with crude aqueous extracts ofbrbccoli, broccoli sprouts, watercress, and brussel sprouts for 48 hours. A dose dependent inhibition in cell growth in the 78 presence of each of the extracts compared to the control cell culture media-only samples was observed. Overall, the broccoli sprout extract had the greatest effect on growth inhibition followed by the watercress extract, then brussel sprout and broccoli (Figure 2.4-Figure 2.6). The inhibitory effects of the extract varied within each cell line. PC-3 cell growth was more sensitive at lower extract concentrations than LNCaP or DU145 cells. LNCaP cells were most sensitive to the broccoli sprout extract, followed by the watercress extract, while brussel sprout and broccoli extracts had similar inhibitory effects. DU145 cells were also most sensitive to the broccoli sprout and watercress extract. At high extract concentrations the decrease in cell growth was similar in each of the cell lines. We observed the broccoli sprout extract to inhibit cell proliferation to the greatest degree in each of the cell lines. We also observed that its HPLC profile contained more components than each of the other extracts. A HPLC preparatory column was used to isolate and evaluate the potencies of the specific components in cell culture. A total of 13 components were isolated and collected and made to similar concentrations (Figure 2.7). Each was then separately added to PC-3 cell culture. The greatest degree of antiproliferation (>70% at highest dose) were observed in components# 8,10-13. Components # 8 and 9 were less effective causing greater than 50% inhibition at highest dose. Components #3 and #7 caused greater than 10% cell growth inhibition and the remaining components had nonsignificant effects at their highest doses. We have identified components # 8 ,9,10, and 11 as erucin, mdole-3-carbinol, iberin, and sulforaphane respectively based upon generated on-line UV spectra and co-elution with authentic standards. Other components #2,4-6 are possibly glucosinolates since they are sensitive to added myrosinase. 79 2.4.3 Effects of Cnicfferous Vegetable Extracts oa Cell Cycle Progression Cell cycle analyses via flow cytometry were used to identify at which phase of cell cycle the extracts exerted their antiproliferative effects. PC-3 and Dul45 cell lines were tested with the broccoli, BroccoSprout® and watercress extracts. The extracts had differing effects on cell cycle progression in the cell lines. Cells were seeded, exposed to extracts, and made ready for cell cycle analysis for 0-72 hours. The broccoli and BroccoSprout® extract caused a buildup of cell population in the S phase of cell cycle and a decrease in cell population in the Gi/Go and G%M phases of cell cycle at 24 hours (Figure 2.8 and Table 2.1). After 48 hours, there was a decrease in the proportion of cells in the S phase and a buildup of Sub Go particles. The watercress extract caused an increase in the proportion of cells in the S and G%M phase (Figure 2.9). After 48 hours, the proportion of cells m the GzM phase drops (by 13.3%) and there is a simultaneous increase in sub-Go particles (26.3%) relative to control. 2.4.4 Effects of Extracts on Apoptosis (Programmed Cell Death) Induction In the presence of the BroccoSprout and watercress extracts, we observed an increase in sub-Go particles and fragments consistent with DNA fragmentation during apoptosis via flow cytometry. We further evaluated apoptosis on the basis of DNA strand break labeling by TdT en^one and phosphatidylserine fluorescent staining with FTTC. In the presence of the broccoli sprout and the watercress extract there was an increase m FTTC staining. The percentage of apoptotic cells was much larger in the watercress treated samples than in the BroccoSprout® treated samples. After 72 hours, there were 45.9% 80 apoptotic cells in the watercress treated sample versus 10.4 % in the BroccoSprout® sprout treated sample (Figure 2.12). 2.5 DISCUSSION Several studies have suggested the reduction of cancer risks by consumption of cruciferous vegetables^'^ Cruciferous vegetable extracts and pure components such as isothiocyanates have been shown to be potent inducers of detoxification and antioxidant enzymes which may be involved in carcinogen metabolism®. Our endeavors were to test the efiects whole aqueous extracts from commonly consumed cruciferous vegetables on cell growth and viability in three well established malignant prostate cell lines. While using solvents such as methanol, acetonitrile, or methylene chloride would have been more efGcient extraction solvents, our endeavors were to mimic human mastication and consumption of these vegetables^^'^°. Thus water was chosen as our extraction solvent and was effective in extracting glucosinolates, isothiocyanates, and indoles from the vegetables. There are several different varieties of each vegetable available to consumers and a limitation of this study is that the data shown can only be valid for the noted varieties of broccoli, brussel sprouts, watercress and BroccoSprouts® used in this study. Vegetables of the same variety were obtained locally over a period of 16 months. The component profiles from each vegetable were quite consistent within this period. There was some variability as far as specific component concentrations within the vegetables 81 during this time possibly due to changes in climate, plant age, soil composition, etc. Thus each vegetable extract was normalized to the same concentration, using UV absorbance throughout this study. From the HPLC chromatograms, we observe unique component profiles with each vegetable. Initially the chromatograms were monitored between wavelengths of200 and 600 nm. Almost all of the components firom the aqueous extracts were detected between 225 and 255 nm, consistent with glucosinolate and isothiocyanate detection^’®. Indeed, each aqueous extract was largely composed glucosinolates and isothiocyanates as we observed several components to be sensitive to myrosinase and have identified several isothiocyanates and indoles by co-elution with authentic standards. There is also the possibility that the extracts could contain glucosinolate hydrolysis products such as thiocyanates and nitriles, which are not detectable by UV absorbance^’'*’®’^^*^®. Nitriles and thiocyanates are cytotoxic (however to a lesser degree than isothiocyanates) and could have had an effect on our cell proliferation assays. However, these products appear at low pH and higher ion concentrations'*’®. Our aqueous extracts were normalized to pH 7.0 for cell culture. Each of the extracts inhibited cell growth in the cell lines with increasing dose. The difference in potency of the extracts was attributed to the specific components in the extracts as each extract was made to a similar concentration before addition to cell culture. The aqueous BroccoSprout® extract caused the greatest amount of growth inhibition in each of the cell lines studied. From the HPLC chromatogram, there was a large concentration of isothiocyanates erucin, iberin, sulforaphane, and indole-3-carbinol. Each of these components have been shown to mhibit in vitro cell growth in malignant 82 cell lines suggesting why we observed a greater degree of proliferation with the Brocco Sprout® extract relative to the other extracts used. Furthermore, we detected several more components in the Brocco Sprout® extract than any of the other extracts that may have contributed to the growth inhibitory effects. From our fractionation study of Brocco Sprouts®, we observed components that had very little effect on cell proliferation. These components were also myrosinase sensitive suggesting that they are glucosinolates. Glucosinolates have been shown to have a very weak effect on cell proliferation in other in vitro studies®’®. Components #12 and #13 have not been identified in this study, however we have observed that components #12 and #13 are degradation products of iberin and sulfbraphane respectively from our pure standards. They are perhaps sulfhydryl reduction products (i.e. removal of oxygen atom from the sdfur atom in the carbon chain) of iberin and sulfbraphane. Components #12 and #13 elute after iberin and sulfbraphane indicating that they are more lipophilic, which is consistent with the conversion of a sulfrnyl or sulfone group to a sulfhydryl group. We also detected isothiocyanates phenethyl isothiocyanate and allyl isothiocyanate in the watercress and brussel sprout extracts respectively. But these isothiocyanate concentrations were to a lesser degree than isothiocyanates detected in the broccoli sprout extract. The least potent in inhibiting growth was the broccoli extract although it showed activity in all three cell lines. The broccoli extract largely consisted of glucosinolates, as many of the components were myrosinase sensitive. The difference in isothiocyanate concentration between mature broccoli and broccoli sprouts is large in that mature broccoli consists of approximately 98% glucosinolates and broccoli sprouts can be 10-100 times more concentrated with isothiocyanates^. Furthermore, glucosinolates have been shown in 83 other studies to not influence malignant cell growth up to 500 pM concentrations®, again suggesting why the broccoli extract was the least potent in inhibiting cell growth. Both normal and malignant cell lines do not posses myrosinase activity, thus the in vitro conversion of glucosinolates in the extracts to indoles or isothiocyanates is insignificant®’®. Since the broccoli sprout and watercress extract were the most potent in inhibiting cell growth and PC-3 cells were most sensitive, we used PC-3 cells to evaluate the effects of the extracts on cell cycle progression. The Brocco Sprout®, broccoli, and watercress extracts had different effects on cell cycle progression. The broccoli and Brocco Sprout® extracts caused a build-up and arrest in the S phase of cell cycle while the watercress and brussel sprout caused a build-up in the GzM phase of cell cycle. While the broccoli and Brocco Sprouts® were of different varieties, there were some consistent components in the chromatograms, indole-3-carbinol, small amounts of iberin and sulfbraphane, and unidentified compounds, which suggests why these extracts both had an efiect on the S phase of cell cycle. The effects of the extracts were cytotoxic rather than cytostatic as the cells began to die after the addition of the extracts. From the flow cytometry histograms, we observed sub-Go particles suggesting DNA fragmentation associated with apoptosis. We further explored the ability of the extracts to induce an apoptotic death in the cell lines m by DNA stand breaks staining with FTTC labeling. There was more cell death in the Brocco Sprout® treated sample than m the watercress treated sample however there was much more apoptosis in the watercress treated sample. This is best explained by the components in each extract. La. the Brocco Sprout® extract there were several 84 components which were potent in inhibiting ceil proliferation and causing cell death, whereas in the watercress extract, much of the cytotoxic potency resided in a single component, which was determined to be phenethyl isothiocyanate ôom fractionation studies. In the presence of the Brocco Sprout® extract, the cells were most Ukely overwhelmed with the large concentrations of cytotoxic components, which can explain the large degree of cell death and smaller degree of apoptosis. In the watercress treated sample, there was essentially one major active component (phenethyl isothiocyanate) that could exert pro-apoptotic effects on the cell lines. We observed that the extracts could induce apoptosis (approximately 10 to 45%) in PC-3 and DU145 cell lines. This is particularly important, as PC-3 cells are p53 deleted, and the extracts can induce apoptosis independent of p53. Induction of programmed cell death, in p53 null cells is highly significant in cancer prevention as greater than 70% of all cancer cells have abnormal or altered p53 genes'^ 2.6 CONCLUSIONS This study has shown that extracts from cruciferous vegetables posses bioactivity in cell culture and are able to influence proliferation, cell cycle, and programmed cell death in malignant prostate cell lines. The most potent extracts ^rocco Sprout® and watercress) were highly concentrated with isothiocyanates, whereas the least potent, broccoli, was not. This suggests that isothiocyanates maybe responsible for the bioactivity observed. 85 2.7 REFERENCES 1. Cancer Facts and Figures, American Cancer Society 2001 2. van Poppel G, Verfaoeven D, Verhagen H, Goldbohm BL Brassica vegetables and cancer prevention, in Advances in Nutrition and cancer 2. Zappia: 1999: New York. 3. Cohen JH, Kristal AR, Stanford JL. Fruit and vegetable intakes and prostate cancer risk. 2000;92:61-68. 4. Fenwick GR, Heaney RK, Muilin WJ. Glucosinolates and their breakdown products in food and food plants. Crit. Rev. Food Set & Nutr. 1983;18:123-201. 5. Mithen R, Dekker, M., Verkerk, R., Rabot, S., Johnson, I. The nutritional signiScance, biosynthesis and bioavailability of glucosinolates in human foods. /. Sci. FoodAgr. 2000;80:967-984 6. Zhang Y, Talalay P, Cho CG, Posner GH. A major inducer of anticarcinogenic protective eotymes from broccoli: isolation and elucidation of structure. Proc. Nat. Acad. Sci. 1992;89:2399-403. 7. Fahey JW, Zhang Y, Talalay P. Broccoli sprouts: an exceptionally rich source of inducers of enzymes that protect against chemical carcinogens. Proc. Nat. Acad. Sci. 1997;94:10367-10372. 8. Nastruzzi C, Cortesi, Rita., Esposito, E., Menegatti, E., Leoni, 0., lori, R., Palmieri, S. In vitro antiproliferation activity if isothiocyanates andnitriles generated by myrosinase-mediated hydrolysis of glu cosinolates from seeds of cruciferous vegetables./. Agr. Food Chem. 2000;48:3572-3575. 9. Leoni 0, lori R, Palmieri S, et al. Myrosinase-generated isothiocyanate from glucosinolates: isolation, characterization and m vitro antiproli&rative studies. Bioorg.&Med. Chem. 1997;5:1799-1806. 86 10. Sasaki T, Keita, K., Yasushi, U., Ozawa, Y., Shimizu, J., Kanke, Y., Takita, T. Effects of isothiocyanates on growth and metastaticity on BI6-FI0 melanoma cells. M 11. Gamet-Payrastre L, Li P, Lumeau S, etal. Sulfbraphane, a naturally occurring isothiocyanate, induces cell cycle arrest and apoptosis in HT29 human colon cancer cells. Cancer Res, 2000;60:1426-33. 12. Huang C, Ma, W., Li, J., Hecht, S., Dong, Z. Essential role of p53 in phenethyl isothiocyanate induced apoptosis. Cancer Res. 1998;58:4102-4106. 13. Uda Y, Kurata T, Arakawa N. Effects of pH and ferrous ions degradation of glucosinolates by myrosinase. AgricBiol CAem. 1986;50:2735-2740. 14. Challenger F, The natural mustard oil glucosides and the related isothiocyanates and nitriles.1959. In: Aspects of the Organic Chemistry of Sulfur, Butterworths, London.pp. 115-161. 15. Palmieri S, lori R, Leoni 0. Myrosinase from Sinapis alba L : A new method of Purification fbrglucosinolate analysis Fbotf CAem. 1986.;34:138-140. 16. Rosa E, Heaney R, Fenwick G, Portas C. Glucosinolates in crop plants. Horticult rev.l997;19:99-2l5. 17. Bennet R, Dawson G, Hick A, Wallsgrove R. Glucosinolate biosynthesis: further characteristic of the aldoxime-forming microsomal monooxygenases in oilseed rape leaves. Plant PhysioLl995;l09:199-305. 18. Cover C, Hsieh, S., Cram, E., Hong, C., Riby, L, Bjeldanes, L., Firestone, G. fridole-3-carbinol and tamoxifen cooperate to arrest cell cycle of MCF-7 human breast cancer cells. Cancer Res. 1999;59:1244-1251. 19. Fenwick G, Griffrths N, Heaney R. Bitterness in brussel sprouts {Brassica oleracea L vargemmifera): the role of glucosinolates and their breakdown products. JScL Food Xgr.l982;34:73-80. 20. van Doom H, van der Kruk G, van Holst G, Raaijmakers-Ruijs N, Postma E, Groeneweg B, Jongen W. The glucosionaltes sinigrin and progoitrin are important determinants for taste preference and bitterness ofbrussel sprouts. J Sci. Food Agr.l998;78:30-38. 87 Broccoli Spout Extract Phase Control 48 hours 72 hours Sub'Go 0.8+/-0.3 5.8 +/-1.2 36.5+/-4.6 Gi/Go 60.2+/-3.5 48.0+/-2.9 34.7+/-63 S 11.5+/-3.9 26.7+/-4.8 8.7+/-23 GzM 20.7+/-5.6 25.9+/-6.2 13.7+/-4.4 Broccoli Extract Phase Control 48 hours 72 hours Sub'Gg 0.8+/-03 14.4+/-6.8 36.8 +/- 7.8 Gi/Go 60.2+/-3.5 413+/-9.6 26.3 +/- 7.6 S 11.5+/-3.9 24.1+/-5.3 73+/-3.4 GiM 20.7+/-5.6 14.3+/-33 17.4+/-4 J Watercress extract Phase Control 48 hours 72 hours Sub-Go 0.8+/-0.3 14.8+/-3.4 26.3+/-3.7 G i/G q 60.2+/-3.5 39.6+/-4.6 22.4+/-5.1 s 11.5+/-3.9 16.1+/-4.9 15.9+/-3.3 GzM 20.7+/-5.6 33.4+/-5.2 20.2 + / - 6.8 Table 2.1: Percentages of Cells in Each Phase of Cell Cycle (means +/- SE) 88 Broccoli Sprout Extract Control 48 Hours 72 Hours % apoptotic cells 3 .2 +/- 0 .6 8.7+/-2.1 10.4+/-2.6 Broccoli Extract Control 48 Hours 72 Hours % apoptotic cells 3.2 +/- 0.6 6 .6 +/-3.3 12.5+/-4.7 Watercress Extract Control 48 Hours 72 Hours % apoptotic cells 3 .2 +/- 0 .6 24.8+/-9.4 45.9 +/- 10.4 Table 2.2: Percent apoptotic cells in treated samples 89 S-P-D-Glucose / R-C NOSO3- Myrosinase H%0 R-C +D-GIucose NOSO^- -HSO pH 7 Low pH Unclear ▼ 1 r u R-N=C=S R-CN R-S-C=N Isothiocyanate Nitrile Thiocyanate Figure 2.1: Glucosinolate hydrolysis 90 idole-3-carbinol Broccoli Endole-3-carbinol Broccoli Sprouts lUiforaphane Watercress pheaethyl ITC Brussel Sprout 0 Retention time, min 30 Figure 2.2: HPLC chromatograms from cruciferous vegetables 91 Glucosinolates No added myrosinase Excess myrosinase I ' ' I ' ■ I 5.00 10.00 15.00 20.00 25.00 30.00 Minutes Figure 2.3: Effects of added myrosinase on Brocco Sprout® extract 92 120 100 - 80- o Ü 40 - # Broccoli - O - Broccoli sprouts 2 0 - - V " Brussel Sprouts Watercress 0.1 1 10 100 1000 D ilu tio n Figure 2.4: Effects of cruciferous vegetable extracts on DU14S cell growth 93 1 2 0 100 - 8 0 - = 6 0 - 4 0 - - V " Broccoli “ O " Broccoli sprouts 2 0 - Brussel Sprouts Watercress 1 10 100 1000 Dilution Figure 2.5: EfTects of cruciferous vegetable extracts on LNCaP cell growth 94 120 100 8 0 oI 6 0 ■« o 4 0 # Broccoli - O - Broccoli sprouts 20 - V " Brussel Sprouts Watercress 0 0.1 1 10 100 1000 Dilution Figure 2 .6 : Effects of cruciferous vegetable extracts on PC-3 cell growth 95 a No significant growth effects ■ 10-20 % growth inhibition m 40-50% growth inhibition indoIe-S-carbiool ■ 70-80% growth inhibition ^ erucin sulfbraphane Figure 2.7: HPLC preparatory fractionatioa of Brocco Sprout extract 96 Control 24 hours 48 hours Figure 2.8: Effects of Brocco Sprout® extract ou cell cycle progression 97 Control 24 hours 48 hours Figure 2.9: Effects of watercress extract ou cell cycle progression 98 ‘f n - T I Control T'T 1 24 hours 48 hours Figure 2.10: Effects of broccoli extract ou cell cycle progression 99 Control Viable cells □MA L IN H-il Apoptotic cells extract Um 9 DNA LIN Si Apoptotic cells 72 Hr Watercress g extract -• e Figure 2.11: Apoptotic effects of watercress extract on PC-3 cells 1 0 0 CHAPTERS THE EFFECTS OF NATURAL ISOTHIOCYANATES, SINIGRIN, AND INDOLE COMPOUNDS FROM CRUCIFEROUS VEGETABLES ON PROSTATIC AND MALIGNANT PROSTATE EPITHELIAL CELL PROLIFERATION, CELL CYCLE, AND CELL VIABILITY Corey E. Scott\ Steven K. Clinton*, Steven J. Schwartz' 'Department of Food Science and Technology Parker Hall 2015 FyfFe Court Columbus OH, 43210 The Ohio State University ^Department of Internal Medicine College of Medicine and Public Health Division of Hematology and Oncology Starling-Loving Hall Columbus OH 43210 The Ohio State University K ^vords: isothiocyanates, sulfbraphane, mdole~3-carbmol, glucosinolates, prostate cancer 1 0 1 3.1 ABSTRACT Cruciferous vegetables contain relatively high concentrations of a class of compounds called glucosinolates, which can be hydrolyzed enzymatically to yield isothiocyanates (ITCs) and indoles when the plant is harvested or consumed. The effects of eight naturally occurring ITCs from cruciferous vegetables (sulfbraphane, iberin, iberverin, phenethyl isothiocyanate, erysolin, allyl isothiocyanate, benzyl isothiocyanate, and erucin), an isothiocyanate cysteine conjugate (benzyl Cys) two indole compounds, brassicin and indole-3-carbinol, and a natural glucosinolate (sinigrin), on in vitro cell growth and alterations of ceUcycle in three human prostate cancer cell lines, PC-3, LNCaP, and DLT145 was studied. Cell proliferation was analyzed using an MTS- tetrazolium colorimetric assay. Each of the ITCs, conjugates, and indole compounds were found to have an inhibitory effect on the growth of each cell line. The concentration of ITCs which caused 50% cell growth inhibition (IC 50) ranged from 23 pM to 59.4 uM, with benzyl isotfiiocyanate, phenethyl isothiocyanate, and sulfbraphane having the most potent effects on inhibiting cell proliferation. Proliferation of each of the cell lines was significantly decreased (80-95%) at ITC concentrations above 200 pM. The degree of cell antiproliferation by ITCs varied among the three cell lines. Cell cycle analyses via flow cytometry suggest an effect on the GiM phase of cell cycle. The indole compounds also had an effect on cell proliferation but to a much lesser extent than the isothiocyanates. The IC 50 for the mdole compounds were as much as 923 pM and 128.3 102 jiM for mdoIe-3-carbmoI and brassicin. respectively. The glucosinolate, sinigrin, showed no effects on inhibiting cell growth or effecting cell viability at concentrations up to 250 [iM. Interestingly, non-malignant prostatic epithelial cells were much more resistant to the isothiocyanates, indole compounds, and glucosinolates. At 50 piM concentrations of each isothiocyanate the normal prostate maintained more than 80 % of their cell growth and viability compared to the media only control. The direct antiproliferative activity shown for several isothiocyanates and indoles for the malignant cell lines and relative resistance to isothiocyanates and indole compounds of the normal prostate cells suggests another role to help prevent prostate cancer by diet or chemoprevention. 3.2 INTRODUCTION Strong evidence from both epidemiological and experimental studies has linked lower cancer incidences with diets high in cruciferous vegetable consumption^"^. Cruciferous vegetables contain large quantities of glucosinolates and their hydrolysis products such as indole compounds, nitriles, thiocyanates, and isothiocyanates (Figure 2.1)^ Isothiocyanates, the major hydrolysis product, can participate in carcinogen metabolism in that they have been shown to be very potent Phase H enzyme inducers and can furthermore inhibit Phase I enzymes (Cytochrome P450 enzymes) which can activate procarcinogens^‘°. Approximately 120 glucosinolates have been identified and they vary in there relative bioeffects in Phase H en^me manifestation^^ Some glucosinolates and their other hydrolysis products have either been shown not to induce Phase II en^mes, 103 induce primarily Phase I enzymes, or be very poor inducers of Phase H enzymes. When a cruciferous vegetable is consumed, there are a variety of breakdown products available to the body, and the means of chemoprevention shown in epidemiological studies can be a feature of several mechanisms aside from Phase H enzyme induction^'^\ Isothiocyanates have been shown to participate in chemoprevention blocking mechanisms such Phase H enzyme manifestation, but the effects of glucosinolates, isothiocyanates, and indoles on suppression mechanisms, such as inhibition of cell proliferation, arrest of cell cycle progression, and induction of apoptosis in malignant cell lines has not been well addressed relative to the xenobiotic metabolism studies. Isothiocyanates have been shown to inhibit proliferation in malignant cell lines. For example, several isothiocyanates generated from glucosinolates have been shown to inhibit K562 human leukemic cell lines, B60 melanoma cell lines, and MCF-7 breast cancer cell lines'"*^'*. In a more comprehensive study, sulfbraphane, a naturally occurring isothiocyanate in broccoli, was shown to inhibit DNA, RNA, protein, and phospholipid synthesis by 30% in as little as one hour in HT29 colorectal cancer cells at a 15 pM concentration^^. Sulfbraphane arrested cycle progression at the GzM phase and also caused cell death via apoptosis by activation of p53 and caspase emtymes. Although these studies have addressed the effects of isothiocyanates and nitriles on several different cell lines, the effects of purified components (i.e. isothiocyanates, glucosinolates, and indoles) on normal and malignant prostate cells have not been well documented. In tfus study our aim is to examine the effects of natural purified components from cruciferous vegetables which are; glucosinolate (sinigrin), isothiocyanates (sulfisraphane, iberm, iberverin, phenethyl isothiocyanate, erysolin, allyl isothiocyanate, benzyl isotfiiocyanate, and erucin), benzyl 104 cysteine (major human metabolite of benzyl isothiocyanate), and indoles (indole-3- carbinol and brassicin) on cell viability, proliferation, cell cycle, and induction of apoptosis in non-malignant prostate epithelial cells and three malignant prostate epithelial cell Lines (DUI45, LNCaP, and PC-3). 3.3 Materials and Methods 3.3.1 Analytical HPLC Analysis of Glucosinolates, Isothiocyanates, and Indoles Pure isothiocyanate standards of sulfbraphane, iherin, erucin, phenethyl isothiocyanate, benzyl isothiocyanate, erysolin, and iberverin and benzyl MAC were purchased from LKT BioChem (St. Paul MN). Allyl isothiocyanate was purchased from Aldrich Chemicals (St. Louis MO). See chemical structures in Appendix A. Isothiocyanates were resuspended in DMSG at a concentration of 100 mM and stored at - 40°C. For HPLC analysis each stock was dissolved in 3 ml of 90:10 distilled watenmethanol. A pure glucosinolate, sinigrin, was purchased from Sigma Chemicals (St. Louis MO) as a lyophilized powder and resuspended in water at a concentration of 100 mM and stored at-20°C. The isothiocyanates, glucosinolates, and indoles were subjected to analytical HPLC analysis on a Waters 2690 system equipped with a 996 photodiode array monitoring at 235 nm using Millennium software for purity. A reverse phase Ci8 column (Supelco, 24 cm x 4.6mm, Sum) was eluted isocratically with 90:10 watenmethanol mobile phase for 30 minutes with, a flow rate of I ml/mtn at room 105 temperature. Pure isothiocyanate, glucosinolate, and indole standards were dissolved in 3 mis of mobile phase (methanolzwater) and injected as 50 pL aliquots. 3.3.2 Cell Culture Human prostate cancer cell lines (LNCaP, DU-145, and PC-3) were cultured in RPMI media containing 10% heat inactivated fetal bovine serum, L-glutamine, and antibiotics streptomycin and penicillin in an atmosphere of 5% CO 2/ 95% O2 with fresh media supplied every 48 hours. Human prostatic epithelial cells (PrECs, Clonetics San Deigo, CA) from a 17 year old Caucasian male were maintained in prostate epithelial growth medium (PrEGM). For antiproliferation experiments, cells were seeded at 1x10"^ cells per well in a 96 well plate and incubated as above overnight. One day after seeding, the cells were challenged with, media containing pure glucosinolates, isothiocyanates, or indoles at concentrations from 0 to 50 pM for 48 hours. 3.3 JI n v i tr o Antiproliferatioa Assay Viable cell numbers were microscopically determined by counting cells using trypan blue exclusion and a hemocytometer. Cell proliferation was judged using an MTS- tetrazolium colorimetric assay. The cells mitochondria metabolize the tétrazolium salt which yields a color change in the media proportional to the number of live and growing cells. Dead cells will not metabolize the salt. 4x10^ cells/well were placed into a 96-well micro titer plate for 24 hours. Fresh media containing purified glucosinolates, purified isothiocyanates, or indoles were then added and incubated for 48 hours. After 48 hours, the MTS reagent was added to each well and cultures were incubated an additional 2-4 hours at 37“C before measuring absorbance at A 490 nm. 106 3.3.4 Cell Cycle and Programmed Cell Death (Apoptosis) Analysis Glucosinolates, isothiocyanates, and indole effects on cell cycle progression and programmed cell death were analyzed by using a TUNEL Assay with an Intergen ApoTag Kit (Purchase, NY). Cell cycle determination was based on the measurement of DNA content labeled with propridium iodide. Apoptotic death can often be diSerentiated from necrotic death via the appearance of DNA degradation. The TUNEL assay uses a TdT enzyme and flourescein isothiocyanate (FITC) staining to fluorescently label DNA strand breaks at the 3 ’ hydroxyl group. Human prostate cancer cells (approx 3x10® ) were exposed to control media, isothiocyanates, glucosinolates, or indoles for 0-72 hours then the cells were trypsinized and fixed with 1% paraformaldehyde and stored in 70% ethanol at -20°C. We also evaluated apoptosis on the basis of phosphatidyl serine (PS) staining with Annexin V. PS is a protein that resides on the interior of the cell membrane in a normal, living cell, and translocates to the exterior of the cell membrane when the cell goes through apoptosis. For cell cycle and apoptosis analyses, the cells were resuspended in 150 |xL buffer contaming ElNase, and propridium iodide. Cytometric analysis was performed using a Coulter Elite Flow cytometer through a 630 nm LP filter. 3.3.5 Statistical Analyses Data represent means ± SEM. Data were analyzed using ANOVA using Sigmastat software. 107 3.4 RESULTS 3.4.1 Effects of Isothiocyanates oa Prostatic and Malignant Prostate Cell Proliferation Human prostatic epithelial and human prostate cancer cells were grown and treated with various concentrations (0-200 jiM) of pure isothiocyanates (sulforaphane, iberin, iberverin, erucin, benzyl isothiocyanate, phenethyl isothiocyanate, allyl isothiocyanate, and erysolin) for 0-72 hours. We observed that each of the isothiocyanates inhibited proliferation of each of the cell lines in a dose dependent manner (Figures 3.2-3.4). Benzyl isothiocyanate, phenethyl isothiocyanate, and sulforaphane had the greatest effect on inhibiting proliferation with tCso values ranging from 2.3 pM to 9.8 pM for the malignant cell lines (Table 3.1). Allyl isothiocyanate was the weakest of the isothiocyanates with IC50 values ranging from 48.2 pM-59.4 pM. The indole compounds had a lesser effect on inhibiting proliferation relative to the isothiocyanates with ICso values ranging from 94.3 pM to 128.3 pM. The natural glucosinolate, sinigrin, showed no signifrcant effects of inhibiting cell proliferation in each of the cell lines at concentrations up to 250 pM. The human prostatic cells were much more resistant to glucosinolates, isothiocyanates and indoles relative to the malignant cells. Sulforaphane, iberm, allyl isothiocyanate, and indole-3-carbinol were incubated with the human prostatic cells. At concentrations up to 108 50 pM of each, compound the human prostatic cells retained greater than 80% of their proliferation (Figure 3.5). 3.4.2 Effects of Cysteine Conjugates and Extra-cellular Glutathione on PC-3 Cell Proliferation Benzyl isothiocyanate and the major human metabolite of benzyl isothiocyanate (benzyl cysteine conjugate) were incubated separately with PC-3 cells at concentrations from 0-50 pM. Each compound inhibited cell growth, however free benzyl isothiocyanate was slightly more potent The IC sq values for benzyl isothiocyanate were approximately 2-3-fold less than its cysteine conjugate and are listed in Table 3.1. Glutathione was added to cell culture at a concentration of500 pM along with benzyl isothiocyanate and sulforaphane concentrations up to 50 pM. In the presence of extra cellular glutathione, there was no significant inhibition of growth at benzyl isothiocyanate or sulforaphane concentrations up to 25 pM as observed with the benzyl isothiocyanate only treated samples (Figure 3.6) 3.4.3 Effects of Isothiocyanates and Indole-3-Carbùiol on Cell Cycle Progression fridole-3-carbinol was added to the cells at its respective IC 50 concentrations. After 24 hours there were essentially no effects on cell cycle progression by indole-3-carbinol. However after 48 and 72 hours there was a slight decrease in cell population in the G[/Go phase of cell cycle (ca. 12%) and increases in cell population in S and G 2M phases of cell cycle (ca. 4 and 8 % respectively) (Table 3.2). After 72 hours, there was only a small build-up of sub Go particles, approximately 3%. Isothiocyanates had a much greater eSect on cell cycle than indole-3-carbinol on the malignant cell lines. Sulforaphane, benzyl isothiocyanate, and phenethyl isothiocyanate were added to malignant cell 109 cultures at each of their respective IC 50 values. Sulforaphane, benzyl isothiocyanate, and phenethyl isothiocyanate were added to prostatic cells at a 30 pM concentration. For the malignant cell lines, in the presence of sulforaphane, benzyl ITC, and phenethyl FTC we observed a build-up in the proportion of cells in the G 2M phase of cell cycle and a decrease in cell population in the Gi phase (Figure 3.7). After 48 hours there was a dramatic decrease in the proportion of cells in the G 2M phase a tremendous increase in sub-Go particles (Table 3.2). At this point there was a tremendous amoimt of cell death (ca. 80%) observed by cells losing flask adherence, shriveling, and floating in culture medium (Figure 3.8). Sulforaphane, benzyl isothiocyanate, and phenethyl isothiocyanate had essentially very little effect on cell cycle progression at concentrations of 30 pM in the human prostatic cell line relative to the malignant cell lines. The isothiocyanate negative control (media only) and the media plus 30 pM sulforaphane (48 hours) histograms are similar (Figure 3.9). After a 72-hour incubation in the presence of 30 pM sulforaphane, there was a build-up in the proportion of cells in the G 2M phase of cell cycle and a decrease in cell population in G(/Gi phase. Also, there was very little cell death (less than 5%) observed after the 72 hour time period. 3.4.3 Effects of Isothiocyanates on Programmed Cell Death (Apoptosis) After 48 and 72 hours in the presence of 5 pM sulforaphane, benzyl isothiocyanate, and phenethyl isothiocyanate there was a tremendous amount of cell death observed (ca. 80%). Cell cycle analyses via flow cytometry yielded the appearance of sub Go particles after a 48 and 72-hour incubation in the presence of sulforaphane, benzyl isothiocyanate, and phenethyl isothiocyanate. We addressed apoptosis in two separate assays by 110 incubating isothiocyanate treated cells with. FITC and TdT enzyme which fluorescently labels DNA strand breaks and PS staining. Both are associated with programmed cell death. We observed an increase in FITC and Annexin V staining in the sulforaphane and benzyl ITC treated samples vs the non-treated control samples (Figures 3.10 and 3.11) In the human prostatic cells, there was very little cell death (less than 5%) observed in the presence of 30 piM sulforaphane, benzyl isothiocyanate, or phenethyl isothiocyanate. Data from flow cytometry showed no signifrcant appearance of sub Go particles (less than 3%) after a 72-hour incubation with each of the three isothiocyanates. 3.5 DISCUSSION We have shown a dose dependent inhibition of proliferation in three highly malignant, tumorigenic human prostate cell lines PC-3, DU145, and LNCaP by several naturally occurring isothiocyanates and indoles from cruciferous vegetables. The eSects of isothiocyanates on cell growth were similar in each cell line, however PC-3 cells were slightly more sensitive, but the difièrences were small. These small differences in growth inhibition between the prostate cell Imes suggest that each line used responds similarly to each isothiocyanate. However, the isothiocyanates used differ strikingly in potency as noted by the IC 50 values. We also observed a dose dependent inhibition by indole compounds, indole-3-carbinol and brassicin, but to a much lesser degree as noted by their III much, higher IC 50 values. A natural glucosinolate, sinigrin, had no significant effects on inhibiting proliferation of each malignant cell line at concentrations up to 250 pM in our study, whereas others have shown no significant effects on growth inhibition up to 500 jiM in other malignant cell lines^^’*®- The antiproliferative potencies shown by the isothiocyanates and indoles differ markedly suggesting that lipophilicity, and structure (an indication of reactivity) may play major roles in the ability of these compounds to inhibit proliferation. It has been shown in other studies that the ability of isothiocyanates to influence cellular fimctions resides mostly in its lipophilicity rather than reactivity'^'^*. Lipophilicity is a key attribute for a molecule to effect cell proliferation and function because it can be a measure of the ability of the compound to pass through the cell membrane lipid bilayer. Our results agree somewhat with this argument as the isothiocyanates with the greatest ability to effect cell growth in our study (benzyl and phenethyl isothiocyanate) are extremely lipophilic and have very low watenoil coefBdents**’^®. Isothiocyanates are indeed taken up by cells to high degrees, but at varying rates. Several reports have recently shown that isothiocyanates enter cells in the free form and then are accumulated as glutathione conjugates inside the cell*®'^^ Initial cellular uptake rates therefore have been described to depend largely on glutathione conjugation rates, both enzymatic and non-emqmatic (2"*^ order). Our data agrees somewhat with these observations as well. Bengal isothiocyanate is an excellent substrate for human recombinant glutathione-%5- tranferases and has a rate constant almost twice that of allyl isothiocyanate (130 M*'min‘^ vs 75 Mr^min*^)^°. Accordingly, ben^l isothiocyanate was much more potent in inhibiting proliferation than allyl isothiocyanate in our study. However, sulforaphane and 112 phenethyl isothiocyanate were comparably more effective m inhibiting proliferation than allyl isothiocyanate, yet the glutathione conjugation rate constants of each are much lower ±an allyl isothiocyanate (45 and 35 M"'mm'\ vs 75 M'^min'^ respectively). The specific metabolism and uptake of the isothiocyanates by the cell lines used in this study have not been addressed. Due to the variability of potency for the isothiocyanates used, even within similar-structured isothiocyanates, the ability for an isothiocyanate to influence cell growth perhaps lies in the glutathione conjugation catalyzed by specific glutathione-5-transferases, and uptake by each prostate cancer cell line. Sinigrin, and most glucosinolates are polar and water-soluble as are some indole compounds. Also glucosinolates are not known to conjugate with glutathione or induce glutathione-5^ tranferases®. This can suggest why we observed a lesser effect on inhibiting proliferation by the indoles and glucosinolates relative to the isothiocyanates. It may be more difficult for the indoles and glucosinolates to pass through the cell membrane and be taken up by the cell. This was further confirmed in our cysteine conjugate studies with benzyl isothiocyanate and glutathione studies. The IC 50 for free bentyl isothiocyanate was approximately 2-3 fold less than its cysteine conjugate. Furthermore when 500 [iM glutathione was added to cell culture medium with sulforaphane and benzyl isothiocyanate(up to 25 ptM concentrations) there was no significant inhibition of proliferation versus control cells. N-acetylcysteine and glutathione conjugation renders isothiocyanates more hydrophilic. Thus, the conjugates may have a more difGcult time passmg into the cell. This can suggest the lower potency of the iV-acetyl conjugate and lower potencies of isothiocyanates when exposed to excess extracellular glutathione. 113 Our data indicates structure and reactivity of the added compounds can also play art important role in inhibitmg proliferation. The indole compounds were the least effective in inhibiting proliferation. Indole compounds are first formed from glucosinolates to highly reactive indole isothiocyanates, which then form more stable indole-carbinols. Indoles and glucosinolates are much less reactive than isothiocyanates which also can explain why each was less effective in inhibiting proliferation'*’*’®. Our isothiocyanate studies suggest that reactivity is a governing factor more so than lipophilicity. For example we observed sulforaphane to have similar ICso values to phenethyl and benzyl isothiocyanate however sulforaphane is water-soluble whereas the latter two are not. Furthermore, allyl isothiocyanate is extremely lipophilic and its ICso values of were approximately 10 times greater than that of sulforaphane. The isothiocyanates used in this study belong to essentially three groups; benzene containing side chains (benzyl isothiocyanate and phenethyl isothiocyanate), sulfur containing side chains with various oxidation states (sulforaphane, iberin, iberverin, erucin, erysolin), and an alkenyl side chain (allyl isothiocyanate). Reactivity is significant inside the cell as isothiocyanates are accumulated as glutathione conjugates which are then metabolized and excreted.'®'^' When isothiocyanates conjugate with glutathione, the highly electrophilic carbon in the N=C=S group is reduced to NH-C(S)=S and is less reactive. Glutathione-isothiocyanate conjugates react with macromolecules, however free isothiocyanates are known to react strongly with oxygen, nitrogen, and sulfur atoms; and also particularly with cysteine residues on proteins.^* The efficacy of isothiocyanates to decrease cell proliferation and viability possibly rests in its ability to free itself from the conjugate and attack macromolecules such as enzymes. Non-enzymatic dissociation rates have been studied 114 for benzyl, phenethyl, and allyl isothiocyanate, and sulforaphane.^® The dissociation rate constants for non-enzymatic cleavage are greatest for ben^l isothiocyanate (6.4), followed by sulforaphane (4.4), then phenethyl isothiocyanate (0.89)^®. Surprisingly, there was little to no allyl isothiocyanate dissociation under the same assay conditions. This phenomenon is consistent with our results in that allyl isothiocyanate is the least potent in inhibiting proliferation, it may not be readily released inside the cell to attack cellular machinery. Isothiocyanate dissociation is accelerated in the presence of glutathione-5'-tranferases and follows differing trends as far as isothiocyanate dissociation'*. This suggests that metabolism of specific G 6 Ts endemic to specific cell types may be a factor in isothiocyanate dissociation and overall potency. Ultimately, the identity of the final reactive species be it free isothiocyanate, glutathione-isothiocyanate conjugate, mixture of both, or other unknown metabolite remains obscure. Future studies should elucidate the identity of GSTs involved, ultimate metabolism, and specific target sites in the cell lines studied. The reactivity of the proton adjacent to the N=C=S group has been studied as well'^. However the degree of reactivity of this methylene proton as measured by 'H NMR chemical shifts was similar for several isothiocyanates studied'®. The benzyl containing side chains had the greatest effects on inhibiting proliferation with IC50 values ranging from 2.3 piM to 9.8 pM. The sulfur containing side chains followed in potency, although there was greater variability, with IC 50 values ranging from 4.2 to 29.4 pM. Finally the alkenyl side chain isothiocyanate allyl isothiocyanate had the least potency in efiecting proliferation relative to the other two structure groups with IC 50 values ranging from 48 JZ to 59.4 pM. Other studies have shown aUyl isothiocyanate to be the most potent 115 isothiocyanate versus several other isothiocyanates studied^^. Our studies conflicting results regarding allyl isothiocyanate and the variability with similar-structures sulfur containing isothiocyanates again suggest that the potency of isothiocyanates possibly rests in specific metabolism and/or dissociation rates. We observed that each of the isothiocyanates and indoles inhibited cell proliferation to varying degrees. Our next endeavors were to evaluate the phase of cell cycle that the isothiocyanates and indoles exerted their effects. From the group of isothiocyanates used we chose sulforaphane (sulfur side chain) phenethyl isothiocyanate (beru^l side chain) and indoIe-3-carbinoI. Since there was little difference in the [C50 values of each cell line for each isothiocyanate we used the PC-3 cell line to evaluated the effects of isothiocyanates and indoles on cell cycle progression. However, initially each cell line was used separately and cell cycle effects were similar. To study cell cycle effects, we used an isothiocyanate concentration of 5-10 pM, which was slightly larger than each IC50 for 0 to 72 hours to observe an effect. In the presence of sulforaphane, we observed a decrease in the population of cells in the Go/Gi phase and simultaneously observe an increase in the proportion of cells in the S and G%M phases (Table 2.2). After 48 hours the proportion of cells in the GzM phase of cell cycle drops, suggesting that the cell division cycle is arrested at the GzM phase. Arrest of cell cycle progression in at the GzM phase of cell cycle has also been shown in malignant HT29 colorectal cells as well as a high level of expression of cyclins A and BI which regulate cdc2 kinase activity during the GzM phase^^. Also m the presence 10 pM sulforaphane there was an increase in sub- Go particles. The appearance of sub Go particles is consistent with fragmented DNA and cell death by apoptosis. The benzyl contaming side chain isothiocyanate, benzyl 1 1 5 isothiocyanate caused cell cycle arrest in the GzM phase of cell cycle as well. Indole-3- carfainol caused a modest buildup in the S and GzM phases, suggesting that cell cycle arrest can occur in one of these phases. We observed a tremendous amount of cell death in the presence of sulforaphane, benzyl ITC, and phenethyl ITC. We evaluated cell death as natural programmed cell death or as a general toxic or necrotic death. Apoptosis was evaluated in several ways. We observed sub-Go particles with cell cycle analyses that are consistent with fragmented DNA associated with apoptosis. A necrotic death involves cell swelling and lysis with release of DNA that does not necessarily result in fragmentation. We used FITC labeling and TdT enzyme to flourescently label DNA strand breaks. We observed a greater amount of FITC labeling, an indication of DNA degradation in the sulforaphane, benzyl isothiocyanate, and phenethyl isothiocyanate treated samples vs. controls. Furthermore, we stained cells with Annexin V, which labels PS translocation during apoptosis, and we again observed an increase in FTTC staining isothiocyanate treated samples vs. controls. These apoptotic phenomena were observed at very low levels ( 117 3.6 CONCLUSIONS This study has shown that several natural components from cruciferous vegetables posses bioactivity and can inhibit malignant cell proliferation, arrest cell cycle progression, and induce apoptosis at achievable physiological levels, Isothiocyanates were the most potent followed by indole compounds, with glucosinolates possessing essentially no significant bioactivity at the concentrations used. Prostatic (non- malignant) cells were much more resistant to the growth inhibitory efiects and pro- apoptotic effects of isothiocyanates than malignant cells. This suggests that a diet high in cruciferous vegetables can lower prostate cancer risks and be less toxic to normal cells. 118 3.7 REFERENCES L Steinmetz K, Potter J. Vegetables, fruit, and cancer. Cancer Causes and Control. 1991;2:325-442. 2. Wattenberg, L. Inhibition of carcinogenesis by minor dietary constituents. Cancer /îes.(Suppl) I992;52,2085s-209ls. 3. Block G, Patterson B, Subar, A. Fruit, vegetables, and cancer prevention: A review of epidemiological evidence. Nutr. Cancer. 1992; 18,1-29. 4. Cohen JH, Kristal AR, Stanford JL. Fruit and vegetable intakes and prostate cancer risk. JNCL 2000;92:61-68. 5. Fenwick GR, Heaney RK, Muliin WJ. Glucosinolates and their breakdown products in food and food plants. Crit. Rev. in FoodSci. & Nutr. I983;18:I23- 201. 6. Zhang Y, Talalay P, Cho CG, Posner GH. A major inducer of anticarcinogenic protective enzymes from broccoli: isolation and elucidation of structure. Proc. m . Acad, o f Sci. 1992;89:2399-403. 7. Fahey JW, Zhang Y, Talalay P. Broccoli sprouts: an exceptionally rich source of inducers of enzymes that protect against chemical carcinogens. Proc. Mat. Acad, o f Sci. 1997;94:10367-10372. 8. Fahey J, Talalay, P. Antioxidant functions of sulforaphane: a potent inducer of Phase H detoxification enemies. F o o t / Tox. 1999;37:973-979. 9. Zhang Y, Kensler TW, Cho CG, Posner GH, Talalay P. Anticarcinogenic activities of sulforaphane and structurally related synthetic norfaomyl isothiocyanates. Proc. Mat. Acad, o f Sci. 1994;91:3147-50 10. Posner C®, Cho CG, Green JV, Zhang Y, Talalay P. Design and synthesis of bifimctional isothiocyanate analogs ofsulAraphane: correlation between structure and potency as inducers of anticarcinogenic detoxication enzymes./. Med. Chemi. 1994;37:170-176. 119 IL Mithea R, Dekker, M., Verkeck, R., Rabot, S., Johnson, I. The nutritional significance, biosynthesis and bioavailability of glucosinolates in human foods. J, Sci. Food and Agr.2QQ0;S0:967-9S4 12. Nastruzzi C, Cortesi, Rita., Esposito, E., Menegatti, E., Leoni, 0., lori, R., Palmieri, S. In vitro antiproliferation activity of isothiocyanates and nitriles generated by myrosinase-mediated hydrolysis of glucosinolates from seeds of cruciferous vegetables./. Agric. Food Chem. 2000;48:3572-3575. 13. Sasaki T, Keita, K., Yasushi, U., Ozawa, Y., Shimizu, J., Kanke, Y., Takita, T. Efiects of isothiocyanates on growth and metastaticity on BI6-F10 melanoma cells. Ifutr. and Cancer 1999;33:76-81. 14. Cover C, Hsieh, S., Cram, E., Hong, C., Riby, J., Bjeldanes, L., Firestone, G. Indole-3-carbinol and tamoxifen cooperate to arrest cell cycle of MCF-7 human breast cancer cells. Cancer Res. 1999;59:1244-1251. 15. Gamet-Payrastre L, Li P, Lumeau S, et al. Sulforaphane, a naturally occurring isothiocyanate, induces cell cycle arrest and apoptosis in HT29 human colon cancer cells. Cancer Res. 2000;60:1426-33. 16. Nastruzzi C, Cortesi R, Esposito E, Menegatti E, Leoni 0, lori R, Palmieri S. In vitro cytotoxicity of some glucosinolate-derived products generated by myrosinase hydrolysis. / Fbo/ Ckem. 1996;44:1014-1021. 17. Leoni 0, lori R, Palmieri S, et al. Myrosinase-generated isothiocyanate from glucosinolates: isolation, characterization and in vitro antiproliferative studies. Bioorg. &Med. Chem. 1997;5:1799-1806. 18. Zhang Y. Role of glutathione in accumulation of anticarcinogenic isothiocyanates and their glutathione conjugates in murine hepatoma cells. Carcinogenesis. 2000;2l: 1175-1182. 19. Jiao D, Eklind K, Choi C, Desai D, Amin S, Chung F. Structure-activity relationship of isothiocyanates as mecham'sm-based inhibitors of 4- (methylnitrosoamino)-l-(3-pyridyl)-I-butanone-induced lung tumorigenesis in A/J mice. Cancer Res.l994;54:4327-4333. 20. Zhang Y, Kolm R, Mannervik B, Talalay P. Reversible conjugation of isothiocyanates with glutathione catalyzed by human glutathione transferases. Biochem. and Biophys. Res. Comm. 1995;206:748-755. 21. Zhang Y. Molecular mechanism of rapid cellular accumulation of anticarcinogenic isothiocyanates. Carcinogenesis. 2001;22:425-431. 120 Glucosinolate IC«, PC3 DU14S LNCaP Sinigrin >250 pM >250 pM >250 pM Indoles IndoleB-carbinol 83.6+/-2.3"’^ 92.3 +/-2.6'’’® 89.5+/-3.4^'^^ Brassicin 101.4+/-3.8"*^ 98.5+/-7.4"'^ 123.8 +/-14.3*^^^ isothiocyanates Allyl 51.3 +/-4.I*’^ 482 +/-3.4*’‘° 59.4+/-4J'-‘* Benzyl 1.5+1-02^'* 4.8 + /-1.3®’“ 3.2+/-0.3"’*® Erysolin 24.5+/-3.2^ 29.3+/-2>‘- 25.6 +/- 3.r-^° Iberin U.3+/-0.4'’® 20.1 +/-1.9"^" 29.4+/-3.6'-“ Iberverin 5.4+/-0.6"^^ 7.6+/-1.7"^'^ 17.3 +/-2.7°’^‘ Phenethyl 2 J+/-O.4 P* 7.3+/-0.6’’‘^ 8.4+/. 0.4'-- Sulforaphane 4.8 +/-1.9*’^ 4.2+/-0.3*-“ 5.3 +/-1.2'-'“ Cysteine con jugate Benzyl Cysteine conjugate 7.3+/-1.4“*^ 9.4+/-2.1“’^^ 5.7+/-0.8“’^’ Table 3.1: IC 5 0 Values for glucosinolates, isothiocyanates, and indoles Each measurement represents the means +/- SE of 9 independent assays. Statistical analyses were determined by ANOVA with significance determined using Fisher PLSD test (p<0.05). Letters denote significant differences between cell lines for each compound. Numbers denote significant differences between compounds within each cell line. 121 Sülfbraphane Phase Sub Go Gi/Go S GzM Control 1.2+/-0.3 58.6+/-2.3 15.6+/-2.8 19.2+/-3.1 24 hours 2.3 +/- L4 523 +/-1.8 18.6+/-3.2 25.6+/-2.8 48 hours 3.9+/-0.2 40.5+/-4.7 24.L+/-6.1 393+/-2.7 72 hours 3L4+/-7.4 52.3 +/-10.9 6.7+/-4.6 63+/-3.5 Benzyl isothiocyanate Phase Sub Go Gi/Go S GzM Control 1.6+/-0.6 55.3+/-4.3 18.6+/-3.5 22.4+/-3.6 24 hours 8.8 +/-1.4 19.6+/-3.2 15.7+/-3.5 47.6 +/- 3.4 48 hours 18.5+/-4.1 19.8+/-2.8 21.2+/-3.1 27.4+/-1.4 72 hours 36.7+/-3.5 26.3+/-4.2 12.9+/-5.6 16.4+/-3.1 Indole-3-carbinol Phase Sub Go Gi/Go S GzM Control 2.3 + /-12 52.5+/-3.6 20.6+/-5.8 24.8+/-2.8 24 hours 1.7+/-0.3 60.1+/-3.8 17.8+/-2.4 16.7+/-43 48 hours 3.1 +/-1.2 50.0+/-4.8 21.7+/-4.7 20.5+/-6.4 72 hours 3.2+/-0.6 48.8+/-6.4 20.9+/-53 223+/-2.5 Table 3.2: Percentage of cells in cell cycle for sulforaphane, benzyl, and indole-3- carbinol (DUI45 cells) 122 / S-P-D-Glucose R-C NOSOj- ( glucosinolate) Myrosinase HzO / R-C +D-GIucose NOSOj- -HSO R-N=C=S Isothiocyanate Figure 3.1: Enzymatic hydrolysis of glucosinolates to isothiocyanates 123 120 c 100 8 (3 P Allyl ITC I o Benzyl ITC Erysolin ~ô 0 —V Iberin Iberverin 1 —a • Indole 3 Carbinol S (D • Phenethyl ITC CL Sulforaphane 1 10 1000 Concentration, pM Figure 3.2: Effects of isothiocyanates and indoles on PC-3 cell growth 124 c 8 Im g) Allyl ITC I R 0 0 Benzyl ITC Erysolin Ô) ü - 9 Iberin c Iberverin <0 - a - Indole 3 Carbinol Phenethyl ITC 1 Sulforaphane 0-1 1 10 100 1000 Concentration, gM Figure 3.3: ECTects of isothiocyanates and indoles on DU145 ceil growth 125 § u > m S £ Allyl ITC ü1 Benzyl ITC Erysolin ÔJ o Iberin c Iberverin (D Indole 3 Carbinol B Phenethyl ITC ir Sulforaphane 1 10 1000 Concentration, nM Figure 3.4: Effects of isothiocyanates and indoles on L^iCaP ceil growth 126 120 2 c 8 100 • 2 1 I 80 - £ Human Prostatic Cells a 60 ■ O 0U145 cells (malignant) LNCaP cells (malignant) o PC3 cells (malignant) 40 - S. 0 10 20 30 40 SO 60 [Sulforaphane], )iM Figure 3.5: Effects of sulforaphane on prostatic and malignant cell growth 127 120 Plus Glutathione No Glutathione 100 - £ (9I ® 6 0 - c 9 Ô 40 - CL 20 - 0 10 20 30 40 50 60 [Sulforaphane], Figure 3 .6 : Effects of added glutathione on PC-3 cell proliferation 128 T “ l I ' ( 1 1 No Sulforaphane T - — I I I I 24 Hr Sulforaphane 48 Hr Sulforaphane 72 Hr Sulforaphane Figure 3.7: Effects of sulforaphane on PC-3 cell cycle 129 LNCaP Control PC-3 Control Live cells attached 5 piM Benzyl isothiocyanate 5 |iM Benzyl isothiocyanate Dead cells floating 25 (iM Benzyl isothiocyanate 25 pM Benzyl isothiocyanate Figure 3.8: Effects of benzyl Isothiocyanate on LNCaP and PC-3 cells 130 No sulforaphane control 48 hour sulforaphane 72 hour sulforaphane Figure 3.9: Effects of sulforaphane on prostatic cell cycle progression 131 Control Necrotic cells ^ate apoptotic cells Carly apoptotic cells -Viable cells rhiiii uiiiii mm F IT C LOG S |xM Benzyl ITC 1 iiiia~ i i imii 1«O0 FITC LOG 5 ^iM Phenethyl ITC r tiiM I UIIIË leao FITC LOG 5 {iM Sulforaphane I mill iirrui leaa FITC LOG Figure 3.10: Annexin V staining of apoptotic PC-3 cells exposed to isothiocyanates 132 5 {xM Sulforaphane 5 Benzy ITC Apoptotic cell^‘ Necrotic cell DMA L IN BNA L IN 30 pM Sulforaphane 30 pM Benzyl ITC Necrotic cells SNA LIN DNA LIN Figure 3.11: Effects of low (5 pM) and high (30 pM) levels of sulforaphane and benzyl ûothlocyanate on apoptosis in PC-3 cells 133 CHAPTER 4 THE EFFECTS OF SINIGRIN, INDOLE-3-CARBINOL, ISOTHIOCYANATES, AND A BROCCOLI SPROUT EXTRACT ON IGF-1 STIMULATED AT6.3 CELL PROLIFERATION AND SURVIVAL Corey E. Scott\ Steven L Schwartz^ and Steven K. Clinton" 'The Department of Food Science and Technology Parker Hall 2015 FySe Court Columbus OH, 43210 The 0 ^0 State University ^Department of Internal Medicine College of Medicine and Public Health Division of Hematology and Oncology Starling-Loving Hall Columbus OH 43210 The Ohio State University Keywords: rat prostate, IGF-I, cell culture, isothiocyanatet, glucosinolates, mdole-3- carbinol, sulforaphane, MAP Kinase, signal transduction 134 4.1 ABSTRACT Cancer of the prostate is the second leading cause among cancer deaths in men in the United States. Recently a family of anti-apoptotic, mitogenic growth factors called insulin-like growth factors (IGF) has been implicated in prostate carcinogenesis. IGF, the most prevalent of which is IGF-I are known to enhance proliferation and inhibit programmed cell death in mammalian cells. Several studies have shown protective effects of consuming a diet high in cruciferous vegetables with a reduction in prostate cancer risks, mainly through Phase H enzyme manifestation. The purpose of this research is to determine the effects of broccoli sprout extract (containing glucosinolates and isothiocyanates) and purified glucosinolates, indoles, and isothiocyanates on IGF-1 stimulated cell proliferation. Rat prostate (AT6.3) cells were grown in serum-firee media (control) and media containing 50 ng/ml IGF-1. There was a 3.41 fold increase in AT6.3 cell proliferation in the presence on IGF-1. A broccoli sprout extract and natural components, sulforaphane, allyl isothiocyanate, benzyl isothiocyanate, and phenethyl isothiocyanate (isothiocyanates), sinigria (glucosinolate), and indole-3-carbinoI (indole) were incubated in cell cultures containing IGF-l. We observed a dose-dependent inhibition to IGF-l stimulated growth, by the broccoli sprout extracts and each of the isothiocyanates, the most potent of which were the broccoli sprout extract causing a 72.8% reduction and benzyl isothiocyanate, causing a 68.2% reduction in proliferation at a 5 pM concentration. A western blot was used to determine the levels of Akt activation 135 associated with apoptosis resistance. Isothiocyanate and broccoli sprout treated samples with IGF-I decreased Akt activation more so than both IGF-I treated samples and non IGF-I treated samples by as much as 85.7% and induced apoptosis. The IGF-I inhibitory mechanisms shown suggest another means of chemoprotection by consuming a diet high ia cruciferous vegetables. 4.2 INTRODUCTION Prostate cancer, a disease that typically affects men over the age of 65, accounts for approximately 40% of new cancers diagnosed each year in the United States^ The etiology of this type of cancer is not entirely understood, however diet, male hormones, and tobacco use are known to play major roles in prostate carcinogenesis.^ Also implicated in cancer of the prostate are insulin-like growth factors (IGFs).^"^ IGFs are single-chain peptides of approximately 70 amino acids that are 40%-50% identical to insulin and largely produced in the liver. IGF play an essential role in growth stimulation and organ development. The most prevalent IGF found in serum is IGF-I. Insulin-like growth factors increase cell proliferation and inhibit apoptosis (programmed cell death) in mammalian cells. Recently, insulin like growth factors (IGFs), have been shown to be involved in prostate cell proliferation and cancer, as men with prostate cancer had higher circulating levels of IGF-l than men with no evidence of prostate cancer^^°. ICTs have also been shown to have mitogenic and anti-apoptotic eSects on prostate epithelial cells 136 in Biological activity of IGFs is mediated through a heterotetrameric tyrosine kinase receptor which is similar to an insulin receptor (Figure 4.1). IGFs influence cell survival and proliferation by binding to receptors causing autophosphorylation and activation of intrinsic tyrosine kinase which then goes on to phosphorylate intracellular substrates such as IRS-1 and Shc.^^''^ These events lead to activation of mitogen- activated protein, kinases (MAPK), extracellular signal related kinases (ERK), and phospholidylinositol 3-kmase (P13K) and Akt pathways. MAPK and ERK are both involved in cell proliferation. Akt is involved in cell survival and is mediated by several growth factors. Akt can phosphorylate a regulatory member of the Bcl-2 family, BAD, which can remove BAD from a death suppressor Bcl-xL. Bcl-xL can then remain associated with the caspace activator Apaf-1, thus preventing activation of caspace enzymes and preventing onset of programmed cell death (apoptosis). IGF-1 receptor binding and cell proliferation effects can be inhibited by insulin-like growth factor binding proteins (IGFBPs), specifically IGFBP-3."'" Both epidemiological and experimental studies have shown a reduction in cancer at various organs by consuming a diet high in fruits and vegetables^'*'^®. Recently, of particular attention in prostate cancer prevention are cruciferous vegetables which include cabbage, broccoli, kale, brussel sprouts, etc^°"^\ A recent study found a 41% decrease in men who consumed, at least 3 servings of cruciferous vegetables per week versus those who consumed less than one^. Cruciferous vegetables contain relatively large amounts of glucosinolates and their hydrolysis products isothiocyanates and indoles‘°’^^’^. Isothiocyanates can participate m blocking mechanisms in cancer prevention by inducing Phase H detoxification enzym es^. Induction of Phase H enzymes can detoxify and 137 block chemical carcinogens &om reacting with cellular DNA and transforming a normal cell into a malignant Recent studies have shown that isothiocyanates can act via suppression mechanisms in cancer prevention as well. These include proliferation inhibition, arrest of cell cycle progression, and induction of apoptosis in malignant cell lines^^l The methods in which compounds from cruciferous vegetables can help inhibit prostate cancer are complex and probably involve several collaborative processes rather than residing in a sole mechanism. Our efforts in this research are to evaluate the eOects of a broccoli sprout extract and a purified glucosinolate (sinigrin), isothiocyanates, and an indole compound (indoIe-3-carbinol) on inhibition of IGF-l stimulated prostate cancer cell proliferation. 4.3 MATERIALS AND METHODS 4.3.1 Glucosinolates, Isothiocyanates, Indole and IGF-1 Preparation Pure isothiocyanate standards were purchased from LKT BioChem (St. Paul MN) and resuspended in OMSO at a concentration of 100 mM and stored at -40°C. For HPLC analysis each stock was dissolved in 3 ml of 90:10 distilled watenmethanol. A pure glucosinolate, sinigrin, and mdole-3-carbinol was purchased from Sigma Chemicals as a lyophilized powder and resuspended in water at a concentration of 100 mM and stored at -20“C. lGF-1 was also purchased fi»m Sigma 138 Chemicals as a lyophilized powder. IGF-l was resuspended in water to a concentration of 50 jig/ml in water and stored as 20 pL aliquots at -40 °C. 4.3.2 HPLC Analysis of Glucosinolates, Isothiocyanates, Indoles, and Broccoli Sprouts For purity, isothiocyanates, glucosinolates, and indoles were subjected to analytical HPLC analysis on a Waters 2690 system equipped with a 996 photodiode array monitoring at 235 nm using Millennium software for purity. A reverse phase Cig column (Supelco, 24 cm x 4.6mm, Sum) was eluted isocratically with 90:10 waternnethanol mobile phase for 30 minutes with, a flow rate of 1 ml/min at room temperature. Pure isothiocyanate, glucosinolate, and indole standards were dissolved in 3 mis of mobile phase (methanolrwater) and injected as 50 pL aliquots. Broccoli sprouts were purchased from local supermarkets and washed in three volumes of 70% and ten volumes of distilled water. The sprouts were then homogenized in distilled water for 20 minutes. The resultant soup was extracted with methylene chloride 3 times. Organic extracts were pooled and evaporated under nitrogen. The resultant residue was extracted with watenmethanol (90:10) and analyzed via HPLC. 4.3.3 Cell Culture Rat prostate cancer cells (AT6.3) were purchased from American Type Culture Collection (ATCC) and cultured in RPMI media containing 10% heat inactivated fetal bovine serum, L-glutamine, and antibiotics streptomycin and penicillin in an atmosphere of 5% COz/ 95% 0% with fresh media supplied every 48 hours. For antiproliferation experiments, cells were seeded at IxIO"^ cells per well in a. 96 well plate and incubated as above overnight. One day after seeding, the cells were challenged with media contaniing 139 the broccoli sprout extract, pure glucosinolates, isothiocyanates, or indoles at concentrations from 0, InM, 10 nM, LOG nM, I ^iM, and 10 pM for 48 hours. 4.3.4 In v itro IGF-1 Growth Inhibition Assay. Viable cell numbers were microscopically determined by counting cells using trypan blue exclusion and a hemocytometer. Cell proliferation was judged using an MTS- tetrazolium colorimetric assay. 4x10^ cells/well were placed into a 96-well microtiter plate for 24 hours. The negative control was cells in serum-free media and the positive control was cells plus 50 ng/ml IGF-l. Fresh media containing the broccoli sprout extract purified glucosinolates, purified isothiocyanates, and indoles plus 50 ng/ml IGF-l were then added and incubated for 0-72 hours. After the respective time point, the MTS reagent was added to each well and cultures were incubated an additional 2-4 hours at 37°C before measuring absorbance at A 490 nm. 4.3.5 Akt (threonine serine kinase) Western Blot We evaluated Akt phosphorylation (activation) by Western blotting using anti-phospho Akt antibodies. AT6.3 cells (approximately 4x10^ were grown in 100 mm x 22 mm round cell culture dishes in serum-free media or media plus 50 ng/ml IGF-1. Cells were incubated in serum-free media for 24 hours. Next the serum free media was removed and cells were challenged with fresh media containing isothiocyanates, glucosinolates, indoles, and IGF-1. The negative control was media with no added IGF-1. Cells were grown for 72 hours as stated in cell culture. Cells were then harvested by scraping the cells from the plates. Cells were lysed in an ice-cold lysis buffer containing Tris, EGTA, Leupeptine, Aprotinin, and Triton X-100 (See Appendix). Homogenates were centrifuged at 12,000g and the triton-X fraction collected. Protein concentrations in lysates were 140 determined according to the BioRad Protein assay kit Proteins were separated by 12% SDS PAGE. Protein samples were transferred onto a PVDF membrane overnight at 4°C. The membrane was then incubated with I jig/ml anti-phospho-AJcT ( 1° antibody) in PEST 5% milk for 24 hours. The membrane was washed (4X) and incubated with anti rabbit IgG-HRP (2° antibody) for 40 minutes and washed in PEST ( 6 X). The membrane was incubated in luminol for 1 minutes and exposed to film. Intensity of blots within lanes was quantified using Scion imaging software. 4 J.6 Statistical Analyses Data represent means + SEM. Data were analyzed using ANOVA using Sigmastat software. 4.4 RESULTS 4.4.1 Effects of Sinigrin, Isothiocyanates, Indole-3-carbinol, and Broccoli Sprout Extract on ATdJ Cell Proliferation AT6.3 cells were exposed to smigrm, isothiocyanates, and indole-3-carbinol at concentrations of 0-100 pM for 72 hours. Sinigrin and indole-3-carbincl had no significant inhibitory effects on proliferation up to 100 pM concentrations (Figure 4J2). Isothiocyanates were more potent in inhibiting growth of the AT6.3 cells. Of the isothiocyanates used, sulforaphane and benzyl isothiocyanate were the most potent having similar IC 50 values (11.3 and 11.8 pM respectively) followed by phenethyl 141 isothiocyanate, then allyl isothiocyanate- ICso values are listed in Table 4.1. At isothiocyanate concentrations above 25 ^ (except for allyl), there was excessive cell death as cells began to loose flask adherence, shrivel, and fragment. The broccoli sprout extract was serial diluted 3-fbld and caused a dose dependent inhibition of cell growth (Figure 4.3). At very low extract dilutions cell death also occurred. From the HPLC analyses we observed indoIe-3-carbinol, and isothiocyanates, erucin, iberin, and sulforaphane in the extract based on co-elution with authentic standards and comparison with on-line UV/vis spectra (Figure 4.4). 4.4.2 Effects of IGF-1 on AT6.3 Cell Proliferation AT6.3 cells were exposed to serum-free media (no added growth factors) and media plus SOng/ml IGF-1. We observed an increase in cell proliferation in the presence of SOng/ml IGF-1 over time (Figures 4.5 and 4.6). After a 72-hour incubation, there was a 3.41-foId increase in AT6.3 cell growth in the presence of IGF-1 vs. serum-free media control (SE +/- 0.48). 4.4.3 Effects of Sinigrin, Indole-3-carbinoi, Isothiocyanates, and Broccoli sprout Extract on IGF-1 Stimulated AT6.3 Cell Growth AT63 cells were simultaneously exposed to 50 ng/ml IGF-1 and 10 nmol, 100 nmol, I pmol or 5 pmol concentrations of sinigrin, indoIe-3-carbinoI, and isothiocyanates for 72 hours. Sinigrin and indole-3-carbinoI had no significant effects on inhibiting IGF-l growth stimulation of AT6.3 cells at concentrations up to 100 pM. Isothiocyanates were able to slow or inhibit IGF-l stimulated cell growth in. AT6.3 cells (Figure 4.7). Benzyl and phenethyl isothiocyanates were the most potent and had similar effects on mhibiting IGF-1 growth stimulation. At 10 nmol, 100 nmol, I pmol, and 5 pmol concentrations of 142 benzyl isothiocyanates there was a 2.1%, 31.5%, 55.3% and 68.2% reduction m IGF-l stimulated cell growth. The 68.2% reduction, in IGF-1 stimulated proliferation coincided with the control (no IGF-1) proliferation samples. Values for other isothiocyanates are listed in Table 4.2. The broccoli sprout extract was also able to inhibit IGF-l stimulated cell proliferation in a dose dependent manner. With the least diluted sample used, there was a 70.3% reduction in IGF-l stimulated cell growth relative to the IGF-l treated samples (see Table 4.2). At higher isothiocyanate concentrations (>10jiM), excessive cell death occurred in the absence of IGF-l. Samples treated with high levels of IGF-l and isothiocyanates showed less cell death initially (Figure 4.8). hi each sample however, there was a noted change in cellular morphology, consistent with death by apoptosis (Figure 4.9). 4.4.4 Effects of Isothiocyanates and broccoli sprout extract on AT6.3 Akt Activation Akt phosphorylation was evaluated by Western blot analysis. There was a 65.6% increase in Akt phosphorylation in the presence of 50 ng/ml IGF-1. Added isothiocyanates affected the levels of Akt activation in the presence of IGF-l. Samples treated with 50 jiM allyl isothiocyanate and 50 ng/ml IGF-1 dropped Akt activation levels down similar to that of control (no added IGF-1) samples (Figure 4.10). Ben^l and phenethyl isothiocyanate (1 pM) were more potent in that Akt activation levels were reduced to 14.4% and 14.2% of the control (Table 5.3). Sulforaphane (2 pM) reduced Akt activation levels to 35% of control levels. The broccoli sprout extract reduced Akt activation to 25.5% of control levels. 143 4.4.5 Effects of Phenethyl Isothiocyanate on Induction of Apoptosis Phenethyl isothiocyanate caused cell death at concentrations above 5 jxM. An Annexin V assay was used to evaluate apoptosis induction by phenethyl isothiocyanate in the presence and absence of IGF-L Phenethyl isothiocyanate was able to induce 14.4% and 17.8% apoptosis in IGF-1 treated and non-IGF-l treated samples respectively. 4.5 DISCUSSION The enhanced proliferative and anti-apoptotic effects of insulin-like growth factors have been implicated to play a possible role in prostate carcinogenesis*^. A major phenomenon in prostate cancer is defective apoptosis, or an inability for cells to die naturally. Regulation of the apoptotic program by phytochemicals can be an appropriate target for chemoprevention through dietary intervention. Studies have shown reduction in cancer risks, including the prostate, by consuming a diet high in cruciferous vegetables, vegetables that contain glucosinolates and their hydrolysis products indoles and isothiocyanates^^'^L Aside from the known mechanisms of glucosinolate hydrolysis products enhancing xenobiotic metabolism, our endeavors were to evaluate the effects of glucosinolates, indoles, isothiocyanates, and a broccoli sprout extract on IGF-l growth stimulation and apoptosis in malignant prostate cell proliferation. Broccoli sprouts were chosen because they are highly concentrated with glucosinolates and isothiocyanates relative to mature broccoli. From HPLC analysis we observed mdole-3-carbinol, and isothiocyanates ibetm, sulforaphane, and erucm. Rat prostate cells (AT53) cells were 144 used ia this study, however our initial efforts involved the use of human prostate cancer cell lines, PC-3, DU145, and LNCaP as well. The human malignant prostate cell lines responded very poorly to IGF-1 (up to 250 ng/ml concentrations), as there were no significant difierences in cell growth in the presence and absence of IGF-1 (not shown). AT6.3 cells are more differentiated than the human malignant cells used and had an improved response to IGF-1. Several concentrations of IGF-1 were used and 50 ng/ml caused an optimal increase in AT6.3 proliferation consistent with other studies. We observed greater than a 3-fold increase in AT6.3 cell growth in the presence of 50 ng/ml lGF-1 . IGF-1 has two primary roles in cellular function, which are enhanced proliferation and decreased apoptosis. We addressed the ability for sinigrin, indole-3-carbinol, and several isothiocyanates to inhibit IGF - 1 proliferation enhancement via an MTS proliferation assay. Glucosinolates and indole-3-carbinol had very little eSects on inhibiting AT6.3 cell growth at concentrations up to 100 pM. However isothiocyanates and the broccoli sprout extract, which contained isothiocyanates, caused a dose dependent inhibition in AT6.3 cell growth and caused cell death, consistent with our human malignant cell studies. However, AT6.3 cells were less sensitive to the human prostate cancer cell lines, having higher IC 50 values. Under 5 pM isothiocyanate concentrations, there were little différences in AT6 J cell proliferation or cell death versus control, as the cell retained greater than 90% of their normal growth. Thus, to elminate the dose effects of isothiocyanate cell growth inhibition, isothiocyanate concentrations less than 5 pM were chosen to address their effects on inhibition of IGF-l enhanced proliferation effects. A broccoli sprout concentration was also chosen that was not cytotoxic as well. Alone, 145 IGF-l caused a 3.41-fold increase m. cell growth but in the presence of isothiocyanates and the broccoli sprout extract, the effects of IGF-1 were decreased. At the broccoli sprout and isothiocyanate concentrations used (under 5 pM), the observed IGF-l inhibition eSect was cytostatic rather than cytotoxic as the cells remained viable, yet failed to grow in the presence of IGF-l. The isothiocyanates differed in. ability to inhibit IGF-1 growth enhancement. Phenethyl and benzyl isothiocyanate were similar in potency, whereas at I pM and 5 pM isothiocyanate concentrations, IGF-l stimulated cell growth was reduced to approximately control levels (i.e. no added IGF-l). Phenethyl and benzyl isothiocyanate are similar in structure (both contain aromatic rings), which can explain their similar potency. Sulforaphane was less effective than tlie aromatic isothiocyanates and allyl isothiocyanate was the least effective. The broccoli sprout extract inhibited IGF-l stimulated cell growth to a high degree. We found the broccoli sprout extract to contain, large concentrations of several isothiocyanates, which can explain its potency and possible synergism in inhibiting proliferation. IGF-l is involved in cell proliferation and cell survival by influencing multiple signaling pathways such as mitogen activated protein kinase (MAPK), extracellular signal-related kinase (ERK), and phosphatidylinsitol-3-kinase (PI3) pathways. In our studies, IGF-l had the ability to protect AT6.3 cells somewhat early on from the pro- apoptotic effects of isothiocyanates, as there was less observed cell death in IGF-l and isotfiiocyanate treated samples vs. isothiocyanate-only treated samples after 24 hours. However, isothiocyanates and the broccoli sprout extract were able to override the anti- apoptotic effects of IGF-l as we observed cellular morphology changes and apoptotic bodies consistent with death via apoptosis. This was also observed in our Annexin V 146 studies in that there were no significant differences in apoptosis induced by phenethyl isothiocyanate between treated and non-treated IGF samples (both ca. 17%). We addressed the ability of the isothiocyanates and broccoli sprout extract to lower IGF-l enhanced cell survival by evaluating Akt activation. Akt is mediated through a PDK signaling pathway, which is involved in cell survival decreased apoptosis. From Western blot analysis, there was a decrease in Akt activation in the presence of isothiocyanates and the broccoli sprout extract. Phenethyl and ben^l isothiocyanate showed similar effects on inhibiting Akt phosphorylation, with a severe inactivation. The broccoli sprout extract also showed very low levels of Akt activation. Sulforaphane and allyl isothiocyanate also reduced Akt activation, with sulforaphane being more potent and allyl isothiocyanate reducing Akt phosphorylation down similar to control (no added IGF-l) levels. The effects of isothiocyanates on Akt inactivation and IGF-l enhanced proliferation inhibition follow a trend in that benzyl and phenethyl isothiocyanate are the most potent inhibitors of IGF-l enhanced growth and Akt activation and allyl is the least. This suggests that the mechanisms in which isothiocyanates influence both IGF-l growth inhibition and Akt inactivation are possibly related. The degree to which isothiocyanates can influence IGF-l stimulated growth and cell survival possibly depends on uptake and metabolism of each isothiocyanate by AT6.3 cells, which has yet to be determined. We have shown that naturally occurring isothiocyanates and a natural broccoli sprout extract can inhibit the proliferative and anti-apoptotic effects of IGF-l in malignant prostate cells at low concentrations. A normal serving of cruciferous vegetables can contain between 20 and 200 pM glucosmolates and isothiocyanates^^This effect may 147 serve as another anti-cancer mechanism from cruciferous vegetable consumption shown in epidemiological studies. 4.6 CONCLUSIONS This study has shown that a broccoli sprout extract containing indoles and isothiocyanates and purified isothiocyanates can inhibit the enhanced proliferative and anti-apoptotic effects of IGF-l on AT6.3 cells. Isothiocyanates with aromatic side chains were the most potent. Each isothiocyanate was able to decrease Akt activation suggesting a possible mechanism by which isothiocyanates can lower the apoptotic threshold of AT6.3 cells in the presence of IGF-1. 148 4.7 REFERENCES 1. Cancer Facts and Figures, American Cancer Society 2001. 2. Clinton, S, Giovanucci, E. Diet, nutrition, and prostate cancer. Annu, Rev. Nutr. 1998; 18:413-40. 3. Powell I, Gelfand D, Parzuchowski J, Heilbrun L, Franklin A. 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Cancer Res. 2000;60:1426-33. 152 Compound ICso Sim'grin >100 |iM IndoIe-3-carfainol >100 (iM Allyl isothiocyanate 98.3 piM Benzyl isothiocyanate 11.8 Phenethyl isothiocyanate 20.3 piM Sulforaphane 11.3 Table 4.1: IC 50 values for glucosmolates, indoles, and isothiocyanates 153 Isothiocyanate 10 nM lOOnM 1 uM 5^M Allyl isothiocyanate 0.5 2.8 16.6 40.8 Benzyl Isothiocyanate 2.1 31.8 55.3 68.2 Phenethyl isothiocyanate 0.4 35.9 58.5 66.6 Sulforaphane 0.1 0.2 38.5 47.8 Broccoli sprout 35.6 43.2 63.4 70.2 Table 4.2: Percent reduction in IGF-1 stimulated cell growth 154 Percent Akt activation vs Control Plus IGF-I + 65.6% Plus IGF-l Plus 50pM Allyl isothiocyanate -16.1 % Plus IGF-l Plus IpM Benzyl isothiocyanate -85.5 % Plus IGF-I Plus I pM Phenethyl isothiocyanate -85.7% Plus IGF-l Plus 2 pM Sulforaphane -64.8 % Plus IGF-l Plus Broccoli Sprout -74.4% Table 4.3: Percent activation of Akt activity by isothiocyanates Percent Apoptosis PIusIGF-l 2.1+/-0.36 Plus 5 Phenethyl ITC 17.8 +/- 3.43 Plus IGF-l plus 5 jxM Phenethyl ITC 14.4 +/- 4.67 Table 4.4 Percentage of apoptotic AT5J cells treated with IGF-1, phenethyl isothiocyanate or both 155 IGF P13K PDK-1 ms Akt PKA BAD Bak (BH3) Bcl-xL Apoptosis Figure 4.1: IGF-1 cell signaling events leading to decreased apoptosis 156 2 (D "âj O I C 0 Aiiyt rrc 2 Q Benzyl ITC 0 indoie-3-carbinol Û- Phenethyl ITC Sulforaphane 0.1 1 10 100 [added compound]. ww Figure 4.2: Effects of isothiocyanates and indole-3-carbinoi on AT6.3 cell proliferation 157 120 100 I 8 0 o 0) 6 0 o c 0) 4 0 a o 20 a . 0 0.1 1 10 100 1000 Inverse Dilution Figure 4.3: Effects of broccoli sprout extract on AT6 J cell growth 158 iberio sulforapban Iodole-3-carbinol erucin Int standard 30.00 15.00 20.00 25.00 5.00 10.00 Minutes Figure 4.4: HPLC chromatogram of broccoU sprout extract 159 8 No lGF-1 7 Q 50 ng/ml IGF-1 CO 0) T" 6 k_ CD 5 4 3 CD O 2 1 0 20 40 60 80 Time, hours Figure 4.5: Effects of IGF-1 on AT6.3 proliferation 160 Control AT6.3 Cells AT6.3 Cells Plus 50 ng/ml EGF-l Figure 4.6: Effects of IGF-1 on AT6.3 cell proliferation 161 0.5 0 45 f * 0 3 5 8 ë ■ 0 3 ■Control ■ IGF-1 alOOnM 0,25 QiOnM ■ 1 uM ■5uM 0,15 0 0 5 % Benzyl ITC Phenelliyt ITC Snlforephane Breccoli Sprout extract Figure 4.7; Effects of isothiocyanates on lGF-1 stimulated AT6.3 cell proliferation Control Plus 15(iM Sulforaphane Plus 15 pM Sulforaphane+ IGF-1 Figure 4.8: Reduced apoptosis in IGF-1 treated samples (24 hours) 163 Apoptoüc bodies Convoluted cell membranes Figure 4.9: iVIorphoiigical changes in IGF-1 treated AT6.3 cells during induction of apoptosis by benzyl isothiocyanate (48 hours) 164 Control IGF-1 Allyl Benzyl Phenethyl Sulforaphane Broccoli Sprout Figure 4.10: Effects of Isothiocyanates on IGF-1 Akt phosphorylation ^T6.3+IGF-1 Necrotic ceils Late apoptotic ceUs Viable cells f -■ arly apoptotic cells rn^inr i tinw i mnit 1«08 F r r c LOG + 5 pA^benethyl isothiocyanate nniA 1 1 nun 11 inni 188* P ttO LOG + 5 pM Ph|nethyl isothiocyanate + IGF-1 1 LMMll I I [[ lilt [ I lllll Pire LOG 1**8 Figure 4.11; Annexin V Staining of apoptotic AT6J ceils induced by phenethyl isothiocyanate 166 APPENDIX A Chemical Structures Glucosinolate l a . Sinîgrîa ■ « a \-0-s03 H 2 OH Indoles [adoIe-3-carbinol B rassicm N—C— SCH3 Isothiocyanates Allyl f=C=S Benzyl =c=s 167 If Erysolin CSi Î f=C=S Enicm CHi f=C=S Ib eria ? '■N=C=S CHi Iberverin CH]' Phenethyl ? Sulforaphane CH3 ' f=C=S 168 APPENDIX B Protocols Rapid Aqueous Vegetable Extraction Purchase organic grown cruciferous vegetables from Wild Oats® Market (Lane Avenue) Purchase Brocco Sprouts® (seasonal) from Wüd Oats® Market, S3.99. If unavailable, call Jed Fahey @ John’s Hopkins University and have Fed Ex delivered Purchase Ohio grown broccoli sprouts from Big Bear® or Kroger® 1. Rinse each vegetable with 70% ethanol and wash with 3 volumes of distilled water 2. Blend 400 g of each vegetable (50 g of broccoli sprouts) with 300 ml of distilled water for five minutes 3. Homogenize the resultant soup at 7000 rpm for 3 minutes each 4. Centrifuge each solution for 5 minutes at 5000 rpm and collect supernatant 5. Pool like supernatants and sterile filter 6. Take 10 ml aliquot for HPLC analysis 7. Store remainder at-40“C Aqueous Vegetable extraction (for cell culture) I. Rinse each vegetable with 70% ethanol and wash with 3 volumes of distilled water 2- Blend 400 g of each vegetable (50 g of broccoli sprouts) with 300 ml of distilled water for five minutes 3. Homogenize the resultant soup at 7000 rpm for 3 minutes each 4. Centrifuge each solution for 5 minutes at 5000 rpm and collect supernatant 5. Add supernatant to commercial freeze drier and lyophilize for 72 hours 6. Extract 5g powder with 10 mis of water on stir plate for 1 hour 7. Store unused powder at -40 “C 8. Sterile filter extract 9. Anaylze profile via HPLC 169 Methanot or Acetonitrile Extractions L Rinse each vegetable with 70% ethanol and wash with 3 volumes of distilled water 2. Blend 400 g of each vegetable (50 g of broccoli sprouts) in. a minimal amount of water 3. Further blend iu 300 ml of methanol OR acetonitrile 4. Homogenize the resultant soup at 7000 rpm for 3 minutes each 5. Centrifuge each solution for 5 minutes at 5000 RPM and collect supernatant 6. Pool like supernatants and sterile filter 7. Take 10 ml aliquot for HPLC analysis 8. Store remainder at-40°C Note: This method is a more efficient and faster extraction for glucosinolates and isothiocyanate extraction however the yields of these verses aqueous extractions are similar. These extracts cannot be added to cell culture. High concentrations of acetonitrile, DMSO, and DMF in extracts will yield poor separations on HPLC. Thus DMSO and DMF are not used. Organic extraction of Cruciferous Vegetables 1. Rinse each vegetable with 70% ethano I and wash with 3 vo lûmes of distilled water 2. Blend 400 g of each vegetable (50 g of broccoli sprouts) in a minimal amount of water 3. Add 200 ml of methylene chloride and stir for I hour 4. Evaporate methylene chloride on a rotary evaporator. 5. Extract residue with 75:25 watemnethanol (to collect isothiocyanates and indoles) 6. Extract residue with 75:25 hexaneracetonitrile (to collect carotenoids) OR 1. Rinse each vegetable with 70% ethanol and wash with 3 volumes of distilled water 2. Blend 400 g of each vegetable (50 g of broccoli sprouts) in 200 ml of water 3. Homogenize the resultant soup at 7000 rpm for 3 minutes each 4. Centrifuge each so lution for 5 minutes at 5000 RPM and co Uect supernatant 5. Add supernatant to seperatory funnel with 100 mis of methylene chloride 6. Extract 3X 7. Collect organic layer contaming isothiocyanates and mdoles 8. Collect aqueous layer containing glucosinolates 9. Concentrate organic layer via rotary evaporation 170 Myrosinase assay 1. Prepare a stock solution of myrosinase 20 units/ml in 33 mM sodium phosphate buffer (pH 7.4). Store at-20"C 2. Prepare a 1 GO mM solution of sinigrin in water (substrate) 3. Prepare 33 mM sodium phosphate buffer containing 2 g/1 ascorbic acid and O.OIM magnesium chloride (reactionbuffer) 4. Add 14 mis of reaction buffer and 100 pL of substrate to 15 ml tube 5. Initiate reaction with addition of 10 pL of enzyme 6. React at 37”C for 10 minutes. 7. Immediately pour tube contents into a 50 ml tube containing 5 ml of methylene chloride to stop reaction. 8. Take 3 mis from aqueous fraction and measure UV absorbance at 227 nm for substrate loss 9. Measure organic fraction at 240 nm for increase in product (allyl isothiocyanate) 10. Substrate and product can also be measured via HPLC Hydrolysis of glucosinolates in aqueous extracts 1. Place 10 ml of aqueous extract into a small beaker with 5 ml of 33 mM sodium phosphat buffer containing 2 g/L ascorbic acid and 0.01 M magnesium chloride and bring pH up to 7.0 2. Add 100 pL of 20 units/gram myrosinase 3. React overnight at 37®C on a stir plate 4. Extract reaction 3X with 10 mis of methylene chloride 5. Evaporate methylene chloride layer and store residue (Containing isothiocyanates and indoles) at -40“C Reverse phase HPLC for standards and extracts 1. Resuspend pure isothiocyanate and indole standards in DMSO 2. Resuspend glucosinolates in water 3. Obtain 3 mis of aqueous extracts 4. Resuspend organic extract residue in 75:25 methanolnvater 5. Place approxfrnately 3 mis of each solution into HPLC injection vials 6. Inject onto a Waters 2690 HPLC system equipped with 996 Photo diode array 7. Flow rate :1 ml/mm, isocratic 90:10 water methanol 8. Wavelength 200-600 nm, monitored at 235 nm 9. Runtime: 30 minutes 10. Column: C[g 5pM monomeric 24 cm x 4.6mm _ 11. Preparatory system: SP 8800 Spectra Physics Pump with a Waters 996 Photo Diode Array 12- Preparatory column: 19 mm x 300 mm 171 13. Flow rate: 7 ml/mm 14. Wavelength 200-600 nm, monitored at 235 nm 15. Run time: 30 minutes TÜNEL Assay (Stains DMA strand breaks associated with Apoptosis) Fix cells 1. Induce cells 2. Resuspend 1-2 million cells in 0.5 ml PBS 3. Place cell suspension into 5 mis 1% paraformaldehyde in PBS (pH 7.4) on ice 4. Fix for 15 minutes 5. Spin down cells and resuspend in 10 ml ice cold PBS 6. Spin down cells and resuspend in 70% ethanol, store at -20°C (up to 6 months) Wash Cells 1. Spin down cells 2. Add I ml PBS and vortex 3. Spin down cells and discard supernatant 4. repeat 5. Resuspend cells in 75 (iL of equilibration buffer Working strength enzyme 1. Spin down cells 2. Remove supernatant 3. Resuspend cells in 50 pL working strength TDT enzyme 4. Incubate at 31°C fo r 30 minutes (avoid light) 5. After 15 minutes resuspend cells that have settled 6. Add I ml of stop wash 7. Spin down cell and remove supematnant DMA staining 1. Add I ml of propidium iodide staining solution 2. Incubate 15 minutes at room temperature (avoid light) Collect Data I. Collect data via Coulter Elite flow cytometer 172 Annexia V Assay (Stains translocated PS protein associated with apoptosis) L Collect 1-2 millioa cells 2. Spin down cells 3. Wash cell in I ml binding buffer 4. Resuspend in 150 pL bindmg buffer 5. Add 5 pL of Annexin V FITC stain 6. Add 10 pL of Propridium iodide stain 7. Incubate 5-15 minutes m dark 8. Immediately co Uect data via flow cytometry 173 P-AKT assay by Western Blot 1. After scraping cells down, cells spinned down at 4C, then wash with ice-cold PBS 2. twice, the cell pellets are lysed in the following ice-cold Lysis Buffer Tris-HCl(pH7.4) 50mM EGTA 2mM EDTA lOmM NaF lOOmM Na4P207 ImM Na3V04 2mM Leupeptine 20pg/ml Aprotinin 20pg/ml Triton X-lOO 0.4%(V/V) PMSF ImM 3. Homogenates were then centrifuged at l2000Xg for min at 4C, collect the triton X-lOO-soluable fractions 4. Determinate the protein concentration according to the BioRad protein assay kit. Protocols for protein concentration assay Add 20pl of reagent S to each ml of reagent A 1. 3 dilution of protein standard (0.2mg/ml to LSmgAnl) in sample bufier 2. pipet 25|xl of standards and samples into clean test tubes 3. add 125jj.l reagent A into each tube 4. add LO ml reagent B into each test tube and votex immediately after 15 minutes, read absorbances at 750nm 5. Resuspended in SDS-sample buffer DTT(dithfothreitol) 0J7g(0.1M) SDS 2g(2%) Tris HCl(pH6.8) 4ml lM(80mM) Bromophenol Blue(0^% in EtOH) SOOpl Volume up to 50 mis with.water(store in 4C) 6. Separated by a 8 or 12% SDS PAGE 7. Transfer onto the PVDF, wash the blotted membrane twice with TBST 8. Block the blotted PVDF in TBST containmg 5% nonfat dry milk for Ihr at room temperature with constant agitation. 174- 9. Incubate the PVDF with I ng/ml of anti-phospho-AKT, diluted in freshly prepared-5%milk TBST overnight with agitation at 4C 10. Wash the PVDF in TBST 15min X4, incubate with Anti-rabbit IgG-HRP (1:3000 dilution) in TBST-5% milk for 40 minutes at room temperature with agitation Wash the PVDF in TBST 15minX6 IIEnhanced chemiluminescence for I minutes, expose to film BuCTers 1. TBST Tris lOXTBS 24.2g Tris Base, 80g NaCl in one liter water 2. IXTBST add Tween-20 to a concentration of 0.1%(V/V) and adjust pH to 7.4 at IX, 3. Running buffer, 14.4 g Glycine (I92mM) 1.0 g SDS (0.1%) 3.0 g Tris base(25mM) in one liter water Transfer buffer (Pre-Made) 175 APPENDIX c Explanation of assays ApoTag® Kit This assay uses Propridium iodide (PI) to stain DNA and fluorescent isothiocyanate (FITC) to stain DNA strand breaks associated with apoptosis. Flow cytometric measurements of cell cycle are made based on the amount on DNA per cell. Cells with 1 compliment of DNA are beginning the cell cycle (Go/Gt) and cells with 2 compliments of DNA are ending the cell division cycle (Gz/M). Cells are fixed in 1% paraformaldehyde and stained with PI and FITC. Cells are sorted and plotted on a histogram based on their amount of DNA. Amount of DNA 176 A Tdt en^one is added to the cell suspension and DNA strand breaks are fluorescently labeled with FITC at the 3’ OH group. Flourescence is measured as log FITC by the flow cytometer. Cells can be viewed and enumerated via a fluorescent microscope. Ofract Ftgur»1B Uninduced cells Endm#ultofapoplo«W:nud#o«om# ■tod ONA.IragmMts n.m iiiiiii luiiii iiii F IT C LOQ Induced cells ApopTkg* SWp IzTMEwNh fluoiMMiiK Now F r r c c j Q Apoptotic cells (above threshold line Q) Necrotic ornon- apoptotic cells DMA LIN 177 Annexin. V Staining This assay is similar to ApoTag® in that fluorescent staining and PI are used however Annexin V yields no cell cycle information. Annexin V can distinguish between early apoptosis, late apoptosis, necrosis, or living cells. The cells are not fixed and this method takes only 10 minutes. Apoptosis is determined by fluorescent staining of phosphatidyl serine (PS) by labeled Annexin V protein. Annexin V protein binds to PS, which normally resides on the inner (cytoplasmic) leaflet of the cell membrane but then translocates to the outer leaflet of the cell during apoptosis. Cells that are stained with Annexin V fluoresce and can be quantified as Log FITC. Cell that are Annexin V stained are apoptotic (14). Cells that take up PI have permeable membranes and are necrotic (II). Cell that stain Annexin V and take up PI are late apoptotic (12). Cells that take up neither are living (13). Necrotic cells ate apoptotic cells arly apoptotic cells Viable cells "I iiiiir-niTjm leoe K T C LOG 178 BIBLIOGRAPHY L Cancer Facts and Figures, American Cancer Society 2001. 2. Clinton, S, Giovanucci, E. Diet, nutrition, and prostate cancer. Anm. Rev. Nutr. 1998; 18:413-40. 3. Powell I, Gelfand D, Parzuchowski J, Heilbnm L, Franklin A. 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