Novel Antiproliferative Flavonoids Induce Cell Cycle Arrest in Human Prostate Cancer Cell Lines

Home , Karanjin

Prostate Cancer and Prostatic Diseases (2006) 9, 68–76 & 2006 Nature Publishing Group All rights reserved 1365-7852/06 $30.00 www.nature.com/pcan ORIGINAL ARTICLE Novel antiproliferative flavonoids induce cell cycle arrest in human prostate cancer cell lines

AQ Haddad1, V Venkateswaran1, L Viswanathan1, SJ Teahan1, NE Fleshner2 and LH Klotz1 1Division of Urology, Sunnybrook & Women’s College Health Sciences Centre, Toronto, ON, Canada and 2Division of Urology, Princess Margaret Hospital, Toronto, ON, Canada

Epidemiologic studies have demonstrated an inverse association between flavonoid intake and prostate cancer (PCa) risk. The East Asian diet is very high in flavonoids and, correspondingly, men in China and Japan have the lowest incidence of PCa worldwide. There are thousands of different naturally occurring and synthetic flavonoids. However, only a few have been studied in PCa. Our aim was to identify novel flavonoids with antiproliferative effect in PCa cell lines, as well as determine their effects on cell cycle. We have screened a representative subgroup of 26 flavonoids for antiproliferative effect on the human PCa (LNCaP and PC3), breast cancer (MCF-7), and normal prostate stromal cell lines (PrSC). Using a fluorescence-based cell proliferation assay (Cyquant), we have identified five flavonoids, including the novel compounds 2,20-dihydroxychalcone and fisetin, with antiproliferative and cell cycle arresting properties in human PCa in vitro. Most of the flavonoids tested exerted antiproliferative effect at lower doses in the PCa cell lines compared to the non-PCa cells. Flow cytometry was used as a means to determine the effects on cell cycle. PC3 cells were arrested in G2/M phase by flavonoids. LNCaP cells demonstrated different cell cycle profiles. Further studies are warranted to determine the molecular mechanism of action of 2,20-DHC and fisetin in PCa, and to establish their effectiveness in vivo. Prostate Cancer and Prostatic Diseases (2006) 9, 68–76. doi:10.1038/sj.pcan.4500845; published online 29 November 2005

Keywords: cell cycle; diet; flow cytometry; flavonoids

Introduction and dietary factors on PCa incidence. A number of case– control studies have correlated increased flavonoid Diets rich in flavonoids have been associated with a intake with a reduced incidence of a number of reduced incidence and mortality of prostate cancer malignancies including PCa.1,7–10 (PCa). The lowest incidence of PCa worldwide is seen Flavonoids comprise over 4000 structurally related in populations consuming the largest amount of flavo- polyphenols,11 which are ubiquitous in plants, and noids.1 In East Asian countries (China and Japan), diets ingested to varying degrees in the diet. The estimated are up to 100 times more abundant in flavonoids than in average daily intake of flavonoids is up to 1 g.12 This by the West, due in part to the consumption of soy and far exceeds the intake of other antioxidants such as green tea.2,3 Correspondingly, the incidence of PCa in vitamin E, and highlights the potential importance of China and Japan is 60- to 80-fold lower than in North flavonoids in the diet. Flavonoids have been shown to America.4 Studies on Japanese migrants to the United possess a wide range of biological activity , including States have shown that migrants born in Japan and living antioxidant (greater than vitamin C),13 anti-inflamma- in the United States have a higher incidence of PCa tory, antithrombogenic, and antiangiogenic activity.14 compared to men living in Japan.5,6 The incidence rates The anticancer properties of flavonoids have been for Japanese Americans born in the United States demonstrated in a variety of cell types in vitro and in increases further, approaching that of American white vivo.15 Despite the large number of flavonoids, studies men. Although these studies are not definitive, they have focused only on a select few. The flavonoids most emphasize the importance of environmental, lifestyle intensely studied in PCa to date are the soy isoflavones (genestein/daidzein),16,17 the green tea catechins (EGCG- epigallocatechin-3-gallate)18,19 and the milk thistle flavo- Correspondence: Dr LH Klotz, Division of Urology, Sunnybrook & nones (silibinin/silymarin).20,21 Little is known of the Women’s College Health Sciences Centre, MG-408, 2075 Bayview biological effect of most other flavonoids.22,23 Avenue, Toronto, Ontario, Canada M4N 3M5. E-mail: [email protected] In an attempt to identify novel flavonoids with Received 5 April 2005; revised 21 September 2005; accepted 22 growth-arresting properties in PCa cells, we have September 2005; published online 29 November 2005 screened a number of compounds from each of the Antiproliferative flavonoids and prostate cancer AQ Haddad et al 69 Table 1 Chemical composition of the flavonoids tested Subgroup Flavonoids Flavonoid side chain position*

20 30 40 50 23 45 6 7 8

Flavones Acacetin OMea OH OH b 6-Aminoflavone NH2 Baicalein OH OH OH Baicalin OH OH O-gluc 7,8-Benzoflavone benzd benzd Chrysin OH OH Diosmin OH OMe OH O-glu Karanjin OMe Furane Luteolin OH OH OH OH 5-Methoxyflavone OMe Primuletin OH

Flavonols Fisetin OH OH OH OH Galangin OH OH OH Geraldol OH OMe OH OH Gossypin OH OH OH OH OH O-glu Kaempferol OH OH OH OH Morin OH OH OH OH OH Myricetin OH OH OH OH OH OH Quercetin OH OH OH OH OH

Isoflavonoids Formononetin OMe OH Prunetin OH OH OMe Chalcones 2,20-DHC OH OH Isoliquiritigenin OH OH OH

Flavonones Pinostrobin OH OMe Catechins Epigallocatechin OH OH OH OH OH OH Anthocyanidins Pelargonidine OH OH OH OH aMethoxyl group. bAmino group. cGlycoside. dBenzene ring. eFuranone. *This figure applies only to flavone and flavonol subgroups. Conventional numbering of the side chains in employed for the remaining flavonoids in the table (ref.11). major flavonoid subgroups (Table 1). We have examined 5-methoxyflavone, acacetin, morin, 6-aminoflavone, 7,8- their antiproliferative effect on the PCa cell lines PC3 benzoflavone, epigallocatechin and epicatechin gallate (androgen independent) and LNCaP (androgen depen- were purchased from Sigma Aldrich (St Louis, MO, dent), MCF-7 breast cancer cell line and a nonmalignant USA). All other flavonoids were procured from Indofine prostate stromal cell line (PrSC). We have identified a Chemical Co. (Hillsborough, NJ, USA). Green tea number of novel flavonoids with antiproliferative and catechin-epigallocatechin (EGC) was dissolved in water. cell cycle effects in human PCa cells in vitro. The All other flavonoids were dissolved in dimethylsulph- compound identified with the greatest antiproliferative oxide (DMSO) to a stock concentration of 100 mM. effect was the synthetic flavonoid 2,20-dihydroxychal- Working standards were made up in serum containing cone (2,20-DHC). media. The final concentration of DMSO in culture did not exceed 0.2%. Flavonoids that were poorly soluble in DMSO were not studied further. These included the Materials and methods flavonoids prunetin, acacetin, formononetin, diosmin and karanjin. Chemicals The flavonoids, which have been included in this study, Tissue culture are presented in Table 1. Quercetin, pinostrobin, kaemp- The human PCa cell lines, LNCaP (mutated androgen ferol, pelargonidin, galangin, formononetin, prunetin, receptor (AR), p53 wild-type), PC3 (AR null, p53 null),

Prostate Cancer and Prostatic Diseases Antiproliferative flavonoids and prostate cancer AQ Haddad et al 70 and the estrogen receptor positive breast cancer cell line concentration of 10–150 mM. Control wells were treated MCF-7 were obtained from the American Type Tissue with vehicle alone (DMSO 0.2%). After 72 h of treatment Collection (ATCC), Rockville, MD, USA. The nonmalig- at 371C, the media was discarded, and the plates frozen nant prostate stromal cell line, PrSC, was obtained from at À801C until use. On the day of the analysis, the plates Cambrex, NJ, USA. LNCaP cells were cultured in RPMI with the adherent cells were thawed and incubated with 1640 medium (Gibco, New York) supplemented with the CyQuant dye for 5 min in the dark. Fluorescence was 10% foetal bovine serum (FBS) and 100 IU/ml penicillin measured on an FL600 fluorescence micro-plate reader and 100 mg/ml streptomycin. PC3 cells were cultured in (Bio-Tek, VT) with filters set at 480 nm excitation and DMEM/F12 medium with 10% FBS and antibiotics. 520 nm emission. The IC50 for each flavonoid was MCF-7 cells were cultured in IMEM media supplemen- determined in the four cell lines tested (Table 2). Each ted with 5% FBS, antibiotics, and insulin. PrSC medium experiment was performed independently at least three consisted of stromal cell basal media, and the manufac- times. turer’s growth factor supplements (Cambrex, NJ, USA). All cells were cultured at 371C with 5% CO . 2 Flow cytometry and cell cycle analysis Cell cycle arrest pattern and S phase enumeration were Cell proliferation assay determined by flow cytometry on cells labelled with anti- BrdU FITC and propidium iodide. Asynchronously Proliferation was assessed using the CyQuant cell 5 proliferation assay (Molecular Probes, OR, USA). In this growing cells (5 Â 10 cells/plate) were plated in 10 cm assay, the proprietary CyQuant dye binds to DNA, and dishes and treated for 72 h with the flavonoid at the IC50 the fluorescence emitted by the dye is linearly propor- concentration as determined earlier. Control plates were tional to the number of cells in the well. Cells were plated treated with vehicle alone (DMSO 0.2%). The cells were in 96-well black fluorescence micro-titre plates, at a pulse labelled with bromodeoxyuridine (BrdU) for 2 h density of 4000 cells/well. At 24 h after plating, triplicate prior to harvesting. As a negative control, a no-BrdU wells were treated with the appropriate flavonoid at a control was also included. Cells were trypsinized, fixed in ice cold 70% ethanol and stored at À201C until further analysis. Cells were then washed in buffer (PBS and 0.5% Tween-20) and treated with 2 N HCl for 20 min to expose labelled DNA. Cells were incubated for 1 h on ice with Table 2 Antiproliferative effect of flavonoids. IC50 (in mM) for anti-BrdU conjugated fluorescein isothiocyanate (Becton flavonoids in human prostate cancer (PC3 and LNCaP), breast cancer (MCF-7) and prostate stromal cells (PrSC) Dickinson, San Jose, CA, USA). Cells were washed, centrifuged, and resuspended in 10 mg/ml propidium LNCaP PC3 MCF7 PRSC iodide, and allowed to incubate for 30 min on ice. Samples were filtered through a nylon mesh and cell 5-Methoxyflavone 25.22 97.31 480 480 cycle analysis performed on the FACSCalibur flow 6-Aminoflavone 4100 4100 Baicalin 52.85 51.58 480 480 cytometer using the Cell Quest Pro software package Baicalein 16.78 64.23 480 480 (Becton Dickinson, CA, USA). 7,8-Benzoflavone 98.04 4100 Chrysin 56.81 4100 2,20-DHC 10.80 10.26 22.99 17.47 Statistical analysis Epigallocatechin 4100 4100 Cell proliferation was determined by Cyquant fluores- Fisetin 22.65 32.50 480 480 cence and expressed as a percentage of untreated control. Galangin 94.92 4100 The percentage value for each treatment was obtained Geraldol 4100 4100 Gossypin 4100 4100 from three replicate experiments. The mean of the three Isoliquiritigenin 17.88 34.43 67.17 45.88 experiments was plotted on a concentration–response Kaempferol 52.24 41.98 curve. A best-fit regression curve was determined by Luteolin 18.22 28.84 29.13 68.37 polynomial third-order equation, and performed indivi- Morin 4100 4100 dually for each flavonoid (GraphPad Prism version 4.03 Myricetin 4100 4100 for Windows, GraphPad Software, San Diego, CA, USA). Pelargonidin 4100 4100 Pinostrobin 4100 4100 We next determined (from the regression curves) the Quercetin 19.44 33.41 480 480 concentration of flavonoid at which there was a 50% reduction in cell number compared to the control (IC50).

Table 3 S-phase reduction Flavonoid LNCaP % reduction PC3 % reduction

Vehicle treated control 22.0874.93* 21.8175.50 w 2,20-DHC 5.1673.08* 76.63 10.1676.98 53.44 Fisetin 9.4371.18* 57.29 6.9373.85* 68.25 Isoliquiritigenin 6.3970.27* 71.07 12.4576.49 42.93 w Luteolin 11.6671.31 47.19 8.47571.72* 61.15 Quercetin 8.1372.04* 63.19 4.072.61* 81.66

The percentage of cells in S phase (7s.d.) in control and flavonoid treated cells at 72 h. Values shown are the mean of 3 experiments. The percentage reduction in flavonoid treated compared to vehicle treated control cells was calculated and unpaired t-test performed to compare each treatment with the control. w *Po0.05, Po0.1.

Prostate Cancer and Prostatic Diseases Antiproliferative flavonoids and prostate cancer AQ Haddad et al

The unpaired t-test (two-tailed) was performed to Flavonoids cause alteration in cell cycle regulation 71 determine statistical significance of S-phase alterations in human PCa cells in vitro in flavonoid treated and control cells (GraphPad Prism Having identified the flavonoids that possessed the version 4.03 for Windows). greatest effect on proliferation, we next examined the effect of these compounds on cell cycle profiles in LNCaP and PC3 cells. Cells were treated with flavonoids for 72 h in vitro, and flow cytometry performed with dual labelling of cells with PI and anti-BrdU (Figures 1 and Results 2). All five flavonoids (2,20-DHC, fisetin, ISLQ, luteolin, and quercetin) caused cell cycle arrest in PC3 and LNCaP Flavonoids exert an antiproliferative effect on human PCa cells. Interestingly, all of these flavonoids caused a cells in vitro similar pattern of cell cycle arrest (G2/M) in PC3 cells The antiproliferative effects of flavonoids were assessed (Figure 1), while LNCaP cells were arrested in both G1 in LNCaP and PC3 cells in vitro. A subset of flavonoids and G2/M (Figure 2). All of the flavonoids tested caused with antiproliferative effect were also tested in the MCF- a reduction in the proportion of cells in the synthesizing 7 breast cancer cell line, and the nonmalignant prostate (S phase) of the cell cycle in both cell lines (Table 3). Time stromal cell line PrSC. Proliferation studies were initially course studies performed demonstrated that the effect of performed by the more commonly used MTT assay. As flavonoid treatment on the cell cycle was observed as many flavonoids are coloured when in solution, the MTT early as 24 h of treatment (data not shown). assay was unsuitable as this interfered with the calori- There was up to a three-fold increase in cells in the metric nature of this assay. In addition, flavonoids are G2/M phase in PC3 cells with flavonoid treatment known to reduce the tetrazolium in the MTT solution, 24 compared to control cultures. Fisetin caused the greatest even in the absence of cells. The CyQUANT assay accumulation of cells in G2/M, with 66.5% (712.6) of overcame these limitations and was used for the high cells in G2/M compared to 13.0% (72.8) in the control. A throughput screening of flavonoids in all further experi- corresponding reduction of S and G1 phase cells was ments. seen in PC3 cells. In terms of S-phase reduction in PC3 Concentration–response curves were generated for cells, the greatest effect was seen with quercetin, fisetin each flavonoid. We determined the concentration at and luteolin (81.7, 68.3 and 61.2% reduction, respec- which flavonoids caused 50% growth inhibition com- tively). 2,20-DHC, the flavonoid with the greatest anti- pared to control (IC50) (Table 2). The five flavonoids with proliferative effect, caused a 53.4% reduction in S phase, 0 the greatest antiproliferative effect in LNCaP were 2,2 - and a three-fold rise in G2/M phase cells in PC3 dihyroxychalcone, baicalein, isoliquiritigenin (ISLQ), (Figure 1). ISLQ caused a smaller reduction in S-phase luteolin and quercetin. In PC3, the most potent com- cell numbers (42.9%) (Table 3). 0 pounds were 2,2 -DHC, luteolin, fisetin, quercetin and A different pattern of cell cycle arrest was observed in 0 ISLQ. 2,2 -DHC caused growth inhibition at low doses in LNCaP (Figure 2). Although LNCaP cells treated with all four cell lines tested, the lowest IC50 observed in PC3 flavonoid demonstrated an increase in G2/M phase cell and LNCaP (10.26 and 10.8 mM, respectively), and the numbers, indicative of a G2/M arrest, the percentage of highest in MCF7 (23 mM) (Table 2). Fisetin had an IC50 of cells in G1 phase did not decrease. This suggests that 22.65 mM in LNCaP and 32.5 mM in PC3. In total, 50% cells were being partially arrested in G1 phase. Com- reduction in cell number at the maximum concentration pared to PC3, the increase in the proportion of G2/M was not achieved in MCF-7 and PrSC. Similarly, ISLQ cells was smaller in LNCaP, with most flavonoids had greater antiproliferative effect in PCa compared to causing around a two-fold increase in G2/M phase non-PCa cell lines (Table 2). Luteolin demonstrated distribution. However, as in PC3, all flavonoids caused a antiproliferative activity in LNCaP (IC50 18.22 mM), PC3 reduction in percentage of cells in S phase in LNCaP (IC50 28.84 mM) and MCF-7 cells (IC50 29.13 mM) and to a cells. Fisetin, at a concentration of 25 mM, caused the lesser degree on PrSC (IC50 68.37 mM). Quercetin had IC50 greatest reduction of cells in S phase in LNCaP cells of 33.41 and 19.44 mM in PC3 and LNCaP, with 50% (76.6% reduction) with a concomitant rise in the reduction in cell numbers compared to control not proportion of cells in G2 phase (19.4%74.9 compared reached in the prostatic stromal or breast cancer cell to 8.7%70.63 in control) and a nonsignificant increase in lines. G1 phase (71.2%76.2 in fisetin vs 69%74.3 in control) Other flavonoids shown to inhibit proliferation inclu- (Table 3). LNCaP cells treated with ISLQ, quercetin, ded 5-methoxyflavone, baicalin, baicalein, chrysin and luteolin and 2,20-DHC also caused reductions in the kaempferol as detailed in Table 2. A greater effect on percentage of cells in the S phase compared to vehicle proliferation of LNCaP than PC3 cells were observed only control (71.1, 63.2, 57.3 and 47.2%, respectively). In with 5-methoxyflavone (IC50 25.22 and 97.31 mM, respec- general, the proportion of cells in S phase in flavonoid tively). Baicalein had a lower IC50 than the glycosylated treated groups was significantly lower than the DMSO baicalin when tested in LNCaP, while baicalin seemed to control, with few exceptions (Table 3). be more potent in PC3 than LNCaP. Kaempferol and chrysin had an IC50 above 40 mM in LNCaP and PC3 cells, a concentration unlikely to be achieved physiologically. Galangin inhibited proliferation in LNCaP only at very Discussion high concentrations, and the remainder of the flavonoids (6-aminoflavone, EGC, geraldol, gossypin, morin, myr- In this study, we have screened a diverse group of icetin, pinostrobin, and pelargonidin) did not inhibit flavonoids for their antiproliferative effect on PCa, breast proliferation at the doses tested. cancer and normal prostate stromal cells in vitro. Most of

Prostate Cancer and Prostatic Diseases Antiproliferative flavonoids and prostate cancer AQ Haddad et al 72

Figure 1 Flow cytometric analysis – PC3. Cells were treated for 72 h with flavonoid or vehicle alone (0.2% DMSO). Cells were then pulse labelled with BrdU for 2 h prior to harvesting, and stained with BrdU-FITC conjugate for determination of DNA synthesis, and with propidium iodide (PI) for determination of total DNA content. (a) A representative of two FACS plots is shown for each flavonoid. The dot plot represents BrdU incorporation (y-axis) vs DNA content, as determined by PI staining (x-axis). The corresponding PI histogram is depicted beneath the dot plots. DNA synthesis (S-phase) was determined by quantifying cells positive for BrdU staining in the dot plots. (b) The bar chart shows the distribution of cells in the different phases of the cell cycle following flavonoid treatment, from three replicate experiments. No BrdU: represents cells treated with vehicle alone (0.2% DMSO) that were not pulse-labelled with BrdU, but were otherwise stained with anti-BrdU-FITC antibody and propidium iodide. C: Vehicle only control (0.2% DMSO) with BrdU pulse labelling; DHC: 2,20- Dihydroxychalcone; Fi: Fisetin; Q: Quercetin; ISLQ: Isoliquiritigenin; LUT: Luteolin.

the flavonoids screened are novel compounds as they 2,20-DHC was observed as early as 24 h following that have not been previously evaluated. We have treatment (data not shown). Whereas most of the identified a number of flavonoids that cause growth flavonoids tested were minimally cytotoxic to the PrSC arrest at low concentrations in PCa, and appear to be less and MCF-7 cell lines, 2,20-DHC was unique in that it was effective on non-PCa cell lines (MCF-7 and PrSC). We effective at low concentrations on PrSC and MCF-7 cells have also demonstrated that these flavonoids caused cell (IC50 17.47 and 22.99 mM respectively). The apparent cycle arrest (G1 and G2/M), with a reduction in the resistance of MCF-7 and PrSC cells to most flavonoids number of cells in the S phase (81% reduction) (Figures 1 may be a true effect; however, this is difficult to ascertain and 2). since all cell lines have been cultured in cell-specific As evident from Table 2, the flavonoid with the lowest media, and differences in growth media differences may 0 IC50 concentration was 2,2 -DHC. This is a synthetic account for some of the observations. Furthermore, it is flavonoid belonging to the chalcone subgroup of not possible to reach conclusions on the cell specificity of flavonoids that are precursors in the flavone synthesis flavonoids based on the limited number of cell lines that pathway in plants.25 The growth inhibitory effect of we have used and this would have to be carried out on a

Prostate Cancer and Prostatic Diseases Antiproliferative flavonoids and prostate cancer AQ Haddad et al 73

Figure 2 Flow cytometric analysis – LNCaP. Cells were treated with flavonoid or DMSO control as for PC3 (see Figure 1). (a) Representative FACS plots for cells treated with flavonoids- BrdU vs PI (top graph) and DNA content (corresponding PI histogram). DNA synthesis (S- phase) was determined by quantifying cells positive for BrdU staining in the dot plots. (b) Bar chart showing the cell cycle distribution of flavonoid treated cells as determined from three independent experiments. No BrdU: cells treated with vehicle alone (0.2% DMSO) with no BrdU pulse labelling; C: Vehicle only control (0.2% DMSO) with BrdU pulse labelling; DHC: 2,20-Dihydroxychalcone; Fi: Fisetin; Q: Quercetin; ISLQ: Isoliquiritigenin; LUT: Luteolin. battery of cell lines. In this study, the PrSC stromal cell been shown to exert antiproliferative effect on a line was preferred to the PREC benign epithelial cell line, number of cell lines.30–32 ISLQ, luteolin and quercetin due to the slow growth kinetics and difficult culture appeared to have greater effect on proliferation in conditions of the latter. LNCaP than in PC3 cells. In addition, two other Other flavonoids identified with antiproliferative flavonoids, 5-methoxyflavone (synthetic) and baicalein effect were fisetin, ISLQ, luteolin, quercetin, baicalein (component of PCSPES herbal remedy), had low IC50 in and 5-methoxyflavone (Table 2). Fisetin, isolated from LNCaP but not PC3 (Table 2). A number of differences the bark of Rhus cotinus,26 demonstrated cytotoxicity in between PC3 and LNCaP cells may account for this both PC3 and LNCaP cells. Fisetin was less cytotoxic in difference in sensitivity. PC3 is a more aggressive cell MCF-7 and PrSC cells (IC50480 mM). ISLQ is a chalcone line, and is null for p53 and androgen receptor (AR). found in liquorice root and is a constituent of a some LNCaP, a much less aggressive cell line possesses a wild- herbal remedies.27 Luteolin is a flavone widely distrib- type p53 and a mutated (AR). ISLQ and quercetin have uted in nature, and found in sources such as parsley, less antiproliferative effect on MCF-7 and PrSC cells. 28 artichoke, and celery. Quercetin is the most prevalent Luteolin has a low IC50 in the MCF-7 breast cancer flavonoid in the Western diet,29 and is ubiquitous in cell line, a property that merits further investigation plants. It is the most widely studied flavonoid and has (Table 2).

Prostate Cancer and Prostatic Diseases Antiproliferative flavonoids and prostate cancer AQ Haddad et al

74 Despite the close similarities in the structure of the enhances the antiproliferative effects of vitamin D by flavonoids, we observed a marked difference in their upregulating vitamin D receptor and p21Cip1/WAF1 pro- antiproliferative activities (Table 1). For example, quer- tein levels.50 This synergistic effect with vitamin D may cetin and gossypin differ by a single hydroxyl substitu- contribute to the reduced incidence of PCa associated tion at the C-8 position. While quercetin caused growth with diets that are rich in both vitamin D and flavonoids, arrest in LNCaP and PC3 cells, gossypin had no such such as the traditional Asian diet. In the present study, action despite high concentrations. Microscopically, at antiproliferative effects were seen in both AR dependent the highest doses tested (100–150 mM), some of the and independent human PCa cell lines, suggesting that flavonoids such as EGC caused minimal cell toxicity the AR is not a critical component for mediating the leading to a high rate of cell survival. Treatment at high growth-arresting properties of flavonoids. concentration with the more toxic flavonoids such as 2,20- Preliminary animal studies investigating the in vivo DHC resulted in morphological changes (viz, rounding effects of flavonoids have been promising, with a and cell detachment). However, even with doses as high number of flavonoids having demonstrated anti-PCa 51,52 as 500 mM of 2,20-DHC, a small number of cells (500–600) activity. For example, TRAMP mice (transgenic remained adherent as detected by the CyQuant assay adenocarcinoma of the mouse prostate) fed genestein (data not shown). showed a reduction in the incidence prostate carcino- Several flavonoids such as myricetin, pelargonidin and mas.51 Gupta et al.52 have shown that TRAMP mice epigallocatechin (EGC) previously identified in epide- placed on a green tea catechin rich extract had reduced miological studies (as being linked to reduced incidence liver metastases. Other investigators have confirmed the of PCa)19,33–38 did not display antiproliferative activity in effect of selected flavonoids in PCa employing xenograft our experiments. These studies, however, were based on models.21,53,54 In these experiments, there have been no retrospective dietary assessments and numerous pro- reports of flavonoid induced systemic toxicity (a desir- blems arise while attempting to examine the effect of able property of an anticancer agent). Since we have flavonoids by this methodology. The in vivo anticancer shown 2,20-DHC, fisetin, luteolin and quercetin to have properties of flavonoids cannot be accurately determined greater in vitro activity than genestein and catechins, we from epidemiologic studies partly due to the confound- feel it would be necessary to examine the effect of these ing effects of the hundreds of other flavonoids present in compounds in vivo. 2,20-DHC and fisetin have demon- the diet. Flavonoids examined in previous in vitro studies strated no toxicity at the concentrations used in this include EGC (IC50 88 mM in DU145 prostate cancer cell experiment (12–14 mM) when tested on isolated rat 55,56 line) and ISLQ (IC50 13 mM in DU145). In our study, EGC hepatocytes, and minimal toxicity would be expected did not inhibit cell growth (50% growth inhibition) in in vivo. LNCaP or PC3 cells, at concentration up to 150 mM. Flavonoids account for many of the beneficial effects In previous studies, flavonoids have been shown to observed with diets rich in fruits and vegetables. We cause alterations in cell cycle regulation in a number of have identified several antiproliferative flavonoids that cell lines. We performed FACS analysis to confirm the cause cell cycle arrest in PCa in vitro. Further studies are alterations in cell cycle regulatory properties of the key underway to explore the molecular mechanisms of action flavonoids identified in this study (Figures 1 and 2). of the novel flavonoids identified in this study, and to Results indicate that 2,20-DHC, fisetin, ISLQ, luteolin and determine their properties in vivo. quercetin all caused a G2/M arrest in both LNCaP and PC3 cells. The percentage of cells in G2 increased with corresponding decrease in the percentage of cells in the S phase cells for all flavonoids in both LNCaP and PC3 Acknowledgements cells. In PC3 cells, the percentage of cells in the G1 phase This work was supported by grant from the Canadian decreased, while in LNCaP, the proportion of cells in G1 Prostate Cancer Research Initiative, and the Canadian remained unchanged compared to the control. The p53 Prostate Cancer Research Bionet. status may partly explain the different cell cycle arrest pattern observed between cell lines. As mentioned earlier, PC3 cells are p53 null, and as a result may be defective in G1 checkpoint control, explaining the lack of References a G1 arrest in this cell line. This hypothesis remains to be proven. Previous studies have shown that quercetin and 1 Ganry O. Phytoestrogens and prostate cancer risk. Prev Med 39–41 2005; 41: 1–6. ISLQ cause cell cycle arrest in PCa cell lines. 2 Morton MS, Chan PS, Cheng C, Blacklock N, Matos-Ferreira A, However, there have been no reports to date on the cell Abranches-Monteiro L et al. Lignans and isoflavonoids in 0 cycle regulatory effects of 2,2 -DHC, fisetin and luteolin. plasma and prostatic fluid in men: samples from Portugal, The precise mechanism by which the flavonoids Hong Kong, and the United Kingdom. Prostate 1997; 32: 122–128. identified in this study exert their action remains to be 3 Adlercreutz H, Markkanen H, Watanabe S. Plasma concentra- determined. Flavonoids have been shown to alter a tions of phyto-oestrogens in Japanese men. Lancet 1993; 342: number of key proteins implicated in growth and 1209–1210. differentiation. These include induction of cyclin-depen- 4 Parkin DM, Bray F, Ferlay J, Pisani P. Global cancer statistics, dent kinase (cdk) inhibitors p21Cip1/WAF1 and 2002. CA Cancer J Clin 2005; 55: 74–108. Kip1 39,42 5 Lyn-Cook BD, Rogers T, Yan Y, Blann EB, Kadlubar FF, p27 , inhibition of phosphorylation of retinoblas- Hammons GJ. Chemopreventive effects of tea extracts and toma (Rb) protein,43 decrease in levels of cyclins B, D and 44–46 47,48 various components on human pancreatic and prostate tumor E and cdk 2,4 and 6, induction of apoptosis, cells in vitro. Nutr Cancer 1999; 35: 80–86. inhibition of topoisomerase II, and alterations to the 6 Maskarinec G, Singh S, Meng L, Franke AA. Dietary soy intake MAPkinase pathway.49 Genestein, a soy isoflavone, and urinary isoflavone excretion among women from a multi-

Prostate Cancer and Prostatic Diseases Antiproliferative flavonoids and prostate cancer AQ Haddad et al 75 ethnic population. Cancer Epidemiol Biomarkers Prev 1998; 7: 31 van der Woude H, Gliszczynska-Swiglo A, Struijs K, Smeets A, 613–619. Alink GM, Rietjens IM. Biphasic modulation of cell proliferation 7 Hirvonen T, Virtamo J, Korhonen P, Albanes D, Pietinen P. by quercetin at concentrations physiologically relevant in Flavonol and flavone intake and the risk of cancer in male humans. Cancer Lett 2003; 200: 41–47. smokers (Finland). Cancer Causes Control 2001; 12: 789–796. 32 Choi JA, Kim JY, Lee JY, Kang CM, Kwon HJ, Yoo YD et al. 8 Knekt P, Kumpulainen J, Jarvinen R, Rissanen H, Heliovaara M, Induction of cell cycle arrest and apoptosis in human breast Reunanen A et al. Flavonoid intake and risk of chronic diseases. cancer cells by quercetin. Int J Oncol 2001; 19: 837–844. Am J Clin Nutr 2002; 76: 560–568. 33 Galvano F, La Fauci L, Lazzarino G, Fogliano V, Ritieni A, 9 Neuhouser ML. Dietary flavonoids and cancer risk: evidence Ciappellano S et al. Cyanidins: metabolism and biological from human population studies. Nutr Cancer 2004; 50: 1–7. properties. J Nutr Biochem 2004; 15: 2–11. 10 Lee MM, Gomez SL, Chang JS, Wey M, Wang RT, Hsing AW. Soy 34 Xue H, Aziz RM, Sun N, Cassady JM, Kamendulis LM, Xu Y and isoflavone consumption in relation to prostate cancer risk et al. Inhibition of cellular transformation by berry extracts. in China. Cancer Epidemiol Biomarkers Prev 2003; 12: 665–668. Carcinogenesis 2001; 22: 351–356. 11 Bohm BA. Structural variation and the flavonoid literature. In: 35 Paschka AG, Butler R, Young CY. Induction of apoptosis in Ravindranath B (ed.), Introduction to Flavonoids, vol. 2, Harwood prostate cancer cell lines by the green tea component, (À)- Academic: New York, 1998, pp. 5–7. epigallocatechin-3-gallate. Cancer Lett 1998; 130: 1–7. 12 Kuhnau J. The flavonoids. A class of semi-essential food 36 Brusselmans K, De Schrijver E, Heyns W, Verhoeven G, Swinnen components: their role in human nutrition. World Rev Nutr Diet JV. Epigallocatechin-3-gallate is a potent natural inhibitor of fatty 1976; 24: 117–191. acid synthase in intact cells and selectively induces apoptosis in 13 Noroozi M, Angerson WJ, Lean ME. Effects of flavonoids and prostate cancer cells. Int J Cancer 2003; 106: 856–862. vitamin C on oxidative DNA damage to human lymphocytes. 37 Saleem M, Adhami VM, Siddiqui IA, Mukhtar H. Tea beverage Am J Clin Nutr 1998; 67: 1210–1218. in chemoprevention of prostate cancer: a mini-review. Nutr 14 Valachovicova T, Slivova V, Sliva D. Cellular and physiological Cancer 2003; 47: 13–23. effects of soy flavonoids. Mini Rev Med Chem 2004; 4: 881–887. 38 Chung LY, Cheung TC, Kong SK, Fung KP, Choy YM, Chan ZY 15 Le Marchand L. Cancer preventive effects of flavonoids – a et al. Induction of apoptosis by green tea catechins in human review. Biomed Pharmacother 2002; 56: 296–301. prostate cancer DU145 cells. Life Sci 2001; 68: 1207–1214. 16 Sarkar FH, Li Y. Soy isoflavones and cancer prevention. Cancer 39 Kobayashi T, Nakata T, Kuzumaki T. Effect of flavonoids on cell Invest 2003; 21: 744–757. cycle progression in prostate cancer cells. Cancer Lett 2002; 176: 17 Castle EP, Thrasher JB. The role of soy phytoestrogens in 17–23. prostate cancer. Urol Clin North Am 2002; 29: 71–81, viii–ix. 40 Knowles LM, Zigrossi DA, Tauber RA, Hightower C, Milner JA. 18 Park OJ, Surh YJ. Chemopreventive potential of epigallocatechin Flavonoids suppress androgen-independent human prostate gallate and genistein: evidence from epidemiological and tumor proliferation. Nutr Cancer 2000; 38: 116–122. laboratory studies. Toxicol Lett 2004; 150: 43–56. 41 Kanazawa M, Satomi Y, Mizutani Y, Ukimura O, Kawauchi A, 19 Adhami VM, Ahmad N, Mukhtar H. Molecular targets for Sakai T et al. Isoliquiritigenin inhibits the growth of prostate green tea in prostate cancer prevention. J Nutr 2003; 133: cancer. Eur Urol 2003; 43: 580–586. S2417–S2424. 42 Choi YH, Lee WH, Park KY, Zhang L. p53-independent 20 Agarwal R. Cell signaling and regulators of cell cycle as induction of p21 (WAF1/CIP1), reduction of cyclin B1 and molecular targets for prostate cancer prevention by dietary G2/M arrest by the isoflavone genistein in human prostate agents. Biochem Pharmacol 2000; 60: 1051–1059. carcinoma cells. Jpn J Cancer Res 2000; 91: 164–173. 21 Tyagi AK, Singh RP, Agarwal C, Chan DC, Agarwal R. Silibinin 43 Tyagi A, Agarwal C, Agarwal R. Inhibition of retinoblastoma strongly synergizes human prostate carcinoma DU145 cells protein (Rb) phosphorylation at serine sites and an increase in to doxorubicin-induced growth Inhibition, G2-M arrest, and Rb-E2F complex formation by silibinin in androgen-dependent apoptosis. Clin Cancer Res 2002; 8: 3512–3519. human prostate carcinoma LNCaP cells: role in prostate cancer 22 Manthey JA, Guthrie N. Antiproliferative activities of citrus prevention. Mol Cancer Ther 2002; 1: 525–532. flavonoids against six human cancer cell lines. J Agric Food Chem 44 Davis JN, Singh B, Bhuiyan M, Sarkar FH. Genistein-induced 2002; 50: 5837–5843. upregulation of p21WAF1, downregulation of cyclin B, and 23 Kawaii S, Tomono Y, Katase E, Ogawa K, Yano M. Antiproli- induction of apoptosis in prostate cancer cells. Nutr Cancer 1998; ferative activity of flavonoids on several cancer cell lines. Biosci 32: 123–131. Biotechnol Biochem 1999; 63: 896–899. 45 Shukla S, Gupta S. Molecular mechanisms for apigenin-induced 24 Bruggisser R, von Daeniken K, Jundt G, Schaffner W, Tullberg- cell-cycle arrest and apoptosis of hormone refractory human Reinert H. Interference of plant extracts, phytoestrogens and prostate carcinoma DU145 cells. Mol Carcinog 2004; 39: 114–126. antioxidants with the MTT tetrazolium assay. Planta Med 2002; 46 Gupta S, Hussain T, Mukhtar H. Molecular pathway for (À)- 68: 445–448. epigallocatechin-3-gallate-induced cell cycle arrest and apop- 25 Bohm BA. Biosynthesis and genetics. In: Ravindranath B (ed.), tosis of human prostate carcinoma cells. Arch Biochem Biophys Introduction to Flavonoids, vol. 2, Harwood Academic: New York, 2003; 410: 177–185. 1998, pp. 299–306. 47 Ikezoe T, Chen SS, Heber D, Taguchi H, Koeffler HP. Baicalin is a 26 Chas E, Sando HHB. The Flavones of Rhus. American J Bot 1918; major component of PC-SPES which inhibits the proliferation of 5: 105–111. human cancer cells via apoptosis and cell cycle arrest. Prostate 27 Takahashi T, Takasuka N, Iigo M, Baba M, Nishino H, Tsuda H 2001; 49: 285–292. et al. Isoliquiritigenin, a flavonoid from licorice, reduces 48 Chen YC, Shen SC, Lee WR, Lin HY, Ko CH, Shih CM et al. prostaglandin E2 and nitric oxide, causes apoptosis, and sup- Wogonin and fisetin induction of apoptosis through activation presses aberrant crypt foci development. Cancer Sci 2004; 95: of caspase 3 cascade and alternative expression of p21 protein 448–453. in hepatocellular carcinoma cells SK-HEP-1. Arch Toxicol 2002; 28 Sampson L, Rimm E, Hollman PC, de Vries JH, Katan MB. 76: 351–359. Flavonol and flavone intakes in US health professionals. JAm 49 Siddiqui IA, Adhami VM, Afaq F, Ahmad N, Mukhtar H. Diet Assoc 2002; 102: 1414–1420. Modulation of phosphatidylinositol-3-kinase/protein kinase B- 29 Hertog MG, Feskens EJ, Hollman PC, Katan MB, Kromhout D. and mitogen-activated protein kinase-pathways by tea poly- Dietary flavonoids and cancer risk in the Zutphen Elderly Study. phenols in human prostate cancer cells. J Cell Biochem 2004; 91: Nutr Cancer 1994; 22: 175–184. 232–242. 30 Trzeciak A. Quercetin: significance in mutagenesis and carcino- 50 Rao A, Coan A, Welsh JE, Barclay WW, Koumenis C, Cramer SD. genesis. Postepy Biochem 2001; 47: 299–306. Vitamin D receptor and p21/WAF1 are targets of genistein and

Prostate Cancer and Prostatic Diseases Antiproliferative flavonoids and prostate cancer AQ Haddad et al 76 1, 25-dihydroxyvitamin D3 in human prostate cancer cells. 54 Singh RP, Sharma G, Dhanalakshmi S, Agarwal C, Agarwal R. Cancer Res 2004; 64: 2143–2147. Suppression of advanced human prostate tumor growth 51 Mentor-Marcel R, Lamartiniere CA, Eltoum IE, Greenberg NM, in athymic mice by silibinin feeding is associated with Elgavish A. Genistein in the diet reduces the incidence of poorly reduced cell proliferation, increased apoptosis, and inhibition differentiated prostatic adenocarcinoma in transgenic mice of angiogenesis. Cancer Epidemiol Biomarkers Prev 2003; 12: (TRAMP). Cancer Res 2001; 61: 6777–6782. 933–939. 52 Gupta S, Hastak K, Ahmad N, Lewin JS, Mukhtar H. Inhibition 55 Moridani MY, Galati G, O’Brien PJ. Comparative quantitative of prostate carcinogenesis in TRAMP mice by oral infusion of green structure toxicity relationships for flavonoids evaluated in tea polyphenols. Proc Natl Acad Sci USA 2001; 98: 10350–10355. isolated rat hepatocytes and HeLa tumor cells. Chem Biol Interact 53 Rice L, Samedi VG, Medrano TA, Sweeney CA, Baker HV, 2002; 139: 251–264. Stenstrom A et al. Mechanisms of the growth inhibitory effects of 56 Sabzevari O, Galati G, Moridani MY, Siraki A, O’Brien PJ. the isoflavonoid biochanin A on LNCaP cells and xenografts. Molecular cytotoxic mechanisms of anticancer hydroxychal- Prostate 2002; 52: 201–212. cones. Chem Biol Interact 2004; 148: 57–67.

Prostate Cancer and Prostatic Diseases