HUMAN BREAST CANCER CELL LINE MDA-MB-231 a Thesis

Home , Hispidulin

SEASONAL VARIATION OF THE CYTOTOXICITY OF Iva hayesiana ON THE

HUMAN BREAST CANCER CELL LINE MDA-MB-231

A Thesis

Presented to the faculty of the Department of Chemistry

California State University, Sacramento

Submitted in partial satisfaction of the requirements for the degree of

MASTER OF SCIENCE

Chemistry

(Biochemistry)

Barbara J. Coulombe

SUMMER 2012

SEASONAL VARIATION OF THE CYTOTOXICITY OF Iva hayesiana ON THE

HUMAN BREAST CANCER CELL LINE MDA-MB-231

A Thesis

Barbara J. Coulombe

Approved by:

______, Committee Chair Dr. Mary McCarthy-Hintz

______, Second Reader Dr. Katherine McReynolds

______, Third Reader Dr. Roy Dixon

______Date

iii

Student: Barbara J Coulombe

I certify that this student has met the requirements for format contained in the

University format manual, and that this thesis is suitable for shelving in the Library and credit is to be awarded for the thesis.

______, Graduate Coordinator ______Dr. Susan Crawford Date

Department of Chemistry

Abstract

SEASONAL VARIATION OF THE CYTOTOXICITY OF Iva hayesiana ON THE

HUMAN BREAST CANCER CELL LINE MDA-MB-231

Barbara J. Coulombe

Iva hayesiana, also known as San Diego poverty weed, is a California native plant that grows in the San Diego coastal region and Baja California. I. hayesiana contains the flavone hispidulin, which has been shown to be cytotoxic to human pancreatic cancer cells. A previous screen indicated that an extract of the leaves and stems of I. hayesiana is cytotoxic to the human breast cancer cell line MDA-MB-231.

However, with repeated extractions over several months, the cytotoxic properties became negligible. The goal of this study was threefold: first, to optimize the extraction conditions for I. hayesiana, second, to determine whether the cytotoxicity is seasonal, and third, to determine whether hispidulin is responsible for the cytotoxicity towards

MDA-MB-231. Results showed that hexane extracts of fresh aerial parts of I. hayesiana were the most effective against MDA-MB-231, with an IC50 of 56 3

µg/mL. Vacuum distillation of the leaves and stems showed that the cytotoxic constituents are not volatile; therefore, HPLC was used for chromatographic analysis.

Monthly extracts from February thru July showed that the cytotoxicity returned in April and dissipated in June, when the plant flowered, which supports the hypothesis that the cytotoxicity is seasonal and is therefore related to the life cycle of the plant. HPLC of these extracts indicate that there are distinct chemical differences between the active and inactive extracts. Most notably, there is one HPLC peak of interest that requires further analysis. Lastly, it was determined that hispidulin is present in I. hayesiana however; it was not found to be responsible for the cytotoxicity seen towards MDA-

MB-231. In fact the IC50 of the pure compound dissolved in ethanol was greater than

100 µg/mL, which was the highest concentration tested.

______, Committee Chair Dr. Mary McCarthy-Hintz

______Date

ACKNOWLEDGEMENTS

There are many people to thank for helping me to complete this journey.

Although this thesis is the culmination of years of work on my part, there were many people who directly and indirectly made this project possible. I would like to start by thanking the Chemistry department here at Sacramento state for allowing me to pursue this goal. Many of the faculty have had a hand in this project but none more so than my research advisor Dr Mary McCarthy- Hintz. This project took seven years, with many drastic changes along the way. Mary challenged me to persevere and encouraged and supported me when I needed it most. This thesis could not have been completed without her. I also want to thank my committee members Dr McReynolds and Dr

Dixon for the many hours of editing. Their unrelenting standards made this paper what it is today.

Next I would like to thank a few of the research students who have shared this journey with me. The friends I have made here, have made the time I spent in the

Masters Program a joy. Himali Somaweera was a member of my research group and we frequently worked side by side, though on different projects. Soraya Ghasemiyeh and I have shared many highs and lows during this process, including the race to our defense dates, which were two weeks apart. Being able to share the stresses has made them all more bearable! A special thank to Michelle Watterson who has been my friend and confidant for years now. She helped me through the thesis writing process even

vii though she finished several years ago and would have liked to never think about it again.

My last thank you is the most important of all - to my family. My husband

Mark has put up with many years of late nights and weekends without me. His love and support have made it possible for me to complete this thesis. My children, Maddy and

Jason who never complained about my absence but were always happy to see me when

I got home. I hope the completion of this project will show you that you can do anything if you set your mind to it. I won't ever be able to truly express how much I love you all! I also need to thank my mother, Pat , my grandmother, Regina and my mother in law Julie for the love and support they gave me. All of you assisted in taking care of my children in my absence for which I cannot thank you enough.

During the seven years of this journey I have achieved many goals and suffered many losses. None so great as the loss of my father, Walt, just six months ago. While I feel elated to have successfully completed my Masters degree, I also feel a little melancholy that he wasn't here to see it. His loss reminded me that you have to live every day like it's your last, and now that I am finished that is exactly what I am going to do.

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TABLE OF CONTENTS

Page

Acknowledgements...... vii List of Tables...... xi List of Figures...... xii Chapter 1. INTRODUCTION...... 1 1.1 Breast Cancer Statistics...... 1 1.2 What is Breast Cancer?...... 1 1.3 Biomarkers for Breast Cancer...... 5 1.4 Breast Cancer Treatments...... 6 1.5 Screening of California Native Plants...... 14 1.6 Botany and Taxonomy of Iva hayesiana...... 16 1.7 Poverty Weed as Herbal Medicine...... 18 1.8 Previous Studies of Iva hayesiana and Related Plants...... 18 1.9 Proposal...... 19 2. MATERIALS AND METHODS...... 21 2.1 Abbreviations...... 21 2.2 Materials...... 21 2.3 Instruments and Apparatus...... 22 2.4 Extraction Methods...... 22 2.4.1 Initial Extraction...... 22 2.4.2 Extraction Solvent Optimization...... 23 2.4.3 Vacuum Distillation...... 23 2.5 Cytotoxicity Assays...... 24 2.5.1 Media Preparation...... 24 2.5.2 Cell Line and Cell Culture...... 24

2.5.3 Cell Viability Assay...... 24

2.5.4 Determination of IC50...... 26 2.6 HPLC Analysis...... 27 3. RESULTS AND DISCUSSION...... 29 3.1 Overview...... 29 3.2 Initial Plant Screening...... 29 3.3 Determination of Optimum Extraction and Analysis Conditions...... 31 3.3.1 Extraction Solvent Optimization...... 31 3.3.2 Determination of Volatility...... 32

3.4 IC50 Determination...... 33 3.5 Large Volume Extraction...... 34 3.6 Investigating the Loss of Cytotoxicity...... 36 3.7 Evaluation of Seasonal Cytotoxicity...... 39 3.8 HPLC Analysis of Monthly Extracts...... 41

3.9 IC50 for Hispidulin...... 46 3.10 HPLC Analysis of Hispidulin...... 47 4. CONCLUSIONS...... 50 5. FUTURE WORKS...... 51 Appendix A. HPLC Chromatograms...... 52 References...... 59

LIST OF TABLES

Tables Page 1. Less common forms of breast cancers and their occurrence rates...... 4 2. TNM staging of breast cancer...... 5 3. Retention times and peak areas from the HPLC chromatogram of I. hayesiana extracted in April...... 42 4. Evaluation of the 7 peaks of interest in the active extracts...... 45 5. Re-evaluation of peaks 9 and 13 via HPLC...... 45 6. Evaluation of the HPLC retention times of hispidulin...... 49

LIST OF FIGURES

Figures Page

1. Anatomy of the female breast...... 2

2. Structure of Taxol...... 9

3. Structure of the monoclonal antibody Herceptin...... 11

4. Enzyme mediated metabolism of Tamoxifen to Endoxifen...... 11

5. The Function of hormone therapy drugs Tamoxifen and Arimidex...... 12

6. The structure of the aromatase inhibitor Arimidex...... 12

7. The structure of Laetrile...... 13

8. Iva hayesiana...... 17

9. Distribution of Iva hayesiana by county...... 17

10. The structures of the flavones hispidulin and axillarin...... 18

11. The cytotoxicity of six native California plants on the human breast cancer cell

line MDA-MB-231...... 30

12. The cytotoxicity of four solvent extracts of I. hayesiana leaves and stems on the

human breast cancer cell line MDA-MB-231...... 32

13. The cytotoxicity of extracts from the I. hayesiana distillate, filtered aqueous

suspension and 2-day extract from vacuum distillation on the human breast

cancer cell line MDA-MB-231...... 33

14. IC50 determination for I. hayesiana extracted in hexanes on the human breast

cancer cell line MDA-MB-231...... 34

xii

15. The IC50 determination for I. hayesiana extracted in hexanes (5mg/mL) on the

human breast cancer cell line MDA-MB-231...... 36

16. The IC50 determination for I. hayesiana extracted in hexanes (10 mg/mL) on the

human breast cancer cell line MDA-MB-231...... 37

17. The cytotoxicity of three new solvent extracts from fresh I. hayesiana (65 and

32.5 µg/mL) on the human breast cancer cell line MDA-MB-231...... 38

18. HPLC investigation of the chemical differences of active and inactive extracts of

I. hayesiana...... 39

19. Results from the cell viability assay of monthly extracts of I. hayesiana

(65 µg/mL) on the human breast cancer cell line MDA-MB-231...... 40

20. HPLC chromatogram of the April extract of I. hayesiana with the 14 peaks of

interest labeled, after comparison to the blank (ethanol) chromatogram...... 42

21. HPLC chromatograms of I. hayesiana extracts from April 28th (red - active) and

Feb 28th (blue - inactive)...... 44

22. HPLC chromatogram of the April extract of I. hayesiana, diluted to a

concentration of 0.1 mg/mL, with peaks 9 and 13 labeled...... 45

23. IC50 assay results for hispidulin dissolved in ethanol (10 mg/mL) on the human

breast cancer cell line MDA-MB-231...... 47

24. Evaluation of the presence of hispidulin in I. hayesiana using HPLC...... 48

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Chapter 1

INTRODUCTION

1.1 Breast Cancer Statistics

Breast cancer is a disease that affects approximately 1 out of every 35 women.1

It is the second most lethal cancer behind lung cancer in women. It is estimated that in

2012, 226,870 women will be diagnosed with an invasive form of breast cancer, and another 63,000 women will be diagnosed with a non-invasive (in situ) form of breast cancer. Men are also susceptible to breast cancer, and it is predicted that 2,190 will be diagnosed in 2012. Some 39,510 deaths are expected in 2012 due to breast cancer or its complications. It has been said that every fifteen minutes, five women will be diagnosed with breast cancer and one woman will die.2 There are approximately 2.5 million women who are breast cancer survivors in the United State today.1 With statistics like these, it is not hard to understand why so much research is being done to find effective treatments, preventative measures, or a cure for this disease. Current treatments usually involve a combination of surgery and an adjuvant therapy such as radiation, chemotherapy, hormone therapy or immunotherapy. The types of therapies used are based on the type and stage of the breast cancer the patient has.

1.2 What is Breast Cancer?

The definition of breast cancer is a malignant growth that inhabits the tissues of the breast.3 The malignant cells form in the breast tissue when replication goes wrong

2 in one cell and that cell multiplies. When these cells build up, a lump or mass called a tumor is formed. A mass in the breast can be benign (non-cancerous) or malignant

(cancerous). Benign masses in the breast are common, and are not life threatening, while malignant masses can be life threatening, depending on the type and stage of the cancer. Some of these malignant cells can invade the surrounding tissues, and then spread (metastasize) to other parts of the body such as the lungs, liver and brain. Most breast cancers form from the cells that line the milk ducts (Figure 1:A) or lobules

(Figure 1:B) of the breast, but a few do form in the fatty or connective tissues (Figure

1:E).4

Breast profile: A. Ducts B. Lobules C. Dilated section of duct to hold milk D. Nipple E. Fat F. Pectoralis major muscle G. Chest wall/rib cage

Enlargement I I - Normal duct cells II II - Basement membrane III III - Lumen (center of duct) Figure 1: Anatomy of the female breast.3

There are fourteen types of breast cancer known to date, but only four are fairly common. The four most common types of breast cancer are identified by the location within the breast tissue and the invasiveness of the tumor. These four are: 1) ductal

3 carcinoma in situ (DCIS), 2) lobular carcinoma in situ (LCIS), 3) invasive ductal carcinoma (IDC), and 4) invasive lobular carcinoma (ILC). Breast cancers are determined to be invasive when they have infiltrated the surrounding tissues or chest wall (Figure 1: E, F and G).4 Once infiltration has occurred, the cancer cells can then metastasize to other parts of the body through the blood or lymph system. DCIS is the most common form of non-invasive breast cancer. It forms in the ducts of the breast and has not spread to the surrounding tissue (in situ). While this is the most commonly diagnosed breast cancer; with one in every five new cases of breast cancer likely to be

DCIS, this form of cancer has a low mortality rate with early detection. LCIS is also a non-invasive form of breast cancer. It begins in the cells of the lobules, which is where milk is produced in the breast. This cancer does not usually progress to an invasive form, but it does indicate a higher risk of developing some form of invasive breast cancer later. IDC is the most common form of invasive breast cancer. This cancer begins in the cells of the milk ducts, invades the surrounding tissue and can ultimately become metastatic. ILC starts in the cells of the lobules of the breast and then infiltrates the surrounding tissues, and can also become metastatic. There are ten additional less commonly diagnosed types of breast cancer, which are listed in Table 1 below. The mortality rates of each of the cancers previously mentioned varies depending on the type and stage (Table 2) of the cancer, as well as how aggressive it is.4 The progression, or stage, of the cancer is also very important when determining treatment protocols. Staging is done using a classification system such as the American

Joint Committee on Cancer (AJCC) classification system. Each of these stages and

4 sub-stages (Table 2) has a different recommended treatment protocol that is dependent on not only the stage of the cancer, but also on the biomarkers found within the cancer cells.

Table 1: Less common forms of breast cancers and their occurrence rates.1 Occurrence rates are based on the percentage of all diagnosed breast cancers. When occurrences are called rare, it indicates a percentage below 1% that has not been calculated.

Type Occurrence Adenocystic Carcinoma 1%

Angiosarcoma Rare

Inflammatory Breast Cancer 1-3%

Medullary Carcinoma 3-5%

Metaplastic Carcinoma Rare

Mucinous Carcinoma Rare

Paget Disease of the Nipple 1%

Papillary Carcinoma >1-2%

Phyllodes Tumor Rare

Tubular Carcinoma 2%

Table 2: TNM staging of breast cancer. This table correlates the stages of cancer to tumor size (T), lymph node involvement (N), and metastasis (M), and the five-year survival rates for each. Tumor classifications range from Tis (in situ) to T4 (tumor size 5 cm or greater). Lymph node involvement ranges from N0 (no involvement) to N3 (multiple lymph node involvement). Metastasis is rated M0 for no metastasis and M1 for metastasis.5

Stage Tumor (T) Node (N) Metastasis (M) 5-Year Relative Survival Rate3 Stage 0 Tis N0 M0 100% Stage I T1 N0 M0 100% Stage IIA T0 N1 M0 92% T1 N1 M0 T2 N0 M0 Stage IIB T2 N1 M0 81% T3 N0 M0 Stage IIIA T0 N2 M0 67% T1 N2 M0 T2 N2 M0 T3 N1, N2 M0 Stage IIIB T4 Any N M0 54% Any T N3 M0 Stage IV Any T Any N M1 20%

1.3 Biomarkers for Breast Cancer

A breast cancer biomarker is a molecule found in the tumor, tissues or fluid of a

tumor, that can be used to predict how well the cancer will respond to a particular

treatment.5 Biomarkers, along with the type and stage of the cancer, are used to map

out a treatment protocol that will give the patient the best outcome possible. There are

currently three biomarkers used to determine the type of adjuvant therapy that will be

6 used for treatment. They are estrogen receptor (ER), progesterone receptor (PR), and human epidermal growth factor receptor two (HER2). Sixty to eighty percent of all breast cancer tumors will test positive for overexpression of ER.6 Cells that are positive for ER and/or PR have receptor proteins that bind estrogen and progesterone, respectively, in the nuclei of the cells. These cells usually need the respective hormone to grow, and therefore respond well to treatments that block the binding sites or make the hormone unavailable. HER2 is a protein found in both normal and cancerous cells.

When HER2 is overexpressed, the cancer is considered HER2+. This protein plays an important role in cell growth and division, and overexpression causes the cells to grow and divide more rapidly, therefore these cancers tend to be more aggressive and are more likely to re-occur. Twenty to forty percent of all breast cancers test positive for

HER2 overexpression.7 A breast cancer can be positive for a combination of these biomarkers, such as ER+ and PR+, or negative for all three, which is called a triple negative. Ten to fifteen percent of all breast cancers are triple negative, with a high occurrence in patients that are positive for the breast cancer 1 gene (BRCA1) mutation.

These cancers are called basal-like because they express basal cell-type cytokeratins

(proteins).8 They tend to be very aggressive and have a poor survival rate. There are no target-specific therapies for hormone insensitive cancers.9

1.4 Breast Cancer Treatments

Treatment protocols for breast cancers in stages 0 - III are targeted to removal of the cancer and then prevention of recurrence. This is usually accomplished with a

7 combination of surgery and adjuvant therapies such as radiation, chemotherapy, hormone therapy, complimentary medicine, and alternative medicine. Surgery is usually the first step in cancer treatment. The patient can have either a lumpectomy or mastectomy along with the removal of one or more of the lymph nodes. A lumpectomy involves the removal of the cancerous mass, while leaving as much of the surrounding breast tissue as possible. This procedure is usually used for smaller masses and is coupled with radiation to ensure that all of the cancer cells are eradicated. A simple mastectomy is the complete removal of all of the breast tissue, and, except in later stages, radiation is not needed. A lymph node close to the tumor is removed (sentinel dissection) and checked for cancerous cells. If the node is positive, additional nodes in the armpit are removed and tested as well (axillary dissection). There is a risk of bleeding or infection with surgery, and arm swelling with lymph node removal. The tumor size and lymph node involvement are determined with surgery, and then used to stage the cancer for further treatment. The excised mass is tested for biomarkers and aggressiveness to give a complete picture.4

Radiation therapy is usually recommended if the tumor is five centimeters or larger, there is chest wall infiltration or multiple lymph node involvement, or the patient has had a lumpectomy. Radiation is also used for inflammatory breast cancer. There are two types of radiation therapy - external radiation and internal radiation. External radiation is delivered to the breast from outside of the body. Internal radiation uses radioactive seeds that are implanted in the affected breast tissue. While radiation is effective at reducing the risk of recurrence, it is not without issues. The treatment takes

8 six to seven weeks to complete, and treatments occur daily. Patients may experience fatigue and skin irritation, and there is an increased risk of secondary cancers, as well as lung or heart damage. If radiation is recommended, treatment usually begins three to six weeks after surgery. If chemotherapy is recommended in addition to radiation, it is usually completed before radiation begins.

Chemotherapy drugs ("chemo") are used to kill fast-growing cells. Chemo is usually recommended for patients with cancers that have metastasized, are re-occurring, or are likely to re-occur. The treatments can last from three to six months in one to three week cycles. The schedule usually includes breaks in treatment to allow for recovery time. Chemotherapy is very effective at killing fast growing cancer cells, but cancer cells are not the only fast growing cells in the body. Hair follicles and nails are examples of other fast growing cells that are killed, which is why many patients who undergo chemo lose their hair.3 Paclitaxel (Taxol) is one of many chemo drugs currently in use. It comes from a class of drugs called taxanes, which disrupt cell division. Taxol (Figure 2A) was originally isolated from the bark of the pacific yew tree (Taxus brevifolia) in 1971.10 The Pacific yew tree is a very slow growing tree that produces very little taxol. Today, the main source of taxol is a semisynthesis from precursors such as baccatin III (Figure 2B), which is found in the needles of the yew tree. The side effects from this drug include nausea, vomiting, diarrhea, decreased blood counts, muscle aches, and hair loss.

A B

Figure 2: Structure of Taxol11 (A), and the precursor baccatin III12 (B) used in the semisynthesis of Taxol.

Another form of adjuvant treatment for breast cancer is hormone therapy.

Hormone therapy is used for breast cancers that have a positive biomarker for HER2,

ER, or PR. Cancers that are ER+, PR+, or ER+/PR+ use the body's estrogen and/or progesterone to promote cell growth. To interfere with the cell cycle of the cancer, drugs are given to block either the production of these hormones or the binding of these hormones to the receptor. Either strategy slows the growth of the cancer, as well as reduces the possibility of recurrence. For cancers that are HER2 positive, there is a targeted form of hormone therapy which specifically affects cancer cells. The drug trastuzumab (Herceptin; Figure 3) is a monoclonal antibody produced by recombinant

DNA technology.13 Herceptin binds to the HER2 receptor on the surface of the cancer cell and blocks the signals for cell growth and division. This drug is frequently given in combination with taxol. Side effects include headache, nausea, vomiting, weakness,

10 breathing difficulty, and skin rashes. There is also an increased risk of congestive heart failure. Herceptin only works for cancers that are HER2+. Other targeted hormone therapy drugs are available for cancers that are ER+ and/or PR+. Tamoxifen (Figure 4) is one of the most commonly used hormone therapy drugs when a cancer is ER+. It is a selective estrogen receptor modulator (SERM) that interferes with the estrogen receptors in the cancer cells (Figure 5A). In the body, tamoxifen requires the action of the cytochrome P450 enzyme family. Specifically, cytochrome CYP-2D6 and cytochrome CYP3A4/5 metabolize tamoxifen to endoxifen (Figure 4). Endoxifen has greater binding affinity for the estrogen receptor and is considered the activated form of tamoxifen. Both cytochromes occur naturally in the human body; however, some patients have flawed versions or take a medication that interferes with their function.14

When this occurs, tamoxifen is no longer effective and an aromatase inhibitor may be used instead.

Aromatase inhibitors (AI) such as anastrozole (Arimidex; Figure 5B, and 6), block the enzyme aromatase, which converts androgens into estrogens.15 While AI's reduce the amount of estrogen the body produces, they do not completely stop estrogen production in the ovaries; therefore, there is significant debate about their effectiveness in premenopausal women. The course of treatment for both types of hormone therapy lasts up to five years. The side effects for tamoxifen are hot flashes, increased risk of cataracts, blood clots, uterine cancer, and osteoporosis. AI are a fairly new breast cancer therapy, so long term side effects are still not known. Short-term side effects

11 include hot flashes, joint pain, muscle aches, vaginal dryness, and increased risk of osteoporosis.

Figure 3 : Structure of the monoclonal antibody Herceptin.13

O H O O CH3 CH3 N N N CH3 CH3 CH3

CYP2D6 CYP3A4/5

OH OH Tamoxifen 4-hydroxytamoxifen Endoxifen

Figure 4: Enzyme mediated metabolism of Tamoxifen to Endoxifen.14

A B

Figure 5: The Function of hormone therapy drugs Tamoxifen and Arimidex. A. Endoxifen binds to an estrogen receptor. This path leads to the inhibition of gene transcription. B. Arimidex blocks the final step in the production of estradiol. The estrogen receptor remains unbound and transcription cannot occur.

Figure 6: The structure of the aromatase inhibitor Arimidex.16

A final class of treatments for breast cancer falls outside of conventional medicine. Complementary and alternative medicine (CAM) is practiced by providers outside of the standard medical community. Some examples of CAM providers are chiropractors, herbalists, and acupuncturists. Complimentary medicine consists of non- conventional treatments that are used along with traditional medicine. These treatments

13 are usually used to treat the side effects caused by conventional medicines such as chemo. Today there are many medical professionals who offer a combination of conventional and complimentary (integrated) treatments. Alternative medicine is non- traditional medicine that is used instead of standard medicine. As such, alternative medicine is generally frowned upon within the standard medical community in the

United States. Many standard medical treatments and practices currently in use were once considered CAM. The use of acupuncture to treat nausea from chemo is a good example of this: With years of scientific proof of the effectiveness of this treatment, it has become an accepted practice in standard medicine.17 Other treatments used as alternative medicine, such as laetrile, were believed to have anti-cancer properties, but were proven to be ineffective and even harmful. Laetrile (Figure 7) is a chemically altered form of amygdalin, which is a glycoside found in the fruit pits and raw nuts.

Laetrile was patented in the US in the 1950’s for cancer treatment. Testing later proved that laetrile had no anticancer properties, and that the drug was potentially lethal. An enzyme in the small intestines of humans catalyzes the release of cyanide from laetrile, causing cyanide poisoning.18 There are many natural products used today that are believed to have anticancer properties that still need to be tested.

Figure 7: The structure of Laetrile.19

While there are many chemotherapeutic drugs in use today, serious side effects are a problem. Some of the side effects are nausea, vomiting, hair loss, blood clots, lung and heart damage, congestive heart failure and even secondary cancers. Targeted therapies can limit some side effects, but there are cancers, such as ER-, that do not respond well to the current treatments available. There are also treatments that become ineffective with prolonged use. The side effects and sometimes ineffectiveness of current treatments indicate the need for novel therapies effective against breast cancer.

In the search for new chemotherapies, plants are proving to be an excellent source for natural therapies that may have less adverse side effects. Thus, there is a renewed interest in investigating plant natural products that might be used against breast cancer.

1.5 Screening of California Native Plants

For centuries, plants have been used for medicinal purposes by different cultures.

In the search for new anti-cancer compounds, these plants represent a vastly under- investigated resource. There are several different ways to identify plant targets for breast cancer research. One method targets plants that are used in traditional indigenous or folk medicine against cancer. One such plant is Vernonia amygdalina, which is used to treat cancer in South Africa. Gresham et al. tested the plant for anticancer activity and found it very effective against ER+ breast cancers.20 A second method targets plants used as traditional medicines, but not necessarily for cancer.

Plants that are used to kill fast-growing organisms such as bacteria or helminthes are possible targets for killing fast growing cancer cells. One such study completed by

Reddy et al. found that Hedychium spicatum, which is used medicinally to treat stomach ailments and other disorders, had good cytotoxic effects against breast, lung, colon and skin cancer.21 A third method is random screening of for cytotoxicity. This method is usually used by pharmaceutical companies and research institutes. An example of this is a study completed by the National Cancer Institute, in which seventy- five hundred South African plants were screened; fifty showed moderate anticancer activity.22 Lastly, screening multiple plants for anticancer activity can also be done in a more targeted fashion. Ramirez-Erosa et al. screened fifteen plants from the Asteraceae family, some members of which are known for anticancer activity, with very positive results. Out of fifteen plants tested, five were very toxic to ER- breast cancer cells.23

The McCarthy research group has also had considerable success with targeted screening.

The McCarthy group has screened over 75 plants for anticancer activity in a targeted manner. The area of focus for this group is California native and naturalized plants that are, or were, used medicinally (in traditional, folk, or modern herbal medicine). Chris Hobbs performed most of the initial research and screening. Of all of the plants screened, six showed moderate to good cytotoxicity against triple negative breast cancer cell line MDA-MB-231: yellow star thistle (Centaurea solstitialis), yellow pond lily (Nuphar luteum), Indian hemp (Apocynum cannabinum), prickly pear cactus

(Opunita ficus-indica), American dogwood (Cornus sericea) and San Diego poverty weed (Iva hayesiana). In the current study, a cytotoxicity assay was performed using water extracts of the medicinal parts of these six plants, as well as ethanol extracts of C.

16 solstitialis, O. ficus-indica, N. luteum. The aqueous extracts of I. hayesiana roots and

C. sericea, N. luteum, C. solstitialis, and O. ficus-indica , and the ethanol extract of C. solstitialis were not cytotoxic against the triple negative-cancer cell line MDA-MB-231.

However, results of this screening confirmed the previous results that showed cytotoxicity for aqueous extracts of I. hayesiana leaves and A. cannabinum, and the ethanol extract of N. luteum. Of these, the cytotoxic compounds in A. cannabinum24, and N. luteum25 have already been isolated and identified. Only I. hayesiana remains to be investigated.

1.6 Botany and Taxonomy of Iva hayesiana

Iva hayesiana (Figure 8), also known as San Diego poverty weed, belongs to the family Asteraceae, the genus Iva, and the tribe Iveae.26 There are 27 species in the genus Iva throughout the United States, Mexico and British Columbia. Common plant names for the Iva genus include Jesuit's bark, copperweed, marshelder, poverty weed, and sumpweed. I. hayesiana is a fast growing evergreen shrub that grows primarily in the San Diego coastal region as well as in Baja California (Figure 9). The plant prefers an alkaline environment below an elevation of 1000 feet. It is highly adaptive and has become a popular plant for ground cover and erosion control. The shrub flowers from

April to September with very small green flowers which are nearly invisible. The plant has a strong odor due to a sesquiterpene lactone (exact structure unknown), which deer and other herbivores do not like.27 The plant also contains two flavones, which were

17 identified as hispidulin (Figure 10A) and axillarin (Figure 10B), both of which have been identified in other plant species in the Asteraceae family.28

Figure 8: Iva hayesiana29

Figure 9: Distribution of I. hayesiana by county.30

A B

Figure 10: The structures of the flavones hispidulin31 (A) and axillarin32 (B).

1.7 Poverty Weed as Herbal Medicine

Plants within the Asteraceae family have been shown to have many medicinal uses. Native American cultures use or used plants from the Asteraceaea family to treat ailments such as stomach and intestinal ailments, skin problems, female problems and cancers. Within the Iva genus specifically, plants such as I. axillaris, which is a close relative of I. hayesiana, were used by the Shoshone Indians to treat stomach aches, cramps, diarrhea, and colds. The Paiute used it for sores, rashes and itching, and the

Mahuna used it as an abortifacient and for birth control.33 The flavone hispidulin, which can be found in I. hayesiana, as well as other plants in the Asteraceae family, is also used in Chinese medicine to treat inflammatory diseases.34

1.8 Previous Studies of Iva hayesiana and Related Plants

To date, very little research has been documented regarding I. hayesiana. The flavones hispidulin and axillarin were both isolated from I. hayesiana and identified by

Herz et al. in 1969.28 There was also a sesquiterpene lactone found that has not been

19 completely characterized due to its polymerization. Axillarin was originally isolated from I. axillaris in 1966. The flavone hispidulin was originally isolated from Ambrosia hispida and identified in 1964. Since then it has been found in other plants within the

Asteraceae family. Hispidulin extracted from Artemisia vesitita has been shown to be effective in killing pancreatic cancer cells as well as ovarian cancer cells. The fact that one of the chemical constituents of I. hayesiana has shown cytotoxicity against several types of cancer cells is promising.

1.9 Proposal

In the search for new chemotherapies for breast cancer, plants are proving to be an excellent source for natural therapies, as they may have less adverse side effects.

Although no documentation was found that I. hayesiana was used for medicinal purposes, it does contain hispidulin, which has been found to have anticancer properties. I. hayesiana was originally screened with ten other plants for cytotoxicity against the human breast cancer cell line MDA-MB-231 for the McCarthy group by

Chris Hobbs in 2004. Based on those positive results, the six plants that showed moderate to good cytotoxicity were re-tested to choose the best target. The results of both screenings coupled with the lack of previous research on I. hayesiana made it a good candidate for study. The presence of a chemical constituent (hispidulin) known to be cytotoxic to pancreatic cancer also makes I. hayesiana a good target. The original objective of this thesis was to identify the cytotoxic constituents of I. hayesiana. While the initial extracts of I. hayesiana showed anticancer activity, some subsequent extracts

20 showed no activity. Upon review of the data, it was noted that the inactive extracts were obtained from leaves harvested at different times of the year than the initial active extracts. Due to these findings, the objectives of this thesis were changed. The new objectives are threefold 1) to determine the best extraction method for future extractions of I. hayesiana, 2) to determine whether the cytotoxic constituent(s) of I. hayesiana are seasonal and 3) to determine whether hispidulin is responsible for the cytotoxicity of I. hayesiana on the human breast cancer cell line MDA-MB-231. Therefore, the hypothesis for this study is that the cytotoxicity of I. hayesiana is related to seasonal variation in the chemical constituents of the plant.

Chapter 2

Materials and Methods

2.1 Abbreviations

[4-(2-hydroxyethyl)-1-piperazine]ethanesulfonic acid, (HEPES); Reverse phase high performance liquid chromatography, (RP- HPLC); Electrical Aerosol Size

Analyzer, (EAA).

2.2 Materials

Three I. hayesiana plants were purchased from Village Nursery in Sacramento,

California. The plants were shipped in from the nursery's San Diego, California, location. Plants were then grown in the greenhouse located at California State

University, Sacramento. Sterile flat bottom tissue culture treated 96 well plates with

360 µL well volume (Corning; Corning, NY), Minimum Essential Media (IMEM) with zinc option containing 2 mg/L L-glutamine, 2 mg L-proline, 50.0 µg/mL L gentamicin sulfate (Mediatech, Inc; Herndon, VI), heat inactivated fetal bovine serum (Anexia

Biologix; Dixon, CA), trypsin-EDTA (Mediatech, Inc; Herndon, VI) and CellTiter 96*

AQueous One Solution Cell Proliferation Assay (Promega; Madison, WI) were used for all cytotoxicity and IC50 assays. All other reagents were purchased through Fisher

Scientific or VWR.

2.3 Instruments and Apparatus

A Labconco (Kansas City, MO) class IIA biosafety hood was used for all cell culture procedures. A Du Pont Instruments (Cincinnati, OH) Sorvall GLC-4B general laboratory centrifuge was used for centrifugation. Cells were kept in an IR Auto Flow

Incubator manufactured by Nuaire (Plymouth, CA). A BioRad (Hercules, CA) microtiter plate reader, model 680, was used for all cytotoxicity assays.

Samples were analyzed using an Agilent (Santa Clara, CA) 1100 HPLC equipped with a binary pump operated using Chem Station software. The system is also equipped with a variable wavelength Agilent UV detector in series with a custom built in-house charged aerosol detector (CAD). The CAD uses the same basic principles as in instruments described by Dixon and Peterson and Gamache et al.35-36 However, it was modified to use nebulization to cause particle charging and is described in more detail by Abhyankar.37 The column used was a Phenomenex (Torrance, CA) C12

Synergi 4 µm diameter MAX-RP 80A with the dimensions 150 x 4.60 mm.

2.4 Extraction Methods

2.4.1 Initial Extraction

All glassware was cleaned in an alcoholic sodium hydroxide bath (10% sodium hydroxide (w/v) in isopropanol ) and rinsed with DI water, until pH of the water was neutral, prior to use. One gram of fresh I. hayesiana leaves and stems were ground in a blender and extracted with 25 mL of hexanes (ACS grade). Extraction was performed at room temperature with stirring over a two day period. The hexane extract was first

23 filtered with a large Buchner funnel and 9.0 cm qualitative grade 1 filter paper

(Whatman) to remove large particulate matter. The extract was then filtered with a 0.45

µm sterilizing polyethersulfone (PES) bench top filter (150 mL). Extracts were next concentrated using a rotary evaporator (Sorvall). The residue was dissolved in ethanol using a volume calculated to bring the sample concentration to 10.0 mg/mL. All extracts were then stored at -20 ºC.

2.4.2 Extraction Solvent Optimization

To determine the best solvent for extracting I. hayesiana, a series of solvents varying from non-polar to polar were tested. The solvents used were water, diethylether

(ethylether anhydrous, ACS grade), hexanes (ACS grade) and ethanol (ACS grade).

Each solvent was used to extract both a fresh sample of I. hayesiana leaves and stems, as well as a dried sample using 1 g of plant material and 25mL of solvent. For dried samples, Iva leaves and stems were collected and dried at room temperature for 3-4 days. All extracts were performed at room temperature with stirring over a two day period. Samples were then filtered and stored as described previously.

2.4.3 Vacuum Distillation

Vacuum distillation was performed using standard vacuum distillation apparatus. One gram of fresh whole I. hayesiana leaves and stems was placed in a round bottomed flask (100 mL) with 5 mL of water. The distillate was collected in a dry flask kept at approximately 0ºC. The I. hayesiana leaves and stems began to distill

24 at 40ºC and continued until the temperature reached 60ºC. At 60ºC the temperature began to drop rapidly. The distillate was extracted three times using equal volumes of hexanes and dried using a rotary evaporator. The leftover aqueous suspension was further extracted with hexanes at room temperature with stirring for 48 hours. This 2 day extract extract was finally filtered and dried using a rotary evaporator and stored as described previously.

2.5 Cytotoxicity Assays

2.5.1 Media Preparation

Growth media was prepared with the addition of 50 mL of fetal bovine serum, 5 mL of 1M HEPES and 5 mL of penicillin/streptomycin to 500mL of IMEM-zinc option.

2.5.2 Cell Line and Cell Culture

Human breast cancer cells from the cell line MDA-MB-231 were purchased from American Type Culture Corporation (ATCC) Rockville, Maryland. The cells were grown in growth media in T-25 and T-75 cell culture flasks. Cells were cultured for 48 hours at 37ºC under a 100% humidified 95%:5% mixture of air and CO2.

2.5.3 Cell Viability Assay

A cell viability assay was used to assess the cytotoxicity of all initial extracts on the human breast cancer cell line MDA-MB-231. The plant extracts tested were

C. solstitialis, N. luteum, A. cannabinum, O. ficus-indica, C. sericea and I. hayesiana.

These extracts tested resulted from the initial plant screening, solvent optimization and vacuum distillation. Using sterile technique, MDA-MB-231 cells were detached from a

T-25 flask by incubating with trypsin:EDTA (0.05%:0.2%). The cells were washed down and collected with 5 mL of warm media and transferred to a 10 mL Falcon tube.

The cells were then pelleted by centrifugation at 1409 X g. The supernatant was removed and replaced with 5 mL of fresh media and the pellet was resuspended with vortexing. Each well of a 96 well plate was inoculated with 100 µL of cell suspension with a cell density of 10,000 cells/mL. The plate was incubated for 48 hours at 37ºC under a 100% humidified 95%:5% mixture of air and CO2. Extracts of interest were normalized at a concentration of 10 mg/mL, and were prepared using 1987 µL of growth media and 13 µL of sample to create a working concentration of 65µg/mL. The negative control contained 13 µL of 95% ethanol and 1987 µL of growth media. The media was aspirated from a set of ten wells (ten replicates) designated for the test, and aliquots of 100 µL from each test sample were added to each well. After 48 hours of incubation, the media was aspirated from wells and replaced with 100 µL of fresh growth media and 20 µL of CellTiter. A blank row also received 100 µL of fresh growth media and 20 µL CellTiter. The plate was incubated for 1-2 hours at 37ºC, then the relative number of cells was quantitated using a microplate reader measuring the absorbance at 490nm. Cell viability was expressed as % of control, which was calculated from the following formula (Equation 1). Cell viability was then plotted to

26 compare the effectiveness of various extracts. The standard deviation was next calculated using Equation 2.

Equation 1

Cell Viability (% Control) = x 100%

= Mean Absorbance of 10 sample wells at 490 nm

= Mean Absorbance of 10 control wells at 490nm

Equation 2

Standard Deviation =

2.5.4 Determination of IC50

Using sterile technique, MDA-MB-231 cells were plated using the same protocol as the cell viability assay. Plates were incubated for 48 hours at 37ºC under a

100% humidified 95%:5% mixture of air and CO2. Working solutions of six concentrations (100, 80, 60 40, 20, 0 µg/mL) for each extract were prepared as follows:

100 µL stock extract in 9.9 mL media, 80 µL extract/20 µL ethanol in 9.9 mL media, 60

µL extract/40 µL ethanol in 9.9mL media, 40 µL extract/60 µL ethanol in 9.9 mL media, 20 µL extract/80 µL ethanol. A negative control using 100 µL ethanol in 9.9 mL media was also prepared. An aliquot of 100 µL of each working solution was added to ten designated replicate wells. The plate was then incubated for another 48 hours at 37ºC under a 100% humidified 95%:5% mixture of air and CO2. The Celltiter

27 procedure and percent control calculations (Equation 1) were completed using the previously defined protocols. Data was graphed in a scatter plot with percent control vs. concentration with error bars to represent the standard deviation (Equation 2). The absorbance data for each of the ten replicate measurements per concentration was then used to calculate the half maximal effective concentration (IC50) using the ED50Plus v1.0 Excel worksheet developed by Dr. Mario H. Vargas at Instituto Nacional de

Enfermedades Respiratorias.38

2.6 HPLC Analysis

An Agilent 1100 HPLC was used to analyze the chemical constituents present in active and inactive extracts. All extracts were diluted in 95% ethanol to a concentration of 1 mg/mL and filtered using a 0.22 µm sterile syringe filter (13mm, Fisherbrand) prior to each run. The injection volume was 5 µl and flow rate was 1 mL/min. The oven temperature was set at 30ºC and the UV detection wavelength was 210 nm. The EAA voltage was set to -0.150 mV. The mobile phase was a binary gradient consisting of nanopure water and acetonitrile. The starting solvent gradient was a 90:10 ratio of water to acetonitrile, which was isocratic for the first five minutes. The gradient then ramped up linearly from 90:10 to a 0:100 ratio of water to acetonitrile over the next five minutes. Following this, the gradient was isocratic at 0:100 for ten minutes, ending with a linear return to 90:10 over the next six minutes. The total run time for each sample was 26 minutes. Chromatograms of the active extracts were overlayed with the chromatograms of the inactive extracts using the Chem Station software. These

28 overlays were evaluated to compare peaks and retention times between the active and inactive fractions.

Chapter 3

Results and Discussion

3.1 Overview

The goals of the work presented in this thesis was to determine if I. hayesiana is cytotoxic to the human breast cancer cell line MDA-MB-231 and to identify the chemical constituents responsible for the cytotoxicity. These goals were derived from the fact that many of the plants in the Asteraceae family, of which Iva is a member, have been used medicinally for centuries. Furthermore, I. hayesiana contains the flavone hispidulin, which is known to be cytotoxic to pancreatic cancer cells. As with all research, this study morphed over the life of the project and the goals changed. As work progressed towards this goal, the cytotoxicity of the extracts disappeared and the focus shifted to determining the cause. This study showed that the cytotoxic compounds in I. hayesiana are seasonal and related to seasonal variation in the chemical constituents of the plant.

3.2 Initial Plant Screening

The extracts of six plants collected and processed by Chris Hobbs in 2004, C. solstitialis, N. luteum, A. cannabinum, O. ficus-indica, C. sericea and I. hayesiana, were screened for cytotoxicity against the ER- cell line MDA-MB-231 using the procedure described in Materials and Methods. The results showed that three plants killed more

30 than 50% of the breast cancer cells (Figure 11). These plants are: I. hayesiana, A. cannabinum, and N. luteum. A literature search indicated that the cytotoxic compounds in both A. cannabinum and N. luteum had been previously isolated and identified.24-25

Therefore, I. hayesiana was chosen for this study.

350

300

250

200

150

100

0 Releative Number (% Control) Cells of of Number Releative

Plant Species

Figure 11: The cytotoxicity of six native California plants on human breast cancer cell line MDA-MB-231. The bars represent the percentage of cells alive compared to the control (cells treated with vehicle only). All extracts used were prepared by Chris Hobbs in 2004. Samples 1-7 were extracted in water and 8-10 in ethanol. Error Bars represent one standard deviation from the average of ten samples.

3.3 Determination of Optimum Extraction and Analysis Conditions

3.3.1 Extraction Solvent Optimization

To determine the optimum extraction method, a series of solvents was used to extract both fresh and dry samples of I. hayesiana leaves and stems. The solvents used were water, ethanol (ACS grade), diethyl ether (ethylether anhydrous, ACS grade), and hexanes (ACS grade). All extracts were then tested against human breast cancer cell line MDA-MB-231 using a cell viability assay (Figure 12). The aqueous extracts of fresh and dry I. hayesiana leaves and stems were not cytotoxic. The ethanol extracts of the dry and fresh samples were somewhat cytotoxic. The most cytotoxicity occurred with both the fresh and dry samples extracted in diethyl ether and hexanes. Due to the fact that the cytotoxicity was so poor in the more polar solvents, coupled with the strong cytotoxicity in hexanes, along with the fact that hexanes are easier to work with, it was determined that hexanes would be the best solvent for all future extracts.

160 140

120 100 80 60

(% control) (% 40 20

0 Relative Number Cells of Number Relative -20

Solvent

Figure 12: The cytotoxicity of four solvent extracts of I. hayesiana leaves and stems (65 µg/mL) on the human breast cancer cell line MDA-MB-231. The bars represent the percentage of cells alive compared to the control (cells treated with vehicle only). The error bars represent one standard deviation from the average of ten samples.

3.3.2 Determination of Volatility

To determine the volatility of the cytotoxic compound(s) found in I. hayesiana, an aqueous vacuum distillation was performed. Using liquid – liquid extraction, the distillate was extracted into hexanes and then concentrated to a concentration of 10 mg/ mL. The remaining water from the aqueous suspension was poured off and filtered for further testing, and the remaining plant material from the aqueous extract was then extracted with hexanes using the initial extraction method discussed in Materials and

Methods. These extracts, along with the (filtered) water from the aqueous suspension, were then tested for cytotoxicity against the human breast cancer cell line MDA-MB-

231 using the cell viability assay. The results from the assay showed that all of the

33 cytotoxicity existed in the aqueous suspension as well as the 2 day extract of the aqueous suspension (Figure 13). The distillate killed approximately 15% of the cells, compared to over 70% cell death from the aqueous suspension and the 2-day extract.

These results indicate that the compounds of interest are not volatile. Therefore, all future extracts were performed using the initial extraction method, and HPLC (rather than gas chromatography) was used for all future chromatographic analysies.

140 120 100 80 60 40 20 0 Relative # of Cells (% Control) (% Cells of # Relative I. hayesiana Distillate Aqueous Suspension 2-Day Hexane Extract of Aqueous Suspension Sample

Figure 13: The cytotoxicity of extracts from the I. hayesiana distillate, filtered aqueous suspension and 2-day extract from vacuum distillation on the human breast cancer cell line MDA-MB-231. The bars represent the percentage of cells alive compared to the control (cells treated with vehicle only). The error bars represent one standard deviation from the average of ten samples.

3.4 IC50 Determination

IC50 stands for the half maximal inhibitory concentration, which in this case means the concentration which causes half the cells to die. This method is commonly

34 used to determine the potency of a drug. To determine the best concentration for future cell viability assays, an IC50 assay was completed on I. hayesiana. The IC50 concentration of the hexane extract on the human breast cancer cell line MDA-MB-231 was calculated to be 56 µg/mL (Figure 14).

170

150

130

110 90 70

(% Control) (% 50 30

Relative Number Cells of Number Relative 10 -10 0 20 40 60 80 100 Concentration (µg/mL)

Figure 14: IC50 determination for I. hayesiana extracted in hexanes on the human breast cancer cell line MDA-MB-231. The error bars represent one standard deviation from the average of ten samples.

All work to this point was completed to address the optimization of future extractions and assays. From here, a large scale extraction was completed to increase the amount of available extract for analysis.

3.5 Large Volume Extraction

To increase the volume of extract available for further analysis, a new extract was prepared using 5 g of chopped fresh I. hayesiana leaves, stems and flowers in 125

35 mL of hexanes (ACS grade), as described in the Materials and Methods section. The extract was brought to a concentration of 5 mg/mL in ethanol and then diluted to working concentrations of 50, 40, 30, 20, and 10 µg/mL in growth media using the IC50 protocol. The concentration change from 10 mg/mL to 5 mg/mL was completed to try to work in a concentration range closer to the calculated IC50 value. The working solutions were then used in an IC50 assay to inoculate the human breast cancer cell line

MDA-MB-231. A solution consisting of growth media and 100 µL of ethanol served as the control. The IC50 results were plotted to determine the potency of the extract

(Figure 15). The results showed almost no activity. At the highest concentration

(50 µg/mL) the cytotoxicity was approximately 21%. At concentrations below 50

µg/mL, the cells actually thrived, with cell growth above 120% of the control cells.

Due to the lack of activity, the IC50 value for this assay was not calculated. It was noted that there were three obvious differences between the initial extract and the new extract: (1) Flowers were used in addition to the leaves and stems used in the first extraction. (2) The hexane extract used in the first solvent screening was extracted from a plant harvest in September of 2010, whereas the scaled-up extraction used new material from the same plant, but harvested in November of 2010. (3) There was also an oily residue in the flask after rotary evaporation of the sample to remove the hexanes.

Based on the first difference, a new extraction was performed using only the leaves and stems. This new extract was tested with a cell viability assay, and it also showed no activity (data not shown). This process of completing new extractions and performing

36 cell viability assays was completed numerous times before it was determined that something within I. hayesiana had changed as evidenced by a lack of cytotoxicity.

140

120

100 80 60

(% Control) (% 40

20 Relative Number Cells of Number Relative 0 0 10 20 30 40 50 60

Concentration µg/mL

Figure 15: The IC50 determination for I. hayesiana extracted in hexanes (5 mg/mL) on the human breast cancer cell line MDA-MB-231. The error bars represent one standard deviation from the average of ten samples.

3.6 Investigating the Loss of Cytotoxicity

To rule out a concentration issue, the initial extraction volume and methods were used to create a new extract. This extract was brought to a stock concentration of

10 mg/mL in ethanol and then diluted to working concentrations of 100, 80, 60, 40 and

20 µg/mL in growth media using the IC50 protocol. A solution consisting of growth media and 100 µL of ethanol served as the control. The working solutions were then used in an IC50 assay to inoculate the human breast cancer cell line MDA-MB-231, this being the concentration used for the first IC50 assay in which I. hayesiana showed

37 strong cytotoxicity. The return to the higher concentration used in the cell viability assay only marginally improved the cytotoxicity (Figure 16). A repeat of the solvent screening assay using new plant material for the extractions also showed almost no cytotoxicity for any of the resultant extracts (Figure 17).

140 120

100 80 60

40 (%Control) 20

Relative Number of Cells Number Relative 0 0 20 40 60 80 100 Concentration (µg/mL)

Figure 16: The IC50 determination for I. hayesiana extracted in hexanes (10 mg/mL) on the human breast cancer cell line MDA-MB-231. The error bars represent one standard deviation from the average of ten samples.

160 140 120 100 80 60

( % Control) % ( 40

Relative # of Cells Cells of # Relative 20 0 65 µg/mL 32.5 µg/mL 65 µg/mL 32.5 µg/mL 65 µg/mL Hexane Hexane Ether Ether Ethanol

Solvent

Figure 17: The cytotoxicity of three new solvent extracts from fresh I. hayesiana (65 and 32.5 µg/mL) on the human breast cancer cell line MDA-MB-231. The bars represent the percentage of cells alive compared to the control (cells treated with vehicle only). The error bars represent one standard deviation from the average of ten samples.

To investigate the chemical differences between the new, inactive extracts and the old, active extracts, both the new and old extracts were compared via HPLC. The chromatograms indicated that there were some differences between the two extracts

(Figure 18). This, coupled with the fact that the extractions were performed during different seasons or plant cycles, led to the hypothesis that the observed cytotoxicity is seasonal.

Norm.

500

400

300

200

100

0 8 10 12 14 16 18 20 22 24 min

Figure 18: HPLC investigation of the chemical differences of active and inactive extracts of I hayesiana. The HPLC chromatogram of the original active extract (extracted September 2010) shown in blue, overlayed with an inactive extract (extracted November 2010) shown in red.

3.7 Evaluation of Seasonal Cytotoxicity

To determine whether the cytotoxic compound(s) in I. hayesiana are seasonal, extractions were performed monthly on the same plant, beginning February 28th, 2011 and recurring on the 28th of every month through July 2011. Extractions were performed using 1 g of I. hayesiana leaves and stems and 25 mL of hexanes (ACS grade), following the initial extraction protocol. Extracts were then evaluated monthly using the cell viability assay, at a concentration of 10 mg/mL. The results shown in

Figure 19 indicate that cytotoxicity returned in April and was present until June. Both

April and May extracts showed almost 100% cell death, while the June extract resulted in approximately 50% cell death. The plant flowered at the end of June. Upon review of previous data, it was noted that the original extract, which showed cytotoxic activity,

40 was completed in September, one month prior to the plant flowering. The second extract was completed in November, after the plant had flowered. This information, coupled with the cell viability assay data from April and May, suggests that cytotoxic constituents are present prior to the flowering of the plant, and that once the plant flowers, these constituents are either no longer present, or are no longer present at high enough concentrations to have an effect on the cancer cells. The data from June, the month the plant flowers, shows a 50% reduction in cytotoxicity, as the compound(s) of interest decreases just before the plant flowers. All active and inactive extracts were evaluated further using HPLC.

135

115

55 (%Control)

35 Relative Number Cells of Number Relative 15

-5 February March April May June July Month of Extraction

Figure 19: Results from the cell viability assay of monthly extracts of I. hayesiana (65 µg/mL) on the human breast cancer cell line MDA-MB-231. The extracts from April and May show 100% cytotoxicity and the extract from June shows ~50% cytotoxicity. All other months are inactive. The bars represent the percentage of cells alive compared to the control (cells treated with vehicle only). The error bars represent one standard deviation from the average of ten samples.

3.8 HPLC Analysis of Monthly Extracts

All seven monthly extracts were analyzed using an Agilent 1100 HPLC.

Extracts of I. hayesiana were prepared using the method described in the initial extraction section of Materials and Methods at a concentration of 10 mg/mL. Samples were further normalized to 1mg/mL and filtered using a 0.22 µm sterile syringe filter

(Fisherbrand) prior to injection. For evaluation purposes, each of the chromatograms of the active extracts was first overlaid with the ethanol control chromatogram. Peaks present in the ethanol control chromatogram were excluded from analysis for all of the extracts. This left 14 distinct peaks that represent the HPLC profile of I. hayesiana and required further analysis (Figure 20). Chromatograms of all monthly extracts are available in Appendix A. The retention times and peak areas for these peaks are shown in Table 3.

Figure 20: HPLC chromatogram of the April extract of I. hayesiana with the 14 peaks of interest labeled, after comparison to the blank (ethanol) chromatogram.

Table 3: Retention times and peak areas from the HPLC chromatogram of I. hayesiana extracted in April. Fourteen distinct peaks were identified for further analysis.

Time Area Peak # (Min) (mV) 1 14.391 1031.6 2 14.641 4454.3 3 15.584 75.1 4 15.761 2674.3 5 17.316 2095.2 6 19.246 862.9 7 20.055 416.8 8 20.411 126.2 9 20.846 29950.9 10 21.197 3105.3 11 21.934 2312.9 12 22.100 780.6 13 22.846 22252.5 14 25.077 2386.1

Once the ethanol peaks were eliminated from further consideration, the remaining peaks were evaluated by comparing retention times of the 14 peaks of interest in the chromatograms of the active samples with those of the inactive samples.

Each active sample chromatograms were overlaid with the inactive sample chromatograms for analysis (for example, Figure 21). Any peaks present in the inactive extracts at similar concentrations (peak areas) to those seen in the active extracts were ruled out. This left 7 peaks of interest for further evaluation. Of these, only peaks 8 and 9 were present exclusively in the active extracts. The 5 remaining

43 peaks (4, 7, 10, 11, and 13) were present in both active and inactive extracts, but the concentration of these constituents was significantly lower in the inactive extracts.

Figure 21: HPLC chromatograms of I. hayesiana extracts from April 28th (red - active) and Feb 28th (blue - inactive). The overlay shows 7 peaks of interest (4, 7, 8, 9, 10, 11 and13).

To complete the analysis, the three chromatograms of the active extracts were individually overlaid with each other. The data from the cell viability assays showed that the extracts from April and May had 100% cytotoxicity while the extract from June had approximately 50% cytotoxicity. This indicates that the concentration of the cytotoxic constituent(s) should show a significant decrease, which should be visible in the peak area of the constituent of interest. Based on this information, the peak areas of the 7 remaining peaks of interest were compared, to determine whether a decrease in concentration was visible for the June extract (Table 4). Peaks 8 and 10 were ruled out

44 due to large increases in concentration in the month of June. Peaks 4, 7 and 11 were ruled out because the concentration in June, when activity was at 50%, was approximately the same as the concentration in April, when activity was 100%. Peak 9 was not present in any of the inactive extracts (February, March and July) and had off- scale peak areas (not quantifiable) for the active extracts. Peak 13 was present in small amounts in the inactive extracts from February and March but not in July. It was not excluded because the concentrations for April, May and June were off scale, and could not be quantified. To further elucidate peaks 9 and 13, the extracts from April, May and June were diluted to 0.1 mg/mL and re-evaluated via HPLC. The resulting chromatograms (Figure 22) show that peaks 9 and 13 were on scale for analysis (Table

5). Based on the new data, peak 13 is ruled out because the concentration for June

(50% activity) is higher than the concentration for May (100% activity). Peak nine appears to be the remaining peak of interest. The areas for peak nine decrease significantly from May to June which indicates a concentration decrease during the time when activity is decreasing. To determine whether the peak of interest might be hispidulin, further HPLC analysis was conducted.

Table 4: Evaluation of the 7 peaks of interest in the active extracts . Peak areas (mV) of the 7 peaks of interest seen in the HPLC chromatograms of three active extracts (April, May and June) of I. hayesiana (* off scale peaks)

Peak # Peak Areas 4/28/2011 5/28/2011 6/28/2011 Peak 4 2674.3 2992.6 2629.2 Peak 7 416.8 505.1 437.9 Peak 8 126.2 179.4 505.9 Peak 9 *29950 *28120 *32157 Peak 10 3105.3 3290.3 4340.2 Peak 11 2312.9 2390.0 3090.5 Peak 13 *22252 *21981 *24161

Figure 22: HPLC chromatogram of the April extract of I. hayesiana, diluted to a concentration of 0.1 mg/mL, with peaks 9 and 13 labeled. . Table 5: Re-evaluation of peaks 9 and 13 via HPLC. Peak areas (mV) of the two peaks of interest (Peaks 9 and 13) seen in the HPLC chromatograms of the three active extracts (April, May and June) diluted to 0.1 mg/mL.

Peak # 4/28/2011 5/28/2011 6/28/2011 Peak 9 3216.6 4485.9 941.0 Peak 13 771.8 537.8 611.3

3.9 IC50 for Hispidulin

To determine whether hispidulin might be the cytotoxic compound responsible for the activity seen in the active I. hayesiana extracts, an IC50 assay was completed using the protocol described in the Materials and Methods section. Commercially obtained hispidulin was brought to an initial concentration of 10 mg/mL before beginning the assay dilutions (100, 80, 60, 40, and 20 µg/mL). A solution consisting of growth media and 100 µL of ethanol served as the control. The working solutions along with the control were used to inoculate the human breast cancer cell line MDA-

MB-231. This concentration range was chosen based on an IC50 of 200 µmol/L reported

34 by Lijun He et al. to be effective against pancreatic cancer cells. The IC50 concentration of the hispidulin sample on the human breast cancer cell line MDA-MB-

231 was greater than 100µg/mL (Figure 23). At the highest concentration of hispidulin

(100µg/mL), only 25% of the cells were killed. This minimal level of cytotoxicity effectively rules out the possibility that hispidulin could be the constituent responsible for the cytotoxicity seen in I. hayesiana. The hispidulin sample was further evaluated using HPLC to determine whether hispidulin could be identified in the I. hayesiana extracts.

140

120

100 80 60

40 (%Control) 20

0 Relative Number ofCells 0 20 40 60 80 100 Concentration (µg/mL)

Figure 23: IC50 assay results for hispidulin dissolved in ethanol (10 mg/mL) on the human breast cancer cell line MDA-MB-231. The error bars represent one standard deviation from the average of ten samples.

3.10 HPLC Analysis of Hispidulin

For the purposes of HPLC, hispidulin was diluted in ethanol and brought to a concentration of 1mg/mL. This sample was sterile filtered using a 0.22µm PTFE syringe filter (FisherBrand). Sample was injected using the method previously described in the HPLC section of Materials and Methods. Solvent peaks were ruled out, which left two remaining peaks, either of which could be hispidulin (Figure 24A). The chromatogram of hispidulin was then overlaid with the chromatogram of the active extract of I. hayesiana (extracted 4/28/11). A comparison of the retention times (Table

6) of the two peaks present in the hispidulin sample with the peaks from the I. hayesiana extract showed that only one of the peaks (peak 2 in Figure 24B) was present in the I. hayesiana extract. The fact the two peaks were identified as possible hispidulin peaks indicates that the sample was not pure or that there were

48 decomposition products present in the hispidulin sample. The data indicates that hispidulin is present in I. hayesiana, as was reported by Hertz et al. (1951), although it has been ruled out as the constituent responsible for the cytotoxicity seen with MDA-

MB-231.28

B Figure 24: Evaluation of the presence of hispidulin in I. hayesiana using HPLC. (A) Chromatogram of hispidulin in ethanol (1 mg/mL) with the hispidulin peaks labeled. (B) Overlay of Active Extract (4/28/11)of I. hayesiana (blue) with hispidulin (red). Peak 2 represents the only peak with a retention time that matches either of the two peaks identified in hispidulin.

Table 6: Evaluation of the HPLC retention times of hispidulin. Retention Times of peaks 1 and 2 represented in Figure 24A and 24B. The difference in retention times for peak one indicate that peak 1 is not hispidulin.

Hispidulin I. hayesiana Peak 1 14.268 min 14.391 min Peak 2 17.310 min 17.316 min

Chapter 4

Conclusions

The purpose of this research was three-fold: 1) To determine the best extraction method for extractions of the cytotoxic component(s) I.hayesiana, 2) To determine whether the cytotoxic constituent(s) occur seasonally in I. hayesiana, and 3) To determine whether hispidulin is responsible for the cytotoxicity of I. hayesiana on the human breast cancer cell line MDA-MB-231. The results showed that the cytotoxic compound(s) in I. hayesiana are non-polar and non-volatile. Therefore, hexanes was determined to be the best solvent for future extractions, and HPLC was the best method for purity analysis. Analysis of monthly extractions showed that the cytotoxicity was seasonal and related to the flowering of the plant. Cytotoxicity appeared approximately two months before the plant flowered and disappeared rapidly once flowering occured.

The IC50 for an active hexane extract of I. hayesiana was determined to be 56

µg/mL. It was also determined that hispidulin was present in I. hayesiana; however, it was not responsible for the cytotoxicity seen in the extracts from April through June.

Chapter 5

Future Work

Based on the results from this study, a number of recommendations can be made for the extension of this project. This study was focused on the cytotoxicity of I. hayesiana extracts towards the triple negative breast cancer cell line MDA-MB-231.

To determine whether I. hayesiana is in fact a viable target for future anticancer research, I. hayesiana must be tested against normal human cells. I. hayesiana should also be tested against other breast cancer cell lines such as MCF-7 and BT-474 to determine whether it might also be effective against ER/PR+ cell lines.

Now that the seasonal nature of the cytotoxicity has been established, large scale extractions can been performed during the appropriate season. Samples from these larger scale extractions can then be used to isolate and identify the cytotoxic constituent(s) present in I. hayesiana, starting with the peak labeled as # 9 in this research. It would also be advisable to attempt to determine the chemical fingerprint

(LC/MS) of I. hayesiana, which is different during the seasonal cytotoxic period.

HPLC Chromatogram of Iva hayesiana Extracted 2/28/11 (1 mg/mL)

Peak # Time Area 1 14.39 362.5 2 14.64 2337 3 -- -- 4 15.76 110.7

HPLC SpectrumHPLC 5 17.31 1290 APPENDIX A 6 19.27 475.5 7 -- -- 8 9

10 21.21 466.5 11 -- -- 12 21.99 161.4 13 22.88 403.2 14 25.08 195.5

HPLC Chromatogram of Iva hayesiana Extracted 3/28/11 (1 mg/mL) # Time Area 1 14.39 756.2 2 14.64 3985 3 15.59 118.1 4 15.76 754.4 5 17.32 2440.1 6 19.27 647.0 7 -- -- 8 -- -- 9 -- -- 10 21.21 383.5 11 -- -- 12 13 22.88 683.0 14 25.07 369.9

HPLC Chromatogram of Iva hayesiana Extracted 4/28/11 (1 mg/mL) Peak Time Area # (Min) (mV) 1 14.391 1031.6 2 14.641 4454.3 3 15.584 75.1 4 15.761 2674.3 5 17.316 2095.2 6 19.246 862.9 7 20.055 416.8 8 20.411 126.2 9 20.846 29950.9 10 21.197 3105.3 11 21.934 2312.9 12 22.100 780.6 13 22.846 22252.5 14 25.077 2386.1

HPLC Chromatogram of Iva hayesiana Extracted 5/28/11 (1 mg/mL)

# Time Area 1 14.39 1138 2 14.64 4843 3 15.58 91.6 4 15.76 2993 5 17.32 2397 6 19.24 868.3 7 20.05 505.1 8 20.41 204.6 9 20.84 28120 10 21.19 3290 11 21.93 2390 12 22.1 986.7 13 22.84 21982 14 25.08 477.2

HPLC Chromatogram of Iva hayesiana Extracted 6/28/11 (1 mg/mL)

# Time Area 1 14.39 685.0 2 14.64 2709.3 3 4 15.76 2629.2 5 17.31 2211.2 6 19.24 809.5 7 20.10 437.9 8 20.41 505.9 9 20.82 32157.2 10 21.18 4340.2 11 21.92 3090.5 12 22.09 2035.7 13 22.82 24161.6 14 25.08 432.5

HPLC Chromatogram of Iva hayesiana Extracted 7/28/11 (1 mg/mL)

# Time Area 1 -- -- 2 -- -- 3 -- -- 4 15.76 81.7 5 17.32 1104.1 6 19.22 285.7 7 20.04 147.6 8 -- -- 9 21.03 88.9 10 21.19 474.3 11 21.95 447.3 12 -- -- 13 22.85 614.1 14 25.10 428.2

HPLC Chromatogram of Iva hayesiana Extracted 4/28/11 (0.1 mg/mL)

# Time Area 1 -- -- 2 -- -- 3 -- -- 4 15.76 81.7 5 17.32 1104 6 19.223 285.7 7 20.04 147.6 8 -- -- 9 21.03 88.9 10 21.19 474.3 11 21.95 447.3 12 -- -- 13 22.85 614.1 14 25.1 428.2

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