Kai Ludwig Principle Investigator: Dr. Manish Patankar Mentor: Dr. Arvinder Kapur

1. Dietary supplements, conjugated linoleic acid (CLA) and ginger extract, as preventive and therapeutic agents for gynecological cancer Kai Ludwig Principle Investigator: Dr. Manish Patankar Mentor: Dr. ArvinderKapur CALS Undergraduate Research Symposium Tuesday, April 17th 2012

2. Overview Background on Gynecological Cancer CLA and effect on Ovarian cancer lines Ginger extract and effect on Endometrial cancer lines Summary/Further Direction/Acknowledgements

3. Gynecological Cancers Constitutes 5 major classes • cervical, vaginal, vulvar, ovarian, and endometrial Estimates for 2012 in US1: • Endometrial = 47,130 new cases; 8,010 deaths • Ovarian =22,280 new cases; 15,500 deaths With advances in radiotherapy, chemotherapy, surgical procedures, what is the problem? 1American Cancer Society. Cancer Facts & Figures 2012. Atlanta: American Cancer Society; 2012

4. Recurrent Cancers with Resistances Leading cause of death among gynecological cancers2 • 10-25% of patients respond to second line anti-neoplastic agents in ovarian cancer3 • 5- and 10-year survival rate was 18 and 12.5%, respectively for recurrent endometrial cancer patients treated with radiotherapy4 Need for development of novel and effective chemopreventive and chemotherapeutic agents 2 Kimio Ushijima. Treatment for Recurrent Ovarian Cancer—At First Relapse, Journal of Oncology, vol. 2010, Article ID 497429, 7 pages, 2010. doi:10.1155/2010/497429 3 MutchDG, Orlando M, Goss T, Teneriello MG, Gordon AN, McMeekin SD, et al. Randomized phase III trial of gemcitabine compared with pegylated liposomal doxorubicin in patients with platinum-resistant ovarian cancer. J ClinOncol2007 Jul 1;25(19):2811-8. 4KutenA, Grigsby PW, Perez CA, Fineberg B, Garcia DM, Simpson JR. Results of radiotherapy in recurrent endometrial carcinoma: A retrospective analysis of 51 patients. Int J RadiatOncolBiol Phys. 1989;17:29–34

5. Dietary Supplementation as Therapeutic Agents • Investigate potential of: • Conjugated Linoleic Acid (CLA) • Extract from rhizomes of ginger • Natural products with minimal side-effects Images from: http://maxcdn.nexternal.com/greenpharm/images/CLA.jpg and http://cancerbattlefield.com/wp-content/uploads/2009/05/ttar_ginger_v.jpg

6. Conjugated Linoleic Acid (CLA) • Fatty acid that constitutes family of 28 isomers found in meat and dairy products • Bioactivity and anti-carcinogenic effects in trans-10,cis-12 (t10:c12) and cis-9,trans-11 (c9:t11) Image from: http://www.jyi.org/articleimages/881/originals/img0.jpg

7. Effects of CLA on Cell Proliferation • Treat Ovarian lines with increasing dosages of CLA for 24, 48, 72, or 96 hours • MTT Assay= cells reduce MTT to purple formazan crystal • -> can be measured by spectrophotometer at 570nm • Optical Density directly measures amount of cells alive • Ovarian cancer cell lines used: • A2780 and SKOV3 Image from: http://en.wikipedia.org/wiki/File:MTT_Plate.jpg

8. Effects of CLA on Cell Proliferation Conclusions: • T10:c12 CLA inhibited ovarian cancer cell proliferation in both time and dose dependent manner. • C9:t11 CLA had no effect on cell growth

9. Effects of CLA on cell cycle • Both A2780 and SKOV subjected to 7µM dosage of t10:c12 CLA for 72 hours • Cellular DNA stained with propidium iodide • Cell cycle analysis with flow cytometer

10. Effects of CLA on cell cycle Conclusions: • Significant increase (12%, p<0.05) in number of cells in G1 phase • Equivalent decrease in number of cells in S phase • G1 phase stalling caused by t10:c12 CLA treatment

11. Western Blot analysis of CLA treatment • b-catenin = oncogene; affects cell cycle and tumor proliferation5 • Negatively regulated by its phosphorylationby active GSK-3b • Western blot analysis done on A2780 after 72 hour CLA treatment • b-actin used as a loading control 5SaldanhaG, Ghura V, Potter L, Fletcher A (July 2004). "Nuclear β-catenin in basal cell carcinoma correlates with increased proliferation". Br. J. Dermatol. 151 (1): 157–64. doi:10.1111/j.1365-2133.2004.06048.x. PMID15270885

12. Western Blot analysis of CLA treatment Conclusion: • T10:c12 CLA resulted in ~50% increase in GSK3bphosphorylation and a ~70% reduction in b-catenin protein levels • C9:t11 CLA had no effect on either proteins.

13. Ginger Extract • Obtained via Clevenger apparatus steam distillation • 250 g ginger rhizome boiled at 60-80˚C for 4-6 hours yielded 300 mg of oil • Oil used for all bioassays Clevenger Apparatus

14. Ginger Extract effects on endometrial cancer cell proliferation Conclusion: • Ginger extract is a potent inhibitor of cell proliferation • Effective on endometrial cancer lines Ishikawa ECC1 MTT Assay performed on Endometrial cell lines Ishikawa and ECC1

15. 0h 0h 24h 24h 48h 48h 72h 72h BAX Bax Bcl2 Bcl2 -Actin -Actin Western Blot analysis of Ginger treatment on protein expression: BAX/Bcl2 • Investigate apoptosis as mechanism of control over cell proliferation • BAX= pro-apoptotic protein • Bcl2= anti-apoptotic protein • BAX/Bcl2 compete6 Ishikawa Treatment with ginger extract • Conclusions: • No effect in Bax expression • Significant decrease in Bcl2 expression • Ratio of BAX/Bcl2 increased • Evidence for Apoptosis 6Oltvai, Z. N.; Milliman, C. L. and Korsmeyer, S. J. (August 1993)Bcl-2 Heterodimerizes In Vivo with a Conserved Homolog, Bax, That Accelerates Programed Cell Death”. Cell 74 (4): 609–619. doi:10.1016/0092-8674(93)90509-O. PMID 8358790.

16. Western Blot analysis of Ginger treatment on protein expression:pP53 • Phosphoprotein 53 (TP53, P53)= tumor suppressor7 • Role in repairing DNA • Increased expression of pP53 leads to stalling and apoptosis 60min 30min control 15min pP53 b-Actin Ishikawa Treatmentwithginger extract • Conclusions: • Increased expression of pP53 • Evidence for Apoptosis 7May P, May E (December 1999). "Twenty years of p53 research: structural and functional aspects of the p53 protein". Oncogene18 (53): 7621–36.

17. Summary • Ginger Extract • Inhibitor of cell proliferation in Endometrial Cancer Cell lines: Ishikawa and ECC1 • Treatment causes an increased pro-apoptotic BAX:Bcl2 ratio and well as increased pP53 expression • Gingerextract treatment mediates proliferation by inducing apoptosis in cancer cells CLA • Inhibitor of cell proliferation in Ovarian Cancer Cell lines: A2780 and SKOV3 • Treatment seems to causes G1 cell cycle arrest coupled with an increased expression of GSK-3b and decreased expression of b-catenin • Cell cycle arrest leads to less cancer proliferation Both treatments show effects as potential therapeutic agents in the treatment of Gynecological cancers

18. Relevance and Future Direction • Further characterization of CLA and ginger extract and its effect on ovarian and endometrial cancers • Does cell cycle arrest lead to apoptosis or autophagy? • What is the potent chemical(s) in the ginger extract? • In vivo mouse models- same effect in immuno-compromised mice? • Use as supplemental therapeutic agent in the treatment of gynecological cancers • Taken as part of diet or alternate application

19. Acknowledgements • PI- Manish Patankar • Mentor- ArvinderKapur • Soma Banerjee, Nick Claussen, Joseph Connor, Mark Cook, Mildred Felder, Yang Liu, BikashPattnaik, Helen Rowland, MianShiazad, Chanel Tyler, Hannah Van Galder, Rebecca Whelan *Partial Funding for this project was given by the Honors and Undergraduate Research Committee of the College of Agricultural and Life Sciences