Date

5-12-2020

Document Type

Thesis

Abstract

Withaferin A (WA), the active-phytochemical of the medicinal plant Withania somnifera, promotes apoptosis in cancer cells. Although its mechanism remains unclear, WA has been shown to upregulate heat shock proteins (Hsps) which protect against diverse cellular insults. The small Hsps, αB-Crystallin and Hsp27, confer resistance to chemotherapy in several cancer cell lines, however, the role of WA on αB-Crystallin and Hsp27 expression in drug resistance remains unknown. Since the majority of ovarian tumors eventually recur in a drug-resistant form leaving patients few treatment options, the goal of this study is to explore the molecular mechanism induced by WA in a cisplatin-resistant ovarian cancer cell line (OVCAR8R) as compared to its cisplatin-sensitive syngeneic counterpart (OVCAR8). Importantly, I found that αB-Crystallin and Hsp27 are constitutively expressed in the OVCAR8R cells, while OVCAR8 cells do not endogenously express these Hsps, suggestingthat overexpression of Hsps may confer resistance to chemotherapy and promote a more aggressive tumor type. To determine the effect of WA, OVCAR8 and OVCAR8R cells were treated with 0.5, 1.0, 2.5, 5.0 or 7.5μMWA. Morphological studies show WA-induced cytotoxicity in both cell lines, however the OVCAR8R cells remain viable at 5.0 and 7.5μM WA while few OVCAR8 cells remain viable at these doses. Flow cytometry supports this as the percent of Annexin positive cells following WA treatment increases in a dose-dependent manner in OVCAR8 cells, while the percent of Annexin positive OVCAR8R cells is negligible compared to controls at high doses of WA. Following WA treatment for 24h, the endogenous level of αB-Crystallin is upregulated in a dose dependent manner in the cisplatin-resistant OVCAR8R cells, while the cisplatin-sensitive OVCAR8 cells upregulate αB-Crystallin and Hsp-27 at 2.5and 5.0μM WA. Experiments are underway to systematically determine the role of αB-crystallin in conferring resistance of ovarian cancer cells to apoptosis. αB-Crystallin will be silenced by CRISPR-Cas9 in the OVCAR8R and clones will be selected to address the role of αB-Crystallin on WA-induced 4apoptosis. Conversely, wildtype αB-Crystallin and αB-Crystallin pseudophosphorylation mutants that alter its chaperone activity will be overexpressed in the OVCAR8 cells to determine the role of this protein in conferring resistance to WA-induced apoptosis. Together, this research aims to elucidate the effectiveness of WA as a potential therapy for ovarian cancer cells that acquire resistance to platinum-based therapies.

Department

Department of Biological Sciences

Thesis Comittee

Dr. Merideth Krevosky, Thesis Advisor

Dr. Kenneth Adams, Committee Member

Copyright and Permissions

Original document was submitted as an Honors Program requirement. Copyright is held by the author.

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