Abstract 4553: Gedunin, a novel HSP-90 inhibitor, synergizes with cisplatin and paclitaxel to inhibit growth of chemoresistant ovarian cancer cell lines
Authors: Johnson Jessica , Venugopal Anand , Kwatra Deep , Roby Katherine , Godwin Andrew , Anant Shrikant 
Published: May 2015

High-grade serous ovarian cancer is the most lethal of all gynecological cancers. It is often not diagnosed until FIGO stage III or IV, at which point the 5-year survival rate is 15%. Although most patients initially respond to the standard of care, 70% of tumors reoccur. Of these, 70-90% are refractory to standard therapies used in the treatment of serous ovarian cancer, cisplatin and paclitaxel. These statistics reveal a need for novel therapeutics that target these tumors. Gedunin, a liminoid from the Neem tree (Azadirachta indica) has been identified as an HSP90-inhibitor. We tested whether gedunin affects the viability of five different ovarian cancer cell lines, ID8, ID8TaxR, A2870, C30 and CP70. We chose these cell lines because cisplatin (C30, CP70) and paclitaxel (ID8TaxR) resistant cells were developed from A2870 and ID8, respectively. Treatment with gedunin (0-30µM) inhibited growth of all five cell lines. In addition, combination indices revealed synergism between gedunin and the two chemotherapeutic agents. More importantly, synergism between gedunin and paclitaxel occurs even at doses (2.5µM for each) that do not have any effect on the cells when treated alone. We next determined the mechanism of gedunin action. Flow cytometry and immunofluorescence microscopy, demonstrated that gedunin induces mitotic arrest between metaphase and anaphase. This was confirmed by western blot analysis of cyclin protein levels. We also observed significant changes to expression of checkpoint kinase-1 (CHK1) and polo-like kinase-1 (PLK1) in the five cell lines. We propose that the misfolding and subsequent degradation of CHK1 and PLK1 leads to the observed mitotic halt, which eventually results in apoptosis. Following cell synchronization, gedunin-treated cells show decreased inhibitory phosphorylation (Y15) of CDK1 and increased levels of cyclin B1 compared to untreated cells. Moreover, immunofluorescence microscopy demonstrated increased monopolar spindle formation. Furthermore, gedunin treatment resulted in formation of double-strand breaks as observed by western blot analysis for gamma-H2AX. This was further verified by TUNEL staining. Finally, apoptosis was observed in gedunin treated cells as measured by increased Bcl-2 to Bax protein ratio and mitochondrial cytochrome c release. These data are in complete agreement with previous studies that show that PLK1 depletion causes spindle abnormalities, as well as increases gamma-H2AX and decreases BCL-2 protein levels. In conclusion, these data suggest that gedunin treatment results in premature mitosis followed by cell cycle arrest and apoptosis. Given that gedunin acts in synergism with both cisplatin and paclitaxel suggests that gedunin is a promising lead compound either alone or in combination with these compounds for the treatment of high-grade serous ovarian carcinoma.

Citation Format: Jessica Johnson, Anand Venugopal, Deep Kwatra, Katherine Roby, Andrew Godwin, Shrikant Anant. Gedunin, a novel HSP-90 inhibitor, synergizes with cisplatin and paclitaxel to inhibit growth of chemoresistant ovarian cancer cell lines. [abstract]. In: Proceedings of the 105th Annual Meeting of the American Association for Cancer Research; 2014 Apr 5-9; San Diego, CA. Philadelphia (PA): AACR; Cancer Res 2014;74(19 Suppl):Abstract nr 4553. doi:10.1158/1538-7445.AM2014-4553