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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

A transient bilateral population of cells immediately rostral to the ferret corpus callosum was examined at weekly intervals between embryonic day 28 and postnatal day 7. This region is tentatively identified as the medial forebrain apoptosis zone (MFAZ) because of the specific nature of cell death, and the limited area or zone in which it was observed. No other region within the brain or retina exhibited a similar pattern or amount of cell death. Only scattered apoptotic cells were found throughout the remainder of the brain-including the cerebral cortical plate, subplate, and white matter-with the exception of the ventricular zone of the lateral ventricles which contained a significant population of apoptotic cells. This study addressed three questions about apoptosis in the MFAZ: (1) does apoptotic cell death in this region signal the appearance of phagocytic macrophages, (2) does cell degeneration and phagocytosis in this region lead to the formation of an extracellular space analogous to the cavum septi pellucidi of rodents, and (3) what is the duration of degeneration, or clearance rate, of cell death in this defined region. The MFAZ was first found to contain apoptotic nuclei and macrophages late in gestation, at E34. Numbers of apoptotic nuclei and macrophages peaked one week later at birth in this area, but disappeared early in postnatal life. During this period, formation of a space rostral to the corpus callosum due to the removal of apoptotic cells was not observed. Finally, presence of apoptotic cells in the MFAZ over a period of > or = 9 days suggests a clearance rate of many hours. The close spatiotemporal correlation of the distribution of apoptotic cells and their removal by macrophages suggests that these are interrelated, and perhaps interdependent events.

Despite research in therapeutic strategies, ovarian cancer (OvCa) remains the most lethal gynecologic malignancy. The anatomical location of the ovaries within the peritoneal cavity facilitates metastasis by allowing tumor cells sloughed from the main tumor on the ovary to bind to the peritoneum or organs within the peritoneal cavity. These tumor cells are released from the main tumor either as single cells or as spheroids, the latter of which have been shown to be more effective at metastasizing and invading into the mesothelial lining. To identify novel tumor markers and therapeutic targets for epithelial cancers, a cDNA library enriched for cancer genes that encode membrane and secreted proteins was generated. The 18th most abundant gene represented in this library was Sushi Domain Containing 2 (SUSD2), which encodes an 822 amino acid membrane surface protein. To begin defining the role of SUSD2 in OvCa, stable SUSD2-expressing and vector control A2780 cell lines were generated. To examine whether SUSD2 affects the growth of OvCa cells, cellular proliferation assays were performed. There was no significant difference in growth rates with SUSD2 over-expression compared to the vector control cell lines. A Boyden chamber assay was performed to analyze the effect of SUSD2 on cell migration. A2780-SUSD2 cell lines and vector-only controls were plated on membranes and allowed to migrate toward a chemoattractant in a lower chamber. SUSD2 over-expression significantly increased migration by greater than 2-fold. In addition, a scratch wound-healing assay was performed, and after 24 hours the A2780-SUSD2 cell line had 2-fold more cells within the wound compared to the A2780-vector control cells (P<0.05). Consistently, agarose beads containing either A2780-SUSD2 or vector cells were grown in chemoattractant containing media. The distance the escaped cells traveled from the bead was measured. A2780-SUSD2 cells traveled farther away from the bead compared to the vector control (717 μm compared to 125 μm, respectively, P<0.05). Spheroid-forming ability was analyzed, and A2780-SUSD2 cells formed smaller and looser spheroids (length and width average of 933 μm) compared to A2780-vector control cells (length and width average of 1237 μm, P=0.025). To correlate OvCa patient outcome with the presence of SUSD2 in the corresponding tumor, a tissue microarray containing high-grade ovarian serous carcinoma core samples from 128 patients was stained with an anti-SUSD2 antibody. The intensity of SUSD2 staining was scored by a pathologist, and results were correlated with patient outcome. A Kaplan-Meier curve indicated a significant separation for patients with low SUSD2 levels, median survival 31.7 months, versus patients with strong SUSD2 staining, median survival 49.1 months (P=0.0083). This data suggests that low SUSD2 levels in OvCa tumors are associated with a poorer prognosis for the patient. Our in vitro assays indicate that expression of SUSD2 in A2780 cells increased the ability of the cells to migrate and caused the formation of smaller spheroids. Because spheroids are better able to resist chemotherapy and invade the mesothelium, the decreased spheroid size of SUSD2-expressing cells may allow for a better patient prognosis. Citation Format: Jennifer A. A. Gubbels, Elizabeth M. Hultgren, Jessica Johnson, Emily Johnson, Jeffrey Sachs, Michelle S. Hirsch, Joyce Liu, Ronny Drapkin, Kristi A. Egland. Expression of Sushi Domain Containing 2 (SUSD2) in high-grade ovarian serous carcinoma correlated with increased longevity of patients. [abstract]. In: Proceedings of the AACR Special Conference on Advances in Ovarian Cancer Research: From Concept to Clinic; Sep 18-21, 2013; Miami, FL. Philadelphia (PA): AACR; Clin Cancer Res 2013;19(19 Suppl):Abstract nr A71.