Abstract B32: Gedunin induces autophagy during mitosis, a novel form of mitotic catastrophe

Background: Breast Cancer is the most prevalent cancer in the world and the second leading cause of cancer death among women in the United States. Triple negative breast cancer (lacking estrogen receptors (ER), progesterone receptors (PR) and human epidermal growth factor receptor 2 (HER2)) can be extremely aggressive and is more likely to recur and metastasize than other subtypes of breast cancer. They are also unresponsive to the most effective receptor targeted treatments. A different treatment strategy is essential to fight against the triple negative breast cancer cells. Heat shock protein 90 (Hsp90) is a molecular chaperone that is required for the stability and function of various signaling proteins that promote the growth and/or survival of cancer cells. Several Hsp90 inhibitors are currently under clinical trial for the treatment of cancer. One natural HSP90 inhibitor is gedunin, which was isolated from the Indian neem tree (Azadirachta indica L.). Here we present our results of studies on the effect of gedunin on BT20 triple negative breast cancer cells and HMLE immortalized breast epithelial cells. Method: BT20 and HMLE cells were grown according to ATCC guidelines. Flow cytometric analyses for cell death was performed using the Apoptosis/Necrosis detection kit (Roche). MDC incorporation and electron microscopic were performed to detect autophagy. BT20 tumor xenografts were used for determining the effect of gedunin in vivo. Real Time PCR, western blot and immunefluorescent studies were performed for determining gene expression. Results: Gedunin induced a dose (0-20 μM) and time (0-72 h) dependent cytotoxicity, with significant inhibition of colony formation of BT20 cells at a concentration of 8 μM. However, gedunin did not affect the viability of HMLE cells. Cell cycle analyses demonstrated G2/M phase arrest of the BT20 cells. Electron microscopy studies revealed that gedunin induced the formation of autophagosomes, which was further confirmed by the monodansylcadaverine (MDC) incorporation. Real time-PCR and Western blot analyses revealed that gedunin induced the expression of autophagy related genes ATG5, ATG7, ATG12 and Beclin1. Furthermore, there was increased cleavage and lipidation of microtubule associated protein 1 light chain 3 (LC3B). Mechanistically, we have identified that gedunin induced phosphorylation of AMP kinase, which induces a signaling casade starting with phosphorylation of ULK1. This was suppressed when cells were either treated with an AMPK inhibitor Compound C or AMPK was downregulated with specific siRNA. There was also a reduction in gedunin-induces cell death. These data suggest that gedunin-mediated induction of autophagy occurs in part via the AMPK pathway. We have confirmed these findings in vivo using BT20 nude mice tumor xenografts. Intraperitoneal gedunin administration (5 mg.Kg bw) significantly decreased tumor growth. Western blot analyses showed increased expression of autophagic markers LC3B and Beclin1 in the gedunin-treated tissues which was further confirmed by the immunohistochemistry. More importantly, we observed increased number of autophagasomes in cells undergoing mitosis (p-Histone H3 staining). Conclusion: Together, these data suggest that gedunin effectively drives triple negative breast cancer cells to an unusual form of mitotic catastrophe by inducing AMPK mediated autophagy during mitosis. Citation Format: Parthasarathy Rangarajan, Prabhu Ramamoorthy, Satish Ramalingam, Dharmalingam Subramaniam, Sivapriya Ponnurangam, Scott Weir, Shrikant Anant, Roy Jensen. Gedunin induces autophagy during mitosis, a novel form of mitotic catastrophe. [abstract]. In: Proceedings of the Twelfth Annual AACR International Conference on Frontiers in Cancer Prevention Research; 2013 Oct 27-30; National Harbor, MD. Philadelphia (PA): AACR; Can Prev Res 2013;6(11 Suppl): Abstract nr B32.