Rangarajan 2013

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

Parthasarathy Rangarajan1, Sydney Byrne1, Satish Ramalingam1,2, Dharmalingam Subramaniam1,2, Sivapriya Ponnurangam1,2, Prabhu Ramamoorthy1, Shrikant Anant1,2 and Roy Jensen2,3

Departments of 1Molecular and Integrative Physiology, 3Pathology and 2University of Kansas Cancer Centre, University of Kansas Medical Centre, Kansas City, KS 66160

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 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 whose association 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. We have determined the effect of Gedunin, a natural HSP90 inhibitor isolated from the Indian neem tree (Azadirachta indica L.) on BT20 triple negative cells. Gedunin induced a dose and time dependent cytotoxicity, with significant inhibition of clonogenicity at a concentration of 8 μM, without affecting HMLE and MCF10A non-transformed cells. Cell cycle analyses demonstrated arrest at the G2/M stage. Electron microscopy studies revealed that gedunin induced the formation of autophagosome structures in the cells, which is further confirmed by the monodansylcadaverine(MDC) incorporation. Real time-PCR and Western blot analysis revealed that gedunin induced the expression of autophagy related markers ATG5, ATG7, ATG12, Beclin1. Furthermore, gedunin induced cleavage and lipidation of microtubule associated protein 1 light chain 3 (LC3). Mechanistically, we have identified that gedunin induced phosphorylation of AMP kinase, which was reduced when cells were pretreated with Compound C, an AMPK inhibitor. These data indicate 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 significantly decreased tumor growth. Western blot analyses showed increased expression of autophagic markers LC3B and beclin1 in the gedunin-treated tissues which was further confimed by the immunohistochemistry. More importantly, we observed increased formation of autophagasomes in cells undergoing mitosis (p-Histone H3 staining) and by electron microscopy. Together, these data suggest that gedunin effectively drives triple negative breast cancer to an unusual form of mitotic catastrophe by inducing AMPK mediated autophagy during mitosis.