Abstract 2566: Critical role of autophagy proteins Beclin 1 and LC3B in deguelin-induced apoptosis in pancreatic tumor cellsin vitroandin vivo: A new paradigm
Authors: Srinivas Reddy Boreddy, Prabodh K. Kandala, Kartick C. Pramanik, Parul Gupta, Sanjay K. Srivastava
Journal: Cancer Research (2012)

Abstract Deguelin is known to inhibit cell growth by inducing apoptosis in various cancer models primarily by inhibiting AKT, but its role in autophagy is largely unknown. In the present study, we evaluated the role of autophagy in deguelin-induced apoptosis in pancreatic cancer. Our results reveal that PanC-1, AsPC-1 and BxPC-3 cells that were treated with deguelin show significant increase in autophagosome vacuoles (AV) formation and increase in the expression of Beclin1, ATG5 and LC3B. In general, induction of autophagy is considered to be a protective mechanism; however, few recent studies have indicated the pro-apoptotic role of autophagy as well. To address this question, cells were pretreated with specific inhibitors of autophagy such as chloroquine and 3-methyladenine prior to deguelin treatment and apoptosis was evaluated. Surprisingly, inhibition of autophagy by all the above-mentioned inhibitors significantly blocked deguelin-induced apoptosis, indicating that apoptosis induction by deguelin was dependent on autophagy. To establish that deguelin-mediated autophagy was involved in apoptosis, Beclin1, ATG5 and LC3B were silenced and treated with deguelin. Consistent with our inhibitor data, silencing of Beclin1 and LC3B made the cells resistant to deguelin-induced apoptosis, however silencing of ATG5 provided modest protection, indicating that Beclin1 and LC3B play a critical role in deguelin-induced apoptosis in pancreatic cancer cells. Previous studies have shown that Beclin1 is cleaved by caspases into N-terminal and C-terminal fragments. C-terminal translocates to mitochondria and cause cytochrome C release to trigger apoptosis. In agreement, our results show that the C-terminal fragment of Beclin1 was increased, whereas cytochrome C levels were decreased in mitochondrial fraction, indicating that Beclin1 acts as a proapoptotic protein in our model. Similarly, LC3B has been shown to induce apoptosis by releasing Fas from caveolin1 to form DISC and trigger extrinsic apoptosis. To test this possibility, DISC formation was evaluated in deguelin treated control and LC3B-silenced PanC-1 cells. Interestingly, DISC formation was significantly reduced in LC3B silenced PanC-1 cells, as compared to LC3B expressing cells. To further validate our observations in vivo, PanC-1-luc cells were orthotopically implanted into the pancreas of nu/nu mice and treated with 5mg/kg deguelin (i.p., daily). After 42 days of treatment, deguelin treated mice showed substantially reduced tumor growth as compared to control mice. In addition, tumors of deguelin treated mice exhibited increased levels of Beclin1 and LC3B. Taken together, our results demonstrate that deguelin-induced apoptosis in pancreatic tumor cells was mediated through autophagy. [Supported in part by R01 grants CA129038 and CA106953 (to S.K.S) awarded by the National Cancer Institute]. Citation Format: {Authors}. {Abstract title} [abstract]. In: Proceedings of the 103rd Annual Meeting of the American Association for Cancer Research; 2012 Mar 31-Apr 4; Chicago, IL. Philadelphia (PA): AACR; Cancer Res 2012;72(8 Suppl):Abstract nr 2566. doi:1538-7445.AM2012-2566