Resveratrol, a naturally occurring phytoalexin, is known to induce apoptosis in multiple cancer cell types, but the underlying molecular mechanisms remain unclear. Here, we show that resveratrol induced p53-independent, X-linked inhibitor of apoptosis protein (XIAP)-mediated translocation of Bax to mitochondria where it underwent oligomerization to initiate apoptosis. Resveratrol treatment promoted interaction between Bax and XIAP in the cytosol and on mitochondria, suggesting that XIAP plays a critical role in the activation and translocation of Bax to mitochondria. This process did not involve p53 but required accumulation of Bim and t-Bid on mitochondria. Bax primarily underwent homo-oligomerization on mitochondria and played a major role in release of cytochrome c to the cytosol. Bak, another key protein that regulates the mitochondrial membrane permeabilization, did not interact with p53 but continued to associate with Bcl-xL. Thus, the proapoptotic function of Bak remained suppressed during resveratrol-induced apoptosis. Caspase-9 silencing inhibited resveratrol-induced caspase activation, whereas caspase-8 knockdown did not affect caspase activity, suggesting that resveratrol induces caspase-9-dependent apoptosis. Together, our findings characterize the molecular mechanisms of resveratrol-induced caspase activation and subsequent apoptosis in cancer cells.Anticancer agents induce cell death in cancer and normal cells via mechanisms including apoptosis and autophagy (1-4). Therefore, there is a need for alternative anticancer agents that can promote cancer cell death while avoiding killing of normal, non-cancerous cells. Resveratrol (trans-3,5,4Ј-trihydroxystilbene) is a naturally occurring polyphenolic phytoalexin found at high levels in the skin of grapes and in red wine. It is also present in peanuts and other plant products. Resveratrol has been shown to possess an apoptosis-dependent anticancer activity and minimal toxicity to normal cells (5-11). How resveratrol induces apoptosis or cancer cell death is not clearly known, but available evidence indicates that resveratrol induces p53-dependent signaling, which leads to cell cycle arrest and apoptosis induction (10, 12, 13). Additionally, resveratrol targets mitochondria to induce cytochrome c release and thereby triggers caspase-dependent apoptotic cell death in multiple types of cancer cells (14 -18). How resveratrol induces cytochrome c release and caspase activation to execute apoptosis remains unclear.Caspases are activated by proteolytic processing and are broadly divided into initiator caspases (e.g. procaspase-8 and -9) and executioner caspases (such as procaspase-3 and -7) (19 -22). During apoptosis, the released cytochrome c from mitochondria triggers caspase-9 activation, whereas ligation of death receptors on the plasma membrane activates caspase-8. Active caspase-8 generated upon death receptor ligation requires Bid-mediated cytochrome c release to execute apoptotic cell death in epithelial cancer cells (22)(23)(24)(25). Proapoptotic BH3-o...

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We recently demonstrated that resveratrol induces caspase-dependent apoptosis in multiple cancer cell types. Whether apoptosis is also regulated by other cell death mechanisms such as autophagy is not clearly defined. Here we show that inhibition of autophagy enhanced resveratrol-induced caspase activation and apoptosis. Resveratrol inhibited colony formation and cell proliferation in multiple cancer cell types. Resveratrol treatment induced accumulation of LC3-II, which is a key marker for autophagy. Pretreatment with 3-methyladenine (3-MA), an autophagy inhibitor, increased resveratrol-mediated caspase activation and cell death in breast and colon cancer cells. Inhibition of autophagy by silencing key autophagy regulators such as ATG5 and Beclin-1 enhanced resveratrol-induced caspase activation. Mechanistic analysis revealed that Beclin-1 did not interact with proapoptotic proteins Bax and Bak; however, Beclin-1 was found to interact with p53 in the cytosol and mitochondria upon resveratrol treatment. Importantly, resveratrol depleted ATPase 8 gene, and thus, reduced mitochondrial DNA (mtDNA) content, suggesting that resveratrol induces damage to mtDNA causing accumulation of dysfunctional mitochondria triggering autophagy induction. Together, our findings indicate that induction of autophagy during resveratrol-induced apoptosis is an adaptive response.

Resveratrol (Resv) selectively kills cancer cells, whereas the molecular mechanisms of Resv-induced cancer cell death are not defined. Here we show that Resv induces mitochondria-dependent but apoptosome-independent apoptosis in cancer cells. Resv inhibits cancer cell growth and proliferation, mitochondrial membrane potential, and ROS production. In contrast, Resv induces dose and time dependent caspase activation, poly (ADP-ribose) polymerase (PARP) cleavage, and thus caspase-dependent apoptosis. Mechanistic analysis reveals that recruitment and oligomerization of Bax on the mitochondria is associated with release of cytochrome c from the mitochondria. Surprisingly, the released cytochrome c fails to induce apoptosome assembly as evidenced by the lack of Apaf-1 oligomerization upon Resv treatment. Interestingly, Apaf-1 was up regulated both at transcriptional and protein levels upon Resv treatment suggesting that the levels of Apaf-1 was not a limiting factor for apoptosome assembly. Subcellular analysis reveals that catalytically active caspase-9 was mostly associated with the mitochondria in Resv-induced apoptotic cells. Reconstitution experiment using recombinant proteins and purified cytosols demonstrates that Resv inhibits apoptosome functions in a dose dependent manner. MALDI-TOF analysis indicates that inhibition of apoptosome formation was not mediated by sequestration of cytochrome c as Resv fails to interact with cytochrome c. Furthermore, Resv also induces autophagy as evidenced by the increase in the levels of LC3-II upon Resv treatment. Inhibition of autophagy either by autophagy inhibitors or by silencing of autophagy related genes (ATG-5 or Beclin-1) further increase caspase activation. Gel filtration and subcellular fractionation analyses demonstrate that caspase-independent apoptosis does not seem to be involved as AIF and endonuclease G was not released from the mitochondrial compartment upon Resv treatment. These findings indicate that Resv targets mitochondria to induce caspase-9 mediated cancer cell death and that induction of autophagy during apoptosis is an adaptive response. Therefore, inhibition of autophagy along with induction of apoptosis could represent a novel strategy for cancer therapy. Citation Format: {Authors}. {Abstract title} [abstract]. In: Proceedings of the 101st Annual Meeting of the American Association for Cancer Research; 2010 Apr 17-21; Washington, DC. Philadelphia (PA): AACR; Cancer Res 2010;70(8 Suppl):Abstract nr LB-47.