Resveratrol Induces p53-independent, X-linked Inhibitor of Apoptosis Protein (XIAP)-mediated Bax Protein Oligomerization on Mitochondria to Initiate Cytochrome c Release and Caspase Activation

Abstract

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...

Curcumin induces Apaf-1-dependent, p21-mediated caspase activation and apoptosis

“…For example, curcumin has been shown to induce apoptosis in a p53-dependent manner in multiple cancer cell types, 11,14,42 and via a p53-independent mechanism in others. 9,13,43,44 We recently demonstrated that the pro-apoptotic function of p53 is inhibited in multiple types of cancer cells 27 due to failure of p53 to activate Bax/Bak and inhibit the anti-apoptotic functions of Bcl-2/Bcl-x L . 27 Findings in this study provide further evidence that p53 is not a critical player in curcumin-induced cytochrome c release and apoptosis.…”

“…9,13,43,44 We recently demonstrated that the pro-apoptotic function of p53 is inhibited in multiple types of cancer cells 27 due to failure of p53 to activate Bax/Bak and inhibit the anti-apoptotic functions of Bcl-2/Bcl-x L . 27 Findings in this study provide further evidence that p53 is not a critical player in curcumin-induced cytochrome c release and apoptosis. p21 protects cells by upregulating the Nrf2 signaling pathway, 45 and is also implicated in regulation of apoptosis.…”

“…As shown in Figure 6B, in untreated cells, cytochrome c showed tubular perinuclear labeling, consistent with mitochondrial localization. 26,27 Figure 2. Curcumin induces caspase-dependent apoptosis.…”

“…Cells were then lysed in protein sample buffer and subjected to protein gel blotting. 27,55 Statistical analysis. Results are presented as mean ± standard deviation (SD) of data from at least three independent experiments.…”

Bim, a Proapoptotic Protein, Up-regulated via Transcription Factor E2F1-dependent Mechanism, Functions as a Prosurvival Molecule in Cancer

“…Whole Cell Lysate Preparation and Western Blotting-Protein extraction and Western blotting were carried out as described previously (10,30). The blots were stripped and reprobed for the indicated proteins in the respective figures.…”