The Bcl-2 Homology Domain 3 Mimetic Gossypol Induces Both Beclin 1-dependent and Beclin 1-independent Cytoprotective Autophagy in Cancer Cells

Abstract

Gossypol, a natural Bcl-2 homology domain 3 mimetic compound isolated from cottonseeds, is currently being evaluated in clinical trials. Here, we provide evidence that gossypol induces autophagy followed by apoptotic cell death in both the MCF-7 human breast adenocarcinoma and HeLa cell lines. We first show that knockdown of the Bcl-2 homology domain 3-only protein Beclin 1 reduces gossypol-induced autophagy in MCF-7 cells, but not in HeLa cells. Gossypol inhibits the interaction between Beclin 1 and Bcl-2 (B-cell leukemia/lymphoma 2), antagonizes the inhibition of autophagy by Bcl-2, and hence stimulates autophagy. We then show that knockdown of Vps34 reduces gossypol-induced autophagy in both cell lines, and consistent with this, the phosphatidylinositol 3-phosphate-binding protein WIPI-1 is recruited to autophagosomal membranes. Further, Atg5 knockdown also reduces gossypol-mediated autophagy. We conclude that gossypol induces autophagy in both a canonical and a noncanonical manner. Notably, we found that gossypol-mediated apoptotic cell death was potentiated by treatment with the autophagy inhibitor wortmannin or with small interfering RNA against essential autophagy genes (Vps34, Beclin 1, and Atg5). Our findings support the notion that gossypol-induced autophagy is cytoprotective and not part of the cell death process induced by this compound.The recent development of small molecule antagonists of prosurvival Bcl-2 family proteins looks promising for the future treatment of cancer. These novel compounds, also known as BH3 3 mimetics, bind to prosurvival Bcl-2 proteins and neutralize them. Gossypol is a natural polyphenol compound isolated from cottonseeds. Various levels of anticancer activity of gossypol have been observed against many different cancer cell lines both in vitro and in vivo (1-3). Evidence is accumulating that gossypol and its derivatives act as BH3 mimetics, killing multiple tumor cell lines, at least in part by activating the Bcl-2-regulated apoptotic pathway (4 -7). Gossypol suppresses both telomerase activity and NF-B activity in human leukemia cells (8, 9). However, the precise mechanisms responsible for the inhibition of cell growth and the stimulation of cell death induced by gossypol are poorly understood. We demonstrate that gossypol potently induces caspase-dependent apoptosis in the absence of Bak and Bax (Bcl-2-associated X protein) by converting Bcl-2 from an inhibitor to an activator of apoptosis (4).Autophagy has only recently become an important area in cancer research and is emerging as a key regulator of death pathways (10 -13). Autophagy is a genetically programmed, evolutionarily conserved process that degrades long-lived cellular proteins and organelles. Autophagy is induced by the formation of the initial membrane nucleation that requires a kinase complex including Beclin 1, a BH3-only protein, and hVps34 to generate the phospholipid PI3P. WIPI-1 (WD repeat domain phosphoinositide-interacting protein 1) binds to PI3P and was found to localize to isolation membranes (14...

Mechanisms associated with resistance to tamoxifen in estrogen receptor-positive breast cancer (Review)

“…In MCF-7 cells, knockdown of Vps34 by RNA interference reduced cytoprotective autophagy mediated by BH3 domain and potentiated apoptosis induction (144). Lastly, Vps34 possesses a tumor suppressor function in MCF-7 mouse xenograft tumors mediated through distinct Beclin-1 binding and enhancement of starvation-induced autophagy (145).…”

The autophagic paradox in cancer therapy

“…Concurrent inhibition of Bcl-2 by ABT-737 and induction of autophagy by rapamycin, a mTOR inhibitor, also significantly enhance the therapeutic effect of ionizing radiation in non-small cell lung tumor xenograft model (Kim et al, 2009b). During pro-survival autophagy, inhibition of Bcl-2 by the natural BH3 mimetic gossypol has also been shown to sensitize cancer cells to apoptosis following suppression of autophagy by wortmannin or Vps34, Atg5 or Beclin 1 short hairpin RNAs (Gao et al, 2010). It is therefore suggested that Bcl-2, in some circumstances, may mask the functional consequences of autophagy, either cell death or survival, with or without the effect on the formation of autophagosomes.…”

“…Impairment of autophagy by dominant-negative Vps34, the class III PI3K, also induces apoptotic cell death in neuroblastoma cells expressing a C98X vasopressin mutant but not in the wild-type vasopressin-expressing counterpart (Castino et al, 2008). Moreover, the pan-Bcl-2 inhibitor (-)-gossypol paradoxically induces cytoprotective autophagy in MCF-7 breast cancer cells but autophagic cell death in glioma cells, in which the effects on cell viability can be blocked by RNA interference against Beclin 1 and Atg5 in both cases (Gao et al, 2010;Voss et al, 2010). The molecular mechanism underlying these disparities is presently unknown, but it is speculated that these cells may differentially express some proteins with cell deathor cell survival-specific function in autophagy.…”

Role of Human WIPIs in Macroautophagy

“…Macroautophagy can also be induced by non-canonical entries, e.g. independent of Beclin 1 [20,21,22]. …”