The Bcl-2-Beclin 1 interaction in (-)-gossypol-induced autophagy versus apoptosis in prostate cancer cells

Abstract

Bcl-2 is a key dual regulator of autophagy and apoptosis, but how the level of Bcl-2 influences the cellular decision between autophagy and apoptosis is unclear. The natural BH3-mimetic (-)-gossypol preferentially induces autophagy in androgen-independent (AI) prostate cancer cells that have high levels of Bcl-2 and are resistant to apoptosis, whereas apoptosis is preferentially induced in androgen-dependent or -independent cells with low Bcl-2. (-)-Gossypol induces autophagy via blocking Bcl-2-Beclin 1 interaction at the endoplasmic reticulum (ER), together with downregulating Bcl-2, upregulating Beclin 1 and activating the autophagic pathway. Furthermore, (-)-gossypol-induced autophagy is Beclin 1- and Atg5-dependent. These results provide new insights into the mode of cell death induced by Bcl-2 inhibitors, which could facilitate the rational design of clinical trials by selecting patients who are most likely to benefit from the Bcl-2-targeted molecular therapy.

Apoptosis and autophagy are two prominent mechanisms of cell selfdestruction. Apoptosis, also called type I programmed cell death, is defined as suicidal cell death and can be determined by a particular morphology involving nuclear chromatin condensation. Autophagy, or type II programmed cell death, is characterized by the sequestration of cytoplasmic material in vacuoles for bulk degradation by lysosomal enzymes. Several stimuli, such as ionizing radiation, ER stress and chemotherapeutic drugs, can induce either apoptosis or autophagy. Whereas a mixed phenotype of autophagy and apoptosis can sometimes be detected in response to these common stimuli, in many other instances, autophagy and apoptosis develop in a mutually exclusive manner, perhaps as a result of variable thresholds for both processes, or as a result of a cellular ‘decision’ between the two responses. Anti-apoptotic Bcl-2 family proteins Bcl-2, Bcl-x_L and Mcl-1 are well-studied inhibitors of cell death. Their cytoprotective function stems from their ability to antagonize Bax and Bak, block MOMP and thus prevent apoptosis. Recently, Bcl-2 and Bcl-x_L have also been shown to inhibit autophagy by antagonizing the BH3-only protein Beclin 1, an essential inducer of autophagy. Hence, Bcl-2 and Bcl-x_L can inhibit both apoptosis and autophagy by binding the BH-3 domains of Bax/Bak/Bad or Beclin 1, respectively.

Our recent studies with human prostate cancer cells show that the level of Bcl-2 is a critical determinant for cells to be driven toward apoptosis or autophagy. Upon BH3-mimetic (-)-gossypol treatment, autophagy is preferentially induced in apoptosis-resistant androgen-independent (AI) prostate cancer cells with high levels of Bcl-2, whereas apoptosis is preferentially induced in androgen-dependent or -independent cells with low Bcl-2. We find that (-)-gossypol induces similar levels of total cell death in prostate cancer cell lines regardless of their expression of the Bcl-2 family of proteins, but in cells with low Bcl-2, more than 80 percent of cells die via apoptotic cell death. Conversely, in cells with high Bcl-2, more than 60 percent of cells die by autophagic cell death. This death can be blocked by the apoptosis inhibitor Z-VAD in low Bcl-2 cells and the autophagy inhibitor 3-MA or Atg5/Beclin 1 siRNAs in high Bcl-2 cells. Thus, the level of Bcl-2 determines which type of cell death will be dominant in prostate cancer cells after treatment with the Bcl-2 inhibitor (-)-gossypol.

We also find that (-)-gossypol induces autophagy via blocking Bcl-2-Beclin 1 interaction at the ER, together with downregulating Bcl-2, upregulating Beclin 1 and activating the autophagic pathway. Our studies indicate that the complex of Bcl-2-Beclin 1 on ER membranes is interrupted by (-)-gossypol prior to the complex of Bcl-2-Bak/Bax on mitochondria when Bcl-2 is overexpressed. After treatment with (-)-gossypol, Beclin 1 is liberated from Bcl-2 in concert with transcriptional upregulation. Together, these events trigger the autophagic cascade. Silencing of endogenous Beclin 1 by RNA interference or overexpressing Bcl-2 decreases the level of (-)-gossypol-induced autophagy, possibly due to the stoichiometric abundance of Bcl-2 sequestering Beclin 1 and inhibiting the induction of autophagy. Autophagic cell death via (-)-gossypol is both Atg5- and Beclin 1-dependent both in vitro and in vivo.

Taken together, our data show that the (-)-gossypol-induced mode of cell death is cellular context-dependent (Fig. 1). When the expression levels of Bcl-2 are low, such as in LNCaP, DU-145 and C4-2B cells, (-)-gossypol preferentially induces apoptosis. On the other hand, when the Bcl-2 protein is present at elevated levels, such as in AI prostate cancer CL-1 and PC-3 cells and xenografts, (-)-gossypol preferentially induces autophagic cell death. The finding that inducing autophagy by Bcl-2 inhibition is a potent means to kill certain tumors will direct future study in other systems where Bcl-2 overexpression drives treatment resistance. Furthermore, clinical trial selection criteria and efficacy studies should be designed carefully with these data in mind since similar results could be expected for other BH-3 mimetic Bcl-2 inhibitors.

Figure 1.

Proposed working model of the mechanisms of action of (-)-gossypol, indicating that (-)-gossypol-induced autophagy versus apoptosis is cellular context-dependent. (A) In apoptosis-sensitive prostate cancer cells such as LNCaP, which have low levels of Bcl-2 and Bcl-x_L and are sensitive to apoptosis, (-)-gossypol potently binds to Bcl-2 and Bcl-x_L at mitochondria, releasing Bax and Bak and inducing apoptotic cell death. (B) In apoptosis-resistant prostate cancer cells such as CL-1 and PC-3, which have high levels of Bcl-2 and Bcl-x_L and are resistant to apoptosis, (-)-gossypol potently binds to Bcl-2 and Bcl-xL and releases Beclin 1 at the ER, thus preferentially inducing autophagic cell death. This figure has been reproduced from Figure 8 in Lian J, et al. Cell Death and Differentiation, advanced publication on-line. Nature Publishing Group 2010; 17.

Punctum to: Lian J, Wu X, He F, Karnak D, Tang W, Meng Y, Xiang D, Ji M, Lawrence TS, Xu L. A natural BH3 mimetic induces autophagy in apoptosis-resistant prostate cancer via modulating Bcl-2-Beclin1 interaction at endoplasmic reticulum. Cell Death Differ. 2010 doi: 10.1038/cdd.2010.74. In press.