A natural BH3 mimetic induces autophagy in apoptosis-resistant prostate cancer via modulating Bcl-2-Beclin1 interaction at endoplasmic reticulum - PubMed (original) (raw)

A natural BH3 mimetic induces autophagy in apoptosis-resistant prostate cancer via modulating Bcl-2-Beclin1 interaction at endoplasmic reticulum

J Lian et al. Cell Death Differ. 2011 Jan.

Abstract

A natural BH3-mimetic, small-molecule inhibitor of Bcl-2, (-)-gossypol, shows promise in ongoing phase II and III clinical trials for human prostate cancer. In this study we show that (-)-gossypol preferentially induces autophagy in androgen-independent (AI) prostate cancer cells that have high levels of Bcl-2 and are resistant to apoptosis, both in vitro and in vivo, but not in androgen-dependent (AD) cells with low Bcl-2 and sensitive to apoptosis. The Bcl-2 inhibitor induces autophagy through blocking Bcl-2-Beclin1 interaction, together with downregulating Bcl-2, upregulating Beclin1, and activating the autophagic pathway. The (-)-gossypol-induced autophagy is dependent on Beclin1 and Atg5. Our results show for the first time that (-)-gossypol can also interrupt the interactions between Beclin1 and Bcl-2/Bcl-xL at endoplasmic reticulum, thus releasing the BH3-only pro-autophagic protein Beclin1, which in turn triggers the autophagic cascade. Oral administration of (-)-gossypol significantly inhibited the growth of AI prostate cancer xenografts, representing a promising new regimen for the treatment of human hormone-refractory prostate cancer with Bcl-2 overexpression. Our data provide new insights into the mode of cell death induced by Bcl-2 inhibitors, which will facilitate the rational design of clinical trials by selecting patients who are most likely to benefit from the Bcl-2-targeted molecular therapy.

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Figures

Figure 1

Figure 1

(−)-Gossypol preferentially induces apoptosis in human prostate cancer cells with low Bcl-2, but is equally potent in inducing non-apoptotic cell death in cells with high levels of Bcl-2. (a) Western blot analysis of the protein levels of Bcl-2, Bcl-xL, Mcl-1, and Beclin1 in prostate cancer cell lines and normal prostate epithelial cells (PrECs); (b) mRNA levels of Bcl-2 and Bcl-xL in these cells by qRT-PCR. (c) (−)-Gossypol dose-dependently induces cells death in prostate cancer cells regardless of their Bcl-2 levels. Cells (1 × 104) were seeded in a 12-well plate overnight, and then treated with different doses of (−)-gossypol. After 24 h, they were trypsinized and counted after Trypan blue staining. Data are presented as percentage of dead cells. Results are mean±S.D. of three independent experiments. (d) MTT-based cytotoxicity assay of (−)-gossypol in prostate cancer cells. Cells were seeded in 96-well plates and treated in triplicates. (e) (−)-Gossypol-induced apoptosis in prostate cancer cells as assayed by sub-G1 analysis. After being treated with (−)-gossypol for 24 h, the cells were fixed by ethanol, stained with PI, and analyzed by flow cytometry. (f) (−)-Gossypol-induced caspase-3 activation in prostate cancer cell lines. Cells (1 × 105) were seeded in a 12-well plate overnight and then treated with (−)-gossypol. After 24 h, caspase-3 activity was measured. (g) PARP cleavage in prostate cancer cells treated with 10 _μ_M (−)-gossypol for 24 h. (h) After overnight culture, cells were treated with DMSO, 10 _μ_M (−)-gossypol, or (−)-gossypol ((−)-G) combined with Z-VAD (10 _μ_M) for 24 h. Cells were then trypsinized and counted after Trypan blue staining. Results are means±S.D. of three independent experiments. **P<0.01 between (−)-G and (−)-G+Z-VAD in indicated cell lines by two-way ANOVA

Figure 2

Figure 2

(−)-Gossypol preferentially induces autophagy in apoptosis-resistant prostate cancer cells with high levels of Bcl-2 but not in cells with low Bcl-2. (a) (−)-Gossypol-induced autophagy in prostate cancer cells as revealed by LC3-II conversion in western blot analysis. Cells were treated with DMSO or 10 _μ_M (−)-gossypol for 24 h, and then lysed for western blot of LC3. 3-MA 5 mM and rapamycin 0.5 _μ_M were used as an inhibitor and an inducer of autophagy, respectively. (b, c) Dose response (b) and time course (c) of (−)-gossypol-induced autophagy in PC-3 and CL-1 cells. (d) Representative electron microscopic images showing autophagic vacuoles with content (black arrows) after (−)-gossypol treatment. The percentage of cells with autophagic vacuoles was quantified in 50 cells each group

Figure 3

Figure 3

(−)-Gossypol preferentially induces autophagy in apoptosis-resistant prostate cancer cells as revealed by LC3-GFP puncta formation. (a) (−)-Gossypol-induced autophagy in prostate cancer cells as analyzed by LC3-GFP. Cells were transfected with LC3-GFP plasmid, treated with DMSO or 10 _μ_M (−)-gossypol for 24 h, and then analyzed under a fluorescent microscope. The yellow arrows indicate the punctate pattern of LC3-GFP in autophagic cells. Treatment with 0.5 _μ_M rapamycin was used as a positive control for autophagy induction. (b) Quantification of data from (a), expressed as percentage of cells with punctate LC3-GFP (50 green fluorescent cells in one field, _n_=5). (c, d) Effects of Atg5 or Beclin1 downregulation or 3-MA on the (−)-gossypol-induced cell death in CL-1(c) and LNCaP cells (d). Cells were transiently transfected either with a control siRNA or siRNA specific to Atg5 or Beclin1 or pre-treated with 5 mM 3-MA, and then treated with DMSO or (−)-gossypol for 24 h. Viability was assessed by Trypan blue staining. Results are means±S.D. of three independent experiments. **P<0.01 or *P<0.05 by two-way ANOVA compared to (−)-G treatment of con-siRNA cells where indicated in panel (c) and (d), respectively

Figure 4

Figure 4

(−)-Gossypol modulates Bcl-2–Beclin1 interaction at the ER. Co-immunoprecipitation (co-IP) pull-down assay shows that (−)-gossypol specifically disrupts Bcl-2–Beclin1 interaction. CL-1 cells were treated with DMSO or 10 _μ_M (−)-gossypol for 6 h at 37°C and subjected to subcellular fractionation and IP with the indicated antibodies

Figure 5

Figure 5

Effects of modulating Bcl-2 or Beclin1 protein levels on (−)-gossypol-induced autophagy and cell death in PC-3 and CL-1 cells. (a–f) Cells were transiently transfected with either control shRNA/siRNA or shRNA/siRNA specific to Bcl-2 (a)/Beclin1 (e), or expression vectors for Bcl-2 (b) or Beclin1 (f). At 24 h after transfection, cells were treated with DMSO or (−)-gossypol for 24 h, and then subjected to either immunoblot analysis for Bcl-2, Beclin1, and LC3 or Trypan blue staining (b, d). *P<0.05 between the indicated groups by two-way ANOVA in panels (b) and (d)

Figure 6

Figure 6

(−)-Gossypol regulates autophagy pathway-associated genes in prostate cancer cells. (a) Human Autophagy PCR Array analysis of the autophagy-associated gene expression levels in CL-1 cells treated with DMSO or 10 _μ_M (−)-gossypol. (_n_=2). (b) qRT-PCR validation of Bcl-2 and Beclin1 expression. (c) Western blot confirmation of the protein-level changes of the most regulated genes identified by PCR Array. (d) PC-3 and CL-1 cells were transiently transfected with either control siRNA or SmartPool Atg5-siRNAs for 24 h, and then treated with DMSO or (−)-gossypol for 24 h. The cells were lysed for immunoblot analysis of Atg5-12 and LC3. (e) Percentage of cells with punctate GFP-LC3 in CL-1 cells transiently transfected with control siRNA or Atg5-siRNA treated with DMSO (control) or 10 _μ_M (−)-gossypol for 24 h. **P<0.01 between the indicated groups by two-way ANOVA

Figure 7

Figure 7

Oral administration of (−)-gossypol inhibited CL-1 and PC-3 xenograft growth and was associated with increased LC3-II conversion in the tumors. (a) (−)-gossypol potently inhibited the CL-1 xenograft tumor growth in nude mice as a single-agent oral therapy. CL-1 cells (2 × 106) were s.c. injected into the flanks on both sides of each mouse. When the tumors reached 100 mm3, the mice were randomized into 5–8 mice per group. (−)-Gossypol was administrated p.o. through oral gavage, daily at a 20 mg/kg dose. The data shown are average tumor size (means±S.E.M., _n_=10). **P<0.01, versus vehicle control, two-way ANOVA (_n_=10). (b) Representative electron micrograph image showing autophagic vacuoles with content after (−)-gossypol treatment in an in vivo CL-1 xenograft model. The percentage of cells with autophagic vacuoles was quantified in 50 cells per group. (c) (−)-Gossypol-induced autophagy in CL-1 xenograft tumors in vivo. CL-1 xenograft tumor tissue lysates from the vehicle control group or (−)-gossypol treatment group were immunoblotted for Beclin1, LC3, and Bcl-2 at the indicated time points. (d) Modulation of the autophagy-associated gene expression in tumor tissues after treatment with DMSO or (−)-gossypol for 2 weeks. Gene expression was detected using Human Autophagy PCR Array and the data are shown as relative mRNA levels (_n_=2). (e) (−)-Gossypol potently inhibited the PC-3 xenograft tumor growth in nude mice as a single-agent oral therapy. Study was conducted as in (a) and tumor size data were collected at 5 weeks. (f) (−)-Gossypol-induced autophagy in vivo in PC-3 xenograft model in nude mice. Immunoblotting for Beclin1, LC3, and Bcl-2 using lysates from PC-3 xenograft tumor tissues treated with vehicle or (−)-gossypol for 3 weeks. *P<0.05 by two-way ANOVA

Figure 8

Figure 8

Model for the mechanisms of action of (−)-gossypol, indicating that the mode of cell death induced by (−)-gossypol is cellular context dependent. (a) In androgen-dependent (AD) prostate cancer cells that have low levels of Bcl-2/xL and are sensitive to apoptosis, for example, LNCaP, (−)-gossypol potently binds to Bcl-2/xL at mitochondria, releasing Bax/Bak and inducing apoptotic cell death. (b) In androgen-independent (AI) prostate cancer cells that have high levels of Bcl-2/xL and are resistant to apoptosis, for example, CL-1 and PC-3, (−)-gossypol potently binds to Bcl-2/xL and releases Beclin1 at ER, and thus preferentially induces autophagic cell death

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