Following cytochrome c release, autophagy is inhibited during chemotherapy-induced apoptosis by caspase 8-mediated cleavage of Beclin 1 - PubMed (original) (raw)

Following cytochrome c release, autophagy is inhibited during chemotherapy-induced apoptosis by caspase 8-mediated cleavage of Beclin 1

Hua Li et al. Cancer Res. 2011.

Abstract

Autophagy is an evolutionarily conserved stress response mechanism that often occurs in apoptosis-defective cancer cells and can protect against cell death. In this study, we investigated how apoptosis and autophagy affect each other in cancer cells in response to chemotherapeutic treatment. We found that specific ablation of the proapoptotic function of cytochrome c, a key regulator of mitochondria-mediated apoptosis, enhanced autophagy following chemotherapeutic treatment. Induction of autophagy required Beclin 1 and was associated with blockage of Beclin 1 cleavage by caspase 8 at two sites. To investigate the role of Beclin 1 cleavage in the suppression of autophagy and cell survival, a caspase-resistant mutant of Beclin 1 was knocked into HCT116 colon cancer cells. Beclin 1 mutant knockin resulted in markedly increased autophagy and improved long-term cell survival after chemotherapeutic treatment but without affecting apoptosis and caspase activation. Furthermore, Beclin 1 mutant tumors were significantly less responsive to chemotherapeutic treatment than were wild-type tumors. These results show that chemotherapy-induced apoptosis inhibits autophagy at the execution stage subsequent to cytochrome c release through caspase 8-mediated cleavage of Beclin 1. If apoptosis fails to execute, autophagy is unleashed due to lack of Beclin 1 cleavage by caspases and can contribute to cancer cell survival and therapeutic resistance. Therefore, Beclin 1 may be a useful target for inhibiting autophagy to sensitize chemotherapy.

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Figures

Fig. 1. Induction of autophagy in cytochrome _c_-mutant knock-in cells

(A) WT and cytochrome _c_-mutant-knock-in (_Cyt c_-KI) HCT116 cells were treated with 500 nM camptothecin (CPT) for 48 hr. Left, apoptosis was analyzed by counting cells with condensed and fragmented nuclei following nuclear staining with Hoechst 33258. **p<0.01. Right, Western blot analysis of caspases 3, 8, and 9. Arrowheads indicate caspase cleavage fragments. (B) Western blot analysis of LC3II accumulation and p62 degradation in WT and _Cyt c_-KI HCT116 cells treated with CPT for 24 hr. (C) Confocal microscopic analysis of WT and _Cyt c_-KI HCT116 cells transfected with GFP-LC3, and then treated with 500 nM CPT for 24 hr. Left, representative confocal images. Scale bar: 5 µm. Right, quantification of GFP-LC3 puncta signals. (D) Transmission electron microscopic analysis of WT and _Cyt c_-KI HCT116 cells treated with 500 nM CPT for 24 hr. Arrowhead indicates an autophagosome with double membrane structure. Arrow indicates an autolysosome with a degraded organelle.

Fig. 2. Inhibition of caspase-mediated Beclin 1 cleavage in _Cyt c_-KI cells

(A) WT and _Cyt c_-KI HCT116 cells were transfected with Beclin 1 or control siRNA, followed by GFP-LC3 transfection, CPT (500 nM) treatment for 24 hr, and then confocal microscopic analysis. Left, Western blot analysis of Beclin 1 at 48 hr after siRNA transfection. * indicates non-specific bands. Right, quantification of GFP-LC3 puncta signals. **p<0.01. (B) Colony formation assay was used to compare the long-term survival of WT and _Cyt c_-KI HCT116 cells transfected with siRNA as in (A), and then treated with 500 nM CPT for 24 hr. Results are presented as ratios of colony numbers formed by treated cells vs. those of untreated cells plated at 1/100 dilutions relative to treated cells. ** p<0.01. (C) Western blot analysis of Beclin 1 in WT and _Cyt c_-KI HCT116 cells treated with 500 nM CPT for 24 hr. FL: full-length Beclin 1. Arrowheads indicate Beclin 1 cleavage fragments. (D) Left, Western blot analysis of Beclin 1 in WT and _BAX_-knockout (_BAX_-KO) HCT116 cells treated with 500 nM CPT for 24 hr. Right, Western blot analysis of Beclin 1 in WT HCT116 cells treated with 500 nM CPT for 24 hr, in the presence or absence of the pan-caspase inhibitor zVAD-fmk at 20 µM.

Fig. 3. Cleavage of Beclin 1 by caspase 8 at two sites in CPT-induced apoptosis

(A) In vitro translated WT and mutant Beclin 1 proteins were incubated with active caspase 3, 8, or 9 at 37°C for 1 hr, and then analyzed for Beclin 1 by Western blotting. Arrowhead indicates the caspase cleavage fragment. (B) Parental and stable _caspase 8_-knockdown (_Casp 8_-KD) HCT116 cells were treated with 500 nM CPT for 24 hr. Left, Western blot analysis of Beclin 1 expression. Right, Western blot analysis of LC3II accumulation and p62 degradation. (C) A schematic representation and amino acid sequences of Beclin 1 mutants analyzed. N-Beclin 1 and C-Beclin 1 correspond to two Beclin 1 fragments generated by caspase cleavage. (D) Left, WT HCT116 cells were transfected with V5-tagged WT or DM (double mutant; D133A/D146A) Beclin 1, and then treated with 500 nM CPT for 24 hr. Transfected Beclin 1 was analyzed by V5 Western blotting. Right, HCT116 cells were transfected with WT or DM Beclin 1, treated with 500 nM CPT for 24 hr, and then analyzed for LC3II accumulation and p62 degradation by Western blotting.

Fig. 4. Knock-in of Beclin 1 double mutant in HCT116 cells

(A) A schematic representation of the Beclin 1 genomic locus and knock-in vector. P1 and P2 represent PCR primers for identifying knock-in clones. (B) DNA sequences of the targeted genomic region in WT and Beclin 1 knock-in (_Beclin 1_-KI) HCT116 cells, with WT and corresponding mutant sequences underlined. (C) Western blot analysis of Beclin 1 expression in WT and _Beclin 1_-KI HCT116 cells. (D) Western blot analysis of Beclin 1 in WT and _Beclin 1_-KI HCT116 cells treated with 500 nM CPT for 24 hr. Arrowheads indicate Beclin 1 cleavage fragments.

Fig. 5. Induced autophagy, unchanged apoptosis, and improved survival of _Beclin 1_-KI cells

(A) WT and _Beclin 1_-KI HCT116 cells were treated with 500 nM CPT for 24 hr. Left, Analysis of LC3II accumulation and p62 degradation by Western blotting. Right, Analysis of autophagy by transmission electron microscopy. Arrowheads indicate autophagosomes with double membrane structure. Arrows indicate autolysosomes with degraded organelles. (B) Confocal microscopic analysis of WT and _Beclin 1_-KI HCT116 cells transfected with GFP-LC3, and then treated with 500 nM CPT for 24 hr. Left, representative confocal images. Scale bar: 5 µm. Right, quantification of GFP-LC3 puncta signals. **p<0.01. (C) WT and _Beclin 1_-KI HCT116 cells were treated with the indicated apoptotic stimuli for 48 hr. Left, analysis of apoptosis by nuclear staining. Right, Western blot analysis of caspases 3, 8, and 9. CPT, 500 nM camptothecin; SUL, 120 µM sulindac sulfide; STS, 100 nM staurosporine; Ad-PUMA, infection with 10 MOI of adenovirus expressing PUMA. (D) Colony formation assay was used to compare the long-term survival of WT and _Beclin 1_-KI HCT116 cells treated with 500 nM CPT for 24 hr. Results are presented as ratios of colony numbers formed by treated cells vs. those of untreated cells plated at 1/100 dilutions relative to treated cells. *p<0.05; **p<0.01.

Fig. 6. In vivo therapeutic resistance of _Beclin 1_-KI tumors

(A) Xenograft tumors established from WT and _Beclin 1_-KI HCT116 cells were treated with 40 mg/kg of irinotecan (CPT-11) by i.p. injection on days 5, 9, 13, 17, and 21, as indicated by arrows. Therapeutic effects were measured by calculating percentage decrease in tumor size of treated tumors compared to untreated tumors. **p<0.01. (B) Upper, Western blot analysis of p62 degradation in WT and _Beclin 1_-KI HCT116 tumors with or without CPT-11 treatment. Lower, quantification of p62 level reduction in CPT-11-treated tumors relative to untreated tumors using Image J software. (C) Apoptosis in WT and _Beclin 1_-KI HCT116 tumors with or without CPT-11 treatment was analyzed by TUNEL staining. Left, representative TUNEL staining pictures. Scale bar: 200 µm. Arrowheads indicate example TUNEL-positive cells. Right, quantification of TUNEL-positive cells, with 300 cells counted for each sample. Values in (A) and (C) were means ± SD (n = 6 in each group).

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Following cytochrome c release, autophagy is inhibited during chemotherapy-induced apoptosis by caspase 8-mediated cleavage of Beclin 1 - PubMed (original) (raw)