Proteasome inhibition can induce an autophagy-dependent apical activation of caspase-8

Abstract

Antiapoptotic Bcl-2 family proteins are often highly expressed in chemotherapy-resistant cancers and impair mitochondrial outer membrane permeabilisation (MOMP), an important requirement for caspase activation via the intrinsic apoptosis pathway. Interestingly, although Bcl-2 overexpression in HeLa cervical cancer cells abrogated caspase processing in response to intrinsic apoptosis induction by staurosporine, tunicamycin or etoposide, residual caspase processing was observed following proteasome inhibition by bortezomib ([(1R)-3-methyl-1-({(2S)-3-phenyl-2-[(pyrazin-2-ylcarbonyl)amino]propanoyl}amino)butyl]boronic acid), epoxomicin (N-acetyl-N-methyl-lisoleucyl-L-isoleucyl-N-[(1S)-3-methyl-1-[[(2R)-2-methyloxiranyl]carbonyl]butyl]-L-threoninamide) or MG-132 (N-(benzyloxycarbonyl)leucinylleucinylleucinal). Similar responses were found in Bcl-2-overexpressing H460 NSCLC cells and Bax/Bak-deficient mouse embyronic fibroblasts. Mild caspase processing resulted in low DEVDase activities, which were MOMP independent and persisted for long periods without evoking immediate cell death. Surprisingly, depletion of caspase-3 and experiments in caspase-7-depleted MCF-7-Bcl-2 cells indicated that the DEVDase activity did not originate from effector caspases. Instead, Fas-associated death domain (FADD)-dependent caspase-8 activation was the major contributor to the slow, incomplete substrate cleavage. Caspase-8 activation was independent of death ligands, but required the induction of autophagy and the presence of Atg5. Depletion of XIAP or addition of XIAP-antagonising peptides resulted in a switch towards efficient apoptosis execution, suggesting that the requirement for MOMP was bypassed by activating the caspase-8/caspase-3 axis. Combination treatments of proteasome inhibitors and XIAP antagonists therefore represent a promising strategy to eliminate highly resistant cancer cells, which overexpress antiapoptotic Bcl-2 family members.

Figure 1

Proteasome inhibition promotes residual processing of procaspase-3 in cells expressing high levels of Bcl-2. (a) Proteasome inhibition induces residual procaspase-3 processing in Bcl-2-overexpressing HeLa cells. Parental and Bcl-2-overexpressing HeLa cells were compared by western blotting for processing of procaspase-3 in response to 1 μM staurosporine (STS) (8 h), 100 nM bortezomib ([(1R)-3-methyl-1-({(2S)-3-phenyl-2-[(pyrazin-2-ylcarbonyl)amino]propanoyl}amino)butyl]boronic acid) (32 h), 3 μM tunicamycin (32 h) or 10 μM etoposide (32 h). Arrows indicate p20 and p19 caspase-3 subunits found in HeLa-Bcl-2 cells owing to residual procaspase-3 processing. Fully mature p17 subunits could not be detected in HeLa-Bcl-2 cells. β-Actin served as loading control. (b–d) Procaspase-3 processing in response to 100 nM bortezomib, 50 nM epoxomicin (N-acetyl-N-methyl-lisoleucyl--isoleucyl-N-[(1S)-3-methyl-1-[[(2R)-2-methyloxiranyl]carbonyl]butyl]--threoninamide) or 5 μM MG-132 (N-(benzyloxycarbonyl)leucinylleucinylleucinal) was compared between parental and Bcl-2-overexpressing HeLa cells. Although p20 and p19 subunits could be detected, fully mature caspase-3 (p17) was not detected in HeLa-Bcl-2 cells. Pan-caspase inhibitor benzyloxycarbonyl-Val-Ala-Asp(O-methyl)-fluoromethylketone (zVAD-fmk) (50 μM) was used in combination with proteasome inhibitors in additional controls. β-Actin served as loading control. (e) Procaspase-3 processing in response to 100 nM bortezomib was analysed in parental and Bcl-2-overexpressing H460 cells. Arrows indicate p20, p19 and p17 subunits. α-Tubulin served as loading control. (f) As in (e), procaspase-3 processing in response to 100 nM bortezomib was analysed in parental or Bax/Bak-deficient mouse embryonic fibroblasts. Porin served as loading control. (g–i) Cell death in response to 100 nM bortezomib was determined by propidium iodide staining. Bcl-2 overexpression or Bax/Bak deficiency significantly reduced cell death. zVAD-fmk (50 μM) was added in additional control groups. Data are shown as means±standard deviation (S.D.). Asterisks indicate significant reduction in cell death (P<0.05, Student's t-test). All experiments were preformed three times with comparable results

Figure 2

Proteasome inhibition induces low levels of DEVDase activity in Bcl-2-overexpressing cells. (a) A cyan fluorescent protein (CFP)-DEVD-yellow fluorescent protein (YFP) Förster resonance energy transfer (FRET) probe was used to measure DEVDase activity in individual cells by flow cytometry. Cleavage of the probe by DEVDases results in a loss in FRET and a concomitant increase in CFP emission. (b) Flow cytometric analysis of parental HeLa cells expressing the DEVD FRET probe. Cells were treated for the indicated times with 100 nM bortezomib ([(1R)-3-methyl-1-({(2S)-3-phenyl-2-[(pyrazin-2-ylcarbonyl)amino]propanoyl}amino)butyl]boronic acid). Benzyloxycarbonyl-Val-Ala-Asp(O-methyl)-fluoromethylketone (zVAD-fmk) co-treated controls ensured caspase specificity of the readout. For comparison, control groups treated with 1 μM staurosporine (STS) (8 h) are shown as well. Data are mean±standard deviation (S.D.) from n=3 experiments. Asterisks indicate significant increase in FRET probe cleavage above control groups (P<0.05; one-way analysis of variance (ANOVA) and subsequent Tukey's test). (c) Flow cytometric analysis of HeLa-Bcl-2 cells expressing the DEVD FRET probe. Cells were treated as in (b). Data are means±S.D. from n=3 experiments. Asterisks indicate significant increase in FRET probe cleavage above control groups (P<0.05; one-way ANOVA and subsequent Tukey's test). (d) As described in (b and c), DEVD FRET probe cleavage was analysed in wild-type and Bax/Bak-deficient mouse embryonic fibroblasts. (e) Scatter plots displaying DEVD FRET probe cleavage in parental HeLa cells. Treatment with 100 nM bortezomib resulted in clearly separable populations of cells with intact or cleaved FRET probe. Caspase inhibition by zVAD-fmk inhibited probe cleavage. (f) Scatter plots displaying DEVD FRET probe cleavage in HeLa-Bcl-2 cells. Following treatment with 100 nM bortezomib populations of cells with intact or cleaved FRET probe were inseparable, indicating slow or incomplete substrate cleavage. Caspase inhibition by zVAD-fmk inhibited probe cleavage. (g and h) Ratiometric analysis of FRET probe cleavage indicates that bortezomib-induced DEVDase activity in parental HeLa cells (g) resulted in separated peaks of intact or cleaved probe. In comparison, probe cleavage was significantly reduced in HeLa-Bcl-2 cells (h), as indicated by a right-shift and reduction in peak height at 24 h and inseparable peaks at 48 h post-addition of 100 nM bortezomib

Figure 3

Time-lapse analysis of DEVDase activation and activity induced by proteasome inhibition. (a) Parental and Bcl-2-overexpressing cells were analysed for DEVDase activation in Förster resonance energy transfer (FRET) imaging experiments. The incidence of DEVDase activation following treatment with 100 nM bortezomib ([(1R)-3-methyl-1-({(2S)-3-phenyl-2-[(pyrazin-2-ylcarbonyl)amino]propanoyl}amino)butyl]boronic acid) was determined from the total number of cells analysed (n=43 parental cells from four experiments and n=27 Bcl-2-overexpressing cells from six experiments). DEVDase activation was evaluated as an irreversible increase of the cyan and yellow fluorescent protein (CFP/YFP) ratio signal above the baseline. Bcl-2 overexpression did not affect the frequency of DEVDase activation. (b) The time from bortezomib addition until activation of DEVDases was determined for all cells measured. DEVDase activity was detected later in Bcl-2-overexpressing cells. Asterisk indicates significant difference (P<0.05, U-test). (c) The duration of DEVD probe cleavage was determined for all cells analysed. Bcl-2 overexpression prolonged the duration of probe cleavage. Asterisk indicates significant difference (P<0.05, U-test). (d) The DEVDase activity was determined as the rate of probe cleavage (change in CFP/YFP emission over time) for all cells investigated. Overexpression of Bcl-2 strongly reduced the DEVDase activity determined in single living cells. Asterisk indicates significant difference (P<0.05, U-test). (e) DEVD FRET probe cleavage in parental HeLa cells. Traces of DEVD FRET probe cleavage are shown for four representative HeLa cells following exposure to 100 nM bortezomib. FRET probe cleavage resulted in an increase in the CFP/YFP emission ratio. Onset of DEVDase activity was set to time zero. Tetramethylrhodamine-methylester (TMRM) fluorescence was plotted to indicate changes in mitochondrial membrane potentials. Mitochondrial depolarisation started shortly before DEVDase activation. Similar results were obtained from n=23 additional cells. (f) DEVD FRET probe cleavage in HeLa cells overexpressing Bcl-2. Experimental traces of DEVD FRET probe cleavage are shown for four representative HeLa-Bcl-2 cells following exposure to 100 nM bortezomib. Onset of DEVDase activity was set to time zero. FRET probe cleavage displayed in increases in the CFP/YFP emission ratio that were lower and prolonged when compared with parental HeLa cells (e). In contrast to parental HeLa cells, changes in mitochondrial membrane potentials were not detectable at the time DEVDase activation was measured. Late changes in TMRM signals resulted from cells rounding up and detaching. Similar results were obtained for n=12 additional cells. (g) DEVD FRET probe cleavage in parental HeLa cells was observed in parallel with the release of red fluorescent protein (mCherry) from the mitochondrial intermembrane space (IMS-RP). The release of IMS-RP results in a fluorescence re-distribution towards a homogeneous signal across the cell (see also Materials and Methods section). The start of IMS-RP release was therefore defined as the time at which the cellular fluorescence standard deviation (S.D.) started to drop. IMS-RP release preceded DEVDase activation. Similar results were obtained from n=5 additional cells. (h) In HeLa cells overexpressing Bcl-2, DEVDase activation resulted in slow and prolonged substrate cleavage. No release of IMS-RP could be detected, indicating that DEVDase activity was established independent of membrane permeabilisation (MOMP). Similar results were obtained for n=14 cells. (i) α-Spectrin cleavage after bortezomib treatment. Prolonged exposure to bortezomib resulted in the detection of the caspase-specific 120 kDa cleavage product and loss of full-length α-spectrin in parental cells, which was strongly diminished in HeLa cells overexpressing Bcl-2. α-Tubulin served as loading control

Figure 4

DEVDase activity in response to proteasome inhibition does not arise from caspase-3 or -7 activity. (a) Depletion of procaspase-3 by small interfering RNA (siRNA) transfection. HeLa-Bcl-2 cells were analysed for procaspase-3 expression at the indicated times following siRNA transfection. β-Actin served as loading control. A scrambled siRNA (scr) was transfected in control cells. (b) HeLa-Bcl-2 cells depleted of procaspase-3 expression were analysed for DEVD Förster resonance energy transfer (FRET) probe cleavage by flow cytometry following treatment with 100 nM bortezomib ([(1R)-3-methyl-1-({(2S)-3-phenyl-2-[(pyrazin-2-ylcarbonyl)amino]propanoyl}amino)butyl]boronic acid). siRNA transfection preceded drug addition by 24 h to provide efficient target depletion 24 h subsequent to bortezomib addition (see Supplementary Figure 8). Depletion of caspase-3 did not reduce FRET probe cleavage. Bars display changes in FRET substrate cleavage (%) above untreated controls. Data represent mean±standard deviation (S.D.) from n=9 values obtained from bootstrap resampling of independent treatment and control triplicates. Experiment was repeated with similar results. (c) Depletion of procaspase-7 by siRNA transfection in Bcl-2-overexpressing MCF-7 cells. Cells were analysed for procaspase-7 expression at the indicated times following siRNA transfection. β-Actin served as loading control. Scrambled siRNA (scr) was transfected in control cells. (d) MCF-7-Bcl-2 cells depleted of procaspase-7 and expressing the DEVD FRET probe were exposed to 100 nM bortezomib for 48 h and analysed by flow cytometry. siRNA transfection preceded drug addition by 48 h to provide efficient target depletion 24 h subsequent to bortezomib addition (see Supplementary Figure 8). A continuous spectrum of substrate cleavage, which could not be inhibited by procaspase-7 depletion, was detected. Experiment was repeated with similar results

Figure 5

DEVDase activity in response to proteasome inhibition arises from Fas-associated death domain (FADD)-dependent caspase-8 activation. (a) Time-resolved analysis of procaspase-8 processing into p43/41 and p18 subunits induced by 100 nM bortezomib ([(1R)-3-methyl-1-({(2S)-3-phenyl-2-[(pyrazin-2-ylcarbonyl)amino]propanoyl}amino)butyl]boronic acid) in parental HeLa cells. Porin served as loading control. (b) As in (a), procaspase-8 processing was investigated in HeLa cells overexpressing Bcl-2. Procaspase-8 was only residually processed (arrows) and processing occurred at later times than in parental HeLa cells. Porin served as loading control. All experiments were preformed three times with comparable results. (c) Bid processing was observed by western blotting in whole-cell extract of HeLa or HeLa Bcl-2 cells treated with 100 nM bortezomib for the indicated times. Corresponding to data on caspase-8 processing (a and b), Bid cleavage in HeLa Bcl-2 cells was delayed. Arrow and high-contrast panel indicate truncated Bid. Porin served as loading control. (d) Depletion of caspase-8 by small interfering RNA (siRNA) transfection. HeLa-Bcl-2 cells were analysed for procaspase-8 expression at the indicated times following siRNA transfection. β-Actin served as loading control. A scrambled siRNA (scr) was transfected in control cells. (e) HeLa Bcl-2 cells depleted of procaspase-8 expression were analysed for DEVD Förster resonance energy transfer (FRET) probe cleavage by flow cytometry following treatment with 100 nM bortezomib. siRNA transfection preceded drug addition by 24 h to provide efficient target depletion 24 h subsequent to bortezomib addition (see Supplementary Figure 8). Bars display changes in FRET substrate cleavage (%) above untreated controls. Data represent mean±standard deviation (S.D.) from n=9 values obtained from bootstrap resampling of independent triplicates. Depletion of procaspase-8 significantly reduced FRET probe cleavage (P<0.05, U-test). Experiment was repeated with similar results. (f) Procaspase-8 depletion by siRNA delivery impairs DEVDase activation in HeLa Bcl-2 cells after exposure to bortezomib. After depletion of procaspase-3 or -8, cells were analysed for DEVDase activation by time-lapse FRET imaging. Scrambled siRNA was transfected in control cells. Procaspase-8 depletion resulted in significantly less cells displaying DEVDase activity. Data are from n=27 (scr), 27 (procaspase-3) and 40 (procaspase-8) cells from three to four independent experiments per group. (g) Depletion of procaspase-8 reduced caspase-3 processing as evidenced by a lower conversion of procaspase-3 into p20/p19 subunits. HeLa Bcl-2 cells were treated with 100 nM bortezomib for 48 h. α-Tubulin served as loading control. (h) Cell death in response to 100 nM bortezomib was determined by propidium iodide staining. Depletion of procaspase-8 significantly reduced cell death. siRNA transfection preceded drug addition by 24 h to provide efficient target depletion 24 h subsequent to bortezomib addition (see Supplementary Figure 8). Data are shown as mean±S.D., asterisks indicate significant differences (P<0.05, Student's t-test). (i) Depletion of FADD by siRNA transfection. As in (d), HeLa-Bcl-2 cells were analysed for FADD expression at the indicated times following siRNA transfection. (j) As in (e), HeLa Bcl-2 cells depleted of FADD expression with 100 nM FADD siRNA were analysed for DEVD FRET probe cleavage by flow cytometry following treatment with 100 nM bortezomib. siRNA transfection preceded drug addition by 24 h to provide efficient target depletion 24 h subsequent to bortezomib addition (see Supplementary Figure 8). Bars display changes in FRET substrate cleavage (%) above untreated controls. Data represent mean±S.D. from n=9 values obtained from bootstrap resampling of independent triplicates. Depletion of FADD significantly reduced FRET probe cleavage (P<0.05, U-test). Experiment was repeated with similar results

Figure 6

Apical caspase-8 activation in response to proteasome inhibition is independent of death receptor ligands. (a) Neutralisation of the extrinsic pathway in parental HeLa cells. At 2 h pretreatment with 5 μg/ml tumour necrosis factor-related apoptosis-inducing ligand-receptor 1 (TRAIL-R1):Fc+5 μg/ml TRAIL-R2:Fc, 5 μg/ml tumour necrosis factor-receptor 1 (TNF-R1):Fc or 10 μg/ml Fas:Fc antibodies, respectively, completely abolished extrinsically induced caspase-3 processing. Cells were treated for 6 h with 10 ng/ml TRAIL plus 1 μg/ml cycloheximide (CHX) or 100 ng/ml Fas ligand (FasL) plus 1 μg/ml CHX or for 8 h with 100 ng/ml TNF-α plus 1 μg/ml CHX. α-Tubulin served as loading control. (b) Inhibition of death receptors did not diminish submaximal procaspase-3 processing in response to proteasome inhibition in HeLa-Bcl-2 cells. Cells were pretreated as in (a), and subsequently exposed to 100 nM bortezomib ([(1R)-3-methyl-1-({(2S)-3-phenyl-2-[(pyrazin-2-ylcarbonyl)amino]propanoyl}amino)butyl]boronic acid) for 32 or 48 h. α-Tubulin served as loading control. (c) Procaspase-8 and -3 processing was investigated when using a combination of all neutralising antibodies. β-Actin served as loading control

Figure 7

Autophagy strongly contributes to caspase-8 processing in response to proteasome inhibition. (a) Whole-cell extracts of HeLa Bcl-2 cells treated with 100 nM bortezomib ([(1R)-3-methyl-1-({(2S)-3-phenyl-2-[(pyrazin-2-ylcarbonyl)amino]propanoyl}amino)butyl]boronic acid) for 32 h in the presence or absence of inhibitors of lysosomal proteases were probed for LC3 and p62. Asterisk indicates an unspecific band. β-Actin served as loading control. (b) Whole-cell extracts of HeLa Bcl-2 cells treated with 100 nM bortezomib for the indicated times were probed for the accumulation of p62, Fas-associated death domain (FADD) or receptor-interacting protein kinase 1 (RIPK1). Porin served as loading control. (c) HeLa Bcl-2 cells expressing an mCherry-green fluorescent protein (GFP)-LC3 fusion protein were analysed for the induction of autophagy subsequent to addition of 100 nM bortezomib by confocal microscopy. GFP punctae represent autophagosomes, mCherry punctae represent both autophagosomes and autolysosomes. (d) Procaspase-8 processing and LC3 accumulation and conversion was analysed in HeLa Bcl-2 cells treated with 5 mM 3-methyl adenine (3-MA), 10 μM necrostatin, 50 nM botezomib or combinations thereof for 32 h. 3-MA significantly reduced bortezomib-induced procaspase-8 processing and LC3-I to LC3-II conversion. β-Actin served as loading control. (e) Cell death of HeLa Bcl-2 cells in response to treatments as in (d) was determined by propidium iodide staining. Data are shown as means±standard deviation (S.D.) from n=3 parallel independent cultures. Asterisk indicates significant difference (P<0.05, Student's t-test). Experiment was repeated with similar results. (f) Atg5 depletion blocks procaspase-8 processing. Atg5 was depleted in HeLa Bcl-2 cells using short hairpin RNA (shRNA) transfection. At 48 h subsequent to transfection, cells were exposed to 50 nM bortezomib for 40 h. Whole-cell lysates were analysed for the amount of Atg12-Atg5 conjugates, LC3-I accumulation and conversion to LC3-II, and procaspase-8 processing. β-Actin served as loading control. (g) Cell death of HeLa Bcl-2 cells in response to treatments as in (f) was determined by propidium iodide staining. Data are shown as mean±S.D. from n=3 parallel independent cultures. Asterisk indicates significant difference (P<0.05, Student's t-test). All experiments were repeated at least once and similar results were obtained

Figure 8

X-linked inhibitor of apoptosis (XIAP) depletion and Smac-derived XIAP-antagonising peptides enhance proteasome inhibitor-induced DEVDase activation and apoptosis in Bcl-2-overexpressing cells. (a) Depletion of XIAP by small interfering RNA (siRNA) transfection. HeLa-Bcl-2 cells were analysed for XIAP expression at the indicated times following siRNA transfection. Asterisk indicates an unspecific band. β-Actin served as loading control. A scrambled siRNA (scr) was transfected in control cells. (b) HeLa Bcl-2 cells depleted of XIAP expression were analysed for DEVD Förster resonance energy transfer (FRET) probe cleavage by flow cytometry following treatment with 100 nM bortezomib ([(1R)-3-methyl-1-({(2S)-3-phenyl-2-[(pyrazin-2-ylcarbonyl)amino]propanoyl}amino)butyl]boronic acid). siRNA transfection preceded drug addition by 24 h to provide efficient target depletion 24 h subsequent to bortezomib addition (see Supplementary Figure 8). Depletion of XIAP significantly enhanced FRET probe cleavage. Data represent mean±(S.D.) from n=9 values obtained from bootstrap resampling of independent treatment and control triplicates. Experiment was repeated with similar results. (c) Cell death in response to 100 nM bortezomib was determined by propidium iodide staining. Depletion of XIAP significantly enhanced cell death following treatment of HeLa Bcl-2 cells with 100 nM bortezomib. Data are shown as means±standard deviation (S.D.), asterisks indicate significant differences (P<0.05, Student's t-test). (d) Smac-derived XIAP-antagonising AVPIA peptides enhance apoptosis execution in response to proteasome inhibition in HeLa Bcl-2 cells. Apoptotic cell death was measured by flow cytometry using AnnexinV-fluorescein isothiocyanate (FITC) and propidium iodide staining. Mild responses were detected following proteasome inhibition by bortezomib (100 nM, 32 h). Addition of AVPIA peptide (100 μM) significantly enhanced cell death. Untreated cells or cells receiving only AVPIA peptide did not undergo apoptosis execution. Experiment was repeated with similar results. (e) As in (d), H460 cells overexpressing Bcl-2 were exposed to bortezomib, AVPIA peptide or both. Co-treatment strongly enhanced cell death following 32 h of treatment. Experiment was repeated with similar results