Oncogenic transformation confers a selective susceptibility to the combined suppression of the proteasome and autophagy - PubMed (original) (raw)

Oncogenic transformation confers a selective susceptibility to the combined suppression of the proteasome and autophagy

Wen-Xing Ding et al. Mol Cancer Ther. 2009 Jul.

Abstract

The proteasome and the autophagy systems are two evolutionarily conserved mechanisms for degrading intracellular materials. They are functionally coupled and suppression of the proteasome promotes autophagy. Although suppression of the proteasome leads to cell death, suppression of autophagy can be either prodeath or prosurvival. To understand the underlining mechanism of this dichotomy and its potential clinical implications, we treated various transformed and nontransformed human cells with proteasome inhibitors. We found that whether autophagy served a prosurvival role in this scenario was contingent on the cellular oncogenic status. Thus, autophagy suppression enhanced apoptosis induced by proteasome inhibitors in transformed cells, but not in nontransformed cells. Oncogenic transformation enhanced the ability of cells to initiate autophagy in response to stress, reflecting a stronger dependence of transformed cells on autophagy for survival. Indeed, a combined use of bortezomib, the only Food and Drug Administration-approved proteasome inhibitor for clinical use, and chloroquine, which inhibits autophagy by disturbing lysosomal functions, suppressed tumor growth more significantly than either agent alone in a xenograft model. These findings indicate that suppression of both intracellular degradation systems could constitute a novel strategy for enhanced cancer control in a tumor-specific way.

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Figures

Figure 1

Figure 1. Induction of autophagy by Bortezomib

(A). Bax-positive and Bax–negative HCT116 cells were treated with different doses of Bortezomib for 16 hours. Total lysates were prepared and subjected to immunoblot analysis. (B). Bax-negative HCT116 stably expressing GFP-LC3 were treated with vehicle control or Bortezomib (20 nM) in the presence or absence of E64D (10 μM) and pepstatin A (10 μM) for 16 hours. Total cell lysates were subjected to immunoblot analysis using an anti-GFP antibody. Digital data (mean± SD) were from densitometry analysis of the GFP band from at least three independent experiments. (C-D). The same cells were treated with vehicle control (a), Bortezomib (20 nM, b), chloroquine (10 μM, d) or the combination of the two (c) for 16 hours and then examined by fluorescence microscopy (C). The numbers of GFP-LC3 dots per cell in each condition were quantified (D). Data (mean± SD) are representative of at least three independent experiments. *: p<0.01; #: p<0.05 (one way ANOVA with Scheffe's post-hoc test).

Figure 2

Figure 2. Bortezomib-induced autophagy is cytoprotective

(A). Bax-positive HCT116 cells were either untreated (a) or treated with Bortezomib (20 nM) alone (b) or in the presence of 3-MA (10 mM, c), CQ (10 μM, d), or z-VAD (50 μM, e) for 24 hours. Cell death was determined by nuclear staining with Hoechst 33342 for apoptotic cells with fragmented or condensed or nuclei (arrows), and by propidium iodide staining for cytoplasmic membrane permeability change. (B) Bax-positive and Bax-negative HCT116 cells were treated as in panel A and the percentages of cells with defined changes were quantified. Upper panel: apoptotic cells with fragmented or condensed or nuclei; lower panel; propidium iodide staining positive cells. (C). Bax-deficient HCT116 cells were transfected with a negative siRNA (Neg) or Beclin-1 (Bec) or LC3B-specific siRNA (120 nM) for 48 h and analyzed by immunoblot with indicated antibodies. (D). siRNA-transfected cells were then treated with Bortezomib (20 nM) for another 24 hours. Cell death was determined as in panel A. a: Neg siRNA only; b: Neg siRNA plus Bortezomib; c: siRNA against Beclin 1 plus Bortezomib and d: siRNA against LC3B plus Bortezomib. Arrows indicate fragmented or condensed nuclei. (E). siRNA-transfected cells were treated with Lactacystin (5 μM), ALLN (10 μM) or Bortezomib (20 nM) for another 24 hours. Cell death was determined as in panel A. Data (mean± SD) are representative of at least three independent experiments. *: p<0.01 (z test).

Figure 2

Figure 2. Bortezomib-induced autophagy is cytoprotective

(A). Bax-positive HCT116 cells were either untreated (a) or treated with Bortezomib (20 nM) alone (b) or in the presence of 3-MA (10 mM, c), CQ (10 μM, d), or z-VAD (50 μM, e) for 24 hours. Cell death was determined by nuclear staining with Hoechst 33342 for apoptotic cells with fragmented or condensed or nuclei (arrows), and by propidium iodide staining for cytoplasmic membrane permeability change. (B) Bax-positive and Bax-negative HCT116 cells were treated as in panel A and the percentages of cells with defined changes were quantified. Upper panel: apoptotic cells with fragmented or condensed or nuclei; lower panel; propidium iodide staining positive cells. (C). Bax-deficient HCT116 cells were transfected with a negative siRNA (Neg) or Beclin-1 (Bec) or LC3B-specific siRNA (120 nM) for 48 h and analyzed by immunoblot with indicated antibodies. (D). siRNA-transfected cells were then treated with Bortezomib (20 nM) for another 24 hours. Cell death was determined as in panel A. a: Neg siRNA only; b: Neg siRNA plus Bortezomib; c: siRNA against Beclin 1 plus Bortezomib and d: siRNA against LC3B plus Bortezomib. Arrows indicate fragmented or condensed nuclei. (E). siRNA-transfected cells were treated with Lactacystin (5 μM), ALLN (10 μM) or Bortezomib (20 nM) for another 24 hours. Cell death was determined as in panel A. Data (mean± SD) are representative of at least three independent experiments. *: p<0.01 (z test).

Figure 3

Figure 3. Combined suppression of the proteasome and autophagy enhances the inhibition of tumor growth in vivo

(A). Balb/c nude mice were implanted with 4 × 106 Bax-positive HCT116 cells on the right flank. Fourteen days after inoculations, mice were grouped and intraperitoneally given saline, Bortezomib (Bort, 0.33 m/kg), Bortezomib plus chloroquine (CQ, 45 mg/kg) or CQ alone every 3 days for 6 times as indicated by the arrows. The first treatment day is designated as day 0. Tumor volume (mm3) was determined every two days after the first treatment (day 0) until day 16 and the mean tumor volumes were calculated (n=5-6 per group). *: p<0.01 (Bortezomib/CQ group versus the saline group, one way ANOVA with Scheffe's post-hoc test). (B). Total lysates were prepared from each tumor sample and pooled in equal amount of protein within each group. The lysates were then analyzed for effector caspase activities using Ac-DEVD-AFC as the substrate. Data (mean± SD) of independent triplicate measurements were expressed as fold of increase over the saline control. (C). Tumor samples from each groups were subjected to TUNEL staining and the percentage of positive cells (mean± SD) were determined *: p<0.01 (z test). (D). Electron microscopic examination of tumor samples recovered from nude mice treated for 16 days with saline (a), Bortezomib (b), Bortezomib+CQ (c), or CQ only (d). Arrows: autophagic vesicles; N: nuclei. Scale bar: 1 μm. (E). The number of autophagic vesicles per 100 μm2 area was determined (means ± SD). *: p<0.01, #: p<0.02 (one way ANOVA with Scheffe's post-hoc test). (F). An equal amount of protein from each tumor sample within the same group (n=5-6) was combined and analyzed by immunoblot assay with anti-LC3 and anti-β-actin antibodies. Digital data (mean± SD) were from densitometry analysis of the LC3-II band from at least three independent experiments.

Figure 3

Figure 3. Combined suppression of the proteasome and autophagy enhances the inhibition of tumor growth in vivo

(A). Balb/c nude mice were implanted with 4 × 106 Bax-positive HCT116 cells on the right flank. Fourteen days after inoculations, mice were grouped and intraperitoneally given saline, Bortezomib (Bort, 0.33 m/kg), Bortezomib plus chloroquine (CQ, 45 mg/kg) or CQ alone every 3 days for 6 times as indicated by the arrows. The first treatment day is designated as day 0. Tumor volume (mm3) was determined every two days after the first treatment (day 0) until day 16 and the mean tumor volumes were calculated (n=5-6 per group). *: p<0.01 (Bortezomib/CQ group versus the saline group, one way ANOVA with Scheffe's post-hoc test). (B). Total lysates were prepared from each tumor sample and pooled in equal amount of protein within each group. The lysates were then analyzed for effector caspase activities using Ac-DEVD-AFC as the substrate. Data (mean± SD) of independent triplicate measurements were expressed as fold of increase over the saline control. (C). Tumor samples from each groups were subjected to TUNEL staining and the percentage of positive cells (mean± SD) were determined *: p<0.01 (z test). (D). Electron microscopic examination of tumor samples recovered from nude mice treated for 16 days with saline (a), Bortezomib (b), Bortezomib+CQ (c), or CQ only (d). Arrows: autophagic vesicles; N: nuclei. Scale bar: 1 μm. (E). The number of autophagic vesicles per 100 μm2 area was determined (means ± SD). *: p<0.01, #: p<0.02 (one way ANOVA with Scheffe's post-hoc test). (F). An equal amount of protein from each tumor sample within the same group (n=5-6) was combined and analyzed by immunoblot assay with anti-LC3 and anti-β-actin antibodies. Digital data (mean± SD) were from densitometry analysis of the LC3-II band from at least three independent experiments.

Figure 4

Figure 4. Suppression of autophagy in normal human peripheral blood mononuclear cells does not enhance the toxicity of proteasome inhibitors

(A). Human PBMC were incubated with MG132 (0.5 μM) or Bortezomib (20 nM) in the presence or absence of 3-MA (10 mM) for 24 hours. Immunoblot was then performed using the anti-LC3 antibody. (B-D). Human PBMC were treated with MG132 (0.5 μM), ALLN (10 μM) or Bortezomib (20 nM) in the presence or absence of 3-MA for 24 hours. Cell death was determined by propidium iodide staining (B) or by nuclear Hoechst 33342 staining for fragmented or condensed nuclei (C). Effector caspase activities were determined using Ac-DEVD-AFC as the substrate (D).

Figure 5

Figure 5. Transformed cells can initiate a more potent autophagic response than the matched non-transformed cells

(A). T-29 and T-29-K-Ras cells were first infected with Ad-GFP-LC3 for 24 hours and then treated with vehicle control, Bortezomib (20 nM), Bortezomib plus CQ (10 μM) or CQ alone for 24 hours. Cells were then analyzed by fluorescence microscopy for LC3 translocation. (B) The percentages (mean ± SD) of GFP-LC3-positive cells showing puncta were determined. *: p<0.01 (z test). (C) The numbers of GFP-LC3puncta per cells (mean ± SE) were determined. *: p<0.05 (Student's t test).

Figure 6

Figure 6. Suppression of autophagy promotes proteasome inhibitor-induced cell death in transformed but not in matched non-transformed cells

(A). T-29 and T-29-K-Ras cells were first transfected with a negative siRNA (Neg) or Beclin-1 specific siRNA (120 nM) for 48 hours. The total lysates were analyzed by immunoblot for the expression of Beclin 1 and β-actin. (B-C). siRNA-transfected cells were then treated with Bortezomib (40 nM) for another 24 hours. Cell death was then determined by nuclear Hoechst 33342 staining for fragmented or condensed nuclei (B). *: p<0.01 (z test). Effector caspase activities were measured using Ac-DEVD-AFC as the substrate (C). *: p<0.01 (one way ANOVA with Scheffe's post-hoc test).

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