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. 2018 May 22;9(6):604.
doi: 10.1038/s41419-018-0642-6.

The Proton Pump Inhibitor Pantoprazole Disrupts Protein Degradation Systems and Sensitizes Cancer Cells to Death Under Various Stresses

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Free PMC article

The Proton Pump Inhibitor Pantoprazole Disrupts Protein Degradation Systems and Sensitizes Cancer Cells to Death Under Various Stresses

Yu Cao et al. Cell Death Dis. .
Free PMC article

Abstract

Proton pump inhibitors (PPIs) play a role in antitumor activity, with studies showing specialized impacts of PPIs on cancer cell apoptosis, metastasis, and autophagy. In this study, we demonstrated that pantoprazole (PPI) increased autophagosomes formation and affected autophagic flux depending on the pH conditions. PPI specifically elevated SQSTM1 protein levels by increasing SQSTM1 transcription via NFE2L2 activation independent of the specific effect of PPI on autophagic flux. Via decreasing proteasome subunits expression, PPI significantly impaired the function of the proteasome, accompanied by the accumulation of undegraded poly-ubiquitinated proteins. Notably, PPI-induced autophagy functioned as a downstream response of proteasome inhibition by PPI, while suppressing protein synthesis abrogated autophagy. Blocking autophagic flux in neutral pH condition or further impairing proteasome function with proteasome inhibitors, significantly aggravated PPI cytotoxicity by worsening protein degradation ability. Interestingly, under conditions of mitochondrial stress, PPI showed significant synergism when combined with Bcl-2 inhibitors. Taken together, these findings provide a new understanding of the impact of PPIs on cancer cells' biological processes and highlight the potential to develop more efficient and effective combination therapies.

Conflict of interest statement

The authors declare that they have no conflict of interest.

Figures

Fig. 1
Fig. 1. Pantoprazole induced autophagosome biogenesis via TM9SF4-mTOR pathway.
a AGS cells treated with 120 μg/ml PPI for 48 h in pH 7.4 condition, were imaged by transmission electron microscopy. Representative micrographs showed increased abundance of lysosomes in cells after PPI treatment compared with control cells. Scale bar: 500 nm. b AGS and HeLa cells transfected with GFP-LC3B plasmid were treated with 120 μg/ml PPI for 48 h in pH 7.4 condition. Scale bar: 20 μm. c The number of autophagic vacuoles and GFP-LC3B dots in each cell were quantified. Data were presented as mean ± SD from three independent experiments (**p < 0.01, identified by Student’s t-test). d, e AGS and HeLa cells were treated for 48 h in pH 7.4 condition with indicated concentrations of PPI, or treated with 60 and 120 μg/ml PPI for the indicated times. Levels of LC3B-II and SQSTM1 protein were analyzed by western blot. β-actin served as an internal control. f mTOR pathway proteins levels in AGS cells after treated with PPI for 48 h both in pH 7.4 and 6.5 conditions, were detected by western blot analysis. g, h AGS cells were transfected with TM9SF4 siRNA for 48 h, and then incubated with 80 and 100 μg/ml PPI for another 48 h. The level of indicated proteins were analyzed by western blot (g). TM9SF4 knockdown efficiency was verified by Q-PCR (h). i Data showing the positive correlation between TM9SF4 and related autophagy genes mRNA expression in TCGA STAD corhort, were generated by GEPIA (http://gepia.cancer-pku.cn/detail.php?clicktag=correlation). Pearson’s coefficient tests were performed to assess statistical significance
Fig. 2
Fig. 2. PPI didn’t inhibit the fusion of autophagosomes and lysosomes in neutral pH condition.
a AGS cells were either untreated or treated with PPI (60–140 μg/ml) for 24 h in pH 7.4 condition in the absence or presence of classical autophagic flux inhibitor baf-A1 (100 nM). The indicated protein levels were analyzed by western blot. b AGS cells were transiently infected with GFP-mRFP-LC3B adenoviral particles for 48 h and subsequently treated with PPI (120 μg/ml), baf A1 (100 nM), Torin 1 (500 nM) for 48 h in pH 7.4 condition. The change of both green and red fluorescence was observed using a confocal microscope. Scale bar: 20 μm. Lower panel, the numbers of acidified autophagosomes (GFPRFP+) versus neutral autophagosomes (GFP+RFP+) per cell in each condition were quantified. Data were presented as mean ± SD from three independent experiments (n.s. not significant; **p < 0.01, identified by two-way ANOVA with Dunnett’s multiple comparison test). c AGS cells were either untreated or treated with PPI (60–140 μg/ml) for 24 h in pH 7.4 condition in the absence or presence of rapamycin (1 μM) or baf-A1 (100 nM), then stained with LysoTracker Red DND-99 dye (50 nM for 15 min) for FACS analysis. Representative results of three independent experiments were shown. d, e qRT-PCR analysis of various V-ATPase subunits (d) and lysosomal genes (e) in AGS cells treated with 100 and 120 μg/ml PPI for 48 h in pH 7.4 condition. Data were presented as mean ± SD. f Different cancer cells were either untreated or treated with 100 μg/ml PPI for 48 h in pH 7.4 condition, and then stained with LTR for FACS analysis. Representative results of three independent experiments were shown. g AGS and MKN45 cells were pretreated with indicated concentrations of PPI for 24 h in pH 7.4 condition, and then incubated with or without rapamycin (1 μM) for another 24 h. The change pattern of SQSTM1 protein was determined by western blot
Fig. 3
Fig. 3. PPI upregulated SQSTM1 mRNA via NFE2L2.
a AGS, HGC27, and HeLa cells were treated with 60 and 100 μg/ml PPI for 48 h in both pH 6.5 and pH 7.4 conditions, respectively. The SQSTM1 mRNA levels were assessed (**p < 0.01, for each cell line using one-way ANOVA with Dunnett’s multiple comparisons test). b AGS cells were treated with PPI for 48 h in both pH 6.5 and pH 7.4 conditions, and then stained with DCFHDA (5 μM) for 30 min. Intracellular ROS was reflected by the fluorescence intensity via flow cytometry analysis. Data presented were representative of three independent experiments (**p < 0.01, identified by one-way ANOVA with Dunnett’s multiple comparisons test). c, d AGS cells were treated with PPI in the absence or presence of GSH (4 mM) (c) or NAC (5 mM) (d) for 48 h in pH 7.4 condition. The expression level of SQSTM1 was measured by western blot. e, f AGS cells were exposed to various concentrations of PPI for 48 h in both pH 6.5 and pH 7.4 conditions. The levels of Nrf2 and its targets such as GCLC, GCLM, NQO1, and HO-1 were assessed by western blot analysis (e) and qRT-PCR analysis (f). Data were presented as mean ± SD (**p < 0.01, for each gene using one-way ANOVA Dunnett’s multiple comparison test). gi AGS and HGC27 cells were reversely transfected with Nrf2 specific siRNA for 48 h, and then exposed to indicated concentrations of PPI for 48 h in both pH 6.5 and pH 7.4 conditions. Knockdown efficiency of Nrf2 was confirmed by western blot analysis (g, h), and the change pattern of SQSTM1 was evaluated by both western blot (g, h) and qRT-PCR analysis (i). Data were presented as mean ± SD (**p < 0.01, identified by two-way ANOVA with Tukey’s multiple comparisons test)
Fig. 4
Fig. 4. PPI-induced accumulation of exogenous SQSTM1 via ubiquitin-proteasome pathway.
a AGS cells were transfected with HA-tagged SQSTM1 plasmid for 48 h, and then treated with PPI for another 48 h in both pH 6.5 and pH 7.4 conditions. The exogenous SQSTM1 protein was detected with antibody for HA. b AGS cells were treated with baf A1 (50 and 100 nM) and HCQ (10, 50, and 100 μM) for 24 h. c AGS cells were pretreated with baf A1 (100 nM) for 30 min, followed with 1 μM rapamycin or amino acid starvation by HBSS for another 24 h. d Different concentrations of two classical proteasome inhibitors Bortezomib and MG132 were added. e AGS cells were either untreated or treated with Bortezomib (25 nM) or MG132 (0.1 μM) for 24 h in the absence or presence of baf A1 (100 nM). f AGS cells transfected with HA-tagged SQSTM1 plasmid, were treated with 25 μg/ml cycloheximide (CHX) over a 240-min time period (left) or treated with 100 μg/ml PPI for 48 h in pH 7.4 conditions, and then followed by 25 μg/ml CHX over a 240-min time period (right). Cells were lysed at the indicated time points (0, 60, 120, and 240 min). Right panel showed the half-life of HA, which reflected the stability of exogenous SQSTM1 protein. gi The accumulated SQSTM1 by either proteasome inhibitors or PPI was sensitive to autophagy mediated degradation. AGS cells were pretreated with 25 and 50 nM bortezomib for 1 h, and then incubated with 500 nM torin 1 for 24 h (g). After pretreatment with PPI for 24 h in pH 7.4 or pH 6.5 condition, rapamycin (1 μM) or torin 1 (500 nM) was added for another 24 h (h). The protein level of SQSTM1 was measured by western blot analysis (g, h), and the change pattern of SQSTM1 mRNA was confirmed by qRT-PCR (i) (**p < 0.01, identified by one-way ANOVA with Dunnett’s multiple comparison test)
Fig. 5
Fig. 5. PPI reduced proteasome expression and induced ER stress in cancer cells.
a AGS cells were treated with indicated concentrations of PPI for 48 h in pH 7.4 condition, followed by measuring proteasome function via western blot analysis using specific antibody to ubiquitin. b The mRNA expression level of the 20S proteasome subunits were measured after PPI treatment for 48 h in pH 7.4 condition. Data were presented as mean ± SD (*p < 0.05, **p < 0.01, for each gene using one-way ANOVA with Dunnett’s multiple comparisons test). c Western blot analysis of ER stress-related proteins after PPI treatment for 48 h in both pH 7.4 and pH 6.5 conditions. Cells treated with Thapsigargin (TG, 0.5 and 1 μM) or Tunicamycin (Tu, 2.5, 5 and 10 μg/ml) for 24 h served as positive controls. d qRT-PCR analysis of UPR genes after 48 h of PPI (100 and 120 μg/ml) treatment in both pH 7.4 and pH 6.5 conditions. Data were presented as mean ± SD (**p < 0.01, for each gene using one-way ANOVA with Dunnett’s multiple comparisons test). e ER-Tracker Red (500 nM) staining of PPI treated cells in pH 7.4 condition was performed. The quantification of ER Tracker fluorescence were accomplished by FACS. Data presented were representative of three independent experiments (**p < 0.01, for each cell line using one-way ANOVA with Dunnett’s multiple comparisons test). f After treated with PPI (60–140 μg/ml) for 24 h in pH 7.4 condition, cells were then incubated with 5 μM Fluo-4/AM and detected by flow cytometry. Data presented were representative of three independent experiments (**p < 0.01, identified by one-way ANOVA with Dunnett’s multiple comparisons test). g, h PPI-induced ER stress contributed to the acitivation of autophagy. AGS and HeLa cells transfected with CHOP-specific siRNA, were treated with 100 and 120 μg/ml PPI for 24 h in both pH 7.4 and pH 6.5 conditions (g). AGS cells were treated with the indicated concentrations of PPI in the absence or presence of 2-APB (20 μM) for 24 h in both pH 7.4 and pH 6.5 conditions (h). The autophagy marker LC3B-II was determined
Fig. 6
Fig. 6. Proteasome inhibitors aggravated while protein synthesis suppression ameliorated the UPR and cell death by PPI.
a, b AGS cells were pretreated with PPI (100 μg/ml) for 24 h in pH 7.4 condition, followed by combination with or without 50 nM Bortezomib or 0.1 μM MG132 for another 24 h. Western blot analysis of apoptosis related protein cleaved-PARP levels (a) and poly-ubiquitinated proteins (b). c, d AGS and HeLa cells were pretreated with CHX (250 ng/ml) for 2 h, and then incubated with PPI (100 μg/ml or 120 μg/ml) for 48 h in pH 7.4 condition. Levels of apoptosis related protein cleaved-PARP, UPR marker CHOP (c), and poly-ubiquitinated proteins (d) were analyzed. e AGS and HeLa cells were pretreated with torin 1 (500 nM) for 2 h, and then incubated with PPI (100 μg/ml or 120 μg/ml) for 48 h in pH 7.4 condition. Indicated proteins were analyzed by western blot. f AGS cells were treated as described in (a). The cell viability was determined by CCK8 assay (left) (**p < 0.01, identified by two-way ANOVA with Sidak’s multiple comparisons test). qRT-PCR analysis of UPR genes was performed (right) (**p < 0.01, for each gene using one-way ANOVA with Tukey’s multiple comparisons test). g AGS cells were treated as described in (c). The cell viability was determined by CCK8 assay (left) (**p < 0.01, identified by two-way ANOVA with Sidak’s multiple comparisons test). qRT-PCR analysis of UPR genes was performed (right) (**p < 0.01, for each gene using one-way ANOVA with Tukey’s multiple comparisons test). h AGS cells were treated as described in (e). The cell viability was determined by CCK8 assay (left) (**p < 0.01, identified by two-way ANOVA with Sidak’s multiple comparisons test). qRT-PCR analysis of UPR genes was performed (right) (**p < 0.01, for each gene using one-way ANOVA with Tukey’s multiple comparisons test)
Fig. 7
Fig. 7. Bcl-2 inhibitors synergized the cytotoxicity of PPI.
a, b The Bcl-2 family members were analyzed by western blot (a) and qRT-PCR (b). Data were presented as mean ± SD (**p < 0.01, for each gene using one-way ANOVA with Dunnett’s multiple comparisons test). c AGS cells were pretreated with various concentrations of PPI for 24 h in pH 7.4 condition, and then incubated with two different doses of ABT-263/ABT-737 for another 24 h. Data were presented as percentages of cell viability as determined by CCK8 assays (upper panel). Synergisms of cell viability inhibition by the combination therapy were analyzed by Combination Index Value (lower panel). d Cell viability of AGS cells after treatment with combination of 100 μg/ml PPI and 4 μM ABT-263/10 μM ABT-737 in pH 7.4 condition. The combination indexes (CI) were calculated as described in Methods section. Data were presented as mean ± SD (***p < 0.001, indentified by one-way ANOVA Dunnett’s multiple comparison test). e AGS cells were treated as described in (c), and the changes in mitochondrial membrane potential (Δψm) were analyzed by JC-1 assay. Ratio of green to red fluorescence was depicted (left panel). Data were representative of three independent experiments. Right panel showed the quantification of the Δψm in AGS cells upon combinational treatment. Data were presented as mean ± SD (***p < 0.001, indentified by one-way ANOVA Tukey’s multiple comparisons test). f AGS cells were treated as described in (c), and then stained with MitoTracker green (100 nM) and MitoTracker red (400 nM). The dysfunctional mitochondria accumulation was analyzed by FACS. Dot plots of subpopulation are depicted (left panel) and percent of dysfunctional mitochondria were shown as mean ± SD (right panel) (***p < 0.001, indentified by one-way ANOVA Tukey’s multiple comparisons test). g AGS cells were treated as described in (c). The apoptosis related proteins were detected
Fig. 8
Fig. 8. Schematic illustration of proposed mechanisms.
PPI inhibited proteasome function, and induced SQSTM1 elevated autophagy as a compensatory response for impaired proteasomal degradation. PPI promotes autophagic flux in neutral pH condition while blocks autophagic flux in low pH condition. When cancer cells were under proteins overload stress caused by proteasome inhibitors and autophagic flux blockers, or mitochondrial stress caused by Bcl-2 inhibitors, the cytotoxicity of PPI would be significantly increased

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