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. 2017 Aug 11;49(8):e365.
doi: 10.1038/emm.2017.112.

Nutlin-3 enhances the bortezomib sensitivity of p53-defective cancer cells by inducing paraptosis

Dong Min Lee  1   2 , In Young Kim  1   2 , Min Ji Seo  1   2 , Mi Ri Kwon  1   2 , Kyeong Sook Choi  1   2
Affiliations

Nutlin-3 enhances the bortezomib sensitivity of p53-defective cancer cells by inducing paraptosis

Dong Min Lee et al. Exp Mol Med. .

Abstract

The proteasome inhibitor, bortezomib, is ineffective against many solid tumors. Nutlin-3 is a potent antagonist of human homolog of murine double minute 2/p53 interaction exhibiting promising therapeutic anti-cancer activity. In this study, we show that treatment of various p53-defective bortezomib-resistant solid tumor cells with bortezomib plus nutlin-3 induces paraptosis, which is a cell death mode accompanied by dilation of the endoplasmic reticulum (ER) and mitochondria. Bortezomib alone did not markedly alter cellular morphology, and nutlin-3 alone induced only a transient mitochondrial dilation. However, bortezomib/nutlin-3 co-treatment triggered the progressive fusion of swollen ER and the formation of megamitochondria, leading to cell death. Mechanistically, proteasomal-impairment-induced ER stress, CHOP upregulation and disruption of Ca2+ homeostasis were found to be critically involved in the bortezomib/nutlin-3-induced dilation of the ER. Our results further suggest that mitochondrial unfolded protein stress may play an important role in the mitochondrial dilation observed during bortezomib/nutlin-3-induced cell death. Collectively, these findings suggest that bortezomib/nutlin-3 perturbs proteostasis, triggering ER/mitochondria stress and irrecoverable impairments in their structure and function, ultimately leading to paraptotic cell death.

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Conflict of interest statement

The authors declare no conflict of interest.

Figures

Figure 1
Figure 1
Bortezomib and nutlin-3 synergistically induce the cell death accompanied by cytoplasmic vacuolation in various cancer cells. (a, j) Cells were treated with the indicated concentrations of bortezomib and/or nutlin-3 for 24 h and cellular viability was assessed using calcein-AM and EthD-1. The percentage of live cells was normalized to that of untreated control cells (100%). Data represent the means±s.d. One-way ANOVA and Bonferroni’s post hoc test. *P<0.005, **P<0.01 vs untreated cells. The experiment was repeated at least three times with similar results. (b) Isoboles for the combination of bortezomib and nutlin-3, which were isoeffective (IC50) for inhibition of cell viability, are shown. (c) Two HCT116 isogenic cell lines were treated with bortezomib and/or nutlin-3 at the indicated concentration in the absence or presence of 20 μM pifithrin-α for 24 h. Cellular viability was assessed using calcein-AM and EthD-1. Data represent the means±s.d. Kruskal–Wallis test was performed followed by Dunn’s test. *P<0.005 vs untreated cells, P<0.005, ##P<0.01 vs cells treated with 20 μM nutlin-3, NS, not significant. (d, e, i) Cells were treated with the indicated concentration of bortezomib and/or nutlin-3 for 24 h and observed under the phase-contrast microscope. Scale bars, 10 μm. (f) MDA-MB 435S cells were treated with the indicated concentrations of carfilzomib and/or nutlin-3 or MG132 and/or nutlin-3 for 24 h and cellular viability was assessed using calcein-AM and EthD-1. Data represent the means±s.d. One-way ANOVA and Bonferroni’s post hoc test. *P<0.005, **P<0.01 vs untreated cells. (g) Isoboles for the combination of carfilzomib and nutlin-3 or isoboles for the combination of MG132 and nutlin-3, which were isoeffective (IC50) for inhibition of cell viability, are shown. (h) MDA-MB 435S cells were treated with the indicated concentrations of carfilzomib and/or nutlin-3 or MG132 and/or nutlin-3 for 24 h and observed under the phase-contrast microscope. Scale bars, 10 μm.
Figure 2
Figure 2
Vacuolation induced by bortezomib/nutlin-3 is derived from the dilation of both the ER and mitochondria. (a, c) YFP-ER/435S cells expressing the fluorescence selectively in the ER (a) or YFP-Mito/435S cells expressing the fluorescence selectively in mitochondria (c), were treated with 5 nM bortezomib and/or 30 μM nutlin-3 for the indicated time points and observed under the fluorescent and phase-contrast microscope. Scale bars, 20 μm. (b) The average widths of the vacuoles originated from the ER and the numbers of the vacuoles per cells were measured in YFP-ER cells treated with bortezomib and/or nutlin-3 for the indicated time points using AxioVision Rel. 4.8 software (Zeiss). (d) Mitochondrial morphology of ‘elongated’, ‘fragmented’ and ‘dilated’ was defined as the mitochondria with the length longer than 6 μm, those with shorter than 6 μm, and those with widths longer than 1 μm. Representative mitochondrial morphologies of YFP-Mito/435S cells treated as indicated are shown. (e) YFP-Mito/435S cells with mitochondrial dilation were quantified following treatment with 5 nM bortezomib and/or 30 μM nutlin-3 for the indicated time points. (f) YFP-ER/435S cells treated with 5 nM bortezomib and 30 μM nutlin-3 for 8 h were stained with 100 nM MitoTracker-Red (MTR) and observed under the phase-contrast and fluorescence microscope. Scale bar, 20 μm. (g) MDA-MB 435S cells were treated with 5 nM bortezomib and/or 30 μM nutlin-3 for 16 h, fixed, and subjected to the immunocytochemistry of PDI and COX II. Scale bar, 20 μm. (h) MDA-MB 435S cells were treated with 5 nM bortezomib and/or 30 μM nutlin-3 for 8 h, fixed, and subjected to electron microscopy. Scale bars, 0.5 μm.
Figure 3
Figure 3
CHX pretreatment effectively blocks the vacuolation and cell death induced by bortezomib/nutlin-3. (a) The respective cells pretreated with or without CHX were further treated with the indicated concentrations of bortezomib plus nutlin-3 for 24 h. Cell viability was assessed using calcein-AM and EthD-1. Data represent the means±s.d. One-way ANOVA and Bonferroni’s post hoc test. *P<0.005 vs untreated cells; #P<0.005 vs cells treated with bortezomib/nutlin-3. The experiment was repeated at least three times with similar results. (b) MDA-MB 435S cells pretreated with CHX at the indicated concentrations were further treated with 5 nM bortezomib plus 30 μM nutlin-3 for 24 h and observed under the phase-contrast microscope. Scale bar, 10 μm. (c) T98G cells treated with 3 nM bortezomib plus 30 μM nutlin-3, DLD-1 cells treated with 20 nM bortezomib plus 30 μM nutlin-3 and HeLa cells treated with 10 nM bortezomib plus 30 μM nutlin-3 for 16 h in the absence or presence of CHX were observed under the phase-contrast microscope. Scale bars, 10 μm. (d) YFP-ER/435S cells were pre-treated with or without 2 μM CHX and further treated with 5 nM bortezomib and 30 μM nutlin-3 for 8 h. Treated cells were stained with 100 nM MTR and observed under the phase-contrast and fluorescence microscope. Scale bars, 20 μm.
Figure 4
Figure 4
CHOP induction critically contributes to the dilation of the ER and subsequent cell death by bortezomib/nultin-3. (a) Cell extracts were prepared from MDA-MB 435S cells treated with the indicated concentrations of bortezomib and/or nutlin-3 for 8 h and western blotting of the proteins associated with ER stress was performed. β-actin was used as a loading control in western blots. (b) Cell extracts were prepared from MDA-MB 435S cells treated with 5 nM bortezomib plus 30 μM nutlin-3 for indicated time points and western blotting of ubiquitin and CHOP was performed. β-Actin was used as a loading control in western blots. (c–e) MDA-MB 435S cells were infected with the lentivirus containing non-targeting (NT) shRNA or a CHOP-targeting shRNA (CHOP shRNA) for 24 h. Infected cells were treated with 5 nM bortezomib plus 30 μM nutlin-3 for 24 h (c, d) or for 16 h (e). (c) Cell viability was assessed using calcein-AM and EthD-1. The percentage of live cells was normalized to that of cells transfected with shNT without treatment (100%). Data represent the means±s.d. One-way ANOVA and Bonferroni’s post hoc test. *P<0.005 vs cells transfected with shNT without treatment, #P<0.005 vs cells transfected with shNT and further treated with bortezomib/nutlin-3. The experiment was repeated at least three times with similar results. (d) Treated cells as indicated were observed under the phase-contrast microscope. Scale bar, 10 μm. (e) Treated cells as indicated were fixed and subjected for immunocytochemistry of PDI and COX II. Scale bars, 20 μm.
Figure 5
Figure 5
Disruption of intracellular Ca2+ homeostasis critically contributes to the dilation of the ER and subsequent cell death by bortezomib/nultin-3. (a) MDA-MB 435S cells treated with 5 nM bortezomib plus 30 μM nutlin-3 for the indicated time points were subjected to flow cytometry using 1 μM Fluo-3 and changes in Ca2+ levels were compared among the treatment groups. (b) MDA-MB 435S cells were pretreated with BAPTA, BAPTA-AM or BAPTA plus BAPTA-AM at the indicated concentrations and further treated with 5 nM bortezomib plus 30 μM nutlin-3 for 24 h. Cell viability was measured using calcein-AM and EthD-1. Data represent the means±s.d. One-way ANOVA and Bonferroni’s post hoc test. *P<0.005 vs untreated cells, #P<0.005 vs bortezomib/nutlin-3-treated group. The experiment was repeated at least three times with similar results. (c) MDA-MB 435S cells were pretreated with 10 μM BAPTA, 10 μM BAPTA-AM or 10 μM BAPTA plus 10 μM BAPTA-AM and further treated with 5 nM bortezomib plus 30 μM nutlin-3 for 16 h. Treated cells were stained with 1 μM Fluo-3 and observed under the phase-contrast and fluorescence microscope. Scale bar, 20 μm. (d) YFP-ER/435S cells were pretreated with 10 μM BAPTA plus 10 μM BAPTA-AM and further treated with 5 nM bortezomib plus 30 μM nutlin-3 for 16 h. Treated cells were stained with 100 nM MTR and observed under phase-contrast and the fluorescence microscope. Scale bar, 20 μm.
Figure 6
Figure 6
MCU-mediated mitochondrial Ca2+ overload is important for the cell death but not for vacuolation induced by bortezomib/nutlin-3. (a) MDA-MB 435S cells treated with 5 nM bortezomib and 30 μM nutlin-3 for the indicated time points were stained with 1 μM Rhod-2 and processed for FACS analysis. Rhod-2 fluorescence intensities (FI) in cells treated with 5 nM bortezomib plus 30 μM nutlin-3 for the indicated time points were compared with those of untreated cells. (b) MDA-MB 435S cells were pretreated with or without Ru360 at the indicated concentrations and further treated with 5 nM bortezomib plus 30 μM nutlin-3 for 24 h. Cellular viability was assessed using calcein-AM and EthD-1. Data represent the means±s.d. Kruskal–Wallis test was performed followed by Dunn’s test. *P<0.005 vs untreated cells, #P<0.005 vs cells treated with bortezomib/nultin-3. (c) MDA-MB 435S cells were untreated or treated with 5 nM bortezomib and 30 μM nutlin-3 in the absence or presence of 20 μM Ru360 for 24 h. Cells were observed under the phase-contrast microscope. Scale bar, 10 μm. (d, e) MDA-MB 435S cells (d) or YFP-ER/435S cells (e) were treated with 5 nM bortezomib and 30 μM nutlin-3 for 8 h in the absence or presence of Ru360. Treated cells were stained with 1 μM Rhod-2 and 100 nM MTG (d) or stained with 100 nM MTR (e) and observed under the phase-contrast and fluorescence microscope. Scale bars, 20 μm. (f, g) MDA-MB 435S cells were transfected with the fluorescent oligonucleotide (FO) or a MCU-targeting siRNA (siMCU) for 24 h and further treated with 5 nM bortezomib plus 30 μM nutlin-3 for 24 h. (f) Cell viability was assessed using calcein-AM and EthD-1. Data represent the means±s.d. Kruskal–Wallis test was performed followed by Dunn’s test. *P<0.005 vs cells transfected with FO, #P<0.005 vs cells transfected with FO and further treated with bortezomib/nutlin-3. Knockdown of MCU was confirmed by western blotting and β-actin was used as a loading control in western blots. (g) Transfected and treated cells were observed under the phase-contrast microscope. Scale bars, 10 μm. (h) Transfected cells were treated with 5 nM bortezomib plus 30 μM nutlin-3 for 16 h, fixed, and subjected for immunocytochemistry of COX II and PDI. Scale bars, 20 μm. (i) MDA-MB 435S cells were pretreated with or without 10 μM BAPTA plus 10 μM BAPTA-AM for 30 min and further treated with 5 nM bortezomib and 30 μM nutlin-3 for 16 h. Treated cells were stained with 1 μM Rhod-2 and 100 nM MTG and observed under the phase-contrast and fluorescence microscope. Scale bar, 20 μm.
Figure 7
Figure 7
Bortezomib/nutlin-3 may trigger mitochondrial unfolded protein response (mtUPR) during mitochondrial dilation. (a) MDA-MB 435S cells treated with 5 μg ml−1 EtBr for the indicated time points were stained with 100 nM MTR and observed under the phase-contrast and fluorescence microscope. Scale bars, 20 μm. (b) MDA-MB 435S cells treated with 5 μg ml−1 EtBr for 24 h or 30 μM nutlin-3 for 16 h were fixed and subjected for immunocytochemistry of COX II and mtHsp70. Scale bars, 20 μm. (c) Plots of the intensity profile of a set of pixels distributed on a line drawn across the indicated mitochondria (shown in mtHsp70+COX II panels) are shown at different emission wavelengths corresponding to the signals of mtHsp70 (green) and COX II (red). (d–g) After treatment of MDA-MB 435S cells with 5 μg ml−1 EtBr for the indicated time points, culture media were changed with the fresh ones and cells were further treated with 5 nM bortezomib for 24 h. (d) Cell viability was assessed using calcein-AM and EthD-1. Data represent the means±s.d. One-way ANOVA and Bonferroni’s post hoc test. *P<0.005 vs cells treated with EtBr alone for the indicated time points. (e) Treated cells were observed under the phase-contrast microscope. Scale bar, 10 μm. (f, g) Treated cells were fixed, and subjected for immunocytochemistry of COX II and PDI (f) or COX II and mtHsp70 (g). In addition, immunocytochemistriy of COX II and mtHsp70 was performed in MDA-MB 435S cells treated with 5 nM bortezomib and/or 30 μM nutlin-3 for 16 h. Scale bars, 20 μm.
Figure 8
Figure 8
Upregulation of CHOP and the increase in Ca2+ levels precede the ER dilation induced by bortezomib/nutlin-3. (a) MDA-MB 435S cells pretreated with or without 2 μM CHX and further treated with 5 nM bortezomib plus 30 μM nutlin-3 for 16 h. Cell extracts were prepared for the western blotting of ubiquitin and CHOP. β-actin was used as a loading control in western blots. (b, c) MDA-MB 435S cells were pre-treated with or without 2 μM CHX for 30 min further treated with 5 nM bortezomib and 30 μM nutlin-3 for 16 h. (b) Treated cells were stained with 1 μM Fluo-3 and observed under the phase-contrast and fluorescence microscope. Scale bar, 20 μm. (c) Treated cells were stained with 1 μM Rhod-2 and 100 nM MTG and observed under the phase-contrast and fluorescence microscope. Scale bar, 20 μm. (d) MDA-MB 435S cells pretreated with or without 2 μM CHX followed by treatment with 5 μg/ml EtBr for 24 h. After culture media was changed with the fresh ones, cells were further treated with 5 nM bortezomib for 12 h. Treated cells were fixed, and subjected for immunocytochemistry of COX II and mtHsp70. Scale bars, 20 μm. (e) Plots of the intensity profile of a set of pixels distributed on a lline drawn across the indicated mitochondria (as shown in mtHsp70+COX II panels) at differnet emission wavelengths corresponding to the signals of mtHsp70 (green) and COX II (red). (f–h) MDA-MB 435S cells were infected with the lentivirus containing non-targeting (NT) shRNA or a CHOP-targeting shRNA (CHOP shRNA) for 24 h. Infected cells were treated with 5 nM bortezomib and 30 μM nutlin-3 for 16 h. (f) Treated cells were stained with 1 μM Fluo-3 and observed under the phase-contrast and fluorescence microscope. Scale bars, 20 μm. (g) Treated cells were stained with 1 μM Rhod-2 and 100 nM MTG and observed under the phase-contrast and fluorescence microscope. Scale bars, 20 μm. (h) Cell extracts were prepared for western blotting of CHOP and ubiquitin. β-actin was used as a loading control in western blots. (i) MDA-MB 435S cells pretreated with 10 μM BAPTA and 10 μM BAPTA-AM were further treated with 5 nM bortezomib plus 30 μM nutlin-3 for 8 h and then western blotting of the indicated proteins was performed. β-actin was used as a loading control in western blots. (j) Hypothetical model of the underlying mechanism of paraptosis induced by bortezomib/nutlin-3 in p53-defective cancer cells. Combination of bortezomib and nutlin-3 triggers ER stress induction, CHOP upregulation, and the increase in cytosolic Ca2+ levels, leading to ER dilation. In parallel to ER dilation, mitochondria are also dilated possibly via the mechanism related to mitochondrial unfolded protein stress, contributing to the paraptotic cell death induced by bortezomib/nutlin-3. Mitochondrial Ca2+ overload, which is preceded by increased cytosolic Ca2+, may also play a role in the cytotoxicity of bortezomib/nutlin-3.
Figure 8
Figure 8
Upregulation of CHOP and the increase in Ca2+ levels precede the ER dilation induced by bortezomib/nutlin-3. (a) MDA-MB 435S cells pretreated with or without 2 μM CHX and further treated with 5 nM bortezomib plus 30 μM nutlin-3 for 16 h. Cell extracts were prepared for the western blotting of ubiquitin and CHOP. β-actin was used as a loading control in western blots. (b, c) MDA-MB 435S cells were pre-treated with or without 2 μM CHX for 30 min further treated with 5 nM bortezomib and 30 μM nutlin-3 for 16 h. (b) Treated cells were stained with 1 μM Fluo-3 and observed under the phase-contrast and fluorescence microscope. Scale bar, 20 μm. (c) Treated cells were stained with 1 μM Rhod-2 and 100 nM MTG and observed under the phase-contrast and fluorescence microscope. Scale bar, 20 μm. (d) MDA-MB 435S cells pretreated with or without 2 μM CHX followed by treatment with 5 μg/ml EtBr for 24 h. After culture media was changed with the fresh ones, cells were further treated with 5 nM bortezomib for 12 h. Treated cells were fixed, and subjected for immunocytochemistry of COX II and mtHsp70. Scale bars, 20 μm. (e) Plots of the intensity profile of a set of pixels distributed on a lline drawn across the indicated mitochondria (as shown in mtHsp70+COX II panels) at differnet emission wavelengths corresponding to the signals of mtHsp70 (green) and COX II (red). (f–h) MDA-MB 435S cells were infected with the lentivirus containing non-targeting (NT) shRNA or a CHOP-targeting shRNA (CHOP shRNA) for 24 h. Infected cells were treated with 5 nM bortezomib and 30 μM nutlin-3 for 16 h. (f) Treated cells were stained with 1 μM Fluo-3 and observed under the phase-contrast and fluorescence microscope. Scale bars, 20 μm. (g) Treated cells were stained with 1 μM Rhod-2 and 100 nM MTG and observed under the phase-contrast and fluorescence microscope. Scale bars, 20 μm. (h) Cell extracts were prepared for western blotting of CHOP and ubiquitin. β-actin was used as a loading control in western blots. (i) MDA-MB 435S cells pretreated with 10 μM BAPTA and 10 μM BAPTA-AM were further treated with 5 nM bortezomib plus 30 μM nutlin-3 for 8 h and then western blotting of the indicated proteins was performed. β-actin was used as a loading control in western blots. (j) Hypothetical model of the underlying mechanism of paraptosis induced by bortezomib/nutlin-3 in p53-defective cancer cells. Combination of bortezomib and nutlin-3 triggers ER stress induction, CHOP upregulation, and the increase in cytosolic Ca2+ levels, leading to ER dilation. In parallel to ER dilation, mitochondria are also dilated possibly via the mechanism related to mitochondrial unfolded protein stress, contributing to the paraptotic cell death induced by bortezomib/nutlin-3. Mitochondrial Ca2+ overload, which is preceded by increased cytosolic Ca2+, may also play a role in the cytotoxicity of bortezomib/nutlin-3.
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