All data were presented as mean ± S.D. (standard deviation) from at least three separate experiments. Student’s t test was applied to evaluate the differences between treated and control groups with cell viability. Data from multiple groups were analyzed by one-way ANOVA, followed by Bonferroni multiple comparison test. For all the tests, the level of significance was values of P < 0.05.
M. Isobologram analysis
To determine the effect of combination of bortezomib and nutlin-3 on MDA-MB 435S, DLD-1, HeLa, and T98G cells, dose-dependent effects were determined for each compound and for one compound with fixed concentrations of another. The interaction of bortezomib and nutlin-3 was quantified by determining the
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combination index (CI), in accordance with the following classic isobologram (Chou TC & Talalay P., 1984). The equation for the isobologram is shown as CI = (D)1/(Dx)1 + (D)2/(Dx)2, where (Dx)1 and (Dx)2 indicate the individual dose of bortezomib and nutlin-3 required to produce an effect, and (D)1 and (D)2 are the doses of bortezomib and nutlin-3, respectively, in combination that produce the same effect. From this analysis, the combined effects of the two drugs can be summarized as follows: CI < 1 indicates synergism; CI = 1 indicates summation (additive and zero interaction); and CI > 1 indicates antagonism.
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III. RESULTS
1. Bortezomib and nutlin-3 synergistically induce cell death in various cancer cell lines with defective p53
First, we investigated whether nutlin-3 could overcome the resistance of various cancer cell lines with defective p53 to bortezomib. For this purpose, we used MDA-MB 435S (breast cancer cells, G266E mutant p53, Gartel et al., 2003), T98G cells (glioma cells, M237I mutant p53, Villalonga-Planells et al., 2011), DLD-1 (colon cancer cells, S241P mutant p53, Li et al., 2005), and HeLa (cervix cancer cells, non-functional p53 due to E6 expression, Hoppe-Seyler & Butz, 1993)). We treated these cancer cells with various doses of bortezomib and/or nutlin-3 for 24 h and performed cell viability assay using calcein-AM and EthD-1, to detect live and dead cells, respectively. When we examined the effect of bortezomib on these cancer cells, bortezomib treatment did not show dose-dependent cytotoxicity at higher doses exceeding the certain concentrations and more than 70% cells treated with bortezomib of different doses were viable (Figure 1). When we assessed IC50s of bortezomib in these cancer cells, they were higher than 14671.1 nM, 4275.5 nM, 250.7 nM, and 159.5 nM in DLD-1, MDA-MB 435S, HeLa, and T98G cells, respectively (Figure 1). While treatment with nultlin-3 up to 30 µM was not cytotoxic to all the tested cancer cells, co-treatment with bortezomib and nultlin-3 significantly enhanced cell death in all the tested cancer cells, compared to the treatment with bortezomib alone (Figure 2), in spite of the differences in the relative sensitivity to bortezomib (Figure 1). Isobologram analysis showed that bortezomib and nutlin-3 synergistically induces cell death in these cancer cells (Figure 3). MTT assay also showed that bortezomib and nutlin-3 synergistically induced cancer cell death (Figure 4).
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Since nutlin-3 is known to be an MDM2 inhibitor, that can lead to stabilization of p53 (Jones, 2011), we investigated whether the sensitizing effect of nutlin-3 on bortezomib-mediated cell death is associated with MDM2 and/or p53. MDM2, an ubiquitin E3 ligase, is known to be upregulated by p53-mediated transcriptional activation (Miyachi et al., 2009) or proteasome inhibition (Hideshima et al., 2003).
When we first examined the expression of MDM2 and p53 in MDA-MB 435S cells, we found that the protein levels of MDM2 were not affected by nutlin-3, but markedly increased by bortezomib (Figure 5A). p53 proteins were highly expressed in untreated MDA-MB 435S cells and their expression was not altered by treatment with bortezomib and/or nutlin-3, possibly due to the fact that this cancer cell line harbors the mutation in p53 (Gartel et al., 2003). In addition, pifithrin-α, an inhibitor of p53-dependent transcriptional activity (Dagher, 2004) did not block the cell death by bortezomib plus nutlin-3 in MDA-MB 435S cells (Figure 5B).
Therefore, these results suggest that upregulation of MDM2 by bortezomib is independent of p53 and the sensitizing effect of nutlin-3 on bortezomib-mediated cell death may not be associated with its activity as an MDM2 inhibitor to indirectly upregulate p53. In addition, when we performed immunocytochemistry of p53 in MDA-MB 435S cells, high expression of p53 in the nuclei was not further accumulated by bortezomib and/or nultlin-3 (Figure 6). To further confirm whether nutlin-3 could sensitize cancer cells to bortezomib-mediated cell death via p53-independent mechanism, we employed two HCT116 isogenic cell lines differing in the p53 status, HCT116 wild-type (WT) cells and HCT116 p53-null (p53-/-) cells. Ten µM bortezomib was toxic neither to HCT116 WT cells nor to HCT116 p53-/- cells (Figure 7). Interestingly, treatment with nutlin-3 alone reduced the cell viability in HCT116 WT cells in a dose-dependent manner and this nutlin-3-induced cytotoxicity was very effectively inhibited by pifithrin-α pretreatment (Figure 7). In addition, co-treatment with bortezomib enhanced nutlin-3-induced cell death and pifithrin-α pretreatment significantly but partially
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inhibited the cell death by bortezomib plus nutlin-3. These results suggest that p53 may be critically involved not only in nutlin-3-induced cell death but also in the cell death by the combined treatment with bortezomib and nutlin-3 in HCT116 WT cells. In contrast, treatment with nutlin-3 up to 20 µM was not cytotoxic to HCT116 p53-/- cells, but nutlin-3 treatment dose-dependently recovered the cellular sensitivity to bortezomib (Figure 7). However, pifithrin-α pretreatment did not attenuate the cell death by bortezomib plus nutlin-3 in these cells, suggesting that nutlin-3-stimulated bortezomib-mediated cell death is independent of p53.
When we performed western blotting, not only p53 protein levels but also the protein levels of MDM2 and p21, downstream targets of p53, were markedly increased by nutlin-3 (Figure 7). The protein levels of p53, MDM2, and p21 were also increased by bortezomib and they were further increased by the combined treatment with bortezomib and nutlin-3, suggesting the possible involvement of p53 in the cellular response to nutlin-3 and/or bortezomib in HCT116 WT cells. In contrast, the protein levels of MDM2 and p53 were not altered by nutlin-3, but by bortezomib, in HCT116 p53-/- cells and they were rather slightly decreased by bortezomib plus nutlin-3. Taken together, these results suggest that since bortezomib plus bortezomib can induce cell death independently of p53, this combined treatment may provide a way to overcome the resistance of bortezomib in cancer cells harboring defective p53.
To understand the underlying mechanism by which nutlin-3 sensitizes bortezomib-mediated cell death, independently of p53, we first observed the cancer cell morphologies following treatment with bortezomib and/or nultlin-3. We found that treatment with bortezomib or nutlin-3 alone did not noticeably alter the cellular morphologies in MDA-MB 435S, T98G, DLD-1 and HeLa cells, because their subtoxic does were used in this experiment (Figure 8). However interestingly, combination of bortezomib and nutlin-3 at the same concentrations induced extensive cytoplasmic vacuolation prior to cell death in all the tested cancer cells.
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To investigate the cell death mode induced by the combination of bortezomib and nutlin-3, we tested the effects of various inhibitors, including z-VAD-fmk to block caspase-mediated apoptosis, necrostatin-1 to block necroptosis, and 3-MA or bafilomycin A to block autophagy. However, neither the cell death nor cytoplasmic vacuolation induced by bortezomib plus nultlin-3 was inhibited by any tested inhibitors in these cancer cells (Figure 9, 10), suggesting that apoptosis, necrosis, or autophagy is not critically involved in the cell death by bortezomib plus nultlin-3.
Collectively, these results suggest that nutlin-3 overcomes the resistance of these several cells to bortezomib through induction of a novel cell death mode.
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Figure 1. Anti-cancer effect of bortezomib on various cancer cells. MDA-MB 435S, DLD-1, HeLa, and T98G cells were treated with various concentrations of bortezomib for 24 h and then the viability was assessed using calcein-AM and EthD-1 (Live/Dead assay). The values of IC50s of bortezomib were assessed in the tested cancer cells.
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Figure 2. Bortezomib and nutlin-3 synergistically induce the cell death in diverse cell lines. MDA-MB 435S, DLD-1, HeLa, and T98G cells were treated with the indicated concentrations of bortezomib and/or nutlin-3 for 24 h and then the viability was assessed using calcein-AM and EthD-1(Live/Dead assay). *P <
0.05; **P < 0.01; ***P < 0.005 versus bortezomib alone treated groups.
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Figure 3. Synergistic induction of cell death by bortezomib and nutlin-3. The classic Isobologram at IC₅₀ (the concentration of each drug that is required to reduce the viability of treated cells for 24 h to 50%). Diverse cancer cells were treated with bortezomib and nutlin-3 for 24 h. Isoboles for the combination of bortezomib and nutlin-3 that were isoeffective (IC₅₀) for inhibition of cell viability are shown.
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Figure 4. Bortezomib and nutlin-3 synergistically induce cell death in various cancer cell lines. (A) Cells were treated with the indicated concentrations of bortezomib and/or nutlin-3 for 24 h and viability was measured by MTT assay. *P
< 0.05; **P < 0.01; ***P < 0.005 versus bortezomib alone treatment groups. (B) The classic Isobologram at IC₅₀.
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Figure 5. Combined treatment with bortezomib and nutlin-3 induces the cell death independent of p53. (A) MDA-MB 435S cells are treated with combination of bortezomib and/or nutlin-3 at the indicated concentrations for 16 h and then western blotting of MDM2, p53, and β-actin was performed. (B) Cells were untreated or pretreated with indicated concentrations of pifithrin-α and further treated with bortezomib plus nutlin-3 with indicated concentration for 24 h.
Cellular viability was assessed using calcein-AM and EthD-1. *P < 0.005 versus non treated groups.
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Figure 6. High expression of p53 in the nuclei is not further accumulated by bortezomib and/or nultlin-3. MDA-MB 435S cells treated as indicated were fixed and subjected for immunocytochemistry of p53 and DAPI staining. Bars, 20 μm
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Figure 7. Cell death induced by bortezomib plus nutlin-3 is not associated with pro-cell death activity of p53. Cells were untreated or pretreated with indicated concentrations of pifithrin-α and further treated with bortezomib and/or nutlin-3 with indicated concentration for 24 h. Cellular viability was assessed using calcein-AM and EthD-1. *P < 0.005 versus DMSE treated groups; **P < 0.005 versus bortezomib plus nutlin-3 treated groups; ***P < 0.005 versus pifithrin-α alone treated groups. #P < 0.005 versus 20 μM nutlin-3 treated groups. The presence/absence of p53 and its transcription activity were confirmed by western blotting of p53 and p53-related proteins following treatment with indicated concentrations of bortezomib and/or nutlin-3 for 12 h. β-actin was used as a loading control in western blots.
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Figure 8. Combined treatment with bortezomib and nutlin-3 induces cytoplasmic vacuolation followed by cell death in all the tested cancer cells.
Cells were treated with indicated concentrations of bortezomib and/or nutlin-3 for 24 h. And the cells were observed under the phase contrast microscopy. Bars, 10 μm
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Figure 9. Combination treatment with bortezomib and nutlin-3 induces alternative cell death in all the tested cells. Cells were untreated or pretreated with indicated concentrations of the respective inhibitors, including z-VAD-fmk, necrostatin-1, 3-MA, and bafilomycin A, and further treated with bortezomib plus nutlin-3 with indicated concentration for 24 h. Cellular viability was assessed using calcein-AM and EthD-1. *P < 0.005 versus non treated groups.
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Figure 10. Each inhibitor did not alter the cytoplasmic vacuolation induced by bortezomib plus nutlin-3 in all the tested cells. Cells were untreated or pretreated with indicated concentrations of each inhibitor, including z-VAD-fmk, necrostatin-1, 3-MA, and bafilomycin A, and further treated with bortezomib plus nutlin-3 with indicated concentration for 24 h. And the cells were observed under the phase contrast microscopy. Bars, 10 μm
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2. Vacuolation induced by bortezomib plus nutlin-3 is derived from the dilation of both the ER and mitochondria
Recently, we have shown that curcumin or celastrol induces paraptosis, a cell death mode which is accompanied by the dilation of the ER and mitochondria, and proteasome inhibition plays a critical role in this cell death (Yoon et al., 2010;
Yoon et al., 2014). Since combination of bortezomib and nutlin-3 induced non-apoptotic cell death accompanied by extensive vacuolation, we next examined whether the cell death by bortezomib plus nutlin-3 is associated with paraptosis.
For this purpose, we employed the MDA-MB 435S sublines transfected with the YFP-ER plasmid and YFP-Mito plasmid for the labeling of the ER and mitochondria (YFP-ER cells and YFP-Mito cells). When we first performed the fluorescence microscopy in YFP-ER cells, the ER structures of reticular shapes were not noticeably affected by treatment with bortezomib or nultlin-3 alone (Figure 11A). In contrast, at 8 h of the combined treatment with bortezomib and nutlin-3, numerous fluorescent ER-derived vacuoles were generated. And with the increased incubation time, the sizes of the vacuoles were increased, whereas their numbers were reduced, suggesting that the fusion among the swollen ER might be progressed (Figure 11B and 11C). We further examined the changes in mitochondrial structures using the YFP-Mito cells. As shown in Figure 12, while mitochondria in non-treated YFP-Mito cells exhibited filamentous and elongated morphology, combined treatment with bortezomib and nutlin-3 induced mitochondrial dilation around the nuclei at 8 h. The intensity of mitochondrial fluorescence was somewhat weakened at 24 h, when cellular spaces were almost occupied with a few giant ER-derived vacuoles. In contrast, treatment with bortezomib alone induced mitochondrial fragmentation, rather than dilation.
Interestingly, treatment with nultlin-3 alone induced dilation of mitochondria at 8 h, although the sizes of mitochondria-derived vacuoles were smaller than those
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induced by bortezomib plus nutlin-3 at the same time. However, at 24 h of nultin-3 treatment, the sizes of mitochondria-derived vacuoles were noticeably reduced, suggesting that mitochondrial structures might be recovered from dilation, possibly via mitochondrial fission. These results suggest that combination of bortezomib and nultlin-3 induces paraptosis-like morphologies and nultlin-3 may contribute to mitochondrial dilation during this process. Dilation of the ER and mitochondria was further confirmed by the fluorescence microscopy after staining of YFP-ER cells treated with bortezomib and/or nutlin-3 for 8 h with MitoTracker Red (MTR) (Figure 13). To confirm whether dilation of the ER and mitochondria by the combination of bortezomib and nutlin-3 is not restricted to MDA-MB 435S cells, we performed immunocytochemistry using the specific antibodies against protein disulfide isomerase (PDI), an ER-resident protein, and cytochrome oxidase subunit II (COXII), a protein localized in the inner mitochondrial membrane in four different cancer cells, including MDA-M 435S, T98G, DLD-1, and HeLa cells. We found the expression of COXII with the small ring shape in the perinuclear area and PDI expression with larger ring shapes in all the tested cancer cells treated with bortezomib and nutlin-3 for 16 h (Figure 14). These results suggest that combination of bortezomib and nutlin-3 commonly induces paraptosis-like morphologies via dilation of the ER and mitochondria in many cancer cells with defective p53.
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Figure 11. Vacuolation induced by bortezomib plus nutlin-3 is mainly derived from dilation of the ER. (A) MDA-MB 435S sublines (YFP-ER/435S) expressing the fluorescence selectively in the ER were treated with bortezomib and/or nutlin-3 at the indicated concentrations and for the indicated time points and observed under the fluorescent and phase contrast microscope. (B) The average widths of the vacuoles originated from the ER were measured in YFP-ER cells treated with combination of bortezomib and/or nutlin-3 using AxioVision Rel. 4.8 software (Zeiss). (C) The average numbers of the vacuoles per cell were assessed in YFP-ER cells treated with bortezomib and/or nutlin-3. Bars, 20 μm.
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Figure 12. Combined treatment with bortezomib and nutlin-3 also induces the formation of mitochondria-derived vacuoles. MDA-MB 435S sublines (YFP-Mito/435S) expressing the fluorescence selectively in mitochondria were treated with combination of bortezomib and/or nutlin-3 for the indicated concentrations and time points and observed under the fluorescent and phase contrast microscope.
And morphologic difference of mitochondrial was quantified. Bars, 20 μm
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Figure 13. Cytoplasmic vacuolation is derived from dilation of both the ER and mitochondria. YFP-ER cells treated with 5 nM bortezomib and/or 30 μM nutlin-3 for 8 h were stained with 100 nM MitoTracker-red (MTR) and observed under the phase contrast and fluorescence microscopy. Bars, 20 μm
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Figure 14. Dilation of the ER and mitochondria is induced by bortezomib plus nutlin-3 in various cancer cells with defective p53. All the tested cells were treated with the indicated concentrations of bortezomib and/or nutlin-3 for 16 h.
Immunocytochemistry using anti-PDI and anti-COX II was performed and the representative images of cells are shown. Bars, 20 μm
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3. Protein synthesis is required for vacuolation and subsequent cell death by bortezomib plus nutlin-3
Since paraptosis is known to require protein synthesis (Sperandio et al., 2010), we next examined whether the cell death induced by bortezomib plus nutlin-3 is blocked by the protein synthesis inhibitor, cyclohexamide (CHX). We found that CHX pretreatment very effectively and commonly blocked the cell death induced by bortezomib plus nutlin-3 in MDA-MB 435S, T98G, DLD-1, and HeLa cells. In addition, CHX pretreatment almost completely blocked the dilation of both the ER and mitochondria in MDA-MB 435S cells treated with bortezomib and nutlin-3 (Figure 15, 16). These results suggest that the combined treatment with bortezomib and nutlin-3 may induce paraptosis-associated cell death.
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Figure 15. CHX pretreatment effectively blocks the cell death by bortezomib plus nutlin-3. Cells were pre-treated with or without CHX for the indicated concentrations and further treated with bortezomib plus nutlin-3 of indicated concentrations for 24 h. Cell viability was assessed using the Live/Dead assay. *P <
0.05 versus non-treat control group; #P < 0.005 versus bortezomib plus nutlin-3 group.
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Figure 16. CHX pretreatment effectively blocks the dilation of both the ER and mitochondria induced by bortezomib plus nutlin-3. YFP-ER cells were pre-treated with or without 2 μM CHX and further pre-treated with 5 nM bortezomib and 30 μM nutlin-3 for 8 h. Treated cells were stained with 100 nM MitoTracker-red and observed under the phase contrast and fluorescence microscopy. Bars, 20 μm
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4. CHOP induction critically contributes to the ER dilation and the cell death by bortezomib plus nultin-3
In an attempt to understand the underlying mechanisms by which combined treatment with bortezomib and nutlin-3 induces paraptosis-associated cell death, we first examined whether combined treatment induces ER stress in cancer cells, as reflected by extensive ER-derived dilation. We found that the protein levels of GRP78 and GRP94 were not noticeably affected by bortezomib and/or nutlin-3 in all the tested cancer cells (Figure 17). In contrast, while treatment with bortezomib or nutlin-3 alone slightly increased the protein levels of ATF4 and CHOP, combined treatment markedly much further increased their protein levels, suggesting that enhanced ER stress may be associated with this cell death by bortezomib plus nultlin-3 (Figure 17). We next examined whether bortezomib-induced proteasome inhibition is affected by co-treatment with nutlin-3. We found that while polyubiquitinated proteins were progressively accumulated by bortezomib treatment alone, combined treatment markedly increased their accumulation, suggesting that bortezomib-mediated impairment may be aggravated by co-treatment with nultlin-3, contributing to the enhanced ER stress. Since we previously showed that CHOP plays a critical role in ER-originated vacuolation and consequently paraptotic cell death induced by curcumin and dimethoxycurcumin (Yoon et al., 2014a), we tested the effect of CHOP knockdown on vacuolation and cell death by bortezomib plus nutlin-3. When we treated MDA-MB 435S cells with the lentivirus containing non-targeting shRNA (shNT) or CHOP-targeting shRNA (shCHOP) and further treated with bortezomib plus nutlin-3, the cell death by bortezomib plus nutlin-3 was markedly attenuated by CHOP knockdown (Figure 18A). Vacuolation induced by the combined treatment was markedly but not completely inhibited by CHOP knockdown (Figure 18B).
When we further examined the effect of CHOP knockdown on the dilation of the ER and mitochondria by the immunocytochemistry of PDI and COXII, we found
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that CHOP knockdown remarkably inhibited the dilation of the ER but not mitochondrial dilation (Figure 19). Taken together, these results show that increased expression of CHOP may critically contribute to ER dilation and subsequent cell death induced by the combined treatment with bortezomib and nutlin-3 in cancer cells.
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Figure 17. Nutlin-3 enhances bortezomib-induced accumulation of ubiquitin
Figure 17. Nutlin-3 enhances bortezomib-induced accumulation of ubiquitin