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. 2019 Dec 4;20(24):6112.
doi: 10.3390/ijms20246112.

Lercanidipine Synergistically Enhances Bortezomib Cytotoxicity in Cancer Cells via Enhanced Endoplasmic Reticulum Stress and Mitochondrial Ca2+ Overload

A Reum Lee  1   2 , Min Ji Seo  1   2 , Jin Kim  1   2 , Dong Min Lee  1   2 , In Young Kim  1 , Mi Jin Yoon  1 , Hur Hoon  2   3 , Kyeong Sook Choi  1   2
Affiliations

Lercanidipine Synergistically Enhances Bortezomib Cytotoxicity in Cancer Cells via Enhanced Endoplasmic Reticulum Stress and Mitochondrial Ca2+ Overload

A Reum Lee et al. Int J Mol Sci. .

Abstract

The proteasome inhibitor (PI), bortezomib (Btz), is effective in treating multiple myeloma and mantle cell lymphoma, but not solid tumors. In this study, we show for the first time that lercanidipine (Ler), an antihypertensive drug, enhances the cytotoxicity of various PIs, including Btz, carfilzomib, and ixazomib, in many solid tumor cell lines by inducing paraptosis, which is accompanied by severe vacuolation derived from the endoplasmic reticulum (ER) and mitochondria. We found that Ler potentiates Btz-mediated ER stress and ER dilation, possibly due to misfolded protein accumulation, in MDA-MB 435S cells. In addition, the combination of Btz and Ler triggers mitochondrial Ca2+ overload, critically contributing to mitochondrial dilation and subsequent paraptotic events, including mitochondrial membrane potential loss and ER dilation. Taken together, our results suggest that a combined regimen of PI and Ler may effectively kill cancer cells via structural and functional perturbations of the ER and mitochondria.

Keywords: ER stress; bortezomib; lercanidipine; mitochondrial Ca2+ overload; paraptosis.

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Conflict of interest statement

The authors declare no conflict of interest.

Figures

Figure 1
Figure 1
Lercanidipine (Ler) sensitizes various cancer cells, but not normal cells, to proteasome inhibitor (PI)-mediated cell death. (A,C,E) Cells were treated with the indicated concentrations of PIs and/or Ler for 24 h and cellular viability was assessed using the IncuCyte as described in Materials and Methods. The percentage of live cells was normalized to that of untreated control cells (100%). Data represent the means ± S.D. (n = 7). One-way ANOVA and Bonferroni’s post hoc test. * p < 0.001 vs. PI treated cells. (B,D) Isoboles for the combination of PIs and Ler that proved iso-effective (IC50) for inhibiting cell viability.
Figure 2
Figure 2
A combination of a 1,4-dihydropyridines (DHPs) and bortezomib (Btz) selectively induces cancer cell death in breast and liver cells. (A,C,D,F) Cells were treated with the indicated concentrations of Btz and/or DHPs for 24 h and cellular viability was assessed using the IncuCyte as described in Materials and Methods. The percentage of live cells was normalized to that of untreated control cells (100%). Data represent the means ± S.D. (n = 7). One-way ANOVA and Bonferroni’s post hoc test. * p < 0.001 vs. PI treated cells. (B,E) Isoboles for the combination of Btz and DHPs that proved iso-effective (IC50) for inhibiting cell viability.
Figure 3
Figure 3
Btz/Ler induces the cancer cell death accompanied by cytoplasmic vacuolation. (A–C) Cellular morphologies were observed by phase-contrast microscopy. Bars, 20 μm. (A) Cells were treated with 4 nM Btz and/or 10 μM for 24 h. (B) Cells were treated with 20 nM Btz and/or 10 μM for 24 h. (C) Cells were treated with PIs (for MDA-MB 435S and MCF-10A cells, 20 nM Cfz, 100 nM Ixz, or 4 nM Btz; for SNU-475 and Chang cells, 20 nM Cfz, 100 nM Ixz, or 20 nM Btz) and/or 10 μM DHPs for 24 h. (D,F) MDA-MB 435S cells were treated with 5 μg/mL doxorubicin or 4 nM Btz and/or 10 μM Ler for 24 h. Western blotting of the indicated proteins was performed, with β-actin used as a loading control. (E,G) Cells were pretreated with the indicated inhibitors and further treated with the indicated concentrations of Btz/Ler for 24 h. (E) Cellular viability was assessed using the IncuCyte system. Data represent the means ± S.D. (n = 7). One-way ANOVA and Bonferroni’s post hoc test. * p < 0.001 vs. untreated cells. (G) Cellular morphologies were observed by phase-contrast microscopy. Bars, 20 μm.
Figure 4
Figure 4
Btz/Ler induces the features of paraptosis in MDA-MB 435S cells. (A,B) YFP-ER or YFP-Mito cells were treated with 4 nM Btz and/or 10 μM Ler for the indicated time durations and then stained with MitoTracker-Red (MTR) or TMRM. Cells were observed by confocal microscopy. Bars, 20 μm. White arrowheads indicate the dilated mitochondria whose mitochondrial membrane potential (MMP) is maintained and blue arrows denote the dilated mitochondria whose MMP is lost. (C) Cells were pretreated with cycloheximide (CHX) and further treated with the indicated concentrations of Btz/Ler for 24 h. Cellular viability was assessed using the IncuCyte system. Data represent the means ± S.D. (n = 7). One-way ANOVA and Bonferroni’s post hoc test. * p < 0.001 vs. untreated cells; # p < 0.05 vs. Btz/Ler-treated cells. (D) MDA-MB 435S cells were untreated or pretreated with 2 μM CHX and further treated with 4 nM Btz and/or 10 μM Ler for 12 h. Cellular morphologies were observed by phase-contrast microscopy. Bars, 20 μm. (E) YFP-Mito and YFP-ER cells were pretreated with 2 μM CHX and further treated with 4 nM Btz plus 10 μM Ler for 12 h. Cells were observed by confocal microscopy. Bars, 20 μm.
Figure 5
Figure 5
Ler enhances Btz-mediated ER stress. (A) MDA-MB 435S cells were treated with 4 nM Btz and/or 10 μM Ler for the indicated time durations. Western blotting of the indicated proteins was performed, with β-actin used as a loading control. (B) MDA-MB 435S cells were treated with 4 nM Btz and/or 10 μM Ler for 16 h. Immunocytochemistry of Ub was performed. Bar, 20 μm.
Figure 6
Figure 6
Mitochondrial Ca2+ overload triggers mitochondrial dilation in Btz/Ler-induced paraptosis. (A,B) Cells were treated with 4 nM Btz and/or 10 μM Ler for the indicated time points. Treated cells were stained with Rhod-2 and subjected to confocal microscopy (A) and flow cytometry (B). Bars, 20 μm. (C) MDA-MB 435S cells were pretreated with the indicated Ca2+ antagonists, and further treated with 4 nM Btz and/or 10 μM Ler for 24 h. Cellular viability was assessed using the IncuCyte as described in Materials and Methods. Data represent the means ± S.D. (n = 7). One-way ANOVA and Bonferroni’s post hoc test. * p < 0.001 vs. untreated cells; # p < 0.05 vs. Btz/Ler-treated cells. (D) YFP-Mito cells were untreated or pretreated with 20 μM Ru360, 50 μM dantrolene (Dant), or 40 μM 2-APB, and further treated with 4 nM Btz plus 10 μM Ler for 8 h. Treated cells were stained with Rhod-2 and subjected to confocal microscopy. Bars, 20 μm. (E) YFP-Mito and YFP-ER cells were untreated or pretreated with 20 μM Ru360, 50 μM Dant, or 40 μM 2-APB, and further treated with 4 nM Btz plus 10 μM Ler for 12 h. Treated cells were observed by confocal microscopy. Bars, 20 μm.
Figure 7
Figure 7
A combination of Btz and Ler induces disruption of Ca2+ homeostasis during paraptosis. (A,B) Cells were treated with 4 nM Btz and/or 10 μM Ler for the indicated time points. Treated cells were stained with Fluo-3 and subjected to confocal microscopy (A) and flow cytometry (B). Bars, 20 μm. (C) MDA-MB 435S cells were pretreated with the 10 μM BAPTA-AM, and further treated with 4 nM Btz and/or 10 μM Ler for 24 h. Cellular viability was assessed using the IncuCyte as described in Materials and Methods. Data represent the means ± S.D. (n = 7). One-way ANOVA and Bonferroni’s post hoc test. * p < 0.001 vs. untreated cells; # p < 0.05 vs. Btz/Ler-treated cells. (D) YFP-Mito and YFP-ER cells were pretreated with 10 μM BAPTA-AM and further treated with 4 nM Btz plus 10 μM Ler for 12 h. Cells were observed under the confocal microscope. Bars, 20 μm.
Figure 8
Figure 8
Differences in the unfolded protein response and Ca2+ modulation may contribute to the differential effect of Btz/Ler in cancer cells and normal cells. (A) Cells were treated with 4 nM Btz and/or 10 μM Ler for 8 h. Western blotting of the indicated proteins was performed, with β-actin used as a loading control. (B,C) Cells were treated with 4 nM Btz and/or 10 μM Ler for 8 h (B) or 24 h (C). Treated cells were stained with NAO/Rhod-2 (B) or Fluo-3 (C) and subjected to confocal microscopy. Bars, 20 μm.
Figure 9
Figure 9
Mitochondrial Ca2+ overload and enhanced ER stress are critical for Btz/Ler-induced paraptosis. (A–C) YFP-Mito cells were pretreated with 20 μM Ru360 or 2 μM CHX and further treated with 4 nM Btz plus 10 μM Ler for 8 h (A,B) or 24 h (C). (A,B) Cells were stained with Rhod-2 and subjected to confocal microscopy (A) or flow cytometry (B). Bars, 20 μm. (C) Treated cells were stained with TMRM and observed by confocal microscope. Bars, 20 μm. (D) MDA-MB 435S cells were pretreated with 2 μM CHX, 20 μM Ru360, or 10 μM BAPTA-AM and further treated with Btz/Ler for 8 h. Western blotting of the indicated proteins was performed, with β-actin used as a loading control. (E) MDA-MB 435S were pretreated with 20 μM Ru360 or 2 μM CHX and further treated with Btz/Ler for 24 h. Treated cells were stained with Flou-3 and subjected to flow cytometry. (F) Hypothetical model of the signaling pathways involved in Btz/Ler-induced paraptosis. The blue arrows pointing up, the red “T” lines, the grey dotted arrows, and the grey arrows indicate increase, inhibition, possible regulation, and activation, respectively.
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