Figure 3 from Acetalax (Oxyphenisatin Acetate, NSC 59687) and Bisacodyl Cause Oncosis in Triple-Negative Breast Cancer Cell Lines by Poisoning the Ion Exchange Membrane Protein TRPM4

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posted on 2024-08-14, 10:08 authored by Makito Mizunuma, Christophe E. Redon, Liton Kumar Saha, Andy D. Tran, Anjali Dhall, Robin Sebastian, Daiki Taniyama, Michael J. Kruhlak, William C. Reinhold, Naoko Takebe, Yves Pommier

Plasma membrane permeabilization and mitochondrial changes caused by Acetalax. A, Representative flow cytometry data showing plasma membrane permeabilization induced by Acetalax within 5 minutes as measured by PI assays. Acetalax concentrations are color-coded as indicated. The x-axis shows PI absorbance, and the y-axis shows cell counts. The right shifts of the peaks in the MDA-MB468, BT549, and Hs578T sensitive cell lines indicate increased permeability. B, Flow cytometric mitochondrial membrane potential (MT-1) assay. Acetalax treatments were at 1 μmol/L for 5 and 60 minutes. Carbonyl cyanide-p-trifluoromethoxyphenylhydrazone (2 μmol/L for 60 minutes) was used as the positive control. The x-axis shows mitochondrial membrane potential, and the y-axis shows cell counts. The left shifts of the peaks seen in the sensitive cell lines indicate decreased mitochondrial membrane potential. Carbonyl cyanide-p-trifluoromethoxyphenylhydrazone, an uncoupler of oxidative phosphorylation, was used as the positive control. C, ATP depletion. Cells were treated with Acetalax 60, 120, and 240 minutes at 1 μmol/L. ATP depletion was calculated as the percentage change compared with the untreated group as measured by CellTiter-Glo 2. D, Mitochondrial fragmentation. Cells were treated with Acetalax for 4 hours at 10 μmol/L. Fragmentation volume percentage was calculated as the total fragmented surface volume divided by the total mitochondrial volume per cell. Percentage of fragmented mitochondria on the y-axis represents the comparison between control and Acetalax-treated groups. A, Significant change in mitochondrial fragmentation is indicated by the brackets. *, P < 0.01; **, P < 0.005; ***, P < 0.0001 (using the Mann–Whitney U test, calculated in Prism 9.0). Center lines indicate medians. FCCP, Carbonyl cyanide-p-trifluoromethoxyphenylhydrazone.

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ARTICLE ABSTRACT

Triple-negative breast cancer (TNBC) is clinically aggressive and relatively unresponsive to current therapies. Therefore, the development of new anticancer agents is needed to satisfy clinical needs. Oxyphenisatin acetate (Acetalax), which had been used as a laxative, has recently been reported to have anticancer activity in murine models. In this study, we demonstrate that Acetalax and its diphenolic laxative structural analogue bisacodyl (Dulcolax) exhibit potent antiproliferative activity in TNBC cell lines and cause oncosis, a nonapoptotic cell death characterized by cellular and nuclear swelling and cell membrane blebbing, leading to mitochondrial dysfunction, ATP depletion, and enhanced immune and inflammatory responses. Mechanistically, we provide evidence that transient receptor potential melastatin member 4 (TRPM4) is poisoned by Acetalax and bisacodyl in MDA-MB468, BT549, and HS578T TNBC cells. MDA-MB231 and MDA-MB436 TNBC cells without endogenous TRPM4 expression as well as TRPM4-knockout TNBC cells were found to be Acetalax- and bisacodyl-resistant. Conversely, ectopic expression of TRPM4 sensitized MDA-MB231 and MDA-MB436 cells to Acetalax. TRPM4 was also lost in cells with acquired Acetalax resistance. Moreover, TRPM4 is rapidly degraded by the ubiquitin–proteasome system upon acute exposure to Acetalax and bisacodyl. Together, these results demonstrate that TRPM4 is a previously unknown target of Acetalax and bisacodyl and that TRPM4 expression in cancer cells is a predictor of Acetalax and bisacodyl efficacy and could be used for the clinical development of these drugs as anticancer agents. Acetalax and bisacodyl kill cancer cells by causing oncosis following poisoning of the plasma membrane sodium transporter TRPM4 and represent a new therapeutic approach for TNBC.