Figure 2 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

Morphologic changes caused by Acetalax in TNBC cell lines with differential sensitivity. A, Representative microscopy images of cell lines treated with Acetalax (10 μmol/L for 30 minutes) and stained in blue with DAPI (binds DNA) and green with phalloidin (stains actin). Control, without Acetalax treatment. Scale bars, 50 μm. (s), most Acetalax-sensitive cell lines; (r), most Acetalax-resistant cell lines (Fig. 1B). B, Top, Violin plots for the quantitation of Acetalax-induced cell size increase at 5 and 30 minutes. The numbers represent the number of cells analyzed. The lines indicate medians. Each dot is a cell. MDA-MB468, BT549, and Hs578T have significant changes in cell size. Bottom, Violin plots for the quantitation of Acetalax-induced nuclear size increase at 5 and 30 minutes. Each dot is a nucleus. The numbers represent the number of nuclei analyzed. Nuclear size increase between the control and Acetalax-treated cells significantly for MDA-MB468, BT549, and Hs578T. C, Microscopy of membrane blebbing caused by Acetalax (10 μmol/L for 30 minutes). Control, untreated cells; red arrows, cell membrane blebs. The images are chosen to fill the frames for improved visibility without normalization by scale. Scale bars, 50 μm. D, Quantitation of membrane blebbing in multiple cells as shown in C. In the most sensitive (s) cell lines, MDA-MB468, BT549, and Hs578T, Acetalax treatment significantly increased the frequency of bleb cells. Statistical significance: *, P < 0.05; **, P < 0.01; ***, P < 0.005; ****, P < 0.001; *****, P < 0.0005; ******, P < 0.0001 (using the Mann–Whitney U test, calculated in PRISM 9.0).

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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.