Methylated Actinomycin D Induces Endoplasmic Reticulum Stress and Caspase-Dependent Apoptosis in Human Breast Cancer Cells

<title>Abstract</title> <p> Actinomycin D (Act D) is a clinically established antitumor agent used for treating highly malignant tumors. However, its severe cytotoxicity limits its therapeutic application. Methylated actinomycin D (mAct D), a novel analog derived from <italic>Streptomyces sp.</italic> KLBMP 2541, exhibits reduced cytotoxicity and induces apoptosis of HepG2 cells via Fas- and mitochondria-mediated apototic pathways. Despite these findings, the precise anticancer mechanisms of mAct D in breast cancer (BC) cells remain unclear. So, we investigated the effects of mAct D on human BC cells, focusing on its ability to induce endoplasmic reticulum stress (ERS) and Caspase-dependent apoptosis in this study. Cell viability was detected using alamar blue assay and colony formation test. Cell apoptosis was measured through PI staining and Flow cytometry. To determine the changes in the expression of key factors in signaling pathway, Western blot was performed. Our results demonstrated that mAct D remarkably suppressed the viability of MDA-MB-231 and MCF-7 cells in a manner dependent on both dose and treatment duration. As determined by Flow cytometry and Western blot analysis, mAct D triggered apoptosis through the downregulating Bcl-2, activating Caspase-3/9, and cleaving PARP. Moreover, the expression of ERS markers including p-elF2α, GRP78, and CHOP was upregulated by mAct D. The cytotoxic effects of mAct D were substantially diminished by pretreatment with Caspase inhibitors (Ac-DEVD-CHO and Z-LEHD-FMK) and ERS inhibitor (4-PBA), confirming the role of ERS and Caspase-dependent pathways. mAct D exerts potent antitumor effects in BC cells by inducing ERS and activating Caspase-dependent apoptotic pathways, suggesting its potential as a therapeutic agent for breast cancer treatment. </p>