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Background/Objectives: Cancer persists as a leading concern in the current medical field. As such, scientists are continuously researching new compounds with anticancer potential. In this study, we explored fifteen new 4-thiazolidinone derivatives as potential anticancer compounds. 4-Thiazolidinones are a well-established group of active structures, most commonly applied for the treatment of Parkinson's disease and diabetic neuropathy. However, they are actively researched as potential anticancer agents. A number of derivatives have qualified for Phase II and III clinical trials as antitumor agents. Methods: MTT cytotoxicity assay was applied to identify the most active compounds. Three out of the fifteen tested structures displayed a significant inhibitory effect on the MCF-7 and MDA-MB-231 cell lines. To further investigate the influence of compounds on breast cancer cells, we analyzed their capability to induce apoptosis using flow cytometry assessment with Annexin V and propidium iodide dyes. Next, flow cytometry analysis of JC-1 dye was utilized to research their capability to affect mitochondrial membrane. Afterwards, concentrations of important proapoptotic proteins such as Bax and cytochrome C were assessed with a highly sensitive ELISA method. Results: Further analysis with a fluorescent microscope displayed that novel compounds significantly increase the generation of reactive oxygen species. Conclusions: The results represented in this article displayed that the most active compounds positively affected the activation of the intrinsic apoptotic pathway in the tested breast cancer cells.
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