Mdivi-1 augments chlorpromazine's anticancer effect via modulation of mitochondrial dynamics, hallmarks of cancer-related cellular behavior, and oxidative stress.

The multifaceted therapeutic potential of phenothiazines was previously documented. This study investigated the anticancer effect of chlorpromazine (CPZ) in hepatoma and breast cancer cell lines and further explored the implications of mitochondrial dynamics. To establish this aim, the cytotoxic and antimitotic effects of CPZ combined with the mitochondrial fission inhibitor (mdivi-1) were assessed in HepG2 and MDA-MB-231 cells using MTT and clonogenic assays, respectively. Apoptosis and nuclear changes were evaluated with Annexin V/PI, acridine orange/ethidium bromide, and DAPI staining. Moreover, cell migration was examined using a wound-healing assay and the expression of epithelial-mesenchymal transition (EMT) markers. Additionally, 2',7'-dichlorodihydrofluorescein diacetate staining was used to assess cellular ROS, whereas the expression of mitochondrial fusion- and fission-related genes was assessed by RT-PCR. The results addressed CPZ's cytotoxic effect in both cancer cell types. The IC50 decreased when cells were concomitantly treated with CPZ and mdivi-1, where HepG2 cells were more responsive than MDA-MB-231 cells. Additionally, dual treatment impaired cell membrane integrity, caused chromatin condensation, increased apoptosis, reduced colony count, restricted cell migration, and downregulated the expression of the mesenchymal markers (vimentin and DDR2 genes). Although mdivi-1 alone upregulated mitochondrial fusion genes (MFN1/MFN2/OPA), combined treatment with CPZ, however, dysregulated both fusion and fission-related genes. The mechanisms underlying the anticancer activity of CPZ and the enhancing potential of mdivi-1 involved chromatin condensation, reduction of the antioxidant capacity, overproduction of cellular ROS, downregulation of Nrf2, and subsequent apoptosis. In conclusion, this work emphasizes the anticancer effect of CPZ and highlights the involvement of mdivi-1 in promoting the anticancer potential against liver and breast cancers.