The Mechanism Of Pimozide Inhibits Proliferation In Breast Cancer Cells

As an antipsychotic,pimozide has been widely confirmed to inhibit a variety of cancers.Previous studies have shown that pimozide can inhibit breast cancer cell proliferation.However,as far as we know,pimozide has not been reported to function through the RAF/ERK signaling pathway,and the effect of pimozide on autophagy in breast cancer cells.Objective: The purpose of this study was to determine whether pimozide exerts anticancer activity via the RAF/ERK pathway and to investigate whether pimozide promotes apoptosis in breast cancer cells by inducing autophagy.Methods: The UALCAN and Kaplan-Meier databases were used to screen for genes associated with breast cancer prognosis,and pimozide was further docked to the selected genes based on Schrodinger software.The effects of pimozide on proliferation,migration and invasion of breast cancer cells were examined by 4,5-dimethylthiazol-2-yl-3,5-diphenylformazan(MTT),wound healing,colony formation,transwell assay and flow cytometry.Using JC-1 analysis,we investigated the changes in membrane potential of breast cancer cells by pimozide.Identification of pimozide-induced breast cancer cells in the apoptotic phase based on acridine orange and ethidium bromide(AO/EB)staining.Transmission electron microscopy(TEM)and monodansylcadaverine(MDC)staining were used to observe autophagosomes.The FRET system detected the effect of pimozide on cyclic adenosine monophosphate(cAMP)in breast cancer cells.Western blot investigated the effect of pimozide on the expression of apoptosis-and autophagy-related proteins in breast cancer cells.Results: Pimozide inhibited the proliferation,migration,and invasion of breast cancer cells,promoted the decrease of mitochondrial membrane potential and apoptosis of breast cancer cells.Pimozide promoted apoptotic activity by down-regulating the phosphorylation of RAF1 and ERK 1/2,resulting in decreased expression of anti-apoptotic proteins Bcl-2 and Bcl-xl,up-regulation of the pro-apoptotic protein Bax,and activation of the apoptosis-initiating signaling protein caspase-9.The Hippo pathway proteins YAP/TAZ,MST1,and MST2 were not regulated by pimozide.Pimozide also promoted autophagy in breast cancer cells by up-regulating cAMP,resulting in increased conversion of LC3-I to LC3-II and down-regulation of P62 expression,but mTOR signaling was not involved in this process.After chloroquine(CQ)blocked autophagy in breast cancer cells,pimozide-induced apoptosis was impaired.Conclusions: These data show that pimozide inhibits breast cancer by regulating the RAF/ERK signaling pathway,and also relies on cAMP-induced autophagy to promote apoptosis in breast cancer cells.These results suggest that pimozide may be a potential drug for breast cancer treatment.