Study On The Mechanism Of Antipsychotic Drug Pimozide Inhibiting The Proliferation Of Human Osteosarcoma Cells

Background: Osteosarcoma(OS),a kind of primary malignant bone tumor,is usually happened in adolescents.It can become a very aggressive cancer and universally fatal if it is left untreated.Distant metastases developed subsequent to surgery for OS which make it difficult to treat and confer a poor prognosis.As well,it has been reported that the activation of STAT3 signaling was positively associated with poor prognosis and aggressive progression in OS patients.Targeting the activation of STAT3 signaling appeared to be an effective therapy for the treatment of OS.Additionally,drug repurposing,new applications for existing or abandoned pharmacotherapies,is one of the most important strategies used to treat cancer cells.Drug repurposing would bring new advances in drug discovery and development,making pharmaceutical research more predictable and reliable and less costly.Moreover,our previous studies have found that the psychotropic drug pimozide can inhibit STAT3 signaling activation to suppress the cell growth of hepatocellular carcinoma cells.Therefore,pimozide may be a potential drug for the treatment of OS.Objective:The purpose of this study was to investigate whether pimozide had anticancer effects on OS cells through the suppression of STAT3 signaling activity and to determine the possible molecular mechanism underlying.Methods:(1)The OS cell lines U2 OS,MG-63 and SW1353 was cultured in DMEM containing 10% FBS with 5% CO2 at 37°C in incubator,and then treated with different concentrations of pimozide.(2)The expression level of p-STAT3 was analyzed using Western blotting assay.The downstream targets of STAT3 signaling were detected by RT-PCR assay.Luciferase reporter gene system was used for determining cellular STAT3 activity.(3)The capacity of cell proliferation,apoptosis and cell cycle was evaluated by clone-forming,spheroid-forming assay and flow cytometric analysis.(4)ROS generation was detected by using the DCFH-DA and DHE staining.(5)The cell viability assay was used to investigate whether overexpression of Catalase in OS cells rescue the inhibitory effect of pimozide.(6)Ch IP assay was performed to analyze the potential binding site of STAT3 signaling in the promoter of the CAT gene.Results:(1)Pimozide suppressed STAT3 activity in OS cells,displaying downregulated the expression of p-STAT3,decreased STAT3-dependent luciferase activity,and reduced the transcriptional expression levels of the downstream targets of STAT3 signaling such as MYC?BCL-xl and MCL-1.(2)Pimozide inhibited the capacity of stemness in OS cells evaluated by colony-and sphere-formation ability.At the same time,pimozide suppressed the expression of Nanog,Sox2 and c-Myc,enhanced the sensitivity of OS cells to 5-Fluorouracil(5-FU)induced proliferative inhibition and decreased the ratio of side population(SP)in OS cells.(3)Furthermore,pimozide induced OS cell apoptosis and G0/G1 phase cell cycle arrest.(4)Pimozide suppressed the Extracellular signal Regulated Kinase(Erk)signaling to inhibit cell viability of OS cells(5)Pimozide induced ROS generation by downregulating the expression of the antioxidant enzyme CAT.NAC treatment partially reversed the ROS generation and cytotoxic effects induced by pimozide.The over-expression of CAT attenuated the pimozide-induced proliferation inhibition.(6)We searched for the potential binding site of STAT3 in the regulatory regions of the CAT gene and found two putative sites.Ch IP analysis showed that STAT3 was able to bind the promoter of CAT in OS cells.Conclusions:The results suggest that the psychotropic drugs pimozide has anticancer effect on OS cells,showing inhibited intracellular STAT3 signali ng.In addition,the results indicates the underlying molecular mechanism of pimozide.The decrease of CAT expression induced by pimozide was potentially mediated through the suppression of cellular STAT3 activity in OS cells.Thus,pimozide is a new STAT3 inhibitor that could suppress cellular STAT3 activity to inhibit OS cells or stem-like cells and will be a potential anti-cancer agent for OS treatment.