Clohydroxyquine Inhibits The Proliferation And Migration Of Malignant Melanoma By Activating PPAR?

Melanoma accounts for over 80% of skin cancer-deaths,and its incidence is increasing worldwide.Current therapies,especially those involving clinical chemotherapy drugs,have achieved remarkable advances.However,their side effects,such as bone marrow suppression,limit the effectiveness of available pharmacological therapies.Therefore,exploring new antimelanoma drugs is critical for the prevention and treatment of melanoma.Here,based on the principle of drug reposition,we screened 1430 small compounds from the FDA-approved drug library for the ability to repress the proliferation of mouse B16F10 melanoma cells.Among these,cloxiquine(CLQ),a traditional drug used to treat tuberculosis,exhibited strong inhibition effects on the growth of B16F10 cells,then EdU incorporation was used to determine cell proliferation rate,Wound-healing and transwell assay were administered to detect cell migration rate.At the molecular level,the protein expression levels of cell cycle positive regulators CDK/Cyclin complex and negative regulators of the CDKN1family(e.g.p27 and p21)and migration-associated proteins(MMP2/9,ICAM-1/VCAM-1)were analyzed by using western blot.Furthermore,we tried to use the bioinformatic analyses to find the potential drug target of CLQ,and to explore the mechanism of CLQ's inhibition effects with PPAR?'s specific inhibitor.we found that CLQ can inhibite the growth and metastasis of mouse B16F10 cells and human A375 in vitro in a dose-dependent manner,and it did not affect the viability of normal Melan-A and PIG1 melanocytes;Next,we found that CLQ can also inhibite the growth and metastasis of mouse B16F10 cells in vivo by using B16F10 tumor xenograft models and a B16F10 melanoma lung metastasis mouse model.At the molecular level,CLQ can decrease the protein expression levels of cell cycle positive regulators(CDK/Cyclin complex)and migration related protein(MMP2/9,ICAM-1/VCAM-1)in a concentration-dependent manner.In contrast,the protein levels of cell cycle negative regulators(p27/p21)were correspondingly increased by CLQ treatment.Bioinformatics analyses revealed that peroxisome proliferator-activated receptor-gamma(PPAR?)served as a potential CLQ target,and its specific inhibitor GW9662 partially abolished the antimelanoma effects of CLQ.More importantly,treatment with CLQ decreased glycolysis(decreased extracellular acidification rate(ECAR)),thus inhibiting the “Warburg effect” in B16F10 cells.Futhermore,the specific PPAR? inhibitor GW9662 can alleviate the impairment of glycolysis similarly occurred in the CLQ-treated B16F10 cells,suggesting that PPAR?plays an important role in the CLQ's antimelanoma function.Taken together,our findings imply that CLQ has great potential in the treatment of melanoma by inhibiting the “Warburg effect” through the activation of PPAR?signaling.