Proguanil Regulates Autophagy Mediated By The MTOR Pathway To Inhibit The Growth Of Bladder Cancer

Purpose: Proguanil(Pro)is considered to be a common anti-malaria drug.In recent years,due to its anti-tumor activity,biguanides have attracted wide attention and become a research hotspot of anti-tumor drugs.Studies have shown that among the biguanide compounds including metformin,proguanil has the strongest inhibitory effect on tumors.However,the current anti-tumor mechanism of proguanil is still unclear.There is evidence that the autophagy-related m TOR signaling pathway affects bladder tumor growth.Therefore,in this study,we explored the effect of proguanil on autophagy of bladder cancer cells and the relationship between autophagy and the anti-tumor activity of proguanil,and preliminarily explored the anti-tumor mechanism of proguanil to provide a theoretical basis for the clinical treatment of proguanil.Methods: Proguanil alone or together with autophagy inhibitor 3-methylpurine(3MA),chloroquine(CQ)and autophagy inducer rapamycin(Rapa)to treat two types of bladder cancer cells(T24 and J82).The protein expressions of p-m TOR,p-P70S6 K,p-4EBP1,Beclin1,p62 and LC3 were detected by Western blotting;using MTT method,clone formation experiment,transwell experiment to evaluate the activity of bladder cancer cells;Transmission electron microscopy and autophagy staining were used to detect the morphology of autophagosomes;immunofluorescence was used to detect the expression of LC3 protein;After subcutaneous tumor transplantation,changes in tumor volume and weight were measured and tumor marker Ki67 was evaluated to evaluate the therapeutic effect of proguanil.The toxicity of the drug in vivo was evaluated by the changes of mouse body weight and pathological analysis of liver and kidney tissues.The expression of LC3 was detected by immunohistochemistry.Results: We found that proguanil treatment inhibited the growth of bladder cancer cells in a dose-dependent manner.In addition,the presence of autophagosomes was observed after proguanil treatment,and the expression of autophagy-related proteins Beclin1 and LC3 was significantly activated.Immunofluorescence assay further confirmed that proguanil enhanced the expression of autophagy protein LC3,while the autophagy inhibitor 3MA could weaken the increase of LC3 expression induced by proguanil.At the same time,after chloroquine treatment,the degradation of autophagy protein LC3 was blocked,and LC3 protein was further increased.More importantly,proguanil treatment inhibited the proliferation,colony formation and migration of bladder cancer cells,and the inhibition of autophagy by 3MA and CQ reduced the toxicity of proguanil on bladder cancer tumor cells.However,when treated with autophagy inducer,the inhibitory effect on bladder cancer cells was enhanced.In vivo experiments confirmed that proguanil induces autophagy and inhibits the growth of subcutaneously transplanted tumors in nude mice,while 3-MA attenuates this effect.Conclusions: We have confirmed through in vivo and in vitro experiments that autophagy plays a cytotoxic effect in the treatment of bladder cancer cells by proguanil.Our results suggest that proguanil suppresses tumor growth by inducing autophagy,which may be an effective treatment strategy for bladder cancer.