Bafilomycin A1 Targets Hepatocellular Carcinoma

Objectives:Hepatocellular carcinoma(HCC)is the third most common malignant tumor and causes the second most cancer deaths in China.Due to the lack of clinical symptoms and rapid development,more than 90% of HCC patients are diagnosed at advanced stages,leading to low surgical excision rate;recurrence rate is very high even though they have surgery.Limitations of the common used chemotherapy drugs at present include poor sensitivity,high cytotoxicity,and poor tolerance,as most patients with HCC are accompanied with liver cirrhosis,making it is hard to reach optimal therapeutic effect.Therefore,seeking effective drugs with least adverse effects is of great clinical significance.Bafilomycin A1(Baf A1)is a specific vacuolar H + ATPase(V-ATPase)inhibitor that is a marcrolide antibiotic in nature.It is frequently used as autophagy inhibitor at high concentration(0.1-1 μM)to block autophagosomelysosome fusion.Baf A1 has been proven to be cytotoxic to a variety of tumor cells.However,in order to achieve the effective inhibitory effects on cancer cell growth and/or autophagic degradation,BafA1 is usually required at high concentrations(> 0.1μM),which may induce severe acidosis and secondary adverse effects in normal cells,thereby hindering its application in clinical trials.The aim of this study is to explore therapeutic effects of low concentration of Baf A1(5 n M)on HCC cells and the underlying mechanisms,providing experimental and theoretical basis for BafA1 as anew therapy for hepatocellular carcinoma.The main questions we want to solve are as follows:(1)to explore the proliferation inhibition effect of low concentration ofBaf A1;(2)to explore the therapeutic effect of low concentration of BafA1 on xenografts derived from HCC cells;(3)to illustrate the mechanisms underlying BafA1 induced hepatocellular carcinoma cell death.Methods:1.The inhibitory effect of low concentration of BafA1 on HCC cell proliferationMTT and colony formation assays were carried out to determine the effect of low concentration of BafA1 on HCC cell proliferation.HCC cells in 3D culture condition to form spheroids were performed to test the effect of low concentration of Baf A1 on spheroids formation and the cytotoxic effect on spheroids cells.2.The inhibitory effect of low concentration of Baf A1 on HCC growth in xenografts derived from HCC cellsNude mice were subcutaneously inoculated HCC cells to induce tumor development.Tumor-bearing mice were divided into four groups: control,JNK inhibition,Baf A1 alone and JNK inhibitor combined with Baf A1 to investigate the inhibitory effect of Baf A1 on the growth of xenografts according to tumor volume.After treatment,tumor sections were subjected to either HE or TUNEL staining assay to analyze the histological changes.3.The regulatory effect of Baf A1 on cell cycleBaf A1-treated HCC cells were stained with propidium iodide and cell cycle analyzed by FACS.Changes of G1 phase regulatory protein levels were detected by western blot.After using inhibitor or si RNA knockdown,we tested the changes of Cyclin D1 levels via western blot and cell proliferation via MTT assays to investigate the mechanism of BafA1 on HCC cell cycle arrest.4.The effect of BafA1 on HCC cell apoptosisBafA1-treated HCC cells were analyzed by flow cytometry with FITC-conjugated Annexin-V and PI double staining to determine the effect of Baf A1 on HCC cell apoptosis.To confirm the apoptosis type,two classical apoptosis markers,caspase-3activation and Poly(ADP-ribose)polymerase(PARP)cleavage were detected by western blot and the changes of cell death after pre-treatment with a broad-specificity caspase inhibitor were observed.We also examined the expression levels of the pro-survival proteins and pro-apoptotic proteins.5.The effect of BafA1 on HCC cell autophagyWestern blot,Transmission electron microscopy(TEM),confocal microscopy,GFP-LC3 and GFP-m RFP-LC3 transfection of HCC cells,lentivirus mediated table LC3 knockdown cell line construction and MTT assays were performed to explore the effect of low concentration of Baf A1 on cell autophagy and the corresponding mechanisms.6.Cell signaling pathways involved in BafA1 induced HCC cell deathMAPK pathways were examined by western blot,cell apoptosis was measured by flow cytometry after MAPK inhibition and MTT assays were performed to examine the cell proliferation upon p38 knockdown.All these were aimed to understand the effect of BafA1 on MAPK pathways,the signaling pathways that BafA1 targets and the detailed mechanism of MAPK pathways involved HCC cell death.Results:1.5 nM BafA1 was sufficient to inhibit HCC cell growth while there was no effect on normal human liver cells.Moreover,Baf A1-treated HCC cells did not form spheroids under 3D culture conditions and if established spheroids were treated with Baf A1,both the number and HCC spheroid volume were significantly reduced over time and large amounts of the cells in Baf A1-treated HCC spheroids were dead.2.BafA1 markedly abrogated the progression of established xenografts and JNK inhibitor,further enhanced this Baf A1-mediated decrease in tumor size.In addition,harvested tumors showed increased inflammatory cell infiltration and cell death in tumors treated with BafA1 alone,or in combination with JNK inhibitor.3.Baf A1 induces HCC cell G1 phase arrest.Consistently,exposure to Baf A1 induced a time-dependent reduction in G1 phase regulatory proteins such as Cyclin D1,CDC6 and pRb.Notably,adenovirus-mediated overexpression of Cyclin D1 impaired Baf A1-mediated inhibitory effects on the growth of HCC cells.In addition,further study demonstrated that DYRK1 B promotes the turnover of Cyclin D1 in a GSK3β-independent manner.4.Low concentration of BafA1 could induce HCC cell apoptosis but two classical apoptosis markers,caspase-3 activation and PARP cleavage,were not observed and pre-treatment with a broad-specificity caspase inhibitor Z-VAD-FMK had no obvious effect on Baf A1-induced cell death in HCC cells,indicating Baf A1 triggered caspase-independent cell death.Baf A1 treatment downregulated the levels of pro-survival proteins Bcl2 and Bcl-XL in HCC cells and the levels of several pro-apoptotic proteins such as Bax,Bid and Bim,but Puma expression levels were upregulated.5.BafA1 is usually used at high concentration as autophagy inhibitor during late stage of autophagy.Our study found that Baf A1,at low concentrations,blocked autophagosome-lysosome fusion in HCC cells.Besides,Baf A1 treatment(5 n M)inhibited the activation of the class I PI3K/Akt/mTOR/p70S6 K signaling pathway in HCC cell lines,resulting in autophagy induction in HCC cells.Inhibition of autophagosome formation decreased Baf A1-mediated cell death,indicating that low concentration of Baf A1 induced HCC cell death by targeting autophagy.6.In addition to targeting autophagy,Baf A1 also targets p38 MAPK signaling pathway to induce cell death.The three major mitogen-activated protein kinase(MAPK)pathways were all activated after treatment by low concentration of Baf A1.Pre-treatment of HCC cells with JNK inhibitor significantly enhanced Baf A1-induced cell death,suggesting that the combination of Baf A1 with a JNK inhibitor might be a potential strategy for HCC treatment.Moreover,the p38 MAPK inhibitor,consistent with genetic ablation of p38α in HCC cells significantly abrogated this Baf A1-mediated Puma effect on HCC cell death.p38 inhibitor substantially abolished Baf A1-induced upregulation of Puma in HCC cells.In addition,knockdown of Puma in HCC cells significantly inhibited the cytotoxicity exerted by BafA1 while overexpression of Puma enhanced BafA1-mediated inhibitory effect on the growth of HCC cells,indicating that p38 regulated BafA1-mediated cytotoxicity of HCC cells by Puma.Conclusions:BafA1 at nanomolar concentrations substantially inhibits the growth of HCC cells in both 2D and 3D cultures and in mouse models.BafA1 induces caspase-independent HCC cell death by targeting autophagy and MAPK pathways.Baf A1 is a promising drug candidate for HCC treatment.