| Objective:Hepatocellular carcinoma(HCC)is a major global health problem,with approximately 600,000 new cases being diagnosed as HCC each year.In 2018,the global mortality rate was approximately 8.2%,ranking fourth among all cancer deaths.The number of new cases in China each year accounts for more than 50%of the global number of new cases.Early HCC is usually asymptomatic,with symptoms of liver pain,poor appetite,and fatigue in the middle and late stages.Most patients are at the advanced stage at the time of diagnosis,and missed the opportunity for surgery.Paclitaxel is a natural product isolated from taxus pacific,ian antitumor drug with a unique structure and mechanism of action,played a significant effect in clinical treatment.Paclitaxel often causes abnormal activation of intracellular phosphoinositide-3-kinase/threonine protein kinase/mammalian rapamycin(PI3K/Akt/mTOR)pathway.Abnormal activation of Akt and mTOR promoted cell proliferation and anti-apoptosis,thereby reduced the effectiveness of antitumor drugs.Dactolisib(BEZ235),a dual ATP-competitive PI3K and mTOR inhibitor.We studied the effects of Paclitaxel combination BEZ235 on the cell viability,migration,cycle and apoptosis of HCC cells in vivo and in vitro,explored the molecular mechanism of related effects,providing new ideas for clinically improving the sensitivity of paclitaxel chemotherapy.Methods:1.Phosphoprotein microarray analysis detected related signaling pathways activated by paclitaxel on HCC cells.2.MTT measured the proliferation ability of BEZ235 and paclitaxel alone and in combination.3.Scratch healing and Transwell assessed the inhibitory effect of the combination of the two drugs on the migration ability of HCC cells.4.Flow cytometry detecte the cell cycle and evaluate the effect of the combination of two drugs on the cell cycle distribution.5.Flow cytometry and fluorescence microscopy(JC-1 staining)detected the changes in mitochondrial membrane potential,and evaluated the effect of the combination of two drugs on mitochondrial apoptosis.6.Flow cytometry and fluorescence microscopy(Annexin-V FITC/PI staining)detected the effects of the combination of two drugs on the activation of Caspase-dependent pathways.7.Tumor-bearing mice further verified the inhibitory effect of paclitaxel and BEZ235 on tumor growth.Results:1.CSP100 results showed that paclitaxel caused abnormal activation of the PI3K/mTOR pathway in HepG2 cells after 24 h.WB results also confirm that paclitaxel stress activated the PI3K/mTOR pathway in HepG2 cells.2.BEZ235 at different concentrations acted on HepG2 cells for 24 h,significantly inhibited the activation of the PI3K/mTOR pathway and induced apoptosis3.Paclitaxel combined with different concentrations of BEZ235 further inhibited the inhibition of PI3K/mTOR pathway activation and promoted apoptosis.4.Compared with any single drug group,the combined effect of the two drugs on cell proliferation is more significant,and they were time-and concentration-dependent.Compared with paclitaxel and BEZ235 single drug group,different concentrations of the two drugs combined inhibited the colon formation of HepG2 cell clones.5.The migration inhibition rates of paclitaxel at 24 h and 48 h were 66.5%and 49.8%,and the migration inhibition rates of BEZ235 at 24 h and 48 h were 77.2%and 65%,respectively.The combination of the two drugs was more effective further inhibited cell migration(24 h:96.1%,48 h:93.6%).The results of the Transwell experiment showed that the inhibition rates of paclitaxel and BEZ235 on cell migration were 38.8%and 40.0%,respectively,and the inhibition rates of the two drugs combined reached 70.9%.6.Compared with paclitaxel(16.18%)and BEZ235(6.13%)alone,paclitaxel combined with BEZ235(33.39%)significantly blocked the cell cycle at the G2/M phase and down-regulated CyclinBl and pCDKl expression level.7.JC-1 and Annexin V/PI staining results showed that the apoptosis rate of paclitaxel combined with BEZ235 was significantly higher than that of the control group,paclitaxel group and BEZ235 group.Flow cytometry further proved that the green-red fluorescence intensity ratio of paclitaxel combined with BEZ235(0.33%)was also higher than that of the control(0.09%),paclitaxel(0.15%)and BEZ235(0.04%);the apoptosis rate of the two drugs combined was 29.90%,higher than the control(3.56%),paclitaxel(7.62%)and BEZ235(7.19%).Compared with each monotherapy group,the combined of paclitaxel and BEZ235 significantly reduced the mitochondrial membrane potential,up-regulated the expression of mitochondrial pro-apoptotic molecules,increased the activity of Caspase9/3/7 and PARP,promoted cell apoptosis.8.In vivo,the combination of paclitaxel and BEZ235 significantly inhibited tumor growth and the mouse body weight was not significant changes.The two drugs combined inhibited the phosphorylation levels of PI3Kp85,Akt,mTOR,eIF4EBP1 and S6K;increased the expression of pro-apoptotic molecules Bak,CytC,Caspase9 and Caspase3;reduced the expression of cell proliferation marker Ki-67.In addition,the proportion of GPC3-positive cells in HepG2 xenograft tumor tissues with high expression of GPC3 molecules was also significantly reduced.conclusion:BEZ235 reversed the stress-induced activation of the PI3K/mTOR pathway by paclitaxel,inhibited cell proliferation and migration,blocked cell cycle progression,and promoted apoptosis,thereby enhancing the sensitivity of HCC to paclitaxel.Thereby,paclitaxel combined with BEZ235 brings potential benefits to the treatment of liver cancer and is a promising treatment option.Figure[19]Table[2]Reference[77]... |
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