Effects Of Cathepsin S On Drug Susceptibility Of Human Hepatocellular Carcinoma MHCC97H Cells

Background & Aims:Cathepsin S (CTSS) has been reported to increase the expression and activity of many malignant tumors. But the mechanism of CTSS silence inducing apoptosis of liver cancer cells MHCC97H is still unclear.Methods:The hepatocellular carcinoma cell line has been used as the basis of research experimental system to evaluate the roles of CTSS in human hepatocellular carcinoma cells and relative mechanisms. qRT-PCR and Western blot were used to screen the hepatocellular carcinoma cell line with high expression of CTSS. Lentivirus was used to interference the expressions of CTSS and its receptor protease activated receptor-2 (PAR2). Apoptosis of CTSS was detected by flow cytometry. Western blot was adopted to detect changes in NF-κB pathway. With the intervention of melittin and doxorubicin, flow cytometry and Western blot were used to evaluate the effects of CTSS silence on drug sensitivity variations and the activation of Caspase 3 protein. At last, the subcutaneously transplanted tumor model of nude mice was used to evaluate the effects of CTSS silence on the in vivo growth of hepatocellular carcinoma cells and melittin sensitivity. ELISA was used to detect the activity of Caspase 3.Results:Among 13 hepatocellular carcinoma cell lines, CTSS expressed highest in the MHCC97-H. In PLVT1150 expressed by the lentivirus-mediated small molecular interfering technology transfected CTSS silence gene, mRNA and protein content of CTSS gene and PAR2 reduced significantly; on the contrary, in PSB880 with CTSS gene overexpression, mRNA and protein content of CTSS gene and PAR2 increased significantly. This can draw a positive conrrelation between PAR2 and CTSS gene. In CTSS gene silencing MHCC97-H cells, the activity of NF-κB signaling pathway was inhibited; more apoptosis of MHCC97-H cells was observed (30.07%±1.8 vs.18.58%±1.6); but PAR2 with overexpressed silent CTSS could reduce its apoptosis rate (30.07%±1.8 vs. 19.72%±1.5). After treated by melittin (0,4 and 8 g/mL) for 30 minutes and doxorubicin (0 and 5 g/mL) for another 24 hours, the apoptosis rates of MHCC97-H cells in the shCTSS and control groups were measured. Compared with the control group, PLVT1150 cells were more sensitive to melittin (18.87%±2.1 vs.4.27%±1.9,36.72%±2.2 vs.15.32%±1.7,52.98%±1.5 vs. 28.86%±2.2) and doxorubicin (19.13%±2.0 vs.3.84%+1.4,71.83%±2.5 vs. 32.66%+1.2). After the CTSS was silenced, activated Caspase 3 increased significantly. It can be indicated that CTSS was associated with MHCC97-H cell apoptosis. In the in vivo study of nude mice, melittin together with CTSS silence can significantly inhibit the growth of hepatocellular carcinoma, which was more sensitive to the melittin-induced apoptosis. Conclusion: Lentivirus-mediated CTSS silencing can significantly induce the apoptosis of MHCC97-H cells and increase the sensitivity to chemotherapy. This makes CTSS a potential molecular target for the treatment of hepatocellular carcinoma, and provides an attractive anticancer strategy of the treatment of hepatocellular carcinoma.