Protopine Inhibits The Growth Of Hepatocellular Carcinoma Through Mitochondrial Mediated Apoptosis Pathway

Objective:Hepatocellular carcinoma（HCC）is a serious threat for human health;the incidence of HCC in China accounts for more than 50%worldwide.The toxicity and drug resistance limits the effects of chemotherapeutical agents on HCC patients clinically.There is an urgent need to develop new anticancer agents for the treatment of HCC patients.It is reported that Protopine,isolated form Corydalis yanhusuo,an isoquinoline alkaloid with a molecular weight of353.37 Da displays potent anticancer activity.However,the underlying mechanisms of the anticancer effect of protopine on HCC still remain unknown.In the present study,we studied the inhibitory effect of protopine on HCC cancer cells.Methods:CCK-8 analysis was performed to measure the effect of protopine on the growth of HCC cells.The apoptosis of cancer cells was analyzed by Hoechst33342/PI double staining and flow cytometry approaches.JC-1 staining was used to determine the change of mitochondrial membrane potential（MMP）in cancer cells.Western blotting was performed to analyze the expression level of proteins related to mitochondrial apoptotic pathway.The xenograft nude mice model bearing human HCC Hep G2 cells was used to determine the anticancer effect of protopine in vivo.HE staining was carried out to determine the effect of protopine on tumor tissue while the apoptosis rate of tumor tissue was determined by TUNEL analysis and the expression of Bcl-2and Ki67 in tumor tissue were determined by immunohistochemistry.Results:The results of CCK-8 assay indicated that protopine displayed anticancer activities on a lot of cancer cells,and exhibited most potent inhibitory effects on HCC BEL-7402 cells and Hep G2 cells with IC₅₀ value of26.32±9.13μM and 32.91±8.49μM respectively.The results of Hoechst33342/PI double staining showed that cancer cells treated with protopine displayed obvious apoptotic characteristics,including nucleus shrank and apoptotic bodies.Quantitative analysis using flow cytometry confirmed that when treating the BEL-7402 cancer cells with protopine at a concentration of 7.5,15.0 and 30.0μM,the apoptotic rates were 23.51%±4.65%,44.58%±4.48%and 55.16%±7.23%respectively.Similar results were also found in Hep G2 cells;treating Hep G2 cells with the compound at a concentration of 7.5,15.0 and 30.0μM,the inhibitory rates were 15.60%±6.45%,26.94%±3.49%and 40.51%±10.11%respectively.JC-1 staining confirmed that treatment with protopine decreased the MMP of HCC cells in a dose-dependent manner.Western blotting analysis confirmed that protopine was able to up-regulate the expression level of proteins related to mitochondrial apoptotic pathway;the level of Cytochrome C,Apaf-1,Bax,cleaved-PARP,cleaved-caspase-3 and cleaved-caspase-9 were increased while the expression of Bcl-2 was suppressed significantly.In vivo study using xenograft nude mice bearing Hep G2 cells revealed that protopine suppressed the growth of tumors significantly;administration of protopine i.p.at the concentration of 12.5 mg/kg,25 mg/kg and 50 mg/kg resulted in the inhibitory rates of tumor growth of23.19%±10.32%,46.38%±8.47%and 72.46%±7.14%respectively.HE staining and TUNEL analysis confirmed that protopine suppressed the growth of tumors;the apoptosis rates were 17.84%±3.55%,49.63%±8.73%and71.88%±2.53%,in nude mice treated with protopine at the concentration of12.5 mg/kg,25 mg/kg and 50 mg/kg respectively.Immunohistochemical analysis showed that protopine down-regulated the expression level of Bcl-2and Ki67 in tumor tissues;the percentage of Bcl-2 positive cells were 54.16%±2.37%,39.93%±3.88%and 21.82%±3.51%while the ratio of Ki67 positive cells decreased from 78.19%±2.73%to 67.74%±2.37%,48.01%±2.49%and24.10%±4.37%,respectively in tumor tissue treated with protopine group at the concentration of 12.5 mg/kg,25 mg/kg and 50 mg/kg respectively.Conclusion:This study confirmed that protopine is able to induce apoptosis on human hepatocellular carcinoma BEL-7402 cells and Hep G2 cells through mitochondrial pathway.In addition,protopine suppressed the growth of tumors in xenograft nude mice bearing Hep G2 cells significantly and there is potential to develop protopine as a lead compound for the treatment of HCC patients.