| Hepatic carcinoma is one of the malignant tumors with the highest incidence rate,which is growing at the rate of about 500,000 new patients each year in the world.About 110,000 people in China die of liver cancer each year,ranking second in the death rate of malignant tumors,which seriously threatening human health and life.Urgently need to develop new anti-cancer drugs and new treatment methods.Puerarin is a kind of isoflavones extracted from the traditional Chinese medicine Pueraria lobata.Puerarin has a wide range of pharmacological effects such as anti-oxidation,anti-hyperlipidemia,anti-apoptosis and anti-inflammatory.In recent years,the anti-tumor effect of Puerarin was attentioned gradually by the majority of medical workers,and previous studies have found that Puerarin has a certain inhibitory effect on lung cancer,kidney cancer,ovarian cancer,cervical cancer,et al.Whose mechanism perhaps related to its effects of regulating tumor cell cycle,regulating the expression of apoptosis-related genes and protein,improving the body's autoimmune function and improving the chemosensitivity and other pharmacological effects,but whether Puerarin has an inhibitory effect on hepatic carcinoma has not been reported in the literature.In this study,human hepatoma Hep G2 cells were cultured in vitro and logarithmically.The cell proliferation inhibition rate,cell cycle,change,and cell apoptosis were determined,the apoptosis rate was calculated and apoptosis-related genes and protein expression were measured.To study the effect of Puerarin on the growth of hepatocellular carcinoma cells and its possible mechanism.Part one Effect of Puerarin on Proliferation and Apoptosis of Hepatoma Cells and Its MechanismObjective: The proliferation inhibition rate and apoptosis of hepatocellular carcinoma cells were detected,and the expression of apoptosis-related proteins was detected.Then the effects of puerarin on the proliferation and apoptosis of hepatocellular carcinoma cells were analyzed and the mechanism was explored.Methods: The Hep G2 human hepatocellular carcinoma cells were cultured in vitro and the cells in logarithmic growth phase were taked for experimentation.Which were grouped randomly into the blank control group,Puerarin 10?g/ml group,Puerarin 20?g/ml group,Puerarin 40?g/ml group and cisplatin(40?g/ml)group.MTT colorimetric assay was used to determine the proliferation inhibition rate of cells;flow cytometry(FCM)was used to detect cell cycle,cell apoptosis was observed,and cell apoptosis rate was calculated.The Western blotting was used to detect the expression of Bax,bcl-2 and activated caspase-3 protein.Results: 1.Treated for 48 h,the inhibition of Hep G2 cells proliferation was significantly increased in Puerarin(10?g/ml,20?g/ml,40?g/ml)groups or cisplatin 40?g/ml group(P<0.05 or P<0.01),which has a certain dosedependent effect.Compared with the cisplatin 40?g/ml group,the proliferation inhibition rate of human hepatocellular Hep G2 cells in the Puerarin 40?g/ml group was significantly increased,and the difference was significant(P<0.05).2.Treated for 48 h,the proportion of human hepatoma Hep G2 cells in G0/G1 phase was significantly increased in Puerarin(20?g/ml,40?g/ml)groups and cisplatin 40?g/ml group while the in G2/M phase was significantly decreased(P<0.05 or P<0.01),which prompted that Puerarin blocks human hepatoma Hep G2 cell mitosis.Compared with cisplatin 40?g/ml group,the proportion of human hepatoma Hep G2 cells in Puerarin 40?g/ml group G2/M phase ratio was significantly decreased(P<0.05),and G0/G1 phase difference between the two groups had not statistically significant(P>0.05).3.Treated for 48 h,the apoptosis of human hepatocellular Hep G2 cells inPuerarin(10?g/ml,20?g/ml,40?g/ml)groups and cisplatin 40?g/ml group could be obviously induced,and the number of apoptotic cells were obviously increased,which had a certain dose-dependent.The apoptosis rate was calculated: the apoptosis rates of Hep G2 cells in Puerarin(10?g/ml,20?g/ml,40?g/ml)groups were(20.98±5.75)%,(46.01±6.43)%,(71.86±8.50)% and in cisplatin 40?g/ml group was(35.72±5.38)%.Compared with blank control group [(3.69±1.41)%],the differences were statistically significant(P<0.01).Compared with cisplatin 40?g/ml group,the apoptotic rate of Hep G2 cells in Puerarin(20?g/ml,40?g/ml)groups were significantly increased(P<0.05 or P<0.01).4.Compared with the blank control group,the expression of bcl-2 protein were significantly down-regulated and the expression of activated caspase-3 protein were significantly up-regulated in Hep G2 cells treated by Puerarin(10?g/ml,20?g/ml,40?g/ml)for 48h(P<0.05 or P<0.01).Bax m RNA expression was significantly up-regulated and Bax/bcl-2 expression ratio was significantly increased in Puerarin(20?g/ml,40?g/ml)groups(P<0.05 or P<0.01).Compared with the cisplatin 40?g/ml group,the expression of Bcl-2 m RNA of human hepatoma Hep G2 cells in Puerarin 40?g/ml group was significantly down-regulated and the Bax/bcl-2 ratio was significantly increased(P<0.05),the expression of activated caspase-3 protein in Puerarin 40?g/ml group was significantly increased(P<0.05).Summary: Puerarin can increase the inhibition rate and apoptosis rate of Hep G2 cells,up-regulate the expression of Bax and caspase-3 and down-regulate the expression of bcl-2;which suggests that Puerarin could inhibit the proliferation of Hep G2 cells and promote the apoptosis of Hep G2 cells,which may be related to the inhibition of Hep G2 cell mitosis and the regulation of apoptosis-related protein expression.Part two Effects of Puerarin on Chemosensitivity of Hepatocellular Carcinoma Cell Lines and Its MechanismObjective: Through the detection of relevant indicators,the effect ofpuerarin on the chemosensitivity of cisplatin in hepatocellular carcinoma cells was studied and the mechanism was explored.Methods: The Hep G2 human hepatocellular carcinoma cells were cultured in vitro and the cells in logarithmic growth phase were taked for experimentation.Which were grouped randomly into the blank control group,Puerarin 20 ?g/ml group,cisplatin 40 ?g/ml group and combined group(Puerarin 20 ?g/ml+cisplatin 20 ?g/ml).MTT colorimetric assay was used to determine the cell proliferation inhibition rate.The cell cycle was observed by FCM,apoptosis was observed and the apoptosis rate was calculated.The expression of Bax,bcl-2 and activated caspase-3 protein were determined by Western blotting.Results: 1.Treated for 48 h,the inhibition of Hep G2 cells proliferation were significantly increased in Puerarin 20?g/ml group,cisplatin 40?g/ml group and combined group(Puerarin 20?g/ml + cisplatin 20?g/ml)(P<0.01).Compared with the cisplatin group,the inhibition rate of the human hepatoma Hep G2 cells in the combination group was significantly increased(P<0.01).2.Treated for 48 h,the proportion of human hepatoma Hep G2 cells in G2/M phase was significantly decreased in Puerarin 20?g/ml group,cisplatin 40?g/ml group and combined group(Puerarin 20?g/ml + cisplatin 20?g/ml)(P<0.05 or P<0.01).The proportion of human hepatoma Hep G2 cell cycle in S phase was significantly decreased in cisplatin group and combination group(P<0.05 or P<0.01).Compared with the cisplatin group,the proportion of the G0/G1 phase in the human hepatoma Hep G2 cell cycle in the combined group was significantly reduced and the proportion of G2/M phase was significantly decreased(P<0.05).3.Detected by FCM,the number of apoptotic Hep G2 cells was significantly increased in the Puerarin 20 ?g/ml group,cisplatin 40 ?g/ml group and the combined group(Puerarin 20 ?g/ml + cisplatin 20 ?g/ml).Calculated cell apoptosis rate: The apoptosis rate of Hep G2 cells in the Puerarin group was(43.46±6.09)%(39.84±5.47)% in the cisplatin group,(65.19±6.71)% in the combined group,and(3.72±1.38)% in the blank control group,the differences were statistically significant(P<0.01).Compared with the cisplatin group,the apoptotic rate of human hepatoma Hep G2 cells in the combination group was significantly increased(P<0.05).4.Detected by Western blotting,the expression of Bax and activated caspase-3 protein of human hepatocellular carcinoma Hep G2 cells were significantly up-regulated and Bcl-2 protein was significantly down-regulated in Puerarin 20 ?g/ml group,cisplatin 40 ?g/ml group and combined group(Puerarin 20 ?g/ml + cisplatin 20 ?g/ml group)(P<0.05 or P<0.01),Bax/bcl-2 ratio increased significantly(P<0.01).The ratio of Bax/Bcl-2 and activated caspase-3 protein expression in human hepatoma Hep G2 cells in the combined group was significantly increased than that in the cisplatin group(P<0.05).Summary: Puerarin may enhance the chemosensitivity of cisplatin in human hepatoma Hep G2 cells by inhibiting mitosis,changing cell cycle,and regulating the expression of apoptosis-related proteins.Conclusion: Puerarin may inhibit the growth of human hepatoma Hep G2 cells and increase the chemosensitivity of human hepatoma Hep G2 cells by inhibiting cell proliferation and promoting apoptosis.The mechanism may be related to inhibition of mitosis and regulation of apoptosis-related protein expression. |
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