Research On The Different Schedule-dependent Effects Of Ginsenoside Rg3in Combination With Sorafenib And Oxaliplatin On Human Hepatocellular Carcinoma Cell Line

Objective:Observe the effects of ginsenoside Rg3, sorafenib, oxaliplatin single,simultaneous and sequential role on human hepatoma cell line SMMC-7721in vitro, explore its cell proliferation and cell cycle-related mechanisms, to provide a theoretical basis for the sequential application of traditional Chinese medicine, chemotherapy and molecular targeted therapy.Methods:Use various methods to detect the growth condition of human hepatoma cell line SMMC-7721when they are affected by a certain concentration of ginsenoside Rg3, sorafenib, oxaliplatin single,simultaneous and sequential role:(l)Estimating the inhibition of proliferation by MTT test;(2) Analyzing the distribution of cell cycle and apoptosis by flow cytometry;(3)Detecting the expression of cell proliferation associated protein:PCNA by immunofiuorescence technique;(4)Detecting the expression of cell cycle related proteinxyclin D1by Western blot.Results:(1)The inhibition of cell proliferation was significant about administrating the ginsenoside Rg3, sorafenib, oxaliplatin alone, simultaneously and sequentialy on human hepatoma cell line SMMC-7721by MTT test.The synergistic inhibitory effect of combining the ginsenoside Rg3and oxaliplatin was significantly higher than first using ginsenoside (P<0.05),but there was no no significant difference by first using oxaliplatin(P>0.05);the effect of first using oxaliplatin was better than first using oxaliplatin(P<0.05).The inhibition rate of combining the ginsenoside Rg3and sorafenib was significantly higher than first using sorafenib (P<0.05),it performed the additive effect, but there was no no significant difference by first using ginsenoside Rg3(P>0.05);the inhibition rate of combining oxaliplatin and sorafenib was the highest,it performed the synergy effect,using oxaliplatin first was better than using sorafenib first(P<0.05).(2)By flow cytometry,the ginsenoside Rg3blocked cells in G0/G1phase, sorafenib blocked cells in S phase, oxaliplatin arrested cells in S phase, G2/M phase. The apoptosis rate of combining ginsenoside Rg3and oxaliplatin was similar to that of using oxaliplatin first,it blocked cells in G2/M phase,and the apoptosis rate was higher compared with the ginsenoside first using group.Using sorafenib sequential the ginsenoside Rg3had the highest apoptosis rate,it was39.0%,the cells was blocked in G0/G1phase, G2/M phase,and the degree of apoptosis was better than combination group and ginsenoside first using group.The apoptosis rate was the highest when combining sorafenib and oxaliplatin,it was67.9%,the cells was blocked in S phase,Using oxaliplatin sequential sorafenib had higher apoptosis rate than using sorafenib first.(3)The ginsenoside Rg3, sorafenib, oxaliplatin were used in different order on human hepatoma cell line SMMC-7721,the effect was detected by immunofiuorescence technique.It was found that the expressions of PCNA protein in sequential group and the simultaneous group declined statistically significant than single agent group(P<0.05).The declining degree of PCNA when combining the ginsenoside Rg3and oxaliplatin is similar to using oxaliplatin first(P＞0.05),and the two groups declined statistically significant than using the ginsenoside first(P<0.05).The expression of PCNA when combining the ginsenoside Rg3and sorafenib decreased significantly than using sorafenib first(P<0.05),it was similar to using ginsenoside first,and there was no significant difference between the sequential groups(P>0.05).The expression of PCNA when combining sorafenib and oxaliplatin decreased similar to using oxaliplatin first(P>0.05),and better than using sorafenib first(P<0.05).(4)The ginsenoside Rg3, sorafenib, oxaliplatin were used in different order on human hepatoma cell line SMMC-7721,the effect was detected by Western blot.lt was found that the expressions of cyclin D1protein in sequential group and the simultaneous group declined statistically significant than single agent group.The expression of cyclin Dl decreased significantly when using oxaliplatin sequential ginsenoside than using ginsenoside first.The expression of cyclin D1when combining the ginsenoside Rg3and sorafenib decreased significantly than using sorafenib first,it was similar to using ginsenoside first.The expression of cyclin D1when combining sorafenib and oxaliplatin decreased significantly than using sorafenib first,it was similar to using oxaliplatin first.Conclusion:(1)Ginsenoside Rg3, simultaneous and sequential application of oxaliplatin could significant inhibit the proliferation of human hepatoma SMMC-7721, using oxaliplatin sequential ginsenoside had synergistic effect was similar to combining the two drugs,and they were better than the first sequential oxaliplatin after the ginsenoside, its mechanism may be due to oxaliplatin first group and with group cells arrested in S phase, G2/M phase, to increase the apoptosis, also lowered the expression of PCNA, cyclin D1protein.(2) Ginsenoside Rg3, simultaneous and sequential application of sorafenib were better in inhibiting the cell proliferation than using these drugs alone, using ginsenoside Rg3sequential sorafenib and combining the two drugs performed similar additive effect,using sorafenib sequential ginsenoside Rg3was manifested as antagonism, its mechanism may be due to the decline about the expression of PCNA and cyclin D1.(3)Sorafcnib, simultaneous and sequential application of oxaliplatin were better in inhibiting the cell proliferation than using these drugs alone, the two drugs at the same time application was better than the sequential application,showing synergistic effect.Using oxaliplatin sequential sorafenib was better than using sorafenib first, the first using sorafenib group performed as antagonism,this phenomenon may be due to the two kinds of drugs acting on different cell cycle, but there was no specific relationship with the expression of PCNA and cyclin D1protein.