Study The Mechanisms Of Combined Jian Pi Ii Qi Decoction With Gefitinib In Inhibition Of Human Liver Cancer Cell Growth

ObjectiveUsing human liver cancer cell HepG2in vitro experiment, we want to detect the growth inhibition effects of Jianpiliqifang (JPLQF) and its active ingredient ursolic acid (UA) combined with Gefitinib (G) and explore the anti-tumor mechanisms.MethodsHuman liver cancer cell HepG2was selected in this study. This experiment has the following groups:blank group, control group, JPLQF group, UA group, gefitinib group, JPLQF combined with gefitinib group, UA combined with gefitinib group. MTT assay was used to detect the drug effects on cell proliferation. Hoechst33258staining and FCM was used to observe the morphological alteration and the apoptosis rates after drug intervention. Western Blotting was used to detect the expression of DNMT1and EZH2proteins and the phosphorylation of AMPK a and EGFR kinase.Results1. Effect of JPLQF and its active ingredient UA combined with gefitinib on cell proliferation.JPLQF and its active ingredient UA can inhibit the proliferation of human liver cancer cell HepG2. With the increase of drug concentration and time, the inhibition ratio was also increase. And this effect was in a time and dose dependent way. The combination of JPLQF or UA with gefitinib can promote the inhibitory effect on proliferation obviously. UA15μM combined with gefitinib20μM can get the strongest effect in control the growth of human liver cancer cell HepG2.2. Effect of JPLQF and its active ingredient UA combined with gefitinib on morphological alteration and apoptosis rates. JPLQF, UA, gefitinib and JPLQF or UA combined with gefitinib can cause apoptosis-related morphological alteration on HepG2cells and the alteration of JPLQF or UA combined with gefitinib group was most obvious. Compare with control group, the apoptosis rates of the drug group was higher. The apoptosis rate of JPLQF or UA combined with gefitinib group was higher than single JPLQF or UA group and single gefitinib group.3. Effect of JPLQF and its active ingredient UA combined with gefitinib on DNMT1and EZH2protein levels and the phosphorylation of AMPKa and EGFR kinase.JPLQF, UA, gefitinib and JPLQF or UA combined with gefitinib can decrease the expression level of DNMT1and EZH2protein in human liver cancer cell HepG2. The down regulated effect of DNMT1and EZH2protein was lowest when they were used together. Moreover, JPLQF10mg/mL or UA25μM effect on human liver cancer cell HepG2can increase the phosphorylation of AMPKa kinase and decrease the phosphorylation of EGFR kinase.Conclusion1. JPLQF, UA, gefitinib and JPLQF or UA combined with gefitinib advanced on HepG2cells can achieved dose-time dependent growth inhibition. The growth inhibition effects were promoted obviously when they were used together.2. JPLQF, UA, gefitinib and JPLQF or UA combined with gefitinib has apoptosis effects on HepG2cells, of which the apoptosis effects were enhanced when they were used together.3. JPLQF, UA, gefitinib and JPLQF or UA combined with gefitinib can decrease DNMT1and EZH2protein expression levels on HepG2cells. The down regulated effect of DNMT1and EZH2protein was lowest when they were used together.4. JPLQF and UA can increase the phosphorylation of AMPKa kinase and decrease the phosphorylation of EGFR kinase.