Growth Inhibition And Apoptosis Induction Of HepG2 And SMMC-7721 Liver Cancer Cells By EGCG

This study used cell culture,CCK-8,Wound Healing,Transwell,RT-PCR,cell cycle,JC-1,DAPI staining,AV/PI,Western blot analysis,xenografted tumor in nude mice of cell molecular biology techniques,mainly using HepG2 and SMMC-7721two human hepatocarcinoma cell lines were used as experimental subjects to study the effects of EGCG on its proliferation,migration,cell cycle,mitochondrial membrane potential and apoptosis.The results are as follows:1 EGCG inhibits cell proliferation and migrationTo determine the effect of EGCG on HCC cells,three HCC cell lines including HepG2,SMMC-7221 and LO2 were treated with 0,1,5,10,20,50,100,150,200?mol/L EGCG for 24 and 48 hours,respectively.Cell viability was assessed using CCK-8 assay.We then measured the effect of EGCG on HepG2 and SMMC-7721 cell migration by using Wound Healing and Transwell assays.The results showed that the migration strength of the two cell lines in the EGCG-treated group was significantly weaker than that of the control group.Western blot and RT-PCR results showed that the RNA expression levels of MMP-2 and MMP-9 were significantly decreased in the two cell lines(P<0.05).The results showed that both HepG2 and SMMC-7721 cell lines were sensitive to EGCG in a dose-dependent and time-dependent manner,while EGCG had little effect on the cell viability of LO2 cells;EGCG inhibited the migration of HepG2 and SMMC-7721 cells.Tip:EGCG can inhibit cell proliferation and migration.2 EGCG induces cell cycle arrestTo elucidate whether EGCG-induced cell growth inhibition was mediated by changes in cell cycle progression,HepG2 and SMMC-7721 cells were treated with0,50,100,150,200?mol/L EGCG for 24 hours,respectively.We then measured cell cycle distribution using flow cytometry by cell cycle staining.Western blot analysis was used to analyze the expression of related proteins.The results showed that EGCG caused the significant arrest of S in HepG2 and SMMC-7721 cells.The results of Western blot showed that the expression levels of Cyclin D1,Cyclin E,p27and p21were increased in the two cell lines,While the level of Cyclin A protein was decreased.It is suggested that EGCG induces cell cycle arrest in S phase.3 EGCG causes a decrease in mitochondrial membrane potential and induces apoptosisTo verify the apoptosis effect of EGCG on cells,HepG2 and SMMC-7221 cells were treated with different density of EGCG for 24 hours,respectively.Then,JC-1,DAPI,and AV/PI were used to measure the changes of cell state after drug treatment,and the expression of related proteins was analyzed by Western blot.The structure showed that when HepG2 and SMMC-7721 cells were exposed to EGCG,the mitochondrial membrane potential was significantly decreased;cells with nuclear fragmentation and condensation(marks of apoptosis)were detected by DAPI staining;the results of double staining flow cytometry showed that the apoptotic rate(early withering,late withering,necrosis)of hepatoma cells increased with the increase of drug concentration.After treatment,the expression of Bax protein was significantly increased,and the expression of Bcl-2 protein was down-regulated.The expression of the apoptotic gene Caspase-3 and Caspase-9 was also up-regulated.It is suggested that EGCG can decrease??m and induce differential expression of apoptosis-related proteins in two kinds of liver cancer cells,which ultimately leads to apoptosis.4 EGCG inhibits the growth of xenografted tumor in nude miceThe selected 16 female SPF nude mice weighed 14-16g and were 4-6 weeks old.The number of cultured human liver cancer cells HepG2 was adjusted to 1×10~7/mL,and the cells were resuspended in serum-free medium and injected into the hind leg side of nude mice.When the size of the subcutaneous tumor of the mouse reached80-100mm~3,the tumor-bearing mice were injected subcutaneously with EGCG(10,20,40 mg/kg)or saline every other day,five times in total.Observe the tumor growth status and weigh the mouse in the experiment every day.After the experiment was completed,the mice were sacrificed and the tumor pieces were removed,measured and weighed.The results showed that the tumor proliferation rate was slower and the tumor weight was significantly different in the drug-administered group compared with the control group.Tip:EGCG can inhibit the growth of xenografts in nude mice.