Effect Of Baicalein On The Migration And Invasion Of Human Hepatocellular Carcinoma Cell Line SMMC-7721 In Vitro

Abstract: Objective: To investigate the possible mechanism that baicalein inhibits the migration and invasion, by detecting the expressions of Ezrin, matrix metalloproteinase-9 (MMP-9) and vascular endothelial growth factor (VEGF) in human hepatocellular carcinoma cell line SMMC-7721. Methods: 1. SMMC-7721 cells were treated with different baicalein concentration(5.0μmol/L,10.0μmol/L,20.0μmol/L,40.0μmol/L,80.0μmol/L and 160.0μmol/L) for 24h,48h and 72h,water soluble tetrazolium-1 (WST-1) assay were used to measure cell proliferation.The growth inhibition ratio(IR) was calculated by the formulation : IR = (1 - average OD value of experimental group / average OD value of control group)×100% .2. Wound-healing assay was performed to observe the migration of SMMC-7721 cells treated with baicalein of 20.0,40.0μmol/L for 24h,48h.3. Transwell chamber assay was performed to observe the invasion of SMMC-7721 cells treated with baicalein of 20.0μmol/L,40.0μmol/L for 48h in vitro. 4.The expressions of Ezrin, MMP-9 and VEGF mRNA in SMMC-7721 cells treated with baicalein of 20.0μmol/L,40.0μmol/L for 48h was analyzed through reverse transcription- polymerase chain reaction (RT-PCR). 5.The expressions of Ezrin, MMP-9 and VEGF protein in SMMC-7721 cells treated with baicalein of 20.0μmol/L,40.0μmol/L for 48h was detected by means of immuno- cytochemistry technique.6.All the experimental results were analyzed by ststistical software SPSS16.0. Results:1. Compared with the control group, the growth inhibition ratio of SMMC-7721 cells was significantly increased after treated by baicalein at different concentrations (5.0μmol/L, 10.0μmol/L, 20.0μmol/L,40.0μmol/L,80.0μmol/L,160.0μmol/L) for 24h, 48h and 72h (P<0.05); the proliferation of SMMC-7721 cells were significantly inhibited, all the inhabition ratio compared with those cause by different times and drug concentrations, and showed a time and dose dependent manner(P<0.05).2. Wound-healing assays displayed that the percent wound closure was 11.2±1.8, 8.6±1.6,56.8±2.5 and 28.5±1.9,respectively,in SMMC-7721 cells treated with baicalein of 20.0,40.0μmol/L for 24h, 48h, it showed a significant decreased compared with control group (47.1±2.3,90.1±2.1, P<0.05).3.A significant decreased was observed in the number of SMMC-7721 cells per field permeating the artificant membrane in the group treated with bacalein of 40.0μmol/L for 48h compared with the control group(54.7±18.5 vs 131.4±32.9,P<0.05)by means of Transwell assay.4.Reverse transcriptase-polymerase chain reaction detected that the expressions of Ezrin, MMP-9 and VEGF mRNA in SMMC-7721 cells of 20.0μmol/L and 40.0μmol/L group were significantly down-regulated,the relative gray values of Ezrin, MMP-9 and VEGF were significantly decreased compared with the control group(P<0.05).6.The expressions of Ezrin, MMP-9 and VEGF protein in SMMC-7721 cells of 20.0μmol/L and 40.0μmol/L group were significantly down-regulated,the integral optical density of Ezrin, MMP-9 and VEGF were significantly decreased compared with the control group (P<0.05)using immunocytochemistry technique.Conclusions:1. Baicalein may inhibit the proliferation of human hepatocellular carcinoma cell line SMMC-7721 in a time and dose dependent manner;2.Baicalein may inhibit the invasion and migration of human hepatocellular carcinoma cell line SMMC-7721; 3.Baicalein may inhibit the mRNA and protein expressions of Ezrin,MMP-9 and VEGF; 4. Baicalein may induce partly a antineoplastic effectiveness via directly or indirectly inhibiting expressions of Ezrin, VEGF and MMP-9,but the mechanism is unknown.