The Effects Of DHA On Cell Apoptosis And Invasive Ability Of Human Hepatocellular Carcinoma Cells

【Objective】(1) To detect different concentrations of DHA on the proliferation ofhuman hepatoma cell line Bel-7402cells.(2)To investigate the effects of differentconcentrations of DHA on cell apoptosis of hepatocellular Carcinoma (HCC) cells andthe possible mechanism.(3) To analyse the effects of DHA on cell invasion andmigration of HCC cells, as well as detect the expression of matrix metallo preteinases-9(MMP-9) after48h. To provide a new theoretical foundation for HCC metastasis andrecurrence.【Methods】(1) The effects of different concentrations of DHA on cells proliferation ofHCC cell line Bel-7402were assessed by MTT staining method and trypan blue dyeexclusion assay.(2) The effects on cell apoptosis was detected by flow cytometricanalysis, and then real-time PCR and western blotting was used to evaluate theexpression of Bim gene, anti-apoptotic protein Bcl-2and pro-apoptotic protein Bax.Measurement of caspase-3activity was detected by caspase-3Activity Assay Kit.(3)The cell invasion and migration of Bel-7402cells were measured by transwell andwound scratch assay, and then the immunohistochemisty was employed to determine theexpression of MMP-9.【Results】(1) Compared with the control group (0μmol/L DHA), each concentrationof DHA significantly inhibited the proliferation of Bel-7402cells, and this effect wasin a time-and dose-dependent manner (P<0.05).(2) After48h, the differentconcentration of DHA treatment groups（25，50，100，200μmol/L）cause the earlyapoptosis of Bel-7402cells, and their rate of apoptosis were10.28±2.28%，19.49±2.21%，25.20±5.53%and35.01±6.01%, which compared with the control group（0.02±0.01%）, each group difference was statistically significant. Simultaneously, to detect the expression of the proteins that is closely related to cell apoptosis on themitochondrial apoptotic pathway, DHA could cause increased the expression ofpro-apoptotic protein Bim gene in does-dependent, as compared with the control group,and the difference was statistically significant. DHA also made the anti-apoptoticprotein Bcl-2reduced and the pro-apoptotic protein Bax increased (P<0.05). Therelative activity of apoptotic protein caspase-3was increased significantly and in adose-dependent manner, as well, the difference was statistically significant (P <0.05).(3)In the transwell invasion assay, The permeable membrane cells number of each DHA(50,100,200μmol/L) treatment group was compared with the control group, thedifference was statistically significant (P <0.05); In the Cell scratch experiment,dynamically observe for0,12,24h, cells scratches spacing of the control group wassignificantly shorter. And after24h, the scratches basically healing. As DHAconcentration increases, the trend of the shortening of cell scratch distance is weakened.And there are the significant difference between each period of DHA (50,100,200μmol/L) treatments groups and control group. DHA (25μmol/L) treatment groupcompared with the control, the difference was not statistically significant. Cellimmunohistochemical results showed that MMP-9relative expression levels weresignificantly reduced by different concentrations of DHA after48hours (P<0.05), andthe effects were in dose-dependent.【Conclusions】(1) DHA has proliferation inhibitory and apoptosis-inducing effect onBel-7402cells, and the effect was in a time-and dose-dependent manner.(2) Throughtesting the expression of several protein on the mitochondrial apoptosis pathway, all theresults obtained in the present study confirmed that DHA induced apoptosis which mayactivate the mitochondrial apoptosis pathway.(3) To a certain degree, DHA reduced theinvasion and migration of HCC cells in dose-dependent, and the mechanism may berelated to the inhibition of the expression of MMP-9.