Effects Of NF-κB Pathway On Matrine-Induced Apoptosis Of Hepatocellular Carcinoma Cells

Objective:To investigate the effect of matrine (Mat) on nuclear factor-κB activation in human hepatoma cell line HepG2 after Mat stimulation, and to observe the effect of NF-κB inhibition by Pyrrolidine dithiocarbamate(PDTC) on matrine-induced apoptosis of HepG2.Methods: HepG2 cells were stimulated by different concentrations of matrine(0.8,1.0,1.5,2.0,2.5 g/L). HepG2 cells were previously treated by 20μmol/L PDTC for 3 hours before the treatment of Mat.The HepG2 cell survival rates were evaluated by MTT assay. HepG2 cells were treated with 1.5g/L concentration Mat for 3h,6h,12h,24h.The cellular distribution and protein level of NF-κB was detected by immunocytochemistry staining and the DNA-binding activity of NF-κB was determined by electrophoretic mobility shift assay (EMSA).Cultured HepG2 cells were implanted in culture flasks and divided into four groups: control group; PDTC group(20μmol/L);Mat group (1.5g/L);combination group:PDTC(20μmol/L)+ Mat (1.5g/L) combination group. The activation of NF-κB in HepG2 cells was detected by immunocytochemistry staining and EMSA method.Apoptosis of cultured HepG2 cells was analyzed by flow cytometry(FCM) and terminal deoxynucleotidyl transferase-mediated dUTP nick end labeling (TUNEL).Results:(1) Matrine could inhibit the proliferation of HepG2 cells in a time- and dose-dependent manner. PDTC enhanced the inhibition of Mat on cell proliferation.(2) NF-κB was located in the cytoplasm of HepG2 cells in control group. Mat demonstrated a power to stimulate the NF-κB influx into the nucleus from plasma and thus the nucleus and cytoplasm were markedly stained. However, when HepG2 cells were previously cultured with PDTC for 3 hours and then stimulated by Mat, the staining of nucleus and cytoplasm was attenuated. (3). EMSA result: Mat stimulated the NF-κB influx into the nucleus from plasma and enhanced the activition of NF-κB which reached the peak at 6th hour.The activition of NF-κB of HepG2 cells in PDTC group was lower than that in control group(P<0.01),and the level of NF-κB activition in Combination group was higher than that in Control group(P<0.01),but lower than that in Mat group(P<0.01). (4).The apoptosis rate was (2.41±0.74)% in control group; (3.07±0.46)% in PDTC group; (6.11±0.81 )% in Mat group; (12.95±2.26)% in combination group.Conclusion: Matrine could induce apoptosis and trigger activation of NF-κB of HepG2 cells. PDTC inhibited NF-κB activation and enhanced apoptosis induced by matrine in hepatocellular carcinoma cells. Therefore, NF-κB activation during matrine-induced apoptosis may have an antiapoptotic effect.