ABSTRACT
OBJECTIVE To investigate the early events of norcantharidin (NCTD) induced cell apoptosis and cell cycle arrest, the variation of reactive oxygen species (ROS) and the NF-E2-relate? dactor 2/antioxidant response element(Nrf2/ARE) pathway in human HepG2 cells. METHODS The cyto?toxicity was measured by MTT assay. Apoptosis and cell cycle was analyzed by flow cytometry. The intra toxicity ROS production was evaluated by flow cytometry analysis with DCFH-DA probe and the effect of NCTD on Nrf2/ARE pathway was detected by luciferase assay in HepG2C8 cells under the same condition. The mRNA expression of heme oxygenase-1(HO-1) and NAD(P)H: quinone oxidoreductase 1(NQO1) antioxidase gene in Nrf2/ARE pathway downstream was evaluated by quantitative real-time PCR. RESULTS No significant cytotoxicity was detected after HepG2 cells were treated with NCTD 30, 60 and 120 μmol · L- 1 for 3 and 6 h, but cellular viability was inhibited significantly by NCTD 30, 60 and 120μmol·L-1 for 24, 48 and 72 h(P<0.01). Cell apoptosis and G2/M phase arrest occurred after HepG2 cells were treated with NCTD 60μmol · L-1 for 12, 24 and 48 h. The percentage of apoptosis increased from (4.00 ± 1.98)%to (12.10 ± 1.70)%for 12 h, from (4.05 ± 0.21)%to (31.8 ± 6.50)%for 24 h, and from (3.90 ± 0.85)% to (33.30 ± 1.41)% for 48 h, respectively. The percentage of G2/M phase increased from (16.51 ± 1.58)% to (40.89 ± 0.18)% for 12 h, from (16.99 ± 1.32)% to (55.29 ± 3.99)% for 24 h, and from (14.45 ± 0.59)% to (50.66 ± 5.88)% for 48 h, respectively. Compared with cell control group, the percentage of G1 phase had a significant decrease in the group with NCTD treated at different time points(P<0.01). No significant change in ROS in HepG2 cells was detected after the treatment with NCTD 30, 60 and 120μmol · L-1 for 3, 6 and 12 h. Nrf2/ARE pathway in HepG2C8 cells was activated by NCTD 30, 60 and 120μmol·L-1 for 6 and 12 h. mRNA expression of HO-1 and NQO1 had a signifi?cant activation in HepG2 cells after treatment with NCTD 30, 60 and 120 μmol · L-1 for 6 and 12 h (P<0.05). CONCLUSION NCTD can activate Nrf2/ARE pathway in the early stage in HepG2 cells, which may inhibit the intracellular ROS production in the early stage. Activation of ROS may not be the main event in NCTD induced HepG2 cell apoptosis and G2/M phase arrest.