The Effects Of SPK/S1P Signal On The Proliferation, Apoptosis And VEGF Expression In Human Hepatoma Cell HepG2

Objective: Sphingosine-1-phospate and sphingosine kinase are both important signal molecule of cell proliferation and survival. It has been proved that SPK/S1P signal plays an important role in the occurrence and growth of some malignant tumors, however very little is known about its mechanism. Hepatocellar carcinoma is rich in vessels. Although numerous reports have been conducted on the over-expression of vascular endothelial growth factor in HCC, few have focused on the effects of SPK/S1P signal on the VEGF expression in HCC cells. So the effects of SPK/S1P signal on proliferation, apoptosis and VEGF expression in human hepatoma cell are investigated by using cell biological and molecule biological techniques. Methods: Human hepatoma HepG2 celles were grown in DMEM supplemented with 10%FBS at 37℃, 5%CO2 . Treated with concentration range of 5～20μmol/LDMS (Dimethyl sphingosine) in culture medium, the antiproliferative effect of DMS was evaluated by the means of MTT assay . Morphological variations of apoptic cells were observed with invert phase-contrast microscope and with scanning electron microscope. Meanwhile, cell apoptosis was examined by the means of TUNEL.The VEGF protein level was measured by enzyme-linked immunosorbent assay( ELISA),and the relative VEGF mRNA level was evaluated by reverse-transcriptase polymerase chain reaction with usingβ—actin as an internal control standard. Results :1.The effects of SPK/S1P signal on proliferation and survival in human hepatoma cell HepG2.1)The results of MTT assay demonstrated that 5～20μmoL/L DMS inhibits the growth of human hepatocarcinoma HepG2 cells in a time-and-dose-dependent manner, and can induce the apoptosis of human hepatocarcinoma HepG2 cells.2) HepG2 cells showed typical morphological characters of apoptosis: the microvilli of the cell surface disappeared, the cell shrinked and decreased in volume and blebs or ball-like bodies appeared. Typical nuclear condensation, margination and fragmentation were observed. The results of TUNEL demonstrated that the nucleus of cells treated with 10μmoL/L and 20μmoL/L DMS(10μmoL/L and 20μmoL/L) get to henna. The stained cells in each group were (5.89±0.65) percent (control group),(26.31±1.97) percent (10μmoL/L Group), (40.24±5.27) percent (20μmoL/L Group ), respectively. The stained cells of DMS was much higher than that of control group ,and has the significant difference(P <0.01) . 2. The effects of SPK/S1P signal on VEGF expression in human hepatoma cell HepG2.1)The results of ELISA demonstrated that the expression of VEGF was inhibited obviously by 5～20μmoL/L DMS and the inhibition showed dose depended.2)The results of RT-PCR demonstrated that the expression of VEGF gene mRNA was inhibited obviously by DMS.Compared with control group ,the relative expression level of VEGF gene mRNA was decreased with significant difference (P <0.01 ) . Conclusion :1. DMS inhibits the growth of human hepatocarcinoma HepG2 cells in a time-and-dose-dependent manner, and can induce the apoptosis of human hepatocarcinoma HepG2 cells.2. DMS makes the VEGF expression decreased both in the protein and the gene level by inhibiting the SPK/S1P signal . SPK/S1P signal can regulate the expression of VEGF in hepatoma cells.