The Effects Of SPK1/S1P Signal Pathway On The Apoptosis, Invasiveness, Multidrug Resistance Characteristics Of Human Hepatocellular Carcinoma MDR Cell Strain BEL-FU

Objective Ceramide (Cer),Sphingosine (Sp) and Sphingosine-1-phospate (SIP) are signal transduction molecules involved in invasion, proliferation, blood vessel formation of tumors. Sphingosine kinasel (SPK1) regulates the production of S1P, and is a key lipid signal moleculer regulates the balance of Cer/S1P. It has been proved that the SPK1/S1P signal pathway interfered by Dimethyl sphingosine (DMS)can cause the apoptosis and inhibit the expression of VEGF of hepatoma cells. However very little is known about whether it can cause the apoptosis, reduce invasiveness and inhibit the expression of multidrug resistance-related protein of human hepatocellular carcinoma multidrug resistant (MDR) cells. The objective of this study is to elucidate the roles of SPK1/S1P signal in human hepatocellular carcinoma MDR cell strain BEL-FU in terms of apoptosis, invasiveness and multidrug resistance.Methods Human hepatocellular carcinoma MDR cell strain BEL-FU were grown in RPMI1640supplemented with 10% FBS,20000ng/mL 5-FU, 100u/L penicillin and 100μg/L streptomycin at 37℃,5%CO2, and were digested by 0.25% trypsin. Treated with concentration range of 5-20μmoL/L DMS in culture medium, the morphological variations of apoptic cells were observed with invert phase-contrast microscope and with fluorescence microscopy. Meanwhile, cell apoptosis was examined by flow cytometry. The invasion of cells and the expression of multidrug resistance-related protein (MRP1) were detected by transwell chamber assay and Western-blot respectively. Results 1.The effects of SPK1/S1P signal on survival in human hepatocellular carcinoma MDR cell strain BEL-FU.①The our results demonstrated that 5-20μmoL/L DMS can cause the apoptosis of human hepatocellular carcinoma MDR cell strain BEL-FU and was in dose-dependent pattern. The apoptosis rate of every concentration group increased significantly compared with the control groups (P<0.01), while the apoptosis was significantly different among the concentration groups (P<0.01).②Inverted phase contrast microscope observation:The control group cells were clonally growth and were spindle or polygonal. The cells treated with DMS exhibited characteristics of apoptosis including increased intra-cellular granules, increased vacuoles in size, decreased cytoplasm and condensed nucleus with smaller, rounded cells shape. More cells detached from the adherent state and suspended in culture medium. With the prolongation of treated time and DMS concentration increasing, those characteristics become more evident.The early and late stage apoptotic cells increased in concentration groups compared with control group. The late stage apoptotic cells increased with DMS concentration. Some nucleus fragmentation occurred among the apoptotic cells,the nuclear debris was clearly visible.2. The effects of SPK1/S1P signal on invasion in human hepatocellular carcinoma MDR cell strain BEL-FU. The results of transwell chamber assay indicated that 5～20μmoL/L DMS can reduce its invasiveness. The inhibitory rate of invasion of experimental groups were significantly increased (P<0.01) compared with control group, and the inhibition exhibit dose-dependent relationship among the experimental groups (P<0.01) 3.The effects of SPK1/S1P signal on the expression of multidrug resistance-related protein (MRP1) in human hepatocellular carcinoma MDR cell strain BEL-FU. The results showed that the MRP1 expression was significantly decreased in DMS treated groups compared with control group (P<0.05), but there was no significant difference in the MRP1 expression between 10μmoL/L DMS treated group and 20μmoL/L DMS treated group (P>0.05).Conclusion 1.The SPK1/S1P signal pathway interfered by DMS can cause the apoptosis of human hepatocellular carcinoma MDR cell strain BEL-FU.2. The SPK1/S1P signal pathway interfered by DMS can reduce the invasion of human hepatocellular carcinoma MDR cell strain BEL-FU.3. The SPK1/S1P signal pathway interfered by DMS can inhibit the expression of mutidrug resistance-related protein in human hepatocellular carcinoma MDR cell strain BEL-FU and overcome its multidrug resistance.