The Effect Of Sphingosine Kinasel On Biological Characteristics Of Human Hepatocellular Carcinoma Cell Line BEL-FU

Objective:Recent studies show that the sphingolipid metabolites are important signal transduction molecules involved in invasion, proliferation, blood vessel formation of tumors. The dynamic balance among ceramide (Cer), sphingosine (Sph), and sphingosine-1-phosphate (S1P) determines cell proliferation and survival which is regulated strictly by multiple kinase. Sphingosine kinase type 1 (SpK1), the enzyme that phosphorylates Sph to form S1P, is a critical regulator of the sphingolipid rheostat, as it not only produces the pro-growth, anti-apoptotic messenger S1P, but also decreases levels of pro-apoptotic Cer and Sph. The objective of this study is to elucidate the roles of sphingosine kinasel (SPK1) on the apoptosis, invasiveness and multidrug resistance characteristics of human hepatocellular carcinoma cell line BEL-FU infected with adenovirus carrying target genes.Methods:AD293 cell were grown in DMEM supplemented with 10% FBS,100 u/L penicillin and 100 u/L streptomycin at 37℃,5% CO2, and were digested by 0.25% trypsin. The morphological variations of cells were observed with invert phase-contrast microscope and with fluorescence microscopy. The adenovirus carrying the wild type SPK1 gene (SPK1WT) and SiRNASPKl(Ad-H1-SPK1)were constructed respectively; viral infection titer was determined by TCID50 and CPE method. Human hepatocellular carcinoma MDR cell strain BEL-FU were grown in 1640 supplemented with 10% FBS,20000 ng/L 5-FU,100 u/L penicillin and 100 u/L streptomycin at 37℃,5% CO2, and were digested by 0.25% trypsin. The morphological variations of cells were observed with invert phase-contrast microscope. BEL-FU cells were infected with adenovirus carrying SPK1WT gene and SPK1siRNA (Ad-H1-SPK1) gene, their effect on biological characteristics of BEL-FU cells was evaluated by MTT, and cellular SPK enzyme activity was assayed. The cell migration was investigated by Transwell Migration Technology. The expression of multidrug resistance-related protein (MRP1) was observed by Western-blot respectively. Results:1. Re-adenoviruses adenoviruses amplified and purified. After purification the adenoviruses were highly enriched.2. The efficiency of infection was assayed. There was nearly full view of fluorescent when MOI is 150 with Ad-EGFP as a standard.3. Enzyme assay of SPK was tested. A standard curve was drawn according to the statistics which was assayed by multifunctional UV-VIS spectrophotometer. The activity of SPK in BEL-FU cell infected with adenovirus carrying Ad-SPK1WT was obviously higher than the activity of SPK in BEL-FU cell infected with adenovirus carrying Ad-H1-SPK1.4. The effects of SPK1 on survival in human hepatocellular carcinoma MDR cell strain BEL-FU. Our results demonstrated that 5～25μmoL/L DMS can cause the apoptosis of BEL-FU and was in dose-dependent pattern.5. The effects of SPK1 on invasion in human hepatocellular carcinoma MDR cell strain BEL-FU. The results of transwell chamber assay indicated that the migration of BEL-FU infected with adenovirus carrying Ad-SPK1WT was enhanced, while the migration of BEL-FU infected with adenovirus carrying Ad-H1-SPK1 was suppressed.6. The effects of SPK1 on the expression of multidrug resistance-related protein (MRP1) in human hepatocellular carcinoma MDR cell strain BEL-FU. The expression of multidrug resistance -related protein (MRP1) of cells infected with SPK1siRNA (Ad-H1-SPK1) was suppressed significantly compared with the control group, while the expression of MRP1 infected with Ad-SPK1WT was enhanced (P<0.05). Conclusion:1.The adenoviruses carrying target gene were highly enriched.2. The adenoviruses carrying Ad-SPK1WT and Ad-H1-SPK1 can infect BEL-FU cell effectively.3. The activity of SPK in BEL-FU cell infected with Ad-SPK1WT was enhanced, while the activity of SPK in BEL-FU cell infected with Ad-H1-SPK1 was suppressed.4. Over expression of SPK1 suppress the apoptosis induced by DMS (Dimethyl sphingosine,DMS), Whereas the cells infected with SPK1siRNA (Ad-H1-SPK1) increased the apoptosis induced by DMS. 5. BEL-FU cells infected with Ad-SPK1WT enhance migration of BEL-FU cells, whereas the cells infected with SPK1 siRNA (Ad-H1-SPK1) inhibited the migration of human hepatocellular carcinoma cells.6. The expression of multidrug resistance-related protein (MRP1) of cells infected with SPK1siRNA (Ad-H1-SPK1) was suppressed significantly compared with the cells infected with Ad-SPK1WT.