The Effect Of FasL Antisense Oligodeoxynucleotides On Tumor Evasion In Tongue Squamous Cell Carcinoma Cells

ObjectiveStudies have shown that many tumors, including oral squamous cell carcinoma has a high expression of FasL and this high expression has an effect on tumor immune escape. The aim of our study was to investigate the mechanisms of immune escape of oral squamous cell carcinoma(OSCC) through Fas/FasL pathway by detecting the expression of Fas and FasL and the function of FasL in human OSCC. Also we were attempted to evaluate the effect of Fas Ligand(FasL) antisense oligodeoxynucleotide(ASODN) on proliferation and apoptosis of OSCC cells in vitro, and to investigate the inhibit action on T lymphocytes apoptosis induced by OSCC cells and the reverse action of Fas and FasL ASODN on the immune escape by transfecting FasL ASODN in OSCC.Methods1.According to the gene sequence, FasL ASODN complementary to the start codon region of FasL mRNA were designed respectively. The SODN same to the start codon region were designed as control. They were all synthesized and phosphorothioated, and signed with the FAM green fluorescent marker on the5’end. Then specific phosphorothioated ASODN were transfected into tongue squamous cell carcinoma cells with lipofectamineTM2000. We tried to observe the growth and the transfection of Tca8113cells by Inverted microscope and the transfection efficiency was tested by flow cytometry(FCM). The cell viability of tongue squamous cell carcinoma cells was assessed by methyl thiazolyl tetrazolium (MTT) assay and the apoptosis rate was detected by FCM before-and after-transfection.2.The expression of FasL of SCC-9and the expression of Fas of Jurkat were examined by FCM. FasL function of inducing T lymphocytes to apoptosis was assessed by co-culture assays in vitro using SCC-9and Jurkat cells.0.4μmol/L ASODN/SODN were chosen according to the result of pre-experiment. FasL ASODN/SODN was transfected into SCC-9cells with lipofectin. And then the alteration of FasL expression were detected by FCM.The changes of FasL mRNA were measured by real-time polymerase chain reaction(RT-PCR). The changes of Jurkat cells apoptosis induced by SCC-9cells were detected by co-culture assays.Results1.The FasL ASODN inhibited cell proliferation in a dose-and time-dependent manner within limits. The total apoptosis rate was apparently increased(P<0.05) in antisense group compared with the control group, the liposome group and the sense group. Antisense group compared with the control group and liposome group, in addition to early apoptosis rate, the remaining were significant differences(P<0.05), but compared with sense group, only the late apoptotic rate,the total apoptosis rate and cell death were significant differences(P<0.05).2.The Fas expression rate of Jurkat was97.6±2.95%. The apoptotic rate of Jurkat cells in co-culture assays was decreased from61.5±2.44%to6.32±0.56%after blocking with FasL neutralizing antibody NOK-2in SCC-9cells. The rate of FasL expression was decreased to8.5±1.15%after the SCC-9cells were transfected with FasL ASODN at the concentration of0.4μmol/L for24hours. There was significant differences compared with control of27.7±2.54%. The rate of FasL expression in cells transfected with FasL SODN at the same concentration, but was not different from that of control.The results of RT-PCR showed that the level of FasL mRNA of SCC-9cells decreased significantly after transfected with FasL ASODN. The relative expression of FasL mRNA to β-actin gene was0.19±0.01in ASODN group,while that was0.98±0.22in SODN group. The apoptotic rate of Jurkat cells was decreased significantly to8.04±1.01%when co-cultured with SCC-9cells transfected with FasL ASODN, While the apoptotic rate in control and SODN group was57.08±7.43%,57.98±6.74%respectively.Conclusions1.FasL ASODN can suppress proliferation and induce significant apoptosis of tongue squamous cell carcinoma cells.2.Functional FasL expressed by the SCC-9cells could induce Jurkat cells to apoptosis and inhibit the immune function of T lymphocytes indicated that Fas/FasL pathway was one of the mechanisms of immune escape in OSCC cells and maybe become a new target of immunological therapy.3.After SCC-9cells were transfected with FasL ASODN,the FasL mRNA, the rate of FasL expression and the apoptotic rate of Jurkat cells in co-culture assays were all decreased suggested that FasL ASODN could block FasL expression of SCC-9cells effectively, therefore could reduce the attacks of OSCC cells upon T lymphocytes. The results of the study revealed the antitumor action in OSCC cells of FasL ASODN. They provided new theoria and experimental evidences for tumor therapy.