Biological Effect Of CD40 Stimulation On Human Lung Cancer Cells And Its Possible Mechanisms

Objective To investigate the biological effect of CD40 stimulation on human lung cancer cells and its possible mechanisms.Methods Human lung cancer cell lines H460,A549 and SPC-A-1 were chosen as target cells and CD40 signal was stimulated by soluble CD40 Hgand(sCD40L)or agonistic CD40 monoclonal antibody(5C11).MTT assay and 3H-TdR incorporation method were used to determine the proliferation of tumor cells. Immunofluorescence technique and flow cytometry were used to monitor changes of cell surface molecules, cell cycle, TRAFs expression as well as cell apoptosis. Expression changes of Bcl-2 and Bax were observed by RT-PCR. Furthermore, cisplatin (DDP) or mitomycin (MMC) was co-incubated with cell lines after CD40 stimulation. MTT assay was used to determine the cell proliferation inhibition effect of cisplatin or mitomycin after CD40 stimulated. Immunofluorescence technique and flow cytometry were employed to monitor the effect of cytotoxicity of DDP or MMC on lung cancer cell lines after CD40 stimulation.Results (1) There was an inhibition of cell proliferation on CD40 positive cell lines H460 and A549 after CD40 stimulation. (2) 72 hours after CD40 stimulation, the expression of cell surface molecules CD49e, CD54, TNFR I and CD95L were higher than those in the control group, while the expression of TNFR II was lower than those in thecontrol group(P<0.05). The expression of TRAF2, 3, 5, 6 in H460 and A549 were downregulated after CD40 stimulation (PO.05). (3) After CD40 stimulation, the number of cells entering GI phase was increased compared with that in the control group, while the number of cells entering S phase was decreased (PO.05). (4) No significant cell apoptosis was observed after 72-hour CD40 stimulation, but an up-regulation of Bax expression was observed in H460 and A549. (5) After CD40 stimulation, an increase of inhibition on CD40 positive cell line H460 and A549 proliferation and an augment of cytotoxicity of DDP and MMC on H460 and A549 were confirmed when DDP or MMC was added (PO.05). (6) No such changes were observed on CD40 negative cell line SPC-A-1.Conclusions On CD40 positive cell lines H460 and A549, stimulation of CD40 signal could inhibit cell proliferation and cause changes of phenotype, cell cycle and TRAFs expression. Meanwhile stimulation of CD40 could alter gene expression that associated with cell apoptosis and upregulate cell sensitivity to chemotherapy on CD40 positive cell lines H460 and A549. These changes indicated that CD40 signal had important adjustive function on CD40 positive cell lines.