Role And Mechanism Of CD40 Signal On Lung Cancer Cell Line H1299 Proliferation,Migration And Invasion

Objective: CD40 signal has a dual effect on malignant tumor. In previous studies,we found that stimulating CD40 on lung cancer cell line H460 and A549 had a growth inhibitory effect, recently in the pretest we observe that activating CD40 on the lung cancer cell line H1299 promotes its proliferation. This experiment focus on the proliferation,migration,invasion and the mechanism of H1299 after CD40 signal activating,which will provide theoretical support for the rationality of immunotherapy of lung cancer.Methods: Human lung cancer cell line H1299 was chosen as a target cell line and CD40 signal was stimulated by agonistic CD40 monoclonal antibody (5C11) or rhuCD40L. Flow cytometry and RT-PCR method were used to detect the expression of CD40.CCK-8 assay method was used to detect the proliferation of tumor cells. Immunofluorescence technique and the changes of cell cycle were monitored by flow cytometry. Scratch assay and Transwell chamber were applied to detect the migration and invasion ability of H1299.Western blot analysis was utilized to examine the expression of ERK1/2,p- ERK1/2,AKT and p-AKT. EILSA was employed to quantitatiate the concentration of MMP-9.Results: (1)Activating the signal of CD40 on H1299 cells has an accelerating and concentration-dependent effect of proliferation, which is prominent when using the concentration of 5C11 with 10μg/ml or rhuCD40L with 2μg/ml compared to the control group(p<0.05). (2)After CD40 signal stimulation, the number of cells entering G1 phase decreases and the proportion of the phase of S increases compared to the control group(p<0.05), the effect is obvious when using the concentration of 5C11 with 20μg/ml. (3)In the two dimension environment, the ability of migration of H1299 cells increases after stimulated by 5C11 especially using the concentration of 10μg/ml, the scarification is filled with cells compared to the control group which still has a distance between the scratch line(p<0.05). (4)In the three dimension enviroment, the number of cells adhesion to the low bottom of transwell chamber increase when cells were treated by 5C11 compared to the control group (p<0.05). (5) After activating the signal of CD40 on H1299, the transcription of phosphorylation of akt protein increases especially at the time of thirty minutes, and the phosphorylation of ERK1/2 protein has no significantly change. The inhibitor of PI3K-akt pathway LY294002 can block the transcription of phosphorylation-akt protein. (6) 5C11 can promote the secretion of MMP-9, which can be blocked by LY294002.The inhibitor of ERK1/2 pathway PD98059 has no influence on the effect of 5C11 (p<0.05)Conclusions: The ability of proliferation,migration and invasion of H1299 can be accelerated after activating the CD40 signal, the effect of promoting the secretion of MMP-9 by 5C11 can be inhibited by blocking the PI3K-AKT pathway. This suggests that activating CD40 signal on different tumor cell has diverse direct effects. Using the CD40 signal as a target for immunotherapy of cancer in clinical trials still needs more exploration, which requires devising more reasonably program and more effective drug of CD40 signal.