Effects Of Nicotine On Promoting The Proliferation,Migration And Invasion Of Oral Squamous Cell Carcinoma Cells Through The Activation Of The TAK1/NF-κB Signaling Pathway

Objective:To investigate the effects of nicotine on the proliferation,migration,and invasion of oral squamous cell carcinoma cell line Cal27 cells and the role of TAK1/NF-κB signaling pathway in it.Methods:1.Cal27 cells were cultured with different concentrations of nicotine.After a certain period of time,the proliferation,migration and invasion ability of Cal27 cells were detected by CCK-8,scratch and Transwell invasion assay.2.The expressions of TAK1,p-TAK1,p65 and p-p65 related proteins of TAK1/NF-κB pathway in Cal27 cells treated with different concentrations of nicotine were detected by Western blot3.Cal27 cells were cultured with different concentrations of TAK1 inhibitor(Celastrol),and the IC50 value was detected by CCK-8 test.4.Cal27 cells were co-cultured with Celastrol and nicotine to observe the changes of the above experimental results.Result:1.Cal27 Cells were incubated with 0.25,0.5 and 1 μg/mL nicotine solution,and the cell activity was detected by CCK-8 test at 24,48 and 72 h.The results showed that the number of Cal27 cells increased with the increase of nicotine concentration,and at 24h and 48h,there was a difference between 1 μg/mL nicotine and control group(p<0.05).At 72h,compared with the blank control group,the three nicotine groups showed statistically significant differences(p<0.05).Scratch test results showed that after 48h,the cell migration rate of 0.5 and 1 μg/mL nicotine group was significantly increased compared with the control group,and the difference was statistically significant(p<0.05).The results of Transwell experiment showed that after 48h,the cells invasion number of 0.25 and 0.5 μg/mL nicotine group were significantly increased compared with the control group,the difference was statistically significant(p<0.05).2.The results of protein immunoblotting experiment showed that there was no difference in the expression levels of TAK1/NF-κB signaling pathway related proteins TAK1 and p65 among the three nicotine groups compared to the control group,while the expression levels of p-TAK1 and p-p65 proteins were significantly increased compared to the control group,the difference was statistically significant(p<0.05),among which the 0.5 μg/mL nicotine group had the most significant difference(p<0.01).3.Cal27 cells were treated with 0.5,1 and 2 μM Celastrol.At 48h,the cell activity was detected by CCK-8 test,The results showed that Celastrol had cytotoxic effect on Cal27 cells,and the IC50 value was about 0.8 μM.4.Cal27 cells were co-cultured with 0.5 μg/mL nicotine and 0.5 μg/mL Celastrol,and the above experiments were repeated,the results showed that Celastrol could reverse the biological characteristics and molecular changes induced by nicotine.Conclusion:Nicotine in a certain dose range could promote the proliferation,migration and invasion of oral squamous cell carcinoma.The mechanism may involve the activation of TAK1/NF-κB signaling pathway.