Correlation Study On The Biological Behaviors, Autophagy And Apoptosis Of Oral Squamous Cell Carcinoma

Objective:To investigate the effect of an autophagy inhibitor,3-Methyladenine?3-MA?and a chemotherapy drug,cisplatin?DDP?on proliferation,migration,invasion and apoptosis of oral squamous cell carcinoma CAL-27 cells,and the expression of the autophagy associated protein Beclin1,P62 and anti-apoptotic protein Bcl-2 before and after using the ABT-263 on CAL-27 cells,to explore the correlation amomg cellular biological behaviors,autophagy and apoptosis of CAL-27 cells.To detect the expression levels of autophagy associated protein LC3 and Beclin1 in 53 cases of oral squamous cell carcinoma paraffin specimens by immunohistochemical method,and evaluate the autophagy expression levels in oral squamous cell carcinoma and its relationship with clinicopathological features,in order to provide a new direction for the treatment of oral squamous cell carcinoma.Methods:1.The CAL-27 cell growth was detect using CCK-8 assay with different concentrations of 3-MA and DDP treatment at different time points.The suitable concentration of 3-MA required for the experiment was selected.And the 50%inhibitory concentration(IC₅₀)of DDP was calculated.The cell inhibition rate was detected after 24 hours treatment among the 3-MA group,the DDP group,the 3-MA+DDP group and the blank control group.2.The number of migration,invasion cells and the migration,invasion indexs in CAL-27 cells were detected using transwell experiment after 24 hours treatment among the 3-MA group,the DDP group,the 3-MA+DDP group,and the control group.3.The cell apoptosis was detected by the flow cytometry,after 24 hours treatment,respectively,in the 3-MA group,the DDP group,the 3-MA+DDP group and the control group.4.The expression levels of autophagy related protein LC3,Beclin1,P62 in CAL-27 cells before and after using 3-MA,ABT-263 were detected by western blotting.5.The expression levels of LC3 and Beclin1 in 53 cases of oral squamous cell carcinoma paraffin specimens were detected by immunohistochemistry.Results:1.3-MA and DDP were applied to CAL-27 cells 24 h,48 h and 72 h respectively,the proliferation of CAL-27 cells was inhibited,and the inhibition rate was increased with time and drug concentration,which indicated that the inhibition was time and concentration dependent.There was no significant difference in cell viability between the control group and the 3-MA group,but the cell viability of DDP group and 3-MA+DDP group was decreased significantly.The proliferation activity of CAL-27 cells in 3-MA+DDP group?OD value=0.445±0.029?was significantly lower than that in DDP group?OD value=0.657±0.038,P=0.000?.2.Transwell migration experiment was used to measure the amount of cells transmigrated in control group,3-MA group,DDP group,3-MA+DDP group after24 h:compared with the control group?127.25±4.03?,the number of transmigrated cells in 3-MA group was not significantly decreased?119.75±5.12?,but the number of transmigrated cells in DDP group?77.5±6.19?and 3-MA+DDP group?51.75±7.68?was significantly decreased.And the 3-MA+DDP group was significantly less than the DDP group?P<0.05?.Compared with the blank control group,the migration index of 3-MA group,DDP group,3-MA+DDP group was 94.11%,60.90%and 40.67%respectively.Transwell invasion experiment was used to measure the amount of cells in control group,3-MA group,DDP group,3-MA+DDP group after 24 h:compared with the control group?183.25±4.11?,the number of perforated cells was no significant decrease in 3-MA group?178.25±2.22?,but the number of perforated cells of DDP group?84.25±6.40?and 3-MA+DDP group?51.50±4.20?was significant decrease.And the 3-MA+DDP group was significantly less than the DDP group?P<0.05?.Compared with the control group,the invasion index of 3-MA group,DDP group,3-MA+DDP group was 94.11%,60.90%and 40.67%respectively.3.Apoptotic cells were detected by flow cytometry:no obvious change in apoptosis could be seen in the control group and 3-MA group,and the apoptosis rate of the DDP group?31.1%?was significantly higher than that in the control group.The apoptosis rate of the 3-MA+DDP group was significantly increased from 31.1%to 57.6%.4.Western blotting results:after the application of 3-MA,the expression of autophagy associated protein LC3 decreased.After using Bcl-2 inhibitor ABT-263,the expression of P62 was not significantly changed,but Beclin1 expression level was decreased.5.Immunohistochemical results showed:LC3 and Beclin1 are mainly expressed in cytoplasm.The positive expression rate of LC3 and Beclin1 in oral squamous cell carcinoma tissues?73.58%,43.40%?were significantly higher than that in the adjacent tissues respectively?7.14%,P<0.05?.The results showed:the expression of LC3 and Beclin1 were not significantly correlated with age,sex,smoking and alcohol consumption in patients with oral squamous cell carcinoma?P<0.05?,but were correlated with lymphatic metastasis,histological differentiation.The lower the degree of tumor differentiation,the lower the positive expression rate of LC3 and Beclin1.LC3 and Beclin1 proteins expression were more lower in tumors with lymph node metastasis than in those without?P<0.05?.Conclusion:1.DDP can induce the protective autophagy of CAL-27 cells,and 3-MA inhibited autophagy levels in a time-and dose-dependent manner.2.Combining 3-MA with DDP may reduce the IC₅₀0 of DDP,in other words,3-MA can enhance the sensitivity of the CAL-27 cells to chemotherapy drug cisplatin,however,further animal model experiments and clinical trials are needed.3.LC3 and Beclin1 protein were highly expressed in oral squamous cell carcinoma.The expression levels of LC3 and Beclin1 protein were associated with lymph node metastasis and histological differentiation.4.Beclin1 autophagy signaling pathway is associated with Bcl-2-mediated apoptosis.It can be used as a new direction for the treatment of oral squamous cell carcinoma.