| Objective: To investigate the effect of rapamycin(RAPA)combined with cisplatin(DDP)on apoptosis,proliferation and invasion of tongue squamous cell carcinoma cell line p160,To explore the expression of MAP1LC3,PTEN and m TOR in the lesion and lymph node specimen of human tongue squamous cell carcinoma,and explore its correlation with the clinicopathological features.In order to explore the possible of autophagy-related proteins and pathways as a new target for tongue cancer treatment.Methods: 1.The cell growth inhibition rate of different concentrations of RAPA and DDP on tongue cancer p160 cells was measured by CCK-8 method,and calculated the inhibitory concentration of cisplatin.The RAPA,DDP and RAPA+DDP were used to detect the growth of the cells after 24 hours of p160 cells.2.The expression of autophagy-related protein LC3 was detected by Western-blot after using different concentrations of RAPA in p160 cells.3.Using flow cytometry to measure apoptosis of p160 cells treated with RAPA,DDP and RAPA + DDP.4.The migration ability of RAPA,DDP and RAPA+DDP on p160 cells was detected by scratches experiments after 6 h,18 h and 24 h respective-ly.5.The expression of MAP1LC3,PTEN and m TOR protein was determined using immunohistochemistry,and the relationship between the expression of MAP1LC3,PTEN and m TOR protein was analyzed.Results: 1.Cell growth was increased first and then inhibited after 24 h and 48 h,but cell growth was inhibited and increased with increasing concentration after 72 h.RAPA acts on p160 cells as protector at low concentrations,but can cause autophagic death when the concentration is high enough.2.After DDP effects on cells,the cell growth was inhibited,and with a time and concentration-dependent.3.CCK-8 method showed that the differenton of cell viability between the control group,RAPA group,DDP group and RAPA + DDP group.There was no significant difference between the control group and the RAPA group,and the cell viability of DDP+RAPA group(OD=0.81± 0.13)was significantly lower than that of DDP group(OD=1.14±0.16,P= 0.008).4.The results showed that there was no apoptosis in the control group and RAPA group,and the apoptosis rate in the DDP group(22.3 %)was significantly higher than that in the control group.The apoptosis rate of the RAPA+DDP group was significantly increased from 22.3 % to 47.0 %.5.The results showed that the difference of the distance between the control group,the RAPA group,the DDP group and the DDP + RAPA group after 6 h,18 h and 24 h.After 6 h,the distance of control group was 0.56±0.09 cm,the scratch distance of the RAPA group was 0.54±0.09 cm,the scratch distance of the DDP group was 0.18±0.14 cm,the scratch distance of the DDP + RAPA group was 0.09±0.07 cm;after 18 h,the control group's distance was 0.61±0.18 cm,the scoring distance was 0.84±0.11 cm in the RAPA group,0.52 ± 0.23 cm in the DDP group and 0.42±0.05 cm in the DDP + RAPA group.After 24 h,the scoring distance of the control group was 1.29±0.21 cm,the scratch distance of RAPA group was 1.16±0.04 cm,the scratch distance of DDP group was 0.64±0.26 cm,and the range of DDP+RAPA was 0.54±0.03 cm.DDP group and RAPA+DDP group were significantly different from those in the control group(P <0.05).6.The results of immunohistochemistry showed that PTEN,LC3 and m TOR were mainly expressed in the cytoplasm,and the expression rate of PTEN(34.78 %)was significantly lower than that of precancerous lesion(P=0.007).The positive expression rate of MAP1LC3 in tongue squamous cell carcinoma was 28.26 %,which was significantly lower than that in precancerous lesion and paracancerous tissue(P =0.008).The positive expression rate of m TOR was 66.7 %,and the expression rate in tongue cancer tissues was higher than that in precancerous lesion and paracancerous tissue(P=0.007).The expression of MAP1LC3,PTEN and m TOR was significantly correlated with Lymph node metastasis and clinical stage(P<0.05),but not correlated with the age,sex,smoking,alcohol consumption and histological type of tongue cancer patients(P(29)0.05).With the lymph node metastasis and the clinical stage,the positive expression rate of PTEN protein and LC3 decreased,and the positive rate of m TOR was higher(P<0.05).Conclusion: 1.Rapamycin can induced cell autophagy.A certain degree of autophagy has a cytoprotective effect,but excessive autophagy can cause apoptosis,and this apoptosis may be autophagic death.2.Rapamycin may be used as a chemotherapeutic sensitizer,which still need further study of animal experiments.3.The expression of PTEN,LC3 and m TOR protein is related to the clinical stage and lymph node metastasis.The adnormal level expression of PTEN,LC3 and m TOR may suggest poor prognosis.4.m TOR signaling pathway involved in cell autophagy process,and associated with apoptosis,can be used as a new therapeutic target for the treatment of tongue cancer. |
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