| Objective:Cutaneous squamous cell carcinoma(CSCC)is a malignant tumor originated from the epidermis or adnexal keratinocytes,which is one of the common skin malignant tumor in clinical.The incidence is rising in recent years,and the therapy of cutaneous squamous cell carcinoma in clinical is mainly by surgery,radiation,photodynamic,cryotherapy or combining several methods,but the effect of oral and external drugs is not good.The new anticancer drugs and new targets have been found with the continuous in-depth study.The metformin,which is a old drug for the first line of use of diabetes,has been used as a new one for tumour.Metformin is continuously found to act on various tumor cells,such as breast cancer,ovarian cancer,colon cancer,prostate cancer,malignant melanoma and squamous cell carcinoma,and so on.The antitumor mechanism of metformin is multiple.It was found that metformin can regulate cell energy metabolism,inhibit protein synthesis,induce cell cycle arrest and induce apoptosis through multiple signaling pathways,and can also be combined with other antineoplastic drugs,enhance the cytotoxicity of antineoplastic drugs,so as to promote the apoptosis of tumor cells and inhibit the growth,invasion and metastasis of tumors by domestic and foreign research.Focal adhesion kinase(FAK)is a cytoplasmic non-receptor protein tyrosine kinase which is a member of focal adhesion complexes family.Paxillin is a key adaptor protein in the cytoplasm and it is one of the downstream focal adhesion regulatory proteins of FAK.It is mainly located in adhesive plaques.Focal adhesion kinase and paxillin both can regulate multiple downstream signal pathways,including P13K/Akt,p53,RAS/Erk and so on,so FAK and Paxillin are the centers of intracellular and extracellular signal transduction.Inhibiting the function of FAK and Paxillin can block multiple tumor-associated signaling pathways,then inhibiting the expression of FAK and Paxillin and reducing its activity.So it become a new target for cancer therapy.Metformin has been shown to inhibit the growth,invasion and metastasis of squamous cell carcinoma cells A431 through the signaling pathways such as P13K/Akt and RAS/Erk.This study was designed to investigate the inhibitory effect of metformin on cutaneous squamous cell carcinoma cells A431 and the effects of phosphorylation and transcriptional expression of FAK and Paxillin,to explore the new mechanism.Methods:1.Cell source: Human cutaneous squamous carcinom a cell A431 cell line2.Experimental grouping: According to different drug concentration,the cells was divided into 4 experimental groups,which were 1.25 mM,2.5 mM,5 mM,and 10 mM in 4 experimental groups.The non-drug group was used as the control group.3.The transwell migration and invasion experiments was used to detect the migration and invasion ability of cells in each group after 24 hours of drug treatment.4.The protein expression of FAK,p-FAK,Paxillin and p-Paxillin in the total protein of each group was detected by western blot.5.Real-time fluorescence quantitative PCR(qRT-PCR)technology was used to detect the transcription of FAK and Paxillin genes in each group of cells.6.The expression of phosphorylated FAK and Paxillin in the control and experimental groups was detected by cellular immunofluorescence.7.Statistical analysis: Statistical analysis was performed with SPSS19.0.obey normal distribution were expressed as mean ± standard(?±s)deviation,one-way ANOVA test was used to analyze statistical differences between groups.Measurement data disobeying normal distribution was distributed with median and interquartile range(m±q),nonparametric tests was used to analyze statistical differences between groups.LSD test the difference between the two groups and P<0.05 was statistically significant difference.Results:1.The migration and invasion of A431 cells treated with metformin were weakened by transwell migration and invasion experiments.The number of transmembrane cells in each group was 113.0±14.32,85.33±6.46,83.44±7.47,71.89±7.22 and 60.56±4.21 respectively with different metformin concentrations(0 mM,1.25 mM,2.5 mM,5 mM,10 mM)for 24 hours in the migration experiment,then the difference between any two groups was statistically significant except for the group of 1.25 mM and 2.5 mM(P<0.01).The number of transmembrane cells in each group was 91.44±4.10,78.78±4.99,67.11±4.83,58.22±5.43 and 38.22±8.67 respectively with different metformin concentrations(0 mM,1.25 mM,2.5 mM,5 mM,10 mM)for 24 hours in the invasion experiment,then there was a statistically significant difference between any two groups of this experimence(P<0.01).The number of cells passing through the cell membrane in both groups showed a tendency to decrease as the drug concentration increased.2.The expression of p-FAK and p-Paxillin in the cytoplasm of A431 cells treated with metformin was observed by immunofluorescence.After 12 hours of metformin treatment,the decrease of expression of p-FAK and p-Paxillin in the cytoplasm was observed with increase of drug concentration.3.The relative expression levels of p-FAK protein in each group of cells were 0.65±0.14,0.49±0.16,0.39±0.21,0.36±0.23 and 0.16±0.09 respectively with different drug concentrations(0 mM,1.25 mM,2.5 mM,5 mM,10 mM)for 12 h and the relative expression levels of p-Paxillin protein were 0.56±0.07,0.45±0.09,0.29±0.14,0.19±0.08 and 0.11±0.1 respectively.The differences between the groups were statistically significant(P<0.01).It was suggested that the relative expression of p-FAK and p-Paxillin in total protein showed a decreasing trend with the increase of drug concentration and a certain concentration dependence.4.The relative expression levels of FAK protein in each group were 0.98±0.21,0.97±0.19,0.99±0.11,0.95±0.20 and 0.99±0.09 respectively with different drug concentration(0 mM,1.25 mM,2.5 mM,5 mM,10 mM)for 12 h and the relative expression levels of Paxillin protein in each group were 1.01±0.17,1.08±0.16,0.98±0.12,0.95±0.09 and 1.12±0.21;There was no significant difference between the two groups(P>0.05),indicating that the relative expression of FAK and Paxillin in total proteins did not change significantly with the increase of drug concentration,suggesting that there is no significant effect on its expression in A431 cells.5.The relative expression levels of FAK mRNA and Paxiilin mRNA at different concentrations of metformin(0 mM,1.25 mM,2.5 mM,5 mM,10 mM)were detected by qRT-PCR: the relative expression levels of FAK mRNA were 1.00±0.00,1.02±0.06,0.98±0.06,1.05±0.08 and 0.99±0.11,the difference among the groups was not statistically significant(P> 0.05);the relative expression levels of Paxillin mRNA were 1.00±0.00,1.05±0.05,1.03±0.08,1.04±0.04 and 1.09±0.12,there was no significant difference among groups(P>0.05).It was suggested that there was no significant difference in the relative expression of FAK and Paxiilin mRNA with different drug concentration.The effect of metformin on its transcriptional expression was not obvious.Conclusion:1.The migration and invasion of A431 cells was inhibited by metformin.2.the phosphorylation of FAK and Paxillin was inhibited by metformin,but its transcription and expression were not significantly affected. |
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