Effect Of Procaine On Inhibiting Proliferation Of Human Tongue Squamous Cell Carcinoma CAL27 Cells And Its Mechanism

Purpose:To explore the effects of procaine?PCA?on proliferation,migration,cell cycle,apoptosis and autophagy of human tongue squamous cell carcinoma CAL27 cells,and to clarify the underlying mechanism of PCA in inhibiting CAL27 cells of human tongue squamous cell carcinoma.To provide data support for the local anaesthesia developed as a drug for the treatment of oral squamous cell carcinoma and other new uses.Methods:The CAL27 cells were treated with different concentrations of PCA?0,0.5,1.0and 2.0 mg/mL?,and the cells were divided into control group and 0.5,1.0 and 2.0mg/mL PCA group.The proliferation rates of the CAL27 cells in various groups were detected by CCK-8 assay and clone formation method.The morphology of CAL27cells in various groups were observed by crystal violet staining.The percentageS of CAL27 cellS healing area in various groups were determined by the scratch method.The mitochondrial membrane potential?MMP?of CAL27 cell in various groups were detected by laser confocal high-intension screening platform.The reactive oxygen species?ROS?levels,percentages of CAL27 cells in different cell cycles and the apoptotic rates in various groups were detected by flow cytometry.The expression levels of cyclin-dependent kinase inhibitor p21 mRNA in the CAL27 cells in various groups were analyzed by Real-time fluorescence quantitative PCR.The expression levels of cyclin dependent kinase inhibitor p21,anti-apoptotic protein Bcl-2 and survivin,apoptosis promoting protein Bax,autophagy signature proteins LC3 and p62,extracellular regulated protein kinase ERK/p-ERK,phosphatidylinositol 3-kinase PI3K and serine/threonine kinase AKT/p-AKT in the CAL27 cells in various groups were determined by Western blotting method.Results:PCA can significantly inhibit the proliferation of CAL27 cells with a concentration dependence.Compared with the control group,the proliferation rates of CAL27 cells in 0.5,1.0 and 2.0 mg/mL PCA groups were significantly decreased,85.9%,75.0%and 42.0%,respectively?*P<0.05,**P<0.01?.And the cell colonies of each PCA group decreased obviously.PCA can change the morphology of CAL27cells.Compared with the control group,CAL27 cells in each group were wrinkled to different degrees,and cells in 1.0 mg/mL and 2.0 mg/mL PCA groups showed intercellular connections loss,cavitation change and even rupture.PCA can significantly inhibit the migration of CAL27 cells.With the increase of concentration,cell scratch healing rates became slower,especially at 24 h and 48 h?*P<0.05,**P<0.01?.PCA can reduce the levels of MMP in CAL27 cells and promote the production of ROS in CAL27 cells.PCA can induce the G₂/M phase cycle arrest of CAL27 cells.Compared with the control group,the percentages of CAL27 cells in G₂/M phase increased significantly in the 0.5 and 1.0 mg/mL PCA groups,and the expression levels of mRNA and protein of p21 increased significantly?*P<0.05,**P<0.01?.PCA can promote the apoptosis of CAL27 cells.Compared with the control group,the apoptosis rates in 1.0 and 2.0 mg/mL PCA groups were 12.2%and17.0%.Bcl-2 protein expression levels were significantly decreased,while Bax protein expression levels were significantly increased.PCA can induce autophagy,but at the same time damage autophagy flux.Compared with the control group,the LC3and p62 protein expression levels were significantly increased.PCA combined with autophagy inhibitor CQ has a stronger inhibitory effect on cell proliferation than PCA or CQ alone.PCA can inhibit the activation of ERK pathway and PI3K/AKT pathway.Conclusion:PCA can inhibit the proliferation and migration of CAL27 cell.PCA can up-regulate the mRNA and protein expression levels of p21,and induce G₂/M cycle arrest.PCA can down-regulate the expression levels of Bcl-2 and up-regulate the expression levels of Bax to promote apoptosis.PCA can damage autophagy flux and further promote apoptosis after inhibiting autophagy.PCA can partially inhibit the phosphorylation of ERK pathway and PI3K/AKT pathway.That is,PCA can significantly inhibit the activity of OSCC,and have significant therapeutic effects.The experimental results provide a new theoretical basis for the chemotherapy of oral tongue squamous cell carcinoma.