The Antitumor Effect And Its Mechanism Of Luteolin On Tongue Squamous Cell Carcinoma SCC15 Cells

Background and objective The head and neck cancer is the sixth most common tumor in the world,and oral cancer accounts for the majority of head and neck cancers.About 90% of oral cancers are squamous cell carcinomas,and tongue squamous cell carcinoma(TSCC)has the highest incidence.TSCC has high growth rate and strong invasiveness,local lymph node metastasis is prone to occur early and distant metastases may develop over time.The clinical comprehensive treatment effect of TSCC is not ideal,such as the side effects of radiotherapy and chemotherapy,easy to tolerate and relapse and so on,which brings a huge economic burden,painful physical and mental torture to patients.Therefore,finding effective and low-toxic drugs to prevent and treat TSCC is of great significance for improving the quality of life and the survival rate of patients.Luteolin is effective against a variety of malignant tumors.The mechanism is related to inhibiting tumor proliferation,inducing tumor apoptosis,inhibiting angiogenesis,and inhibiting migration and invasion.Luteolin can also reverse tumor resistance to radiochemotherapy.In addition,luteolin also has the advantages of low toxicity,wide sources,and low cost,so it is suitable for the chemoprevention of tumors.However,the effect and mechanism of luteolin on TSCC are rarely studied.Therefore,this study aims to investigate the effects of luteolin on proliferation,apoptosis,migration and invasion of TSCC and related mechanisms in order to provide scientific basis for the development of new drugs.Methods 1.MTT assay was carried out to observe the proliferation of SCC15 cells after treatment with luteolin at the concentration of 5?g/ml,10?g/ml,15?g/ml,20?g/ml,40?g/ml,60?g/ml for 24 h,48h and 72 h,respectively.2.Hoechst 33258 staining was carried out to observe the apoptosis of SCC15 cells after treatment with luteolin at concentrations of 10?g/ml,20?g/ml,and 40?g/ml for 12 h.3.Scratch assay was carried out to observe the migration of SCC15 cells after treatment with luteolin at concentrations of 1?g/ml,5?g/ml,10?g/ml for 12 h,and 24 h,respectively.4.Transwell migration/invasion assays was carried out to observe the migration and invasion of SCC15 cells after treatment with luteolin at concentrations of 5?g/ml,10?g/ml for 24 h.5.SCC15 cells were treated with luteolin at concentrations of 10?g/ml,20?g/ml,and 40?g/ml for 24 h and the conditional medium was collected.The gelatinases-matrix metalloproteinase-2 and-9(MMP-2,-9)in the conditional medium were detected by gelatin zymography assay.6.The expression of apoptosis-related proteins Survivin,caspase-3,and cleaved caspase-3 in SCC15 cells after treated with luteolin at concentrations of 10?g/ml,20?g/ml,and 40?g/ml for 24 h was detected by Western blotting,and the expression levels of MMP-2 and MMP-9 proteins were also detected by this method.Results 1.MTT assay showed that luteolin had obvious proliferation inhibition effect on SCC15 cells in a concentration-dependent and time-dependent manner.The IC50 values of SCC15 cells at 24 h,48 h,and 72 h were 31.2?10.2and 10.6 ?g/ml,respectively.2.Hoechst 33258 nuclear staining showed that the number of apoptotic nuclei increased significantly compared with the control group after treatment with luteolin for 12 h.With the increase of drug concentration,the proportion of apoptosis in SCC15 cells also gradually increased.The apoptotic rate of SCC15 cells increased from 2.81% to 9.69% when the drug concentration increased from 0?g/ml to 40?g/ml.3.After 12 h and 24 h of scratching,luteolin exhibited a significant inhibitory effect on SCC15 cells compared with the control group.When the concentration was only 1?g/ml,the inhibitory effect was obvious.And when the drug concentration was increased to 5 ?g/ml and 10 ?g/ml,SCC15 cells hardly migrated.4.The results of Transwell migration/invasion assays showed that with the increase of drug concentration,the number of cells that migrated to the lower chamber was less compared with the control group,indicating that luteolin inhibits the migration and invasion of cells more strongly.There were(340.0±22.94),(52.67±6.936)and(6.57±0.80)transmembrane cells in the three groups of the migration assay,and(85.67±5.18),(39.67±4.63)and(2.67±0.29)in the three groups of the invasion assay,respectively(P<0.001).5.Gelatin zymography results showed that the activity of gelatinase MMP-2 and MMP-9 in supernatant of SCC15 cells decreased with the increase of luteolin concentration.6.Western blot results showed that the expression of MMP-2 and MMP-9 were down-regulated in the SCC15 cells treated with luteolin.And the expression levels of apoptosis-related proteins Survivin and Caspase-3 were decreased while the expression of Cleaved caspase-3 was increased.Conclusion 1.Luteolin can inhibit the proliferation and induce the apoptosis of SCC15 cells,the mechanism may be related to the down-regulation of Survivin.2.Luteolin inhibited the migration and invasion of SCC15 cells,and the mechanism may be related to inhibiting the activities of gelatinase MMP-2 and MMP-9 in the cell supernatant and down-regulating the expression of MMP-2 and MMP-9 protein.