Effects Of Chloroquine Combined With Bleomycin And Nedaplatin On Apoptosis And Autophagy Of Tongue Squamous Cell Carcinoma SCC25 Cells And Its Mechanism

Objective: To investigate the effects of chloroquine combined with bleomycin and nedaplatin on apoptosis and autophagy in tongue squamous cell carcinoma SCC25 cells and its mechanism.It provides a certain research basis for the clinical use of chloroquine as a chemotherapy-assisted drug for tongue squamous cell carcinoma.Methods: 1.The growth inhibitory effect of selected drugs on SCC25 cells was detected by MTT assay,and the concentration of selected drugs was determined.2.Hoechst33258 fluorescence staining was used to observe the apoptosis of cells after drug treatment.Flow cytometry was used to observe the effect of drug treatment on apoptosis and cycle of SCC25 cells.4.Autophagosomes were observed under a fluorescence microscope.5.Transwell chamber invasion and migration experiments,to observe the effects of different drugs on the invasion and migration of SCC25 cells.Western blot was used to detect the expression of Bax,BcL-2,NFkB,LC3,Beclin-1and P62 proteins in SCC25 cells.Results: 1.MTT assay detected different concentrations of chloroquine and bleomycin and nedaplatin treatment of SCC25 cells for different time,the cell survival rate decreased gradually,in a concentration-and time-dependent manner.The concentration of 5 mg/L chloroquine combined with bleomycin in IC50(5 mg/L)and nedaplatin at IC50(7 mg/L)was compared with bleomycin treatment alone and nedaplatin treatment alone.The survival rate of SCC25 cells was significantly reduced(p<0.05).2.The morphology of cells observed by Hoechst 33258 fluorescence staining showed that the proportion of chloroquine combined with bleomycin group was higher than that of bleomycin group,and the proportion of apoptotic cells in chloroquine combined with nedaplatin group was higher than that of nedaplatin group.(p<0.05).3.Apoptosis: Compared with the control group,theapoptosis rate of SCC25 cells increased significantly after 48 h of drug treatment(p<0.05).Compared with the bleomycin group,chloroquine combined with bleomycin group The mortality rate increased significantly(p<0.05);the apoptosis rate of chloroquine in the nedaplatin group was significantly increased compared with the nedaplatin group(p<0.05).4.Cell cycle distribution: After 48 hours of treatment with SCC25 cells,the proportion of G0/G1 phase in SCC25 cells increased,the proportion of S phase and G2/M phase decreased,and chloroquine combined with G0/G1 of bleomycin group.The proportion of cells in the period was higher than that in the bleomycin group(p<0.05);the proportion of G0/G1 phase cells in the chloroquine-linked nedaplatin group was higher than that in the nedaplatin group(p<0.05).5.Transwell chamber invasion and migration experiments showed that compared with the control group,the number of SCC25 cells treated in each group migrated to the opposite side of the basement membrane and the number of particles moving across the basal membrane to the opposite side of the basement membrane decreased.The drug can have a significant effect on the migration and invasion of SCC25 cells(p<0.05).5.Observation of autophagosomes under a fluorescence microscope The total sum of yellow spots and red spots in each drug group was reduced relative to the control group.Compared with the control group,the expression of Bax and P62 increased after 48 h of drug treatment;the expression levels of BcL-2,NFkB,LC3 and Beclin-1 were decreased(p<0.05).Compared with the bleomycin group,chloroquine combined with bleomycin group increased Bax and P62 protein expression levels,and BcL-2,NFkB,LC3 and Beclin-1 expression levels decreased;chloroquine compared with nedaplatin group The expression levels of Bax and P62 proteins in the nedaplatin group were increased,and the expression levels of BcL-2,NFkB,LC3 and Beclin-1 were decreased(p<0.05).Conclusion: Compared with bleomycin or nedaplatin,the effect of chloroquine combined with bleomycin and nedaplatin on tongue squamous cell carcinoma SCC25 cells is reflected in its growth inhibition,invasion,migration and apoptosis.Cycle and autophagy regulation and other aspects,and combined drugs have stronger inhibition of proliferation and induction of apoptosis than chemotherapy alone.Chloroquine combined with chemotherapy drugs can induce more obvious cycle arrest in G0/G1 phase and inhibit the invasion and migration ability of SCC25 cells.This combined effect may be achieved by inhibiting autophagy to increase the chemosensitivity of SCC25 cells to bleomycin and nedaplatin.This study provides a certain research basisfor chloroquine as a chemotherapy-assisted drug for tongue squamous cell carcinoma and improving clinical treatment.