Effects of 3MA on apoptosis and autophagy of Ce6-PDT-induced colorectal cancer cells SW620

YANG Kaizhen; Gulinuer·Abudureheman; LUO Mengyu; KANG Ling

doi:10.3969/j.issn.1002-266X.2017.25.001

Abstract：Objective To investigate the effects of autophagy inhibitor 3-methyladenine (3MA)combined with Ce6-PDT on the apoptosis and autophagy of human colorectal cancer SW620 cells,and to provide theoretical and practical basis for autophagy inhibitors improving sensitivity of Ce6-PDT in treatment of colorectal cancer. Methods SW620 cells in the logarithmic phase were divided into the blank control group,negative control group,Ce6-PDT group,and 3MA-Ce6-PDT group. Cells in the Ce6-PDT group were treated with different concentrations of Ce6 (0. 125,0. 25,0. 5,1. 0,2. 0,and 8. 0 μg/ mL),and cells in the 3MA-Ce6-PDT group were treated with 1. 0 μg/ mL Ce6 + 5 mmol/ mL 3MA. Cell viability was detected by using MTT. The apoptosis and cell mitochondrial membrane potential changes were detected by using An-nexinV-PE/ 7-ADD and Rho123 combined with flow cytometry. Formation of autophagosome was observed by using fluores-cence microscopy with Acridine orange staining. The expression of apoptosis-related proteins Caspase-3,B-cell lymphoma/leukemia-2 (Bcl-2)and autophagy-related protein LC3Ⅱ/ Ⅰ was detected by Western blotting. Results In the Ce6-PDT group,the cell viability was negatively correlated with Ce6 concentration (r = - 0. 820,P < 0. 01);the apoptosis rate was positively correlated with the concentration of Ce6 (r = 0. 845,P < 0. 01);When the cell survival rate and apoptosis rate were 50%,1 μg/ mL Ce6 was taken as the concentration of the subsequent administration. When the Ce6 concentration reached 1 μg/ mL,the mitochondrial membrane potential decreased with the increase of Ce6 concentration (P < 0. 01).There was almost no bright red fluorescence in the blank control group and the negative control group. There was a large a-mount of bright red fluorescence in the Ce6-PDT group. The number of bright red fluorescence in the 3MA-Ce6-PDT group was significantly lower than that in the Ce6-PDT group. Compared with the Ce6-PDT group,the cell viability decreased (P< 0. 05),the apoptosis rate increased (P < 0. 05),mitochondrial membrane potential decreased (P < 0. 05),the expres-sion of autophagy-related protein decreased (P < 0. 05),and the expression of apoptosis-related protein increased in the 3MA-Ce6-PDT group (P < 0. 05). Conclusions Ce6-PDT induces the apoptosis and autophagy of colorectal cancer cells SW620. 3MA may promote the apoptosis of Ce6-PDT-induced colorectal cancer cells.