Inhibition of autophagy enhances apoptosis induced by Ce6-photodynamic therapy in human colon cancer cells

Objective: To evaluate the therapeutic effect of Chlorin e6 photodynamic therapy (Ce6-PDT) in human colorectal Cancer cells and investigate the role of Autophagy in Ce6-PDT.

Methods: SW480 cells underwent Ce6-PDT with and without pretreatment with the Autophagy Inhibitor 3-methyladenine (3MA). Cell viability was assessed using 3-(4,5-dimethylthiazol-2-yl)-2, 5-diphenyltetrazolium bromide (MTT) assay. Apoptosis was evaluated using an Annexin V assay, using a rhodamine 123 (RH123) assay to evaluate mitochondrial membrane potential (MMP), and by measuring Caspase-3 and Bcl-2 protein expression using western blotting. Autophagy was evaluated by directly visualizing acridine orange-stained acidic vesicular organelles (AVOs) using fluorescent microscopy and by measuring LC3Ⅰ/Ⅱand Atg5 expression using western blotting.

Results: Ce6-PDT decreased SW480 viability in a dose-dependent manner. Ce6-PDT induced Apoptosis in SW480 cells via the mitochondrial Apoptosis pathway as indicated by decreased mitochondrial membrane potential, increased Annexin V staining, and increased Caspase-3 expression. Ce6-PDT was also shown to induce Autophagy as demonstrated by increased acridine-orange stained AVOs as well as increased expression of the autophagy-associated proteins Atg5. Inhibition of Autophagy with 3MA potentiated SW480 cell response to Ce6-PDT and increased the rate of Apoptosis in the treated cells.

Conclusions: Ce6-PDT induces Autophagy and Apoptosis of SW480 cells in a dose-dependent manner. Inhibition of Autophagy increases the Apoptosis induced by Ce6-PDT. Modulation of Autophagy may be a potential therapeutic target for colon Cancer cells treated with Ce6-PDT.