Figure 2.

Induction of prosurvival autophagy in chemoresistant cells and pharmacological inhibition of autophagy augments the sensitivity of chemoresistant breast cancer cells

(A) Basal level of autophagy was increased in chemoresistant cells as LC3-II, ATG5 and Beclin-1 proteins expression were increased in chemoresistant cells compared to their respective parental cells in western blot analysis. (B) Autophagic flux was determined by the accumulation of LC3-II and p62 after combined treatment of DOX (10 ng/ml) for 72 h and autophagic flux inhibitor Baf A1 (25 nM) for 4 h in both the BT-549Par and BT-549^rDOX²⁰ cells and (C) similarly in MDA-MB-468Par and MDA-MB-468^r5-FU²⁰⁰⁰ cell lines after combined treatment of 5-FU (1 μg/ml) for 72 h and Baf A1 (25 nM) for 4 h. GAPDH was used as loading control. (D) Breast cancer cell lines BT-549Par and BT-549^rDOX²⁰ were treated with 10 ng/ml and 80 ng/ml of DOX for 72 h with or without Baf A1 (25 nM for 4 h), (E) similarly MDA-MB-468Par and MDA-MB-468^r5-FU²⁰⁰⁰ cell lines were treated with 1 μg/ml and 18 μg/ml of 5-FU for 72 h with or without Baf A1 (25 nM for 4 h). Total cell death was quantified by Annexin V/PI double staining followed by flow cytometry. Columns represent means of three independent experiments performed in triplicate ± SEM. Statistical significance: * p<0.05, ** p<0.01, *** p<0.001 and ns not significant compared to respective controls (Ctrls); # p<0.05, ## p<0.01 and ns not significant with Baf A1 treatment compared to without Baf A1 treatment.