Figure 5.

HSP70/BAG3 interaction inhibitor YM-1 and HSF-1 inhibitor KRIBB11 sensitize the chemoresistant breast cancer cells

(A) Dissociation of the HSP70/BAG3 complex by YM-1 (5 μM) decreased Mcl-1 expression in BT-549^rDOX²⁰ cells after 4 h, 18 h, 24 h, 48 h treatment in western blot analysis. 0.1% DMSO for 48 h was used as Control (Ctrl) for the solvent, whereas Sorafenib (Sora) 5 μM for 48 h was used as positive control. GAPDH was used as loading control in western blot. (B) % of cell viability was analyzed by MTT assay in BT-549Par and BT-549^rDOX²⁰ cells after 2 h pre-treatment of 5 μM of YM-1 for 48 h followed by DOX for 72 h. DMSO 0.1 % for 48 h was used as control (Ctrl) for the solvent. (C) 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 YM-1 (5 μM, 48 h). 0.1 % DMSO for 48 h was used as control (Ctrl) for the solvent. Then cell death was analyzed by Annexin-V positive staining in flow cytometry. Columns represent means of three independent experiments ± SEM (n = 3). Statistical significance: * p<0.05, ** p<0.01, *** p<0.001 and ns not significant compared to ctrls (0.1 % DMSO); # p<0.05, ## p<0.01 and ns not significant with combined treatment of YM-1 and DOX compared to DOX treatment alone. (D) Combined treatment of KRIBB11 (20 μM, 48 h) and DOX significantly augment the total cell death in BT-549^rDOX²⁰/sh Ctrl cells compared to DOX treatment alone. 0.1 % DMSO for 48 h was used as control (Ctrl) for the solvent. Cell death was determined by Annexin V/PI double staining followed by flow cytometry. Columns represent means of three independent experiments ± SEM (n = 3). Statistical significance: * p<0.05, ** p<0.01, *** p<0.001 and ns not significant compared to ctrls (0.1 % DMSO); # p<0.05, ## p<0.01 and ns not significant with combined treatment of KRIBB11 and DOX compared to DOX treatment alone. (E) KRIBB11 treatment decreased BAG3, HSP70, Mcl-1 and LC3 II in a dose dependent manner in BT-549^rDOX²⁰ and (F) MDA-MB-468^r5-FU²⁰⁰⁰ cells respectively. Cells were treated with 5 μM, 10 μM and 20 μM of KRIBB11 for 48 h. DMSO (0.1 %, 48 h) was used as control (Ctrl). GAPDH served as loading control for western blot.