Salinomycin induces cell death with autophagy through activation of endoplasmic reticulum stress in human cancer cells

Abstract

Salinomycin is perhaps the first promising compound that was discovered through high throughput screening in cancer stem cells. This novel agent can selectively eliminate breast and other cancer stem cells, though the mechanism of action remains unclear. In this study, we found that salinomycin induced autophagy in human non-small cell lung cancer (NSCLC) cells. Furthermore, we demonstrated that salinomycin stimulated endoplasmic reticulum stress and mediated autophagy via the ATF4-DDIT3/CHOP-TRIB3-AKT1-MTOR axis. Moreover, we found that the autophagy induced by salinomycin played a prosurvival role in human NSCLC cells and attenuated the apoptotic cascade. We also showed that salinomycin triggered more apoptosis and less autophagy in A549 cells in which CDH1 expression was inhibited, suggesting that the inhibition of autophagy might represent a promising strategy to target cancer stem cells. In conclusion, these findings provide evidence that combination treatment with salinomycin and pharmacological autophagy inhibitors will be an effective therapeutic strategy for eliminating cancer cells as well as cancer stem cells.

Keywords: MTOR; apoptosis; autophagy; endoplasmic reticulum stress; salinomycin.

Figure 1. Salinomycin induces autophagy in human NSCLC cells. (A) The indicated cells were seeded in 96-well cell culture plates and treated with 1.25 μM, 2.5 μM and 5 μM of salinomycin on the second day. After treatment for another 48 h, the cells were fixed and subjected to estimate the cell number using the sulforhodamine B assay. (B) A549, Calu-1 and H157 cells were treated with 2.5 μM salinomycin for the indicated times. Levels of protein expression were analyzed by western blot using antibodies against MAP1LC3B and ACTB. (C) A549, Calu-1 and H157 cells were treated with the indicated concentrations of salinomycin and incubated for 24 h. Levels of protein expression were analyzed by western blot using antibodies against MAP1LC3B and ACTB. (D) Calu-1-EGFP-MAP1LC3B cells were treated with 2.5 μM salinomycin for the indicated period of time, and then EGFP-MAP1LC3B puncta were quantified. (E) Calu-1-EGFP-MAP1LC3B cells were treated with the indicated concentrations of salinomycin for 24 h, and then EGFP-MAP1LC3B puncta were quantified. Columns: mean of triplicate treatments; bars: ± SD. The statistical differences between the two treatments were analyzed by two-sided unpaired Student’s t-tests (*p < 0.05; **p < 0.01; *** p < 0.001).

Figure 2. Salinomycin induces autophagic flux in human NSCLC cells. (A and B) Calu-1 cells were transfected with ATG5 siRNAs (#1 and #2) or ATG7 siRNAs (#1 and #2). 48 h later, cells were treated with 2.5 μM salinomycin or DMSO for 12 h, and then the samples were subjected to western blot analysis. Calu-1-EGFP-MAP1LC3B cells were transfected with ATG5 siRNA (#1) or ATG7 siRNA (#1). 48 h later, cells were treated with 2.5 μM salinomycin for 12 h, and then EGFP-MAP1LC3B puncta were quantified. (C) Calu-1 cells were pretreated with 3-MA (10 mM) for 1 h, and then coincubation with salinomycin (2.5 μM) for another 12 h. Levels of protein expression were analyzed by western blot using antibodies against MAP1LC3B and ACTB. Calu-1-EGFP-MAP1LC3B cells were pretreated with 3-MA (10 mM) for 1 h, and then coincubation with salinomycin (2.5 μM) for another 12 h, and then EGFP-MAP1LC3B puncta were quantified. (D) Calu-1 cells were pretreated with LY294002 (10 μM) for 1 h, and then coincubation with salinomycin (2.5 μM) for another 12 h. Levels of protein expression were analyzed by western blot using antibodies against MAP1LC3B and ACTB. Calu-1-EGFP-MAP1LC3B cells were pretreated with LY294002 (10 μM) for 1 h, and then coincubation with salinomycin (2.5 μM) for another 12 h, and then EGFP-MAP1LC3B puncta were quantified. (E) Calu-1 cells were pretreated with bafilomycin A₁ (20 nM) for 1 h, and then coincubation with salinomycin (2.5 μM) for another 12 h. Levels of protein expression were analyzed by western blot using antibodies against MAP1LC3B and ACTB. Calu-1-EGFP-MAP1LC3B cells were pretreated with bafilomycin A₁ (20 nM) for 1 h, and then coincubation with salinomycin (2.5 μM) for another 12 h, and then EGFP-MAP1LC3B puncta were quantified. (F) Calu-1 cells were pretreated with CQ (3 μM) for 1 h, and then coincubation with salinomycin (2.5 μM) for another 12 h. Levels of protein expression were analyzed by western blot using antibodies against MAP1LC3B and ACTB. Calu-1-EGFP-MAP1LC3B cells were pretreated with CQ (3 μM) for 1 h, and then coincubation with salinomycin (2.5 μM) for another 12 h, and then EGFP-MAP1LC3B puncta were quantified. The average number of punctate structures per cell was quantified by confocal microscopy. Differences between groups were evaluated by Student's t-test. Columns: mean of triplicate treatments; bars: ± SD. The statistical differences between the two treatments were analyzed by two-sided unpaired Student’s t-tests (*p < 0.05; **p < 0.01; *** p < 0.001).

Figure 3. Salinomycin induces ER stress in human lung cancer cells. (A) A549, Calu-1 and H157 cells were treated with the given concentrations of salinomycin for 24 h. Levels of protein expression were analyzed by western blot using antibodies against ERN1, p-EIF2A, ATF4, DDIT3, MAP1LC3B and ACTB. (B) A549 and Calu-1 cells were treated with salinomycin (2.5 μM) for the indicated period of time. Levels of protein expression were analyzed by western blot using antibodies against ERN1, p-EIF2A, ATF4, DDIT3, MAP1LC3B and ACTB. (C) XBP1 cDNA was amplified by PCR followed by incubation with PstI. The products were separated by electrophoresis on a 2% agarose gel and visualized by ethidium bromide staining. Treatment of cells with 1 μM thapsigargin for 24 h was used as a positive control.

Figure 4. Salinomycin induces autophagy via ER stress-evoked ATF4-DDIT3 upregulation. (A) Calu-1 cells were transfected with control siRNA or DDIT3 siRNAs (#1 and #2) for 48 h and then treated with salinomycin (2.5 μM) for 12 h. Levels of protein expression were analyzed by western blot using antibodies against DDIT3, MAP1LC3B and ACTB. (B) Calu-1 cells were transfected with control siRNA or ATF4 siRNAs (#1 and #2) for 48 h and then treated with salinomycin (2.5 μM) for 12 h. Levels of protein expression were analyzed by western blot using antibodies against ATF4, DDIT3, MAP1LC3B and ACTB. (C) A549 and Calu-1 cells were pretreated with 4-PBA (1 mM) for 30 min and then cotreated with salinomycin (2.5 μM) for another 12 h. Levels of protein expression were analyzed by western blot using antibodies against ATF4, DDIT3, MAP1LC3B and ACTB.

Figure 5. Salinomycin induces autophagy via the ATF4-DDIT3-TRIB3-AKT1-MTOR axis. (A) A549, Calu-1 and H157 cells were treated with the indicated concentration of salinomycin for 24 h. Levels of protein expression of p-AKT1, p-RPS6KB1, p-EIF4EBP1 and ACTB were measured by western blot analysis. (B) A549, Calu-1 and H157 cells were incubated with the given concentration of salinomycin for 24 h. Level of protein expression of TRIB3 was measured by western blot analysis. (C) A549 and Calu-1 cells were treated with salinomycin (2.5 μM) for the indicated period of time. Levels of protein expression were analyzed by western blot using antibodies against ATF4, DDIT3, TRIB3 and ACTB. (D) Calu-1 cells were transfected with control siRNA or DDIT3 siRNAs (#1 and #2) for 48 h and then treated with salinomycin (2.5 μM) for 12 h. Levels of protein expression were analyzed by western blot using antibodies against DDIT3, TRIB3 and ACTB. (E) Calu-1 cells were transfected with control siRNA or TRIB3 siRNAs (#1 and #2) for 48 h and then treated with salinomycin (2.5 μM) for 12 h. Levels of protein expression were measured by western blot analysis using antibodies against TRIB3, p-AKT1, AKT1, p-RPS6KB1, RPS6KB1, p-EIF4EBP1, MAP1LC3B and ACTB. (F) Calu-1 cells were transfected with control siRNA or ATF4 siRNAs (#1 and #2) for 48 h and then treated with salinomycin (2.5 μM) for 12 h. Levels of protein expression were measured by western blot analysis using antibodies against ATF4, DDIT3, TRIB3, MAP1LC3B, p-AKT1, p-RPS6KB1, p-EIF4EBP1 and ACTB.

Figure 6. Autophagy plays a protective role in salinomycin-treated human NSCLC cells. (A) A549 cells were transfected with control siRNA, ATG5 siRNAs (#1 and #2) or ATG7 siRNAs (#1 and #2) for 48 h and then treated with salinomycin (5 μM) for another 48 h. Levels of protein expression were measured by western blot analysis using antibodies against ATG5, ATG7, CASP3, PARP1 and ACTB. (B) A549 cells were transfected with control siRNA, ATG5 siRNA (#1) or ATG7 siRNA (#1) for 48 h. After treatment with salinomycin (5 μM) for 72 h, cells were measured for DNA fragmentation by ELISA assay. (C) A549 cells were transfected with control siRNA, ATG5 siRNA (#1) or ATG7 siRNA (#1) for 48 h. After treatment with salinomycin (5 μM) for 72 h, cells were stained with ANXA5-PE/7-AAD and detected by flow cytometry analysis. (D) A549 cells were pretreated with CQ (3 μM) for 1 h, then cotreated with salinomycin (5 μM) for 72 h and were stained with ANXA5-PE/7-AAD and detected by flow cytometry analysis. Columns: mean of triplicate treatments; bars: ± SD. The statistical differences between the two treatments were analyzed by two-sided unpaired Student’s t-tests (*p < 0.05; **p < 0.01; *** p < 0.001).

Figure 7. Salinomycin increases sensitivity to apoptosis in human NSCLC cells in which CDH1 expression is inhibited. (A) A549/shCtrl cells and A549/shCDH1 cells were treated with salinomycin (5 μM) for 48 h. Levels of protein expression were measured by western blot analysis using antibodies against CDH1, MAP1LC3B, ATF4, DDIT3, p-RPS6KB1, p-EIF4EBP1, CASP8, CASP3, PARP1 and ACTB. (B) A549/shCtrl cells and A549/shCDH1 cells were treated with salinomycin (5 μM) for 72 h, then cells were measured for DNA fragmentation by the ELISA assay. (C) A549/shCtrl cells and A549/shCDH1 cells were treated with salinomycin (5 μM) for 72 h, then cells were stained with ANXA5-PE/7-AAD and detected by flow cytometry analysis. (D) A549/shCtrl-EGFP-MAP1LC3B and A549/shCDH1-EGFP-MAP1LC3B cell lines were treated with salinomycin (2.5 μM) for 12 h. The average number of punctate structures per cell was quantified by confocal microscopy. Columns: mean of triplicate treatments; bars: ± SD. The statistical differences between the two treatments were analyzed by two-sided unpaired Student’s t-tests (*p < 0.05; **p < 0.01; *** p < 0.001; ns: no significant difference).