Salinomycin suppresses TGF-β1-induced EMT by down-regulating MMP-2 and MMP-9 via the AMPK/SIRT1 pathway in non-small cell lung cancer

Abstract

Salinomycin (Sal) is a recently identified anti-tumor drug for treating several types of solid tumor; however, its effects on the migratory and invasive properties of non-small cell lung cancer (NSCLC) remain unclear. This study investigated the inhibitory effect underlying mechanisms of Salon transforming growth factor-β1 (TGF-β1)-induced epithelial-to-mesenchymal transition (EMT) and cell migration. Sal solidly blocked cell migration and invasion enhancement by TGF-β1-induced EMT, through recovering E-cadherin loss and suppressing mesenchymal markers induction, as well as TGF-β1-mediated AMPK/SIRT signaling activity upregulation. The pharmacologic inhibition or knockdown of AMPK or SIRT1 can act synergistically with Sal to inhibit TGF-β1-induced MMP-2 and MMP-9. In contrast, AMPK or SIRT1 upregulation can protect against TGF-β1-induced MMP-2 and MMP-9 inhibition by Sal. Next we demonstrated that the MMP-2 and MMP-9 knockdown can act synergistically with Sal to inhibit TGF-β1-induced EMT. Moreover, treatment of PMA of MMP activator increased TGF-β1-induced MMP-2 and MMP-9, even with Sal. Our results demonstrate that Sal suppresses TGF-β1-induced EMT by downregulating MMP-2 and MMP-9 through the AMPK/SIRT pathway, thereby inhibiting lung cancer cell migration and invasion.

Keywords: AMPK; EMT; Lung cancer; MMP; SIRT; Salinomycin; TGF-β1.

Figure 1

Sal's effects on NSCLC cells growth. (A) A549 and H460 cells were treated with different concentrations of Sal for 24 or 48 h, and viability was determined using the MTT assay. The viability of control cells was set at 100%, and survival relative to the control is presented. The data represent the means ± S.D. of three independent experiments. ^*, p < 0.05, compared with the control. (B) Athymic nude mice were subcutaneously injected with 2 × 10⁶ H460 cells (0.2 mL cell suspension) in both hind legs. When the implanted tumors reached a volume of 90-130 mm³, mice were assigned randomly to one of the following experimental groups (n = 5 each): no treatment, TGF-β1, and TGF-β1 plus Sal (5 mg/kg, intraperitoneally, daily for three weeks). Tumor dimensions were measured three times a week. The tumor volume was estimated using the formula: volume = L × W²/2. Points, mean of five animals; bars, SD. ^*, p < 0.01, compared with the control.(C)The tumors were weighed. The data represent the mean ± SD of five animals. ^*, p < 0.01, compared with the control. (D) Body weights of the mice were insignificantly different among all groups.

Figure 2

Sal's effects on TGF-β1-induced A549 and H460 cell migration and invasion. (A) Cell migration was evaluated by scratch assay. The confluent A549 and H460 monolayer was scratched with a pipette tip and washed to remove debris. Fresh medium containing 0.5% serum was then added. Red lines indicate cell edges at the T₀ point. Representative pictures are shown. (B) For the ECIS migration assay, A549 cells were stimulated with 5 ng/mL TGF-β1 for 2 h and then incubated with 0, 2.5, 5, and 10 µM Sal for 48 h. Then, cell migration was assessed by continuous resistance measurements for 40 h. (C) Effects of Sal on A549 and H460 cell invasion in a 200× light microscope after crystal violet staining by Matrigel invasion assay, as described in Materials and Methods. Matrigel invasion of A549 and H460 cells, as counted in five random views. The data represent the mean ± SD of three independent experiments. ^*, p < 0.01, compared with the control;^**, p < 0.05, compared with the TGF-β1 group. (D) For the ECIS invasion assay, resistance changes in the impedance were recorded at 4 kHz, as confluent layers of HUVECs were challenged with A549 cell suspensions. The control curve represents HUVECs that received media without A549 cells. A549 cells were treated as described in Material and Methods, and resistance changes were monitored for 40 h.

Figure 3

Effects of Sal on TGF-β1-induced EMT through the AMPK/SIRT1 signaling pathway. (A) A549 and H460 cells were treated with 5 ng/mL TGF-β1 and 10 µM Sal for 48 h. Cell lysates were used to measure E-cadherin, N-cadherin, and vimentin level. (B) The cells were treated with 5 ng/mL TGF-β1 and Sal (5 or 10 µM), and AMPK/SIRT1-related protein levels were examined through immunoblotting. Similar data were obtained from three independent experiments.

Figure 4

AMPK involvement in TGF-β1-induced AMPK/SIRT1 signaling by Sal. (A, B) The Effects of AMPK inhibition on TGF-β1-induced AMPK/SIRT1 signaling. A549 and H460 cells were treated with an AMPK inhibitor, Compound C, or transfected with AMPK siRNA, and then further incubated in the presence of Sal for 24 h. The cell lysates were routinely prepared, and changes in signaling-mediated hallmarks were determined through Western blotting. (C) The cells were treated with an AMPK activator, AICAR, and then further incubated in the presence of Sal for 24 h.

Figure 5

SIRT1 involvement in TGF-β1-induced AMPK/SIRT1 signaling by Sal. (A, B) Effects of SIRT1 inhibition on TGF-β1-induced AMPK/SIRT1 signaling. The cells were treated with a SIRT1 inhibitor, sirtinol, or transfected with SIRT1 siRNA, and then further incubated in the presence of Sal for 24 h. (C) Effects of SIRT1 activation on TGF-β1-induced AMPK/SIRT1 signaling. The cells were transfected with Ad-lacZ or Ad-SIRT1 and then further incubated in the presence of Sal for 24 h. The cell lysates were routinely prepared, and alterations in signaling-mediated hallmarks were determined through Western blotting.

Figure 6

Sal's effects on TGF-β1-induced EMT through MMP-2 and MMP-9 signaling. (A) A549 and H460 cells were stimulated with 5 ng/mL TGF-β1 for 2 h and then incubated with 10 µM Sal for 24 h. The supernatants were analyzed by gelatin zymography to measure MMP-2 and MMP-9 expression. (B, C) Effects of MMP-2 or MMP-9 inhibition on TGF-β1-induced EMT by Sal. The Cells were transfected with MMP-2 or MMP-9 siRNA and then further incubated in the presence of Sal for 24 h. (D) The cells were treated with an MMP total activator, PMA, and then incubated further in the presence of Sal for 24 h. The cell lysates were routinely prepared, and alterations in EMT hallmarks were determined through Western blotting. Similar data were obtained from three independent experiments.