Metformin inhibits growth of human non-small cell lung cancer cells via liver kinase B-1-independent activation of adenosine monophosphate-activated protein kinase

Abstract

Metformin, the most widely administered oral anti‑diabetic therapeutic agent, exerts its glucose-lowering effect predominantly via liver kinase B1 (LKB1)-dependent activation of adenosine monophosphate-activated protein kinase (AMPK). Accumulating evidence has demonstrated that metformin possesses potential antitumor effects. However, whether the antitumor effect of metformin is via the LKB1/AMPK signaling pathway remains to be determined. In the current study, the effects of metformin on proliferation, cell cycle progression, and apoptosis of human non‑small cell lung cancer (NSCLC) H460 (LKB1‑null) and H1299 (LKB1‑positive) cells were assessed, and the role of LKB1/AMPK signaling in the anti‑growth effects of metformin were investigated. Cell viability was determined using a 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide assay, cell cycle distribution and apoptosis were assessed by flow cytometry, and protein expression levels were measured by western blotting. Metformin inhibited proliferation, induced significant cell cycle arrest at the G0‑G1 phase and increased apoptosis in NSCLC cells in a time- and concentration-dependent manner, regardless of the level of LKB1 protein expression. Furthermore, knockdown of LKB1 with short hairpin RNA (shRNA) did not affect the antiproliferative effect of metformin in the H1299 cells. Metformin stimulated AMPK phosphorylation and subsequently suppressed the phosphorylation of mammalian target of rapamycin and its downstream effector, 70‑kDa ribosomal protein S6 kinase in the two cell lines. These effects were abrogated by silencing AMPK with small interfering RNA (siRNA). In addition, knockdown of AMPK with siRNA inhibited the effect of metformin on cell proliferation in the two cell lines. These results provide evidence that the growth inhibition of metformin in NSCLC cells is mediated by LKB1‑independent activation of AMPK, indicating that metformin may be a potential therapeutic agent for the treatment of human NSCLC.

Figure 1

Metformin inhibited proliferation of human non-small cell lung cancer cells. (A) The protein expression of LKB1 in H1299 and H460 cells was analyzed by western blotting. β-actin served as a loading control. (B) H1299 and H460 cells were seeded at 4×10³ cells/well in 96-well plates. After 24 h, the culture medium was replaced with fresh culture medium containing 0, 5, 10 or 20 mM metformin for 24, 48 and 72 h. Cell viability was determined by 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide assay at the indicated time-points. Data from three independent experiments are presented as the mean ± standard error of the mean. ^*P<0.05 vs. the control. LKB1, liver kinase B1.

Figure 2

Metformin arrested the cell cycle at the G₀–G₁ phase, and induced apoptosis in the H460 and H1299 cells. (A) Cells were seeded in 6-well plates (3×10⁵ cells/well), incubated for 24 h, exposed to 0, 5 or 10 mM metformin for another 24 h, and were subjected to flow cytometry for analysis of cell cycle distribution. (B) Cells were subjected to flow cytometry to analyze apoptosis following treatment with 0, 5 or 10 mM metformin for 48 h. Data from three independent experiments are presented as the mean ± standard error of the mean. ^*P<0.05 vs. the control.

Figure 3

Silencing LKB1 did not alter the antiproliferative effects of metformin. (A) The protein expression level of LKB1 was reduced in H1299 cells following transfection with lentiviral vectors carrying shRNA-LKB1. β-actin served as a loading control. (B) H1299 cells were treated with 0, 5, 10 or 20 mM metformin for 48 h after transfection with shRNA-LKB1 or shRNA-NC, and a 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide assay was performed to assess cell viability. Data from three independent experiments are presented as the mean ± standard error of the mean. ^*P<0.05 vs. the shRNA-NC group. LKB1, liver kinase B1; sh, short hairpin; NC, negative control.

Figure 4

Metformin activated AMPK and inhibited the mTOR signaling pathway in H460 and H1299 cells. The H460 and H1299 cell lines were treated with 10 mM metformin for the indicated times or treated with the indicated concentrations of metformin for 6 h. Following treatment, protein extracts were examined by western blot for p-AMPK, AMPK, p-mTOR, mTOR, p-p70S6K, p70S6K and β-actin protein expression levels. Data are representative of a minimum of three independent experiments. p, phosphorylated; AMPK, adenosine monophosphate-activated protein kinase; mTOR, mammalian target of rapamycin; p70S6K, 70-kDa ribosomal protein S6 kinase.

Figure 5

Knockdown of AMPK with siRNA reversed the effects of metformin on non-small cell lung cancer cells. (A) Cells were treated with 10 mM metformin for 6 h after transfection with si-AMPK, and examined by western blot for p-AMPK, AMPK, p-mTOR, mTOR, p-p70S6K, p70S6K and β-actin protein expression levels. Data are representative of a minimum of three independent experiments. (B) Cells were transfected with si-AMPK or si-NC. Following transfection, at 24 h, cells were treated with 0, 5, 10 or 20 mM metformin for 48 h, and a 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide assay was performed to assess cell viability. Data from three independent experiments are presented as the mean ± standard error of the mean. ^*P<0.05 vs. the si-NC group. p, phosphorylated; AMPK, adenosine monophosphate-activated protein kinase; met, metformin; mTOR, mammalian target of rapamycin; p70S6K, 70-kDa ribosomal protein S6 kinase; si, small interfering; NC, negative control.