Metformin Inhibits TGF-β1-Induced Epithelial-to-Mesenchymal Transition via PKM2 Relative-mTOR/p70s6k Signaling Pathway in Cervical Carcinoma Cells

Abstract

Background: Epithelial-to-mesenchymal transition (EMT) plays a prominent role in tumorigenesis. Metformin exerts antitumorigenic effects in various cancers. This study investigated the mechanisms of metformin in TGF-β1-induced Epithelial-to-mesenchymal transition (EMT) in cervical carcinoma cells.

Methods: cells were cultured with 10 ng/mL TGF-β1 to induce EMT and treated with or without metformin. Cell viability was evaluated by CCK-8 (Cell Counting Kit 8, CCK-8) assay; apoptosis were analyzed by flow cytometry; cell migration was evaluated by wound-healing assay. Western blotting was performed to detect E-cadherin, vimentin, signal transducer and activator of transcription 3 (STAT3), snail family transcriptional repressor 2 (SNAIL2), phosphorylation of p70s6k (p-p70s6k) and -Pyruvate kinase M2 (PKM2) Results: TGF-β1 promoted proliferation and migration, and it attenuated apoptosis compared with cells treated with metformin with or without TGF-β1 in cervical carcinoma cells. Moreover, metformin partially abolished TGF-β1-induced EMT cell proliferation and reversed TGF-β1-induced EMT. In addition, the anti-EMT effects of metformin could be partially in accord with rapamycin, a specific mTOR inhibitor. Metformin decreased the p-p70s6k expression and the blockade of mTOR/p70s6k signaling decreased PKM2 expression.

Conclusion: Metformin abolishes TGF-β1-induced EMT in cervical carcinoma cells by inhibiting mTOR/p70s6k signaling to down-regulate PKM2 expression. Our study provides a novel mechanistic insight into the anti-tumor effects of metformin.

Keywords: PKM2; epithelial-mesenchymal transition; mammalian target of rapamycin; metformin.

Figure 1

TGF-β1 induces EMT in cerivical cancer cells. (A,B) Photomicrographs of the morphological change in HeLa (A) and SiHa (B) cells. The number of hours indicates the period since EMT induction was initiated (scale bar, 50 μm). Western blot assays of E-cadherion, vimentin, and β-actin are shown in comparison with those in the pre-EMT condition; and (C) schematic representation of the procedure for EMT induction. The cells incubated for 48 h after seeding are defined as pre-EMT, and the cells cultured with 10 ng/mL TGF-β1 are defined as post-EMT. ** p < 0.01.

Figure 2

Metformin inhibits TGF-β1-induced proliferation, migration, and induces apoptosis in HeLa cells. (A) HeLa cells were treated with metformin (0, 1, 2.5, 5, 10 and 15 mM) or TGF-β1 with or without 10 mM metformin. Cell numbers were measured by CCK-8 assays at indicated times; (B) wound-healing assays. Representative images were obtained at 40× magnification. Graphs show the relative migration distance after 24 h incubation; (C) annexin V-FITC apoptosis assay. Cells were harvested and stained with Annexin V-FITC/PI (propidium iodide, PI), and cell apoptosis was analyzed using flow cytometry. Representative images are shown. TGF-β1: transforming growth factor β1; Met: metformin. * p < 0.05, ** p < 0.01.

Figure 3

Metformin inhibits TGF-β1-induced proliferation, migration, and induces apoptosis in SiHa cells. (A) SiHa cells were treated with metformin (0, 1, 2.5, 5, 10, and 15 mM) or TGF-β1 with or without 10 mM metformin. Cell numbers were measured by CCK-8 assays at indicated times; (B) wound-healing assays. Representative images were obtained at 40× magnification. Graphs show the relative migration distance after 24 h incubation; (C) annexin V-FITC apoptosis assay. Cells were harvested and stained with Annexin V-FITC/PI (propidium iodide, PI), and cell apoptosis was analyzed using flow cytometry. Representative images are shown. TGF-β1: transforming growth factor β1; Met: metformin. ** p < 0.01.

Figure 4

EMT condition stimulates an increase in PKM2. (A) HeLa and (B) SiHa cells were detected E-cadherin, vimentin, SNAIL2, and PKM2 expression by Western blot between pre-EMT and post-EMT state. Columns represent the average of at least three independent experiments; error bars represent the SD of the mean from triplicate results. ** p < 0.01.

Figure 5

mTOR/p70s6k signaling involved in regulating PKM2 in the EMT condition. HeLa (A) and SiHa (B) cells were treated with TGF-β1, with or without rapamycin. Rapamycin was dissolved in DMSO and the same dose of DMSO was used as a control, and the p-p70s6k and PKM2 expressions were detected by Western blot. DMSO: dimethylsulfoxide; Rapa:rapamycin. ** p < 0.01.

Figure 6

Metformin reverses TGF-β1-induced EMT in HeLa cells involving mTOR/p70s6k/PKM2 signaling pathways. (A) Cells were treated with TGF-β1, metformin, or both agents for 48 h. The protein expression levels of E-cadherin, vimentin, SNAIL2, STAT3, PKM2, p-p70s6k, and β-actin were detected by Western blot. β-actin was used as a loading control; and (B) the morphology of HeLa cells were treated with TGF-β1 and with or without metformin for 48 h. The cells were observed using phase contrast microscopy at 200× magnification. Scale bar: 50 μm. The data are presented as the mean ± SD of three replicates per group. TGF-β1: transforming growth factor β1; Met: metformin. ** p < 0.01.

Figure 7

Metformin reverses TGF-β1-induced EMT in SiHa cells involving mTOR/p70s6k/PKM2 signaling pathways. (A) Cells were treated with TGF-β1, metformin, or both agents for 48 h. The protein expression levels of E-cadherin, vimentin, SNAIL2, STAT3, PKM2, p-p70s6k, and β-actin were presented by Western blot. β-actin was used as a loading control; and (B) the morphology of SiHa cells were treated with TGF-β1 with or without metformin for 48 h. The cells were observed using phase contrast microscopy at 200× magnification. Scale bar: 50 μm. The data are presented as the mean ± SD of three replicates per group. TGF-β1: transforming growth factor β1; Met: metformin. ** p < 0.01, * p < 0.05.

Figure 8

Schematic representation of metformin roles in TGF-β1-induced epithelial-to-mesenchymal transition in cervical carcinoma cells. EMT: Epithelial-to-Mesenchymal Transition; PKM2: Pyruvate kinase M2; P: STAT3: signal transducer and activator of transcription 3; SNAIL2: snail family transcriptional repressor 2; P: phosphorylation.