Metformin attenuates ovarian cancer cell growth in an AMP-kinase dispensable manner

Abstract

Metformin, the most widely used drug for type 2 diabetes activates 59 adenosine monophosphate (AMP)-activated protein kinase (AMPK), which regulates cellular energy metabolism. Here, we report that ovarian cell lines VOSE, A2780, CP70, C200, OV202, OVCAR3, SKOV3ip, PE01 and PE04 predominantly express -α(1), -β(1), -γ(1) and -γ(2) isoforms of AMPK subunits. Our studies show that metformin treatment (1) significantly inhibited proliferation of diverse chemo-responsive and -resistant ovarian cancer cell lines (A2780, CP70, C200, OV202, OVCAR3, SKVO3ip, PE01 and PE04), (2) caused cell cycle arrest accompanied by decreased cyclin D1 and increased p21 protein expression, (3) activated AMPK in various ovarian cancer cell lines as evident from increased phosphorylation of AMPKα and its downstream substrate; acetyl co-carboxylase (ACC) and enhanced β-oxidation of fatty acid and (4) attenuated mTOR-S6RP phosphorylation, inhibited protein translational and lipid biosynthetic pathways, thus implicating metformin as a growth inhibitor of ovarian cancer cells. We also show that metformin-mediated effect on AMPK is dependent on liver kinase B1 (LKB1) as it failed to activate AMPK-ACC pathway and cell cycle arrest in LKB1 null mouse embryo fibroblasts (mefs). This observation was further supported by using siRNA approach to down-regulate LKB1 in ovarian cancer cells. In contrast, met formin inhibited cell proliferation in both wild-type and AMPKα(1/2) null mefs as well as in AMPK silenced ovarian cancer cells. Collectively, these results provide evidence on the role of metformin as an anti-proliferative therapeutic that can act through both AMPK-dependent as well as AMPK-independent pathways.

Fig 1

Metformin inhibits proliferation of ovarian cancer cell lines. Various ovarian cell lines (A2780, CP70, C200, OV202, SKOV3ip, OVCAR3, PE01 and PE04) were treated with metformin with indicated doses. Cells were counted from days 0 to 7, on alternate days by trypan blue staining. The data represent three separate experiments done in triplicates. ***P < 0.001; **P < 0.01, *P < 0.05; NS: not significant, compared to untreated cells at respective time-point.

Fig 2

Metformin inhibits colony formation of ovarian cancer cells. 2000 cells/well (A2780, CP70, C200, SKOV3ip, PE01 and PE04) in 6-well plates were treated with indicated concentrations of metformin, every third day for 2–3 weeks until colonies were formed. The colonies were stained with MTT and counted. The data represent three separate experiments done in triplicates. ***P < 0.001; **P < 0.01, *P < 0.05; NS: not significant compared to untreated cells.

Fig 3

Metformin causes cell cycle arrest in G1-phase. (A) A2780, CP70, C200 and SKOV3ip cells were treated with metformin with indicated concentrations for 24 hrs. Cells were fixed overnight, stained with propidium iodide and flow-sorted. The data represent three separate experiments. ***P < 0.001; **P < 0.01, *P < 0.05; NS: not significant compared to untreated cells. (B) Immunoblot analysis of ovarian cancer cells treated with metformin showing reduced cyclin D1 and up-regulated p21 levels. Blots are representation of two separate experiments.

Fig 4

Metformin treatment activates AMPK in ovarian cancer cell lines. (A) Ovarian cancer cells (A2780, CP70, C200, SKOV3ip and PE04) were treated with metformin with indicated concentrations for 12 hrs. Protein lysates were immuno-blotted against phospho-ACC and phospho-AMPK antibodies. Same blot was stripped and probed for b-actin to show equal protein loading. (B) A2780, CP70, C200 and SKOV3ip cells were treated with indicated concentrations of metformin for 8 hrs. Cells were pulsed with 6 mM [1-¹⁴C] palmitic acid. After partition of cells, the upper aqueous phase was taken for measurement of radioactivity. ***P < 0.001; **P < 0.01, *P < 0.05; NS: not significant compared to untreated cells.

Fig 5

Metformin restricts mTOR-translation pathway in ovarian cancer cells. (A) A2780 and CP70 cells were treated with metformin (10 mM) at indicated time-points. Cells lysates were prepared and analysed for Akt (Ser-473), mTOR (Ser-1448) and S6-kinase phosphorylation by Western blot. The blots are representative of three independent experiments. (B) A2780, CP70, C200 and SKOV3ip cells were seeded and treated with metformin with indicated concentrations for 18 hrs followed by C¹⁴-methionine (20 mCi/mL) labelling for 4 hrs. C¹⁴ radioactivity incorporated was assessed as described in material and methods. The data are representative of three separate experiments. ***P < 0.001; **P < 0.01, *P < 0.05; NS: not significant compared to untreated cells.

Fig 6

Metformin inhibits lipid biosynthesis in ovarian cancer cells. (A) A2780, CP70, C200 and SKOV3ip cells were treated with indicated concentrations of metformin. After 12 hrs treatment, cells were pulsed with 1 mCi of [¹⁴C] acetate for an additional 4 hrs. Incorporation of labelled acetate into non-polar lipids was examined by HPTLC followed by 3 days exposure on X-ray film at –708C. Densitometric analysis was performed on the films and represented as bar graphs. ***P < 0.001; **P < 0.01, *P < 0.05; NS: not significant, compared to untreated cells.

Fig 7

Metformin mediated its action via LKB1. (A) LKB null (LKB^2/2) and wild-type (LKB^+/+) mefs were treated with metformin (5–10 mM) and analysed by immunoblot for AMPK and ACC phosphorylation and b actin for equal protein loading. (B) LKB^2/2 and wild-type mefs were treated with metformin with indicated concentrations for 24 hrs. After overnight fixation and propidium iodide staining cells were flow-sorted. The data are representations of two separate experiments. (C) Immunoblot analysis revealed down-regulation of LKB1 in untransfected (C), siRNA (Si) and non-target siRNA control (NT) transfected A2780 cells at 48 hrs. (D) After 48 hrs transfection, similar sets were treated with metformin for 12 hrs and processed for immunoblot for p-ACC and total ACC. (E) A2780 untransfected (C), siRNA (Si) and non-target siRNA control (NT) transfected cells were treated with indicated concentrations of metformin for 48 hrs and assessed for proliferation by MTT. **P < 0.01, siRNA (Si) compared to C and NT.

Fig 8

Metformin can inhibit growth independent of AMPK. (A) Immunoblot analysis revealed the down-regulation of AMPKα₁ in untransfected (C), siRNA (Si) and non-target siRNA control (NT) transfected A2780 cells at 48 hrs. Similar sets were treated with metformin (10 mM) for 12 hrs and processed for immunoblot for p-ACC and β actin (lower panel). (B) A2780 untransfected, siRNA and non-target siRNA transfected cells were treated with indicated concentrations of metformin for 48 hrs and assessed for cell number. **P < 0.01, *P < 0.05 compared to siRNA (Si) and non-target siRNA control (NT) transfected cells. (C) AMPK null (AMPK^+/+) and wild-type (AMPK^+/+) mouse embryo fibroblast (mefs) were treated with metformin (5–10 μM) and analysed by immunoblot for pAMPK and pACC, as well as for status of proteins of mTOR pathway. (D) AMPK^+/+ and wild-type mefs were treated with metformin (5–10 μM) for 72 hrs. MTT assay was performed to estimate cell viability. The data represent three separate experiments. **P < 0.01, *P < 0.05 compared to untreated AMPK null and wild-type mefs. (E, F) AMPKα null (AMPK^+/+) and wild-type (AMPK^+/+) mefs were transfected with CyclinD1 and p21 reporter-luciferase constructs (0.2 μg/24 well). The next day, the cells were treated with 10 μM metformin and 24 hrs later, luciferase activity was determined using dual luciferase assay system. ***P < 0.001, **P < 0.01 AMPKα_1/2^–/– mefs compared to AMPKα^+/+ mefs (black bars). ^‡P < 0.001, ^†P < 0.01 between metformin treated AMPKα^–/– mefs and AMPKα_1/2^+/+ mefs.