Metformin inhibition of mTORC1 activation, DNA synthesis and proliferation in pancreatic cancer cells: dependence on glucose concentration and role of AMPK

Abstract

Metformin, a widely used anti-diabetic drug, is emerging as a potential anticancer agent but the mechanisms involved remain incompletely understood. Here, we demonstrate that the potency of metformin induced AMPK activation, as shown by the phosphorylation of its substrates acetyl-CoA carboxylase (ACC) at Ser(79) and Raptor at Ser(792), was dramatically enhanced in human pancreatic ductal adenocarcinoma (PDAC) cells PANC-1 and MiaPaCa-2 cultured in medium containing physiological concentrations of glucose (5 mM), as compared with parallel cultures in medium with glucose at 25 mM. In physiological glucose, metformin inhibited mTORC1 activation, DNA synthesis and proliferation of PDAC cells stimulated by crosstalk between G protein-coupled receptors and insulin/IGF signaling systems, at concentrations (0.05-0.1 mM) that were 10-100-fold lower than those used in most previous reports. Using siRNA-mediated knockdown of the α(1) and α(2) catalytic subunits of AMPK, we demonstrated that metformin, at low concentrations, inhibited DNA synthesis through an AMPK-dependent mechanism. Our results emphasize the importance of using medium containing physiological concentrations of glucose to elucidate the anticancer mechanism of action of metformin in pancreatic cancer cells and other cancer cell types.

Conflict of interest statement

Conflict of Interest Statement: The authors declare no conflicts of interest

Copyright © 2012 Elsevier Inc. All rights reserved.

Figures

Fig. 1. Enhanced metformin-induced AMPK activation in PDAC cells cultured in medium containing glucose at a physiological concentration

A. Confluent Mia PaCa-2 and PANC-1 cells were incubated with increasing concentrations of metformin for 16 h in DMEM containing either 5 mM or 25 mM glucose, as indicated. Cell lysates were analyzed by immunoblotting for phosphorylation of ACC at Ser79 (pACC) and Raptor at Ser792 (pRaptor). Total ACC was used to verify equal gel loading. Similar results were obtained in three independent experiments. B. Results are expressed as the percentage of maximum mean ±SEM; n=3.

Fig. 2. Enhanced metformin-induced mTORC1 inhibition in PDAC cells exposed to physiological glucose concentrations

A) Cultures of MiaPaca-2 and PANC-1 cells were incubated without (−) or with 1 mM metformin for 16 h in DMEM containing 5 mM or 25 mM glucose, as indicated. Then, the cells were stimulated for 2 h with 5 nM neurotensin (NT), 10 ng/ml insulin (Ins) or their combination (NT+Ins), lysates were analyzed by immunoblotting with the following phospho antibodies: S6K Thr389 (pS6K), S6 Ser235/236 (pS6) and ACC Ser79 (pACC). Total ACC was used to verify equal gel loading. Similar results were obtained in 3 independent experiments. B). Mia PaCa-2 cells were incubated with DMEM containing 5 mM glucose either in absence or presence of 0.05mM or 0.1 mM metformin for 16 h. Then, the cells were treated with NT+Ins, as in panel A, and lysates were analyzed by immunoblotting. Similar results were obtained in 6 independent experiments. Results are expressed as the percentage of maximum mean ±SEM, n=6 (S6K) or as the fold increase over basal as indicated. P values were determined using the t-test (Sigma Plot 12.) *p<0.05; **p<0.001; n=6.

Fig. 3. Metformin potently inhibits DNA synthesis and proliferation in PDAC cells incubated in 5 mM glucose

A) Dose-response effect of metformin on DNA synthesis induced by neurotensin and insulin in MiaPaca-2 cells. Cultures in serum-free medium containing 5mM (closed circles) or 25 mM glucose (open circles) were stimulated with 5 nM neurotensin and 10 ng/ml insulin in the presence of metformin (0.025–1 mM). Results are expressed as the percentage of maximum mean ±SEM, n=6. B) Effect of metformin on DNA synthesis induced by IGF in hTERT-HPNE cells. The hTERT-HPNE cell cultures were stimulated with 10 ng/ml IGF-1 in the absence or presence of 1mM metformin (as indicated). Results are expressed as the percentage of maximum mean ±SEM; n=6. C) Low concentrations of metformin inhibit DNA synthesis in MiaPaca-2 and PANC-1 cells cultured in DMEM containing 5 mM glucose. Cultures were incubated in medium containing 5mM glucose either in the absence (grey bars) or the presence of 0.05 mM or 0.1 mM metformin (black bars) and 5nM neurotensin and 10 ng/ml insulin (NT + Ins) as indicated. Results are expressed as the percentage of maximum mean ±SEM, n=6. D), Low concentrations of metformin inhibit proliferation of MiaPaca-2 and PANC-1 cells cultured in DMEM containing 5 mM glucose. Single-cell suspensions of MiaPaca-2 and PANC-1 cells were plated at a density of 2 × 104 cells per dish. After 4 h, the cultures were shifted to media containing 1% FBS without (−) or with 10 nM neurotensin and 10 ng/ml insulin (NT + Ins) the absence (grey bars) or presence (closed bars) of 0.05 mM or 0.1 mM metformin. After 4 days, cell numbers were determined from 6 plates per condition. Results are presented as mean ± SEM. Similar results were obtained in two independent experiments. P values were determined using the t-test (SigmaPlot 12.) *p<0.05; **p<0.01; n=6.

Fig. 4. Knockdown of the α subunits of AMPK abrogates ACC and Raptor phosphorylation and reverses the inhibition of DNA synthesis induced by metformin

A). PANC-1 cells were transfected with either non-targeting negative control (N. Targ siRNA.) or 75 nM AMPK siRNA (AMPK siRNA) in media containing 5 mM glucose. Cells were incubated in the absence or presence of 1 mM metformin for 16h, lysates were analyzed by immunoblotting with the following phospho-antibodies: ACC Ser79 (pACC) and Raptor Ser792 (pRaptor), α1/α2 subunits of AMPK and total ACC. Similar results were obtained in four independent experiments. Results are expressed as the percentage of maximal mean ±SEM, n=16. B). PANC-1 cells were transfected with either transfection reagent alone (open bars) non-targeting negative control (grey bars) or 75 nM AMPK siRNA (black bars) in DMEM/FBS containing 5 mM glucose. After 3 days the cells were incubated for 6 h in serum-free medium containing 5mM glucose and stimulated with 5nM neurotensin and 10 ng/ml insulin in the absence or presence of 1 mM metformin (Met). Results are expressed as the percentage of maximal mean ±SEM, n=6.