Metformin inhibits P-glycoprotein expression via the NF-κB pathway and CRE transcriptional activity through AMPK activation

Abstract

Background and purpose: The expression of P-glycoprotein (P-gp), encoded by the multidrug resistance 1 (MDR1) gene, is associated with the emergence of the MDR phenotype in cancer cells. We investigated whether metformin (1,1-dimethylbiguanide hydrochloride) down-regulates MDR1 expression in MCF-7/adriamycin (MCF-7/adr) cells.

Experimental approach: MCF-7 and MCF-7/adr cells were incubated with metformin and changes in P-gp expression were determined at the mRNA, protein and functional level. Transient transfection assays were performed to assess its gene promoter activities, and immunoblot analysis to study its molecular mechanisms of action.

Key results: Metformin significantly inhibited MDR1 expression by blocking MDR1 gene transcription. Metformin also significantly increased the intracellular accumulation of the fluorescent P-gp substrate rhodamine-123. Nuclear factor-κB (NF-κB) activity and the level of IκB degradation were reduced by metformin treatment. Moreover, transduction of MCF-7/adr cells with the p65 subunit of NF-κB induced MDR1 promoter activity and expression, and this effect was attenuated by metformin. The suppression of MDR1 promoter activity and protein expression was mediated through metformin-induced activation of AMP-activated protein kinase (AMPK). Small interfering RNA methods confirmed that reduction of AMPK levels attenuates the inhibition of MDR1 activation associated with metformin exposure. Furthermore, the inhibitory effects of metformin on MDR1 expression and cAMP-responsive element binding protein (CREB) phosphorylation were reversed by overexpression of a dominant-negative mutant of AMPK.

Conclusions and implications: These results suggest that metformin activates AMPK and suppresses MDR1 expression in MCF-7/adr cells by inhibiting the activation of NF-κB and CREB. This study reveals a novel function of metformin as an anticancer agent.

© 2011 The Authors. British Journal of Pharmacology © 2011 The British Pharmacological Society.

Figures

Figure 1

Characterization of MCF-7 and MCF-7/adriamycin (MCF-7/adr) cells. (A) The protein product of the multidrug resistance 1 (MDR1) gene in MCF-7/adr and MCF-7 cells was analysed by Western blotting. MDR1 was overexpressed in the resistant (MCF-7/adr) cells. (B) Cell viability was determined by the 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide assay after adriamycin treatment in MCF-7 (sensitive) and MCF-7/adr (resistant) cells for 48 h. Each point shows the mean ± SD of three independent experiments, performed in triplicate. (A, B) Representative results are shown for experiments that were repeated independently 3 times.

Figure 2

Metformin enhanced the cytotoxicity of adriamycin in MCF-7/adriamycin (MCF-7/adr) cells. (A) The cytotoxicity of metformin (0.5–10 mM) in MCF-7/adr was determined by 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide (MTT) assay. Each column shows the mean ± SD of three independent experiments, performed in triplicate. *Significantly different from untreated cells (P < 0.01). (B–E) Effects of metformin and adriamycin in MCF-7/adr (B, C) and MCF-7 cells (D, E). Cells were pretreated with or without 3 mM metformin, followed by incubation with various concentrations of adriamycin for an additional 48 h. Cell viability was determined by MTT and lactate dehydrogenase (LDH) assays. Each column/point shows the mean ± SD of three independent experiments, performed in triplicate. *Significantly different from untreated cells (P < 0.01). (A–E) Representative results are shown for experiments that were repeated independently 3 times.

Figure 3

Effect of metformin on multidrug resistance 1 (MDR1) mRNA and protein expression in MCF-7/adriamycin (MCF-7/adr) cells. (A) Cells were treated with metformin (1–10 mM) for 24 h or with 10 mM metformin for 3–48 h. Total RNA was extracted. MDR1 expression was analysed by semiquantitative RT-PCR. The β-actin band confirms the integrity and equal loading of RNA. (B) Under identical conditions, cells were lysed, and total RNA was prepared to analyse MDR1 gene expression by real-time PCR. For normalization, β-actin was amplified in each sample. MDR1 mRNA expression was compared between metformin-treated and untreated cells by real-time PCR at each time point. Each column shows the mean ± SD of three independent experiments, performed in triplicate. *Significantly different from untreated cells (P < 0.01). (C) Lysates of cells treated with various concentrations of metformin for 24 h or with 10 mM metformin for 3–48 h were analysed by Western blotting with a MDR1-specific antibody. The β-actin band confirms the integrity and equal loading of protein. (D) Effect of metformin on intracellular rhodamine-123 (Rh-123) accumulation in MCF-7/adr cells. Cells were treated with vehicle, 1–10 mM metformin, or 20 µM verapamil (positive control) for 48 h and then exposed to 5 µM Rh-123 for 90 min. Intracellular Rh-123 accumulation was then measured. Each column shows the mean ± SD of three independent experiments, performed in triplicate. *Significantly different from untreated cells (P < 0.01). (A–D) Representative results are shown for experiments that were repeated independently 3 times.

Figure 4

Metformin suppressed the multidrug resistance 1 (MDR1) gene through NF-κB activation. Cells were transiently transfected with plasmids harboring MDR1 and NF-κB reporter genes and treated with 1–10 mM metformin and 0.1–1 µg·mL−1 adriamycin for 24 h. The cells were lysed, and MDR1 (A) and NF-κB (B) activities were measured by luciferase assay. Each column shows the mean±SD of three independent experiments, performed in triplicate. *Significantly different from untreated cells (P < 0.01). **Significantly different from adriamycin−treated cells (P < 0.01). (C) Cells were incubated for 24 h with metformin (0.5–10 mM). The cells were lysed and subjected to Western blot analysis using anti-phospho-IκB-α, anti-IκB-α and anti-β-actin antibodies. Metformin dose-dependently inhibited the TNF-α-induced phospho-IκB-α level. (D) MCF-7/adriamycin (MCF-7/adr) cells were incubated with 10 mM metformin for 5–120 min. The cells were lysed and subjected to Western blot analysis using anti-phospho-IκB-α, anti-IκB-α and anti-β-actin antibodies. Metformin time-dependently inhibited phospho-IκB-α levels. (E) MCF-7/adr cells were incubated with metformin. The cells were lysed, and nuclear extracts were prepared for Western blot analysis using antibodies against nuclear p65 and lamin B. Under identical conditions, the cells were lysed and subjected to Western blot analysis using anti-p65 and anti-β-actin antibodies. (F, G) Cells were transfected with p65-GFP plasmid, incubated with metformin for 24 h, and lysed. MDR1 protein expression was determined by Western blot analysis, and MDR1 activity was determined by luciferase activity. Each column shows the mean ± SD of three independent experiments, performed in triplicate. *Significantly different from untreated MCF-7/adr cells (P < 0.01). **Significantly different from metformin-treated MCF-7/adr cells (P < 0.01). (A–G) Representative results are shown for experiments that were repeated independently 3 times.

Figure 5

Involvement of the AMP-activated protein kinase (AMPK) pathway in multidrug resistance 1 (MDR1) expression induced by metformin. (A) Metformin stimulated AMPK and acetyl-CoA carboxylase (ACC) phosphorylation in MCF-7/adriamycin (MCF-7/adr) cells at the indicated times. (B) MCF-7/adr cells were treated for 30 min with 10 µM compound C (AMPK inhibitor) and then for 30 min with 3 mM metformin. Phosphorylated AMPK and AMPK were detected by Western blot analysis. (C) The effect of metformin and compound C on MDR1 expression in MCF-7/adr cells. Cells were incubated for 24 h with 3 mM metformin and 10 µM compound C, lysed, and subjected to Western blot analysis using anti-MDR1 and anti-β-actin antibodies. (D) Effect of metformin and compound C on MDR1 promoter activity in MCF-7/adr cells. Cells were transiently transfected with a MDR1 promoter reporter gene and then treated with 3 or 10 mM metformin and 10 µM compound C for 24 h. The cells were lysed, and luciferase activity was measured. Each column shows the mean ± SD of three independent experiments, performed in triplicate. *Significantly different from untreated cells (P < 0.01). **Significantly different from compound C-treated cells (P < 0.01). (E) MCF-7/adr cells were transfected with AMPK small interfering (siRNA) or control siRNA for 24 h. The transfected cells were treated with 10 mM metformin for 24 h. MDR1 expression and phosphorylation of AKT, AMPK, and MDR were measured by Western blot analysis. The cells were lysed, and nuclear extracts were prepared for Western blot analysis using antibodies against nuclear p65 and lamin B. (F) After transfection with AMPK siRNA or control siRNA for 24 h, cells were treated with 10 mM metformin for 48 h and then exposed to 5 µM rhodamine-123 (Rh-123) for 90 min. The intracellular Rh-123 accumulation was measured. Each column shows the mean ± SD of three independent experiments, performed in triplicate. *Significantly different from untreated cells (P < 0.01). **Significantly different from metformin-treated cells (P < 0.01). (A–F) Representative results are shown for experiments that were repeated independently 3 times.

Figure 6

Metformin inhibited the multidrug resistance 1 (MDR1) gene via cAMP response element (CRE) transcriptional activity. (A) MCF-7/adriamycin (MCF-7/adr) cells were treated with 3 mM metformin for 15–360 min, and cell extracts were analysed by Western blotting with antibodies against phospho-AMP-activated protein kinase (AMPK), AMPK, phospho-acetyl-CoA carboxylase (ACC), ACC, phospho-glycogen synthase kinase (GSK)-3β, GSK-3β, phospho-cAMP-responsive element binding protein (CREB) (Ser 133), CREB and β-actin. (B) MCF-7/adr cells were transfected with dominant-negative AMPK (DN-AMPK) or a pcDNA 3.1 control for 6 h. The transfected cells were treated with 3 mM metformin for 6 or 24 h. MDR1 expression and phosphorylation of CREB, AMPK, ACC and GSK-3β were measured by Western blot analysis. (C) After transfection with DN-AMPK or the pcDNA 3.1 control for 6 h, cells were treated with 3 mM metformin or 20 µM verapamil for 48 h and then exposed to 5 µM rhodamine-123 (Rh-123) for 90 min. The intracellular Rh-123 accumulation was measured. Each column shows the mean ± SD of three independent experiments, performed in triplicate. *Significantly different from untreated cells (P < 0.01). **Significantly different from metformin-treated cells (P < 0.01). (A–C) Representative results are shown for experiments that were repeated independently 3 times.