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

Abstract

BACKGROUND AND PURPOSEThe 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 APPROACHMCF-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 RESULTSMetformin 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-kB (NF-kB) activity and the level of IkB degradation were reduced by metformin treatment. Moreover, transduction of MCF-7/adr cells with the p65 subunit of NF-kB 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 IMPLICATIONSThese results suggest that metformin activates AMPK and suppresses MDR1 expression in MCF-7/adr cells by inhibiting the activation of NF-kB and CREB. This study reveals a novel function of metformin as an anticancer agent. AbbreviationsACC, acetyl-CoA carboxylase; aicar, 5-aminoimidazole-4-carboxamide-1-b-D-ribofuranoside; AMPK, adenosine 5′-monophosphate-activated protein kinase; CRE, cAMP-responsive element; CREB, cAMP-responsive element binding protein; DN-AMPK, dominant negative AMPK; GSK-3b, glycogen synthase kinase-3b; MDR1, multidrug resistance 1; NF-kB, nuclear factor-kB; P-gp, P-glycoprotein; PKA, protein kinase A; Rh-123, rhodamine 123; TNF-a, tumour necrosis factor a

Mechanism of Metformin-dependent Inhibition of Mammalian Target of Rapamycin (mTOR) and Ras Activity in Pancreatic Cancer

“…The anticancer activities of metformin are also similar to * This work was supported, in whole or in part, by National Institutes of Health that observed after knockdown of Sp1 or all three Sp proteins by RNA interference in cancer cells, and this includes growth inhibition, induction of apoptosis, reversal of epithelial to mesenchymal transition, and decreased migration/invasion (32)(33)(34)(35)(36). Metformin also inhibits NFB and decreases cyclin D1 and ErbB2 in cancer cell lines (13,20,27,28), and these gene products are also decreased after Sp1, Sp3, and Sp4 silencing by RNAi or by other drugs that down-regulate Sp proteins (32)(33)(34)(35)(36). However, the relationship between metformin-induced down-regulation of Sp1, Sp3, and Sp4 and modulation of mTOR signaling has not been reported, except that total mTOR protein expression was unaffected by silencing of Sp transcription factors in pancreatic cancer cells (31).…”

“…Recent studies report that diabetic cancer patients who take metformin exhibit improved outcomes as compared with patients taking other antidiabetic drugs (10), and this has spurred interest in possible clinical applications of metformin for cancer therapy. One of the hallmarks of metformin action is associated with inhibition of the mTOR signaling in both cancer and non-cancer tissues and cells (15)(16)(17)(18)(19)(20)(22)(23)(24)(25). For exam- A, RNA interference with siRNA against EGFR (siEGFR) decreased cell proliferation.…”

“…The antineoplastic activities of metformin in cancer cell lines includes the inhibition of several pathways and genes that are important for cancer cell proliferation, survival, migration, and invasion (13)(14)(15)(16)(17)(18)(19)(20)(21)(22)(23)(24)(25)(26)(27)(28). Metformin down-regulates expression of Sp1, Sp3, and Sp4 and several pro-oncogenic Sp-regulated genes, and this study shows that inhibition of mTOR signaling and RAS activation by metformin in pancreatic cancer cells is also due to decreased expression of the Sp-regulated upstream receptor tyrosine kinases (RTKs) IGF-1R and EGFR, respectively.…”

“…Metformin inhibits growth and induces apoptosis and other antineoplastic responses in multiple cancer cell lines, and this is accompanied by modulation of different pathways and genes. Several studies demonstrate that metformin activates AMP-activated protein kinase␣ (AMPK␣), which results in the inhibition of the mTOR 2 signaling pathway and downstream effects (15)(16)(17)(18)(19)(20)(22)(23)(24)(25), and this compliments one of the proposed mechanisms of action of metformin as an antidiabetic drug (29,30).…”

“…The potential clinical applications for metformin in cancer chemotherapy also stems from studies on the anticancer activities of this drug in cancer cells in culture and in in vivo models (1)(2)(3)(4)(13)(14)(15)(16)(17)(18)(19)(20)(21)(22)(23)(24)(25)(26)(27)(28). Metformin inhibits growth and induces apoptosis and other antineoplastic responses in multiple cancer cell lines, and this is accompanied by modulation of different pathways and genes.…”