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

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Graphical AbstractHighlights d LKB1-deficient T reg cells produce Th1 and Th17 cytokines d The loss of LKB1 compromises the mevalonate pathway in T reg cells d Mevalonate or GGPP treatment restores function and stability in LKB1-deficient T reg cells d LKB1-mediated regulation of T reg cells is independent of AMPK SUMMARY The function of regulatory T (T reg ) cells depends on lipid oxidation. However, the molecular mechanism by which T reg cells maintain lipid metabolism after activation remains elusive. Liver kinase B1 (LKB1) acts as a coordinator by linking cellular metabolism to substrate AMP-activated protein kinase (AMPK). We show that deletion of LKB1 in T reg cells exhibited reduced suppressive activity and developed fatal autoimmune inflammation. Mechanistically, LKB1 induced activation of the mevalonate pathway by upregulating mevalonate genes, which was essential for T reg cell functional competency and stability by inducing T reg cell proliferation and suppressing interferon-gamma and interleukin-17A expression independently of AMPK. Furthermore, LKB1 was found to regulate intracellular cholesterol homeostasis and to promote the mevalonate pathway. In agreement, mevalonate and its metabolite geranylgeranyl pyrophosphate inhibited conversion of T reg cells and enhanced survival of LKB1-deficient T reg mice. Thus, LKB1 is a key regulator of lipid metabolism in T reg cells, involved in optimal programming of suppressive activity, immune homeostasis, and tolerance.

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