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Metformin Attenuates ROS via FOXO3 Activation in Immune Cells.

ABSTRACT: Forkhead box O 3 (FOXO3) is a transcription factor involved in cell metabolism, inflammation and longevity. Here, we investigated if metformin can activate FOXO3 in human immune cells and affects the subsequent level of reactive oxygen/nitrogen species (ROS/RNS) in immune cells. AMP-activated protein kinase (AMPK) and FOXO3 activation were investigated by immunoblot or flow cytometry (FC) analysis, respectively. FOXO3 target gene expression was quantified by real-time PCR. ROS/RNS measurement using dichlorodihydrofluorescein diacetate (DCFH-DA) dye was investigated by FC. The role of the FOXO3 single nucleotide polymorphisms (SNPs) rs12212067, rs2802292 and rs12206094 on ROS/RNS production was studied using allelic discrimination PCR. Metformin induced activation of AMPK (pT172) and FOXO3 (pS413). ROS/RNS level was reduced in immune cells after metformin stimulation accompanied by induction of the FOXO3 targets mitochondrial superoxide dismutase and cytochrome c. Studies in Foxo3 deficient (Foxo3^-/- ) mouse splenocytes confirmed that metformin mediates its effects via Foxo3 as it attenuates ROS/RNS in myeloid cells of wildtype (WT) but not of Foxo3^-/- mice. Our results suggest that FOXO3 can be activated by metformin leading to reduced ROS/RNS level in immune cells. This may add to the beneficial clinical effects of metformin observed in large cohort studies on longevity, cardiovascular and cancer risk.

SUBMITTER: Hartwig J

PROVIDER: S-EPMC8089390 | biostudies-literature |

REPOSITORIES: biostudies-literature

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Project description:Acetylshikonin is a shikonin derivative originated from Lithospermum erythrorhizon roots that exhibits various biological activities, including granulation tissue formation, promotion of inflammatory effects, and inhibition of angiogenesis. The anticancer effect of acetylshikonin was also investigated in several cancer cells; however, the effect against renal cell carcinoma (RCC) have not yet been studied. In this study, we aimed to investigate the anticarcinogenic mechanism of acetylshikonin in A498 and ACHN, human RCC cell lines. MTT (3-(4,5-Dimethylthiazol-2-yl)-2,5-Diphenyltetrazolium Bromide), cell counting, and colony forming assay showed that acetylshikonin induced cytotoxic and antiproliferative effects in a dose- and time-dependent manner. Cell cycle analysis and annexin V/propidium iodide (PI) double staining assay indicated the increase of subG1 phase and apoptotic rates. Also, DNA fragmentation was observed by using the TUNEL and comet assays. The intracellular ROS level in acetylshikonin-treated RCC was evaluated using DCF-DA. The ROS level was increased and cell viability was decreased in a dose- and time-dependent manner, while those were recovered when cotreated with NAC. Western blotting analysis showed that acetylshikonin treatment increased the expression of FOXO3, cleaved PARP, cleaved caspase-3, -6, -7, -8, -9, γH2AX, Bim, Bax, p21, and p27 while decreased the expressions of CYP2J2, peroxiredoxin, and thioredoxin-1, Bcl-2, and Bcl-xL. Simultaneously, nuclear translocation of FOXO3 and p27 was observed in cytoplasmic and nuclear fractionated western blot analysis. Acetylshikonin was formerly identified as a novel inhibitor of CYP2J2 protein in our previous study and it was evaluated that CYP2J2 was downregulated in acetylshikonin-treated RCC. CYP2J2 siRNA transfection augmented that apoptotic effect of acetylshikonin in A498 and ACHN via up-regulation of FOXO3 expression. In conclusion, we showed that the apoptotic potential of acetylshikonin against RCC is mediated via increase of intracellular ROS level, activation of FOXO3, and inhibition of CYP2J2 expressions. This study offers that acetylshikonin may be a considerable alternative therapeutic option for RCC treatment by targeting FOXO3 and CYP2J2.

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Metformin Attenuates ROS via FOXO3 Activation in Immune Cells.

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