Metformin Inhibits Proliferation And Proinflammatory Cytokines Of Human Keratinocytes In Vitro Via MTOR Signaling Pathway

Metformin is an insulin sensitizer, which is the first-line treatment method for type 2 diabetes. Retrospective epidemiological investigations and basic studies have shown that metformin has anti-tumor effects in humans and experimental animal models of colon, breast, prostate and lung cancers. Interestingly, it has been previously reported that diabetics receiving metformin treatment have a lower occurrence of psoriasis than other insulin sensitizing agents. One case-control study on 36,702 patients showed that long-term use of metformin was associated with a reduced risk of psoriasis.The antidiabetic drug metformin exhibits antiproliferative. and pro-apoptotic effects in various cells, suggesting its potential to treat a variety of malignant and nonmalignant hyperplastic diseases. The mechanism underlying this phenomenon is thought to be through the activation of AMP-activated protein kinase (AMPK), which is a master sensor and regulator of energy homeostasis in mammol/Lalian cells and also a therapeutic target for metabolic disorders. AMPK activation strongly inhibits cell growth and proliferation in both malignant and nonmalignant cells through inhibition of mTOR signaling and stimulation of the p53-p21 axis. In addition, the cell growth regulatory effects of metformin were associated with a reduction in mTOR signaling independent of AMPK.The mTOR pathway has been identified as a key intracellular signaling pathway for important cellular functions. It regulates cell growth and proliferation largely by promoting key anabolic processes, sensing nutrition levels and growth factors, as well as responding to various environmental cues. mTOR has been implicated in the development and progression of various skin diseases including melanoma, acne vulgaris, and psoriasis.Recent findings have suggested that the mTOR pathway may play an important role in the pathogenesis of psoriasis by mediating epidermal hyperplasia, immune response, and/or angiogenesis in psoriasis Inhibition of the mTOR pathway can inhibit multiple proinflammatory cytokines including IL-6, TNF-a and VEGF, which play important roles in the pathogenesis of psoriasis. Our previous study showed that metformin could inhibit the proliferation of human keratinocytes in association with the activation of the AMPK signaling pathway. However, whether metformin suppresses human keratinocyte proliferation by specifically targeting the mTOR signaling pathway has not been studied.The present study evaluates the antiproliferative activity and anti-inflammatory responses of metformin in human keratinocytes in vitro and explores the underlying mechanisms.Part 1. The effect of metformin on the proliferation of Hacat eells by MTT and flow cytometry incorpration.Objective:To study the effect of metformin on the proliferation of Hacat cells.Materials and Methods:Materials and methods:HaCaT cells were collected and inoculated in 96-well (1 X 10’cells/well) during the logarithmic growth phase. The culture media in the metformin groups were replaced with metformin at 0,25,50 and 100 mmol/L for 24,48,72and 96 h. Antiproliferative activity was evaluated by MTT and apoptotic response was examined by flow cytometry.Results:Compared to typical healthy HaCaT cells in the untreated group, the cells treated with metformin became small and deformed, with nuclear pyknosis and fragmentation. With the increase in concentration of metformin, adherent cells appeared to be shrunken and round with reduced cytoplasm. Compared to the non-treated group, inhibition of cell proliferation is closely associated with the concentration of metformin. Metformin at higher concentrations over 50 mmol/L significantly inhibited proliferation of HaCaT cells after 24 h treatment. Similar to the findings for growth inhibition, cell viability inhibition by metformin was also dose-dependent. The survival rates of HaCaT cells treated with metformin at 50 mmol/L were 75.6,59.4,40.7 and 30.3% at 24,48,72 and 96 h, respectively.To examine if metformin can induce HaCaT cell apoptosis, we performed flow cytometry and apoptotic analysis. Compared to non-treated cells, the numbers of apoptotic (annexin+) HaCaT cells increased at 25 mmol/L and significantly increased at 50 mmol/L metformin after 48 h treatment.100 mmol/L metformin treatment induced apoptosis in almost half of the cells.Conclusion:The results of the study showed that metformin can inhibit the proliferation of Hacat cells and induce Hacat cells apoptosis in a dose-dependent manner.Part 2. Metformin inhibits pro inflammatory cytokines of human keratinocytes in vitroObjective:To investigate the effect of metformin on the cytokines that were secreted by Hacat cells.Materials and Methods:HaCaT cells were collected and inoculated in 6-well (1 × 10-1 cells/well) during the logarithmic growth phase. The culture media in the metformin groups were replaced with metformin at 0, 25,50 and 100 mmol/L for 48 h. An equal volume of DMEM containing 3% fetal bovine serum was added to the control group.The culture supernatant was used for cytokine examination. We used ELISA to evaluate changes in inflammation-related cytokine expression in protein levels.Results:Metformin reduced IL-6, TNF-α and VEGF levels at 48h. The expression of IL-6, TNF-a and VEGF reduced with the increase in concentration of metformin. The difference between the metformin and control groups was found to be statistically significant.Conclusion:The results of the study showed that metformin can inhibit IL-6, TNF-a and VEGF levels that were secreted by Hacat cells.Part 3. Metformin inhibits proliferation and proinflammator y cytokines of human keratinocytes in vitro via mTOR signaling pathwayObjective:To study the effect of metformin on mTOR signaling pathway in Hacat cells.Materials and Methods:HaCaT cells grown in 60 mm plates were treated with metformin (0,25,50 and 100 mmol/L) for 48h. Total protein extracts (20 μ g) were electrophoresed on 10% SDS polyacrylamide gels and transferred to nitrocellulose membranes. Membranes were blocked with 5% non-fat dried milk in Tris-buffered saline and incubated for 2 h at room temperature with appropriate primary antibodies against mTOR, p-mTOR (ser2448), p70S6K and p-p70S6K. After washing with TBST, the membranes were incubated for 1h with appropriate horseradish peroxidase-conjugated secondary antibody and the immune-complex was detected with chemiluminescent substrate. The developed films were scanned using the AlphaImager gel imaging systems.. β-actin was used as an internal control.Results:mTOR, a central protein in the mTOR signaling pathway, plays a pivotal role in regulating protein and nucleotide synthesis through its main effectors, p70S6K and eIF4E binding protein 1 (4E-BP1). We further examined if the effect of metformin on HaCaT cells is through the inhibition of mTOR and p70S6K. Western blot shows that metformin at 50 mmol/L significantly reduced the phosphorylation of mTOR and p70S6K (49.0% and 62.1%, respectively) with an amplified reduction at 100 mmol/L with no discrepancy in total mTOR and p70S6K. The results indicate that down-regulation of mTOR signaling may contribute to the inhibition of metformin induced proliferation and apoptosis in HaCaT cells.Conclusion:In the current study, we found that metformin not only inhibited cell proliferation, but also induced HaCaT cell apoptosis. Met formin has further been shown to inhibit the expression of IL-6, TNF-a and VEGF proteins in HaCaT cells, indicating that metformin may attenuate inflammation in psoriasis patients. The current study reveals insight into the potential underlying antiproliferative and anti-inflammatory mechanisms of metformin, which may contribute to the observed clinical benefits in psoriasis patients.