Metformin inhibits proliferation of human keratinocytes through a mechanism associated with activation of the MAPK signaling pathway

Abstract

In the present study, the effects of metformin on the proliferation of human immortalized keratinocytes (HaCaTs) and the underlying mechanisms were investigated. HaCaT cells in the logarithmic growth phase were treated with 50 mM metformin for 24, 48 and 72 h. Cell morphology after 24 h of treatment was observed under a microscope. Cell proliferation was detected using a colorimetric cell proliferation and cytotoxicity assay kit. Western blot analyses were performed to detect the protein phosphorylation levels of adenosine monophosphate-activated protein kinase (AMPK) and extracellular signal-related kinase 1/2 (ERK1/2). Metformin treatment resulted in morphological changes of the HaCaT cells. The survival rates of HaCaT cells treated with metformin were 36.18, 12.70 and 10.12% at 24, 48 and 72 h, respectively. As the treatment time extended, the survival rates of HaCaT cells decreased. Western blot analysis results showed that the mean level of phosphorylated (p)-AMPK in the HaCaT cells without metformin treatment was 2.856±0.323. However, the mean p-AMPK level following metformin treatment for 24 h increased to 5.198±0.625, indicating a significant difference between these two groups (P<0.05). The mean absorbance ratio of p-ERK1/2 was 7.550±1.087 for the untreated cells, but the levels in cells following metformin treatment for 24 h increased to 10.430±1.217, indicating a significant difference between the two groups (P<0.05). In conclusion, metformin treatment upregulated the levels of p-AMPK and p-ERK1/2 in HaCaT cells, and significantly inhibited HaCaT cell proliferation in vitro by a mechanism associated with activation of the mitogen-activated protein kinase signaling pathway.

Keywords: HaCaT cells; adenosine monophosphate-activated protein kinase; extracellular signal-related kinase 1/2; metformin; proliferation; psoriasis.

Figures

Figure 1

Morphology analysis of HaCaT cells following metformin treatment. Cells were observed under an inverted microscope (magnification, ×200). (A) Untreated HaCaT cells and (B) HaCaT cells treated with 50 mM metformin for 24 h. HaCat, human immortalized keratinocytes.

Figure 2

Cell proliferation analysis of HaCaT cells following metformin treatment. After treatment with 50 mM metformin for 24, 48 and 72 h, cell viability was detected by a CCK-8 kit. The OD values at 450 nm of each group were measured and the cell viability was calculated. OD value at 450 nm of each group at (A) 24, (B) 48 and (C) 72 h. (D) Cell viability of each group at 24, 48 and 72 h following metformin treatment. Cell survival rate (%) was calculated using the following formula: (ODmetformin - ODcontrol)/(ODcontrol - ODmetformin) × 100. Experiments were conducted three times and data are expressed as the mean ± standard deviation. *P<0.05, vs. metformin group. HaCat, human immortalized keratinocytes; OD, optical density; CCK-8, cell proliferation and cytotoxicity assay kit-8.

Figure 3

Analysis of p-AMPK and p-ERK1/2 expression in HaCaT cells following metformin treatment. Expression of p-AMPK and p-ERK1/2 in HaCaT cells was evaluated by western blot analysis 24 h after metformin treatment. β-actin was used as the internal control. (A) Western blot analysis results of p-AMPK and p-ERK1/2 expression levels and the relative absorbance ratio of (B) p-AMPK to β-actin of each group and (C) p-ERK1/2 to β-actin of each group. Data are presented the mean ± standard deviation of three independent experiments. *P<0.05, vs. the control group. p, phosphorylated; HaCat, human immortalized keratinocytes; AMPK, adenosine monophosphate-activated protein kinase; ERK1/2, extracellular signal-related kinase 1/2.