Metformin Promotes HaCaT Cell Apoptosis through Generation of Reactive Oxygen Species via Raf-1-ERK1/2-Nrf2 Inactivation.

Abstract

Although metformin (MET) may be useful for the treatment of psoriasis, the mechanisms underlying its method of action have yet to be fully elucidated. Here, the relationship between MET function and reactive oxygen species (ROS) levels and the underlying mechanism were explored in human immortalized keratinocyte cell line (HaCaT). HaCaT cells were incubated with MET at 0, 10, 20, 40, and 60mM for 24h. The cell viability was evaluated by the CCK-8 assay. The cell apoptosis rate and intracellular ROS levels were examined using flow cytometry. The protein expression and the phosphorylation levels of nuclear factor erythroid-derived 2 related factor 2 (Nrf2), Raf-1, and ERK1/2 were assessed by Western blot. The specific ROS scavenger N-acetyl-cysteine (NAC) and the specific Nrf2 agonist Oltipraz (OPZ) were used to analyze the effect of MET. MET decreased HaCaT cell proliferation and induced HaCaT cell apoptosis in a dose-dependent manner. MET was found to elevate intracellular ROS levels in a dose-dependent manner, while pretreatment with NAC attenuated these effects. MET inhibits the protein expression and the phosphorylation levels of Nrf2. The combination of OPZ and MET can significantly increase the cell viability, decrease the rate of apoptosis, and attenuate the intracellular ROS levels relative to MET alone. MET inhibits the protein expression and the phosphorylation levels of Raf-1 and ERK1/2. MET was found to attenuate Raf-1-ERK1/2 signaling in HaCaT cells to suppress the expression and phosphorylation levels of Nrf2, which contributed to the intracellular generation of ROS and the pro-apoptotic effects of MET.