Nitrogen mustard inhibits keratinocyte proliferation by activating AMPK/autophagy pathway

Abstract

Objective To explore the role of AMP-activated protein kinase (AMPK)/ autophagy signaling pathway in nitrogen mustard (NM)-induced cytotoxicity in cultured keratinocytes. Methods HaCaT cells were treated with different concentrations of NM(1, 5, 10 and 20 μmol/L) in the presence or absence of 5 mmol/L 3-methyladenine (3-MA, a specific inhibitor of autophagy), 20 nmol/L rapamycin (RAPA, a specific activator of autophagy), or 10 μmol/L compound C (a specific inhibitor of AMPK) for 24 h. CCK-8 assay was used to assess the cell viability, and the expression of p62, LC3, AMPK and pAMPK in the cells were detected using Western blotting; laser confocal microscopy was performed to detect the formation of autophagosomes in the cells after the treatments. Results Treatment with NM significantly decreased the viability of HaCaT cells in a dose-dependent manner, and NM at 20 μmol/L markedly decreased the cell viability to 60% of the control level. NM also dose-dependently up-regulated the phosphorylation of AMPK and LC3-B2, down-regulated the expression of p62, and significantly promoted the formation of autophagosomes in HaCaT cells. The application of 3-MA and compound C both abolished the effects of NM (P 0.05). Conclusion NM induces cytotoxicity in cultured keratinocytes by activating AMPK/autophagy signaling pathway to inhibit the cell proliferation.

Keywords

• nitrogen mustard
• autophagy
• amp-activated protein kinase
• keratinocyte
• cell proliferation