Induction of Cellular Senescence and Autophagy in Oxidative Stressed Keratinocytes

Objectives: To elucidate whether autophagy is required for oxidative stress-induced premature senescence, we examined induction of both senescence and autophagy in H₂O₂-stimulated human keratinocytes.
Methods: HaCaT cells, human keratinocytes, were used in this study. Cells were treated with H₂O₂ in the range of concentrations from 200 mM to 2000 mM. The viability of cells was assessed by the CCK-8 assay. In certain experiments, H₂O₂ treatment was preceded with 5 mM chloroquine (CQ) and 5 mM 3-methyl-adenine (3-MA). Induction of premature senescence was evaluated by cell staining of SA-b-Gal activity and immunoblotting of p21 and RB expression. Detection of autophagy in H₂O₂-treated cells was examined by immunocytochemistry and immunoblotting of LC3 and beclin-1.
Results: H₂O₂-treated cells showed positive staining of SA-b-Gal activity, a classic biochemical marker for cellular senescence. We found that H₂O₂-stimulated cells consistently induced p21 expression, but not RB, suggesting a potential involvement of p21 in cellular senescence. Treated cells induced conversion of LC3-I to LC3-II and punctate distribution of LC3-II, indicating an induction of autophagy in those cells. Senescent cells also showed beclin-1 expression, which was known as another marker for autophagy. These results showed that exposure of HaCaT cells to H₂O₂ induced both cellular senescence and autophagy. We next examined whether suppression of autophagy using pharmacologic inhibition would interfere with H₂O₂-induced cellular senescence. The impact of inhibition of autophagy by 3-MA, a phosphatidylinositol 3-kinase/Akt inhibitor or CQ, a late-stage autophagic inhibitor was observed in treated cells. Furthermore, immunoblotting analyses showed that both 3-MA and CQ treatment produced a reduction in p21 level.
Conclusions: In this study, we demonstrated that oxidative stress could induce both cellular senescence and autophagy in HaCaT cells. From the results of pharmacological inhibition of autophagy, we suggest that autophagy may be responsible for regulating p21-mediated cellular senescence in human keratinocytes.