Induction of Apoptosis and Autophagy in UVB-Treated HaCaT Cells

Yoon, Sang Don; Baek, Won Ki; Kim, Sang Pyo; Lee, Kyu Suk; Cho, Jae We

Abstract

BACKGROUND
UVB irradiation induces apoptosis or/and autophagy through several molecular pathways in keratinocytes. However, the precise molecular mechanism of UVB-induced autophagy is largely unknown in keratinocytes.
OBJECTIVE
The purpose of this study was to investigate the molecular mechanisms of UVB-induced apoptosis and autophagy in HaCaT cell lines.
METHODS
Cells were irradiated by UVB (Westinghouse FS-40 sunlamps) with various doses (0, 30, 60, 120, 240 mJ/cm2). The expression levels of caspase-3, Bax, Bcl2, Bcl-X(L) and LC3 were confirmed by Western blot analysis in UVB-irradiated HaCaT cell lines. Apoptotic cells were analyzed by PI staining, and autophagy cells were analyzed by immunofluorescent staining.
RESULTS
The expression of Bcl-X(L) decreased from UVB 60 mJ/cm2 and Bcl2 decreased from UVB 240 mJ/cm2. The expression of caspase-3 was increased from UVB 120 mJ/cm2. These data showed that UVB-induced apoptosis is mediated by up-regulation of caspase-3 and down-regulation of Bcl2 and Bcl-X(L). Furthermore, the expression of LC3 increased from UVB 120 mJ/cm2. In addition, autophagy formation was observed in few fractions of apoptotic HaCaT cells in immunofluorescent staining; most apoptotic cells did not show autophagy formation. Moreover, autophagy formation inhibitor treatment induced a slight increment of apoptotic cell population under UVB irradiation.
CONCLUSION
UVB irradiation induces not only apoptotic cell death but also autophagy formations; these events may create a defense mechanism for the prevention of apoptosis in UVB-treated HaCaT cells.