This study was aimed to determine the ultraviolet (UV: 235-310nm) action spectrum for killing normal human epidermal keratinocytes (NHEK) and to investigate the preventive effect of EPC-K1, a phosphate diester of vitamin C and vitamin E on UV radiation-induced NHEK injury. NHEK were cultured in EpiLife medium supplemented with Human Keratinocyte Growth Supplement Kit. NHEK viability was determined by crystal violet (CV) staining 48 h after the UV irradiation. The mRNA expressions of the C/EBP homologous protein (Chop) transcription factor and endoplasmic reticulum-resident molecular chaperone, Bip, were determined by RT-PCR analyses. UV was especially effective in killing NHEK when applied in the wavelength region of 250-280nm. The minimum exposure dose required to kill 50% of cells (LD50) was 1.64mJ/cm2 at 269nm. At 235 and 310nm, the LD50 for NHEK was 6.62 and 293mJ/cm2, respectively. Irradiation of 660-mJ/cm2 at 310nm significantly decreased the cell viability to 30% of control (without irradiation). The addition of 0.1mM EPC-K1 after irradiation returned the cell viability to 118%. Six hours after the 660-mJ/cm2 irradiation at 310nm, Chop and Bip mRNA levels in NHEK were increased to 487% and 283%, respectively, and were not significantly affected by EPC-K1. Chop and Bip are responsive to ER stress. These results suggested that EPC-K1 exerts a protective effect against UV-induced NHEK injury, and further studies should investigate the molecular mechanism underlying this effect.
Ultraviolet Action Spectrum and Effect of EPC-K1 on Ultraviolet Radiation-induced Injury in Cultured Normal Human Epidermal Keratinocytes
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