Suppression of ultraviolet B exposure-mediated activation of NF-kappaB in normal human keratinocytes by resveratrol

Abstract

Chemoprevention by naturally occurring agents is a newer dimension in the management of neoplasia, including skin cancer. Solar ultraviolet (UV) radiation is the major cause of skin cancer. We recently demonstrated that resveratrol (3,5,4'-trihydroxystilbene), a polyphenolic antioxidant found in grapes and red wine, imparts protection from UVB-mediated cutaneous damages in SKH-1 hairless mice. The mechanism of action of resveratrol is not clearly understood. Here, we investigated the involvement of nuclear factor kappa B (NF-kappaB), which is known to play a critical role in skin biology and the development of skin cancer, as the mechanism of chemoprevention of UV damage by resveratrol. In the normal human epidermal keratinocytes, resveratrol blocked UVB-mediated (40 mJ/cm(2)) activation of NF-kappaB in a dose-dependent (5, 10, and 25 micro M resveratrol for 24 hours) as well as time-dependent (5 micro M resveratrol for 12, 24, and 48 hours) fashion. Resveratrol treatment of keratinocytes also inhibited UVB-mediated 1) phosphorylation and degradation of IkappaBalpha, and 2) activation of IKKalpha. We suggest that NF-kappaB pathway plays a critical role in the chemopreventive effects of resveratrol against the adverse effects of UV radiation including photocarcinogenesis.

Figure 1

Chemical structure of trans-resveratrol.

Figure 2

Effect of resveratrol on UVB-mediated activation of NF-κB/p65: immunoblot analysis. The normal human keratinocytes were treated with resveratrol (5, 10, and 25 µM for 24 hours for dose-dependent studies or 5 µM for 12, 24, and 48 hours for time-dependent studies) after which the cells were washed and exposed to UVB radiation in PBS. Six hours following UVB exposure, the nuclear lysates were prepared. The immunoblot analysis was performed for NF-κB/p65 using appropriate antibodies as described under Materials and Methods. For every immunoblot, equal loading of protein was confirmed by stripping the blot and reprobing with β-actin antibody (data not shown). The data shown here are from representative experiment repeated four times with similar results.

Figure 3

Effect of resveratrol on UVB-induced activation of NF-κB: EMSA. The normal human keratinocytes were treated with resveratrol (5, 10, and 25 µM for 24 hours for dose-dependent studies or 5 µM for 12, 24, and 48 hours for time-dependent studies) after which the cells were washed and exposed to UVB radiation in PBS. Six hours following UVB exposure, the nuclear lysates were prepared. The EMSA was performed as detailed under Materials and Methods. C1, C2 and C3 represent controls; biotin-EBNA control DNA, biotin-EBNA control DNA+EBNA extract, biotin-EBNA control DNA+EBNA extract +200-fold molar excess of unlabeled EBNA DNA, respectively. The data shown here are from representative experiment repeated three times with similar results.

Figure 4

Effect of resveratrol on UVB-induced activation of NF-κB/p65: ELISA. The normal human keratinocytes were treated with resveratrol (5, 10, and 25 µM for 24 hours for dose-dependent studies or 5 µM for 12, 24, and 48 hours for time-dependent studies) after which the cells were washed and exposed to UVB radiation in PBS. Six hours following UVB exposure, the nuclear lysates were prepared for ELISA. The effect on NF-κB/p65 was evaluated using ELISA as detailed in Materials and Methods. The data are expressed as mean±SEM from four experiments, conducted at least in triplicate.

Figure 5

Effect of resveratrol on UVB-induced degradation and phosphorylation of IκBα. The normal human keratinocytes were treated with resveratrol (5, 10, and 25 µM for 24 hours for dose-dependent studies or 5 µM for 12, 24, and 48 hours for time-dependent studies) after which the cells were washed and exposed to UVB radiation in PBS. Six hours following UVB exposure, the postnuclear lysates (cytosol) were prepared. The immunoblot analysis was performed for IκBα or phospho-IκBα using appropriate antibodies as described under Materials and Methods. For every immunoblot, equal loading of protein was confirmed by stripping the blot and reprobing with β-actin antibody (data not shown). The data shown here are from representative experiment repeated four times with similar results.

Figure 6

Effect of resveratrol on UVB-induced activation of IKKα. The normal human keratinocytes were treated with resveratrol (5, 10, and 25 µM for 24 hours for dose-dependent studies or 5 µM for 12, 24, and 48 hours for time-dependent studies) after which the cells were washed and exposed to UVB radiation in PBS. Six hours following UVB exposure, the postnuclear lysates (cytosol) were prepared. The immunoblot analysis was performed using appropriate antibodies as described under Materials and Methods. For every immunoblot, equal loading of protein was confirmed by stripping the blot and reprobing with β-actin antibody (data not shown). The data shown here are from representative experiments repeated four times with similar results.

Figure 7

Schematic representation of the mechanism of the chemopreventive effects of resveratrol against UVB-mediated damages.