Solar UV radiation-induced skin cancer is increasing at an alarming rate in the United States and other parts of the world, especially among Caucasian populations (1
). Therefore, additional efforts are needed to define novel agents-approaches to prevent skin cancer and other cutaneous damage that result as a consequence of UVB exposure. For this reason, it is important to identify mechanism-based photo-protective agents. We have shown previously that PFE treatment to mice before single UVB exposure resulted in inhibition of UVB-induced early biomarkers of carcinogenesis (28
). The present study was designed to investigate the photo-protective effects of PFE against multiple UVB exposure (cumulative UVB dose: 1260 mJ/cm2
) on markers of inflammation, proliferation and oxidative stress. In this study, we used seven exposures as an intermediate point between chronic and acute exposure that may possibly reflect the early effect of persistent damage, and still photo-protective effect of PFE was observed in mouse skin. UVB radiation has both direct and indirect effects on the skin that includes protein oxidation, DNA damage, lipid peroxidation, generation of ROS, inflammation, proliferation, oxidative stress and immunosuppression (2
). We determined whether oral feeding of PFE, after multiple UVB exposure to mice, inhibits epidermal hyperplasia and infiltration of leukocytes. Our data clearly demonstrate that oral feeding of PFE significantly reduced multiple UVB-induced epidermal hyperplasia and infiltration of leukocytes. Persistent damage to critical cellular molecules, such as, proteins, lipids and DNA can result in oxidative stress that may lead to development of skin cancers (2
). The oxidation of amino acids leads to the formation of carbonyl groups in proteins and has become a widely accepted measure of oxidative damage of proteins. Since proteins have different and distinctive biological functions, oxidative alterations to proteins can lead to diverse functional consequences. The formation of lipid peroxides in biological membrane is a ROS–mediated process and their enhanced level is associated with disruption of cellular membrane (36
). Multiple exposures of the mouse skin to UVB radiation enhanced the formation of protein carbonyls and MDA in comparison to non-UVB exposed mouse skin. Oral feeding of PFE significantly inhibited multiple UVB irradiation-induced protein oxidation and lipid peroxidation in mouse skin.
UVB radiation is known to known to promote tumor growth and development by activating multiple cellular signaling pathways (2
). UVB-induced responses depend on dose, cell type, duration of activation of the pathways and crosstalk between pathways that determine cell survival, inflammation, proliferation and apoptosis (5
). MAPK are serine/threonine kinases that regulate the activity of NF-κB and AP-1 in response to wide variety of extracellular stimuli including UVB radiation (13
). The MAPK is divided into the extracellular signal-regulated kinases (ERK1/2), the c-Jun N-terminal kinases (JNK), and the p38 kinases. Earlier, we have shown that UVB exposure to NHEK results in an increased production of hydrogen peroxide that in turn activates the MAPK signaling pathway (39
). In the present study, we observed that oral feeding of PFE significantly reduced multiple UVB-mediated phosphorlation of ERK1/2, JNK1/2 and p38. UVB radiation results in the activation of NF-κB by a series of upstream kinases, including MAPK. Studies have demonstrated that MAPK family proteins such as ERK1/2 and p38 regulate the activation of NF-κB via the IKK pathway (40
). The transcription factor NF-κB is a critical mediator of the cellular response to inflammation, proliferation, survival and cellular stress (4
). NF-κB proteins (p65/RelA, RelB c-Rel, p50 and p52) form homo- or heterodimers that can bind to consensus DNA sequences of target genes and regulate their transcription. In resting cells, NF-κB is sequestered in the cytoplasm because of its association with inhibitory proteins of the IκB family (43
). Upon stimulation by UVB radiation, inflammatory cytokines, phorbol esters and lipopolysaccharide, the multisubunit of IκB kinase (IKK) phosphorylates IκB proteins at Ser32/36
, leading to its polyubiquitination and proteasomal degradation, which allows nuclear translocation of NF-κB dimers and activate transcription of target genes (44
). We found that oral feeding of PFE to mice resulted in inhibition of UVB-induced degradation and phosphorylation of IκBα, activation of IKKα/β, nuclear translocation and phosphorylation of NF-κB/p65 at Ser536
. Overall, our studies suggest that PFE modulates UVB-mediated activation of these cellular signaling pathways in vivo
situation leading to photoprotection of skin cells.
Cellular signaling pathways act independently or coordinately to regulate expression of target genes involved in inflammation and proliferation (2
). The expression of COX-2 and iNOS is primarily regulated by MAPK and NF-κB leading to inflammation, proliferation, cell transformation and tumor promotion (45
). COX-2 is a key enzyme required for prostaglandins synthesis and has been linked to the pathophysiology of inflammation and skin cancers (47
). iNOS produces nitric oxide that is associated in the pathogenesis of various inflammatory skin diseases (48
). Studies have shown that p38 MAPK and NF-κB are regulators of iNOS expression in response to UVB radiation in mouse skin (13
). In addition, p38 MAPK pathway is a key regulator of COX-2 both at the transcriptional and translational levels (49
). Studies have shown that UVB-induced inflammatory responses lead to cellular proliferation in skin cells (50
). We found that oral feeding of PFE significantly inhibited mutiple UVB-induced inflammatory responses in terms of inhibition of COX-2 and iNOS expression and reduction in the levels of epidermal cellular proliferation viz PCNA and cyclin D1. These studies demonstrate that the photoprotective effects of PFE are associated with the inhibition of UVB-induced inflammation and proliferation and their associated signaling molecules that regulate these events.
UVB radiation activate AP-1 that results in a series of complex biologic interactions leading to skin cancer (5
). In addition, UV radiation activates AP-l that stimulates transcription of MMP genes encoding MMP-l, MMP-3 and MMP-9 in skin cells. These changes apparently occur through induction of AP-1 that is downstream target of MAPK (51
). Studies have shown that the expression of MMP-2 and MMP-9 are known to play an important role in the degradation of type IV collagen, which is a major constituent of the basement membrane during cancer invasion and metastasis (34
). We found that PFE consumption inhibited UVB-induced phosphorylation of c-Jun and protein expression of MMPs. Our results demonstrate that inhibition of UVB-induced expression of MMPs in mouse skin may be atleast in part be associated due to inhibition in the phosphorylation of c-jun, a member of the AP-1 family.
Taken together, these data show that PFE consumption affords protection to mouse skin against the adverse effects of UVB radiation by modulating UVB-induced signaling pathways. This study suggests the potential efficacy of PFE as a photochemopreventive agent for skin cancer.