Photochemopreventive Effect of Pomegranate Fruit Extract on UVA-mediated Activation of Cellular Pathways in Normal Human Epidermal Keratinocytes

ABSTRACT

UVA is the major portion (90–99%) of solar radiation reaching the surface of the earth and has been described to lead to formation of benign and malignant tumors. UVA-mediated cellular damage occurs primarily through the release of reactive oxygen species and is responsible for im-munosuppression, photodermatoses, photoaging and photocar-cinogenesis. Pomegranate fruit extract (PFE) possesses strong antioxidant and anti-inflammatory properties. Our recent studies have shown that PFE treatment of normal human epidermal keratinocytes (NHEK) inhibits UVB-mediated activation of MAPK and NF-κB pathways. Signal transducers and activators of transcription 3 (STATJ), Protein Kinase B/AKT and Map Kinases (MAPKs), which are activated by a variety of factors, modulate cell proliferation, apoptosis and other biological activities. The goal of this study was to determine whether PFE affords protection against UVA-mediated activation of STAT3, AKT and extracellular signal-regulated kinase (ERK1/2). Immunoblot analysis demonstrated that 4 J/ cm² of UVA exposure to NHEK led to an increase in phosphorylation of STAT3 at Tyr⁷⁰⁵, AKT at Ser⁴⁷³ and ERK1/2. Pretreatment of NHEK with PFE (60–100 μg/mL) for 24 h before exposure to UVA resulted in a dose-dependent inhibition of UVA-mediated phosphorylation of STAT3 at Tyr⁷⁰⁵, AKT at Ser⁴⁷³ and ERK1/2. mTOR, structurally related to PDK, is involved in the regulation of p70S6K, which in turn phosphorylates the S6 protein of the 40S ribosomal sub-unit. We found that UVA radiation of NHEK resulted in the phosphorylation of mTOR at Thr²⁴⁴⁸ and p70S6K at Thr⁴²¹/ Ser⁴²⁴. PFE pretreatment resulted in a dose-dependent inhibition in the phosphorylation of mTOR at Thr²⁴⁴⁸ and p70S6K at Thr⁴²¹/Ser⁴²⁴. Our data further demonstrate that PFE pretreatment of NHEK resulted in significant inhibition of UVA exposure-mediated increases in Ki-67 and PCNA. PFE pretreatment of NHEK was found to increase the cell-cycle arrest induced by UVA in the G1 phase of the cell cycle and the expression of Bax and Bad (proapoptotic proteins), with downregulation of Bcl-X_L expression (antiapoptotic protein). Our data suggest that PFE is an effective agent for ameliorating UVA-mediated damages by modulating cellular pathways and merits further evaluation as a photochemopreventive agent.