Apigenin restores TSP-1 expression in UVB-irradiated keratinocytes

Over two million cases of non-melanoma skin cancer (NMSC) are diagnosed yearly in the United States, and the majority are caused by exposure to ultraviolet B (UVB) radiation. Like all solid tumors, NMSC promotes growth of new blood vessels (angiogenesis) in order to grow and metastasize. Thrombospondin-1 (TSP-1) is the first endogenous inhibitor of angiogenesis to be identified, and its expression is markedly downregulated in epidermis following UVB irradiation and throughout distinct steps of skin carcinogenesis. Apigenin (Api, 4’, 5, 7-trihydroxyflavone), a nonmutagenic flavonoid, inhibits NMSC induced by UV exposure and chemical carcinogens. Multiple anti-cancer and chemopreventive effects of Api are well known, however we are the first to link TSP-1 and Api’s beneficial effects. We found that Api blocks downregulation of TSP-1 by UVB in cultured keratinocytes and in skin in vivo. Our novel preliminary results also indicate that Api regulates TSP-1 post-transcriptionally and this regulation is mediated by RNA-binding protein Human antigen R (HuR). Importantly, we provide a direct link between HuR and TSP-1 whereby siRNA knockdown of HuR abrogates the ability of Api to maintain normal TSP-1 levels in UVB-irradiated keratinocytes. Furthermore, Api inhibited stromal inflammation and microvascular density (MVD) in superficial dermis that occurs in response to UVB. Given the known anti-angiogenic and less studied anti-inflammatory effects of TSP-1, we hypothesize that high TSP-1 expression in Api-treated skin is the major cause of decreased MVD and inflammatory infiltrates. We propose to test the novel hypothesis that Api maintains TSP-1 expression in UVB-irradiated epidermis via upregulation of TSP-1 translation, resulting in inhibition of inflammation and angiogenesis in the stromal compartment, with overall chemoprevention of NMSC. Aim 1 will delineate mechanisms by which RNA-binding protein HuR mediates Api’s ability to block downregulation of TSP1 expression by UVB. Aim 2 will employ wildtype and TSP-1 null mice to investigate the inhibition of UVB-induced inflammation and angiogenesis by Api in cell-based studies and in vivo, and define TSP1-dependent molecular and cellular events. Aim 3 will use wildtype and TSP-1 null mice in a UVB carcinogenesis regimen to investigate the role of TSP1 in development of UVB-induced NMSC. Studies will employ TSP1 peptide mimetic ABT-898 to restore/mimic chemoprevention by Api in TSP1 null mice. Identifying TSP-1 as a key target of apigenin and demonstrating its important role in chemoprevention by Api will provide a compelling rationale to use Api for maintaining the expression of protective TSP-1 in skin as a novel strategy for NMSC prevention