Apigenin restores TSP-1 expression in UVB-irradiated keratinocytes

描述（由申请人提供）：非黑色素瘤皮肤癌（NMSC）是美国的一个主要健康问题，每年诊断出超过200万例新病例，使其成为该国最常见的癌症（1）。紫外线B（UVB）辐射是非黑色素瘤皮肤癌的主要致癌物质。与所有实体瘤一样，NMSC促进新血管的生长（血管生成）以生长和转移。血小板反应蛋白-1（TSP-1）是第一个被鉴定的血管生成蛋白抑制剂（2），并且其表达在UVB照射后和皮肤癌发生的不同步骤中在表皮中显著下调（3-6）。芹菜素（API，4 '，5，7-三羟基黄酮）是一种在水果和蔬菜中发现的非致突变类黄酮，可抑制紫外线暴露和化学致癌物诱导的NMSC（7，8）。我们和其他人已经表明，API在多种细胞类型中诱导许多抗肿瘤和化学预防作用（9-14），然而，TSP-1在API的化学预防中的作用从未报道过。我们的初步研究结果表明，API块下调TSP-1的UVB在体外培养的角质形成细胞和皮肤。我们发现TSP-1在UVB/Api处理的角质形成细胞中的表达调控发生在转录后，并由RNA结合蛋白HuR介导。重要的是，HuR最近被证明在TSP-1 mRNA的翻译中起重要作用。此外，我们已经表明API促进HuR的细胞核至细胞质的易位（15），其中发生TSP-1的多核糖体翻译。此外，我们已经表明，HuR表达的siRNA敲低消除了API恢复UVB照射的角质形成细胞中正常TSP-1水平的能力。我们令人信服的初步结果，在体内建立的UVB诱导的皮肤癌变模型，证实了在体内UVB抑制TSP-1蛋白表达在表皮和API恢复TSP-1的表达。此外，API抑制UVB诱导的间质炎症和真皮浅层微血管密度（MVD）的上调。由于TSP-1具有众所周知的抗血管生成作用和较少研究的抗炎作用，因此API诱导TSP-1表达可能是MVD降低和炎性浸润的原因。基于这些发现，我们提出了新的假设，即API通过上调TSP-1翻译恢复TSP-1在UVB照射的表皮中的表达。我们进一步假设，这种诱导TSP-1表达的API导致抑制炎症和血管生成的间质区室，与NMSC的整体化学预防。我们建议使用暴露于UVB和/或API的野生型和血小板反应蛋白-1-null（TSP-1 -/-）小鼠和角质形成细胞在基于细胞的和体内研究中检验该假设。将TSP-1鉴定为芹菜素的关键靶点并证明其在API化学预防中的重要作用将为NMSC的改善治疗和预防策略提供新的靶点和理论基础。

DESCRIPTION (provided by applicant): Non-melanoma skin cancer (NMSC) is a major health problem in the United States, with over two million new cases diagnosed yearly, making it the most common cancer in this country (1). Ultraviolet B (UVB) radiation is the major carcinogen for non-melanoma skin cancer. 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 protein inhibitor of angiogenesis to be identified (2), and its expression is markedly downregulated in epidermis following UVB irradiation and throughout distinct steps of skin carcinogenesis (3-6). Apigenin (Api, 4', 5, 7- trihydroxyflavone), a nonmutagenic flavonoid found in fruits and vegetables, inhibits NMSC induced by UV exposure and chemical carcinogens (7,8). We and others have shown that Api induces many antitumorigenic and chemopreventive actions in multiple cell types (9-14), however, the role of TSP-1 in chemoprevention by Api has never been reported. Our preliminary results show that Api blocks downregulation of TSP-1 by UVB in cultured keratinocytes and skin in vivo. We show that regulation of TSP-1 expression in UVB/Api-treated keratinocytes occurs post-transcriptionally and is mediated by RNA-binding protein HuR. Importantly, HuR has recently been demonstrated to play an important role in translation of TSP-1 mRNA. In addition we have shown that Api promotes nuclear to cytoplasmic translocation of HuR (15), where polyribosomal translation of TSP-1 occurs. Furthermore, we have shown that siRNA knockdown of HuR expression abrogates the ability of Api to restore normal TSP-1 levels in UVB-irradiated keratinocytes. Our compelling preliminary results in an established in vivo model of UVB-induced skin carcinogenesis, confirms in vivo that UVB inhibited TSP-1 protein expression in epidermis and Api restored TSP-1 expression. Furthermore, Api inhibited UVB-induced up-regulation of stromal inflammation and microvascular density (MVD) in superficial dermis. Given the well-known anti-angiogenic and less studied anti-inflammatory action of TSP-1, it is likely that induction of TSP-1 expression by Api is the cause of decreased MVD and inflammatory infiltrates. Based on these findings, we propose to test the novel hypothesis that Api restores TSP-1 expression in UVB-irradiated epidermis via upregulation of TSP-1 translation. We further hypothesize that this induction of TSP-1 expression by Api leads to inhibition of inflammation and angiogenesis in the stromal compartment, with overall chemoprevention of NMSC. We propose to test this hypothesis in cell-based and in vivo studies using wild- type and Thrombospondin-1-null (TSP-1 -/-) mice and keratinocytes exposed to UVB and/or Api. Identifying TSP-1 as a key target of apigenin and demonstrating its important role in chemoprevention by Api will provide a new target and rationale for improved treatment and prevention strategies for NMSC.