Apigenin restores TSP-1 expression in UVB-irradiated keratinocytes

描述(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，每年有200多万新诊断病例，使其成为美国最常见的癌症(1)。紫外线B(UVB)辐射是非黑色素瘤皮肤癌的主要致癌物。像所有实体肿瘤一样，NMSC促进新血管的生长(血管生成)，以便生长和转移。凝血酶敏感蛋白-1(TSP-1)是第一个被发现的血管生成抑制蛋白(2)，其在紫外线照射后的表皮和皮肤癌发生的不同阶段中的表达显著下调(3-6)。芹菜素(Api，4‘，5，7-三羟基黄酮)是一种存在于水果和蔬菜中的非致突变黄酮类化合物，可抑制紫外线照射和化学致癌物(7，8)诱导的NMSC。我们和其他人已经证明了Api在多种细胞类型(9-14)中诱导了许多抗肿瘤和化学预防作用，然而，TSP-1在Api化学预防中的作用从未报道过。我们的初步结果表明，Api在体内阻断了UVB对培养的角质形成细胞和皮肤中TSP-1的下调。我们发现，在UVB/Api处理的角质形成细胞中，TSP-1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-1mRNA的翻译中起着重要作用。此外，我们还表明，Api促进Hur(15)的核到细胞质的转位，TSP-1的多核糖体翻译发生在那里。此外，我们还发现，在UVB照射的角质形成细胞中，siRNA抑制了Hur的表达，取消了Api恢复正常TSP-1水平的能力。在已建立的中波紫外线诱发皮肤癌变的体内模型中，我们令人信服的初步结果证实，中波紫外线抑制了表皮中TSP-1的蛋白表达，而Api则恢复了TSP-1的表达。此外，Api还可抑制UVB诱导的真皮浅层间质炎症和微血管密度(MVD)的上调。鉴于TSP-1众所周知的抗血管生成和抗炎作用，很可能是Api诱导TSP-1表达是减少MVD和炎症浸润的原因。基于这些发现，我们建议验证这一新的假设，即Api通过上调TSP-1的翻译来恢复UVB照射的表皮中TSP-1的表达。我们进一步假设，Api诱导TSP-1的表达导致抑制间质间质的炎症和血管生成，从而全面地化学预防NMSC。我们建议在基于细胞的研究和活体研究中，使用暴露于UVB和/或Api的野生型和凝血酶反应蛋白-1缺失(TSP-1-/-)小鼠和角质形成细胞来验证这一假设。将TSP-1确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，将为改进NMSC的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.