AMPK as a molecular target for chemoprevention by apigenin in preneoplastic skin

描述（由申请人提供）：在美国，每年诊断出超过200万例UVB诱导的非黑色素瘤皮肤癌新病例，使其成为该国最常见的癌症形式。芹菜素是一种非诱变性的天然类黄酮，存在于各种水果和多叶蔬菜中，可抑制紫外线诱导的皮肤癌。我们的实验室和其他实验室已经表明，芹菜素处理细胞导致多种抗肿瘤和化学预防作用，包括抑制血管生成，抑制考克斯-2表达，诱导细胞周期停滞和细胞凋亡。然而，负责芹菜素的化学预防功能的主要靶标仍然难以捉摸。我们已经获得了初步证据，芹菜素激活角化细胞（小鼠和人角化细胞系，原代正常人表皮角化细胞（NHEK）和小鼠表皮在体内）中的AMP活化蛋白激酶（AMPK）。AMPK的激活导致mTOR信号传导的抑制，这缓解了对自噬途径的阻断，进一步诱导人角质形成细胞中的自噬。自噬途径的激活有望成为一种化学预防策略。我们的初步结果还表明，在UVB辐射后观察到中度AMPK活化，这表明在单独UVB辐射后存在保护性细胞反应以提供有限的保护。更有趣的是，在UVB辐射之前芹菜素的预处理显著增强AMPK活化（体外和体内），从而提供更强的保护。在这个提议中，我们假设AMPK/mTOR轴参与了芹菜素对UVB诱导的皮肤癌的化学预防，并且AMPK的激活进一步诱导自噬以抑制UVB诱导的肿瘤发生。我们建议在使用人类角质形成细胞的细胞研究和使用UVB照射的小鼠模型的体内研究中测试这一假设。目的#1将通过使用培养的人角质形成细胞（HaCaT细胞和NHEK）来研究芹菜素如何激活AMPK并诱导自噬。我们还将确定UVB加芹菜素治疗如何调节AMPK激活，如果UVB辐射本身诱导自噬，如果用芹菜素预处理增强UVB照射的角质形成细胞的自噬。UVB诱导的肿瘤前表皮细胞突变p53簇是一种早期形态学变化，已被证明是UVB诱导的皮肤癌的非常早期的事件。因此，在目标#2中，我们将使用该模型来研究局部芹菜素是否在体内激活AMPK，以及AMPK活性的抑制是否会抑制芹菜素对UVB诱导的肿瘤前表皮细胞突变p53簇的化学预防功能。确定AMPK作为芹菜素的关键靶点并靶向自噬途径进行癌症化学预防将为开发更好的治疗和预防皮肤癌提供新的靶点和策略。

DESCRIPTION (provided by applicant): Over two million new cases of UVB-induced non-melanoma skin cancer are diagnosed yearly in the USA, making it the most common form of cancer in this country. Apigenin, a nonmutagenic, naturally occurring flavonoid found in a variety of fruits and leafy vegetables, inhibits UV-induced skin cancer. Our laboratory and others have shown that apigenin treatment of cells results in a wide variety of antitumorigenic and chemopreventive actions, including inhibition of angiogenesis, suppression of COX-2 expression, and induction of cell cycle arrest and apoptosis. However, the primary target(s) responsible for apigenin's chemopreventive function remains elusive. We have obtained preliminary evidence that apigenin activates AMP-activated protein kinase (AMPK) in keratinocytes (mouse and human keratinocyte cell lines, primary normal human epidermal keratinocytes (NHEKs) and mouse epidermis in vivo). The activation of AMPK results in inhibition of mTOR signaling, which relieves the block to the autophagy pathway, further inducing autophagy in human keratinocytes. Activation of the autophagy pathway holds promise as a chemoprevention strategy. Our preliminary results also show that a moderate AMPK activation was observed after UVB radiation, suggesting that there is a protective cellular response after UVB radiation alone to provide limited protection. More intriguingly, pretreatment of apigenin prior to UVB radiation enhances AMPK activation dramatically (both in vitro and in vivo), thus providing much stronger protection. In this proposal, we hypothesize that the AMPK/mTOR axis is involved in chemoprevention of UVB-induced skin cancer by apigenin, and mechanistically the activation of AMPK further induces autophagy to suppress UVB-induced tumorigenesis. We propose to test this hypothesis both in cell-based studies using human keratinocytes and in vivo using a UVB-irradiated mouse model. Aim #1 will investigate how apigenin activates AMPK and induces autophagy by using cultured human keratinocytes (both HaCaT cells and NHEKs). We will also determine how UVB plus apigenin treatment modulates AMPK activation, if UVB radiation by itself induces autophagy, and if pretreatment with apigenin enhances autophagy in UVB-irradiated keratinocytes. UVB-induced mutant p53 clusters of preneoplastic epidermal cells are an early morphologic change that has been demonstrated to be a very early event in UVB-induced skin cancer. Therefore, in Aim #2, we will use this model to investigate if topical apigenin activates AMPK in vivo, and if inhibition of AMPK activity will suppress apigenin's chemopreventive function against UVB-induced mutant p53 clusters of preneoplastic epidermal cells. Identifying AMPK as a key target of apigenin and targeting the autophagy pathway for cancer chemoprevention would provide a new target and strategy for the development of better treatment and prevention of skin cancer.