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

描述(申请人提供)：美国每年诊断出超过200万例紫外线诱导的非黑色素瘤皮肤癌新病例，使其成为美国最常见的癌症形式。芹菜素是一种在各种水果和叶类蔬菜中发现的非诱变、自然存在的类黄酮类化合物，它可以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证明，芹菜素处理细胞可以产生广泛的抗肿瘤和化学预防作用，包括抑制血管生成，抑制COX-2的表达，诱导细胞周期停滞和凋亡。然而，负责芹菜素化学预防功能的主要靶点(S)仍然难以捉摸。我们已获得初步证据表明，芹菜素能激活角质形成细胞(小鼠和人角质形成细胞系、原代正常人表皮角质形成细胞和小鼠表皮)中的AMP激活蛋白激酶(AMPK)。AMPK的激活导致mTOR信号的抑制，从而解除了对自噬途径的阻断，进一步诱导了人角质形成细胞的自噬。激活自噬途径有望成为一种化学预防策略。我们的初步结果还显示，在UVB辐射后观察到适度的AMPK激活，这表明单独UVB辐射后存在保护性细胞反应，提供有限的保护。更有趣的是，在UVB辐射之前对芹菜素进行预处理可以显著增强AMPK的激活(无论是在体外还是在体内)，从而提供更强的保护。在这个方案中，我们假设AMPK/mTOR轴参与了通过芹菜素对UVB诱导的皮肤癌的化学预防，从机械上讲，AMPK的激活进一步诱导自噬来抑制UVB诱导的肿瘤的发生。我们建议在使用人类角质形成细胞的基于细胞的研究中和在体内使用UVB辐射的小鼠模型来验证这一假设。目的研究芹菜素如何通过培养的人角质形成细胞(HaCaT细胞和NHEK细胞)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的激活，如果UVB辐射本身诱导自噬，以及是否用芹菜素预处理增强UVB照射的角质形成细胞的自噬。UVB诱导的癌前表皮细胞突变型P53簇是一种早期的形态变化，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，我们将使用这个模型来研究局部应用芹菜素是否在体内激活AMPK，以及抑制AMPK活性是否会抑制芹菜素对UVB诱导的癌前表皮细胞突变型P53簇的化学预防功能。将AMPK确定为芹菜素的关键靶点，并将自噬途径作为癌症化学预防的靶点，将为皮肤癌的更好治疗和预防提供新的靶点和策略。 与公共健康相关：人们普遍认为，长期紫外线暴露是美国大多数非黑色素瘤皮肤癌的原因，芹菜素是一种存在于各种食物来源中的天然化合物，已被证明可以抑制紫外线诱导的皮肤癌。这项提议将检验一种创新的假设，即一种酶-AMPK(AMP激活的蛋白激酶)参与了芹菜素对紫外线诱导的皮肤癌的化学预防。将AMPK确定为芹菜素的靶点将在皮肤癌领域产生重大影响，并为更好地治疗和预防皮肤癌提供新的策略。

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. PUBLIC HEALTH RELEVANCE: Chronic UV exposure is widely believed to be the cause of most non-melanoma skin cancer in the United States, and apigenin, a naturally occurring compound present in a wide variety of food sources, has been shown to inhibit UV-induced skin cancer. This proposal will test the innovative hypothesis that an enzyme - AMPK (AMP-activated protein kinase) is involved in chemoprevention of UV-induced skin cancer by apigenin. Identifying AMPK as a target for apigenin would have a significant impact in the skin cancer field and provide a new strategy for better treatment and prevention of skin cancer.