自噬及相关AMPK/mTOR和ERK1/2双重信号通路与洋地黄类药物抗肿瘤作用关系研究

Autophagy and autopahgy-related signaling pathways have been reported to be involved in the mechansims of many anti-cancer agents, thus attracting more and more attentions of oncologists in recent years. Digitalis drugs, represented by digoxin and ouabain, are a diverse family of naturally derived compounds that bind to and inhibit the pumping function of Na+/K+-ATPase (NKA). The class of compounds have been in clinical use for many years to treat heart failure. They are also reported to have selective killing activity in cancer cells and the anti-tumor activity is found to have little correlation with NKA inhibition function. Although multiple anti-cancer mechanisms for the compounds have been revealed, the role of autophagy and related molecular signaling pathways in their anti-cancer activity is not systematically examined. Our preliminary data have shown that autophagic cell death is induced, and may play an important role in digoxin or ouabain-induced toxicity in A549 and H460 cell lines at IC50 level. More importantly and interestingly, AMPK mediated down-regulation of mTOR signaling pathway, as well as activation of ERK1/2 pathway, are both found to be involved in the autophagy regulation. Based on above observations, this project aims to systematically study the correlation between autophagy as well as related signaling pathways and anti-cancer effects of the compounds with the use of multiple cellular and molecular biotechnology. Specifically, we will study whether autophagy is universally induced and its role in the anti-cancer effects of the compounds, and how autophagy-related signaling pathways are mechanistically regulated by the compounds.The role of NKA in the regulation of autophagy and autophagy-related signaling pathways will also be examined. Taken together, the project will help thoroughly understand the molecular action of NKA inhibitors towards cancer.

自噬是近年来国内外研究的热点，围绕自噬及其信号进行抗肿瘤药物机制研究成为一种新颖策略。以哇巴因和地高辛为代表的洋地黄类药物作为Na+/K+-ATPase（NKA）抑制剂，临床上广泛用于心力衰竭的治疗。此类药物对肿瘤也具有强效的选择性杀伤活性，且其抗肿瘤效应与NKA抑制作用并不相关。目前有关自噬及相关分子信号通路与洋地黄药物抗肿瘤作用的关系，国内外报道甚少。我们前期工作首次发现此类药物可诱导人肺癌细胞自噬性死亡，并调节自噬相关的AMPK/mTOR以及ERK1/2双重信号通路变化。基于此，本项目拟综合应用多项生物学技术，系统研究自噬及其激酶信号与该类药物抗肿瘤作用的关系，包括研究药物诱导肿瘤细胞自噬的普遍性以及自噬对药物选择性抗肿瘤作用的影响；药物调节自噬相关AMPK等激酶信号的确切分子机制；NKA对自噬及其信号的影响等。本研究属于"老药新用"，将有助于全新阐释靶向NKA的抗肿瘤药物作用机理。

洋地黄类药物是一种细胞内Na+/K+-ATPase的特异性配体，临床上广泛用于心力衰竭的治疗。近年研究表明该类药物有抗肿瘤作用。本研究选用人非小细胞肺癌细胞A549和人乳腺癌细胞MCF-7作为研究对象，以哇巴因为代表药物，探讨了洋地黄类药物诱导肿瘤细胞发生自噬的条件及相关AMPK、Src及ERK1/2等激酶在其中的调控作用。结果发现，哇巴因在纳摩尔水平能够诱导人肿瘤细胞A549及MCF-7发生自噬性死亡，但对正常细胞系MRC5无明显自噬作用。药物还诱导细胞内活性氧自由基（ROS）水平升高，提示ROS介导自噬的发生。Western Blot检测到哇巴因能引起AMPK、Src、ERK1/2、PERK及eIF2α磷酸化水平的上调，并且检测到体外AMPK活性的升高，提示洋地黄类药物同时激活AMPK及Src/ERK1/2双重信号通路。该化合物还可抑制Na+/K+-ATP酶α1亚型（NKAα1）的表达。采用抑制剂PP2或siRNA-Src分别抑制Src活性或敲低Src蛋白后发现，药物引起的ERK1/2活化显著降低，自噬和ROS水平及细胞毒作用均减弱，提示Src在药物诱导的自噬中起正调控作用。研究还发现，哇巴因能够时间依赖性地降低MCF7细胞内ATP水平，剂量依赖性地降低线粒体呼吸作用，同时促进葡萄糖吸收，提示其促进有氧糖酵解。综上，洋地黄类药物可激活ATP/AMPK及Src/ERK1/2双重信号通路，导致肿瘤细胞发生自噬。该类药物还可影响细胞ROS水平和线粒体呼吸作用。本研究为深入理解此类药物的抗肿瘤作用机制提供了实验依据。

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