基于Akt/mTOR信号通路干预肿瘤细胞能量代谢异常的寒凉中药白头翁中皂苷类成分抗癌机制研究

Warburg found tumor tissue metabolic anomalies in the early 20 's and therefore won the Nobel Prize in 1931. When Robert and Weinberg reported that energy metabolism is one of the ten characteristics of tumor cells in the journal Cell once again in 2011, the feature of tumor cell metabolic anomalies has attracted a great deal of scholarly attention.This feature suggests that it could become the new means of treatment to inhibit glycolytic pathway of tumor cells and cut off the tumor's "nutrition" to "starve" tumor. Our previous studies have found that pulsatilla saponin had a regulating effect on energy metabolism of human colon cancer cell（HT-29）.It could significantly reduce the consumption of glucose ，lactic acid and ATP in order to inhibit glycolysis of cancer cell. It and its components also might inhibit p-AKT and p-mTOR protein expression of HT-29 differently, and different components have different effects on related genes expression.Our group study found that pulsatilla saponin constituents have a regulating effect on energy metabolism in colon cancer cell, and different ingredients might have different targets on regulation of AKT/mTOR signal pathway in order to achieve synergistic antitumor effect.Based on this, pulsatilla saponin and its components would be studied on AKT/mTOR signal pathway and related targets by the methods of molecular biology on colon cancer animal and cell experiment. It might clarify multi-target combined effect of anti-tumor mechanism of pulsatilla saponin components.

Warburg 早在上世纪20年代就发现肿瘤代谢异常现象，为此1931 年获诺贝尔奖。直到2011年Weinberg在Cell上报道能量代谢异常是肿瘤细胞十大特征之一，又引起全球再次高度重视。这一特征表明，抑制肿瘤细胞糖酵解而切断肿瘤的"营养"从而"饿死"肿瘤，可能成为新的治疗手段。本课题组前期研究发现，寒凉中药白头翁中皂苷类成分对结肠癌能量代谢有调控作用，主要表现为能显著降低肿瘤细胞对葡萄糖消耗及乳酸产生，抑制糖酵解，减少ATP等；并能抑制HT-29细胞p-AKT、p-mTOR蛋白表达，且不同成分对相关调控基因影响也不同。因此，白头翁不同成分可能作用于Akt/mTOR信号通路不同靶点而达到协同抗肿瘤作用。基于此，本课题在前期基础上，拟通过结肠癌动物与细胞实验，利用分子生物学技术系统研究白头翁皂苷类成分对Akt/mTOR信号通路及各靶点作用特点，揭示白头翁总皂苷抗肿瘤多靶点综合效应机制。

能量代谢异常是肿瘤细胞的一大特征，本课题组在前期研究基础发现中药白头翁皂苷类成分对能量代谢有一定调控作用，主要表现为能显著降低肿瘤细胞对葡萄糖消耗及乳酸产生，抑制糖酵解等作用。本课题通过细胞和动物实验开展白头翁总皂苷、白头翁有效成分及有效成分配伍组对不同人癌细胞HT-29、NCI-H460、SW480、A549能量代谢的影响及干预机制，结果显示白头翁有效成分能降低肿瘤细胞的葡萄糖摄取及乳酸生成，对糖酵解的关键酶GLUT1，HK2，PKM2，LDHA，MCT4有显著抑制作用，其机制可能与白头翁皂苷干预肿瘤细胞有氧糖酵解调控因子c-Myc及HIF-1α有关，但研究中发现有效成分α-常春藤皂苷对肿瘤细胞糖酵解调节因子AKT，p-Akt，P53蛋白的表达量无明显影响；同时本课题开展了基于能量代谢机制的白头翁皂苷有效成分协同抑制NCI-H460人肺癌细胞增殖作用研究，采用质谱技术探讨白头翁皂苷有效协同抑制NCI-H460肺癌细胞增殖的分子机制，结果认为白头翁皂苷有效成分（PSD、R13、PSA）联用具有协同抑制NCI-H460人肺癌细胞肿瘤细胞增殖及诱导其凋亡作用，可显著的干预LDHA, PI3K, EEF1D, NOL3 和Caspase-3蛋白表达，协同作用可能通过PI3K/AKT/mTOR信号通路来进行。本研究采用多种技术如nano LC-LTQ-Orbitrap-MS/MS、Annexin-V/PI染色流式细胞术、Western blotting等开展中药有效部位、有效成分及有效成分配伍组抗肿瘤作用及机制的研究，较全面对中药不同层次的药效物质基础进行研究，并运用多种软件如Calcusyn3.0、Protein Discovery 1.4充分分析实验数据，阐明有效成分配伍具有协同抗肿瘤作用研究，同时预测抗肿瘤作用途径和靶点，为具有复杂性特点中药的抗肿瘤作用研究提供了新思路。