高良姜素诱导肝癌细胞自噬死亡的途径及相关分子机制的研究

We have originally discovered that galangin, the active ingredient of Zhanjiang province-featured Chinese traditional medicine galangal, elevate the AMP concentration in hepatocellular carcinoma cells, up-regulate expression of p53, and induce autophagic death by inhibition of mTOR pathway through AMPK activation.However, the underlying mechanisms referring to galangin-induced increase of AMP concentration in hepatocellular carcinoma cell remain elusive. Elucidation of the underlying mechanisms will contribute to clinical treatment for hepatocellular carcinoma. In the in vitro experiments, the expressions and functions of glucose transporter protein 1 (GLUT1) and pyruvate kinase isomer M2 (PKM2) pre- and post-treatment of galangin were analyzed, clarifying the influence of galangin in glucose uptake and glycolysis, and energy metabolism of hepatocellular carcinoma. .The role/function of galangin in the activation of p53, thus inhibiting the expressions and functions of GLUT1 and PKM2, is determined by ChIP assay and co-immunoprecipitation technique. Finally the in vitro results will be confirmed by animal experiments. The proposed project will clarify the molecular mechanisms of autophagy induced by galangin through inhibition of energy metabolism of hepatocellular carcinoma, and also the effects of p53 on the galangin-induced inhibition of expressions of GLUT1 and PKM2. These forthcoming findings will provide a theoretical basis for the application of galangin in clinical treatment for hepatocellular carcinoma.

我们首次发现高良姜素(湛江特色中药高良姜的有效成分)可上调肝癌细胞内AMP浓度和p53表达，激活AMPK抑制mTOR通路，引发自噬而抑制肝癌增殖，但是高良姜素升高肝癌细胞内AMP浓度的机制不明，该机制的阐明有助于肝癌的治疗。因此，本项目首先以肝癌细胞为研究对象，通过检测高良姜素处理前后葡萄糖转运蛋白1（GLUT1）和丙酮酸激酶M2(PKM2)的表达和功能的变化，分析其能否通过抑制肝癌细胞糖摄取和酵解，升高胞内AMP浓度，从而对肝癌能量代谢产生影响。然后采用ChIP技术检测高良姜素如何激活p53调控GLUT1和PKM2表达，IP技术分析高良姜素激活p53结合和抑制两者功能。最后通过动物实验确证。本项目将阐明高良姜素升高肝癌细胞内AMP浓度诱导自噬的机制，明确p53在高良姜素抑制GLUT1和PKM2表达及功能过程中的作用。结果有助于指导利用高良姜素调控自噬治疗肝癌，为高良姜素应用奠定理论基础。

药物调控自噬治疗肿瘤具有重要意义，研究药物诱导自噬治疗肿瘤的机制是当前关注的热点。我们在研究湛江特色中药高良姜抗肿瘤作用工作基础上首次发现，高良姜的活性成分高良姜素诱导肝癌自噬死亡（Ⅱ型程序性细胞死亡）。这提示高良姜素是一种具有应用前景的抗肝癌化合物，研究它诱导肝癌自噬死亡的机制可为应用高良姜提供理论基础。项目研究了高良姜素是否抑制肝癌HepG2细胞GLUT1功能，减少摄入葡萄糖。肝癌HepG2细胞经高良姜素诱导后，取样检测细胞内乳酸水平变化；分析PKM2表达变化和活性变化；探讨P53对PKM2和GLUT1的影响，检测细胞内AMP、ADP和ATP水平变化， AMPK活性和SIR家族成员表达的影响；高良姜素对裸鼠肝癌移植瘤葡萄糖代谢的影响。高良姜素可直接抑制GLUT1转运葡萄糖活性，诱导自噬死亡的机制；高良姜素可激活P53和DAPK，抑制PKM2表达，上调PKM1活性和表达水平，逆转肝癌细胞糖酵解，促进糖有氧氧化。通过基因芯片检测发现了高良姜素抑制肝癌细胞糖代谢和三羧酸循环的关键酶的表达。高良姜素不仅可以抑制柠檬酸合酶的表达，同时影响ATP合酶的亚基表达。确定高良姜素可抑制HepG2细胞摄取葡萄糖的转运，造成细胞能量代谢不足，激活AMPK，促进细胞发生自噬的机制。并确定高良姜素诱导自噬促进肝癌细胞死亡，所用的剂量低于诱导细胞凋亡剂量，并能增强顺铂的作用。高良姜素通过激活MAPK系统（p38MAPK、JNK、ERK）引起肝癌细胞（HepG2细胞、Hep3B细胞、PLC/5rf细胞）内质网应激，抑制肝癌细胞增殖。内质网应激也是诱发细胞发生自噬的原因。高良姜素可通过TGF-B通路影响肝癌细胞自噬抑制细胞增殖。高良姜素通过上调SIRT1表达，通过去乙酰化LC3促进高良姜素诱导的HepG2细胞自噬。筛选了一批可能影响高良姜素诱导细胞自噬和凋亡相关的microRNA。我们研究结果提示高良姜素是一种具有应用前景的抗肝癌化合物，本项目研究结果有助于指导如何利用高良姜素调控自噬治疗肝癌，降低用药浓度，减轻毒副作用，为拓展高良姜应用范围，增加高良姜的附加值提供理论基础。