基于HIF-1α介导的糖酵解探讨小豆蔻明抑制乳腺癌作用机制

Breast cancer is the leading cause of cancer-related mortality worldwide in females. In view of the existing chemotherapy drugs, including molecular targeted drugs, the serious side effects, recurrence and drug resistance still results in the low 5-year survival rate. Thus, chemotherapy drug development with low toxicity and high efficience still faces severe challenges. Different from normal cells, tumor cells have high rates of glucose consumption associated with lactate production through glycolysis but not mitochondrial aerobic phosphorylation, then acidify tumor environment and provide a large number of metabolic intermediate for rapid cell proliferation and tumor growth. HIF - 1α mediated glycolysis is very important in the energy metabolism of breast cancer, and a variety of inflammatory factors involved in the regulation of HIF - 1 alpha mediated glycolysis in tumor cells. Our preliminary results obtained by dual luciferase report gene screening showed that cardamonin with anti-inflammatory activity could inhibit the activity of HIF - 1α, also could inhibit the tumor growth of breast cancer in vivo, which could be reversed by HIF- 1α overexpression. Moreover, cardamonin could suppress the gene expression of LDHA which is one target gene of HIF-1α associating with glycolysis, without toxicity of normal cells, indicating that cardamonin would inhibit breast cancer growth through inhibiting HIF-1α mediated glycolysis. This study aims to clarify the effect and molecular mechanism of cardamonin on growth of breast cancer, based on HIF - 1α mediated glycolysis.

乳腺癌的发病率和致死率均高居女性肿瘤第一。鉴于现有化疗药物（包括分子靶向药物）的严重毒副作用，易耐药复发等致使患者的5年生存率仍然较低，研发低毒高效的化疗药物仍然面临严峻的挑战。与正常细胞主要通过线粒体介导的有氧磷酸化不同，肿瘤细胞主要通过糖酵解分解葡萄糖获取能量，排出乳酸酸化肿瘤微环境，提供大量的代谢中间产物用于肿瘤细胞快速增殖和生长。HIF-1α介导的糖酵解在乳腺癌能量代谢中非常重要，而多种炎症因子参与调控肿瘤细胞HIF-1α介导的糖酵解。我们前期通过双荧光素酶报告基因筛选得到抑制HIF-1α活性的中药抗炎活性分子小豆蔻明，前期研究发现，小豆蔻明抑制乳腺癌作用与其抑制HIF-1α有关，其也能抑制HIF-1α的糖酵解相关靶基因如LDHA的基因表达，且对正常乳腺细胞无毒，提示其可能通过抑制HIF-1α介导的糖酵解起效。本研究旨在基于HIF-1α介导的糖酵解探讨小豆蔻明抑制乳腺癌作用机制。

乳腺癌的发病率和致死率均高居女性肿瘤第一。鉴于现有化疗药物（包括分子靶向药物）的严重毒副作用，易耐药复发等致使患者的5年生存率仍然较低，研发低毒高效的化疗药物仍然面临严峻的挑战。小豆蔻明是一种从中药草豆蔻中分离出来的查耳酮，具有抗炎和抗肿瘤活性。然而，其抑制乳腺癌作用的分子机制仍有待确定。本项目通过CCK-8法和Hoechst 33258染色法分别用于检测细胞生长和凋亡；通过荧光素酶报告基因法测量HIF-1α驱动的转录调控活性；用2-NBDG和L-乳酸测定试剂盒检测葡萄糖摄取和乳酸含量；在Agilent Seahorse Bioscience XF96细胞外通量分析仪上进行细胞代谢测定；用JC-1探针测量线粒体膜电位；DCFH-DA用于测量ROS水平；通过western blotting法测定蛋白表达；采用免疫组织化学法测定肿瘤组织中HIF-1α，LDHA和CD31的表达。研究结果显示，小豆蔻明在体内外均有显著抑制三阴性乳腺癌MDA-MB-231细胞的增殖，促进乳腺癌细胞凋亡作用。小豆蔻明通过抑制mTOR/P70S6K信号通路的激活，从而抑制HIF-1α的mRNA和蛋白表达，调控HIF-1a的转录调控活性，抑制HIF-1a/PDHK1轴的活性，进而下调其能量代谢靶基因PDHK1、LDHA、MCT4、GLUT1、GLUT3的基因和蛋白表达，抑制乳腺癌细胞葡萄糖摄取，促进乳腺癌细胞线粒体氧化磷酸化，降低糖酵解，抑制乳酸生成和外排，降低Nrf2抗氧化系统的活性，共同促进活性氧ROS在肿瘤细胞内的过度累积，从而降低乳腺癌细胞线粒体膜电位，促进肿瘤细胞凋亡，抑制乳腺癌细胞增殖，最终发挥乳腺癌抑制作用。本研究阐明中药草豆蔻黄酮小豆蔻明抑制乳腺癌作用机制与其下调乳腺癌细胞HIF-1a/PDHK1介导的糖酵解，促进线粒体有氧磷酸化有关，为其临床用于乳腺癌治疗提供理论依据，同时为靶向能量代谢调控抗肿瘤药物的研发提供参考。