异甘草素调控糖代谢诱导黑色素瘤细胞凋亡机制研究

Our previous research has shown that isoliquiritigenin could induce apoptosis in melanoma cells, which accompanied by down regulation of hypoxia-inducible factor (HIF - 1 alpha) mRNA expression, the decrease of content of lactate and glucose intake, and accompanied by increase of ATP content reactive oxygen species (ROS) level; and potential mitochondrial depolarization phenomenon. We hypothesized that isoliquiritigenin may be targeted HIF - 1, lower levels of aerobic glycolysis, raised oxidative phosphorylation metabolic pathway, causing increased formation of ROS, activation of mitochondrial or endoplasmic reticulum stress apoptosis pathway. To verifying our hypothesis, two lines of tumor cells (mouse melanoma B16F0, human melanoma A875) and following technology, such as RT-PCR, Western blot, gene silencing, chromatin immune co-precipitation (ChIP), laser confocal and flow cytometry etc. will be used in this research protocol. First, we will find out whether isoliquiritigenin have effects on the glucose metabolism in melanoma cells, then we will clear and definite whether these effects on glucose metabolism is through targeted HIF - 1, and definite the relationship between glucose metabolism changes and elevated ROS level. Finally, we will determine the mechanism of apoptosis in melanoma cells induced by isoliquiritigenin. For clinical application of such kind of compound laid a foundation, in order to find out HIF-1 inhibitor from natural products for treatment of cancer provided beneficial references, for further development and utilization of isoliquiritigenin and its analogues provide the basis of theory and practice.

课题组前期研究表明，异甘草素能诱导黑色素瘤细胞凋亡，并伴随低氧诱导因子（HIF-1α）下调，葡萄糖摄取量降低，乳酸含量降低，ATP和活性氧（ROS）水平升高，线粒体电势去极化等现象，推测异甘草素可能靶向HIF-1，下调丙酮酸脱氢酶激酶1（PDK1） 表达，降低有氧糖酵解水平，上调氧化磷酸化代谢途径，ROS生成增多，活化线粒体凋亡途径或者产生内质网胁迫引起细胞凋亡。为验证上述假设，本研究拟采用小鼠黑色素瘤B16F0、人黑色素瘤A875两种黑色素瘤细胞，用RT-PCR、Western blot、基因沉默、染色质免疫共沉淀（ChIP）、激光共聚焦、流式细胞等技术，首先弄清异甘草素如何影响肿瘤细胞糖酵解，明确对糖代谢的影响是否通过靶向HIF-1起作用；接下来阐明糖代谢改变与ROS升高的关系，最后确定异甘草素诱导黑色素瘤细胞凋亡的机制。本研究将为筛选靶向肿瘤能量代谢的天然产物研究提供有益尝试。

主要采用MTT法检测异甘草素对黑色素瘤（A375，B16F0）细胞增殖的抑制作用，集落形成实验检测A375细胞的分化程度，Annexin V FITC/PI双染和PI染色法检测细胞的凋亡率和周期变化，Hoechst 33258染色法检测细胞核内染色质的变化，mitoTracker green和JC-1染色法检测线粒体分布及线粒体膜电位，DCFH-DA和mitoSox red染色法检测细胞内总活性氧及线粒体内活性氧含量，比色法检测线粒体呼吸链复合物I~IV的活性，酶联免疫吸附法检测细胞色素C及ATP含量，实时荧光定量PCR检测细胞黑色素合成、糖酵解途径关键酶及mTOR复合物mRNA表达水平，蛋白质免疫印迹法检测mTOR2-AKT-GSK3β信号通路蛋白、凋亡蛋白以及mitoNEET的表达。采用Western Blot 检测对黑色素瘤细胞HIF1α蛋白的影响。采用实时荧光定量 PCR 和RT-PCR检测对HIF1α靶基因的影响。.异甘草素能够使黑色素瘤细胞周期阻滞在G2/M期，明显抑制细胞增殖却并未导致细胞凋亡。异甘草素作用后使细胞黑色素生成及酪氨酸酶活力增加，黑色素合成途径关键酶TYR及小眼相关转录因子MITF的mRNA明显增加，葡萄糖摄取、乳酸释放、糖酵解途径关键酶GLUT1和HK2的mRNA表达以及细胞内ATP含量明显下降，而细胞氧耗率升高，使RICTOR、mTOR、p-mTOR、p-AKT和p-GSK3β表达下降，异甘草素通过激活mTORC2-AKT-GSK3β信号通路诱导人黑色素瘤细胞重编程。异甘草素也能够抑制细胞增殖，使细胞核染色质皱缩、形态出现损伤特征、细胞凋亡率以及cleaved PARP和cleaved caspase 3蛋白表达明显增加；使细胞线粒体分布改变、线粒体膜电位下降、线粒体呼吸链复合物I~IV活性下降，并导致胞浆细胞色素C升高及细胞内ATP含量下降。异甘草素作用后使细胞内及线粒体内ROS水平明显升高，mitoNEET蛋白表达显著下降；而构建mitoNEET重组载体使mitoNEET过表达后发现，细胞内总活性氧含量下降，cleaved caspase 3蛋白表达下降，细胞增殖率增加并且线粒体膜电位升高，说明40 μg/mL异甘草素通过抑制mitoNEET表达，使细胞内ROS水平升高而诱导细胞凋亡及线粒体功能障碍。