天然小分子化合物对肠癌及胶质瘤细胞的抗肿瘤作用及潜在机制研究

发布时间：2018-06-24 17:58

  本文选题：胶质瘤 + 能量代谢　； 参考：《浙江大学》2014年博士论文

【摘要】：一基于胶质瘤代谢网络的小分子化合物抗肿瘤作用及机制研究目的：胶质瘤是一类常见的原发性颅内肿瘤,发病率约占原发性恶性脑肿瘤的50%以上。尽管现代医学技术有了极大发展,但是胶质瘤患者平均生存时间不超过15个月,而且手术过后易复发,现在仍然没有理想的药物及治疗方法。有氧糖酵解是肿瘤细胞主要的能量代谢方式,本实验主要研究受试药物ZZZ-1针对胶质瘤细胞代谢网络的抗肿瘤作用及其机制。实验设计：本研究用SRB法及集落形成实验检测受试药物ZZZ-1对一系列胶质瘤细胞株的抗肿瘤药效,研究其对胶质瘤细胞能量代谢过程中葡萄糖消耗、丙酮酸消耗、乳酸产生、细胞内ATP的产生及氧气消耗比例(OCR)的影响,进一步研究药物对胶质瘤细胞线粒体膜电位ΔΨm、ROS产生、细胞衰老、细胞凋亡及其相关蛋白、干细胞标志Bmi-1、正常星形胶质细胞标志GFAP及糖酵解过程中关键蛋白PFKFB3、LDH5表达的影响。用氧化磷酸化过程中ATP合成酶抑制剂寡霉素研究ZZZ-1的潜在作用机制。本实验进一步研究ZZZ-1在动物体内实验中对胶质瘤增殖抑制、血管内皮生长因子VEGF表达、糖酵解过程中关键酶、干细胞指标和分化指标的影响。实验结果：1)ZZZ-1具有强效的抗胶质瘤药效学作用：可显著抑制人及大鼠胶质瘤细胞增殖,抑制胶质瘤细胞集落形成,并良好的呈剂量依赖性；2)诱导胶质瘤细胞死亡及分化：诱导胶质瘤细胞AΨm去极化及ROS过度产生,抑制PCNA、 survivin的表达,诱导大鼠胶质瘤C6细胞凋亡及人胶质瘤细胞U-87MG细胞衰老,抑制干细胞指标Bmi-1的表达,促进胶纸瘤细胞向正常星形胶质细胞分化；3)诱导胶质瘤细胞能量代谢方式发生改变：ZZZ-1处理胶质瘤细胞24 h可诱导细胞葡萄糖及丙酮酸消耗及乳酸产生增多,可显著提高细胞内ATP产生量和氧气消耗量,药物处理细胞72 h可降低糖酵解过程中关键蛋白PFKFB3、LDH5的表达,寡霉素可降低ZZZ-1引起的胶质瘤细胞增殖抑制,抑制△Ψm的去极化及ROS的产生,减少葡萄糖的消耗及ATP的产生；ZZZ-1可减少寡霉素引起的代偿性乳酸的产生；4)抑制裸小鼠移植瘤生长：在U-87MG裸小鼠移植瘤模型中,ZZZ-1可选择性的抑制肿瘤细胞的生长,并且动物体重无降低,具有很高的安全性；可降低PCNA. survivin及糖酵解过程中关键蛋白PFKFB3、LDH5的表达；促进胶质瘤细胞向正常星形胶质细胞、神经细胞及小胶质细胞分化。结论：结果提示,ZZZ-1对胶质瘤细胞具有显著而稳定的抗肿瘤作用,可通过多层次、多途径调控胶质瘤细胞能量代谢、诱导细胞凋亡及衰老、促进胶质瘤细胞向正常神经细胞、星形胶质细胞及小胶质细胞分化等方面起到抑制胶质瘤细胞增殖的作用。二小分子化合物CADPE对肠癌多靶点调控作用及机制研究目的：结直肠癌是一类严重影响全球人类健康的疾病,每年世界范围内大约有一百万人被诊断出患有肠癌,同年有超过五十万人死于肠癌。本实验主要研究咖啡酸3,4-二羟基苯乙酯(CADPE)对结直肠癌的抗肿瘤药效、作用机制及可能的作用靶点。实验设计：本研究用SRB法及集落形成实验检测CADPE对四株人结直肠癌细胞的抗肿瘤药效,并研究CADPE对肠癌细胞的细胞周期、凋亡及周期和凋亡相关蛋白的影响。用MAPK信号通路的四种抑制剂研究CADPE对肠癌细胞的潜在作用机制。本实验进一步在体内动物实验中研究CADPE对肠癌的增殖抑制、细胞凋亡及细胞周期相关蛋白的影响,并评估CADPE对人正常肠癌组织细胞的影响。实验结果：本研究发现CADPE可显著抑制人结直肠癌细胞增殖和集落形成能力,并诱导肠癌细胞G0/G1期细胞周期阻滞和细胞凋亡；CADPE对肠癌的抑制作用与p38 MAPK、p53、p21及p16等信号通路的激活、核转录因子κB及其下游靶基因信号转导子和转录激活因子3 (STAT3)和c-Myc的抑制有关；CADPE还可以下调生存素survivin及细胞周期相关蛋白(如cyclin D1、CDK4、CDK6、p-Rb和E2F-1)的表达；P38 MAPK的抑制剂可减少CADPE引起的细胞增殖抑制、细胞周期阻滞及细胞凋亡,但是ERK和JNK抑制剂没有此类作用。在SW620裸小鼠移植瘤模型中,CADPE同样可抑制肿瘤生长及诱导细胞凋亡,并降低PCNA、survivin及细胞周期相关蛋白(cyclin D1、CDK4、CDK6、p-Rb)的表达,并且抗肿瘤作用浓度范围内的CADPE对正常细胞无细胞毒作用,在体内及体外实验中均具有很高的安全性。结论：实验结果提示CADPE在调控结直肠癌细胞增殖方面起着重要的作用,并可通过多靶点进行调控,从而抑制肠癌细胞增殖、引起肠癌细胞周期阻滞、诱导肠癌细胞凋亡。
[Abstract]:A study on the anti-tumor effect and mechanism of small molecular compounds based on the metabolic network of glioma: glioma is a common type of primary intracranial tumor, which accounts for more than 50% of the primary malignant brain tumors. Although modern medical technology has developed greatly, the average survival time of the patients with glioma is not more than 15 months. It is easy to relapse after the operation, and there is still no ideal medicine and treatment. Aerobic glycolysis is the main energy metabolism mode of tumor cells. This experiment mainly studies the anti-tumor effect and mechanism of drug ZZZ-1 on the metabolic network of glioma cells. Experimental design: This study was tested by SRB method and colony formation test. The antitumor effect of drug ZZZ-1 on a series of glioma cell lines, and the effects of glucose consumption, pyruvate consumption, lactic acid production, production of intracellular ATP and oxygen consumption ratio (OCR) on the energy metabolism of glioma cells, and further research on the mitochondrial membrane potential of glioma cells, delta m, ROS production, cell senescence, and cell aging Apoptosis and its related proteins, stem cell marker Bmi-1, normal astrocyte marker GFAP and the influence of the key protein PFKFB3, LDH5 expression in glycolysis process. The potential mechanism of ZZZ-1 was studied by oligomycin, a ATP synthetase inhibitor in the process of oxidative phosphorylation. This experiment studied the glioma of ZZZ-1 in animal experiments. Proliferation inhibition, expression of vascular endothelial growth factor VEGF, the influence of key enzyme, stem cell index and differentiation index in glycolysis. Experimental results: 1) ZZZ-1 has a potent anti glioma pharmacodynamic effect: it can significantly inhibit the proliferation of glioma cells in human and rat, inhibit glioma cell colony formation, and have a good dose dependence; 2) induced death and differentiation of glioma cells: inducing glioma cell A m depolarization and ROS overproduction, inhibiting the expression of PCNA, survivin, inducing apoptosis of C6 cells in glioma and senescence of human glioma cell U-87MG cells, inhibiting the expression of stem cell index Bmi-1, promoting the differentiation of gelatinous tumor cells to normal astrocytes; 3) lure. Changes in the energy metabolism of glioma cells: ZZZ-1 treatment of glioma cells 24 h can induce the consumption of glucose and pyruvic acid and the increase of lactic acid, which can significantly increase the amount of intracellular ATP production and oxygen consumption. Drug treatment cells 72 h can reduce the expression of PFKFB3, LDH5, the key protein in glycolysis. Oligomycin can be reduced. Low ZZZ-1 induced proliferation inhibition of glioma cells, depolarization of delta m depolarization and ROS production, reducing glucose consumption and ATP production; ZZZ-1 can reduce the production of compensatory lactic acid induced by oligomycin; 4) inhibit the growth of xenografts in nude mice: ZZZ-1 can selectively inhibit tumor cells in the nude mice model of U-87MG Growth, and the animal weight has no decrease, and has high safety; it can reduce the expression of PFKFB3, LDH5, the key protein in PCNA. survivin and glycolysis; promote the differentiation of glioma cells to normal astrocytes, nerve cells and microglia. Conclusion: the results suggest that ZZZ-1 has a significant and stable resistance to glioma cells. Tumor action can regulate the energy metabolism of glioma cells by multilevel and multiple pathways, induce apoptosis and senescence, and promote the inhibition of glioma cells to normal nerve cells, astrocytes and microglia differentiation. Two small molecule compound CADPE regulates the multiple target of colorectal cancer. Objective: colorectal cancer is a kind of disease that seriously affects human health worldwide. About one million people are diagnosed with colorectal cancer worldwide every year. More than five hundred thousand people died of cancer in the same year. This experiment mainly studied the antitumor effect of caffeic acid 3,4- two hydroxybenzene ethyl ester (CADPE) on colorectal cancer. Possible target targets. Experimental design: This study detected the antitumor efficacy of CADPE on four human colorectal cancer cells by SRB method and colony formation test, and studied the effect of CADPE on the cell cycle, apoptosis, cycle and apoptosis related proteins of colorectal cancer cells. The potential of CADPE to the potential of colorectal cancer cells was studied with four inhibitors of MAPK signaling pathway. The effect of CADPE on proliferation inhibition, apoptosis and cell cycle related proteins in human colon cancer was further studied in this experiment. The effect of CADPE on human normal colon cancer tissue cells was evaluated. Experimental results showed that CADPE could significantly inhibit the proliferation and colony formation ability of human colorectal cancer cells and induce the formation of colorectal cancer cells. Cell cycle arrest and apoptosis in G0/G1 phase of colon cancer cells; the inhibition of CADPE on colorectal cancer is related to the activation of p38 MAPK, p53, p21 and p16 signaling pathways, and the inhibition of nuclear factor kappa B and its downstream target gene signal transducer and transcription activator 3 (STAT3) and c-Myc; CADPE can also down regulate survivin survivin and cell weeks The expression of related proteins (such as cyclin D1, CDK4, CDK6, p-Rb and E2F-1); the inhibitors of P38 MAPK can reduce the proliferation inhibition, cell cycle arrest and apoptosis induced by CADPE, but ERK and JNK inhibitors do not have such effects. In the xenograft model of nude mice, the tumor also inhibits tumor growth and induces apoptosis and decreases. The expression of low PCNA, survivin and cell cycle related proteins (cyclin D1, CDK4, CDK6, p-Rb), and CADPE against normal cells in the range of anti-tumor concentration, has high safety in both in vivo and in vitro experiments. Conclusion: experimental results suggest that CADPE plays an important role in the regulation of the proliferation of colorectal cancer cells. It can regulate the proliferation of colon cancer cells through multitarget regulation, induce cell cycle arrest and induce apoptosis of colon cancer cells.
【学位授予单位】：浙江大学
【学位级别】：博士
【学位授予年份】：2014
【分类号】：R96
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本文编号：2062442