Tirapazamine与拓扑异构酶Ⅰ抑制剂在人肝癌中合用的药效学及机制研究

发布时间：2018-09-09 20:07

【摘要】：研究目的：肝细胞癌(HCC)是全球癌症中致死率较高的一种恶性肿瘤,在中国HCC的发病率呈逐年递增的趋势。目前临床上用于治疗肝癌的药物主要有索拉菲尼、舒尼替尼、贝伐单抗、西罗莫司等,但这些药物单独治疗晚期肝癌的效果依然十分有限,新的药物治疗策略亟待开发。拓扑异构酶Ⅰ抑制剂是一类临床具有广谱抗肿瘤作用的药物,在体外具有非常强的抗肝癌活性,但其在肝癌临床治疗中疗效有限。目前对引起体内外之间巨大差异的机制还尚不清楚。肝癌微环境中氧压较其他实体瘤更低,本研究结果表明低氧环境下HIF-1α的高表达是引起人肝癌对拓扑异构酶Ⅰ抑制剂敏感性降低的重要因素之一。Tirapazamine (TPZ)是一种低氧选择性药物,课题组前期实验成果发现其在低氧下可抑制HIF-1α蛋白的合成。本文将研究TPZ和拓扑异构酶Ⅰ抑制剂合用在体内外人肝癌中的抗肿瘤活性,并探讨其合用的具体机制。研究方法：(1)SRB法检测在5种不同的人肝癌细胞株和1种正常的人肝细胞中TPZ和四种拓扑异构酶Ⅰ抑制剂(SN-38、TPT、HCPT、MONCPT)联合用药抑制人肿瘤细胞增殖的能力,并计算了合用指数(CI)。(2)利用shRNA技术沉默HIF-1α基因表达。(3) Western Blotting法检测体内外相关蛋白的表达情况。(4)双荧光素酶报告基因技术检测HIF-1α转录因子的转录活性。(5)克隆形成实验检测TPZ和四种拓扑异构酶Ⅰ抑制剂联合用药的抗肝癌活性。(6)建立人源性肿瘤裸小鼠Bel-7402移植瘤模型,检测TPZ和CPT-11(SN-38前药)合用对裸小鼠移植瘤的实验治疗作用。(7) DAPI染色法检测给药前后人肝癌细胞中凋亡小体的产生情况。(8)PI单染或Annexin-V/PI双染结合流式细胞术来检测细胞凋亡的发生。(9)利用JC-1染色法检测线粒体膜电位的变化情况。(10)免疫荧光法检测蛋白在细胞或组织内的分布和表达情况。(11)荧光实时定量PCR法检测mRNA的表达。(12)免疫组化法检测瘤组织切片中蛋白的分布及表达情况。(13)利用质粒转染技术扩增细胞内HIF-1α蛋白的表达。研究结果：(1) Tirapazamine与拓扑异构酶Ⅰ抑制剂在人肝癌中合用的药效学研究：实验结果发现在人肝癌中存在拓扑异构酶Ⅰ抑制剂低氧敏感性降低的现象,而HIF-1α在其中起了关键的作用。TPZ在人肝癌细胞中也可抑制低氧下HIF-1α蛋白的累积。在多株人肝癌细胞中(HepG-2、Bel-7402、Hep3B、Huh7和SMMC-7721)TPZ与拓扑异构酶Ⅰ抑制剂都可协同抑制肿瘤细胞的增殖,合用指数均在0.7以下。而在正常的人肝细胞中,两者合用并未产生协同作用。同时,TPZ与CPT-11合用在人肝癌Bel-7402移植瘤裸小鼠模型上也表现出显著的协同实验治疗作用,且未引起动物体重显著下降。 (2) Tirapazamine与拓扑异构酶Ⅰ抑制剂在人肝癌中发挥协同作用的机制研究：在人肝癌中TPZ与SN-38(CPT-11的主要活性代谢产物)合用可协同诱导细胞产生凋亡,两者通过降低线粒体的膜电位,激活Caspase-3的裂解,引起PARP的切割,最终诱导人肝癌Bel-7402细胞发生凋亡。TPZ与SN-38合用能够协同降低HIF-1α蛋白的合成速率,抑制HIF-1α蛋白的表达水平和转录活性。而HIF-1α表达水平的下调会导致DNA损伤修复水平的降低,引起细胞内DNA损伤的大量累积。RAD51、Chk1磷酸化水平的降低提示RAD51介导的同源重组修复通路在这过程中扮演了重要的角色。结论：(1)HIF-1α是介导拓扑异构酶Ⅰ抑制剂在人肝癌中低氧敏感性降低的关键因素。TPZ与拓扑异构酶Ⅰ抑制剂合用可诱导肝癌细胞发生凋亡,抑制体内外肿瘤的增殖,发挥协同抗肿瘤作用。 (2)在TPZ与SN-38合用过程中HIF-1α蛋白的降低是关键因素,其产生协同作用的机制是：通过协同抑制HIF-1α的蛋白合成途径,引起HIF-1α蛋白表达和转录活性的下调,抑制了Rad51介导的同源重组修复通路,导致药物引起的DNA损伤在细胞内大量累积,最终诱导细胞走向凋亡。
[Abstract]:Objective: Hepatocellular carcinoma (HCC) is one of the most lethal cancers in the world. The incidence of HCC in China is increasing year by year. At present, the main drugs used to treat hepatocellular carcinoma are sorafenib, sunitinib, bevacizumab, sirolimus and so on. Topoisomerase I inhibitors are a class of drugs with broad-spectrum anti-tumor effect in clinic and have very strong anti-hepatoma activity in vitro. However, their efficacy in clinical treatment of hepatocellular carcinoma is limited. Tirapazamine (TPZ) is a hypoxic selective drug. Previous studies have shown that it can inhibit the synthesis of HIF-1a protein under hypoxia. In this paper, the antitumor activity of TPZ and topoisomerase I inhibitors in human hepatocellular carcinoma in vitro and in vivo was studied, and the specific mechanism of their combination was discussed.
Methods: (1) SRB assay was used to detect the ability of TPZ and four topoisomerase I inhibitors (SN-38, TPT, HCPT, MONCPT) to inhibit the proliferation of human hepatoma cells in 5 different human hepatoma cell lines and 1 normal human hepatocytes, and the co-use index (CI) was calculated. (2) HIF-1a gene expression was silenced by shRNA technique. (3) Western Blotti. The expression of related proteins in vivo and in vitro was detected by ng method. (4) The transcriptional activity of HIF-1 alpha transcription factor was detected by double luciferase reporter gene technique. (5) The anti-hepatoma activity of TPZ combined with four topoisomerase I inhibitors was detected by cloning formation assay. (6) The human tumor model of Bel-7402 transplanted in nude mice was established, and TPZ and CPT-11 (S) were detected. (7) DAPI staining was used to detect the production of apoptotic bodies in human hepatoma cells before and after administration. (8) PI single staining or Annexin-V/PI double staining combined with flow cytometry was used to detect the occurrence of apoptosis. (9) JC-1 staining was used to detect the changes of mitochondrial membrane potential. (10) Immunoassay. Fluorescence assay was used to detect the distribution and expression of proteins in cells or tissues. (11) Real-time quantitative PCR was used to detect the expression of mRNA. (12) Immunohistochemistry was used to detect the distribution and expression of proteins in tumor tissues. (13) The expression of HIF-1a protein in cells was amplified by plasmid transfection.
Results: (1) Pharmacodynamics of Tirapazamine and Topoisomerase I Inhibitor in Human Hepatocellular Carcinoma: The results showed that the hypoxia sensitivity of topoisomerase I inhibitor was decreased in human hepatocellular carcinoma, and HIF-1a played a key role in it. TPZ could also inhibit HIF-1a protein in human hepatocellular carcinoma cells under hypoxia. TPZ and topoisomerase I inhibitors in several human hepatoma cells (HepG-2, Bel-7402, Hep3B, Huh 7 and SMMC-7721) could synergistically inhibit the proliferation of tumor cells with a combined index below 0.7. In normal human hepatocytes, the combination of TPZ and CPT-11 did not produce synergistic effects. At the same time, TPZ and CPT-11 were combined in human hepatoma Bel-7402 transplantation. The tumor nude mice model also showed significant synergistic experimental effect, and did not cause significant weight loss.
(2) the synergistic effect of Tirapazamine and topoisomerase I inhibitor in human hepatocellular carcinoma.
TPZ combined with SN-38 (the main active metabolite of CPT-11) can induce apoptosis in human hepatocellular carcinoma cells. TPZ and SN-38 can induce apoptosis of human hepatocellular carcinoma Bel-7402 cells by lowering the membrane potential of mitochondria, activating the cleavage of Caspase-3 and inducing the cleavage of PARP. Inhibition of HIF-1a protein expression and transcriptional activity. The down-regulation of HIF-1a expression may result in a decrease in DNA damage repair and accumulation of DNA damage in cells. The decrease of RAD51 and Chk1 phosphorylation suggests that RAD51-mediated homologous recombinant repair pathway plays an important role in this process. TPZ combined with topoisomerase I inhibitor can induce apoptosis of hepatocellular carcinoma cells, inhibit tumor proliferation in vivo and in vitro, and play a synergistic anti-tumor role.
(2) The decrease of HIF-1a protein is a key factor in the combination of TPZ and SN-38. The synergistic mechanism of HIF-1a protein synthesis is that HIF-1a protein expression and transcriptional activity are down-regulated by synergistic inhibition of HIF-1a protein synthesis pathway, which inhibits Rad51-mediated homologous recombinant repair pathway and leads to a large number of DNA damage in cells. Accumulation eventually induces cell apoptosis.
【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】：R735.7;R96

【共引文献】