亚叶酸钙增强硼替佐米在结直肠癌的抗癌效应及分子机制研究

发布时间：2018-05-12 15:27

本文选题：硼替佐米 + 亚叶酸钙　；参考：《吉林大学》2017年博士论文

【摘要】：背景:结直肠癌(CRC)是一种常见的严重威胁人类健康的恶性肿瘤。在美国,结直肠癌的发病率占所有癌症的第四位,死亡率的第二位。其发病率随年龄增长而显着增加,男性高于女性,与生活方式密切相关。近年来,西方一些国家结直肠癌的发病率和死亡率逐步降低,而我国结直肠癌的发病率逐年升高且呈年轻化趋势。结直肠癌是常见的好发于直肠与乙状结肠交界处的消化道恶性肿瘤。CRC的治疗取决于肿瘤部位和疾病阶段,手术治疗是首选方案,配合辅助放、化疗。单纯手术治疗预后不佳,术后约半数肿瘤出现复发、局部浸润和远处转移。近年来,分子靶向药物成为治疗癌症的有效手段,与传统放化疗相比,分子靶向治疗优势明显。靶向药物针对靶向肿瘤细胞,对正常组织影响小,可减轻毒副反应、提高治疗效率。目前,CRC的辅助化疗中仍以5-氟尿嘧啶为主的化疗方案,如FOLFOX和FOLFIRI,然而对5-氟尿嘧啶的耐药性成为了化疗失败的主要原因。同时,大多数接受静脉注射5-氟尿嘧啶的患者出现了明显的副作用。亚叶酸钙,本身并没有抗肿瘤作用,临床多用于5-氟尿嘧啶的增敏剂,由于其类似维生素样功能,几乎没有副作用。因此,临床治疗亟需一种针对于CRC的新型治疗剂或新的化疗组合方案。目的:1进行新的药物方案重组筛选,以FDA批准的抗肿瘤化合物与亚叶酸钙联用,研究对CRC细胞是否具有协同细胞杀伤作用。2研究硼替佐米和亚叶酸钙对结直肠癌细胞HCT116和HT29恶性生物学行为的影响。3探讨硼替佐米和亚叶酸钙诱导细胞凋亡、激活DNA损伤途径的抗癌效应及相关分子机制。方法:1.硼替佐米(0,3,10,30,100和300n M)和/或亚叶酸钙(10μM)处理结肠癌HCT116和HT29细胞24或48小时,采用MTT法检测细胞的存活率;2.Cell Titer-Glo法检测时间梯度和不同浓度硼替佐米联用亚叶酸钙的细胞存活率;3.流式细胞术测经硼替佐米(3或10n M)和/或亚叶酸钙(10μM)处理后HCT116和HT29的细胞凋亡及细胞周期变化;4.Western blot技术检测caspase家族,PARP,Bcl-2,p53,p-p53,H2AX,PUMA,Bax,p21,p-JNK,p-ERK1/2(Thr202/Tyr204),p-Akt和p-Bcl-x L的蛋白表达。5.动物模型:6周领雌性免疫缺陷NSG小鼠皮下注射HCT116细胞,建立人类结直肠癌异种移植肿瘤,通过每周两次腹膜内注射施用治疗。将20只小鼠随机分成四个治疗组(每组n=5)。给予亚叶酸钙和/或硼替佐米作治疗组,PBS用作空白载体对照。在处理16天后,处死小鼠并评估肿瘤指标;6.TUNEL法测定小鼠异种移植肿瘤组织切片并染色,观察两药联用对细胞凋亡的影响。结果:硼替佐米和亚叶酸钙联用可以通过一种涉及caspase依赖性细胞凋亡的机制有效地诱导CRC细胞的杀伤。硼替佐米联用亚叶酸钙可诱导CRC细胞周期G2/M期的阻滞。蛋白质印迹法结果表明两药联用可以强烈诱导剪切体PARP,caspase-3,-8和-9的表达,并下调抗凋亡因子BCL-2的表达,伴随DNA损伤信号通路和JNK信号通路的激活及ERK、Akt通路活化的抑制。体内实验进一步证明硼替佐米和亚叶酸钙可有效诱导细胞凋亡,抑制免疫缺陷小鼠在CRC异种移植肿瘤的生长,两药联用在CRC细胞体外协同作用也在体内实现。这些数据表明凋亡可能是两种药物联用抑制CRC细胞生长和肿瘤发生的主要下游机制,伴随细胞周期进程的抑制、DNA损伤反应和其他途径的激活。结论:本研究证实美国FDA批准的119种抗肿瘤药物中有13种化合物与亚叶酸钙联用对CRC细胞具有协同抗肿瘤效应。在CRC细胞中硼替佐米和亚叶酸钙的联用组合相比单独任一药剂可增加25%以上的细胞凋亡。硼替佐米联用亚叶酸钙协同诱导抗肿瘤效应是通过介导半胱天冬酶活化的增加及DNA损伤途径的活化,伴随细胞周期进程阻滞和其他途径的激活。小鼠CRC异种移植肿瘤中也观察到凋亡的协同诱导和肿瘤生长的抑制。这些数据表明硼替佐米联用亚叶酸钙可能成为的一种新型治疗CRC的辅助化疗方案,值得在临床进一步推广应用。
[Abstract]:Background: colorectal cancer (CRC) is a common malignant tumor that is a common serious threat to human health. In the United States, colorectal cancer accounts for fourth of all cancers, second of the mortality rates. The incidence of colorectal cancer increases with age, men are higher than women and closely related to lifestyle. In recent years, colorectal cancer in some western countries The incidence and mortality rate gradually decrease, and the incidence of colorectal cancer in China is increasing year by year and the trend is younger. The treatment of colorectal cancer is a common treatment of digestive tract malignant tumor.CRC, which is located at the junction of rectum and sigmoid colon, which depends on the tumor location and stage of disease. Surgical treatment is the first choice, combined with adjuvant radiotherapy, chemotherapy and simple hand. In recent years, molecular targeted drugs have become an effective means to treat cancer. Compared with traditional chemoradiotherapy, molecular targeting therapy has obvious advantages. The target drug targeting the tumor cells, which has little influence on the normal tissue, can reduce the toxic and side effects and improve the treatment. Efficiency. At present, CRC's adjuvant chemotherapy is still based on 5- fluorouracil, such as FOLFOX and FOLFIRI. However, the resistance to 5- fluorouracil is the main cause of chemotherapy failure. At the same time, most patients receiving intravenous injection of 5- fluorouracil have obvious side effects. Calcium folate itself has no anti tumor effect. Clinical use of the sensitizer of 5- fluorouracil has almost no side effects because of its similar vitamin like function. Therefore, a new therapeutic agent or a new combination chemotherapy regimen for CRC is urgently needed in clinical treatment. Objective: 1 a new drug regimen is screened for the combination of FDA approved antitumor compounds with calcium folate, and the study of CRC Whether cells have synergistic cytotoxicity.2 to study the effects of bortel Zomi and calcium folate on the malignant biological behavior of HCT116 and HT29 in colorectal cancer cells.3 explore the apoptosis induced by bortezen and calcium folate and activate the anticancer effect of DNA damage pathway and related molecular mechanisms. Methods: 1. bortel Zomi (0,3,10,30100 and 300N M) And / or calcium folate (10 M) treatment of colon cancer HCT116 and HT29 cells for 24 or 48 hours, MTT method was used to detect the cell survival rate; 2.Cell Titer-Glo method was used to detect the time gradient and the cell survival rate of different concentrations of bortezomib combined with calcium folate; 3. flow cytometry was treated with bortezoso (3 or 10N M) and / or calcium folate (10 u M) treated HCT116. Caspase family, PARP, Bcl-2, p53, Bcl-2, p53, p-p53, H2AX, PUMA, PARP, and protein expression animal model: 6 weeks of 6 weeks leading female immune deficiency mice subcutaneous injection of cells to establish human colorectal cancer xenotransplantation The tumor was treated by intraperitoneal injection of two times a week. 20 mice were randomly divided into four treatment groups (each group of n=5). Calcium folate and / or bortezomib were given as the treatment group. PBS was used as a blank vector control. After 16 days of treatment, the mice were killed and the tumor markers were evaluated. 6.TUNEL method was used to determine the tumor tissue section and stain in the xenotransplantation of mice. The effect of combined use of two drugs on apoptosis. Results: bortezomizomi and calcium folate can effectively induce the killing of CRC cells through a mechanism involving caspase dependent apoptosis. Bortezomizomi combined with calcium folate can induce CRC cell cycle G2/M phase block. The results of protein imprinting showed that the combination of two drugs could be stronger. Strongly induced expression of PARP, Caspase-3, -8 and -9, and down regulation of the expression of anti apoptotic factor BCL-2, accompanied by the activation of DNA damage signal pathway and JNK signaling pathway, and the inhibition of ERK and Akt pathway activation. In vivo experiments further demonstrated that bortezomizomi and folate can effectively induce apoptosis and inhibit the xenotransplantation of immunodeficiency mice in CRC. The growth of the tumor, the synergistic effect of the two drugs combined with the CRC cells in vitro is also realized in the body. These data suggest that apoptosis may be the main downstream mechanism of the two drugs in combination with the inhibition of CRC cell growth and tumorigenesis, with the inhibition of the cell cycle process, the DNA damage response and the activation of other pathways. Conclusion: This study confirmed the approval of American FDA. 13 of the 119 antitumor drugs have synergistic antitumor effects on CRC cells combined with calcium folate. In CRC cells, the combination of bortezomizomias and folate can increase more than 25% of apoptosis compared with a single agent. The antitumor effect of bortezomizomib combined with calcium folate is mediated by mediating cysteine The increase of aspartic enzyme activation and the activation of DNA damage pathway, accompanied by cell cycle progression and activation of other pathways. The synergistic induction of apoptosis and inhibition of tumor growth are also observed in mouse CRC xenotransplantation tumors. These data indicate that bortezomizomib may be a new adjuvant chemotherapy for CRC with calcium folate calcium. The case is worth further popularization and application in clinical practice.

【学位授予单位】：吉林大学
【学位级别】：博士
【学位授予年份】：2017
【分类号】：R735.34

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