氯硝柳胺对肾上腺皮质癌SW-13细胞的抑制作用和机制研究

发布时间：2018-06-23 16:35

本文选题：氯硝柳胺 + 肾上腺皮质癌　；参考：《第二军医大学》2017年硕士论文

【摘要】：研究背景:肾上腺皮质癌(Adrenocortical carcinoma,ACC)是一种来源于肾上腺皮质的上皮性恶性肿瘤,临床罕见,每年的发病率约为1-2/100万人,发病多见于两个年龄高峰,5岁的儿童和50岁左右的成人,平均发病年龄45岁,女性发病率略高于男性。ACC的病理生理机制仍未能明确,目前认为ACC的病理是一个多因素参与的复杂过程,染色体畸变、基因突变、DNA的甲基化,胰岛素样生长因子(IGF)和Wnt/β-catenin信号通路的功能异常都对ACC的发生发展起重要作用。ACC恶性程度高,多数肿瘤直径5cm,平均10cm,瘤体内多伴有出血及坏死,病程进展迅速且隐匿,大多数患者就诊时已属癌症晚期,伴发有淋巴或远处器官转移,最常见于肺、肝、腹膜后淋巴结和骨转移。ACC的临床表现由肿瘤的功能和体积决定,大多数成人ACC具有内分泌功能,分泌皮质醇和性激素的库欣综合症(Cushing'syndrome,CS)以及男性化在临床中最为常见,约占ACC的40%,单纯CS和男性化表现分别占30%和20%,女性化约占10%,分泌醛固酮的ACC临床中较为少见,约2%。儿童ACC90%以上具有内分泌功能,临床表现多为男性化或假青春期表现,其中以分泌雄激素的肿瘤最为多见,约占儿童ACC的55%以上,混合分泌皮质醇和雄激素的占30%,单纯分泌皮质醇的肿瘤较少见,不足5%。非功能性的ACC因起病隐匿,一般临床表现多以肿瘤进展后引起的腹部症状为主。影像学和内分泌检查是临床诊断ACC的主要手段。目前ACC的治疗由于缺乏特效的化疗和靶向治疗药物,手术仍是其主要的治疗手段,但30%-85%患者就诊时肿瘤已处于进展期,伴发有淋巴或其它脏器转移,无法进行有效的手术切除或根治性手术切除,平均生存期仅为3～9个月,大部分生存时间不超过1年,5年生存率不到15%,即使手术治疗后的患者5年生存率也不足30%。所以对于无法进行手术或无法进行根治性手术的患者仍须考虑药物治疗,或是术后辅以药物治疗来延长期生存时间。ACC药物治疗国外推荐使用米托坦,但其有效性及安全性仍存在争议。EDPM(顺铂、依托泊甙、多柔比星、米托坦)和Sz/m(链霉素、米托担)是米托坦联合细胞毒性药物作为进展期ACC的一线治疗方案,但总的疗效也非常有限。放疗、射频消融、介入栓塞只能使少数患者受益,因此为了有效提高ACC患者的生存率,手术联合药物治疗仍是一项重要的研究课题。由于ACC对传统的化疗药物不敏感,所以寻找有效的、安全的、高选择性药物也是当今研究ACC治疗的重点。氯硝柳胺(Niclosamide),又名灭绦灵,化学名:N-(2'-氯-4'-硝基苯)-5-氯水杨酰胺,分子式C13H8C12N2O4,临床中用来治疗人肠道寄生虫病,是美国食品药品监督管理局(FDA)推荐治疗蠕虫的药物。作用机制通过抑制虫体细胞内氧化磷酸化过程和线粒体功能、阻断NADPH向NADP+的转化、抑制ATP的产生、改变细胞能量代谢发挥治疗作用。近年来,在抗肿瘤药物的的研发过程中,通过大量的药物筛选实验发现氯硝柳胺还具有潜在的抗肿瘤作用,相对于新研究合成的抗肿瘤药物,氯硝柳胺具有细胞毒性强,人体毒副作用小的优势。WANG于2009报道氯硝柳胺可以抑制白血病细胞K562靶基因蛋白的表达,随后其他学者在研究中发现氯硝柳胺可以通过影响 Wnt/β-catenin、mTORC1、STAT3、NF-κB、Notch、Hedgehog 等信号通路和肿瘤细胞内线粒体、溶酶体来产生抑制肿瘤细胞增殖和诱导凋亡的作用,进一步研究发现氯硝柳胺不仅作用于肿瘤细胞,还可以作用于拥有自我更新能力并能产生异质性细胞的肿瘤干细胞。除此之外,在另一些研究中还发现,氯硝柳胺能够增加肿瘤细胞对化疗和放疗的敏感性,甚至可以逆转肿瘤细胞对化疗和放疗的耐受性。氯硝柳胺对正常细胞的能量代谢和信号传导影响甚微,作为潜在的抗肿瘤药物展现出良好的应用前景。研究目的:1.研究氯硝柳胺对人肾上腺皮质癌SW-13细胞株细胞增殖、周期、凋亡和迁移力、侵袭力的影响并初步探讨其机制。2.研究氯硝柳胺对肾上腺皮质癌SW-13细胞裸鼠皮下移植瘤生长的抑制作用和机制。研究方法:1.通过体外培养SW-13细胞,采用CCK-8法和iCELLigence实时无标记细胞增殖监测仪检测不同浓度氯硝柳胺对SW-13细胞增殖的影响。2.采用流式细胞技术检测氯硝柳胺作用SW-13细胞后对细胞周期分布率的影响。3.采用AO/EB染色法和Annexin V/PI染色流式细胞技术检测氯硝柳胺作用SW-13细胞后对细胞凋亡率的影响。4.采用Transwell实验和细胞划痕实验检测氯硝柳胺作用SW-13细胞后对细胞侵袭力、迁移能力的影响。5.采用Western Blotting检测氯硝柳胺作用SW-13细胞后对β-catenin、LRP6、vimentin、E-cadherin 表达的影响。6.建立SW-13细胞裸鼠移植瘤模型,用随机数字表法设对照组、DMSO组,氯硝柳胺低浓度组、氯硝柳胺高浓度组。经腹腔注药,动态监测瘤体直径、瘤重,观察氯硝柳胺作用裸鼠后对肿瘤体积和瘤重的影响,并计算生长抑制率。部分瘤体行HE染色,镜下观察组织形态。7.设顺铂组为阳性对照,各组裸鼠处死前通过眼眶内眦静脉采血,对血液样本进行血细胞、生化分析。8.采用TUNEL法检测氯硝柳胺作用裸鼠后对移植瘤组织细胞凋亡率的影响。9.采用免疫组织化学染色法检测氯硝柳胺作用裸鼠后对移植瘤组织中β-catenin、LRP6、vimentin、E-cadherin、Bc 1-2,caspase-3 表达的影响。结果:1.不同浓度的氯硝柳胺分别作用SW-13细胞24、48、72、96、120h后,呈时间和浓度依赖性抑制SW-13细胞增殖(F=157.93,P0.001)。2.流式细胞仪细胞周期检测结果显示24、48、72h后,与对照组相比氯硝柳胺组将SW-13 细胞周期阻滞在 G0/G1 期(P=0.001、0.005、0.008)。3.AO/EB染色法和Annexin V/PI染色法结果均显示氯硝柳胺组的凋亡率高于照组(均 P0.001)。4.Transwell实验结果示氯硝柳胺组SW-13细胞的侵袭力低于对照组(P0.001),细胞划痕实验结果显示氯硝柳胺组SW-13细胞的迁移能力低于对照组(P=0.002)。5.Western Blotting 结果示氯硝柳胺组 SW-13 细胞 β-catenin、LRP6、vimentin 的表达低于对照组(均P0.001),而E-cadherin的表达高于对照组(P=0.004)。6.HE染色组织切片证实成功建立SW-13细胞裸鼠移植瘤模型,氯硝柳胺低浓度组、氯硝柳胺高浓度组的终末肿瘤体积和瘤重,低于与对照组(均P0.001),对照组与DMSO组差异无统计学意义(均P0.05)。7.顺铂组的白细胞低于氯硝柳胺组、对照组(均P0.001),氯硝柳胺组与对照组比较差异无统计学意义(P0.05)。氯硝柳胺组、顺铂组的红细胞和血小板与对照组之间比较差异无统计学意义(均P0.05)。顺铂组的尿素氮、肌酐、总胆红素、谷丙转氨酶均高于氯硝柳胺组、对照组(均P0.001),氯硝柳胺组与对照之间比较差异无统计学意义(均P0.05)。8.移植瘤组织TUNEL检测结果示氯硝柳胺低浓度组、氯硝柳胺高浓度组的细胞凋亡率高于对照组(P=0.004,P=0.005)。9.移植瘤组织免疫组织化学染色结果显示氯硝柳胺低浓度组、氯硝柳胺高浓度组的β-catenin、LRP6、vimentin、Bcl-2 的表达低于对照组(均 P0.001),E-cadherin、caspase-3的表达高于对照组(均P0.001)。结论:1.氯硝柳胺呈时间-浓度依赖性抑制人肾上腺皮质癌SW-13细胞增殖,阻滞细胞周期,抑制侵袭力和迁移能力,促进细胞凋亡;抑制SW-13细胞裸鼠皮下移植瘤生长,其机制可能涉及Wnt/β-catenin信号通路,上皮间质转化(EMT)和凋亡相关蛋白表达异常。2.氯硝柳胺对血细胞、肝肾功能的影响小于顺铂。
[Abstract]:Background: Adrenocortical carcinoma (ACC) is an epithelial malignant tumor of the adrenal cortex. It is rare in the adrenal cortex. The incidence of the adrenal cortical carcinoma is about 1-2/100 million per year. The incidence of adrenal cortical cancer is about two age peaks, 5 years old and 50 years old, with an average age of 45 years. The incidence of women is slightly higher than that of male.ACC. The pathophysiological mechanism is still not clear. At present, the pathology of ACC is a complex process involving multiple factors. Chromosomal aberration, gene mutation, DNA methylation, insulin like growth factor (IGF) and Wnt/ beta -catenin signaling pathway are important for the development of ACC, and the malignant degree of.ACC is high, and most of the tumor diameter 5cm is 5cm. The average 10cm, with more bleeding and necrosis in the tumor, the course of the disease progresses rapidly and occult. Most patients have advanced cancer, with lymphatic or distant organ metastasis. The most common clinical manifestations of the lung, liver, retroperitoneal lymph nodes and bone metastases are determined by the ability and volume of the tumor, and most of the adult ACC has endocrine function. Cushing'syndrome (Cushing'syndrome, CS), which secretes cortisol and sex hormones, is the most common in the clinic, accounting for about 40% of ACC, 30% and 20% for simple CS and masculine, 10% in feminization, and rare in the ACC secreting aldosterone. About 2%. children have endocrine function above ACC90%, and the clinical manifestations are mostly male. It is the most common manifestation of the androgenic hormone, accounting for more than 55% of the children's ACC, 30% of the mixed secretion of cortisol and androgens, and a rare tumor that secretes cortisol only, and the 5%. nonfunctional ACC is concealed because of the onset of the disease, and the general clinical manifestations are mainly abdominal symptoms after the tumor progresses. Study and endocrine examination are the main means of clinical diagnosis of ACC. Currently, the treatment of ACC is still the main treatment because of the lack of effective chemotherapy and targeted therapy, but the tumor in 30%-85% patients is in progress, with lymph or other organ metastasis, and no effective surgical resection or radical operation can be carried out. The average survival time is only 3~9 months, most of the survival time is not more than 1 years, and the 5 year survival rate is less than 15%. Even if the 5 year survival rate of the patients after the operation is less than 30%., the patients who are unable to perform the operation or do not have a radical operation still have to consider the drug treatment or the postoperative adjuvant treatment to prolong the long-term survival. Inter.ACC drug therapy is recommended for the use of mitoxan, but its effectiveness and safety are still controversial.EDPM (cisplatin, etoposide, doxorubicin, mitoxan) and Sz/m (streptomycin, mitoxin) are the frontline treatments for the combined cytotoxic drugs of mitoxan as advanced ACC, but the total efficacy is very limited. Radiotherapy, radiofrequency ablation, intervention Embolization can only benefit a small number of patients, so in order to effectively improve the survival rate of ACC patients, operation combined with drug therapy is still an important research topic. Because ACC is insensitive to traditional chemotherapeutic drugs, the search for effective, safe and high selective drugs is also the focus of the study of ACC today. Niclosamide, N- (2'- chlorine -4'- nitrobenzene) -5- chlorosalicamide, molecular C13H8C12N2O4, used in the clinical treatment of human intestinal parasitic diseases, is the American food and Drug Administration (FDA) recommended for the treatment of worms. The mechanism of action blocks NADPH to NADP+ by inhibiting the phosphorylation and mitochondrial function of the insect body cells. Transformation, inhibiting the production of ATP and changing cell energy metabolism play a therapeutic role. In recent years, in the process of research and development of antitumor drugs, a large number of drug screening experiments have found that niclosamide also has potential antitumor effects. Compared with the newly synthesized antitumor drugs, niclosamide has a strong cytotoxicity and human toxicity. Using small advantage.WANG in 2009 reports that niclosamide inhibits the expression of K562 target gene protein in leukemia cells, other scholars have found that niclosamide can inhibit tumor cells by affecting Wnt/ beta -catenin, mTORC1, STAT3, NF- kappa B, Notch, Hedgehog and other signaling pathways and tumor cell mitochondria, lysosomes Further studies have shown that niclosamide not only acts on tumor cells but also acts on tumor stem cells that have self renewal capacity and can produce heterogeneous cells. In addition, in other studies, niclosamide can increase the sensitivity of tumor cells to chemotherapy and radiotherapy, even in some other studies. It can reverse the tolerance of tumor cells to chemotherapy and radiotherapy. The effect of niclosamide on the energy metabolism and signal transduction of normal cells is very little. As a potential antitumor drug, it can be used as a potential antitumor drug. Objective: 1. to study the proliferation, cycle, apoptosis and migration of niclosamide on human adrenocortical carcinoma SW-13 cell lines. The effect of attack and preliminary study on its mechanism.2. to study the inhibitory effect and mechanism of niclosamide on the growth of subcutaneous xenografts in nude mice of adrenocortical cancer SW-13 cells. 1. through the culture of SW-13 cells in vitro, CCK-8 and iCELLigence real-time unmarked cell proliferation monitoring apparatus were used to detect the effect of different concentrations of niclosamide on SW-13 cells Effects of proliferation on.2. using flow cytometry to detect the cell cycle distribution of niclosamide using SW-13 cells.3. AO/EB staining and Annexin V/PI staining flow cytometry were used to detect the effect of niclosamide on the apoptosis rate of SW-13 cells..4. using Transwell test and cell scratch test to detect clonite Effect of salamine on SW-13 cells on cell invasiveness and mobility,.5. using Western Blotting to detect the effects of nitramine on SW-13 cells on the expression of beta -catenin, LRP6, vimentin and E-cadherin.6. to establish a SW-13 cell xenografts in nude mice. The random number table method was used to set up the control group, DMSO group, nitramine low concentration group and clonite. In the Liu Angao concentration group, the diameter of the tumor and the weight of the tumor were monitored dynamically by intraperitoneal injection. The effects of nude mice on tumor volume and weight were observed and the growth inhibition rate was calculated. Part of the tumor body was stained with HE, and the tissue morphology.7. was observed in the group of cisplatin as positive control. Blood cells, biochemical analysis of.8., TUNEL method was used to detect the effect of niclosamide on nude mice. The effect of.9. on the expression of beta -catenin, LRP6, vimentin, E-cadherin, Bc 1-2 and Caspase-3 in transplanted tumor tissues by immunohistochemical staining. Results: 1. After the same concentration of niclosamide acted on SW-13 cell 24,48,72,96120h respectively, the time and concentration dependent inhibition of SW-13 cell proliferation (F=157.93, P0.001).2. flow cytometry showed 24,48,72h, and the nitramine group blocked the SW-13 cell cycle in G0/G1 phase (P=0.001,0.005,0.008).3.AO/EB, compared with the control group. The results of staining and Annexin V/PI staining showed that the apoptosis rate of the niclosamide group was higher than that of the irradiated group (P0.001).4.Transwell experimental results showed that the invasiveness of SW-13 cells in the niclosamide group was lower than that of the control group (P0.001). The results of cell scratch test showed that the migration ability of SW-13 cells in the niclosamide group was lower than that of the control group (P=0.002).5.Western Blott. The results of ing showed that the expression of beta -catenin, LRP6 and vimentin in the SW-13 cells of the niclosamide group was lower than that of the control group (P0.001), while the expression of E-cadherin was higher than that of the control group (P=0.004).6.HE staining tissue section confirmed the successful establishment of the tumor model of nude mice in SW-13 cells, the low concentration group of niclosamide, the final tumor volume and tumor of the high concentration group of niclosamide. Weight, lower than the control group (all P0.001), the control group and the DMSO group had no statistically significant difference (all P0.05) the white cells in the.7. cisplatin group were lower than the niclosamide group, the control group (P0.001), the niclosamide group and the control group had no significant difference (P0.05). The difference between the red cells and the platelets in the niclosamide group and the cisplatin group was different from the control group. No statistical significance (all P0.05). The urea nitrogen, creatinine, total bilirubin and alanine transaminase in the cisplatin group were higher than the niclosamide group and the control group (P0.001). There was no significant difference between the niclosamide group and the control group (all P0.05) the TUNEL test results of.8. transplanted tumor showed the low concentration group of niclosamide, the cells of the high concentration group of niclosamide The apoptosis rate was higher than that of the control group (P=0.004, P=0.005).9. transplantation tumor tissue immunohistochemical staining results showed the low concentration group of niclosamide, the expression of beta -catenin, LRP6, vimentin, Bcl-2 in the high concentration group of niclosamide was lower than that of the control group (P0.001), E-cadherin, caspase-3 expression was higher than that of the control group (P0.001). Conclusion: 1. niclosamide was present. The proliferation of SW-13 cells in human adrenocortical cancer cells, inhibition of cell cycle, inhibition of invasion and migration, and apoptosis, and the inhibition of the growth of subcutaneous xenografts in nude mice of SW-13 cells may involve the Wnt/ beta -catenin signaling pathway, epithelial mesenchymal transition (EMT) and apoptosis related protein expression of.2. niclosamide The effect on blood cells, liver and kidney function is less than cisplatin.
【学位授予单位】：第二军医大学
【学位级别】：硕士
【学位授予年份】：2017
【分类号】：R736.6

【参考文献】