高通量药物筛选识别秋水仙碱作为甲状腺癌新型抑制剂的研究

发布时间：2018-08-12 12:42

【摘要】：背景与目的:甲状腺癌已成为常见的、多发性疾病。其中甲状腺乳头状癌是发病率最高的一种,占所有类型甲状腺癌的85-90%。BRAFV600E基因突变是BRAF基因最常见的一种突变,可改变癌细胞生物学状态,加速了恶性肿瘤的进展期,在甲状腺癌、结肠癌以及黑色素瘤等恶性肿瘤中发生率较高,是目前新型的肿瘤标志物,提示临床预后较差。而BRAFV600E基因突变在甲状腺乳头状癌中发生率最高,在其它类型的甲状腺癌也已发现,如甲状腺未分化癌。虽然目前临床上治疗甲状腺乳头状癌的主要方式为手术切除,内分泌治疗和放射性碘治疗,然而对于侵袭性甲状腺癌,放射性碘治疗耐受的患者,这些传统治疗方式的预后效果仍然不理想。虽然针对BRAFV600E基因突变的靶向药物在临床前期和临床治疗中已经应用,然而经过临床观察BRAFV600E抑制剂例如PLX4032(vemurafenib)在治疗BRAFV600E基因突变的黑色素瘤时展示了强效的抗肿瘤特性,而对BRAFV600E基因突变的甲状腺癌的治疗中,效果一般。近年来研究发现BRAFV600E抑制剂可激活BRAFV600E基因突变的结肠癌细胞中EGFR磷酸化高表达,从而导致其对BRAF抑制剂产生耐药性,目前已证实EGFR抑制剂和BRAF抑制剂联合应用治疗可产生很好的协同作用。然而由于EGFR抑制剂和BRAF抑制剂价格昂贵,以及其自身毒副作用,极大的限制其在临床应用的可行性,因此寻找新型的治疗甲状腺癌细胞的药物代替BRAFV600E抑制剂,研究其生物学活性和抗肿瘤的机制,为其在今后临床应用打下基础。方法:为了寻找能抑制甲状腺癌细胞活性的小分子化合物,我们采用高通量药物筛选方法,将2种含有BRAFV600E基因突变的人甲状腺癌细胞株8505C和KTC-1作为筛选对象,而将另外1种非甲状腺癌并具有BRAFV600E基因突变的人黑色素瘤细胞株Malme-3M做为参照。筛选5种药库共包含了约5200种小分子化合物,对筛选出的候选药物药物秋水仙碱进行重新定位及其机理的研究。通过Alamar Blue染色法检测细胞活力,细胞计数方法检测其细胞增殖能力,通过流式细胞术测定细胞周期确定药物的生物学特性;通过westernblot方法和Annexin V-FITC与PI双染色结合流式细胞术测定细胞凋亡;通过Westernblot方法和建立稳定秋水仙碱耐药细胞克隆的方法寻找其诱导凋亡的机制。建立小鼠甲状腺癌细胞(8505C和WRO)接种模型,确定秋水仙碱在体内动物试验的有效性,以及其在小鼠体内有无毒副作用。结果:高通量药物筛选成功发现了74个候选药物,将候选药物之一AV-412与针对Malme-3M的药物PLX4032进行验证试验,结果与高通量筛选实验相符,证实该系统的可靠性及可信性。秋水仙碱作为候选药物之一作为本次课题主要研究对象。通过Alamar Blue染色实验和细胞计数实验,秋水仙碱可明显抑制BRAFV600E(8505C、KTC-1)和BRAFWT(WRO、TPC-1)甲状腺癌细胞活力和细胞增殖能力,流式细胞术测定发现秋水仙碱可诱导8505C和WRO细胞阻滞在G2/M期,而进入G1期细胞明显减少。Annexin V-FITC与PI双染检测秋水仙碱诱导8505C和WRO细胞凋亡,与浓度和时间相关。蛋白免疫印迹试验表明,在8505C细胞中,秋水仙碱以浓度依赖和时间依赖的方式诱导PARP切割形成凋亡片段,并同时激活AKT,MEK/ERK,P38和JNK/c-Jun通路。然而通过应用选择性抑制剂U0126和SP600125分别对MEK1/2和JNK通路进行抑制,干扰了秋水仙碱诱导凋亡的能力,导致其细胞活力增高,同时激活Caspase3和诱导PARP形成切割片段的能力明显降低,凋亡细胞数显著减少。相反,SB203580和LY294002分别抑制p38 MAPK和AKT通路后,对秋水仙碱诱导细胞凋亡作用无影响。在WRO细胞中,秋水仙碱以浓度依赖和时间依赖的方式诱导PARP切割形成凋亡片段,MEK,P38和JNK/c-Jun的磷酸化水平明显上调。AKT磷酸化在秋水仙碱处理24小时表达下调,而在48和72小时无明显变化,ERK也无明显变化,通过应用选择性抑制剂U0126和SP600125分别对MEK1/2和JNK进行抑制,干扰了秋水仙碱诱导凋亡的能力,导致其细胞活力增高,Caspase 3和PARP形成切割片段的能力明显降低,凋亡细胞数量显著减少。SB203580抑制p38 MAPK通路后,只轻度干扰了秋水仙碱诱导凋亡的能力。而LY294002抑制AKT通路后,对秋水仙碱诱导细胞凋亡作用无明显影响。通过建立8505C秋水仙碱耐药细胞株和蛋白免疫印迹检测表明,MEK1/2,ERK1/2和JNK的磷酸化水平在亲代细胞中表达明显增高,而在耐药细胞R2和R4中几乎未见激活。不同的是p38的磷酸化在耐药细胞株中表达仍增高,与亲代细胞相似。通过建立小鼠移植瘤8505C和WRO模型,每日注射不同剂量的秋水仙碱,共2周,发现治疗组的肿瘤体积和肿瘤重量较对照组明显降低,并具有浓度和时间依赖性;通过免疫组化PHH3染色发现经秋水仙碱治疗后,有丝分裂的细胞数较对照组相比明显减少,具有浓度依赖性。通过TUNEL染色发现治疗组的肿瘤凋亡细胞与对照组相比明显增多,具有浓度依赖性。同时,对小鼠重要脏器进行HE染色镜下观察,秋水仙碱治疗组与对照组均无组织损伤和毒性作用;体重监测也表明秋水仙碱在小鼠动物模型试验的安全性及耐受性。结论:1、高通量药物筛选作为一种有效方法对药物的生物学活性进行重新定性提供了新的平台。高通量筛选方法成功筛选出了热点药物秋水仙碱,并为甲状腺癌药物治疗的选择提供了新的思路和方法。2、秋水仙碱不仅能对具有BRAFV600E基因突变的甲状腺癌有明显的抑制作用,而且对其他BRAF野生型的甲状腺癌同样有强效的抗肿瘤的功能。3、秋水仙碱通过对细胞周期G2/M阻滞和诱导凋亡抑制了多种甲状腺癌细胞的生长,具有浓度和时间相关性。4、秋水仙碱通过激活MEK/ERK和JNK/c-Jun磷酸化途径从而诱导细胞凋亡,p38的磷酸化表达仅部分的导致了WRO细胞的凋亡,而在8505C细胞中无明显的作用。AKT磷酸化表达并未参与细胞凋亡的过程。5、秋水仙碱在抑制甲状腺癌8505C和WRO小鼠移植瘤的生长展现了良好的抗肿瘤效果,而且对动物模型无明显的毒副作用。同时秋水仙碱作为一种已知常见的广泛治疗痛风的药物,值得在其他临床领域例如甲状腺癌中进一步关注和研发。
[Abstract]:BACKGROUND & OBJECTIVE: Thyroid carcinoma has become a common and multiple disease. Papillary thyroid carcinoma is the most common type of thyroid cancer, accounting for 85-90% of all types of thyroid cancer. Colorectal cancer, melanoma and other malignant tumors have a high incidence and are new tumor markers, suggesting a poor clinical prognosis. BRAFV600E mutation has the highest incidence in papillary thyroid cancer and has been found in other types of thyroid cancer, such as undifferentiated thyroid cancer. Although the current clinical treatment of papillary thyroid cancer. Surgical resection, endocrine therapy, and radioiodine therapy are the main modalities of carcinomas. However, the prognosis of these traditional therapies is still unsatisfactory for patients with invasive thyroid cancer who are tolerant to radioiodine therapy. BRAFV600E inhibitors such as PLX4032 (vemurafenib) have shown potent antitumor properties in the treatment of melanoma with BRAFV600E gene mutation, while BRAFV600E gene mutation has been shown to be effective in thyroid cancer. Recent studies have shown that BRAFV600E inhibitors can activate BRAFV600E gene mutation in colorectal cancer cells. High phosphorylation of EGFR leads to resistance to BRAF inhibitors. It has been proved that EGFR inhibitors combined with BRAF inhibitors can produce a good synergistic effect. However, the high price of EGFR inhibitors and BRAF inhibitors, as well as their own toxic and side effects, greatly limit the feasibility of clinical application. To find a new drug to replace BRAFV600E inhibitor and study its biological activity and anti-tumor mechanism, so as to lay a foundation for its clinical application in the future.Methods: In order to find a small molecule compound which can inhibit the activity of thyroid cancer cells, we used high-throughput drug screening method to select two kinds of drugs containing BRAFV600E. Human thyroid cancer cell lines 8505C and KTC-1 were selected as the screening targets, while another non-thyroid cancer cell line Malme-3M with BRAFV600E gene mutation was used as the reference. Cell viability was detected by Alamar Blue staining, cell proliferation was detected by cell counting, and the biological characteristics of the drug were determined by flow cytometry; apoptosis was detected by Western blot, Annexin V-FITC and PI double staining combined with flow cytometry; and apoptosis was detected by Western blot. Methods The mechanism of apoptosis induced by colchicine-resistant cells was investigated by establishing stable clone of colchicine-resistant cells. The inoculation model of mouse thyroid cancer cells (8505C and WRO) was established to determine the validity of colchicine in vivo animal test and its toxicity in mice. AV-412, one of the candidate drugs, was validated with PLX4032 for Malme-3M. The results were in good agreement with high throughput screening experiments, which confirmed the reliability and reliability of the system. Colchicine as one of the candidate drugs was the main research object of this project. Colchicine was cocoa through Alamar Blue staining experiment and cell counting experiment. The viability and proliferation of BRAFV600E (8505C, KTC-1) and BRAFWT (WRO, TPC-1) thyroid cancer cells were significantly inhibited by colchicine. Flow cytometry showed that colchicine could induce 8505C and WRO cells to block at G2/M phase, but the number of cells entering G1 phase was significantly decreased. The apoptosis of 8505C and WRO cells induced by colchicine was detected by Annexin V-FITC and PI staining. In 8505C cells, colchicine induced PARP cleavage to form apoptotic fragments in a concentration-and time-dependent manner, and activated AKT, MEK/ERK, P38 and JNK/c-Jun pathways simultaneously. However, selective inhibitors U0126 and SP600125 were used to inhibit MEK1/2 and JNK pathways respectively, interfering with them. In contrast, SB203580 and LY294002 inhibited p38 MAPK and AKT pathways respectively, but had no effect on colchicine-induced apoptosis in WRO cells. The phosphorylation levels of MEK, P38 and JNK/c-Jun were significantly up-regulated. AKT phosphorylation was down-regulated after colchicine treatment for 24 hours, but did not change significantly at 48 and 72 hours, and ERK did not change significantly. The selective inhibitors U0126 and SP600125 were used to treat MEK, respectively. 1/2 and JNK inhibited the ability of colchicine to induce apoptosis, resulting in increased cell viability, decreased the ability of caspase 3 and PARP to form cleavage fragments, and decreased the number of apoptotic cells. SB203580 inhibited the p38 MAPK pathway, only slightly interfered with the ability of colchicine to induce apoptosis. The phosphorylation levels of MEK1/2, ERK1/2 and JNK in the parental cells were significantly higher than those in the drug-resistant cells R2 and R4. The phosphorylation of p38 was different in the drug-resistant cells. The tumor volume and weight in the treatment group were significantly lower than those in the control group, and the tumor volume and weight were in a concentration-and time-dependent manner. The number of mitotic cells in the treatment group was significantly lower than that in the control group, which was concentration-dependent. TUNEL staining showed that the number of apoptotic cells in the treatment group was significantly higher than that in the control group. Conclusion: 1. High-throughput drug screening provides a new platform for characterizing the biological activity of drugs as an effective method. Colchicine, a hot drug, was successfully screened out by high-throughput screening method, and it is also a drug for thyroid cancer. Colchicine can inhibit not only BRAFV600E gene mutation but also other BRAFV600E wild-type thyroid cancers. 3. Colchicine inhibits a variety of thyroid cancers by blocking G2/M cell cycle and inducing apoptosis. Colchicine induces apoptosis by activating MEK/ERK and JNK/c-Jun phosphorylation pathways. The phosphorylation of p38 only partially induces apoptosis in WRO cells, but not in 8505C cells. AKT phosphorylation does not participate in the process of apoptosis. Colchicine has been shown to be effective in inhibiting the growth of transplanted thyroid cancer in 8505C and WRO mice, and has no significant toxic and side effects on animal models.
【学位授予单位】：吉林大学
【学位级别】：博士
【学位授予年份】：2016
【分类号】：R736.1

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