多靶点酪氨酸激酶抑制剂AL3810实验治疗甲状腺癌及机制研究

发布时间：2018-11-13 10:54

【摘要】：甲状腺癌(Thyroid Cancer,TC)是内分泌腺系统中最常见的恶性肿瘤,目前其治疗主要以手术治疗、手术后放射性碘(Radioactive Iodine,RAI)治疗以及促甲状腺激素(Thyroid Stimulating Hormone,TSH)释放抑制治疗为主。尽管在过去的几十年研究表明,恶性甲状腺癌的治疗有新突破,已经有新的有效的药物和制剂出现,目前大部分晚期甲状腺癌病人对常规的放射性碘131的治疗并不敏感,且可供选择的治疗有限。因此,积极寻找有效的、新的治疗药物和治疗策略对提高甲状腺癌的疗效、改善患者的生存率及提高他们的生活质量具有重大意义。本课题通过体内外不同模型系统评价了小分子多靶点酪氨酸激酶抑制剂AL3810对甲状腺癌的实验治疗效果。结果显示,该化合物能显著抑制多种甲状腺癌细胞的体内外增殖。AL3810作用72或120小时后,可以剂量依赖性的抑制甲状腺癌细胞TT、TPC-1和SW579的增殖,IC50值分别为2.56?M、7.03?M和590 nM。在体内实验中AL3810对细胞株来源的人源甲状腺癌SW579、TT等裸小鼠皮下移植瘤也均具有显著的抑制作用。SW579模型中,AL3810以5 mg/kg连续口服给药三周抑制率可达97.57%左右,与索拉菲尼60 mg/kg实验治疗效果相当。更令人欣喜的是,在该模型中连续实验治疗三周后,停药两周该组肿瘤仍未有明显生长。我们还尝试模拟肿瘤后期给药,即当肿瘤生长至2000 mm3左右开始实验治疗,AL3810在此方案下依然能发挥很好的抑制肿瘤生长的效果。AL3810 10 mg/kg实验治疗组给药后小鼠所荷肿瘤明显缩小,五周时该组肿瘤平均体积已缩至300 mm3左右。同样,在另一个甲状腺癌TT模型中,AL3810 10mg/kg也能显著抑制TT裸小鼠皮下移植瘤的生长,给药处理28天后抑制百分数为66.15%,抑制作用优于Sorafenib 60 mg/kg和XL184 30 mg/kg。我们前期的研究结果表明AL3810是一个新生血管抑制剂,因此在以上甲状腺肿瘤的实验治疗中,我们以内皮细胞表面标记物CD34为检测指标,免疫组化(Immunohistochemistry,IHC)考察其对肿瘤内新生血管生成的影响。结果显示AL3810能够显著降低瘤组织内微小血管的数目:当SW579裸小鼠移植瘤组织内AL3810给药剂量为5 mg/kg和10 mg/kg时,瘤组织CD34表达量分别下降了81.88%和92.83%;TT模型中结果类似,AL3810给药剂量为0.4、2和10 mg/kg时,瘤组织内CD34表达量分别下降了59.06%、63.78%和81.10%。这些结果显示该化合物的抗甲状腺肿瘤活性与其抗血管新生能力密切相关。流式细胞仪的检测结果展示,AL3810可浓度依赖和时间依赖地阻滞甲状腺癌细胞于G1期,并诱导细胞发生凋亡,最终导致甲状腺癌细胞死亡。0.5?M AL3810作用48小时即可诱导约20%的SW579细胞发生凋亡。我们进一步检测裸小鼠甲状腺癌皮下移植瘤在AL3810作用后的情况。免疫组化染色凋亡标记物TUNEL结果显示,无论是TT模型还是SW579模型中,AL3810作用后,瘤组织中TUNEL染色阳性率具有显著升高。在SW579模型中,当AL3810剂量为0.4、2和10 mg/kg时,凋亡率分别为4.66%、22.00%和36.00%;TT模型中,凋亡率则分别为6.66%、13.66%和22.66%。再次证实AL3810确实可引起甲状腺癌细胞发生凋亡。同样该化合物还可引起细胞周期阻滞的发生,AL3810 1?M作用24小时,64.2%的SW579细胞被阻滞处于G1期。相应的Western Blot结果则表明,在AL3810处理后,与G1期阻滞相关的周期调控蛋白如p21和p27的表达量显著上升,而蛋白CyclinD1、CDK2和P-Rb的表达量则显著下调,这与流式周期的结果一致。AL3810的前期研究结果提示,该化合物在分子水平可抑制RET磷酸化的发生。鉴于RET在甲状腺癌发生发展中的重要作用,我们进一步考察该化合物是否通过抑制RET信号通路而抑制RET依赖的人甲状腺癌细胞的体内外生长。我们首先选用转染RET的Ba F3工具细胞考察。结果表明,AL3810能显著抑制转染RET的BaF3的体外增殖,可阻滞细胞于G1期,并诱导细胞发生凋亡,但对亲本细胞BaF3的生长则无明显影响。Western Blot结果证实,该化合物能显著抑制BaF3-RET以及人源甲状腺癌细胞TT和TPC-1细胞内RET磷酸化的发生,同时下调其下游蛋白AKT、ERK1/2以及STAT3的磷酸化。以上结果表明,该化合物在分子水平和细胞水平对RET信号通路均有抑制作用。综上,AL3810能够显著抑制RET依赖和RET非依赖的甲状腺癌,抗肿瘤疗效显著,是一个有前景的抗肿瘤药物。这些结果为AL3810的临床应用于甲状腺癌的治疗提供了扎实的实验依据,也使我们更加确信AL3810是一个值得进一步研究的光明的多靶点酪氨酸激酶类抗肿瘤药物。
[Abstract]:Thyroid cancer (TC) is the most common malignant tumor in the system of endocrine gland. At present, the treatment of thyroid carcinoma (TC) is the most common malignant tumor in the endocrine system. Although new breakthroughs have been made in the treatment of malignant thyroid cancer in the past few decades, new and effective drugs and formulations have emerged, and most of the patients with advanced thyroid cancer are not sensitive to the treatment of conventional radioiodine 131 and are available for limited treatment. Therefore, it is of great significance to actively seek effective and new therapeutic drugs and treatment strategies to improve the curative effect of thyroid cancer, to improve the survival rate of patients and to improve their quality of life. The effect of AL3810 on the treatment of thyroid carcinoma was evaluated by different models in vivo. The results show that the compound can significantly inhibit the in vitro proliferation of various thyroid cancer cells. After the action of AL3810 for 72 or 120 hours, the proliferation of TT, TPC-1 and SW579 of the thyroid cancer cells can be inhibited in a dose-dependent manner, with an IC50 value of 2.56? M, 7.03? M, and 590 nM, respectively. In vivo, AL3810 has a significant inhibitory effect on the human source of human thyroid cancer SW579, TT and other nude mice. In the SW579 model, the inhibition rate of AL3810 at 5 mg/ kg for three weeks was 97. 57%, which was comparable to that of the experimental treatment with solani 60 mg/ kg. It was more gratifying to note that after three weeks of continuous experimental treatment in this model, no significant growth of the group of tumors was observed for two weeks. We also tried to simulate the post-treatment of the tumor, that is, when the tumor grows to about 2000 mm3 to start the experimental treatment, the AL3810 can still play a very good effect in inhibiting the growth of the tumor under the scheme. The average volume of tumor of the group was reduced to about 300 mm3 at five weeks after the treatment group of AL3810 mg/ kg. Similarly, in another TT model of thyroid carcinoma, AL3810 mg/ kg could significantly inhibit the growth of the subcutaneous graft of TT nude mice, and the inhibition percentage after 28 days of administration was 66. 15%, which was superior to that of Sorafenib 60 mg/ kg and XL184 30 mg/ kg. Our previous study shows that AL3810 is a new blood vessel inhibitor. Therefore, in the above-mentioned experimental treatment of thyroid tumor, we use the endothelial cell surface marker CD34 as the detection index and immunohistochemistry (IHC) to investigate the effect of the tumor on the angiogenesis in the tumor. The results showed that AL3810 could significantly reduce the number of microvessels in the tumor tissue: the expression of CD34 in the tumor tissue decreased by 81.88% and 92.83%, respectively, when the dose of AL3810 was 5 mg/ kg and 10 mg/ kg in the nude mice of SW579 nude mice, and the results of the TT model were similar, and when the dose of AL3810 was 0. 4, 2 and 10 mg/ kg, The expression of CD34 in the tumor tissues decreased by 59. 06%, 63. 78% and 81. 10%, respectively. These results show that the anti-thyroid tumor activity of the compound is closely related to its anti-angiogenic ability. The results of flow cytometry show that the concentration of AL3810 can block thyroid cancer cells in G1 phase and induce apoptosis, which leads to the death of thyroid cancer cells. The apoptosis of about 20% of SW579 cells can be induced by 0. 5? M AL3810 for 48 hours. We further examine the effect of subcutaneous transplantation of nude mouse thyroid carcinoma on the role of AL3810. The TUNEL results showed that the positive rate of TUNEL staining in the tumor tissues was significantly higher in the tumor tissues, either in the TT model or in the SW579 model. In the SW579 model, when the dose of AL3810 was 0. 4, 2 and 10 mg/ kg, the apoptosis rate was 4.66%, 22.00% and 36.00%, respectively. In the TT model, the apoptosis rate was 6.66%, 13.66% and 22.66%, respectively. Again, it is confirmed that AL3810 can cause apoptosis in the thyroid cancer cells. The compound can also cause cell cycle arrest, AL3810 1-M acts for 24 hours, and 64. 2% of the SW579 cells are blocked in the G1 phase. The corresponding Western Blot results showed that the expression of cyclin D1, CDK2 and P-Rb decreased significantly after the treatment with AL3810, while the expression of Cyclin D1, CDK2 and P-Rb was significantly reduced, which was consistent with the results of the flow cycle. The early results of AL3810 suggest that the compound can inhibit the occurrence of RET phosphorylation at the molecular level. In view of the important role of RET in the development of thyroid cancer, we further investigate whether the compound inhibits the in vitro growth of RET-dependent human thyroid cancer cells by inhibiting the RET signal pathway. We first selected the BaF3 tool cells transfected with RET. The results showed that AL3810 can significantly inhibit the in vitro proliferation of BaF3 transfected with RET, can block the cells in G1 phase and induce apoptosis, but has no obvious effect on the growth of BF3 in the parent cell. The results of Western Blot confirmed that the compound could significantly inhibit the phosphorylation of RET and the phosphorylation of RET, ERK1/ 2 and STAT3 downstream of BaF3-RET and human thyroid cancer cells TT and TPC-1 cells. The results show that the compound has an inhibitory effect on the RET signal pathway at both the molecular level and the cell level. In general, AL3810 can significantly inhibit RET dependence and RET's non-dependent thyroid cancer. It is a promising anti-tumor drug. These results provide a solid basis for the clinical application of AL3810 in the treatment of thyroid cancer, and also make us more convinced that AL3810 is a promising multi-target tyrosine kinase anti-tumor drug that is worth further study.
【学位授予单位】：南昌大学
【学位级别】：硕士
【学位授予年份】：2017
【分类号】：R96

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