多靶點(diǎn)酪氨酸激酶抑制劑AL3810實(shí)驗(yàn)治療甲狀腺癌及機(jī)制研究
發(fā)布時(shí)間：2018-11-13 10:54
【摘要】：甲狀腺癌(Thyroid Cancer,TC)是內(nèi)分泌腺系統(tǒng)中最常見的惡性腫瘤,目前其治療主要以手術(shù)治療、手術(shù)后放射性碘(Radioactive Iodine,RAI)治療以及促甲狀腺激素(Thyroid Stimulating Hormone,TSH)釋放抑制治療為主。盡管在過去的幾十年研究表明,惡性甲狀腺癌的治療有新突破,已經(jīng)有新的有效的藥物和制劑出現(xiàn),目前大部分晚期甲狀腺癌病人對(duì)常規(guī)的放射性碘131的治療并不敏感,且可供選擇的治療有限。因此,積極尋找有效的、新的治療藥物和治療策略對(duì)提高甲狀腺癌的療效、改善患者的生存率及提高他們的生活質(zhì)量具有重大意義。本課題通過體內(nèi)外不同模型系統(tǒng)評(píng)價(jià)了小分子多靶點(diǎn)酪氨酸激酶抑制劑AL3810對(duì)甲狀腺癌的實(shí)驗(yàn)治療效果。結(jié)果顯示,該化合物能顯著抑制多種甲狀腺癌細(xì)胞的體內(nèi)外增殖。AL3810作用72或120小時(shí)后,可以劑量依賴性的抑制甲狀腺癌細(xì)胞TT、TPC-1和SW579的增殖,IC50值分別為2.56?M、7.03?M和590 nM。在體內(nèi)實(shí)驗(yàn)中AL3810對(duì)細(xì)胞株來源的人源甲狀腺癌SW579、TT等裸小鼠皮下移植瘤也均具有顯著的抑制作用。SW579模型中,AL3810以5 mg/kg連續(xù)口服給藥三周抑制率可達(dá)97.57%左右,與索拉菲尼60 mg/kg實(shí)驗(yàn)治療效果相當(dāng)。更令人欣喜的是,在該模型中連續(xù)實(shí)驗(yàn)治療三周后,停藥兩周該組腫瘤仍未有明顯生長。我們還嘗試模擬腫瘤后期給藥,即當(dāng)腫瘤生長至2000 mm3左右開始實(shí)驗(yàn)治療,AL3810在此方案下依然能發(fā)揮很好的抑制腫瘤生長的效果。AL3810 10 mg/kg實(shí)驗(yàn)治療組給藥后小鼠所荷腫瘤明顯縮小,五周時(shí)該組腫瘤平均體積已縮至300 mm3左右。同樣,在另一個(gè)甲狀腺癌TT模型中,AL3810 10mg/kg也能顯著抑制TT裸小鼠皮下移植瘤的生長,給藥處理28天后抑制百分?jǐn)?shù)為66.15%,抑制作用優(yōu)于Sorafenib 60 mg/kg和XL184 30 mg/kg。我們前期的研究結(jié)果表明AL3810是一個(gè)新生血管抑制劑,因此在以上甲狀腺腫瘤的實(shí)驗(yàn)治療中,我們以內(nèi)皮細(xì)胞表面標(biāo)記物CD34為檢測(cè)指標(biāo),免疫組化(Immunohistochemistry,IHC)考察其對(duì)腫瘤內(nèi)新生血管生成的影響。結(jié)果顯示AL3810能夠顯著降低瘤組織內(nèi)微小血管的數(shù)目:當(dāng)SW579裸小鼠移植瘤組織內(nèi)AL3810給藥劑量為5 mg/kg和10 mg/kg時(shí),瘤組織CD34表達(dá)量分別下降了81.88%和92.83%;TT模型中結(jié)果類似,AL3810給藥劑量為0.4、2和10 mg/kg時(shí),瘤組織內(nèi)CD34表達(dá)量分別下降了59.06%、63.78%和81.10%。這些結(jié)果顯示該化合物的抗甲狀腺腫瘤活性與其抗血管新生能力密切相關(guān)。流式細(xì)胞儀的檢測(cè)結(jié)果展示,AL3810可濃度依賴和時(shí)間依賴地阻滯甲狀腺癌細(xì)胞于G1期,并誘導(dǎo)細(xì)胞發(fā)生凋亡,最終導(dǎo)致甲狀腺癌細(xì)胞死亡。0.5?M AL3810作用48小時(shí)即可誘導(dǎo)約20%的SW579細(xì)胞發(fā)生凋亡。我們進(jìn)一步檢測(cè)裸小鼠甲狀腺癌皮下移植瘤在AL3810作用后的情況。免疫組化染色凋亡標(biāo)記物TUNEL結(jié)果顯示,無論是TT模型還是SW579模型中,AL3810作用后,瘤組織中TUNEL染色陽性率具有顯著升高。在SW579模型中,當(dāng)AL3810劑量為0.4、2和10 mg/kg時(shí),凋亡率分別為4.66%、22.00%和36.00%;TT模型中,凋亡率則分別為6.66%、13.66%和22.66%。再次證實(shí)AL3810確實(shí)可引起甲狀腺癌細(xì)胞發(fā)生凋亡。同樣該化合物還可引起細(xì)胞周期阻滯的發(fā)生,AL3810 1?M作用24小時(shí),64.2%的SW579細(xì)胞被阻滯處于G1期。相應(yīng)的Western Blot結(jié)果則表明,在AL3810處理后,與G1期阻滯相關(guān)的周期調(diào)控蛋白如p21和p27的表達(dá)量顯著上升,而蛋白CyclinD1、CDK2和P-Rb的表達(dá)量則顯著下調(diào),這與流式周期的結(jié)果一致。AL3810的前期研究結(jié)果提示,該化合物在分子水平可抑制RET磷酸化的發(fā)生。鑒于RET在甲狀腺癌發(fā)生發(fā)展中的重要作用,我們進(jìn)一步考察該化合物是否通過抑制RET信號(hào)通路而抑制RET依賴的人甲狀腺癌細(xì)胞的體內(nèi)外生長。我們首先選用轉(zhuǎn)染RET的Ba F3工具細(xì)胞考察。結(jié)果表明,AL3810能顯著抑制轉(zhuǎn)染RET的BaF3的體外增殖,可阻滯細(xì)胞于G1期,并誘導(dǎo)細(xì)胞發(fā)生凋亡,但對(duì)親本細(xì)胞BaF3的生長則無明顯影響。Western Blot結(jié)果證實(shí),該化合物能顯著抑制BaF3-RET以及人源甲狀腺癌細(xì)胞TT和TPC-1細(xì)胞內(nèi)RET磷酸化的發(fā)生,同時(shí)下調(diào)其下游蛋白AKT、ERK1/2以及STAT3的磷酸化。以上結(jié)果表明,該化合物在分子水平和細(xì)胞水平對(duì)RET信號(hào)通路均有抑制作用。綜上,AL3810能夠顯著抑制RET依賴和RET非依賴的甲狀腺癌,抗腫瘤療效顯著,是一個(gè)有前景的抗腫瘤藥物。這些結(jié)果為AL3810的臨床應(yīng)用于甲狀腺癌的治療提供了扎實(shí)的實(shí)驗(yàn)依據(jù),也使我們更加確信AL3810是一個(gè)值得進(jìn)一步研究的光明的多靶點(diǎn)酪氨酸激酶類抗腫瘤藥物。
[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.
【學(xué)位授予單位】：南昌大學(xué)
【學(xué)位級(jí)別】：碩士
【學(xué)位授予年份】：2017
【分類號(hào)】：R96
本文編號(hào)：2328888
[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.
【學(xué)位授予單位】：南昌大學(xué)
【學(xué)位級(jí)別】：碩士
【學(xué)位授予年份】：2017
【分類號(hào)】：R96
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