發(fā)布時(shí)間：2019-06-20 06:42

【摘要】：Sorafenib(索拉菲尼,Bay43-9006)是第一個(gè)口服的小分子多激酶抑制劑,廣泛應(yīng)用于晚期腎細(xì)胞癌、不可切除的肝癌患者。另外,在治療乳腺癌、白血病、甲狀腺癌等癌癥類型中也具有很好的臨床優(yōu)勢(shì)。到目前為止,索拉菲尼被推薦為治療晚期肝癌的一線藥物,晚期腎細(xì)胞癌的二線治療藥物和其它晚期或轉(zhuǎn)移性甲狀腺癌的推薦藥物。索拉菲尼一方面通過直接靶向抑制絲氨酸／蘇氨酸(Raf-1, B-Raf V600E, wild-type B-Raf and C-Raf),阻斷腫瘤細(xì)胞增殖信號(hào)沿Raf/MEK/ERK通路傳導(dǎo)；另一方面,通過抑制某些受體酪氨酸激酶(PDGFR-β, EGFR、VEGF、VEGFR、Flt-3、c-Kit等),抑制實(shí)體瘤血管內(nèi)皮細(xì)胞形成血管,阻斷腫瘤的血液營(yíng)養(yǎng)供給,間接地抑制腫瘤的生長(zhǎng)。然而在臨床治療中,sorafenib治療導(dǎo)致的不良反應(yīng)也不能忽視,使得治療被迫中斷或者降低劑量。常見的不良反應(yīng)包括中性粒細(xì)胞減少、高血壓、肝功能異常、腎功能損傷等,會(huì)影響患者的生活質(zhì)量。同時(shí),索拉菲尼是國(guó)外的專利保護(hù)藥物,在中國(guó)售價(jià)昂貴,增加了中國(guó)患者治療癌癥的費(fèi)用。因此,開發(fā)索拉菲尼衍生物中抗腫瘤活性高且低毒的多激酶抑制劑是臨床急需的。鑒于Sorafenib在癌癥治療中較好的臨床優(yōu)勢(shì)和在臨床治療中發(fā)生的不良反應(yīng)等問題,李文保教授實(shí)驗(yàn)室在索拉菲尼結(jié)構(gòu)的基礎(chǔ)上,合成了一系列的索拉菲尼衍生物。在初期化合物活性篩選實(shí)驗(yàn)中,采用MTT實(shí)驗(yàn)評(píng)價(jià)了17個(gè)化合物對(duì)5種腫瘤細(xì)胞增殖的抑制作用。以抑制率和IC50值作為參考,我們篩選了6個(gè)具有研究潛力的化合物(1118-20、1124-21、1124-15、1125-67、1125-4、1118-41),都對(duì)5種腫瘤細(xì)胞的增殖具有較強(qiáng)的抑制作用。其中1118-20對(duì)上述5種腫瘤細(xì)胞抑制活性尤為明顯,故選擇1118-20作為研究對(duì)象,研究其抑制人肝癌細(xì)胞HepG2增殖的作用和機(jī)制。MTT和克隆形成實(shí)驗(yàn)表明,1118-20比sorafenib更能顯著地抑制HepG2細(xì)胞的增殖；而1118-20對(duì)人正常肝細(xì)胞HL-7702的抑制作用較弱,與sorafenib無顯著性差異；統(tǒng)計(jì)分析還發(fā)現(xiàn),1118-20對(duì)肝癌細(xì)胞HepG2的細(xì)胞毒作用強(qiáng)于人正常肝細(xì)胞HL-7702。流式細(xì)胞儀檢測(cè)細(xì)胞周期結(jié)果顯示,1118-20可將HepG2細(xì)胞阻滯在S期。Hoechst 33258染色發(fā)現(xiàn)1118-20可誘導(dǎo)HepG2細(xì)胞發(fā)生細(xì)胞核濃縮、分裂、并邊緣化等凋亡現(xiàn)象；Annexin-V/PI雙染法發(fā)現(xiàn)1118-20可顯著提高細(xì)胞凋亡的數(shù)量；western blotting檢測(cè)發(fā)現(xiàn)1118-20可上調(diào)cleaved caspase-9、cleaved caspase-3及其底物裂解帶cleaved PARP表達(dá)水平；JC-1染色和western blotting檢測(cè)結(jié)果顯示線粒體膜電勢(shì)(△Ψm)下降,Mcl-1蛋白表達(dá)下調(diào),Bax/Bcl-2比例上升,表明Bcl-2家族介導(dǎo)的線粒體凋亡途徑被激活。進(jìn)一步分析顯示1118-20對(duì)HepG2細(xì)胞內(nèi)增殖信號(hào)Wnt/β-catenin傳導(dǎo)通路,EGFR/PI3K/Akt傳導(dǎo)通路,Ras/ERK傳導(dǎo)通路以及p53蛋白皆有影響。結(jié)果顯示,1118-20不但上調(diào)抑癌蛋白p53的表達(dá),還能抑制這三條信號(hào)通路,阻斷增殖信號(hào)的傳導(dǎo),從而使腫瘤細(xì)胞的增殖阻滯。腫瘤細(xì)胞如果發(fā)生增殖或轉(zhuǎn)移,甚至進(jìn)一步惡化,腫瘤血管的形成是很有必要的。沒有腫瘤血管,腫瘤細(xì)胞就不能獲得足夠的營(yíng)養(yǎng)和氧氣,也不能將無用的代謝物排泄出去,將極大地限制腫瘤的惡化和轉(zhuǎn)移。MTT實(shí)驗(yàn)數(shù)據(jù)顯示1118-20顯著地抑制了人臍靜脈血管內(nèi)皮細(xì)胞HUVEC的增殖,且抑制效應(yīng)比索拉菲尼更強(qiáng)。劃痕試驗(yàn)表明1118-20可以減弱HUVECs的遷移能力；Western blotting結(jié)果顯示1118-20可下調(diào)active-MMP-2、active-MMP-9的表達(dá)；體外管形成實(shí)驗(yàn)表明1118-20可抑制血管內(nèi)皮細(xì)胞聚集形成血管管狀結(jié)構(gòu);并且發(fā)現(xiàn)1118-20顯著地降低血管形成相關(guān)蛋白(FGF-2、VEGF、VEGFR-2、EGFR)的表達(dá),并能抑制VEGFR-2、EGFR的磷酸化,從而減弱形成血管的分子基礎(chǔ)。綜上所述,1118-20是一個(gè)抗腫瘤活性優(yōu)于索拉菲尼,毒性較低的新化合物。在肝癌細(xì)胞中,1118-20不但激活Bcl-2家族介導(dǎo)的線粒體凋亡途徑,還可調(diào)節(jié)Wnt/β-catenin通路、EGFR/PI3K/Akt通路、Ras/ERK通路抑制增殖信號(hào)的傳導(dǎo)。1118-20還可通過下調(diào)蛋白抑制血管形成,間接限制腫瘤細(xì)胞的生長(zhǎng)。這些數(shù)據(jù)表明1118-20可能是一個(gè)癌癥治療的潛在藥物,具有較強(qiáng)的開發(fā)潛能。
[Abstract]:Sorafenib (Sorafenib, Bay43-9006) is the first oral micromolecule multi-kinase inhibitor, which is widely used in patients with advanced renal cell carcinoma and non-resectable liver cancer. In addition, the invention also has a good clinical advantage in the treatment of cancer types such as breast cancer, leukemia, thyroid cancer and the like. So far, Solani is recommended for the treatment of first-line drugs for advanced liver cancer, second-line treatment for advanced renal cell carcinoma and other advanced or metastatic thyroid cancer. so as to block the proliferation of tumor cells along the Raf/ MEK/ ERK pathway and, on the other hand, by inhibiting certain receptor tyrosine kinases (PDGFR-1, EGFR, VEGF, VEGFR, Flt-3, c-Kit, etc.), on the one hand, by direct targeting of a serine/ threonine (Raf-1, B-Raf V600E, a-type B-Raf and C-Raf); on the other hand, by inhibiting certain receptor tyrosine kinases (PDGFR-1, EGFR, VEGF, VEGFR, Flt-3, c-Kit, etc.), Inhibiting the formation of blood vessels of the vascular endothelial cells of the solid tumor, blocking the blood nutrient supply of the tumor, and indirectly inhibiting the growth of the tumor. However, in the clinical treatment, the adverse reaction caused by the treatment of the sorafenib cannot be ignored, so that the treatment is forced to be interrupted or the dose is reduced. Common adverse reactions include neutropenia, hypertension, liver function abnormality, renal function injury, etc., which may affect the quality of life of the patient. At the same time, Solani is a foreign patent protection drug, which is expensive in China and increases the cost of Chinese patients for cancer treatment. Therefore, the development of a multi-kinase inhibitor with high anti-tumor activity and low toxicity in the solani derivative is in urgent need. In view of the better clinical advantage of Sorafenib in the treatment of cancer and the adverse reactions that have occurred in clinical treatment, Professor Li Wenbao's laboratory has synthesized a series of solani derivatives on the basis of the structure of Sorafini. The inhibitory effect of 17 compounds on the proliferation of five tumor cells was evaluated by MTT assay in the initial compound activity screening experiment. As a reference to the inhibition and IC50 values, we screened six compounds (1118-20,1124-21,1124-15,1125-67,1125-4,1118-41) with the potential to study and have a strong inhibitory effect on the proliferation of 5 tumor cells. The inhibitory activity of 1118-20 on the above-mentioned five tumor cells was particularly obvious, and 1118-20 was selected as the research object to study the effect and mechanism of inhibiting the proliferation of human liver cancer cells HepG2. The results of MTT and clone formation showed that 1118-20 was more effective in inhibiting the proliferation of HepG2 cells than sorafenib, but the inhibitory effect of 1118-20 on human normal liver cells HL-7702 was weak and no significant difference with sorafenib. The statistical analysis also found that the cytotoxic effect of 1118-20 on HepG2 cells was stronger than that of human normal liver cells HL-7702. The cell cycle results were detected by flow cytometry, and the HepG2 cells were blocked in the S phase at 1118-20. Hoechst 33258 staining showed that 1118-20 could induce the nuclear concentration, division, and marginalization of HepG2 cells. The Annexin-V/ PI double-staining method found that 1118-20 could significantly increase the number of cell apoptosis. The western blotting assay found that 1118-20 could increase the level of clear PARP expression in clear caspase-9, clear caspase-3 and its substrate. The results of JC-1 staining and western blotting showed that the mitochondrial membrane potential decreased, the down-regulation of the Mcl-1 protein and the increase of the ratio of Bax/ Bcl-2, indicating that the Bcl-2 family-mediated mitochondrial apoptosis pathway was activated. Further analysis showed that 1118-20 had an effect on the proliferation signal Wnt/ P-cata conduction pathway, the EGFR/ PI3K/ Akt conduction pathway, the Ras/ ERK conduction pathway, and the p53 protein in HepG2 cells. The results show that 1118-20 not only upregulates the expression of the tumor suppressor protein p53, but also can inhibit the conduction of the proliferation signal, thereby blocking the proliferation of the tumor cells. The formation of a tumor vessel is necessary in the event of proliferation or metastasis or even further deterioration of the tumor cells. Without tumor blood vessels, the tumor cells can not obtain enough nutrients and oxygen, and the useless metabolites can not be discharged out, and the deterioration and metastasis of the tumor can be greatly limited. The MTT assay showed that 1118-20 significantly inhibited the proliferation of HUVEC in human umbilical vein endothelial cells, and the inhibitory effect was stronger. The scratch test shows that 1118-20 can reduce the migration ability of HUVECs; Western blotting results show that 1118-20 can downregulate the expression of active-MMP-2, active-MMP-9; in vitro tube formation experiments show that 1118-20 can inhibit the aggregation of vascular endothelial cells to form a vessel-like structure; and it has been found that 1118-20 significantly reduces angiogenesis-related proteins (FGF-2, VEGF, VEGFR-2, The expression of EGFR can inhibit the phosphorylation of VEGFR-2 and EGFR, thereby reducing the molecular basis for forming blood vessels. In conclusion,1118-20 is a new compound that has an anti-tumor activity that is superior to that of Solani and has a lower toxicity. In the liver cancer cell,1118-20 not only activates the Bcl-2 family-mediated mitochondrial apoptosis pathway, but also regulates the Wnt/ P-catenin pathway, the EGFR/ PI3K/ Akt pathway, and the Ras/ ERK pathway inhibits the conduction of the proliferation signal. These data suggest that 1118-20 may be a potential drug for cancer treatment and has a strong potential for development.
【學(xué)位授予單位】：山東大學(xué)
【學(xué)位級(jí)別】：碩士
【學(xué)位授予年份】：2015
【分類號(hào)】：R96

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