eIF4E磷酸化促翻譯及與蛋白酶體共抑制致結(jié)腸癌細胞凋亡的機制研究
發(fā)布時間：2018-08-02 17:30
【摘要】：背景結(jié)腸癌是惡性腫瘤中發(fā)病率及病死率最高的疾病之一,然而近年來結(jié)腸癌患者的存活率并無太大改善。結(jié)腸癌患者往往對傳統(tǒng)治療產(chǎn)生耐藥性,因此新的治療策略迫在眉睫。蛋白質(zhì)翻譯起始為一類進化保守的細胞生物過程,受營養(yǎng)/生長因子和細胞能量水平影響而調(diào)控細胞生長和新陳代謝。腫瘤細胞快速生長增殖伴隨PI3K/Akt/m TOR和Ras/Raf/MEK/ERK等信號通路的激活,蛋白質(zhì)翻譯起始處于這些信號通路的下游樞紐位置。其中,e IF4E是翻譯起始復合物中受到多層面精確調(diào)控的一個蛋白,其mRNA和蛋白質(zhì)表達水平在多種腫瘤細胞中上調(diào)。除了在轉(zhuǎn)錄和翻譯水平受到調(diào)控,e IF4E還在翻譯后修飾水平受到更加精細和快速的調(diào)節(jié)。在某些腫瘤模型中,只有磷酸化修飾的e IF4E才能發(fā)揮其最大活性并誘導腫瘤發(fā)生發(fā)展。在此基礎(chǔ)上,我們推測e IF4E S209磷酸化修飾在結(jié)直腸癌發(fā)生發(fā)展中發(fā)揮重要的作用,為了證實這一假設(shè),本研究擬構(gòu)建eIF4E S209A KI結(jié)腸癌細胞株,并比較e IF4E KI和WT兩種HCT116結(jié)腸癌細胞生長和新陳代謝情況;同時使用polysome實驗比較兩種細胞中分子水平的變化并探討其調(diào)控機制,從而系統(tǒng)性闡述eIF4E S209磷酸化修飾在結(jié)腸癌發(fā)生發(fā)展中的作用和機制,為靶向eIF4E磷酸化修飾的抗癌新藥研發(fā)奠定理論基礎(chǔ)。由于多種癌基因和抑癌基因調(diào)控蛋白質(zhì)翻譯起始過程,因此這一生物過程的失調(diào)表現(xiàn)在多種癌癥和疾病中。目前靶向蛋白質(zhì)翻譯起始的治療策略已成為一個引人注目的焦點。一些臨床前期研究和臨床研究表明真核細胞起始因子e IF4A抑制劑Episilvestrol及其類似物Silvestrol均可殺傷多種癌細胞,其缺陷表現(xiàn)為蛋白質(zhì)翻譯起始也參與正常的細胞生物過程,高濃度翻譯起始抑制劑可引發(fā)嚴重的副反應(yīng)。因此聯(lián)合應(yīng)用蛋白質(zhì)翻譯起始抑制劑和其他藥物成為結(jié)腸癌治療的一個選擇。Bortezomib是常用的藥物增敏劑,在本研究中我們發(fā)現(xiàn)Bortezomib也可協(xié)同Episilvestrol誘導更加顯著的結(jié)腸癌細胞凋亡。目的:本研究旨在探索e IF4E磷酸化修飾在蛋白質(zhì)翻譯中的重要作用并研究靶向蛋白質(zhì)翻譯抑制劑Episilvestrol協(xié)同蛋白酶體抑制劑Bortezomib誘導結(jié)腸癌細胞凋亡的作用及機制。方法和材料:(1)通過腺病毒系統(tǒng)構(gòu)建e IF4E磷酸化修飾KI細胞系,并利用帽依賴性報告基因、免疫共沉淀和m7 pull down實驗驗證eIF4E KI細胞系對mRNA翻譯的影響;(2)利用MTT、2D/3D克隆形成實驗、Brd U摻入實驗、ds RED、GFP-LC3、流式細胞、裸鼠實驗等研究eIF4E KI對體內(nèi)外細胞生長和新陳代謝的影響;(3)利用polysome實驗研究e IF4E KI對細胞內(nèi)mRNA翻譯的影響;(4)通過MTS,Crystal violet和克隆形成試驗研究不同濃度Episilvestrol對HCT116結(jié)腸癌細胞的生長抑制和殺傷作用。(5)通過定量PCR和Western blot實驗研究高濃度Episilvestrol對凋亡信號通路和線粒體應(yīng)激反應(yīng)的誘導作用。(6)通過MTS,結(jié)晶紫染色和克隆形成試驗研究低濃度Episilvestrol協(xié)同Bortezomib誘導結(jié)腸癌細胞生長抑制和凋亡的作用。(4)使用Z-VAD抑制劑證實Episilvestrol協(xié)同Bortezomib誘導結(jié)腸癌細胞凋亡的作用。(5)通過定量PCR和Western blot實驗研究Episilvestrol協(xié)同Bortezomib誘導細胞凋亡/線粒體應(yīng)激反應(yīng)和CHOP介導DR5上調(diào)的作用。(6)利用FADD敲除細胞研究Episilvestrol協(xié)同Bortezomib誘導外源性細胞凋亡。(7)通過MTS,結(jié)晶紫染色,克隆形成,定量PCR和Western blot試驗研究Episilvestrol協(xié)同Bortezomib誘導其他結(jié)腸癌細胞凋亡的作用。(8)通過裸鼠移植瘤實驗驗證Episilvestrol協(xié)同Bortezomib誘導結(jié)腸癌細胞凋亡的作用。結(jié)果:(1)通過腺病毒系統(tǒng)成功構(gòu)建eIF4E磷酸化修飾KI細胞系;并證實eIF4E KI抑制m RNA翻譯;(2)e IF4E KI抑制體內(nèi)外結(jié)腸癌細胞生長和新陳代謝反應(yīng);(3)e IF4E與p-eIF4E優(yōu)先翻譯不同群體的m RNA;(4)2.5nM Episilvestrol對HCT116結(jié)腸癌細胞無作用,5n M,10n M,15n MEpisilvestrol可引起HCT116結(jié)腸癌細胞的生長抑制作用,20n M,25n M Episilvestrol可誘導HCT116結(jié)腸癌細胞凋亡。(5)25n M Episilvestrol可抑制HCT116結(jié)腸癌細胞克隆形成,并誘導caspase-3/8,DR5,CHOP的表達。(6)2.5nM Episilvestrol聯(lián)合5nMBortezomib可有效引發(fā)HCT116結(jié)腸癌細胞凋亡,細胞凋亡抑制劑Z-VAD可抑制這一作用。(7)2.5nM Episilvestrol聯(lián)合5nMBortezomib在4h即可上調(diào)HCT116結(jié)腸癌細胞中DR5,CHOP,GADD34的表達,24h作用更加顯著。(8)DR5的上調(diào)為CHOP介導的,使用CHOP siRNA下調(diào)(knockdown)CHOP表達可抑制DR5的上調(diào)作用。(9)FADD敲除細胞可完全抑制Episilvestrol聯(lián)合Bortezomib引發(fā)的細胞凋亡作用。(10)2.5nM Episilvestrol聯(lián)合5n MBortezomib可在其他結(jié)腸癌細胞中發(fā)揮類似的殺傷作用。(11)在裸鼠移植瘤試驗中,單藥Episilvestrol,Bortezomib對移植瘤的生長影響甚微,二者聯(lián)合可有效抑制移植瘤生長。結(jié)論:這些結(jié)果表明腫瘤細胞利用超過正常生理水平的eIF4E磷酸化來達到在營養(yǎng)缺失等應(yīng)激條件下的生存。聯(lián)合使用蛋白質(zhì)翻譯抑制劑Episilvestrol和蛋白酶體抑制劑Bortezomib可作為治療結(jié)腸癌患者的一種策略。
[Abstract]:Background colon cancer is one of the highest morbidity and fatality rates in malignant tumors. However, the survival rate of colon cancer patients has not been greatly improved in recent years. Patients with colon cancer often have resistance to traditional treatment, so the new treatment strategy is imminent. Cell growth and metabolism are regulated by growth factors and cell energy levels. The rapid growth and proliferation of tumor cells are associated with the activation of signal pathways such as PI3K/Akt/m TOR and Ras/Raf/MEK/ERK, and protein translation begins at the downstream hub of these signaling pathways. Among them, e IF4E is a multidimensional precision in the starting complex of translation. A protein that regulates the expression level of mRNA and protein in a variety of tumor cells. In addition to the regulation of transcriptional and translation levels, e IF4E is also more finely and quickly regulated after the translation. In some tumor models, only phosphorylated e IF4E can play its maximum activity and induce tumor hair. On this basis, we speculate that e IF4E S209 phosphorylation modification plays an important role in the development of colorectal cancer. In order to confirm this hypothesis, this study intends to construct a eIF4E S209A KI colon cancer cell line and compare the growth and metabolism of two HCT116 colon cancer cells of E IF4E KI and WT; meanwhile, the polysome experiment ratio is also used. The changes in the molecular level of the two cells and its regulatory mechanism are discussed, which systematically expounds the role and mechanism of eIF4E S209 phosphorylation in the development of colon cancer. It lays a theoretical basis for the development of a new anticancer drug targeted by eIF4E phosphorylation. The maladjustment of this biological process is manifested in a variety of cancers and diseases. The therapeutic strategy targeting the initiation of protein translation has become a noticeable focus. Some preclinical and clinical studies have shown that the eukaryotic initiation factor E IF4A inhibitor Episilvestrol and its analogous Silvestrol can kill a variety of cancer cells. The defect shows that the protein translation initiation is also involved in normal cell biological processes, and the high concentration translation initiation inhibitor can cause serious side effects. Therefore, the combination of protein translation initiation inhibitor and other drugs as a choice for colon cancer treatment is a common drug sensitizer. In this study, we found that.Bortezomib is a common drug sensitizer. Bortezomib can also induce more significant apoptosis of colon cancer cells in conjunction with Episilvestrol. Purpose: This study aims to explore the important role of E IF4E phosphorylation in protein translation and to study the role and mechanism of the protein translation inhibitor Episilvestrol synergistically with proteasome inhibitor Bortezomib to induce the apoptosis of colon cancer cells. Methods and materials: (1) construct e IF4E phosphorylated KI cell lines through adenovirus system, and use caps dependent reporter gene, immunoprecipitation and M7 pull down test to verify the effect of eIF4E KI cell line on mRNA translation; (2) MTT, 2D/3D clone formation experiments, Brd experiments, flow cells, nude mice experiments and so on The effect of eIF4E KI on cell growth and metabolism in vitro and in vivo; (3) the effect of E IF4E KI on intracellular mRNA translation was studied by polysome experiment. (4) the inhibitory and killing effects of different concentrations Episilvestrol on the growth of colorectal cancer cells were studied by MTS, Crystal violet and clone formation. (5) The effect of high concentration of Episilvestrol on the apoptosis signaling pathway and mitochondrial stress response was investigated. (6) the effects of low concentration of Episilvestrol on growth inhibition and apoptosis induced by Bortezomib were studied by MTS, crystal violet staining and clone formation. (4) Z-VAD inhibitors were used to confirm that Episilvestrol was induced with Bortezomib. The role of inducing apoptosis of colon cancer cells. (5) the effect of Episilvestrol synergistic Bortezomib induced apoptosis / mitochondrial stress response and CHOP mediated up regulation of DR5 was studied by quantitative PCR and Western blot. (6) Episilvestrol synergistic Bortezomib induced exogenous apoptosis using FADD knockout cells. (7) through MTS, crystal violet staining, Clone formation, quantitative PCR and Western blot test to study the effect of Episilvestrol synergistic Bortezomib on the apoptosis of other colon cancer cells. (8) the effect of Episilvestrol on the apoptosis of colon cancer cells induced by Bortezomib in nude mice was verified by Bortezomib. Results: (1) the KI cell line modified by eIF4E phosphorylation was successfully constructed by adenovirus system. And eIF4E KI inhibits m RNA translation; (2) e IF4E KI inhibits the growth and metabolism of colon cancer cells in vivo and in vivo and in vivo; (3) e IF4E and p-eIF4E give priority to the translation of M RNA in different groups; (4) 25N M Episilvestrol can induce apoptosis in HCT116 colon cancer cells. (5) 25N M Episilvestrol can inhibit the formation of HCT116 colon cancer cells, and induce caspase-3/8, DR5, CHOP expression. (6) the apoptosis of colorectal cancer cells can be induced by the joint 2.5nM. (7) 2.5 NM Episilvestrol combined with 5nMBortezomib in 4H can increase the expression of DR5, CHOP, GADD34 in HCT116 colon cancer cells, and the effect of 24h is more significant. (8) the up regulation of DR5 is mediated by CHOP. Cell apoptosis. (10) 2.5nM Episilvestrol combined with 5N MBortezomib can play a similar killing effect in other colon cancer cells. (11) in nude mice transplantation tumor test, the single drug Episilvestrol and Bortezomib have little influence on the growth of the transplanted tumor. The two combination can effectively inhibit the growth of the transplanted tumor. Conclusion: These results indicate that the tumor cells are beneficial. The use of eIF4E phosphorylation, which is more than normal physiological level, is used to survive under stress conditions such as nutritional deficiency. The combination of protein translation inhibitor Episilvestrol and proteasome inhibitor Bortezomib can be used as a strategy for the treatment of colon cancer patients.
【學位授予單位】：第三軍醫(yī)大學
【學位級別】：博士
【學位授予年份】：2016
【分類號】：R735.35
[Abstract]:Background colon cancer is one of the highest morbidity and fatality rates in malignant tumors. However, the survival rate of colon cancer patients has not been greatly improved in recent years. Patients with colon cancer often have resistance to traditional treatment, so the new treatment strategy is imminent. Cell growth and metabolism are regulated by growth factors and cell energy levels. The rapid growth and proliferation of tumor cells are associated with the activation of signal pathways such as PI3K/Akt/m TOR and Ras/Raf/MEK/ERK, and protein translation begins at the downstream hub of these signaling pathways. Among them, e IF4E is a multidimensional precision in the starting complex of translation. A protein that regulates the expression level of mRNA and protein in a variety of tumor cells. In addition to the regulation of transcriptional and translation levels, e IF4E is also more finely and quickly regulated after the translation. In some tumor models, only phosphorylated e IF4E can play its maximum activity and induce tumor hair. On this basis, we speculate that e IF4E S209 phosphorylation modification plays an important role in the development of colorectal cancer. In order to confirm this hypothesis, this study intends to construct a eIF4E S209A KI colon cancer cell line and compare the growth and metabolism of two HCT116 colon cancer cells of E IF4E KI and WT; meanwhile, the polysome experiment ratio is also used. The changes in the molecular level of the two cells and its regulatory mechanism are discussed, which systematically expounds the role and mechanism of eIF4E S209 phosphorylation in the development of colon cancer. It lays a theoretical basis for the development of a new anticancer drug targeted by eIF4E phosphorylation. The maladjustment of this biological process is manifested in a variety of cancers and diseases. The therapeutic strategy targeting the initiation of protein translation has become a noticeable focus. Some preclinical and clinical studies have shown that the eukaryotic initiation factor E IF4A inhibitor Episilvestrol and its analogous Silvestrol can kill a variety of cancer cells. The defect shows that the protein translation initiation is also involved in normal cell biological processes, and the high concentration translation initiation inhibitor can cause serious side effects. Therefore, the combination of protein translation initiation inhibitor and other drugs as a choice for colon cancer treatment is a common drug sensitizer. In this study, we found that.Bortezomib is a common drug sensitizer. Bortezomib can also induce more significant apoptosis of colon cancer cells in conjunction with Episilvestrol. Purpose: This study aims to explore the important role of E IF4E phosphorylation in protein translation and to study the role and mechanism of the protein translation inhibitor Episilvestrol synergistically with proteasome inhibitor Bortezomib to induce the apoptosis of colon cancer cells. Methods and materials: (1) construct e IF4E phosphorylated KI cell lines through adenovirus system, and use caps dependent reporter gene, immunoprecipitation and M7 pull down test to verify the effect of eIF4E KI cell line on mRNA translation; (2) MTT, 2D/3D clone formation experiments, Brd experiments, flow cells, nude mice experiments and so on The effect of eIF4E KI on cell growth and metabolism in vitro and in vivo; (3) the effect of E IF4E KI on intracellular mRNA translation was studied by polysome experiment. (4) the inhibitory and killing effects of different concentrations Episilvestrol on the growth of colorectal cancer cells were studied by MTS, Crystal violet and clone formation. (5) The effect of high concentration of Episilvestrol on the apoptosis signaling pathway and mitochondrial stress response was investigated. (6) the effects of low concentration of Episilvestrol on growth inhibition and apoptosis induced by Bortezomib were studied by MTS, crystal violet staining and clone formation. (4) Z-VAD inhibitors were used to confirm that Episilvestrol was induced with Bortezomib. The role of inducing apoptosis of colon cancer cells. (5) the effect of Episilvestrol synergistic Bortezomib induced apoptosis / mitochondrial stress response and CHOP mediated up regulation of DR5 was studied by quantitative PCR and Western blot. (6) Episilvestrol synergistic Bortezomib induced exogenous apoptosis using FADD knockout cells. (7) through MTS, crystal violet staining, Clone formation, quantitative PCR and Western blot test to study the effect of Episilvestrol synergistic Bortezomib on the apoptosis of other colon cancer cells. (8) the effect of Episilvestrol on the apoptosis of colon cancer cells induced by Bortezomib in nude mice was verified by Bortezomib. Results: (1) the KI cell line modified by eIF4E phosphorylation was successfully constructed by adenovirus system. And eIF4E KI inhibits m RNA translation; (2) e IF4E KI inhibits the growth and metabolism of colon cancer cells in vivo and in vivo and in vivo; (3) e IF4E and p-eIF4E give priority to the translation of M RNA in different groups; (4) 25N M Episilvestrol can induce apoptosis in HCT116 colon cancer cells. (5) 25N M Episilvestrol can inhibit the formation of HCT116 colon cancer cells, and induce caspase-3/8, DR5, CHOP expression. (6) the apoptosis of colorectal cancer cells can be induced by the joint 2.5nM. (7) 2.5 NM Episilvestrol combined with 5nMBortezomib in 4H can increase the expression of DR5, CHOP, GADD34 in HCT116 colon cancer cells, and the effect of 24h is more significant. (8) the up regulation of DR5 is mediated by CHOP. Cell apoptosis. (10) 2.5nM Episilvestrol combined with 5N MBortezomib can play a similar killing effect in other colon cancer cells. (11) in nude mice transplantation tumor test, the single drug Episilvestrol and Bortezomib have little influence on the growth of the transplanted tumor. The two combination can effectively inhibit the growth of the transplanted tumor. Conclusion: These results indicate that the tumor cells are beneficial. The use of eIF4E phosphorylation, which is more than normal physiological level, is used to survive under stress conditions such as nutritional deficiency. The combination of protein translation inhibitor Episilvestrol and proteasome inhibitor Bortezomib can be used as a strategy for the treatment of colon cancer patients.
【學位授予單位】：第三軍醫(yī)大學
【學位級別】：博士
【學位授予年份】：2016
【分類號】：R735.35
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