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TRIB3 involves in capsaicin - induced apoptosis in gastric cancer cells

By 林榮展 and Rong-Jaan Lin

Abstract

TRIB3是ㄧ個從果蠅到人類都存在的基因,人類細胞中表現的TRIB3蛋白參與了許多重要的細胞流程,包括細胞的生長、分化、發炎反應、移行能力、侵襲力、新陳代謝以及細胞凋亡等,這些機制建立於TRIB3可以調控轉錄因子以及和蛋白質間的交互作用。然而,在過去的研究中顯示,TRIB3在細胞內是抵抗或是促進細胞凋亡仍然是有爭議的,其機轉也尚未清楚,但是許多的研究認為TRIB3是一個壓力感受器蛋白質,常常會被誘導出現在細胞處於壓力的情況下。辣椒素是辣椒中的主要成分,經萃取純化後已經運用在癌細胞的研究上。在過去的研究顯示,辣椒素可以抑制癌細胞的增殖以及促使細胞凋亡,但是其相關機制仍然未完全明瞭。為了研究TRIB3在胃癌細胞內可以被辣椒素誘導表現,並調控辣椒素誘導的細胞凋亡,我們建構TRIB3的表現載體以及利用RNA干擾技術來調控TRIB3在胃癌細胞內的表現,並研究其參與細胞凋亡的路徑。我們發現在七種不同胃癌細胞中的TRIB3蛋白表現量都不一樣,而且都一樣不穩定,並透過26S 蛋白酶體降解。我們使用AGS, MKN-45, SCM-1 and TMK-1處理辣椒素,發現除了SCM-1在50 uM處理24小時就可以誘導TRIB3蛋白增量表現外,其它細胞株均需200 uM處理24小時才會誘導TRIB3蛋白增量表現,並開始產生細胞凋亡的現象,其細胞凋亡的比率和TRIB3的誘導量及辣椒素劑量成正相關。從以上的研究結果,我們推測調控TRIB3蛋白表現可以控制辣椒素誘導細胞凋亡的機制。我們使用TRIB3的表現載體來增量TRIB3表現,並處理辣椒素,結果顯示,增量表現TRIB3後處理辣椒素誘導的細胞凋亡增加,並且可以降低辣椒素處理劑量,其磷酸化AKT的表現量也降低;當利用RNA干擾技術降低TRIB3蛋白表現並處理辣椒素後,結果可以逆轉。從以上的研究結果,我們推測TRIB3蛋白調控辣椒素誘導細胞凋亡的機制是透過抑制AKT磷酸化,進而影響到AKT所調控的路徑,但其調控的分子機制則需更進一步的研究探討。TRIB3 is an evolutionarily conserved protein from Drosophila to human. Human cells express TRIB3 to play an key determinant in a large number of cellular processes, such as control the proliferation, differentiation, inflammatory response, migration, invasion, metabolisms and apoptosis. These mechanisms are established in TRIB3 can regulate transcription factor and protein interactions. However, in previous studies that TRIB3 is anti-apoptoic or pre-apoptoic still controversial, the mechanism remain unclear, but several studies have led to the suggestion that TRIB3 was due to as a stress sensor, and induction in cell stressful condition. Capsaicin is the main ingredient of hot chili peppers, after extraction and purification has been used in cancer research. In previous studies have shown that capsaicin can inhibit cancer cell proliferation and promote apoptosis, but its relevant mechanisms still not fully understood. In order to study the TRIB3 in gastric cancer cells within capsaicin induction and regulation of capsaicin-induced apoptosis, we construct the TRIB3-Myc plasmid and using RNA interference technology to regulate TRIB3 expression in gastric cancer cells, and study that involved in apoptosis pathway. We found that TRIB3 expression in seven different gastric cancer cells is not the same, but are the same unstable, and degradation by the 26S proteasome. We treated capsaicin in AGS, MKN-45, SCM-1 and TMK-1, the data showed that treated capsaicin 50 uM for 24 hours in SCM-1 can induction TRIB3, other cell lines need to be 200 uM for 24 hours, and began to produce the phenomenon of apoptosis, that apoptosis ratio and TRIB3 induction by capsaicin dose dependent. These results suggest that regulation the TRIB3 may control a mechanism of capsaicin-induced apoptosis. We overexpression TRIB3 and treated capsaicin, the data showed that overexpression TRIB3 and treated capsaicin could promoted capsaicin induced apoptosis, and can reduce the dose of capsaicin, the phosphorylated AKT level also reduced; when knockdown TRIB3 and treated capsaicin, the result were reversed. From the above findings, we speculate that the mechanism of TRIB3 regulate capsaicin-induced apoptosis mediate inhibition of AKT phosphorylation, thereby affecting the AKT regulated pathway, but the molecular mechanisms of its regulation will be further investigated.中文摘要 ………………………………………………………………………………………………………………………………………i Abstract …………………………………………………………………………………………………………………………………ii Chapter 1.Research background and aims………………………………………………………1 1.The role of TRIB3 in PI3K pathway……………………………………………………………2 2.The role of TRIB3 in ER stress …………………………………………………………………3 3.The role of TRIB3 in apoptosis……………………………………………………………………4 4.The role of capsaicin in mammalian cell……………………………………………6 5.Aims…………………………………………………………………………………………………………………………………………8 Chapter 2.Materials and Methods…………………………………………………………………………9 1.Cell culture……………………………………………………………………………………………………………………9 2.Chemicals……………………………………………………………………………………………………………………………9 3.Antibodies…………………………………………………………………………………………………………………………9 4.RNA interference………………………………………………………………………………………………………10 5.TRIB3 plasmid DNA construction and cell transfection………10 6.Preparation of cell lysates and Western Blot……………………………11 7.Proliferation assay………………………………………………………………………………………………11 8.Apoptosis Determination……………………………………………………………………………………11 9.Reverse Transcriptase–Polymerase Chain Reaction(RT-PCR)12 Chapter 3.Results……………………………………………………………………………………………………………13 1.Difference TRIB3 expression level in gastric cancer cell lines……………………………………………………………………………………………………………………………………13 2.TRIB3 is an unstable protein and increase stability by treated MG132………………………………………………………………………………………………………………13 3.Capsaicin induces TRIB3 expression in gastric cancer cell lines……………………………………………………………………………………………………………………………………14 4.Capsaicin induces TRIB3 expression and gastric cancer cell apoptosis……………………………………………………………………………………………………………14 5.TRIB3-Myc expression plasmid construction and overexpression in gastric cancer cell lines ……………………………15 6.Overexpression TRIB3 decreased gastric cancer cell proliferation and enhances the capsaicin induced apoptosis ………………………………………………………………………………………………………………………15 7.Knockdown TRIB3 decrease the capsaicin induced gastric cancer cell apoptosis ……………………………………………………………………………………16 8.TRIB3 regulated the capsaicin induced gastric cancer cell apoptosis by inhibition of AKT activation ………………………………16 Chapter 4. Discussion ………………………………………………………………………………………………18 References ……………………………………………………………………………………………………………………………20 Figures ……………………………………………………………………………………………………………………………………2

Topics: 辣椒素, 胃癌, 細胞凋亡, Capsaicin, Gastric cancer, Apoptosis
Publisher: 生命科學院碩士在職專班
Year: 2014
OAI identifier: oai:ir.lib.nchu.edu.tw:11455/81126
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