對抑制表皮生長因子接受器標靶藥物產生繼發性抗藥性之肺腺癌細胞能量代謝特性之探討

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題名:	對抑制表皮生長因子接受器標靶藥物產生繼發性抗藥性之肺腺癌細胞能量代謝特性之探討 Molecular Insights into the Metabolism of Lung Adenocarcinoma Cell with Secondary Resistance to Epidermal Growth Factor Receptor – Tyrosine Kinase Inhibitors (EGFR-TKIs)
作者:	黃中玉 Huang, Chung-Yu
貢獻者:	醫學檢驗暨生物技術學系所高淑慧
關鍵詞:	肺癌;Gefitinib;癌症代謝;表皮生長因子接受器;抗藥性 lung cancer;Gefitinib;cancer metabolism;epidermal growth factor receptor;drug-resistance
日期:	2016
上傳時間:	2021-11-21 20:33:29 (UTC+8)
摘要:	在肺腺癌病患中具有高比例的表皮生長因子接受器(epidermal growth factor receptor, EGFR)基因突變，其主要突變於EGFR exon 19或21。EGFR突變後會促進細胞的生長與分化，活化的EGFR可以藉由活化MAPK、Akt以及JNKpathway來促進DNA的合成以及癌細胞的生長。帶有EGFR基因突變的肺癌，對於EGFR tyrosine kinase inhibitors類的藥物，例如：Gefitinib，具有敏感性；但有些病患會於Gefitinib治療六個月後產生抗藥性。本研究針對具Gefitinib敏感性的肺腺癌細胞(PE089)，以及具有繼發性抗藥性的肺腺癌細胞(Ire)，進行代謝差異實驗分析。我們發現Ire細胞的細胞移行能力大於PE089細胞。同時我們發現PE089和Ire細胞的粒線體和細胞核蛋白質有EGFR以及p-EGFR的存在。在免疫螢光圖中也發現EGFR可轉位分布於粒線體內。為了釐清EGFR對於粒線體功能的影響，我們利用海馬生物能量測定儀偵測兩者細胞的粒腺體耗氧量，結果顯示Ire細胞的粒線體耗氧量是PE089細胞的1.5倍。我們進一步分析糖解作用相關的調控蛋白，發現Ire細胞具有較高的monocarboxylate transporter 1 (MCT-1)、lactate dehydrogenase B、pyruvate dehydrogenase蛋白含量。最後我們給予細胞粒線體呼吸鏈的抑制劑oligomycin或MCT-1的抑制劑AZD3965，結果顯示oligomycin或AZD3965皆可以顯著性抑制PE089及Ire的細胞移行能力及生長速率。因此，我們推測Ire細胞的代謝特性與反向瓦氏效應 (Reverse Warburg Effect)癌症代謝理論有關。這些結果顯示了對於Gefitinib有抗藥性的病人轉而使用粒線體標靶藥物或AZD3965藥物治療的可能性。　Most of lung adenocarcinoma epidermal growth factor receptor (EGFR)-associated mutations are found in exon 19 or exon 21. These mutations cause hyperactivation of the EGFR signaling pathway leading to promoting cell proliferation and DNA synthesis. The patients harboring EGFR gene mutations are superior to the treatment of EGFR-Tyrosine Kinase Inhibitors (EGFR-TKIs), like Gefitinib. However, secondary resistance to EGFR-TKIs sometimes happens after treatment for 6 months. In this study, we examined the different metabolism compared between primary lung adenocarcinoma cancer cell line (PE089) and secondary Gefitinib-resistant lung adenocarcinoma cancer cell line (Ire). In wound healing assay, we demonstrated higher cell migration in Ire than in PE089. Then, we found that both EGFR and p-EGFR located in the mitochondria of PE089 cells and Ire cells. The mitochondrial localization of EGFR in both PE089 cells and Ire cells was also shown in immunofluorescent images. Next, we also found that 1.5 fold increase of mitochondrial oxygen consumption rate in Ire. In addition, we analyzed the levels of glycolysis-associated protein including monocarboxylate transporter 1 (MCT-1), lactate dehydrogenase B and pyruvate dehydrogenase. The results showed all of them increased in Ire cells. Finally, we treated cells with mitochondrial inhibitor (oligomycin) and MCT-1 inhibitor (AZD395) and found both cell migration and cell proliferation were decreased in PE089 cells and Ire cells. Based on our findings, we suggested that Ire cells may rely on oxidative phosphorylation, as “the Reverse Warburg Effect”, to promote tumor progression. These results support the concept of developing mitochondria-targeted therapies or the Reverse Warburg Effect inhibitors for patients with drug resistance to EGFR-TKIs.
描述:	碩士指導教授：高淑慧
資料類型:	thesis
顯示於類別:	[醫學檢驗暨生物技術學系所] 博碩士論文

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