探討 alpha 7 尼古丁型乙醯膽鹼受體(A7-nAChR)表現量與胃癌對於微管抑制劑藥物敏感性的角色

The role of A7-nAChR in the chemosensitivity of gastric cancer cells to microtubule inhibitors

塗昭江(Chao-Chiang Tu)

指導教授 ： 張育嘉

共同指導教授 ： 魏柏立(Po-Li Wei)

臺北醫學大學/醫學院/臨床醫學研究所/博士(2016年)

摘要

根據世界衛生組織的統計資料,胃癌是全世界排名第五位最常見的癌症。經由防治胃癌致癌因子以及推動篩檢計畫,近二十年胃癌的發生率有逐年下降的趨勢,然而胃癌整體預後依舊不佳,其死亡率為所有癌症死亡率排行的第三名。為了改善胃癌病人的預後以及生活品質,建立全世界胃癌治療的共識和個人化的治療刻不容緩。
抽菸是胃癌的致癌因子之一,香菸中的尼古丁或尼古丁衍生物 (NNK)可透過活化細胞上的alpha7尼古丁型乙醯膽鹼受體(A7-nAChR) 進行細胞增生、轉移、血管新生或細胞凋亡等作用。過去研究發現 A7-nAChR的表現量對胃癌細胞增生及轉移上扮演重要角色,但是 A7- nAChR表現量與藥物治療效果的相關性,仍不是很清楚。本研究主要 目標是探討A7-nAChR表現量在微管抑制劑藥物治療胃癌上藥物敏感性的角色。
紫杉烷(taxane)屬抑制微管(microtubule)的抗腫瘤藥物,主要是運用在晚期或轉移性胃癌的化療藥物,而使用後約有三成的胃癌病人將 產生抗藥性。易莎平(ixabepilone)是新一代抑制微管蛋白藥物,結構與 紫杉烷不同,但其作用機轉類似。易莎平主要作為對紫杉烷產生抗藥 性的病人的替代藥物, 但缺點是該藥物毒性大且易產生嚴重副作用。 紫杉烷及易莎平都是治療胃癌的重要藥物,如果可以改善抗藥性或提 升藥物敏感性對於胃癌治療將有莫大的助益。
藉由小干擾RNA(small interferering RNAs)抑制A7-nAChR表現 量,進一步探討A7-nAChR 表現量在胃癌細胞對於微管抑制藥物敏感 性的影響。我們在細胞存活率分析實驗中(MTT assay)發現,A7-nAChR表現量降低時會增加人類胃癌細胞對紫杉烷及易莎平的抑癌反 應。使用流式細胞儀進行細胞週期及細胞凋亡分析結果也發現A7- nAChR表現量降低時會抑制細胞週期、增加紫杉烷及易莎平對於胃癌 細胞毒殺作用及促進細胞凋亡。 最後我們分析細胞凋亡的相關調控因 子發現,抑制A7-nAChR表現量可能藉由減少抑制細胞凋亡蛋白pAkt 誘導人類胃腺癌細胞走向自體凋亡的途徑。我們證實A7-nAChR的表 現量高低會影響紫杉烷及易莎平對於胃癌細胞的毒殺效果。我們的發 現可以提供胃癌治療及藥物運用上的新方向,將來可以藉由偵測病人 胃癌組織A7-nAChR的表現量選擇化療藥物、減少藥物抗性及減少化 療藥物相關併發症,繼而發展胃癌個人化的治療。

關鍵字

胃癌 ； alpha 7 尼古丁乙醯膽鹼受體 ； 紫杉烷 ； 易莎平

並列摘要

According to the latest global cancer statistics, gastric cancer is the fifth most common malignancy in the world. The incidence rate of gastric cancer has gradually declined over the past decades under growing awareness of potential carcinogens and screening programmes for the early detection. Nevertheless, the outcome of gastric cancer still remains unsatisfied and stomach cancer is the third leading cause of cancer death in the world. To improve prognosis and quality of life of gastric cancer patients, both standardization and individualization of treatments are imperative.
Smoking is a well-known risk of gastric cancer. Tobacco-specific mitogen, nicotine and nicotine-derived nitrosamine ketone(NNK) enhance gastric cancer cell metastasis through alpha7 nicotinic acetylchoine receptor(A7-nAChR). Previous study demonstrated that the expression of A7-nAChR was associated with gastric cancer proliferation and metastasis. A7-nAChR could be a potential target for gastric cancer. But the current knowledge between A7-nAChR and the drug efficacy is less discussed. With this study we are going to explore the role of A7-nAChR in the chemosensitivity of gastric cancer cells to microtubule inhibitors.
Taxanes, one of the most famous microtubule inhibitors, are widely used in anti-cancer treatment. Taxane-based regimens are usually used for the treatment of advanced or metastatic gastric cancer patients, which are benefit for overall survival and quality of life. Unfortunately, resistance develops in 30% taxane-treated patients and complex resistant mechanisms are responsible for taxane resistance. Ixabepilone, an analogue of epothilone B, also disrupt microtubule dynamics by binding to the β- tubulin subunit and stabilize microtubules. Ixabepilone had demonstrated clinically efficacy in patients refractory to taxanes. But major adverse effects of ixabepilone such as bone marrow suppression and neurotoxicity often limit the dosage and efficacy.
In our study, gastric cancer cells were subjected to A7-nAChR knockdown using short interfering RNA. The anti-proliferative effects of are assessed via MTT array and the results show silencing of A7-nAChR will enhance chemosensitivity of gastric cancer to taxane and ixabepilone. The cell cycle distribution assay and apoptosis are assessed by flow cytometry, TUNEL assay and annexin V/propidium iodide (PI) apoptotic assay. The results demostate that silencing of A7-nAChR expression will inhibit cell ccycle, enhance cytotoxic effects of taxane and ixabepilone and promote apoptosis. We also analyse the apoptosis related factors and the result demostarte silencing of A7-nAChR expression may induce apoptosis through decreasing pAkt. All these laboratory findings indicate that A7- nAChR-KD cells are more sensitive to microtubule inhibitors. Our findings offer new ways to select optimal drug and to boost drug efficacy for gastric cancer patients. Thus, we can choose the anti-cancer drugs depending on the expression of A7-nAChR in the future to develop personized treatment in gastric cancer patients

並列關鍵字

gastric cancer ； A7-nAChR ； taxane ； ixabepilone

參考文獻

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