- 學位論文
探討粒線體之型態在c-Met誘發之抗藥性肺癌細胞株中的變化
Mitochondria Morphological Change in c-Met Mediated Drug Resistant Lung Cancer Cells
摘要
粒線體是細胞維持生理運作的中樞，在癌症中更是參與了腫瘤增生，對癌細胞適應環境具有支持性的作用。隨著癌症治療中伴隨的抗藥性問題日益嚴重，探討粒線體在癌症抗藥中所扮演的角色有機會為癌症治療提供一個新的標的。本篇研究中我們以肺腺癌細胞作為研究模型，透過粒線體螢光染色進行影像分析，建立一套癌症抗藥性評估用的粒線體監控方法。選用細胞為HCC827及PC9細胞株，帶有表皮生長因子接受器（epidermal growth factor receptor, EGFR）基因突變，而EGFR突變正是發生在亞洲非小細胞肺癌(non-small cell lung cancer, NSCLC)病人中最大比例的突變。臨床上治療帶有EGFR突變的案例通常會給與病人針對EGFR的標靶藥物酪胺酸激酶抑制劑（tyrosine kinase inhibitors, TKI），但是病人多半會於一年左右產生抗藥性，這也成為臨床治療上的困境。已知受體酪胺酸激酶c-Met是造成EGFR-TKI抗藥的其中一條途徑，然而詳細機制目前尚不清楚。本篇研究中我們設計了一套針對c-Met激酶區段外顯子14到21的引子，以期供臨床檢驗c-Met突變檢測使用。另外我們試圖解析c-Met在我們的細胞模型中於抗藥機制所扮演的角色，透過西方點墨法分析發現c-Met在我們的兩種細胞株中可能具有不同功能。同時我們利用前述的粒線體監控方法嘗試分析粒線體與c-Met於抗藥機制中是否有相互調控的關係，然而沒有發現在抗藥性及非抗藥性細胞株中粒線體分型出現差異，免疫螢光染色也沒有觀察到c-Met與粒線體螢光明顯重合的現象。這些結果暗示c-Met在細胞中透過調控粒線體達成細胞對EGFR-TKI抗藥此一假設在實驗室的HCC827及PC9細胞株中並不成立，然而這樣的調控機制是否會存在於其他c-Met有大量表現的細胞中尚需進一步研究。
並列摘要
Mitochondria are important bioenergetic and biosynthetic organelles, providing cell energy, controlling cell metabolism, and mediating cell death. In cancer, mitochondria play an important role in tumorigenesis, supporting cancer cells adapt to the environment. As drug resistance of cancer therapy has become a troubling problem, exploring the role of mitochondria in drug resistance may provide a potential cancer therapy target on mitochondria. Here we used lung adenocarcinoma cell lines as models, accessing to image-dependent mitochondrial screening strategies for cancer drug resistance evaluation. The chosen cell lines HCC827 and PC9 harbor EGFR mutant, which occurs as a majority in non-small cell lung cancer (NSCLC) mutation in Asia. EGFR tyrosine kinase inhibitor (TKI) is a commonly used therapy on patients harboring EGFR-mutation, however also faced with drug-resistance dilemma. One of the pathways to develop EGFR-TKI resistance is through c-Met, however mechanism is still unclear. In this research we designed 7 pairs of primers for c-Met exon 14 to 21 mutation sequencing. On the other hand we tried to examine the role of c-Met in drug-resistance in our cell models, and through Western blotting we found that c-Met may have different roles in the two cell models. Besides, with mitochondria screening strategies described previously, we attempted to resolve the relationship between mitochondria and c-Met in drug-resistance, however we didn’t observe a colocalization between c-Met and mitochondria. These results indicated that our hypothesis didn’t work in our HCC827 and PC9 cells. Whether this hypothesis could be proved in other high c-Met expressing cells is worth investigating.