| Abstract: | 從 1977 年以來肺癌為國人重要的癌病殺手。在台灣每 5 個癌症死亡人口便有一個 死於肺癌。然而雖然隨著近年來治療的發展有顯著進步，肺癌仍然還是維持在預後很差 的疾病。因此提高病人的存活率並降低藥物毒性，一直都是努力的治療方向。肺癌主要 可分為小細胞肺癌 (SCLC) 及非小細胞肺癌(NSCLC)兩大類。非小細胞肺癌包括腺癌、 鱗狀上皮癌與大細胞癌。大多數這類病人發現時都已屬無法開刀的末期，而且對於化療 及放療多不敏感，手術後發生移轉和復發的機率很高。近年台灣肺癌病患以罹患肺腺癌 的比例最高，特別是女性患者中，比例高達七成，肺腺癌已成為婦女的重要死因，尤其 是其抗藥性問題是目前臨床治療的最大困擾。根據臨床經驗，大部份非小細胞肺癌患者 由於藥物無法有效控制，因此從診斷出至死亡一般不會超過一年。其五年的存活率只有 14%，所以發展新的安全又有效的治療方式來做臨床治療和避免或抑制抗藥性的產生就 顯得非常重要。 Pemetrexed愛寧達(Alimta) 注射劑型是目前治療非小細胞肺癌的化療藥物。愛寧達 似乎是對非小細胞肺癌中的非鱗狀上皮癌(non-squmous types)有較佳的療效，因此被推 薦用來治療非鱗狀上皮類之肺癌。愛寧達每 3週 1次的治療比其他的化療藥物方便。接 受愛寧達治療的病人出現血液毒性副作用（嗜中性白血球減少、血小板減少、貧血）的 機率較低，且掉髮機率也少了一半，治療期間也較少需要輸血或輸液等醫療支持。因為 其治療肺癌的效果和一線藥物相同，因此自 2009 年二月起，衛生署已經核准愛寧達可 用於肺腺癌的第一線化療用藥，讓肺癌病患在化療第一時間就可以選用愛寧達，不必像 過去，得等到第一線化療失敗後才能使用。Pemetrexed (愛寧達)為葉酸拮抗劑，作用機 轉是分別抑制 glycinamide ribonucleotide formyltransferase (GARFT)，dihydrofolate reductase (DHFR)與 thymidylate synthase (TS)三個酶，而使其作用更廣泛。這些酵素蛋 白是癌細胞製造合成 DNA及 RNA所需 thymidine及 purine nucleotides的關鍵步驟，因 此可以藉此抑制作用來殺死癌細胞。可是目前對於愛寧達之抗藥性產生之機轉一般皆認 為是因為 TS 蛋白質高量所造成，對其餘的抗藥性基因並無深入的研究，基本上都是以 舊一代的單一作用之葉酸拮抗劑抗癌藥物產生之抗藥性做為論點，這和愛寧達是多重作 用之抗葉酸藥物的本質不相符。因此，研究肺癌對愛寧達之抗藥性產生之機轉就顯得非 常需要。 我們已經建立起兩株抗藥細胞株 (A549/A400 和 CL1-0/A200)並且經過基因微矩陣 分析來做為此次研究的材料和參考。從我們的初期數據顯示，愛寧達之抗藥基因可能依 據細胞株而有異有同，但是兩株抗藥細胞株都對 vincristine有比較高的敏感性。另外還 觀察到 CL1-0 /A200抗藥細胞株的癌症轉移能力會增強。 為此，我們提出三年的研究計畫包含下列三個目標：[壹]、找出重要的抗藥基因並 且抑制其表現或將其過度表達來觀察其與抗藥性產生之關聯。[貳]、探討是否可以使用 vincristine 之化療用藥做為當愛寧達治療抗藥性產生後時之後續藥物，並且使用小鼠模 式來驗證其可適用性。同時也要探討為何 vincristine 敏感性增加的相關機制。[叁]、研 究為何愛寧達之抗藥細胞株的癌症轉移能力會增強的相關機制。我們將從對愛寧達抗藥 細胞株的研究結果來得到如何來反轉其抗藥性的方法，進而延長病人之存活。並且研究 出適合使用愛寧達治療肺癌的參考基因來做為新的治療肺腺癌之策略依據。
In Taiwan, since 1997, lung cancer has been in the first cause of cancer death several times. Small-cell lung carcinoma (SCLC); represents about 15% of primary lung cancers and exhibits the most malignant phenotype of lung cancer. Non-small cell lung cancer (NSCLC) accounts for 85% of lung cancer patients and they are usually associated with poor prognosis, more than one half of NSCLC patients have advanced disease at diagnosis and beyond the limits of surgery. NSCLC include squamous-cell (epidermoid) carcinoma, adenocarcinoma, and large-cell carcinoma; bronchoalveolar carcinoma is generally subclassified under adenocarcinoma. Most individuals with lung adenocarcinoma (especially women) are nonsmokers. In spite of new and expensive chemotherapy treatments, the overall five-year survival rate remains about 14%. Obviously, novel therapeutic strategies to improve efficacy in accord with safety are urgently needed, but the drug resistance remains the important issue of chemotherapy. Since 2009, pemetrexed was approved as a first-line drug for non-squamous type NSCLC therapy in Taiwan. Pemetrexed (ALIMTA, LY231514, MTA, Eli Lilly and Company) is a novel multitargeted antifolate that inhibits one or several key folate-requiring enzymes of the thymidine and purine biosynthetic pathways, in particular, thymidylate synthase (TS), dihydrofolate reductase (DHFR), and glycinamide ribonucleotide formyltransferase (GARFT). These enzymes are involved in the synthesis of nucleotides, ultimately hindering ribonucleic acid (RNA) and deoxyribonucleic acid (DNA) synthesis. previouly studies have demonstrated the cytotoxic activity of this agent in a broad range of tumor types including NSCLC. Pre-clinical and clinical studies have identified a plethora of mechanisms of antifolate resistance with classical single targeted antifolates such as methotrexate (MTX). These include downregulation of the reduced folate carrier (RFC) and various alterations in the target enzymes DHFR, TS and folylpolyglutamate synthase (FPGS). Recently, high TS expression has been demonstrated as major factor for pemetrexed resistance. Whether all of above mentioned alterations would result in pemetrexed-resistance is not well demonstrated. Therefore, a clear characterization of the mechanisms to overcome pemetrexed-resistance is required. We have established two pemetrexed-resistant sublines (A549/A400 and CL1-0 /A200) followed by DNA microarry analysis. From these data, we found that genes associated with pemetrexed resistance are similar but varied in these two sublines. Only the high sensitivity to vincristine is identical. We also observed that higher migration and invasion abilities have been obtained in CL1-0 /A200 subline. Therefore, we propose a three-year research plan as following: (1) Characterization of the pemetrexed-resistance genes (TS, FPGS and others). Apply in vivo animal model for pemetrexed resistance and vincristine sensitivity. (2) Target the NEFL gene as the gene mediates vincristine sensitivity. Further investigate the role of promoter hypermethylation in NEFL expression. (3) Application of in vivo animal model and in vitro studies for pemetrexed resistance and metastasis. By using the pemetrexed-resistant cell lines of A549 and CL1-0, we can further understand the mechanisms of pemetrexed resistance and associated metastasis. Future application of our findings may benefit the management of lung adenocarcinoma therapy with pemetrexed and other drugs. |
