研究抗愛寧達藥物之肺癌細胞的抗藥性及對轉移之影響

Investigation of the drug resistance and migration in Alimta-resistant lung cancer cells

齊婕妤(Jie-Yu Chi)

指導教授 ： 許國堂

中山醫學大學/醫學院/醫學研究所/碩士(2011年)

https://doi.org/10.6834/CSMU.2011.00020

摘要

全世界的死亡率一直以癌症位居第一，其中又以肺癌的死亡率增加最快，雖然隨著醫療的進步發展，但治癒率仍然很低。癌症之所以能夠威脅生命是因為將近90% 以上的癌症死亡是癌細胞轉移擴散所致，其主要原因是對一般的癌症治療產生抗性。一直以來，肺癌病患對化學藥物治療產生抗藥性是臨床上急需解決的問題，而愛寧達 (Alimta) 雖然為近十年來新一代治療肺癌的新興藥物，但仍有抗藥性的問題尚未解決。所以，如何解決肺癌患者產生抗藥性的問題是現今醫學上所關注的。本實驗利用臨床上新興的抗代謝的化療藥物-愛寧達 (Alimta) 的抗藥細胞株，希望藉此了解抗藥機制以及轉移機制。
從感受性的實驗結果首先發現到，CL1-0 Alimta 抗藥株除了 vincristine、5-FU 之外，對大部分的化療藥物有交叉抗性的現象；此結果或許可作為臨床二次用藥上的參考。接著進一步得知，Thymidylate synthase (TS) 高度表現於抗藥株中，是抗藥株對 Alimta 較不敏感的原因，而當利用lentivirus-shRNA system將抗藥株抑制 TS 後即部分恢復對 Alimta 的敏感性；此外，將抗藥株抑制 TS 後並不影響對其他化療藥物的感受性。所以 TS 高度表現於抗藥株中與其化療藥物有交叉抗性的現象無關。另一部分，利用移行試驗往下探討抗藥株的移行能力，發現這三株 Alimta 的抗藥株中，有兩株移行能力較強的細胞株 (CL1-0/A100-3、CL1-0/A200-3) 以及一株移行能力較弱的細胞株 (CL1-0/A200-49)。其中發現到，CL1-0/A100-3、CL1-0/A200-3會透過 epithelial-mesenchymal transition (EMT) 的形式以具有較高的移行能力，然而這樣的機制可能是受到 pErk pathway 所調控；進一步將 CL1-0/A100-3加入 MEK inhibitor (U0126) 處理後，發現似乎有反轉 EMT 的能力。
歸納總結來說，TS 在細胞對 Alimta 感受性上扮演著非常重要的角色；另外具有較高移行能力的抗藥株會透過進行 EMT 的形式以具有較高的移行能力可能是受到 pErk pathway 的調控。

關鍵字

肺癌 ； 愛寧達 ； 抗藥性 ； 轉移

並列摘要

Cancer has ranked the number-one cause of death in worldwide. Among them, lung cancer death rate is the fastest growing. Despite therapeutic advances, the cure rate for lung cancer remains low. Metastasis is responsible for as much as 90% of cancer-associated mortality; the main reason for metastasis is the general resistance to cancer treatment. All along, the lung cancer patients on chemotherapy resistance is an urgent clinical problem. Although Alimta is a new generation antifolate over the past decade and retains activity in many cell lines resistant to other antimetabolites, the cellular resistance mechanisms to Alimta have not been well resolved. In this study, we used the Alimta resistant cell lines, hoping to understand the mechanisms of resistance to Alimta and the mechanisms of metastasis.
From MTT assay, we observed cross-resistance to most chemotherapy drugs in these three CL1-0 Alimta resistant cell lines (CL1-0/A100-3、CL1-0/A200-3 and CL1-0/A200-49), except vincristine and 5-FU. The data may be used as a reference on the choice of the second line of drugs. Furthermore, we also found thymidylate synthase (TS) expression is up-regulated in these three Alimta resistant cell lines, suggested that TS might be associated with the acquired resistance to Alimta. We applied letivirus-shRNA system to knockdown TS expression in these Alimta resistant cell lines and the Alimta cytotoxicity was enhanced when TS was suppressed. Interestingly, knockdown of TS did not affect susceptibility to other chemotherapeutic drugs. The data suggested that TS is not associated with the cross-resistance in Alimta resistant cells. Another part, we utilized migration assay to measure the ability of migration in these Alimta resistant cell lines. We found that two resistant cell lines have higher migration ability (CL1-0/A100-3、CL1-0/A200-3) and one (CL1-0/A200-49) has lower migration ability. Further investigation showed that higer migration ability is due to epithelial-mesenchymal transition (EMT) occurred in Alimta resistant cells. The mechanism of EMT seemed to be regulated by pErk pathway, since when we treated MEK inhibitor (U0126) in CL1-0/A100-3 found that EMT might be reversed.
To summarize, TS overexpression may have an important role in the acquired resistance to Alimta. The resistant cell lines which have higher migration ability are due to EMT and the mechanism may be regulated by pErk pathway.

並列關鍵字

lung cancer ； alimta ； drug-resistance ； metastasis

參考文獻

Alberg, A.J., Brock, M.V., and Samet, J.M. (2005). Epidemiology of lung cancer: looking to the future. J Clin Oncol 23, 3175-3185.

Assaraf, Y.G. (2007). Molecular basis of antifolate resistance. Cancer Metastasis Rev 26, 153-181.

Barr, R.K., and Bogoyevitch, M.A. (2001). The c-Jun N-terminal protein kinase family of mitogen-activated protein kinases (JNK MAPKs). Int J Biochem Cell Biol 33, 1047-1063.

Barrallo-Gimeno, A., and Nieto, M.A. (2005). The Snail genes as inducers of cell movement and survival: implications in development and cancer. Development 132, 3151-3161.

Bertino, J.R. (1993). Karnofsky memorial lecture. Ode to methotrexate. J Clin Oncol 11, 5-14.