第二相抗氧化酵素是A549人類肺腺癌細胞在低氧狀況下抗藥性的重要決定因子

Abstract

肺癌在台灣及全世界的癌症死亡排名一直蟬聯首位。肺癌的治療策略大部份以含鉑藥物的化療配合基因篩檢的標靶治療為主。但是癌細胞對化療藥物的抗藥性以及化療副作用使肺癌的根治困難。因此增加化療藥物療效，或是使用合併藥劑減輕化療藥物使用劑量，一直是大家熱衷的議題。癌細胞在人體內所處的微環境並不一致，在遠離微血管或處於腫瘤中心的微環境通常較缺氧，使得適應惡劣環境的癌細胞更惡化，產生較強的抗藥性，但是對於癌細胞在缺氧環境增加抗藥性的機制並不甚瞭解。因此，在本篇研究中，我們將探討藉由抑制A549非小細胞肺腺癌細胞株適應缺氧的機制的啟動，是否可以回復肺癌常用化療藥物順鉑的療效。研究結果顯示，跟正常氧氣量的細胞相比，缺氧的狀態下的A549細胞對順鉑有更強的抗藥性。接著我們發現可以抵銷活性氧化物質的第二相抗氧化酵素 (phase II antioxidant enzyme) 的製造，如glutathione-cysteine ligase catalytic subunit (GCLC)及 glutathione-cysteine ligase modifier subunit (GCLM)，在8小時內的低氧環境下的表現量均有上升，但是另一個下游酵素NADPH dehydrogenase Quinone 1 (NQO1)則無改變。我們利用GCL的抑制劑buthionine sulfoximine (BSO)或NQO1的抑制劑 Dicoumarol加上順鉑的合併投藥，可以發現順鉑的抗癌效率大大增加，而且上游Nrf2的蛋白質表現量也同樣出現增加的趨勢。Nrf2是個轉錄因子，它的表現可促進下游基因產生可以第二相抗氧化酵素，抵銷細胞在低氧環境壓力產生的活性氧化物質 (ROS) ，使得ROS沒有在24小時之內增加。我們認為，利用Nrf2與其下游第二相抗氧化酵素的抑制劑與化療藥物順鉑的合併投藥的治療方式也許可以改善現今臨床上對於肺癌治療上面臨的抗藥性及副作用的限制。

Lung cancer remains the leading cause of death in Taiwan and worldwide. The main treatment for lung cancer is chemotherapy combined with target therapy, but the drug resistance and side effects make it hard to overcome the disease. Therefore, drug resistance and side effects of chemotherapy still considered as important issues. The tumor microenvironments differ in tumors. Previous researches have shown that cancer cells in hypoxic microenvironment are more aggravated and more resistant to cisplatin, a locally advanced chemotherapy drug used in the first-line treatment of non-small-cell lung cancer (NSCLC). Here we discussed that by inhibiting the mechanism of hypoxic adaptation could we return the efficacy of common anti-cancer drug cisplatin. The result showed that A549 human lung carcinoma cells had a stronger drug resistance under hypoxic condition than normal oxygen state. We used the combination of cisplatin with buthionine sulfoximine (BSO), a glutamine-cysteine ligase (GCL) inhibitor, or dicoumarol, a NQO1 inhibitor, leading more cell death under both hypoxic and normoxic condition. Western blot showed the increased protein expressions of glutamine-cysteine ligase catalytic subunit (GCLC) and glutamine-cysteine ligase modifier subunit (GCLM). However, another phase II antioxidant enzyme, NADPH dehydrogenase Quinone 1 (NQO1), didn’t change in protein level. Also, we spotted the increased protein level of Nrf2, a transcription factor which induce antioxidant pathway, which triggers its downstream genes to synthesize phase II enzymes. The induced Nrf2 and its downstream phase II enzymes might consume the reactive oxygen species (ROS) produced under hypoxia, resulting in steady levels of ROS under hypoxia within 24 hours. To summarize the result above, the present study reveals that using the combination of inhibitor of Nrf2 antioxidant pathway and cisplatin may be a better strategy to decrease the dose usage of anti-cancer drug and the side effects but maintaining the efficacy of chemotherapy.