| 摘要: | 在臨床觀察中發現患有惡性肝細胞癌的病人，容易有葉酸缺乏的情狀。因此本研究第一項目標著重在探討葉酸缺乏所誘導之氧化暨氮化壓力是否能激起氧化還原適應機制 (RA)，以及其對於肝細胞癌之多重抗藥性累積的潛在影響性。本研究發現肝癌細胞株中存在2種不同的氧化還原適應型態之細胞株：Hep G2細胞株被證實為不具氧化還原適應性 (RA-null) 的細胞株，因此當其面臨葉酸缺乏所誘導之氧化暨氮化壓力時，會陷入細胞凋亡的命運。然而，對比Hep G2，其他三種肝癌細胞株，包括：SK-Hep-1、Mahlavu及Hep J5，皆能逃脫葉酸缺乏所誘導之細胞凋亡，並轉變為對於多種抗癌藥物增加了抵抗性，包括：doxorubicin、paclitaxel、cisplatin及sorafenib，此葉酸缺乏誘導的藥物抵抗性被證明是：氧化還原適應機制所調控之葡萄糖調控蛋白78 (GRP78) 及survivin的增加，這些關鍵蛋白藉由降低氧化暨氮化壓力而促進細胞的存活能力。在進一步的研究指出，抑制GRP78及survivin的表達能降低還原穀胱甘肽 (GSH) 的合成酵素 ??-GCSh的表現，此現象意味著此兩種關鍵蛋白能藉由增加GSH的合成進而增加對於藥物的抵抗性，順著這樣的思路，抑制GRP78及survivin的表達能增加活性氧 (ROS) 的產量，也意味著這兩種關鍵蛋白具有ROS清除者的角色。最後，第一項目標的研究貢獻了一個重要的成果：補充葉酸可降低具有氧化還原適應性的 (RA-prone) 肝癌細胞株的抗癌藥物抵抗性，因此意味著微量維生素「葉酸」能增加細胞對於藥物的敏感性。
接下來，我們研究一種多標的型抗葉酸藥物 (Pemetrexed) 和葉酸缺乏對於肺腺癌細胞株，包括CL1-0、CL1-5 及A549的影響。這些細胞在葉酸缺乏的情況下，可顯著地改變細胞的型態，展現了間質細胞及變形蟲的型態，有趣的是，pemetrexed所誘導的適應反應 (模擬了葉酸缺乏的情況)，可藉由活化NF-?羠調控了上皮-間質轉換 (EMT) 相關的生物指標，包括N-cadherin、vimentin、金屬催化型蛋白酶 (MMPs) 和 slug的大量表現。除了型態之改變及EMT相關的生物指標之上昇外，我們更發現無論是葉酸缺乏或pemetrexed處理後的肺腺癌細胞株，在細胞侵襲能力上，皆較未處理的對照組細胞增加了三倍以上。這些有趣的發現促使我們進一步測試處理過pemetrexed的肺腺癌細胞株對於erlotinib及gefitinib的敏感性是否有改變，由於pemetrexed誘導N-cadherin及slug的過度表現，此兩種影響因子已經被報導其功能涉及藥物抵抗性的增加，因此這兩種影響因子可能貢獻於增加對erlotinib及gefitinib的抵抗性。同時藉由降低slug的表達，不只顯著地降低pemetrexed所誘導的侵襲能力，也增加了肺腺癌細胞株對於erlotinib的敏感性。總結第二項目標的研究，我們發現了在非小細胞型肺癌的治療中，當細胞先使用pemetrexed 後，再處理erlotinib或gefitinib時，存在有上述兩種潛在的不利副作用，因此我們建議在先處理pemetrexed，而後使用erlotinib或gefitinib的治療策略，需重新詳細的評估其安全性是必要的。
Patients with hapatocellular carcinoma (HCC) are prone to folate deficiency (FD). Therefore, the first specific aim of this research project is to examine whether or not FD-induced oxidative-nitrosative stress (ONS) can evoke redox adaptation (RA) and its potential impacts on the multi-drug resistance (MDR) acquisition of HCC cells. In this study, two RA-mediated phenotypes of HCC cells have been identified. Hep G2 cells, being classified as a RA-null cell type, underwent apoptosis when exposed to FD-induced ONS. However, in sharp contrast to Hep G2 cells, three other HCC cell lines including SK-Hep-1, Mahlavu and Hep J5 were found to be capable of evading FD-induced apoptosis and becoming MDR against a group of ROS-producing anti-cancer drugs including doxorubicin, paclitaxel, cisplatin and sorafenib. This FD-evoked MDR acquisition was proved to be linked to the RA-mediated upregulation of glucose-regulated protein 78 (GRP78) and survivin, which decreased ONS and promoted survival. Further studies indicated that both GRP78 and survivin silencing were associated with the downregulation of ??-GCSh, a catalytic subunit of GSH biosynthesis implying that both effectors might influence drug resistant attribute by enhancing GSH biosynthesis. Along the same vein, both GRP78 and survivin silencing drastically increased the generation of ROS implying that both effectors functioned as a ROS sinker. Finally, we demonstrated that FD-induced MDR acquisition in RA-prone HCC cells could be restituted either partially or even completely by folate replenishment. This finding implies that folate micronutrient per se can confer cells with the capacity to reverse MDR acquisition.
Next, we investigated the effects of FD or pemetrexed, a multi-targeted antifolate agent, on a group of lung adenocarcinoma cell lines (CL1-0, CL1-5 and A549). Cultivation of these cells under FD condition resulted in drastic change of morphology showing mesenchymal to amoeboid phenotype. Interestingly, pemetrexed could induce adaptation response, in a mimicry of folate depletion episode, by increased activation of NF-?羠-mediated overexpression of epithelial-mesenchymal transition (EMT) biomarkers including N-cadherin, vimentin, metalloproteinases (MMPs) and slug. Besides morphological and EMT biomarkers changes, cell invasion ability of either FD or pemetrexed-treated cells was 3-fold higher than the pair-matched control cells. These interesting findings prompted us to test the sensitivity of these lung cancer cells toward erlotinib and gefitinib following pemetrexed pretreatment. Our data unveiled that pemetrexed could unilaterally trigger the overproduction of N-cadherin and slug, two newly identified resistance-acquiring effectors that contributed to increased gefitinib resistance. Knockdown of slug significantly reduced pemetrexed-induced invasiveness and erlotinib resistance acquisition. Collectively, we uncovered two potential adverse effects that can occur when pemetrexed is used prior to erlotinib and gefitinib in the treatment of non-small cell lung cancer. Thus, we suggest that a detailed reappraisal of the safety of erlotinib and gefitinib after the treatment of pemetrexed is warranted. |
