Epi-Reevesioside F 及其合併Gefitinib 在人類非小細胞肺癌的抗癌機轉研究

Abstract

癌症問題長時間困擾全球人類，成為世界三大死因之一。而其中，肺癌更是造成癌症死亡率居高不下的主因之一。大多數的肺癌可被歸類為非小細胞肺癌(non-small cell lung cancer)，因此，開發出有效的治療策略來對付非小細胞肺癌極為急迫且重要。標靶治療是現今主流的癌症治療策略之一，更是可以用來對付晚期非小細胞肺癌的方法，即便如此，晚期非小細胞肺癌的存活率仍只有6%。此外，標靶藥物依然存在毒性、副作用及潛在抗藥性的問題。因此，合併使用其他藥物或可成為降低使用劑量以降低藥物毒性及副作用的治療策略之一。我們的合作團隊從臺灣梭羅木中萃取出一系列的化合物，進行篩選後，發現Epi-Reevesioside F 對於細胞生長的抑制效果最好。Epi-Reevesioside F可透過抑制鈉鉀幫浦的活性、增加細胞內鈉離子含量、抑制細胞週期、mTOR及其下游的蛋白，來達到抑制癌細胞生長的效果。而後再使用該化合物合併現有臨床藥物進行篩選以選出最佳的合併治療組合。使用流式細胞儀分析PI染色結果後發現，將Epi-Reevesioside F合併使用Gefitinib可以有效地增強Gefitinib所造成的細胞凋亡效果，造成Sub G1的比例顯著增加。相比之下，在單獨使用Epi-Reevesioside F的實驗結果中無法看到細胞凋亡的現象。除此之外，我們也透過西方墨點法來確認合併療法處理過的細胞的蛋白表現，發現Epi-Reevesioside F 確實以透過增強細胞凋亡相關蛋白的表現來增進Gefitinib誘導的細胞凋亡現象，包含caspase級聯反應及Bcl-2家族蛋白的表現。此外，我們也發現合併療法可以透過影響細胞週期來抑制癌細胞的生長。依據caspase 級聯反應及Bcl-2家族蛋白的實驗結果，特定死亡受器DR4及DR5在NCI-H460 細胞株的表現，及鈣離子含量在A549細胞株的表現皆被偵測且表現量明顯上升。與此同時，透過西方墨點法也證實Epi-Reevesioside F可以增強Gefitinib誘導的MAPK訊息傳遞路徑及影響FoxO家族的出核現象。總結來說，透過實驗證實合併療法可以活化caspase聯級反應、Bcl-2蛋白家族、細胞週期蛋白的表現、MAPK訊息傳遞路徑及影響FoxO家族的出核現象。不僅如此，DR4/DR5在NCI-H460 細胞株及鈣離子含量在A549細胞株的增加也已被證實。相較於單獨使用Gefitinib，合併使用Epi-Reevesioside F確實可以增強癌細胞的凋亡現象，證實合併療法的潛力。

Cancer persecutes human for a long time and becomes one of the top three mortalities worldwide. In all types of cancers, lung cancer is one of the main reasons for persistently high lethal rate. Most lung cancers are categorized into non-small cell lung cancer (NSCLC). Thus, developing an effective strategy to treat NSCLC is important and urgent. Targeted therapy is one of the major modalities of cancer treatment, and is also an effective strategy to treat advanced NSCLC. Even so, the survival rate of advanced NSCLC remains 6%. Besides, targeted therapy still exists some problems, including tolerability, adverse effects, and probable resistance. Therefore, the major specific aim of the thesis was doing a combination therapy study in which the therapeutic targeted drug and a potential research compound were combined to lower the given dose of the targeted drug to reduce the side effects. Epi-Reevesioside F, purified and provided by our colleagues, showed the highest efficacy on cell growth inhibition. It inhibited the cancer cell growth via suppressing the activity of Na+/K+-ATPase, increasing the intracellular Na+ concentration, inhibiting the cell cycle, and mTOR and its downstream proteins. Subsequently, Epi-Reevesioside F was used in this study for the combination treatment. The data of flow cytometry with PI staining showed that Epi-Reevesioside F synergistically increased Gefitinib-induced apoptosis identified by an increased Sub-G1 population of the cell cycle. Notably, Epi-Reevesioside F, by itself, did not increase the Sub-G1 population. Furthermore, the examination of protein expression levels of apoptosis-related proteins by Western blot analysis demonstrated that combinatory treatment increased the expression of catalytically active (cleaved) caspases and Bcl-2 family proteins. Combinatory treatment also inhibited cell growth by regulating the cell cycle progression. Besides, the expression of DR4 and DR5 in NCI-H460 cells, and calcium concentration in A549 cells were also detected. Further, Epi-Reevesioside F synergistically potentiated Gefitinib-induced mitogen-activated protein kinase (MAPK) pathway and modified the expression levels of FoxO family proteins. In conclusion, our data suggest that Epi-Reevesioside F synergistically potentiates apoptosis induced by Gefitinib in NSCLC cell lines NCI-H460 and A549. The sensitization effects are evidenced by increased caspase activation, expression of Bcl-2 family members, cell cycle-related proteins, MAPK pathway, and altered FoxO expression. Increased DR4/DR5 expressions in NCI-H460 cells and intracellular concentrations Ca2+ in A549 cells were apparent in the combinatory treatment. Collectively, in contrast to Gefitinib alone, Epi-Reevesioside F could synergistically potentiate Gefitinib-induced anti-NSCLC effects.