| Abstract: | 研究目的：Pemetrexed 為一多標的葉酸拮抗劑抗癌藥物，其作用機轉主要是抑制 TS (thymidylate synthase)、GARFT (glycinamide ribonucleotide formyltransferase)、DHFR (dihydrofolate reductase)三種酵素，這些酵素主導製造細胞DNA及RNA合成所需的thymidine及purine nucleotides，pemetrexed是藉由阻斷這些酵素的活性達到抑制腫瘤細胞生長之目的。目前pemetrexed廣泛地被使用於治療多種癌症，在台灣pemetrexed已經成為標準的晚期肺腺癌第一、二線化學治療藥物。但pemetrexed的抗癌作用機轉，仍有許多未明之處，有待繼續研究。本論文將以pemetrexed處理人類肺癌A549細胞株，探討其對肺癌之抑制效果及機轉。
研究材料及方法：以Pemetrexed處理人類肺癌A549細胞株，看此藥物抑制A459細胞生長的情形，是否造成細胞死亡，進一步以流式細胞儀分析此藥物對細胞週期之影響，發現細胞有S期停滯的情形，並以DAPI染色及TUNEL assay確認pemetrexed處理後的細胞有產生細胞凋亡的情形，之後檢測caspase-2,-3,-8,-9活化的情形，並以caspase抑制劑確定細胞凋亡與內生性(intrinsic pathway)及外緣性(extrinsic pathway)的細胞凋亡路徑有關；接著探討與S期相關的蛋白質分子的表現，發現cyclin A及Cdk2有上升的情形，同時也發現ERK，p53及P53的上游ATM在細胞處理pemetrexed後皆有增加的情形，分別以抑制劑來看對pemetrexed處理後細胞週期及細胞凋亡的影響。
研究結果：Comet彗星拖尾實驗結果顯示，pemetrexed處理造成細胞內DNA損傷，DNA合成受抑制，使細胞週期停滯於S期，抑制細胞的生長。由西方墨點法分析蛋白質的含量，發現調控細胞S期進行的cyclin A/Cdk2蛋白質表現量增加且活性增強。使用小分子干擾核糖核酸（siRNA）抑制cyclin A，及處理flavopiridol抑制Cdk2的活性，可以顯著降低pemetrexed對A549細胞所引起之S期停滯和細胞凋亡。此外，pemetrexed可以長時間的激活細胞外信號調節激?1/2（extracellular signal-regulated kinase-1/2，ERK1/2）之活性。若前處理ERK1/2特定的siRNA及ERK1/2的抑制劑(PD98059及U0126)可有效地抑制cyclin-A和Cdk2的表現，並降低pemetrexed引起的S期停滯和細胞凋亡的現象。表示pemetrexed誘導細胞S期停滯和細胞死亡是與Cdk2和cyclin A的活化和表現增加有關；而Cdk2和cyclin A的表現量增加則和ERK1/2的活化相關。此結果顯示，由ERK信號傳導活化了Cdk2/cyclin-A激?活性在pemetrexed誘導肺癌細胞S期停滯和細胞死亡過程中扮演一個重要的角色。進一步探討pemetrexed促進細胞死亡的作用方式，由TUNEL及DAPI染色結果顯示，處理pemetrexed後DNA產生斷裂，A549細胞會呈現細胞凋亡現象，同時伴隨著ataxia telangiectasia mutated (ATM)及其下游分子p53的磷酸化活化。前處理ATM抑制劑Caffeine後，可以抑制p53的磷酸化活化現象，顯示pemetrexed誘導的p53活化是經由ATM的訊息傳導而來。若處理p53抑制劑PFT之後，A549細胞死亡的現象如預期地被抑制，代表p53在pemetrexed所誘導的細胞凋亡路徑中確實占有重要的角色。接著，本研究發現A549細胞處理pemetrexed後，細胞內促凋亡分子PUMA、Bax、Fas、Fas-L、DR4及DR5大量表現，而且Bax由細胞質轉位至粒線體膜上。同時pemetrexed處理後caspase-3，-8及-9被活化；若前處理caspase抑制劑，可有效的抑制pemetrexed所造成的細胞凋亡現象，表示pemetrexed可透過內生性(Intrinsic pathway)及外緣性(Extrinsic pathway)兩路徑誘導A549之細胞凋亡。
結論：pemetrxed處理A549細胞後先造成DNA損傷，導致ERK1/2刺激的cyclin A/Cdk2活化，使細胞生長停滯於S期。DNA受損斷裂也激活了ATM及其下游轉錄因子p53，進而增加促細胞凋亡的分子PUMA、Bax、Fas及DR4/5等之表現，造成caspase-2、-8及-9的活化，進而活化了caspase-3，最終誘導A549細胞的凋亡。此研究結果顯示降低thymidylate synthase的活性或表現量及增加促凋亡分子(PUMA、Bax、Fas及DR4/5)之蛋白質表現量，可能是評估pemetrxed抗癌療效的標記分子；另外，如果用藥物使細胞無法進入S期，將使pemetrexed造成的細胞凋亡現象減少，所以提示臨床上pemetrexed與其他藥物合併使用時，須考慮藥物使用的順序，若先使用一個會減少細胞進入S期的藥物，之後再使用pemetrexed可能會減低這個藥物的效果。
Objective：Pemetrexed, a multitargeted antifolate with the ability to inhibit several enzymes, including thymidylate synthase、glycinamide ribonucleotide formyltransferase、dihydrofolate reductase, which involved in purine and pyrimidine synthesis, has demonstrated clinical activity in a broad array of solid tumors, and also has demonstrated promising single-agent activity in front-line and second-line treatments of non-small cell lung cancer. However, the molecular mechanism of pemetrexed-mediated antitumor activity remains largely unclear. In this study, the anti-tumor effects and mechanisms of pemetrexed were examined in human lung adenocracinoma A549 cell line.
Methods and Materials：Human lung adenocarcinoma cell line A549 was used to detect the cytotoxicity of pemetrexed. Flow cytometry was used to clarify the influence of cell cycle of A549 cells by treatment of pemetrexed. DAPI staining and TUNEL assay proved cell apoptosis caused by pemetrexed. The activities of caspase-2,-3,-8,-9 were measured and showed apoptosis was related to intrinsic and extrinsic apoptotic pathways. S-phase related proteins were checked and the expression of cyclin-A and Cdk2 increased after A549 cells were treated with pemetrexed. ERK, p53 and ATM activity was also increased by pemetrexed. Inhibitors of these proteins were used to detect their roles in S-phase arrest and apoptosis caused by pemetrexed in A549 cells.
Results：Comet assay showed that pemetrexed-induced DNA damage was accompanied by cell cycle S-phase arrest and cell death. These events were associated with increased cyclin-A and cyclin-dependent kinase 2 (Cdk2) protein levels and Cdk2/cyclin-A kinase activity. Knockdown of cyclin-A using small interfering RNA (siRNA), and inhibiting Cdk2 activity with flavopiridol, strikingly reduced S-phase arrest and apoptosis. Besides, pemetrexed induced sustained activation of extracellular signal-regulated kinase1/2 (ERK1/2). Knockdown of ERK1/2 using specific siRNA, as well as known inhibitors (PD98059 and U0126), effectively suppressed the expression of cyclin-A and Cdk2, as well as reduced S-phase arrest and apoptosis induced by pemetrexed. These data provide the first evidence that pemetrexed-induced S-phase arrest and apoptosis is associated with an increase in Cdk2 and cyclin-A expression and activation, which is ERK-dependent. These findings suggest that the ERK-mediated Cdk2/cyclin-A signaling pathway plays an important role in pemetrexed-induced S-phase arrest and cell death. To characterize the pattern of pemetrexed-triggered cell death in A549 cells, TUNEL and DAPI staining assays were performed. Results revealed that pemetrexed-triggered DNA fragmentation and cell apoptotic cell death. These events were correlated with ataxia telangiectasia mutated (ATM) and p53 phosphorylated activation. Pretreatment with ATM inhibitor caffeine inhibited p53 phosphorylation, indicating that p53 activation was through a ATM-dependent pathway. Moreover, pretreatment with p53 inhibitor PFT attenuated pemetrexed-induced apoptosis, suggesting p53 plays an important role in pemetrexed-mediated apoptosis. Furthermore, treatment with pemetrexed increased the expressed levels of pro-apoptotic molecules, PUMA, Bax, Fas, Fas-L, DR4, and DR5. Additionally, the translocation of Bax from cytosol to mitochondria was observed. Pemetrexed also stimulated caspase-2, -3, -8, and -9 activation in A549 cells and that treatment with caspase inhibitors significantly abolished cell death, suggesting pemetrexed induced both intrinsic and extrinsic apoptotic signaling pathways. Taken together, these finding indicated that treatment of A549 cells with pemetrexed induced DNA damage, subsequently ERK1/2 and cyclin A/Cdk2 activation, leading to growth arrest at S phase. DNA damage also stimulated the activation of ATM and its downstream transcriptional factor p53, up-regulated the expressed levels of anti-apoptotic proteins, such as PUMA, Bax, Fas, DR4, and DR5, and then activating caspase cascade, consequently leading to apoptotic cell death in A549 cells.
Conclusion：Our findings reveal that the decrease of thymidylate synthase and the increase of anti-apoptotic proteins (Bax, PUMA, Fas, DR4, and DR5) may serve as biomarkers for predicting responsiveness to pemetrexed. Besides, pretreatment of cells with drugs which decrease S-phase arrest of cells could reduce apoptosis induced by pemetrexed. This finding implies the sequence of drug combinations may influence the drug efficacy. When using a drug which prevents the cells into S-phase probably diminish the cytotoxicity of followed pemetrexed |
