| 摘要: | 近年來癌症發生率高居國人死亡率第一，根據國民健康局統計，2008年因肺癌喪命，占十大癌症死因第一位。臨床上肺癌可分為小細胞肺癌及非小細胞肺癌(non-small-cell lung cancer，通常稱作NSCLC)，其中後者發生率占85~88%。非小細胞肺癌治療方式主要以化學治療及標靶藥物治療為主。目前第一線化療藥物-順鉑(cis-platin)其作用機制為藥物進入細胞核中與DNA上相鄰的鳥嘌呤(Guanine)-N7形成共價鍵，經由順鉑與DNA交聯結合(cross-linking)造成DNA結構改變，使DNA無法進行複製、轉錄、轉譯等過程，進而抑制DNA與RNA合成。當DNA受損卻無法修復時，將會使細胞走向計畫性死亡，造成細胞凋亡(apoptosis)，如此達到殺死腫瘤細胞之目的。另一方面，非小細胞肺癌常會出現過量的上表皮生長因子受體(EGFR)促使癌細胞快速生長。目前非小細胞肺癌第二、三線標靶治療之藥物艾瑞莎(Iressa)及得舒緩(Tarceva)，皆為上表皮生長因子受體的酪胺酸酶抑制劑，藉由阻斷受體將訊號傳入細胞核中，亦可達到抑制腫瘤細胞增生的目的。本實驗中所使用之PD153035亦屬於酪胺酸脢抑制劑之一，但目前PD153035還未使用於臨床上。
本研究目的將針對非小細胞肺癌之標靶藥物-PD153035與DNA之間的結合作用，利用雷射光鉗系統以單分子層級來釐清藥物與DNA的結合模式；同時，透過改變藥物濃度，測量DNA分子在有、無PD153035時作用力隨拉伸長度的變化，並經由inextensible WLC model藉以分析DNA分子的特徵長度變化包括維持長度(Lp)及輪廓總長(Lc)。當藥物嵌入DNA時會造成DNA結構變得較為鬆軟(soft)，造成維持長度變短，同時藉由B-form DNA輪廓總長的變化量進而求出PD153035平衡結合親和力常數值。並透過van’t Hoff衍生方程式(ΔG = -RTlnKA)求出自由能變化量。實驗結果顯示，PD153035僅於1 mM sodium cacodylate低鹽濃度條件下嵌入DNA結構中，當藥物濃度為200 μM時趨於飽和，此時n = 12 ?b 0.92，代表每隔12個鹼基對即會有一藥物嵌入DNA結構中，平衡結合親和力常數值為1.34 ( ?b 0.53) × 104 M-1，自由能變化量為-5.53 ?b 0.92 kcalmol-1，表示PD153035在此條件下嵌入DNA結構中屬於自發性反應。未來希望利用自行設計之溫控系統，經由吉布斯自由能方程式(ΔG=ΔH-TΔS，G：自由能；H：焓值；S：熵值；T：溫度)，分析完整熱力學參數的變化量(ΔG、ΔH以及ΔS)。
Lung cancer is the most common cause of cancer-related death in human. According to the statistics from Bureau of Health Promotion, lung cancer is the first leading cause of cancer-related death in Taiwan in 2008. The main types of lung cancer are small cell lung cancer (SCLC) and non-small cell lung cancer (NSCLC). The occurrence of NSCLC accounts for as many as 85% to 88% of the cases. The primary treatment for NSCLC usually involves chemotherapy and target therapy. The chemotherapy involves cis-platin, which can get into nucleus, and form a variety of DNA adduct. In this adduct, the platinum is covalently bound to the N7 positions of adjacent Guanine bases, and cross-linked with DNA. For this reason, it is capable of changing DNA conformation, and a number of processes such as regulation, transcription, and translation are not working. When a cell with irrepairable DNA damage which switched on the apoptosis mechanism and, therefore, frequently used to kill the tumor cells. On the other hand, define EGFR is normally found on the surface of epithelial cells and is often over-expressed in NSCLC, which cause promotion of cancer cells proliferation. Presently, Iressa and Tarceva are the second/ third line target therapy. All of them are epidermal growth factor receptor tyrosine kinase inhibitor (EGFR-TKI), and which can block the signal transduction via nucleus. Hence, EGFR-TKI can inhibit the cancer cell proliferation. Besides, PD153035 was used in our study which is also EGFR-TKI but not clinical medicine.
In this study, we investigated PD153035-DNA interaction, and presented a novel single-molecule approach to validate the binding mode using optical tweezers system. In addition, we measured the force-extension curves of single λ-DNA molecule coexisting with PD153035 or not in various concentrations from 0 to 200 μM. We then utilized the inextensible wormlike chain model to determine the DNA persistence length (Lp) and B-form contour length (Lc) in the absence and presence of PD153035. We found that the persistence length was reduced by the presence of the intercalating agent, which suggests that the DNA molecules become softer due to the presence PD153035. On the other hand, the binding affinity constant (KA) ie explicitly related to the B-form contour length of DNA molecule in the absence and presence of PD153035. Furthermore, based on the van’t Hoff equation, ΔG = -RTlnKA, the binding free energy of PD153035-DNA interaction could be dertermined. In this study, we found that there is noticeable increment in Lc only in 1 mM sodium cacodylate, and the binding of PD153035 to λ-DNA began to saturate at about 200 μM. In addition, the exclucion number, the binding affinity constant, and the binding free energy are 12 ?b 0.92, 1.34 ( ?b 0.53) × 104 M-1, -5.53 ?b 0.92 kcalmol-1, respectively, indicating that the PD153035-DNA interaction tends to occur spontaneously. In the future, using our home-made temperature control system, together with utilizing the Gibb’s free energy function (ΔG = ΔH - TΔS, where G, H and S represent free energy, enthalpy, and entropy, respectively), we can determine the thermodynamic characteristics including changes in free energy (ΔG), enthalpy (ΔH), and entropy (ΔS) due to the presence of PD153035. |
