| 摘要: | 順鉑（cis-Pt(NH3)2Cl2）是一種常用的抗癌藥物。順鉑的生物活性作用在DNA上會有強大的共價鍵結合和在雙螺旋結構產生中間單官能基(intermediate monofunctional)和雙官能基產物（bifunctional）並讓結構扭曲。雙官能基產物大部分為intrastrand crosslinks，intrastrand crosslinks也是在DNA上形成。
然而，順鉑是高度的細胞毒性且具有一些副作用，有些副作用會限制了它的應用。故了解DNA結合機制與順鉑的抗癌特性，有利於創造新的更有效的藥物。因此，與順鉑DNA複合物的熱力學和其他研究的特定和鉑化合物等目前已在進行。在報告本研究之前，熱力學研究主要是短鍊的DNA，雖然長鍊的DNA與鉑的結合更接近真實系統：故在此研究裡，我們有針對長鍊 DNA來進行研究。利用微分掃描熱分析儀（DSC）和紫外光譜儀進行研究。利用已開發的電腦軟體來處理實驗數據，並用模型來對實驗結果進行分析解釋。
但在之前的研究裡，有一些錯誤的觀念。如：一、熔化曲線(metling curve), 熔點(metling temperature)，在DSC的實驗結果裡，熔點範圍(metling temperature range)有很強烈地區分。二、熔點和熔點範圍 沒有辦法從多峰 DSC 曲線獲得和使用。對此觀點，卻沒有任何證據。針對此一問題展開了研究。重新去計算”真實”DSC 熔化曲線。利用不同於在DSC軟體中所使用的計算方法，從不同熔化曲線，證明了DSC熔化曲線內的偏差是可以忽略。且發展一套適合的方法去量測熔點和熔點範圍在多峰DSC 曲線中且證實了其合理性。
在DNA和鉑化合物中的實驗，找到新的條件。且從實驗中，提供了兩個有用的優勢：首先，鉑化合物消除了長鍊 DNA的非特異性靜電穩定性(non-specific electrostatic stabilization)。其次，在高溫的熔化實驗（metling experiment）中鉑化合物提供了中間產物(intermediate products)穩定性。由於這些結果，由中間的單官能基(intermediate monofunctional)和最後的雙官能基產物(bifunctional product)所引起的熱穩定性（thermal stability）改變，可在單獨計量(separately measured) 和 對比第一次的無鉑結合的實驗。這表示鉑化合物會減少在雙螺旋線圈變化時(helix-coil transition)焓(enthalpy)和熵（entropy），增加熔點範圍和扭曲DSC熔化曲線。這與其他之前的結果相符合。然而這些破壞結構的因子 (destructive factors)永遠會降低 DNA熔點，但這只對短鍊 DNA如此。對於長鍊DNA卻不是。Transplatin和Pt[(dien)Cl]Cl 會增加DNA 熱穩定性(thermal stability)，雖然他們的雙螺旋被破壞掉。我們證明此不尋常的結果是因為帶有大量正電荷（positive charge）的鉑化合物與雙螺旋結合中而產生的。
有一些鉑化合物和其他抗癌藥物也會形成DNA interstrand crosslinks。為了要解釋我們的實驗結果，我們研究了短鍊 DNA和長鍊 DNA的DNA interstrand crosslinks 熔化結果。這也是第一次針對crosslinking來研究局部不穩定熱因子（thermal impact of local distortions）。
Cisplatin (cis-Pt(NH3)2Cl2) is a commonly used anticancer drug. Biological activity of cisplatin is exerted by strong covalent binding to DNA and distortion of the double helix by formation of intermediate monofunctional and final bifunctional products. The majority of bifunctional products are intrastrand crosslinks. Additionally, interstrand crosslinks are formed in DNA.
However, cisplatin is highly cytotoxic and has a number of side effects that limit its application. Understanding the DNA binding mechanisms and anticancer properties of cisplatin facilitates creation of new more effective drugs. Therefore, a lot of thermodynamic and other investigations of DNA complexes with cisplatin and other platinum compounds have been carried out. However, before the present study, thermodynamic investigations were mainly fulfilled for short DNAs, although long platinated DNAs are much closer to real systems. In this work, studies of long DNAs were carried out using differential scanning calorimetry (DSC) and UV spectroscopy. Computer programs have been developed and used for the processing of these experimental data. Computer modeling was carried out to explain obtained experimental results.
There are widely spread mistaken opinions that 1) Melting curve, melting temperature, temperature melting range obtained in DSC experiments strongly distinguish from their real values
2) Melting temperature and temperature melting range cannot be obtained and used for multi-peak DSC curves. However, there were no evidences or disclaimers of this viewpoint. That issue was studied in the present work. Thereto, a procedure for recalculation of DSC melting curves into "real" ones was worked out. Using different approaches, we have demonstrated that deviation of DSC melting curves from real differential melting curves is negligible. Suitable methods for determination of the melting temperature and temperature melting range in the case of multi-peak DSC curves were developed, and their reasonableness was confirmed.
New conditions for melting experiments of DNA with platinum compounds were found. They provide two useful advantages: firstly, elimination of the non-specific electrostatic stabilization of long DNA caused by platinum compounds and, secondly, stabilization of their intermediate products against high temperature of melting experiments. Thanks to those findings, a change in thermal stability caused by intermediate monofunctional and final bifunctional products was separately measured and compared for the first time.
It was shown that platinum compounds decrease the enthalpy and entropy of the helix-coil transition, increase the temperature melting range and destroy the fine structure of DSC curves. That is in agreement with the impact of other destructive factors known before. However, those destructive factors always decrease DNA melting temperature. In the case of complexes with platinum compounds, it is true only for short DNAs. For long DNAs, this general rule is not always held. Transplatin and Pt[(dien)Cl]Cl increase DNA thermal stability, although they destroy the structure of the double helix. The mechanism of such abnormal melting behavior has been found. We have demonstrated that the unusual effect is caused by large positive charge introduced by platinum compounds into the double helix.
Some platinum compounds and many other anticancer drugs form DNA interstrand crosslinks. To explain our experimental results, the influence of DNA interstrand crosslinks on melting of short and long DNAs was studied. For the first time, the thermal impact of local distortions caused by crosslinking agents was studied besides the crosslinking effect in itself. |
