Cancer Biomarkers and Prevention Group, and Radiation & Oxidative Stress Group, Department of Cancer Studies and Molecular Medicine, University of Leicester, Leicester, United Kingdom.
Chem Res Toxicol. 2010 Aug 16;23(8):1313-21. doi: 10.1021/tx100023c.
Platinum-containing drugs are widely used to treat cancer in a variety of clinical settings. Their mode of action involves the formation of DNA adducts, which facilitate apoptosis in cancer cells. Cisplatin binds to the N7 position of the purine DNA bases forming intrastrand cross-links between either two adjacent guanines [cis-Pt(NH(3))(2)d(pGpG), 1,2-GG] or an adjacent adenine and guanine [cis-Pt(NH(3))(2)d(pApG), 1,2-AG)]. The cytotoxic efficacy for each of the different types of DNA adducts and the relationship between adduct levels in tumor cells and blood are not well understood. By using these Pt-containing adduct species as biomarkers, information on a patient's response to chemotherapy would be directly related to the mode of action of the drug. This type of analysis requires the most sensitive and specific methods available, to facilitate detection limits sufficient to measure the DNA adduct in the limited sample quantities available from patients. This was achieved in the current study by coupling a highly specific enzyme-based adduct isolation method with a sensitive detection system based on HPLC coupled to inductively coupled plasma mass spectrometry to measure the 1,2-GG cisplatin adducts formed in DNA. The method was developed and validated using calf thymus DNA and two different adenocarcinoma cell lines. The values for the limit of detection (LOD) and the limit of quantitation determined for the 1,2-GG cisplatin adduct were 0.21 and 0.67 fmol per microg DNA, respectively. This corresponds to an absolute LOD of 0.8 pg as Pt for the 1,2-GG adduct. Cisplatin-sensitive (H23) and -resistant (A549) tumor cells were exposed to the drug, and the 1,2-GG adduct levels were measured over a 24 h time period. The results showed a statistically significant (P < 0.05) higher concentration in the sensitive cells as compared to the resistant cells after repair for 7 h. Although the adduct concentration present fell at subsequent time points (12 and 24 h), the levels in each cell line were broadly similar. The protocol was then applied to the analysis of patient samples taken before and then 1 h after treatment. The 1,2-GG cisplatin adduct was present in the range from 113 to 1245 fg Pt per microg DNA in all of the patient samples taken after treatment. Although the adduct was not present at levels greater than the LOD in the initial pretreatment samples, trace amounts were discernible in some patient samples on their third treatment cycle.
含铂药物被广泛用于治疗多种临床环境下的癌症。其作用机制涉及 DNA 加合物的形成，这促进了癌细胞的凋亡。顺铂与嘌呤 DNA 碱基的 N7 位结合，形成两条相邻鸟嘌呤之间的链内交叉链接[顺铂（NH3）2d（pGpG），1,2-GG]或相邻腺嘌呤和鸟嘌呤[顺铂（NH3）2d（pApG），1,2-AG]。每种不同类型的 DNA 加合物的细胞毒性功效以及肿瘤细胞中加合物水平与血液之间的关系尚不清楚。通过将这些含铂加合物用作生物标志物，可以将患者对化疗的反应信息直接与药物的作用模式相关联。这种类型的分析需要使用最敏感和最特异的方法，以促进检测极限，从而足以测量从患者获得的有限样本量中的 DNA 加合物。本研究通过将高度特异的基于酶的加合物分离方法与基于 HPLC 与电感耦合等离子体质谱联用的敏感检测系统相结合，从而实现了这一点，以测量 DNA 中形成的 1,2-GG 顺铂加合物。该方法使用小牛胸腺 DNA 和两种不同的腺癌细胞系进行了开发和验证。确定的 1,2-GG 顺铂加合物的检测限（LOD）和定量限（LOQ）值分别为 0.21 和 0.67 fmol/μg DNA。这相当于 1,2-GG 加合物的绝对 LOD 为 0.8 pg 作为 Pt。将顺铂敏感（H23）和耐药（A549）肿瘤细胞暴露于药物中，并在 24 小时的时间内测量 1,2-GG 加合物的水平。结果表明，在修复 7 小时后，与耐药细胞相比，敏感细胞中的浓度具有统计学意义（P <0.05）更高。尽管随后的时间点（12 和 24 小时）的加合物浓度下降，但在每个细胞系中的水平大致相似。然后将该方案应用于分析治疗前后的患者样本。在所有治疗后的患者样本中，1,2-GG 顺铂加合物的含量范围为 113 至 1245 fg Pt/μg DNA。尽管在初始预处理样本中加合物的浓度未超过 LOD，但在一些患者样本的第三个治疗周期中可以发现痕量。
