Ratanaphan Adisorn, Canyuk Bhutorn, Wasiksiri Siriwat, Mahasawat Pawika
Laboratory of Pharmaceutical Biotechnology, Department of Pharmaceutical Chemistry, Faculty of Pharmaceutical Sciences, Prince of Songkla University, Hat-Yai, Songkla 90112, Thailand.
Biochim Biophys Acta. 2005 Sep 15;1725(2):145-51. doi: 10.1016/j.bbagen.2005.07.006.
Carboplatin is an anticancer drug for the treatment of cancers affecting various organs including ovary and testes. It essentially exerts its cytotoxicity against cancerous cells via covalent attachment of platinum atom to DNA, generating various platinum-DNA adducts. Platinum-DNA adducts inhibit biological processes essential for cellular viability. However, carboplatin interacts nonspecifically with DNA, resulting in damaging of normal cell DNA. Potential in vitro interaction of carboplatin with genes encoding tumor suppressor proteins such as human breast cancer suppressor gene 1(BRCA1) was herein investigated. The 696--bp fragment of the 3'-region of BRCA1 gene (nucleotide 4897--5592) was amplified by RT-PCR using mRNA templates isolated from human white blood cells. Retardation of the electrophoretic migration on agarose gel of drug-treated DNA, in the dose-response manner, was observed. Analysis by restriction digestion with PvuII and Eco O 109I suggested that the platination favorably occurred at the dGpG sequence of Eco O 109I-cleaved site. The semi-quantitative PCR-based assay was used to determine the lesion frequencies produced by carboplatin in the 696-bp fragment of the 3'-region of BRCA1 gene and in the 3,426-bp fragment of the BRCA1 exon 11 of human breast adenocarcinoma MCF-7 cells. A significant decrease in DNA amplification was observed at 400 microM of carboplatin with approximately 1--2 platinum atoms per BRCA1 fragment. Carboplatin caused slightly less damage at equimolar concentrations in cells than in cell-free BRCA1 fragment.
卡铂是一种抗癌药物，用于治疗影响包括卵巢和睾丸在内的各种器官的癌症。它主要通过铂原子与DNA的共价连接对癌细胞发挥细胞毒性作用，产生各种铂-DNA加合物。铂-DNA加合物抑制细胞存活所必需的生物学过程。然而，卡铂与DNA非特异性相互作用，导致正常细胞DNA受损。本文研究了卡铂在体外与编码肿瘤抑制蛋白的基因（如人类乳腺癌抑制基因1，BRCA1）的潜在相互作用。使用从人类白细胞中分离的mRNA模板，通过逆转录聚合酶链反应（RT-PCR）扩增BRCA1基因3'区域的696 bp片段（核苷酸4897 - 5592）。观察到药物处理的DNA在琼脂糖凝胶上的电泳迁移以剂量反应方式延迟。用PvuII和Eco O 109I进行限制性消化分析表明，铂化作用有利于发生在Eco O 109I切割位点的dGpG序列处。基于半定量PCR的检测方法用于确定卡铂在人乳腺腺癌MCF-7细胞BRCA1基因3'区域的696 bp片段和BRCA1外显子11的3426 bp片段中产生的损伤频率。在400 microM卡铂处理下，每个BRCA1片段约有1 - 2个铂原子时，观察到DNA扩增显著减少。在等摩尔浓度下，卡铂对细胞造成的损伤略小于对无细胞BRCA1片段造成的损伤。
