Podell E R, Harrington D J, Taatjes D J, Koch T H
Department of Chemistry and Biochemistry, University of Colorado, Boulder, Colorado 80309-0215, USA.
Acta Crystallogr D Biol Crystallogr. 1999 Sep;55(Pt 9):1516-23. doi: 10.1107/s0907444999008161.
Epidoxorubicin and daunorubicin are proposed to be cytotoxic to tumor cells by catalyzing production of formaldehyde through redox cycling and using the formaldehyde for covalent attachment to DNA at G bases. The crystal structure of epidoxorubicin covalently bound to a d(CGCGCG) oligomer was determined to 1.6 A resolution. The structure reveals slightly distorted B-form DNA bearing two molecules of epidoxorubicin symmetrically intercalated at the termini, with each covalently attached from its N3' to N2 of a G base via a CH2 group from the formaldehyde. The structure is analogous to daunorubicin covalently bound to d(CGCGCG) determined previously, except for additional hydrogen bonding from the epimeric O4' to O2 of a C base. The role of drug-DNA covalent bonding in cells was investigated with synthetic epidoxorubicin-formaldehyde conjugate (Epidoxoform) and synthetic daunorubicin-formaldehyde conjugate (Daunoform). Uptake and location of drug fluorophore in doxorubicin-resistant human breast-cancer cells (MCF-7/ADR cells) was observed by fluorescence microscopy and flow cytometry. The fluorophore of Daunoform appeared more rapidly in cells and was released more rapidly from cells than the fluorophore of Epidoxoform over a 3 h exposure period. The fluorophore appeared predominantly in the nucleus of cells treated with both conjugates. The difference in uptake is explained in terms of the slower rate of hydrolysis of Epidoxoform to the species reactive with DNA and a proposed slower release from DNA based upon the crystal structures.
表柔比星和柔红霉素被认为可通过氧化还原循环催化甲醛生成，并利用甲醛与鸟嘌呤碱基的DNA进行共价连接，从而对肿瘤细胞产生细胞毒性。确定了与d(CGCGCG)寡聚物共价结合的表柔比星的晶体结构，分辨率为1.6 Å。该结构显示出B型DNA略有扭曲，两个表柔比星分子对称地插入末端，每个分子通过甲醛的一个CH2基团从其N3'与鸟嘌呤碱基的N2共价连接。该结构类似于先前确定的与d(CGCGCG)共价结合的柔红霉素，不同之处在于差向异构的O4'与胞嘧啶碱基的O2之间存在额外的氢键。使用合成的表柔比星-甲醛缀合物（表柔比星甲醛）和合成的柔红霉素-甲醛缀合物（柔红霉素甲醛）研究了药物-DNA共价键在细胞中的作用。通过荧光显微镜和流式细胞术观察了多柔比星耐药人乳腺癌细胞（MCF-7/ADR细胞）中药物荧光团的摄取和定位。在3小时的暴露期内，柔红霉素甲醛的荧光团在细胞中出现得更快，从细胞中释放得也比表柔比星甲醛的荧光团更快。两种缀合物处理的细胞中，荧光团主要出现在细胞核中。摄取差异的原因是表柔比星甲醛水解为与DNA反应的物种的速度较慢，以及根据晶体结构推测其从DNA释放的速度较慢。
