Optimized sample preparation strategy for the analysis of low molecular mass adducts of a fluorescent cisplatin analogue in cancer cell lines by CE‐dual‐LIF
Optimized sample preparation strategy for the analysis of low molecular mass adducts of a fluorescent cisplatin analogue in cancer cell lines by CE‐dual‐LIF
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通过 CEâdualâLIF 分析癌细胞系中荧光顺铂类似物的低分子量加合物的优化样品制备策略
DOI：
10.1002/elps.201400467
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发表时间：
2015
期刊：
影响因子：
2.9
通讯作者：
Weber G
中科院分区：
文献类型：
--
作者：
Zabel R;Kullmann M;Kalayda GV;Jaehde U;Weber G
已知铂基抗癌药物（如顺铂）在给药后会经历几个（生物）化学转化步骤。与小的亲核试剂和较大的蛋白质的水解和加合物形成是它们在最终反应位点（DNA）的途中最相关的反应，但是关于各种提议的加合物和中间体的身份和药理学相关性仍然有许多开放的问题。此外，在任何类型的分析测量过程中不可避免地添加到样品中的缓冲组分或添加剂的作用在以前的研究中经常被忽视。在这里，我们报告了荧光顺铂类似物二乙酸羧基荧光素铂（CFDA-Pt）在常用缓冲液和细胞培养基中的加合物形成反应。我们的结果表明，与非无害缓冲液（例如，在解释结果时，必须考虑细胞培养/细胞裂解培养基的成分。使用CE-LIF在纳摩尔浓度的CFDA-Pt下跟踪加合物形成动力学长达60小时。CE-MS能够在线鉴定低至纳摩尔浓度范围的此类意外加合物。通过使用优化的样品制备策略，可以避免不需要的加合物，并且可以在敏感和顺铂耐药癌细胞系中检测到CFDA-Pt的几种荧光加合物。通过在孵育后快速处理样品，我们甚至可以将细胞中最初但短暂的Pt物质鉴定为脱乙酰化的CFDA-Pt，Pt处的络合环境未改变。总的来说，所提出的方法能够非常灵敏和准确地分析癌细胞中的低分子量Pt物质，包括5分钟内的快速CE-LIF检测。
Pt‐based anticancer drugs, such as cisplatin, are known to undergo several (bio‐)chemical transformation steps after administration. Hydrolysis and adduct formation with small nucleophiles and larger proteins are their most relevant reactions on the way to the final reaction site (DNA), but there are still many open questions regarding the identity and pharmacological relevance of various proposed adducts and intermediates. Furthermore, the role of buffer components or additives, which are inevitably added to samples during any type of analytical measurement, has been frequently neglected in previous studies. Here, we report on adduct formation reactions of the fluorescent cisplatin analogue carboxyfluorescein diacetate platinum (CFDA‐Pt) in commonly used buffers and cell culture medium. Our results indicate that chelation reactions with noninnocent buffers (e.g., Tris) and components of the cell culture/cell lysis medium must be taken into account when interpreting results. Adduct formation kinetics was followed up to 60 h at nanomolar concentrations of CFDA‐Pt by using CE‐LIF. CE‐MS enabled the online identification of such unexpected adducts down to the nanomolar concentration range. By using an optimized sample preparation strategy, unwanted adducts can be avoided and several fluorescent adducts of CFDA‐Pt are detectable in sensitive and cisplatin‐resistant cancer cell lines. By processing samples rapidly after incubation, we could even identify the initial, but transient, Pt species in the cells as deacetylated CFDA‐Pt with unaltered complexing environment at Pt. Overall, the proposed procedure enables a very sensitive and accurate analysis of low molecular mass Pt species in cancer cells, involving a fast CE‐LIF detection within 5 min.
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影响因子：
4.1
作者：
Brauckmann, Christine;Faber, Helene;Karst, Uwe
通讯作者：
Karst, Uwe
植物材料中铂形态的检测
影响因子：
2.9
作者：
J. Messerschmidt;F. Alt;G. Tölg
通讯作者：
G. Tölg
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DOI：
10.1016/j.chroma.2012.07.062
发表时间：
2012
期刊：
Journal of chromatography. A
影响因子：
--
作者：
Gerlinde Grabmann;S. Meier;Y. Y. Scaffidi;M. Galanski;B. Keppler;C. Hartinger
通讯作者：
C. Hartinger
DOI：
10.1039/c4mt00033a
发表时间：
2014-05
期刊：
Metallomics : integrated biometal science
影响因子：
--
作者：
Nowakowski AB;Wobig WJ;Petering DH
通讯作者：
Petering DH
反应活性对奥沙利铂类似物细胞积累和细胞毒性的影响
DOI：
10.1007/s00775-012-0889-9
发表时间：
2012
期刊：
JBIC Journal of Biological Inorganic Chemistry
影响因子：
--
作者：
I. Buss;Ganna V. Kalayda;A. Lindauer;Michael R. Reithofer;M. Galanski;B. Keppler;U. Jaehde
通讯作者：
U. Jaehde