Cancer Research UK Cambridge Institute, University of Cambridge, Li Ka Shing Centre, Box 278, Robinson Way, Cambridge CB2 0RE, UK.
Cold Spring Harbor Laboratory, 1 Bungtown Road, Cold Spring Harbor, NY 11724, USA.
Br J Cancer. 2014 Jul 15;111(2):318-25. doi: 10.1038/bjc.2014.288. Epub 2014 May 29.
The modest benefits of gemcitabine (dFdC) therapy in patients with pancreatic ductal adenocarcinoma (PDAC) are well documented, with drug delivery and metabolic lability cited as important contributing factors. We have used a mouse model of PDAC: KRAS(G12D); p53(R172H); pdx-Cre (KPC) that recapitulates the human disease to study dFdC intra-tumoural metabolism.
LC-MS/MS and NMR were used to measure drug and physiological analytes. Cytotoxicity was assessed by the Sulphorhodamine B assay.
In KPC tumour tissue, we identified a new, Kennedy pathway-linked dFdC metabolite (gemcitabine diphosphate choline (GdPC)) present at equimolar amounts to its precursor, the accepted active metabolite gemcitabine triphosphate (dFdCTP). Utilising additional subcutaneous PDAC tumour models, we demonstrated an inverse correlation between GdPC/dFdCTP ratios and cytidine triphosphate (CTP). In tumour homogenates in vitro, CTP inhibited GdPC formation from dFdCTP, indicating competition between CTP and dFdCTP for CTP:phosphocholine cytidylyltransferase (CCT). As the structure of GdPC precludes entry into cells, potential cytotoxicity was assessed by stimulating CCT activity using linoleate in KPC cells in vitro, leading to increased GdPC concentration and synergistic growth inhibition after dFdC addition.
GdPC is an important element of the intra-tumoural dFdC metabolic pathway in vivo.
吉西他滨（dFdC）治疗胰腺导管腺癌（PDAC）的疗效有限，已有大量文献记载，其药物递送和代谢不稳定性被认为是重要的影响因素。我们使用一种 PDAC 的小鼠模型：KRAS(G12D); p53(R172H); pdx-Cre (KPC)，该模型可重现人类疾病，用于研究 dFdC 的肿瘤内代谢。
采用 LC-MS/MS 和 NMR 测量药物和生理分析物。采用磺基罗丹明 B 测定法评估细胞毒性。
在 KPC 肿瘤组织中，我们鉴定出一种新的、与 Kennedy 途径相关的 dFdC 代谢物（吉西他滨二磷酸胆碱（GdPC）），其与前体物，即公认的活性代谢物吉西他滨三磷酸（dFdCTP）的含量相等。利用其他皮下 PDAC 肿瘤模型，我们证明了 GdPC/dFdCTP 比值与胞苷三磷酸（CTP）之间存在反比关系。在体外肿瘤匀浆中，CTP 抑制 dFdCTP 形成 GdPC，表明 CTP 和 dFdCTP 与 CTP：磷酸胆碱胞苷转移酶（CCT）竞争。由于 GdPC 的结构阻止其进入细胞，因此通过在线粒体细胞中使用亚油酸刺激 CCT 活性来评估其潜在的细胞毒性，导致 GdPC 浓度增加，并在添加 dFdC 后协同抑制生长。
GdPC 是体内肿瘤内 dFdC 代谢途径的重要组成部分。
