Shahi Sanjay, Ang Ching-Seng, Mathivanan Suresh
Department of Biochemistry and Genetics, La Trobe Institute for Molecular Science, La Trobe University, Melbourne, VIC 3086, Australia.
The Bio21 Molecular Science and Biotechnology Institute, University of Melbourne, Parkville, VIC 3010, Australia.
Biology (Basel). 2020 May 6;9(5):96. doi: 10.3390/biology9050096.
Currently, 5-fluorouracil (5-FU)-based combination chemotherapy is the mainstay in the treatment of metastatic colorectal cancer (CRC), which benefits approximately 50% of the patients. However, these tumors inevitably acquire chemoresistance resulting in treatment failure. The molecular mechanisms driving acquired chemotherapeutic drug resistance in CRC is fundamental for the development of novel strategies for circumventing resistance. However, the specific phenomenon that drives the cancer cells to acquire resistance is poorly understood. Understanding the molecular mechanisms that regulate chemoresistance will uncover new avenues for the treatment of CRC. Among the various mechanisms of acquired chemoresistance, defects in the drug metabolism pathways could play a major role. In the case of 5-FU, it gets converted into various active metabolites, which, directly or indirectly, interferes with the replication and transcription of dividing cells causing DNA and RNA damage. In this project, we developed a high-resolution mass spectrometry-based method to effectively extract and quantify levels of the 5-FU metabolites in cell lysates and media of parental and 5-FU resistant LIM1215 CRC cells. The analysis highlighted that the levels of 5-FU metabolites are significantly reduced in 5-FU resistant cells. Specifically, the level of the nucleotide fluorodeoxyuridine monophosphate (FdUMP) is reduced with treatment of 5-FU clarifying the compromised 5-FU metabolism in resistant cells. Corroborating the metabolomic analysis, treatment of the resistant cells with FdUMP, an active metabolite of 5-FU, resulted in effective killing of the resistant cells. Overall, in this study, an effective protocol was developed for comparative quantitation of polar metabolites and nucleotide analogues from the adherent cells efficiently. Furthermore, the utility of FdUMP as an alternative for CRC therapy is highlighted.
目前，基于5-氟尿嘧啶（5-FU）的联合化疗是转移性结直肠癌（CRC）治疗的主要手段，约50%的患者从中获益。然而，这些肿瘤不可避免地会产生化疗耐药性，导致治疗失败。驱动CRC获得性化疗耐药性的分子机制是开发规避耐药性新策略的基础。然而，促使癌细胞获得耐药性的具体现象仍知之甚少。了解调节化疗耐药性的分子机制将为CRC的治疗开辟新途径。在获得性化疗耐药的各种机制中，药物代谢途径的缺陷可能起主要作用。就5-FU而言，它会转化为各种活性代谢物，这些代谢物直接或间接干扰分裂细胞的复制和转录，导致DNA和RNA损伤。在本项目中，我们开发了一种基于高分辨率质谱的方法，以有效提取和定量亲本及5-FU耐药的LIM1215 CRC细胞的细胞裂解物和培养基中5-FU代谢物的水平。分析表明，5-FU耐药细胞中5-FU代谢物的水平显著降低。具体而言，在用5-FU处理后，核苷酸氟脱氧尿苷单磷酸（FdUMP）的水平降低，这表明耐药细胞中5-FU代谢受损。与代谢组学分析结果一致，用5-FU的活性代谢物FdUMP处理耐药细胞可有效杀死这些耐药细胞。总体而言，在本研究中，开发了一种有效的方案，用于从贴壁细胞中高效地比较定量极性代谢物和核苷酸类似物。此外，还强调了FdUMP作为CRC治疗替代物的效用。
