A High-Resolution Mass Spectrometry-Based Quantitative Metabolomic Workflow Highlights Defects in 5-Fluorouracil Metabolism in Cancer Cells with Acquired Chemoresistance

Sanjay Shahi,Suresh Mathivanan,Ching-Seng Ang

doi:10.3390/biology9050096

Abstract

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.

Highlights

Colorectal cancer (CRC) is the third most common cancer and has the fourth highest mortality rate worldwide [1,2,3] The incidence of CRC is highest in Australia/New Zealand and Western Europe.Surgery is the main treatment option for early stage CRC patients [4]
Follow up cell death analysis confirmed the induction of acquired resistance in the 5-FU resistant LIM1215 CRC cells (Figure 1B)
5-FU metabolites andstudies their quantification cultures and human samples has absence of standards to suitbyalldifferent adherent cell types andInatthis thestudy, same time would be aeffective been previously described studies we developed protocolfor in the the quantitative comparison of the metabolites among multiple groups

Summary

Introduction

Colorectal cancer (CRC) is the third most common cancer and has the fourth highest mortality rate worldwide [1,2,3] The incidence of CRC is highest in Australia/New Zealand and Western Europe.Surgery is the main treatment option for early stage CRC patients [4]. The response rate to 5-FU when used alone is approximately 15% [7,9] Most of these patients eventually acquire resistance to 5-FU, resulting in treatment failure and patient death. While a combination of 5-FU with other anti-cancer drugs (irinotecan and oxaliplatin) has resulted in higher response rates (40–50%), it is crucial to understand the molecular mechanisms regulating chemoresistance to 5-FU in order to predict or overcome drug resistance. Unravelling the mechanisms of 5-FU resistance could result in the stratification of patients likely to respond to this agent. It may aid in the identification and development of therapeutic strategies aimed at preventing or overcoming resistance

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