A Direct and Sensitive Method for Determination of 5-Fluorouracil in Colorectal Cancer Cells: Evaluating the Effect of Stromal Cell on Drug Resistance of Cancer Cells.

Fengjing Xu,Mengwei Zhang,Hongxia Yan,Yanping Liu,Zhipeng Wang,Xinhua Song,Wansheng Chen,Yan Liu,Lili Cui,Shouhong Gao,María José Trujillo-Rodríguez

doi:10.1155/2021/6689488

Abstract

Fibroblasts in the stroma play a critical role in tumor evolution. In this study, we assessed the influence of colonic fibroblasts on colon cancer cells treated with 5-fluorouracil (5-FU), and mouse colon cancer cell lines MC38 and colonic fibroblasts NIH3T3 were used in this study. A sensitive and rapid UHPLC-MS/MS method for the quantitation of 5-FU from the cell and their medium has been successfully developed and validated. The cells were lysed with methanol, and the mixture was evaporated and then redissolved to extract intracellular 5-FU. The analysis was performed on UHPLC-MS/MS using an Atlantis T3-C18 column (3 μm, 2. 1 ∗ 100 mm) and gradient elution with acetonitrile and 0.1% formic acid in water. Method validation included the following parameters: the matrix effect range 88.82%–93.64% and the recovery range 93.52%–94.56%. The intraday and interday precision and accuracy were <11% and within ±6%, and the stability, specificity, carry-over, dilution effect, and linearity all conformed to the criteria. The method was applied to detect the concentration of 5-FU inside cells and cell culture medium. The preliminary results present that NIH3T3 could enhance the drug resistance of MC38 to 5-FU with a decreased intracellular concentration of 5-FU in MC38, which showed a positive relationship with NIH3T3 number.

Highlights

Colorectal cancer (CRC) is one of the most common cancers worldwide
To find better parameters that suit our analyte, a series of tests were performed. e detections of 5FU and IS by mass spectrometry were optimized under electrospray ionization (ESI) mode, which showed superiority in ionization efficiency compared to atmospheric pressure chemical ionization (APCI) sources [15]
For instance, ZORBAX SB-C18, Waters Atlantis T3-C18, Xselect BEH, Xbridge BEH, and Eclipse PLUS-C18, the results found that the 5-FU has better peak shape and retention time in the Waters T3-C18 column, and the other columns did not show satisfying retention and symmetrical peaks

Summary

Introduction

Colorectal cancer (CRC) is one of the most common cancers worldwide. Its incidence ranks thirdly after prostate cancer and lung cancer among males and is the second most common cancer in females. Chemotherapy intervention combined with surgery is one of the main treatments for advanced CRC. In several decades after its discovery, 5-FU has been the backbone in adjuvant and palliative treatment for colorectal cancer (CRC) [1, 2]. Despite advances in new treatment drugs in recent years, 5-FU is still a first-line agent for CRC patients [3]. During the treatment process, most patients will develop drug resistance to 5-FU, which is a major obstacle for CRC treatment [4]. Drug resistance has stirred up much attention of researchers on the mechanism exploration and resistance elimination

Methods

Results

Conclusion