LONG-TERM RESISTANCE TO 5-FLUOROURACIL ACTIVATES DISTINCT CELLULAR EVENTS UNDERLYING DRUG RESISTANCE ON COLON CANCER CELLS

INTRODUCTION AND OBJECTIVES: Drug-resistant cell line models are crucial for understanding resistance mechanisms and developing new therapeutics. However, little is published about these cell lines, including the methodology and development process. The present work aimed at the establishment and characterization of a long-term drug resistance model in an effort towards exploring the cellular mechanisms underlying drug resistance. MATERIAL AND METHODS: The colon cancer cell line HCT-116 was treated with intermittent, repeated, and increasing 5-fluorouracil (5-FU) doses followed by cell recovery in a drug-free medium. To assess the acquired resistance, cell viability, thymidylate synthase (TS), and efflux pump levels were measured. The cell cycle distribution and proliferation were determined using flow cytometry and clonogenic assays. In addition, apoptosis was evaluated by flow cytometry and caspase 3/7 detection, while autophagy was determined by LC3B detection. Moreover, the activation of epithelial-mesenchymal transition (EMT) was evaluated by mRNA and protein measurement using epithelial and mesenchymal markers, as well as by migration and invasion transwell assays. Analysis of drug resistance and mesenchymal gene signatures their correlation, and prognostic values were performed using the GEPIA database. RESULTS AND CONCLUSION: The 5-FU-treated cells (HCT-116 5FUR) using a prolonged treatment protocol were significantly more resistant than parental cells. Likewise, cell viability and IC50 values were also observed to increase in HCT-116 5FUR cells when treated with increasing doses of oxaliplatin, indicating a cross-resistance mechanism to other cytotoxic agents. Moreover, HCT-116 5FUR cells exhibited metabolic and molecular changes, as evidenced by increased TS levels and upregulated mRNA levels of ABCB1. HCT-116 5FUR cells were able to overcome S phase arrest and evade apoptosis, as well as activate autophagy, as indicated by increased LC3B levels. Cells treated with low and high doses displayed EMT features, as observed by decreased E-cadherin and claudin-3 levels, increased vimentin protein levels, and increased SLUG, ZEB2, and TWIST1 mRNA levels. Furthermore, HCT-116 5FUR cells displayed enhanced migration and invasion capabilities. Also, resistant cells developed cell heterogeneity and acquired a significant mesenchymal-like population, indicated by quantification of the morphometric parameters as circularity index. Interestingly, we found that the 5-FU drug-resistance gene signature is positively associated with the mesenchymal signature in colorectal cancer (CRC) samples, and that ABCB1 and ZEB2 co-expressed at high levels could predict poor outcomes in CRC patients. Overall, the 5-FU long-term drug-resistance model established here induced various cellular events, and highlighted the importance of further efforts to identify promising targets involved in more than one cellular event to successfully overcome drug resistance.