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Pharm Sci. 2024;30(1): 116-128.
doi: 10.34172/PS.2023.19

Scopus ID: 85181579677
  Abstract View: 433
  PDF Download: 342

Research Article

Effects of 5-Fluorouracil on the Expression of Epigenetic Enzymes and Promoter Methylation of Selected Genes in Monolayer and Spheroid Cultures of Colorectal Cancer Cells

Maryam Niknam 1 ORCID logo, Masoumeh Varedi 2, Mozhdeh Zamani 3, Pooneh Mokarram 3, Fakhraddin Naghibalhossaini 1,4* ORCID logo

1 Department of Biochemistry, School of Medicine, Shiraz University of Medical Sciences, Shiraz, Iran.
2 Department of Physiology, School of Medicine, Shiraz University of Medical Sciences, Shiraz, Iran.
3 Autophagy Research Center, Department of Biochemistry, Shiraz University of Medical Sciences, Shiraz, Iran.
4 Autoimmune Diseases Research Center, School of Medicine, Shiraz University of Medical Sciences, Shiraz, Iran.
*Corresponding Author: Email: fakhraddin.naghibalhossaini@mail.mcgill.ca

Abstract

Background: Emerging evidence suggests that epigenetic mechanisms contribute to 5-fluorouracil (5-FU) resistance in colorectal cancer (CRC). However, there is limited research on the direct impact of 5-FU on epigenetic alterations in CRC. This study aimed to investigate how 5-FU treatment affects the expression of enzymes involved in epigenome regulation and promoter DNA methylation in human CRC cells.

Methods: The viability of CRC cell lines (SW48, HCT116, LS180, and HT29) was evaluated after 48 hours of 5-FU treatment using MTT assay in both monolayer and hanging drop spheroid cultures. The cells were treated with an IC20 concentration of 5-FU and then the relative expressions of histone deacetylases (HDAC) and DNA methyltransferas1 (DNMT1) in 5-FU-treated and untreated cells were measured by quantitative RT-PCR (qRT-PCR). The status of promoter methylation of selected genes was analyzed using the methylation-specific PCR (MSP) method.

Results: The 3D cultures of cells were more resistant to 5-FU than their 2D counterparts. The effect of 5-FU on HDAC1 expression was greater in 3D cultures compared to 2D cultures. 5-FU downregulated SIRT1 and DNMT1 in 2D culture of HCT116 and SW48 and upregulated them in 3D cultures of HT29 and LS180 cells. In both monolayer and spheroid cultures, 5-FU downregulated HDAC2 in HCT116, LS180, and HT29 and HDAC4 in HCT116, LS180, and SW48 cells. 5-FU primarily changed promoter methylation in monolayer cultures.

Conclusion: The epigenetic response to 5-FU is cell line-specific and depends on the culture method. 5-FU modulates epigenome in CRC cells by regulating DNMT1 and HDAC expressions. 3D cultures were found to be considerably more resistant to 5-FU-induced cytotoxicity and promoter DNA methylation changes than 2D cultures. 5-FU downregulated HDAC and DNMT1, particularly in the drug-sensitive cells, and increased the levels of DNMT1 in the drug-resistant cells.

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Submitted: 12 Aug 2023
Revision: 24 Sep 2023
Accepted: 25 Sep 2023
ePublished: 27 Nov 2023
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