Abstract 3730: Folic acid has a cell type- and dose-dependent effect on the genome and the epigenome of colorectal cancer cells
Authors: Zsigrai Sára , Kalmár Alexandra , Barták Barbara Kinga , Nagy Zsófia Brigitta , Szigeti Krisztina Andrea , Valcz Gábor , Dankó Titanilla , Sebestyén Anna , Barna Gábor , Tulassay Zsolt , Igaz Péter , Takács István , Molnár Béla 
Published: June 2022

Abstract
Synthetic vitamin B9, also known as folic acid (FA), is an extensively used nutritional supplement as well as an adjunctive medication in cancer therapy. However, there is increasing evidence that FA can promote the progression of established colorectal cancers (CRC); therefore, great care is required in the case of its application. In order to gain knowledge about the underlying mechanisms, we analyzed the genomic and the epigenomic effect of different FA supplies on two CRC cell lines with distinct molecular backgrounds. HT-29 and SW480 cells were kept in FA-free media (0 ng/mL) or treated with 100 ng/mL and 10000 ng/mL FA for 72 hours. Firstly, cell proliferation and cell viability alterations were determined with Supforhodamine B and AlamarBlue assays; then, cell cycle analysis was performed using fluorescence-activated cell sorting (FACS). Global DNA methylation level was investigated with the pyrosequencing of long interspersed nuclear element 1 (LINE-1) retrotransposons. Micronucleus scoring performed on DAPI- and anti-γ-H2AX-stained slides, as well as Comet assay, were used to detect the impact of FA on genome integrity. Finally, we analyzed gene expression alterations with Human Transcriptome Array (HTA) 2.0. Our data revealed that 100 ng/mL FA induced significant (p≤0.05) elevation (HT-29100: 128.4±24.9%) of HT-29 cell proliferation compared to the other two circumstances (HT-290: 101.3±13.5%; HT-2910000: 86.1±20.8%). The tendency of cell viability was analogous to the results detected during cell proliferation analyses (HT-290: 91.6±13.3%; HT-29100: 115.8±30.9%; HT-2910000: 64.1±20.2%). The genomic stability of FA-supplemented HT-29 samples was improved in a significant manner (p≤0.05), based on the results of micronucleus scoring (HT-290: 0.56±0.05%; HT-29100: 0.17±0.05%; HT-2910000: 0.25±0.09%) and Comet assay (HT-290: 37.4±3.5%; HT-29100: 31.2±3.4%; HT-2910000: 20.1±3.6%). However, in SW480 cells, remarkable alterations were not detected concerning these parameters. Fundamental differences were observed between the two cell lines in the case of cell cycle (HT-29: G0/1 phase dominance; SW480: S phase dominance) and global DNA methylation analysis (HT-29: 59.1±1.0; SW480: 49.0±0.2), but exposing them to different FA doses did not lead to significant changes. Gene expression alterations were more diverse, as genes involved in carcinogenesis were either up- (HES1, SLC7A11) or downregulated (CCL2) for FA supplementation. We concluded that the effect of FA was considerably influenced by the cell type and the applied FA concentration. Thereby, translating our in vitro results to patient care, we would emphasize the importance of genetic and epigenetic investigations coupled with the choice of proper FA dose upon CRC diagnosis to achieve the best disease outcome.
Citation Format: Sára Zsigrai, Alexandra Kalmár, Barbara Kinga Barták, Zsófia Brigitta Nagy, Krisztina Andrea Szigeti, Gábor Valcz, Titanilla Dankó, Anna Sebestyén, Gábor Barna, Zsolt Tulassay, Péter Igaz, István Takács, Béla Molnár. Folic acid has a cell type- and dose-dependent effect on the genome and the epigenome of colorectal cancer cells [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2022; 2022 Apr 8-13. Philadelphia (PA): AACR; Cancer Res 2022;82(12_Suppl):Abstract nr 3730.