the use of 24 - well plates, instead of the standard 96 - well format recommended in the kit manual, helped minimize several technical issues encountered during long - term culture, including high cell density effects, difficulties in handling very small volumes, and uneven medium evaporation at the plate edges. in addition, the larger well format reduced the risk of cell detachment during medium changes, which can easily occur in smaller wells due to suction effects. rna seq or microarray data used to assess the role of ythdf2 in autoimmune diseases were originally found using the adex: autoimmune diseases explorer database ( https: / / adex. genyo. es ). data files were accessed and downloaded using the gene expression omnibus ( geo ). data was imported into prism for statistical analyses. datasets analyzed were gse110169 _ sle ( microarray ), gse50772 ( microarray ), gse72509 ( rna - seq ), gse11907 _ t1d ( microarray ), gse110169 ra ( microarray ), gse56649 ( microarray ), gse45291 ( microarray ), gse89408 ( rna - seq ), gse104174 ( rna - seq ), gse124073 ( rna - seq ), and gse51092 ( microarray ). statistical analyses were performed using prism v10 ( graphpad software ). data are presented as the mean of at least three independent experiments and were analyzed using a two - tailed unpaired student ’ s t - test or a mann – whitney u test, as indicated. error bars indicate the sds or ses of the means as specified. p < 0. 05 was considered statistically significant. all experiments were independently repeated at least three times with similar results. representative images ( e. g., micrographs, blots ) are shown from experiments that were repeated independently with comparable outcomes. sample sizes ( n ) refer to biologically independent replicates ( e. g., independent mice, cell cultures, or tissue samples ) unless otherwise indicated. further information on research design is available in the nature portfol io reporting summary linked to this article. this study aimed to evaluate the potential effects of agmatine on cell viability, migration, invasion, apoptosis, and the expression of the abcb1, abcc1, and abcg2 genes in the caco - 2 colon cancer cell line. agmatine efficacy was assessed thruogh proliferation, migration, and invasion assays at various concentrations. the apoptotic index was determined using apoptosis - related markers ( bax, bcl - 2, csp - 3 ) via apoptosis assays, quantitative real - time pcr ( qrt - pcr ), and western blot analysis. expression levels of the abcg2, abcb1, and abcc1 genes were measured by qrt - pcr in agmatine - treated caco - 2 cells. oxidative stress markers, including glutathione peroxidase ( gpx ) and catalase ( cat ), were evaluated by qrt - pcr. cell viability analysis revealed that agmatine exerted its most pronounced effects at 72 h, with significant reductions at concentrations of 6, 7. 3, and 9 mm in caco - 2 cells and 6, 6. 25, and 9 mm in l929 cells ( p < 0. 05 ). at these concentrations, migration and invasion assays showed dose - dependent decreases in cell motility and invasiveness in caco - 2 cells. apoptosis analysis demonstrated a significant increase in the apoptotic index with rising agmatine concentrations. significant decreases in gpx and cat were observed in all three agmatine - treated caco - 2 groups compared to untreated controls ( p < 0. 01 ). however, the expression levels of abcg2, abcb1, and abcc1 showed no significant changes following agmatine treatment ( p > 0. 05 ). these findings indicate that agmatine exerts antiproliferative, anti - migratory, anti - invasive, and pro - apoptotic effects in caco - 2 colon cancer cells, potentially through the modulation of apoptosis - and oxidative stress – related pathways. the lack of significant impacts on abc transporter gene expression suggests that agmatine may be a promising candidate molecule for further translational studies in colorectal cancer. worldwide, the incidence of colorectal cancer ( crc ) is increasing each year, and is becoming a significant public health concern. currently, chemotherap y protocols based on the combination of fluorouracil ( 5 - fu ) and oxaliplatin ( oxa ) constitute one of the backbones of crc treatment. nevertheless, because currently used therapeutic agents also penetrate healthy tissues, patients may experience many kinds of side effects. yet, all these treatment options may not be equally effective for every patient and can entail serious side effects as well as resistance - related challenges. consequently, it is of great importance to explore new therapeutic and supportive agents for crc therapy. polyamines can interact with dna, rna, atp, proteins, and phospholipids under physiological conditions. polyamine synthesis is essential for sustaining the continuous proliferation characteristic of cancer cells. notably, the genes encoding two rate limiting enzymes in polyamine biosynthesis, ornithine decarboxylase ( odc; encoded by odc1 ) is often dysregulated in various cancers. agmatine has been reported to confer protective effects against ischemic injury and chronic neuropathic pain in mammals. it has also been identified as having neuroprotective and antioxidant properties in pathophysiological conditions such as anxiety disor ders and depression. in contrast to the proliferative effects of the aforementioned polyamines, the polyamine agmatine has been shown to reduce odc activity and the intracellular uptake of polyamines. furthermore, emerging evidence suggests that agmatine exerts antiproliferative and pro - apoptotic effects in various cancer models such as prostate hepatocellular carcinoma and glioma primarily by interfering with polyamine metabolism and modulating key signaling pathways [ 9 – 11 ]. in colorectal cells, agmatine has been shown to inhibit cell proliferation and induce cell cycle arrest, potentially through the suppression of odc activity and the disrup tion of polyamine homeostasis. these findings highlight agmatine ’ s potential to serve as a selective therapeutic against in cancer, particularly in tumors with high polyamine requirements, while sparing normal cells in which it exhibits protective and antioxidant properties. collectively, these findings position agmatine as a promising therapeutic candidate for colorectal cancer, warranting further investigation into its clinical potential. one of the principal mechanisms underlying resistance to anticancer drugs is multidrug resistance ( mdr ), in which cancer cells overexpress the atp - binding cassette ( abc ) transporter proteins that actively secrete various chemotherap eutic agents from the cell. in particular, the transporters encoded by abcb1 ( mdr1 / p - gp ), abcc1 ( mrp1 ), and abcg2 ( bcrp ) play crucial roles in pumping numerous chemotherap y drugs out of the cell, thus limiting intracellular drug accumulation and enabling tumor cell survival. this study aimed to examine whether agmatine has potential regulatory effects on cell viability, migration and invasion, apoptosis, reactive oxygen species ( ros ), and the gene expression levels of abcb1, abcc1, and abcg2 transporters, which play important roles in drug resistance in the human colon carcinoma cell line caco - 2. the caco - 2 and l929 cell lines were obtained from the american type culture collection ( manassas, va, usa ). caco - 2 cells were cultured in high - glucose dmem ( ecotechbio, turkiye ) mixed with 10 % fetal bovine serum ( fbs ) capricorn ( ebsdorfergrund, germany ) and 1 % penicillin / streptomycin ( gibco waltham, usa ) at 37 °c and 5 % co₂. all experiments were performed using cells at passages 17 – 27. experiments were performed in triplic ate. untreated caco - 2 cells were used as the control group in all experiments.