caco - 2 and l929 cells were seeded in 96 - well plates ( 20 × 10³ cells / well, 100 µl medium ) and incubated for 24 h. caco - 2 cells were treated with agmatine at concentrations of 1, 3, 6, 9, 12, 15, 18, and 21 mm, while l929 cells were treated with 6, 9, 12, 15, 18, and 21 mm for 24, 48, and 72 h. cell viability was assessed using the mtt assay. briefly, 5 mg / ml mtt solution was added to the cells and incubated for 4 h. the medium was then removed, and 100 µl of dmso was added to each well and incubated for 30 min. absorbance was measured at 570 nm. all experiments were performed in triplic ate. transwell assays were performed using 8 μm pore inserts ( corning, ny, usa ). for migration, 2 × 10⁴ caco - 2 cells were seeded in the upper chamber with serum - free medium and treated with agmatine ( 6, 7. 3, and 9 mm ) for 72 h, while 750 µl supplemented medium was added to the lower chamber. non - migrated cells were removed, and migrated cells were fixed ( 4 % paraformaldehyde, 100 % methanol ), stained with 0. 1 % crystal violet, and counted in three random fields under an inverted microscope. for invasion, 2 × 10⁴ cells were seeded in matrigel - coated upper chambers, incubated for 24 h, and then treated with agmatine ( 6, 7. 3, and 9 mm ) for 72 h. the lower chamber contained medium with 10 % fbs as a chemoat tractant. non - invasive cells were removed, and invasive cells were fixed, stained, and quantified as above. caco - 2 and l929 cells were seeded in 96 - well plates ( 2 × 10⁴ cells / well ) and incubated for 24 h. after pbs wash, caco - 2 cells were treated with agmatine ( 6, 7. 3, and 9 mm ) and l929 ( 6 mm, 6. 25 mm, and 9 mm ) for 72 h. cells were then stained with dapi ( 1. 8 µl, 1. 11 mg / ml ) and propidium io dide ( pi; 1. 17 µl, 0. 85 mg / ml ) for 25 min at 37 °c in the dark, washed with pbs, and analyzed for apoptosis using an fluorescence microscope. caco - 2 cells ( 2 × 10⁵ / well ) were seeded in 6 - well plates and incubated for 24 h, then treated with agmatine ( 6, 7. 3, and 9 mm ) for 72 h. cells were harvested with 0. 25 % trypsin - edta, pelleted ( 300 rpm, 5 min ), resuspended in 5 % tca, and incubated at 4 °c for 15 min. after centrifugation ( 12, 000×g, 10 min, 4 °c ), supernatants were collected. for the assay, aliquots were mixed with 2 % ninhydrin ( bio basic inc., canada ) and heated at 100 °c for 5 min, and absorbance was measured at 440 nm. total rna was extracted using trizol reagent ( biobasic, markham, canada ) according to the manufacturer ’ s instructions. the concentration and purity of rnas were determined with spectrophotometry using nanodrop ( thermo fisher scientific, maryland, usa ). cdna synthesis was conducted using the onescript plus cdna synthesis kit ( applied biological materials inc., richmond, bc, canada ) according to the manufacturer ’ s instructions. quantitative real - time pcr ( qrt - pcr ) was performed using the blastaq™ 2x qpcr master mix kit ( applied biological materials inc., bc, canada ) according to the manufacturer ’ s instructions. the relative expression levels of abcc1, abcb1, abcg2, bax, bcl - 2, caspase - 3 ( csp - 3 ), glutathione peroxidase ( gpx ) and catalase ( cat ), with beta - actin ( sentebiolab, ankara, turkiye ) as an internal control, were evaluated with qrt - pcr in a t100™ thermal cycler ( bio - rad, usa ). caco2 and l929 cell lines were seeded with a density of 3 × 10 cells / well in a 6 - well plate. the following day, caco2 cells were treated with 6 mm, 7. 3 mm, and 9 mm agmatine, and l929 cells were treated with 6 mm, 6. 25 mm, 9 mm agmatine. after 72 h, whole - cell lysates were prepared using the mammalian total protein extraction kit ( serva, germany ), and protein concentrations were determined using the bca protein assay macro kit ( serva, germany ) according to the manufacturer ’ s instructions. thirty µg proteins were run on 12 % sds - page and transferred by using the semi - dry system to a 0. 22 - µm pvdf membrane. membranes were blocked in 5 % non - fat milk for 1 h and incubated with primer antibod ies against bcl - 2 ( cst, usa ), bax ( cst, usa ), cleaved csp - 3 ( cst, usa ) and gapdh ( abclonal, usa ) overnight at 4 °c. the following day, membranes were washed three times with 1x tbs - t and probed with hrp - conjugated secondary antibod ies ( anti-rabbit, igg, abclonal, usa ) for 2 h at room temperature. after incubation, membranes were washed three times with 1x tbs - t and protein bands were detected with clarity ecl substrate ( bio - rad, usa ) using the chemi - doc mp imaging system ( bio - rad, usa ). signals were normalized to housekeeping gapdh, and intensities were calculated using image lab software ( version 6. 1 ). all performed experiments were repeated three times, and the graphical presentations were performed using graphpad prism 10. 0 ( san diego, ca, usa ). data are presented as the means ± standard deviation and were analyzed using spss 22. 0 software ( chicago, il, usa ). target gene expression was normalized to the corresponding b - actin value, and δct values were calculated accordingly. the normality of δct distributions was assessed using the shapiro - wilk test. as the δct values did not deviate significantly from a normal distribution, parametric statistical tests were applied. group comparisons were performed using one - way analysis of variance. when a significant overall group effect was detected, post hoc pairwise comparisons were conducted using tukey ’ s honestly significant difference test. statistical analyses were carried out on δct values, whereas relative gene expression levels were calculated using the 2⁻δδct method for graphical presentation and descriptive purposes. a two - sided p value of < 0. 05 was considered statistically significant. a schematic representation of the anti-tumor effects of agmatine in caco - 2 cells is illustrated in fig. 1a. as shown in fig. 1b, the viability of both caco - 2 and l929 cells decreased in a concentration - dependent manner with increasing agmatine concentrations. the half - maximal inhibitory concentration ( ic₅₀ ) of agmatine at 72 h were determined to be 7. 3 mm for caco - 2 cells and 6. 25 mm for l929 cells. overall, a significant increase in inhibition rate was observed in caco - 2 cells following agmatine treatment. therefore, agmatine doses corresponding to concentrations below and above the ic₅₀ values ( 6, 7. 3, and 9 mm for caco - 2 cells; 6, 6. 25, and 9 mm for l929 cells ) were selected for subsequent experiments. fig. 1c presents the logarithmic representation of the mtt assay results for the cells. the effects of agmatine on the migration and invasion of caco - 2 cells were evaluated using transwell migration and matrigel invasion assays. as shown in fig. 2a the number of caco - 2 cells migrating through the membrane into the lower chamber was significantly and dose - dependently reduced in the agmatine - treated groups compared with control groups. when caco - 2 cells were treated with agmatine at concentrations of 6, 7. 3, and 9 mm for 72 h, their relative migration rates decreased from 61. 2 % to 46. 3 % and further to 30. 02 %, respectively ( fig. 2a; p < 0. 05 ). similarly, agmatine was observed to inhibit caco - 2 cell invasion in a dose - dependent manner. at 72 h, treatment with 6, 7. 3, and 9 mm agmatine resulted in a reduction of the relative invasion rate from 54. 6 % to 33. 4 % and further to 18. 8 %, respectively ( fig. 2b; p < 0. 05 ). collectively, both assays demonstrated that the numbers of migrated and invaded caco - 2 cells were significantly decreased in a dose - dependent manner in agmatine - treated groups ( 6, 7. 3, and 9 mm ) compared with untreated caco - 2 cells ( control ). the effects of agmatine on the apoptosis of caco - 2 and l929 cells were evaluated by fluorescence microscopy using dapi and pi staining.