dapi / pi staining revealed that after agmatine treatment with 6, 7. 3, or 9 mm in caco - 2 and 6, 6. 25, or 9 mm in l929 for 72 h, apoptosis rates were increased dose - dependently when compared to the control cells ( fig. 3a ). in caco - 2 cells treated with 6, 7. 3, or 9 mm agmatine for 72 h, the apoptotic indices were 46 % ( ns ), 50 % ( p < 0. 05 ), and 36 % ( ns ), respectively, compared to the untreated control. in contrast, agmatine treatment in l929 cells induced a dose - dependent increase in the apoptotic index, reaching 40 % ( ns ), 46 % ( ns ), and 66 % ( p < 0. 001 ) at 6, 6. 25, and 9 mm concentrations, respectively ( fig. 3b ). fig. 1 ( a ) schematic figure of anti-tumor effect of agmatine in caco - 2 cells. ( figure was created using biorender under an academic license ). ( cell viability ( % ) of agmatine on the caco - 2 and l929 cells. caco - 2 was treated with different concentrations of agmatine 1, 3, 6, 9, 12, 15, 18, and 21 mm and l929 cells were treated with 6, 9, 12, 15, 18, and 21 mm for 24, 48, and 72 h. ( c ) logarithmic representation of the mtt assay results for the l929 and caco - 2. error bars show standard error. * also indicates that statistically significant differences ( * p < 0. 05, * * p < 0. 01, * * * p < 0. 001 ) ( a ) schematic figure of anti-tumor effect of agmatine in caco - 2 cells. ( figure was created using biorender under an academic license ). ( cell viability ( % ) of agmatine on the caco - 2 and l929 cells. caco - 2 was treated with different concentrations of agmatine 1, 3, 6, 9, 12, 15, 18, and 21 mm and l929 cells were treated with 6, 9, 12, 15, 18, and 21 mm for 24, 48, and 72 h. ( c ) logarithmic representation of the mtt assay results for the l929 and caco - 2. error bars show standard error. * also indicates that statistically significant differences ( * p < 0. 05, * * p < 0. 01, * * * p < 0. 001 ) fig. 2agmatine inhibited invasion and migration of caco - 2 cells. after 72 h of agmatine treatment, the effect on the migration ability of caco - 2 cells was evaluated by transwell migration assay. ( the relative migration ratios of migration cells. ( after 72 h of agmatine treatment, the effect on the invasion ability of caco - 2 cells was evaluated by transwell invasion assay. ( the relative invasion ratios of migration cells. data were obtained from triplic ate experiments and expressed as the means ± standard deviation. * p < 0. 05 agmatine inhibited invasion and migration of caco - 2 cells. after 72 h of agmatine treatment, the effect on the migration ability of caco - 2 cells was evaluated by transwell migration assay. ( the relative migration ratios of migration cells. ( after 72 h of agmatine treatment, the effect on the invasion ability of caco - 2 cells was evaluated by transwell invasion assay. ( the relative invasion ratios of migration cells. data were obtained from triplic ate experiments and expressed as the means ± standard deviation. * p < 0. 05 fig. 3agmatine induced apoptosis in caco - 2 and l929 cells. ( the apoptosis of caco - 2 cells ( 6, 7. 3, and 9 mm ) and l929 ( 6, 6. 25 and 9 mm ) after agmatine treatment was observed by dapi and pi staining for 72 h. ( b ) the apoptotic index ( % ) values of caco - 2 and l929 cells. ( relative expression levels of bax, bcl - 2 and csp - 3 in agmatine - treated caco - 2 and l929 cells versus control groups. data were obtained from triplic ate experiments and expressed as the means ± standard deviation. * p < 0. 05, * * p < 0. 01, * * * p < 0. 001 agmatine induced apoptosis in caco - 2 and l929 cells. ( the apoptosis of caco - 2 cells ( 6, 7. 3, and 9 mm ) and l929 ( 6, 6. 25 and 9 mm ) after agmatine treatment was observed by dapi and pi staining for 72 h. ( b ) the apoptotic index ( % ) values of caco - 2 and l929 cells. ( relative expression levels of bax, bcl - 2 and csp - 3 in agmatine - treated caco - 2 and l929 cells versus control groups. data were obtained from triplic ate experiments and expressed as the means ± standard deviation. * p < 0. 05, * * p < 0. 01, * * * p < 0. 001 the expression levels of the apoptosis - related genes, bax, bcl - 2 and casp - 3 were evaluated in caco - 2 cells across the control, 6 mm, 7. 3 mm, and 9 mm groups using δct values normalized to b - actin ( fig. 3c ). for bax, the mean δct value in the control group was 0. 35 ± 0. 02, compared with 1. 01 ± 0. 19 in the 6 mm group, 1. 18 ± 0. 07 in the 7. 3 mm group, and 1. 12 ± 0. 13 in the 9 mm group, indicating a significant increased difference in the 6 mm, 7. 3 mm, and 9 mm groups compared to the control ( p < 0. 001, p < 0. 01, p < 0. 01 ). when the 6 mm groups were compared to the control group, the expression levels of bcl - 2 increased in caco - 2. however, expression in the 7. 3 and 9 mm groups decreased relative to the 6 mm group cells. the overall difference between groups was statistically significant without 9 mm, compared with the control ( p < 0. 01, p < 0. 01, ns ). the mean δct value of csp - 3 was increased in the 7. 3 mm, and 9 mm groups compared to the control group, with a significant group effect ( p < 0. 05, p < 0. 001 ). qrt - pcr analysis revealed dose - dependent upregulation of bcl - 2 and downregulation of bax and csp - 3 expression in agmatine - treated ( 6, 6. 25, 9 mm ) l929 cells, resulting in significant differences relative to the control ( fig. 3c ). to investigate the effects of agmatine - induced ros formation in caco - 2 and l929 cells, gpx and catalase ( cat ) mrna levels were assessed using qrt - pcr. in contrast to the control group, a significant decreases in gpx and cat expression were observed in all three agmatine - treated groups of caco - 2 cells compared to the untreated controls. in l929 cells, however, gpx and cat mrna levels increased in a dose - dependent ( 6. 25 and 9 mm ) and linear manner relative to the control group ( fig. 4a and b ). fig. 4relative expression levels of gpx, cat, abcg2, abcc1, and abcb1 in l929 and caco - 2 cells treated with agmatine. ( the expression levels of gpx and cat in l929 cells were modulated by agmatine in a dose - dependent manner ( b ) the expression levels of gpx and cat in caco - 2 cells were modulated by agmatine in a dose - dependent manner c ) relative expression levels of abcg2, abcc1, and abcb1 in caco - 2 cells treated with 7. 3 mm agmatine for 72 h compared to control. data were obtained from triplic ate experiments and expressed as the means ± standard deviation. * p < 0. 05, * * p < 0. 01, * * * p < 0. 001 relative expression levels of gpx, cat, abcg2, abcc1, and abcb1 in l929 and caco - 2 cells treated with agmatine. ( the expression levels of gpx and cat in l929 cells were modulated by agmatine in a dose - dependent manner ( b ) the expression levels of gpx and cat in caco - 2 cells were modulated by agmatine in a dose - dependent manner c ) relative expression levels of abcg2, abcc1, and abcb1 in caco - 2 cells treated with 7. 3 mm agmatine for 72 h compared to control. data were obtained from triplic ate experiments and expressed as the means ± standard deviation. * p < 0. 05, * * p < 0. 01, * * * p < 0. 001 the relative expression levels of the abcg2, abcc1, and abcb1 genes were evaluated with qrt - pcr after 7. 3 mm agmatine treatment of caco - 2 cells. qrt - pcr data revealed no significant differences between the abcg2, abcc1, and abcb1 expression levels and agmatine - treated caco - 2 cells ( p > 0. 05; fig. 4c ). western blot analyses performed to assess apoptotic pathways ( bax, bcl - 2, and csp - 3 ) in caco - 2 cells treated with agmatine at concentrations of 6 mm, 7. 3 mm, and 9 mm revealed no detectable bands for these proteins, despite showing clear and consistent gapdh bands as the internal loading control ( data not shown ).