Molecular Basis for the Antiproliferative Effect of Agmatine in Tumor Cells of Colonic, Hepatic, and Neuronal Origin

Abstract

The aim of the present study was to challenge potential mechanisms of action underlying the inhibition of tumor cell proliferation by agmatine. Agmatine inhibited proliferation of the human hepatoma cells HepG2, the human adenocarcinoma cells HT29, the rat hepatoma cells McRH7777, and the rat pheochromocytoma cells PC-12. Inhibition of proliferation of HepG2 cells was associated with an abolition of expression of ornithine decarboxylase (ODC) protein and a doubling of mRNA content encoding ODC. In HepG2 cells, silencing of ODC-antizyme-1, but not of antizyme inhibitor, by RNA interference resulted in an increase of agmatine's antiproliferative effect. Thus, the distinct decrease in intracellular polyamine content by agmatine was due to a reduced translation of the synthesizing protein ODC but was not essentially mediated by induction of ODC-antizyme or blockade of antizyme inhibitor. In interaction experiments 1 mM L-arginine, 1 mM D-arginine, 1 mM citrulline, 100 M N -nitro-L-arginine methyl ester, 1 and 10 M sodium nitroprusside, and 1 M N 1 -guanyl-1,7-diaminoheptane failed to alter agmatine's antiproliferative effect. Hence, the antiproliferative effect of agmatine in HT29 and HepG2 cells is due to an interaction with neither the NO synthases, the hypusination of eIF5A, nor an agmatine-induced reduction in availability of intracellular L-arginine. L-Arginine and citrulline, but not D-arginine, inhibited tumor cell proliferation by themselves. Their inhibitory effect was abolished after silencing of arginine decarboxylase (ADC) expression by RNA interference indicating the conversion to agmatine by ADC. Finally, in the four cell lines under study, agmatine-induced inhibition of cell proliferation was paralleled by an increase in intracellular caspase-3 activity, indicating a promotion of apoptosis.Agmatine, a cationic amine formed by decarboxylation of L-arginine by the mitochondrial enzyme arginine decarboxylase, initially attracted attention as an endogenous ligand at imidazoline receptors (Li et al., 1994). However, independent of binding to those receptors, agmatine induces a variety of physiological and pharmacological effects (Raasch et al., 2001). In particular, the antiproliferative effect of agmatine has aroused interest as a new alternative in the treatment of neoplasms. Agmatine administration to tumor cells in vitro results in a suppression of cell proliferation (Satriano et al., 1998;Choi and Cho, 1999;Vargiu et al., 1999;Babal et al., 2001;Dudkowska et al., 2003;Higashi et al., 2004;Kribben et al., 2004;Molderings et al., 2004;Mayeur et al., 2005). At the intracellular level, agmatine-induced decrease of cell proliferation was shown to be due to a decrease in the intracellular levels of the polyamines putrescine, spermidine, and spermine (Satriano et al., 1998;Choi and Cho, 1999;Vargiu et al., 1999;Babal et al., 2001;Dudkowska et al., 2003;Higashi et al., 2004;Mayeur et al., 2005), which are pivotal for cell growth (Igarashi and Kashiwagi, 2000). The intracellular concentration of polyam...

Ornithine decarboxylase and glutamate decarboxylase 65 as prognostic markers of gallbladder malignancy: A clinicopathological study in benign and malignant lesions of the gallbladder

“…It has been reported that the overexpression of ODC may lead to increased polyamine levels and thus induce the overproliferation and malignant transformation of cells (4,(16)(17)(18)(19). It contributes to the development, progression, metastasis and surrounding invasion of malignant tumors, and serves as a useful biological marker for certain precancerous lesions of digestive malignancies (4)(5)(6)(7)(8)16,17,20). A previous study demonstrated that Helicobacter pylori upregulates the expression of ODC, resulting in gastric precursor lesions and cancer (21).…”

“…Carcinomas with highly expressed ODC are more progressive and poorly differentiated, with increased metastasis, surrounding invasion and poor prognosis. Hence, ODC may be a novel target for use in the development of new anticancer drugs and as an indicator for the efficacy of treatments (4)(5)(6)(7)(8)16,17,20). Tissue-specific markers are useful for the identification of tumor type in advanced cancers of unknown origin.…”

Modulation of L-Arginine-Arginase Metabolic Pathway Enzymes: Immunocytochemistry and mRNA Expression in Peripheral Blood and Tissue Levels in Head and Neck Squamous Cell Carcinomas in North East India

“…Both RNA and cDNA concentrations were determined spectrophotometrically (Biorad Spectronanodrop). The primer sequences included sense 5'-ATTGTTCCGTTCTTCTTGACTT-3' and antisense 5'-AGTGTGATGTGAAGGATTATG-3' for ARG1 (148bp), sense 5'-GGTCCCGCTGCCATAAGAG-3' and anti-sense 5'-CGTGGATTCACTATCAGGTTGTTG-3' f o r A R G 2 ( 1 3 7 b p ) , s e n s e 5'-GGATCCAAATGTGGCTGCGTTCAT-3' and antisense 5'-AACAGCCAGCCATCTACCAGTTCT-3' f o r O A T ( 1 7 5 b p ) , s e n s e 5'-GTGGGTGATTGGATGCTCTTTG-3' and anti-sense 5'-AGGCCCTGACATCACATAGTAG-3' for ODC (108bp), sense 5'-GAAATCCCATCACCATCTTCC-3' and anti-sense 5'-GAGCCCCAGCCTTCTCCATG -3 ' f o r G A P D H ( 1 2 0 b p ) , a n d s e n s e 5'-TGGATCAGCAAGCAGGAGTATG-3' and antisense 5'-AAGAAAGGGTGTAACGCAACTAAG-3' for β-actin (98bp) (Bergeron et al, 2007;Wolf et al, 2007;Haenisch et al, 2008;Kitowska et al, 2008;Kim et al, 2012;Mondal et al, 2013). For all analysis, sqPCR assays were performed in a 25 µL system containing PCR master mix, 30ng first-strand cDNA template and 0.4 µM appropriate primers.…”

Administering the Optimum Dose of l-Arginine in Regional Tumor Therapy

“…The ability of Arg to inhibit the tumor growth based on the metabolism of Arg or based on its chemical structure has been verified and confirmed. Arg when incubated in medium for 24-72 h had been shown to kill gastric cancer cells through apoptosis based on the metabolism of Arg [7,8]. Yurtcu et al [4] have reported also that based on the metabolism Arg could induce DNA damage with rising of NO in lymphocytes.…”