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L-glutamine And L-arginine UTILIZATION BY WALKER 256 TUMOR CELLS
Rosilene Rebeca1,2; Lívia Bracht1; Guilhermina Rodrigues Noleto1; Maria Eliane Merlin Rocha1; Maria Benigna Martinelli de Oliveira1
1Departamento de Bioquímica e Biologia Molecular, Setor de Ciências Biológicas da Universidade Federal do Paraná (UFPR), Curitiba-PR; 2Departamento de Ciências Biológicas, Centro de Ciências Agrárias e Ambientais (CAA) da Universidade Estadual do Centro-Oeste (UNICENTRO), Guarapuava-PR.
Cancer cells frequently use amino acid as an energy substrate, glutamine and arginine being important in catabolic states, being administered to patients as nutritional supplements (Yoshida et al., Nutrition, 17:766-68, 2001). Several tumor lineages use L-glutamine by its conversion to lactate, a pathway known as glutaminolysis. L-Arginine is another key amino acid in tumor cells, and can be converted via arginase to L-ornithine and urea. L-Arginine is also substrate for the nitric oxide synthase (NOs) giving rise to nitric oxide (NO) and citrulline. These metabolic pathways were now established in the ascitic form of the Walker 256 tumor, a recognized model for cancer cachexia. Cell suspensions from four or six days developed ascitic tumors, free of erythrocytes and macrophages, were used in all experiments. Glutaminolysis was evaluated by lactate production by intact cells and the activity of glutaminase was characterized in cell free extracts. Lactate production, enzymatically evaluated in supernatants of cell suspensions, incubated (30 min at 37oC) in the presence of L-glutamine (10mmol.L-1), was 43.4% higher than in controls. In addition, the activity of the phosphate dependent glutaminase occurred in cell free extracts (11.69 ± 6.5 µmol.min-1.mg-1protein), the enzyme showing Kmap of 4.69 ± 0.81 mmol.L-1. Utilization of L-arginine by the ascite Walker 256 cells was evaluated, based on two possibilities, via arginase and via NOs. Intact cell suspensions from six days developed ascitic tumors, when incubated with L-arginine (25 mmol.L-1), produced 17 or 25 times more urea after 30 or 45 min incubation respectively, in comparison with controls. It was meaningful that the presence of arginase in cell free extracts was dependent on the time of the development of the tumor. The activity of the enzyme after four days development of tumors was ~36 % lower than that present in cell extract of six days, when Kmap was 9.66.10-2 ± 2.39 mol.L-1. Ascites Walker 256 cells were also able to utilize L-arginine to produce NO, as verified with cell suspensions incubated for different times in the presence or absence (control) of 10 mmol.L-1 L-arginina. Since NO is also an important mediator of cachexia, these results suggest that the behavior of Walker 256 cells concerning with the utilization of L-arginine, would be an important determining factor for manifestation of cachexia. Support: UNICENTRO, CNPq, PRONEX.
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