P052 Walker factor (proteolysis induced factor-like) affects the vero cells activity, and these changes can be modulated by leucine treatment

Abstract

Introduction The cachexia process is investigated in experimental models and also in vitro models, evaluating the muscle protein synthesis and degradation. The molecular and biochemical effects of the Walker Factor, a glycosylated peptide similar to proteolysis induced factor (PIF), on the cachexia mechanism are the main subject for researchers in this area. The Vero cells are widely studied in assays involving cellular cytotoxicity, growth and differentiation, transformation in vitro, and experimental carcinogenesis, induced by different agents or conditions, therefore, these cells are recommended for standard protocols. The Vero cells have well defined characteristics in culture. Methods Therefore, the main of this study was to investigate the effect of Walker factor exposure (FW) modulated by leucine treatment in Vero cells, observing in these cells modifications on growth properties, differentiation, morphologic and metabolic alterations. We analysed the cellular viability and growth, proteolytic enzymes activities (chymotrypsin like, cathepsin and calpain) and medium cytokines content (INFgamma, IL-10, IL-4, IL-6 and TNFalpha) in Vero cells exposed to different concentration of WF (1, 3, 5, 10, 15, 20 and 25 μg/mL Walker factor for 24 h) associated to leucine treatment (previously to 25 or 50 μM leucine during 2 h (L25 or L50)). Results At higher Walker factor concentrations (WF25μg/mL), there were reduction on the cellular viability and growth. The alkaline phosphatase activity, which suggests the cellular activity, showed that Vero cells presented reduction in this enzyme activity at WF1, WF3 and WF10, which was recovered at L25 and L50 treatment. The chymotrypsin, cathepsin and calpain activities increased especially at WF15, WF20 and WF25 exposition, but these enzymes activities were similar to control cells at L50 treatment. Protein synthesis reduced at WF25 exposure but was maintained by previous leucine treatment (L50). Protein breakdown increased at WF20 and WF25 and also remained similar to control cells after treatment with L25 and L50. The cytokine content in culture medium (DMEM enriched with foetal calf serum and glutamine) was analysed and established as a standard control. The control cells culture medium had similar concentration of INFgamma, IL4 and IL10 and low levels of IL6 and TNF compared to standard medium. The medium from experimental Vero cells exposed to WF showed different cytokines levels compared to control cells and standard medium. After WF exposure, the Vero cells medium cytokine concentration reduced, especially in INFgamma, IL-10, IL-6 and IL-4 content, which showed deep decrease at WF25 treatment. The leucine treatment modulated and recovered these parameters. Conclusion This is an important point to be investigated in nearly future as the results could be related to cell signalling and the mechanism involved in up-regulation or down-regulation of proteolytic pathway, induced by WF, which can be leaded by these proinflammatory cytokines (IL-6, INFgamma and TNFalpha). New experiments are now underway in our laboratory to address these questions. Statistical support: Dr. J Marcondes; Financial support: FAPESP #2006/6003-7, #2010/11328-9.