Extracellular adenine nucleotide hydrolysis is mediated by action of the e-NTPDase and of the 5´-nucleotidase. Among others properties described for adenine nucleotides, an anticancer activity is suggested, once the ATP is considered cytotoxic in several tumor cell systems. Some studies demonstrate that adenosine presents a tumor-promoting activity. We show that NTPDases expression profile and extracellular ATP hydrolysis in Walker 256 tumor-bearing rats are modified during in vivo growth.
Male Wistar rats were inoculated at a single dorsal subcutaneous site in dorsolumbar region with an Walker 256 tumor cell suspension. 6, 10 and 15 days after inoculation, the total RNA from rat solid tumor was isolated with Trizol LS reagent in accordance with the manufacturer's instructions and cDNA species were synthesized with SuperScript II for NTPDase 1-VI and 5´-nucleotidase. 10ml of the PCR reaction was analyzed on a 1.3% agarose gel. For quantitative expression, the total RNA and cDNA generated were submitted in an SYRB Green I-based real-time PCR carried out on MJ Research DNA Engine OpticonTM Continuous Fluorescence Detection System as described. For extracellular ATP hydrolysis, the solid tumor was dissociated with collagenase and the cellular suspension was incubated with ATP 0,2 mM. The reaction was finished in ice, the samples centrifuged and the supernatant was analyzed by HPLC as described. The suspension cells were 98% viable. We identify all six NTPDase subtypes and a 5´-nucleotidase in solid tumor from 6, 10 and 15 days in vivo growth.
The quantitative expression showed an increase on expression of NTPDase I and II and 5´-nucleotidase after 10 days in vivo growth. The HPLC analysis could show that the extracellular ATP cascade was more quickly terminate with adenosine and inosine production in cells obtained from solid tumor of 6 and 10 days in vivo growth. The cells obtained from solid tumor of 15 days growth had AMP as final product after 90 min of incubation. In a future, these results can generate news approach for protection mechanism against the tumoral process in circulation.