Extracellular ATP modulates several biological processes via activation of plasma membrane receptors (P2Rs) in normal human thyrocytes (NT). We characterized P2Rs expression and function in two thyroid cancer cell lines: FB1 (anaplastic cancer) and FB2 [papillary cancer (PTC)]. P2Rs expression was evaluated by RT-PCR and WB, intracellular Ca2+changes by fluorimetric technique (Fura2-AM), IL-6 release by ELISA, intracellular [i(ATP)] and extracellular ATP [e(ATP)] concentration by luminometry.
FB1 and FB2 showed significantly higher e(ATP) and i(ATP) concentration than NT (P<0.001 for both). [Ca2+] fluxes induced by e(ATP) (1 mM, in the presence of external Ca2+) were higher in both FB1 and FB2 than NT cells, (P<0.01). Moreover, the addition of ATP (0.25 and 1 mM) induced a significantly higher IL-6 release respect to NT (P<0.001at both ATP concentrations) in both cell lines. The P2X7 agonist BzATP, almost ineffective in NT, induced a huge IL-6 release in FB2 (from 6315±328 to 11764±1652 and to 25661±2815 pg/ml/1.5x105 cells with BzATP 0.25 and 1 mM, respectively) and FB1, although at a lesser extent (from 7388±170 to 8721±1332 and to 10620±2216, respectively). Moreover, IL-6 release was prevented either by oxidized-ATP or KN-62, selective blockers of human P2X7. Accordingly, FB2 cells showed a strong expression of P2X7, less evident in FB1 cells. These findings demonstrated an enhanced expression of functional P2X7 receptors in thyroid cancer cell lines. Therefore, we checked P2X7 expression in 33 human PTC histological samples, confirming an increased P2X7 expression in cancer than in normal thyroid tissue both by RT-PCR (P<0.0001) and immunostaining (avidin-biotin method) (72±15% Vs 8±3% of cells, respectively).
In conclusion, human thyroid cancer is characterized by an enhanced P2XRs function; specifically, PTC shows a strong P2X7 expression in comparison to normal thyroid tissue. The increased P2X7R function may play a role in the modulation of the inflammatory response to neoplasia.