Intracellular signalling in Te85 osteosarcoma cells expressing P2X7R isoforms A and B

Background. Osteosarcoma is the most frequent primary malignant bone cancer. The natural history of osteosarcoma is sill mostly unknown and genetic complexity is a hallmark of high grade osteosarcoma so that, currently, no ideal target has been identified. Several efforts are thus in progress to identify new biomarkers as possible targets of efficacious therapies, especially in counteracting pathologic bone remodelling and alleviating associated pain. The P2X7 receptor (P2X7R), a receptor for extracellular ATP widely distributed in human tissues, has recently been shown to have a central role in carcinogenesis, as it enhances in vivo tumor cell growth and cancer-associated angiogenesis (Adinolfi E et al, Cancer Res. 2012) as well as in vitro cancer cell invasiveness (Adinolfi E et al, FASEB J. 2010). P2X7R activity is tightly regulated by extracellular ATP. Indeed, low ATP levels induce the ion channel formation, whereas higher concentrations (nM) provoke opening of a pore permeable to high molecular mass molecules. Among the nine different naturally occurring splice variants known, P2X7RA is thefull-length and full-functioning isoform, whereas P2X7RB is a shorter isoform which lacks the pore forming ability while retaining channel properties. Co-expression of the A and B isoforms in HEK293 cells has been demonstrated to potentiate receptor functions (Adinolfi E et al, FASEB J. 2010). P2X7Rrelated intracellular signalling includes various events such as activation of NFATc1 and PKC/MAPK pathways, as well as activation of PI3K/Akt signalling (Amoroso F et al, Cell Death Dis, 2012; Grol MW et al, Am J Physiol, 2012). Aim of the study. Investigate the role of A and B isoforms of the P2X7R in intracellular signaling linked to receptor activation in Te85 human osteosarcoma cells. Methods. Te85 osteosarcoma cells, which lack endogenous P2X7R expression, were transfected with P2X7RA and P2X7RB, separate or in combination. Proliferation was assessed as a kinetic up to 72 hours in serum starvation conditions. Intracellular calcium levels following stimulation with P2X7R agonists were measured by fluorimetry. NFATc1 levels were assessed by ELISA on nuclear extracts. Total and phosporylated Akt were detected by WB. Results and conclusion. P2X7R expression conferred a proliferative advantage in absence of serum as shown by increased cell growth rate of all Te85 transfectants compared to Te85 wt. All transfected clones showed increased Ca2+ mobilization, the highest response found in Te85 co-transfected with both P2X7RA and B. Nuclear levels of NFATc1 were increased in all transfectants. Finally, higher Akt phosphorylation levels were found in Te85-P2X7RA clones. In conclusion, P2X7R expression in osteosarcoma cells induce augmented proliferation and activation of specific intracellular signalling pathways susceptible of targeting for cancer therap