The anti-tumor effect of A3 adenosine receptors is potentiated by pulsed electromagnetic fields in cultured neural cancer cells

Abstract

A(3) adenosine receptors (ARs) play a pivotal role in the development of cancer and their activation is involved in the inhibition of tumor growth. The effects of pulsed electromagnetic fields (PEMFs) on cancer have been controversially discussed and the detailed mechanisms are not yet fully understood. In the past we have demonstrated that PEMFs increased A(2A) and A(3)AR density and functionality in human neutrophils, human and bovine synoviocytes, and bovine chondrocytes. In the same cells, PEMF exposure increased the anti-inflammatory effect mediated by A(2A) and/or A(3)ARs. The primary aim of the present study was to evaluate if PEMF exposure potentiated the anti-tumor effect of A(3)ARs in PC12 rat adrenal pheochromocytoma and U87MG human glioblastoma cell lines in comparison with rat cortical neurons. Saturation binding assays and mRNA analysis revealed that PEMF exposure up-regulated A(2A) and A(3)ARs that are well coupled to adenylate cyclase activity and cAMP production. The activation of A(2A) and A(3)ARs resulted in the decrease of nuclear factor-kappa B (NF-kB) levels in tumor cells, whilst only A(3)ARs are involved in the increase of p53 expression. A(3)AR stimulation mediated an inhibition of tumor cell proliferation evaluated by thymidine incorporation. An increase of cytotoxicity by lactate dehydrogenase (LDH) release and apoptosis by caspase-3 activation in PC12 and U87MG cells, but not in cortical neurons, was observed following A(3)AR activation. The effect of the A(3)AR agonist in tumor cells was enhanced in the presence of PEMFs and blocked by using a well-known selective antagonist. Together these results demonstrated that PEMF exposure significantly increases the anti-tumor effect modulated by A(3)ARs.

Figure 1. PEMFs increased A_2A and A₃AR mRNA levels.

Relative mRNA expression of A₁, A_2A, A_2B and A₃ARs in rat cortical neurons, untreated or NGF-treated PC12 cells and U87MG cells in the absence or in the presence of PEMFs. Values are the mean (n = 6) ± SEM.

Figure 2. cAMP modulation by PEMFs up-regulated A_2A and A₃ARs.

Effect of a well-known A_2AAR agonist and antagonist (CGS 21680, 100 nM; SCH 58261, 1 µM) or A₃AR agonist and antagonist (Cl-IB-MECA, 100 nM; MRS 1523, 1 µM) in rat cortical neurons, untreated or NGF-treated PC12 cells and U87MG cells in the absence or in the presence of PEMFs on cAMP production. Values are the mean (n = 6) ± SEM.

Figure 3. Inhibition of NF-kB activation by the co-presence of PEMFs and A_2A or A₃AR agonists.

Effect of a well-known A_2AAR agonist and antagonist (CGS 21680, 100 nM; SCH 58261, 1 µM) or A₃AR agonist and antagonist (Cl-IB-MECA, 100 nM; MRS 1523, 1 µM) in rat cortical neurons, untreated or NGF-treated PC12 cells and U87MG cells in the absence or in the presence of PEMFs on NF-kB activation which was evaluated by detecting phosphorylated p65 proteins in nuclear extracts. Values are the mean (n = 6) ± SEM.

Figure 4. A₃AR agonist and PEMFs increased p53 levels.

Effect of a well-known A_2AAR agonist and antagonist (CGS 21680, 100 nM; SCH 58261, 1 µM) or A₃AR agonist and antagonist (Cl-IB-MECA, 100 nM; MRS 1523, 1 µM) in rat cortical neurons, untreated or NGF-treated PC12 cells and U87MG cells in the absence or in the presence of PEMFs on p53 levels. Values are the mean (n = 6) ± SEM.

Figure 5. Synergistic effect of A₃AR stimulation and PEMFs in the inhibition of tumor cell proliferation.

Effect of a well-known A_2AAR agonist and antagonist (CGS 21680, 100 nM; SCH 58261, 1 µM) or A₃AR agonist and antagonist (Cl-IB-MECA, 100 nM; MRS 1523, 1 µM) in rat cortical neurons, untreated or NGF-treated PC12 cells and U87MG cells in the absence or in the presence of PEMFs on thymidine incorporation. Values are the mean (n = 6) ± SEM.

Figure 6. Cytotoxicity of A₃AR activation and PEMFs in tumor cells.

Effect of a well-known A_2AAR agonist and antagonist (CGS 21680, 100 nM; SCH 58261, 1 µM) or A₃AR agonist and antagonist (Cl-IB-MECA, 100 nM; MRS 1523, 1 µM) in rat cortical neurons, untreated or NGF-treated PC12 cells and U87MG cells in the absence or in the presence of PEMFs on lactate dehydrogenase (LDH) levels. Values are the mean (n = 6) ± SEM.

Figure 7. PEMF exposure enhanced the A₃AR agonist-induced apoptosis in tumor cells.

Effect of a well-known A_2AAR agonist and antagonist (CGS 21680, 100 nM; SCH 58261, 1 µM) or A₃AR agonist and antagonist (Cl-IB-MECA, 100 nM; MRS 1523, 1 µM) in rat cortical neurons, untreated or NGF-treated PC12 cells and U87MG cells in the absence or in the presence of PEMFs on active caspase 3 levels. Values are the mean (n = 6) ± SEM.