Phosphorylation of S338 of c-Raf is reduced by NECA and forskolin.

A time-dependent inhibition of phosphorylation of c-Raf at S338 is observed by stimulation of the A2B receptor with NECA (A). A similar effect was seen with forskolin (B). Western blots show a representative experiment, the columns represent mean values from n = 5 (A) and 10 (B) independent experiments (* p < 0.001, significantly different from control).</p

Effect of cycloheximide on ERK-1/2 phosphorylation and MKP-1 expression.

The inhibition of ERK1/2 phosphorylation by NECA was blocked by 10 μg/ml of cycloheximide (CHX) suggesting that protein synthesis is mandatory for the NECA effect (A). Stimulation of A2B receptors in MDA-MB-231 with NECA cells causes and increase in MKP-1 expression (B), treatment with 1 μM forskolin shows the same effect (C). The increase caused by NECA is blocked by cycloheximide (D). Western blots show a representative experiment, the columns represent mean values from n = 7 (A), 4 (B), 7 (C), and 6 (D) independent experiments (* p < 0.001, significantly different from control).</p

Ca²⁺ effect on MKP-1 expression.

The NECA-induced increase of MKP-1 expression was blocked by the Ca2+-chelator BAPTA (addition of 30 μM BAPTA-AM). The Western blot shows a representative experiment, the columns represent mean values from n = 4 independent experiments (* p < 0.001, significantly different from control).</p

Reduction of ERK1/2 phosphorylation by UTP compared to NECA.

The effect of 100 μM UTP was consistently lower than of 100 nM NECA. Addition of 30 nM forskolin to 100 μM UTP increased the UTP effect although forskolin at this concentration had no effect on its own. The Western blot shows a representative experiment, the columns represent mean values from from n = 7 independent experiments (* p < 0.001, significantly different from control; # p < 0.05, significantly different from each other).</p

Effect of the PKA inhibitor H-89 on NECA-mediated reduction of ERK1/2 phosphorylation.

Preincubation (30 min) of MDA-MB-231 cells with 10 μM of the PKA inhibitors H-89 prevented the NECA effect on ERK1/2 phosphorylation. The Western blot shows a representative experiment, the columns represent mean values from n = 7 independent experiments (* p < 0.001, significantly different from control).</p

Overview of A_2B signaling pathways.

The scheme summarizes signaling pathways contributing to a reduction of ERK1/2 phosphorylation through stimulation of A2B adenosine receptors in MDA-MB-231 breast cancer cells. p-Raf denotes c-Raf phosphorylated at S338.</p

Reduction of ERK1/2 phosphorylation by the adenosine receptor agonist NECA.

(A) NECA reduced the ERK1/2 phosphorylation in a concentration-dependent manner (EC50 5.85 nM, 95% confidence limit 2.91–11.8). (B) The reduction was time-dependent with a maximum after 30 min. (C) Only NECA as a nonselective agonist showed this effect while activation of A1 with CCPA, A2A (also A1 and A3 to a certain degree) with CGS 21680, and A3 with HEMADO showed no effect. Panel A and the Western blots in B and C show representative experiments, the columns in B and C show data from n = 7 and 5 independent experiments, respectively (* p < 0.001, significantly different from control).</p

NECA-induced reduction of ERK1/2 phosphorylation and effect of adenosine receptor antagonists.

The nonselective antagonist EFA (10 μM) blocked the NECA (100 nM) effect (A) and so did DPCPX at 10 μM, a concentration high enough to block A2BAR (B). In contrast, the A2A selective antagonist SCH 58261 (C) and the A3 selective antagonist MRS 1220 (D), both at 10 μM, had no effect. Western blots show a representative experiment, the columns represent mean values of n = 8 (A), 6 (B), 4 (C), and 5 (D) independent experiments (* p < 0.001, significantly different from control).</p

Role of Ca²⁺ for reduction of ERK1/2 phosphorylation.

The NECA-induced reduction of ERK1/2 phosphorylation was blocked by the Ca2+-chelator BAPTA intracellularly released from 30 μM of the cell-penetrating derivative BAPTA-AM (A). Consequently, an intracellular increase of Ca2+ triggered by 100 μM UTP (B) resulted in a reduction of ERK1/2 phosphorylation similar to increasing levels of cAMP (C). Western blots and the Ca2+ trace in B show a representative experiment, the columns represent mean values from n = 7 (A) and 8 (C) independent experiments (* p < 0.001, significantly different from control).</p

Reduction of ERK1/2 phosphorylation by cAMP.

The effect of 100 nM NECA on ERK1/2 phosphorylation was mimicked by compounds increasing intracellular cAMP, like 1 μM forskolin (A), 100 μM cAMP-AM (“caged cAMP”, B), or the PDE inhibitor Ro20-1724 (C) at 100 μM. Western blots show a representative experiment, the columns represent mean values from n = 6 (A), 4 (B), and 5 (C) independent experiments (* p < 0.001, significantly different from control).</p