Fig 1.
Effect of Ado on proliferation of MDA-MB-231 cells.
(A) Incorporation of [3H]-thymidine into cells incubated for 48–72 h in the absence (Control) or the presence of Ado (50 μM). (B) Dose dependence of the effects of Ado on the incorporation of [3H]-thymidine. Cells were cultured for 48–72 h in the absence (Control) or presence of various concentrations of Ado. (C) Effect of Ado on cell growth. Cells were plated and after 48or 72 h of incubation, attached cells were trypsinized and counted with the aid of a using a handheld automated cell counter (Scepter 2.0, Millipore).
Fig 2.
Effect of Ado on migration of MDA-MB-231 cells.
(A) Wound-healing migration assay for MDA-MB-231 cells in the absence and presence of 50 μM Ado (left panel). The percentage of migration in the transwell assay is shown in right panel. (B) The cell migration was assessed by the transwell migration assay in MDA-MB-231 cells incubated in the absence (control) or presence of 50 μM Ado. Shown are representative micrographs of the migrated cells stained with crystal violet (left panel) and the percentage of migrated cell (right panel). The results represent the means ± SE of three independent experiments performed in triplicate. Asterisks denote significant differences (p<0.05) compared to control.
Fig 3.
Expression of Ado receptors in MDA-MB-231 cells.
(A). Percentage of MDA-MB-231 cell proliferation in the presence of Ado with or without dipyridamole (Dipy) an inhibitor of nucleoside transport. (B) RT-PCR of Ado receptor mRNAs in MDA-MB-231 cells. A representative gel is shown (upper panel). Levels of Ado receptor mRNAs were estimated from gene quantitative RT-PCR data and expressed as a percentage of β-actin mRNA expression (lower panel). Cells were incubated for 72 h in the absence (Control) or presence of Ado (50 μM), or the A2B receptor agonist Bay 60–6583 (1 μM). (C) Immunocytochemistry for the A2B receptor was performed to verify expression at the protein level in the absence (Ab -) or the presence of specific antibodies (Ab +).
Fig 4.
Ado increases proliferation in MDA-MB-231 cells via A2B receptor activation.
The activation of the A2B receptor increases [3H]-thymidine incorporation (A) and cell migration expressed as a percentage of the control (B). In contrast, incubation for 72 h in the presence of an A2B receptor antagonist (GS6201; 100 nM) prevented the Ado- and Bay 60-6583-mediated enhancement of [3H]-thymidine incorporation and migration in MDA-MB-231 cells.
Fig 5.
The adenylate cyclase/PKA pathway is involved in the effects of Ado in MDA-MB-231 cells.
Incubation of MDA-MB-231 cells in the presence of an adenylate cyclase agonist (FSK; 10 μM) mimics the Ado-induced effect on cell proliferation (A) and migration (B). In contrast, incubation in the presence of the antagonist KT-5720 (500 nM) prevented the effect of Ado on MDA-MB-231 cell proliferation and migration. (C) The activation of Ado receptors in MDA-MB-231 cells causes a concentration-dependent increase of [3H]-cAMP.
Fig 6.
Adenosine increases proliferation in triple negative breast cancer cells (TNBC) cells.
Effects of Ado on [3H]-thymidine incorporation (A) and cell number (B) in TNBC (MDA-MB-231 and BT-549) and non-TNBC (MCF-10A and MCF-7) cell lines. Cells were treated for 3 days with 50 μM of Ado. (C) Comparison of the effects of Ado on Ki-67 mRNA expression in breast cancer cell lines as A and B. Cells were treated with 50 μM of Ado for 3 days. Percentages of [3H]-thymidine incorporation, cell number and Ki-67 mRNA expression in treated cells (bars) are compared with that in control cells (dotted lines).
Fig 7.
Ado increases NaV1.5 channel functional expression.
(A) Relative effect of Ado on NaV1.5 mRNA expression in the MDA-MB-231 cells as compared to control. (B) Representative superimposed trace currents through NaV channels in MDA-MB-231 cells in the absence and the presence of Ado (250 μM). The currents were generated by pulsing the membrane potential from a holding voltage of -80 mV to 0 mV for 50 ms. (C) Mean current-voltage relationship obtained from MDA-MB-231 control cells and cells incubated 48 h in the presence of Ado (250 μM). Cells were depolarized from -70 to +60 mV for 50 ms by 10-mV increments from a holding potential of -80 mV.
Fig 8.
Effect of tetrodotoxin (TTX) on migration of MDA-MB-231 cells.
The cell migration was assessed by the transwell migration assay in MDA-MB-231 cells incubated in the absence (control) or presence of 50 μM Ado, as well as 10 μM TTX as indicated. Shown are representative micrographs of the migrated cells stained with crystal violet (A) and the percentage of migrating cells is summarized in the bar chart (B).