| Background and Objective: Adenosine is an ubiquitous endogenous nucleoside, which is not only a metabolite of ATP, but also a procursor of ATP. When oxygen supply is enough, only little adenosine is released into the extracellular space, that is enough to play it's important pathophysiological role; But if there is a disbalance of oxygen-supply, a great quantity of adenosine will be released. Adenosine is widely used in many clinical conditions including arrhythmia, acute myocardial infarction (AMI), inflammation, neuropathic pain, ischemia-reperfusion injuries. Adenosine affects on cells via two pathways: one is adenosine receptor pathway, the other is non-receptor pathway. Most of the previous researches focus on the receptor-media- ted pathway. Four Adenosine receptor subtypes have been identified : A1,A2a,A2b,A3, which are all coupled to G-proteins. Different receptor subtypes lead to different physiological and pathological effects and most of the clinical applications of adenosine is based on the receptor-mediated mechanism. Adenosine can also be transported into intracellular space and play effects on cells, which defined as non-receptor-mediated mechanism. There were lots of reports about daul opposing effects of adenosine but the mechanism is unclear. The aim of our work is to observe the daul opposing effects of adenosine by non-receptor-mediated mechanism and the relationship between effects and intracellular ATP. .Methods:1. Cell culture and treatment 2. manipulaiton of the intracellular ATP2.1 The regulation of the basic intracellular ATP Glucose-free culture medium/ cultrue medium contain D-Glucose culture medium contain 1% FBS/ culture medium contain 10%FBS2.2 The down-regulation of the intracellular ATP(1) Oligomycin: Oligomycin is an oxidateve phosphorylation inhibitor which can block mitochondrial/glycolytic ATP generation.(2) 2-DG: 2-DG is a glycolytic inhibitor which can block glycolytic ATP generation.(3) Dipyridamole (DP): DP is a nucleoside uptake inhibitor, which can block adenosine uptake.3. Intracellular ATP Assay(1) HPLC(2) ATP Assay Kit(firefly luciferase)4. Cell death Assay(1) Flow cytometry(Annexin V-FITC-PI)(2) Morphological observation5. Cell viability Assay and Cell proliferation Assay(1) Alamar blue assay(2) MTT assay(3) Cell counter6. Measurement of PARP, caspase3, caspase8, caspase9 proteins Western BlottingResults:1. Dual opposing effects of adenosine: cytoprotection and cytotoxicity1.1 Adenosine increased intracellular ATP in multiple cell linesMany kinds of cell lines were cultured in mediums with or without D-glucose respectively. The effect of Ade on intracellular ATP was assessed by HPLC and ATP increased in all the cell lines observed to some extent.1.2 Adenosine decreased cell viabilityMany kinds of cell lines were cultured in standard culture conditions and incubated with Ade at various concentrations (0.5~5mM) for 72 hr. The viability of cells was detected using the MTT assay. These results showed that Ade decreased cell viability of all the measured-cell lines and this effect was observed in a dose-dependent manner.1.3 Effect of adenosine on cell survival1.3.1 Adenosine induced apoptosis of Ana-1 cellsAna-1 cells were cultured under standard culture conditions. After exposure to Ade at different concentrations (0.5, 1, 2, 4, 8mM) for 12 hr, cell survival was assessed using the Flow cytometry. Co-incubation with Ade induced apoptosis and this effect was observed in a dose-dependent manner. The cell death rates were 13.1%, 17.6%, 43.4%, 58% and 73.5% of control, respectively. Ana-1 cells were cultured under standard culture conditions. After exposed to Ade at different concentrations (0.5, 0.8, 1, 2, 4mM) for 6 hr, PARP cleavage was detected by Western blotting. These data showed that Ade induced PARP cleavage in a dose-dependent manner.1.4 Effects of adenosine on cell viability and proliferation1.4.1 Protective effect of adenosine on cell viability and cell proliferation in low ATP statesK562 cells were cultured in D-glu-free medium with 1% FBS and incubated with Ade at different concentrations (0~8mM) for 48 hr. Then the cell viability and cell proliferation was quantified by the alamar blue assay. These data showed that Ade increased cell viability and cell proliferation up to approximately 164.93%, 163.80%, 149.13%, 141.12% and 128.72% of control, respectively. K562 cells were cultured in medium without D-glucose, and Ade at different concentrations (0.125~2mM) was added into medium. After co-incubated with Ade for 48 hr, the cell proliferation was count by a cell counter. A pretective effect of Ade on cell proliferation was observed and the maximum value of cell number was 139.62% of control value.1.4.2 Inhibiting effect of adenosine on cell viability and cell proliferationK562 cells were cultured under different conditions (D-glu-free+10%FBS, D-glu+1%FBS or 10%FBS), and exposed to Ade at different concentrations (0~8mM) for 48 hr, alamar blue was used to assay the cell viability and cell proliferation, and the results indicated a inhibiting effect of Ade. The minimum value of cell viability and proliferation was 48.23% of control.K562 cells were cultured in medium with 2g/L D-glucose, and Ade at different concentrations (0.125~2mM) was added into medium. After co-incubated with Ade for 48 hr, the cell proliferation was count by a cell counter. A inhibiting effect of Ade on cell proliferation was seen, and the minimum value of cell number was 53.29% of control value.These results indicated dual opposing effects of adenosine that exhibited respectively with intracellular ATP states which more or less than physio-intracellular. The pretective effect of Ade existed when intracellular ATP was less than physio-intracellular ATP, otherwise the effect of Ade was existed as cytotoxity. 1.5 Effect of adenosine on cell survival under ATP depletion statesK562 cells were cultured in medium without D-glucose, and divided as follows: control group, Ade2mM group, olig (0.5μg/ml) group and Ade+olig group. After co-incubated with drugs for 18 hr, cell apoptosis was detected using flow cytometry. Oligomycin caused a mass of apoptotic cells by exhausing intracellular ATP, and the cell death rate of olig (0.5μg/ml) group was 30.85% of control, the difference between two groups was statistically significant (P<0.05); Co-incubation with Ade (2mM) almost suppressed cell death completely and the cell death rate was inversed nearly 26% of control. The difference between two groups was statistically significant (P<0.05);1.6 DP blocked the effects of adenosine1.6.1 DP supressed inhibition of proliferationK562 cells were cultured in medium with 2g/L D-glucose and divided as follows: control group, Ade (2mM) group, DP (5μM) group and Ade +DP group, with each drug were incubated for 24 hr cell number was assay using a cell counter. The cell number of Ade (2mM) group was 51.72% of control and the difference between two groups was statistically significant (P<0.05). This inhibiting effect of Ade was partly blocked by DP (5μM), the cell number of Ade+DP group was inversed to 73.56% of control. The difference between Ade2mM group and Ade +DP group was statistically significant (P<0.05).1.6.2 DP supressed the cytopretective effect on cell survival K562 cells were cultured in medium without D-glucose and divided as follows: control group, Ade (2mM) group, DP (5μM) group, olig (0.5μg/ml) group, Ade +olig group and olig +Ade +DP group, cell apoptosis was detected 18 hr later using flow cytometry. These data showed that incubation with oligomycin along caused a large cell death, the cell death rate of olig0.5μg/ml group was 30.85% of control, but when cells were co-incubated with Ade and oligomycin, the cell death rate was nearly same with control. The difference between olig0.5μg/ml group and Ade+olig group was statistically significant (P<0.05); This cytopretective effect of adenosine was suppressed by DP (5μM) and the cell death of olig + Ade +DP group increased up to 21.05% of control. The difference between olig +Ade +DP group and Ade +olig group was statistically significant (P<0.05).1.6.3 DP blocked ATP increasingK562 cells were cultured in medium without D-glucose and divided as follows: control group, Ade (2mM) group, DP (5μM) group, olig (0.5μg/ml) group, Ade +olig group and olig +Ade +DP group. Intracellular ATP was quantified using ATP assay kid 3 hr later. These data showed that 0.5μg/mloligomhycin decreased 33.44% intracellular ATP compared with control. Co-incubation with adenosine and oligomycin blocked this sharp decreasing, and the intracellcular ATP recoverd up to 97.91% of control group; DP (5μM) partly blocked Ade-mediated ATP-recovering and the intracellular ATP of olig +Ade +DP group was the same with intracellcular ATP of oligo (0.5μg/ml) group.2. Adenosine sensitized proteasome inhibition-inducd cell death2.1 Cells cultured in normal conditions2.1.1 Cell death by flow cytometryK562 cells were cultured under standard culture condition and divided as follows: control group, MG132 (5μM) group, Ade (0.1, 0.5, 2mM) group, MG132 (0h) +Ade (0.1, 0.5, 2mM) group and MG132 (6h) +Ade (0.1, 0.5, 2mM) group. Flow cytometry was used to assay cell apoptosis 15 hr later. These data suggested that (1) Incubation with Ade at low concentrations (0.1mM, 0.5mM) increased cell death which induced by proteasome inhibition. The cell death rate of MG132 (0h) +Ade0.1mM group and MG132 (0h) +Ade (0.5mM) group was 3.55% and 8.35% of control more than MG132 group , respectivily; But co-incubation with Ade 6 hr after the treatment of MG132 caused more cell death, the cell death rate of MG132 (6h) +Ade (0.1mM) group was 7.9% of control more than MG132 (0h) +Ade0.1mM group and the cell death rate of MG132 (6h) +Ade0.5mM group was 9.75% of control more than the rate of MG132 (0h) +Ade0.5mM group. (2) when cells were treated with Ade (2mM) 6 hr after the treatment of MG132, there were a larger cell apoptosis detectied of MG132 (6h) +Ade2mM group, the cell death rate of MG132 (6h) +Ade2mM group was 23.2% of control more than MG132 group. The difference between two grouops was statistically significant (P<0.05); But when cells were treated with MG132 and Ade (2mM) at the same time, there were litter apoptotic cells contrasted to a MG132 group, the cell death rate of MG132(0h)Ade2mM group was 14.55% of control less than MG132 group.2.1.2 Cell death by morphological observationK562 cells were cultured under standard culture condition and divided as follows: control group, MG132 (5μM) group, Ade (0.1, 0.5, 2mM) group, MG132 (0h) +Ade(0.1, 0.5, 2mM) group and MG132 (6h) +Ade(0.1, 0.5, 2mM) group. PI Staining performed and inverted fluorescence microscope was being used to observe morphology for more than 24 hr. The morphologic images 15 hours later suggested that (1) Incubation with Ade at low concentrations (0.1mM, 0.5mM) increased cell morphological damage slightly which induced by proteasome inhibition, but there were larger cell death and more PI positive cells when Ade was added 6 hr later than MG132. (2) A large upregulation of sensitivity was showed when Ade (2mM) was added 6 hours later than MG132. Cytopretective effect of Ade was showed when Ade (2mM) was added at the same time with MG132, and little mophologic damage was observed; (3) when Ade was added 6 hr later than MG132, cell morphological damage induced by proteasome inhibition was enhanced in a dose-dependent manner.These results suggested that adenosine sensitized proteasome inhibition-inducd cell death under a physio-ATP level.2.2 Cells cultured in glucose-free conditions2.2.1 Cell death by flow cytometryK562 cells were cultured in Glu-free medium, and divided as follows: control group, MG132 (5μM) group, Ade (0.1, 0.5, 2mM) group and MG132 (0h) +Ade (0.1, 0.5, 2mM) group. Flow cytometry was used to detected cell apoptosis 12 hours later. These data showed that adenosine sensitized proteasome inhibition-inducd cell death in a dose-dependent manner. The differences between each two groups were statistically significant (P<0.05)2.2.2 Cell death by morphological observationK562 cells were cultured in Glu-free medium, and divided as follows: control group, MG132 (5μM) group, Ade (0.1, 0.5, 2mM) group and MG132 (0h) +Ade (0.1, 0.5, 2mM) group. PI Staining performed and inverted fluorescence microscope was being used to observe cell morphology for more than 24 hr. The morphologic images of phase contrast at 12 hours and images of PI staining at 24 hour suggested that adenosine enhanced cell morphological damage induced by protersome inhibition in a dose-dependent manner.These results suggested that adenosine sensitized proteasome inhibition-inducd cell death in a dose-dependent manner.2.3 Adenosine-sensitizeded cell death was mediated by intracellular ATP increasing.2.3.1 Regulation by oligomycin2.3.1.1 Cell death by flow cytometryK562 cells were cultured in Glu-free medium, and divided as follows: control group, MG132 (5μM) group, Ade (2mM) group, oligo (0.5μg/ml) group, MG132 (0h) +Ade group and MG132 +oligo (0h) +Ade group. Flow cytometry was used to detected cell apoptosis 18 hours later. The data showed that adenosine obviously enhanced cell death induced by proteasome inhibition but treatment with oligomycin suppressed this upregulation of cell death rate. The cell death rate of MG132+oligo (0h) +Ade group was 33% of control less than the rate of MG132 (0h) +Ade group.K562 cells were cultured in Glu-free medium, and divided as follows: control group, MG262 (1μM) group, Ade (2mM) group, oligo (0.5μg/ml) group, MG262 (0h) +Ade group and MG262 +oligo (0h) +Ade group. Flow cytometry was used to detected cell apoptosis 18 hours later. The data showed that adenosine obviously enhanced cell death induced by proteasome inhibition, the cell death rate of MG262 (0h) +Ade group was 28.8% of control more than the rate of MG262 (1μM) group, but treatment with oligomycin suppressed this upregulation of cell death rate. The cell death rate of MG262 +oligo (0h) +Ade group was 12.1% of control less than the rate of MG262 (0h) +Ade group.2.3.1.2 Cell death by morphologic observationK562 cells were cultured in Glu-free medium, and divided as follows: control group, MG132 (5μM) group, MG262 (1μM) group, Ade (2mM) group, oligo (0.5μg/ml) group, MG132(0h)+Ade group,MG262(0h)+Ade group, MG132 +oligo (0h) +Ade group and MG262 +oligo (0h) +Ade group. PI Staining performed and inverted fluorescence microscope was being used to observe cell morphology for more than 24 hr. The morphologic images at 24 hour showed the same tendency as cell apoptosis detected by flow cytometry.2.3.2 Regulation by 2-DGK562 cells were cultured in Glu-free medium, and divided as follows: control group, MG132 (5μM) group, Ade (2mM) group, 2-DG (0.5mM) group, MG132 (0h) +Ade group and MG132 +2-DG (0h) +Ade2mM group. PI Staining performed and inverted fluorescence microscope was being used to observe morphological changes for more than 24 hr. The morphologic images of PI-staining at 12 hour suggested that MG132 induced less PI-positive cells in the presence of 2-DG.2.3.3 Regulation by DPK562 cells were cultured in Glu-free medium, and divided as follows: control group, MG132 (5μM) group, Ade (2mM) group, DP (5μM) group, MG132 (0h) +Ade group and MG132 +DP (0h) +Ade group. PI Staining performed and inverted fluorescence microscope was being used to monitor cell morphology for more than 24 hr. The morphologic images of phase contrast at 15 hour and PI-staining at 24h suggested that adenosine enhanced cell morphological damage and PI-positive cells induced by protersome inhibition, and DP partially blocked cell death caused by adenosine. There were less cell morphological damages and PI-positive cells observed in MG132 +DP (0h) + Ade group.These results suggested that the adenosine sensitized proteasome inhibition- inducd cell death, which was mediated by ATP.2.4 Caspase activation2.4.1 Cell death rescued by Z-VADK562 cells were cultured under standard culture condition and divided as follows: control group, MG132 (5μM) group, MG132 (6h) + Ade group, Z-VAD100μM group and MG132 +Z-VAD (6h) +Ade2mM group. After treatments, PI Staining performed and inverted fluorescence microscope was being used to observe cell morphology for more than 24 hr. The morphologic images of phase contrast at 15 hours and images of PI staining at 24 hour showed that Z-VAD (a broad caspase inhibitor) counteracted effects of Ade and MG132.2.4.2 Activation of caspases and PARP cleavageK562 cells were cultured under standard culture conditions and divided as follows: control group, MG132 (5μM) group, Ade (0.2, 1.0, 2.0mM) group and MG132 (0h) +Ade. Then western blotting was carried out 12 hr later, as a semiquantitative method to detect caspase proteins cleavage and PARP cleavage.The results suggested that (1) Ade treatments at low concentrations (0.2mM, 1.0mM) caused more caspase protein cleavage in a dose-dependent manner, which were induced by proteasome inhibitors. (2)Ade treatment at high concentration (2mM) induced a decrease of protein cleavage, which was different from effects of Ade at low concentrations.2.4.3 Comparision of the activation of caspases and PARP cleavage in various ATP statesK562 cells were cultured under standard culture condition and in Glu-free medium respectively, and divided as follows: control group, PI group, Ade group, PI (0h) +Ade group and PI (6h) +Ade group. Then western blotting was carride out 12 hr later, as a semiquantitative method to detect changs of cleaved caspase proteins and cleaved PARP. The results suggested that (1) when cells were cultured under standard culture, co-incubation with adenosine and PI at the same time caused a serious decrease of protein cleavage induced by proteasome inhibition, but caused a serious increase of protein cleavage if the treatment of Ade was 6 hours later than the treatment of PI. (2)Co-incubation with adenosine and PI at the same time caused a slight increase of protein cleavage induced by proteasome inhibition when cells were cultured in Glu-free medium, and a serious increase of protein cleavage was caused by the treatment of adenosine 6 hours later than the treatment of PI.Conclusions:1. Adenosine possesses dual opposing effects as cytopretection and cytotoxity; Intracellular ATP concentration determines the bidirectional regulation of adenosine.2. Adenosine sensitized cell death induced by proteasome inhibition; Caspase activation is responsible for the sensitization effect of adenosine. |
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