Abstract
2-Oxoadenosine (2-oxo-Ado), an oxidized form of adenosine, is cytotoxic and induces growth arrest and cell death, which has potential as an anti-cancer drug. However, it is not well understood how 2-oxo-Ado exerts its cytotoxicity. We examined the effects of 2-oxo-Ado on non-tumour cells, namely immortalized mouse embryonic fibroblast lines, and investigated mechanisms by which 2-oxo-Ado exerts its cytotoxicity. We found that cell death induced by 2-oxo-Ado is classical caspase-dependent apoptosis, and requires its sequential intracellular phosphorylation catalysed by adenosine kinase (ADK) and adenylate kinase 2, resulting in intracellular accumulation of 2-oxo-ATP accompanied by accumulation of 2-oxo-Ado in RNA and depletion of ATP. Moreover, we showed that overexpression of MTH1, an oxidized purine nucleoside triphosphatase, prevents 2-oxo-Ado-induced cytotoxicity accompanied by suppression of accumulation of both intracellular 2-oxo-ATP and 2-oxo-Ado in RNA and recovery of ATP levels. We also found that 2-oxo-Ado activates the p38 MAPK pathway. However, siRNAs against Mkk3 and Mkk6, or treatment with several p38 MAPK inhibitors, except SB203580, did not prevent the cytotoxicity. SB203580 prevented intracellular phosphorylation of 2-oxo-Ado to 2-oxo-AMP, and an in vitro ADK assay revealed that SB203580 directly inhibits ADK activity, suggesting that some of the effects of SB203580 may depend on ADK inhibition.
Highlights
1,2-Dihydro-2-oxoadenosine (2-oxoadenosine; 2-oxo-Ado), an oxidized form of adenosine and known as 2-hydroxyadenosine, isoguanosine or crotonoside, has been reported as a naturally generated nucleoside analogue in Croton tiglium[1] and Diaulula sandiegensis2. 2-Oxo-Ado has not been identified in mammals, but some biological activities have been shown in mammals, such as effects on muscle contraction[3] and cAMP accumulation[4]
Values (% control) of cell viability relative to that in the absence of nucleosides are shown as the mean ± SD of three experiments. (b,c) T9 cells were incubated in the presence of 100 μM Ado or 2-oxo-Ado for various times, and the numbers of live and dead cells were determined by the trypan blue exclusion
We examined the effects of Mkk[3] and Mkk[6] knockdown on the cytotoxicity of 2-oxo-Ado (Fig. 5c) and found no suppression of cytotoxicity caused by increased concentrations of 2-oxo-Ado (20–120 μM)
Summary
We showed that exposure of immortalized MEFs to 2-oxo-Ado induces growth arrest and classical caspase-dependent apoptosis, accompanied by accumulation of intracellular 2-oxo-ATP, resulting in accumulation of 2-oxo-Ado in cellular RNA, and depletion of ATP, both of which are dependent on ADK and AK2. Overexpression of human MTH1, an oxidized purine nucleoside triphosphatase, efficiently suppressed 2-oxo-Ado-induced cytotoxicity with significantly reduced accumulation of both intracellular 2-oxo-ATP and 2-oxo-Ado in cellular RNA, as well as recovery of ATP levels. Exposure of T9 cells to 2-oxo-Ado caused intracellular accumulation of 2-oxo-ATP to a level equivalent to the normal level of intracellular ATP and halved the ATP level as early as 6 h after exposure Both of these effects were almost completely prevented by Itu, an ADK inhibitor (Fig. 2). We demonstrated that exposure of T5v cells to 2-oxo-Ado significantly increased the 2-oxo-Ado level in cellular RNA, which was efficiently suppressed by overexpression of hMTH1 (Fig. 2e). For such a combination therapy, a cancer-directed drug delivery system would be very important to suppress side effects Another possible mechanism of 2-oxo-Ado-induced cytotoxicity is via the p38 MAPK pathway. Our finding that ADK is a novel target of SB203580 is helpful for the re-interpretation of the results obtained using SB203580, especially when observations are not consistent with results following suppression of p38 MAPK inhibition
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