Induction of apoptosis by A3 adenosine receptor agonist N-(3-iodobenzyl)-adenosine-5'-N-methylcarboxamide in human leukaemia cells: a possible involvement of intracellular mechanism.
OBJECTIVE
The sensitivity of cancer cells which exhibit multi-drug resistance phenotype to A3 adenosine receptor (A3AR) agonist N(6)-(3-iodobenzyl)-adenosine-5'-N-methylcarboxamide (IB-MECA) was studied.


METHODS
To establish direct relationship between P-glycoprotein (P-gp, ABCB1 and MDR1) expression and IB-MECA induced cell death, a straightforward method for precise estimation of intracellular level of this A3AR agonist was developed.


RESULTS
We subjected three human leukaemia cell lines HL-60, K562 and K562/HHT to treatment with micromolar concentrations of IB-MECA. Although all cell lines used expressed A3AR, there was a large difference in their sensitivity to IB-MECA. While HL-60 and K562 cells were almost equally sensitive, the K562/HHT cells, which exhibit a multi-drug resistance phenotype because of overexpression of P-gp, were significantly more resistant. We found that the intracellular level of IB-MECA in K562/HHT cells was approx. 10 times lower than those in HL-60 or K562 cells. Inhibitors of P-gp, including cyclosporine A (CsA) and verapamil (Vpa), increased the intracellular level of IB-MECA and reversed the resistance of K562/HHT cells to this drug. Accordingly, shRNA-mediated down-regulation of P-gp significantly increased the intracellular level of IB-MECA in K562/HHT cells which simultaneously exhibited reduced resistance to this A3AR agonist. In addition, an in vitro enzyme-based assay provided evidence that IB-MECA might serve as a substrate for P-gp.


CONCLUSIONS
Our results suggest that P-gp overexpression prevents cells from IB-MECA induced apoptosis despite the A3AR expression. Pro-apoptotic effect of IB-MECA seemed to strongly depend on its intracellular accumulation rather than on its interaction with A3AR.
Mlejnek
P
P
Department of Biology, Faculty of Medicine and Dentistry, Palacky University Olomouc, Olomouc, Czech Republic. mlejnek_petr@volny.cz
Dolezel
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P
D020168:ATP Binding Cassette Transporter, Subfamily B, Member 1 / Q000037:antagonists & inhibitors / Q000235:genetics / Q000378:metabolism; D000241:Adenosine / Q000031:analogs & derivatives* / Q000737:chemistry / Q000378:metabolism; D058909:Adenosine A3 Receptor Agonists; D000251:Adenosine Triphosphatases / Q000378:metabolism; D017209:Apoptosis / Q000502:physiology*; D002121:Calcium Channel Blockers / Q000494:pharmacology; D018432:Drug Resistance, Multiple / Q000502:physiology; D018922:HL-60 Cells / Q000187:drug effects* / Q000378:metabolism; D006801:Humans; D020014:K562 Cells / Q000187:drug effects* / Q000378:metabolism; D015398:Signal Transduction / Q000502:physiology; D014700:Verapamil / Q000494:pharmacology
D020168:ATP Binding Cassette Transporter, Subfamily B, Member 1; D058909:Adenosine A3 Receptor Agonists; D002121:Calcium Channel Blockers; C084956:N(6)-(3-iodobenzyl)-5'-N-methylcarboxamidoadenosine; D014700:Verapamil; D000251:Adenosine Triphosphatases; D000241:Adenosine