adenosine-kinase and benzyloxycarbonylvalyl-alanyl-aspartyl-fluoromethyl-ketone

The naturally occurring cytokinin, ortho-topolin riboside (oTR), has been recently reported to have a strong anticancer effect. However, the molecular mechanism has not been elucidated. From our research we found that oTR strongly inhibited the proliferation of SMMC-7721 cells inducing apoptosis. After oTR treatment, up-regulation of the protein levels of pro-apoptotic Bax and the down-regulation of the anti-apoptotic proteins, Bcl-2 and Bcl-xL was observed, leading to the loss of mitochondrial membrane potential, the release of cytochrome c from the mitochondria into the cytosol, the downstream activation of caspase-9 and caspase-3, as well as the cleavage of poly ADP-ribose-polymerase (PARP), the effect of apoptosis could be blocked by the pan-specific caspase inhibitor z-VAD-fmk and caspase-9-specific inhibitor z-LEHD-fmk. Moreover, oTR was shown to inhibit the activation of the extracellular signal-regulated kinase-1/2 (ERK(1/2)) as well as the Akt pathway. These results suggest that oTR interferes with the mitogen-activated protein kinase (MAPK) and Akt pathways and induces the apoptosis of human SMMC-7721 cells through the activation of intrinsic mitochondria-mediated pathways. However, the apoptosis was completely prevented when cells were treated with A-134974, an inhibitor of adenosine kinase, it indicated that the intracellular phosphorylation of oTR is necessary for its cytotoxic effects to SMMC-7721 cells.

Topics: Adenosine Kinase; Amino Acid Chloromethyl Ketones; Apoptosis; Blotting, Western; Caspase Inhibitors; Cell Line, Tumor; Cell Survival; Cytochromes c; Cytokinins; Flow Cytometry; Humans; MAP Kinase Signaling System; Membrane Potentials; Mitochondrial Membranes; Mitogen-Activated Protein Kinases; Nucleosides; Oligopeptides; Oncogene Protein v-akt; Proto-Oncogene Proteins c-bcl-2; Signal Transduction

The in vitro induction of apoptosis by N6-substituted derivatives of adenosine and adenine was investigated in HL-60 cells. Using reversed phase HPLC/MS analysis we demonstrated that both N6-substituted derivatives of adenosine and adenine are phosphorylated within cells to the monophosphate level. While N6-substituted derivatives of adenosine were phosphorylated by adenosine kinase and corresponding mononucleotides were produced in large quantities, N6-substituted derivatives of adenine were converted into the corresponding mononucleotides via the phosphoribosyl transferase pathway, which yielded 50-100 times lower amounts of the mononucleotides than the adenosine kinase pathway. Accordingly, N6-substituted derivatives of adenine were relatively inefficient inductors of apoptosis at the concentrations applied. Inhibitors of adenosine kinase that abrogated the formation of monophosphates from N6-substituted derivatives of adenosine completely prevented cells from going into apoptosis. These results consistently support the idea that pro-apoptotic effects of N6-substituted derivatives of adenosine are related to their intracellular conversion into corresponding mononucleotides which eventually trigger apoptosis when accumulated beyond certain level. Intracellular accumulation of mononucleotides derived from the corresponding N6-substituted derivatives of adenosine led to a rapid decrease in ATP production and consequently to apoptosis induction. Nevertheless, the detailed mechanism is unknown and must be further elucidated. Apoptosis, induced by N6-substituted derivatives of adenosine, was accompanied by a distinct caspase-3 activation. However, a broad spectrum caspase inhibitor, z-VAD-fmk, failed to prevent cells from death, thereby indicating that caspases alone were not mediators of cell death.

Topics: Adenine; Adenine Nucleotides; Adenine Phosphoribosyltransferase; Adenosine; Adenosine Kinase; Adenosine Monophosphate; Amino Acid Chloromethyl Ketones; Apoptosis; Caspase 3; Caspase Inhibitors; Caspases; Enzyme Inhibitors; HL-60 Cells; Humans; Phosphorylation