Adenosine and deoxyadenosine induces apoptosis in oestrogen receptor-positive and -negative human breast cancer cells via the intrinsic pathway

Abstract

In this study we have examined the cytotoxic effects of different concentrations of adenosine (Ado) and deoxyadenosine (dAdo) on human breast cancer cell lines. Ado and dAdo alone had little effect on cell cytotoxicity. However, in the presence of adenosine deaminase (ADA) inhibitor, EHNA, adenosine and deoxyadenosine led to significant growth inhibition of cells of the lines tested. Ado/EHNA and dAdo/EHNA-induced cell death was significantly inhibited by NBTI, an inhibitor of nucleoside transport, and 5'-amino-5'-deoxyadenosine, an inhibitor of adenosine kinase, but the effects were not affected by 8-phenyltheophylline, a broad inhibitor of adenosine receptors. The Ado/EHNA combination brought about morphological changes consistent with apoptosis. Caspase-9 activation was observed in MCF-7 and MDA-MB468 human breast cancer cell lines on treatment with Ado/EHNA or dAdo/EHNA, but, as expected, caspase-3 activation was only observed in MDA-MB468 cells. The results of the study, thus, suggest that extracellular adenosine and deoxyadenosine induce apoptosis in both oestrogen receptor-positive (MCF-7) and also oestrogen receptor-negative (MDA-MB468) human breast cancer cells by its uptake into the cells and conversion to AMP (dAMP) followed by activation of nucleoside kinase, and finally by the activation of the mitochondrial/intrinsic apoptotic pathway.

Figure 1

Effect of adenosine and deoxyadenosine on cell growth in breast cancer cell lines, MCF‐7 and MDA‐MB468. Cells were treated with various concentrations of adenosine and deoxyadenosine for 24, 48 and 72 h, and viability was assessed by MTT assay. Results are expressed as percentage of the corresponding control and represent the mean ± SD of six repeats. The standard deviation bars were omitted in order to preserve the clarity of the figure, however, they never exceeded 9%.

Figure 2

Adenosine deaminase inhibitor (EHNA) strongly potentiates the toxicity of adenosine and deoxyadenosine. MCF‐7 and MDA‐MB468 cells were pretreated with 40 µm EHNA, various concentrations of adenosine and deoxyadenosine for 24, 48 and 72 h; viability was assessed by MTT assay. Results are expressed as percentage of the corresponding control and represent the mean ± SD of six repeats.

Figure 3

The effect of non‐specific Ado receptor antagonist 8‐phenyltheophylline on adenosine and deoxyadenosine on the growth of the breast cancer cell populations. We have tested the effect of non‐specific Ado receptor antagonist 8‐phenyltheophylline (10 µm) on adenosine and deoxyadenosine toxicity (in the presence of 40 µm EHNA) in breast cancer cell lines, MCF‐7 and MDA‐MB468. Pretreatment of cells with 10 µm 8‐phenyltheophylline had no effect upon the Ado/EHNA or dAdo/EHNA‐mediated toxicity for 48 h. Results are expressed as percentage of the corresponding control and represent the mean ± SD of six repeats.

Figure 4

Intracellular but not extracellular adenosine or deoxyadenosine is toxic to these cells. Pretreatment with 20 µm nucleoside transport inhibitor, NBTI, significantly abrogated cytotoxicity of Ado/EHNA or dAdo/EHNA within 48 h. Results are expressed as percentage of the corresponding control and represent the mean ± SD of six repeats.

Figure 5

Adenosine kinase is required for adenosine or deoxyadenosine toxicity. Pretreatment with 5′‐amino‐5′‐deoxyadenosine (20 µm), an inhibitor of adenosine kinase, significantly reduced the growth inhibition induced by the combination of adenosine and EHNA or deoxyadenosine and EHNA within 48 h. Results are expressed as percentage of the corresponding control and represent the mean ± SD of six repeats.

Figure 6

Treatment with adenosine or deoxyadenosine induces typical apoptotic loss of cell membrane symmetry. Cells were treated with various concentrations of adenosine and deoxyadenosine in the presence of adenosine deaminase inhibitor (EHNA) for 48 h, and percentages of early and late apoptotic cells were assessed by flow cytometric analysis of annexin‐V and PI binding. Results are represent the mean ± SD of four repeats.

Figure 7

Cells treated with adenosine or deoxyadenosine have typical apoptotic morphology. The morphology of both the types of breast cancer cells, treated with either adenosine or deoxyadenosine, was examined by microscopy after Hochest 33258 staining.

Figure 8

Adenosine and deoxyadenosine induce DNA‐fragmentation in target cells. Agarose gel electrophoresis of DNA extracted from untreated (C), treated (T) MCF‐7 and MDA‐MB468 cells plus 1 kb DNA marker (M). Cells were treated with 500 µm adenosine or deoxyadenosine in the presence of 40 µm EHNA for 48 h. DNA was then extracted and electrophoresis was performed.

Figure 9

Activation of caspases after adenosine and deoxyadenosine treatment. The enzymatic activity of caspases‐3 and ‐9 was measured in MCF‐7 and MDA‐MB468 cells treated with various concentrations of adenosine and deoxyadenosine in the presence of the adenosine deaminase inhibitor (EHNA) for 48 h. Results represent the mean ± SD of four repeats.