Anti-proliferative effects of cladribine on human breast MCF-7 adenocarcinoma cells

Alex Henson, Claire Simons, Emma J. Kidd

Cardiff University, Cardiff, UK

Cladribine (2CdA) is the 2-chloro analogue of deoxyadenosine and is resistant to deamination by adenosine deaminase. 2CdA is currently used clinically in the treatment of hairy cell leukaemia and other haematological malignancies (Bryson et al. 1993). The cytotoxic effects of 2CdA require it to be transported intracellularly where it undergoes a sequential phosphorylation to 2-chloro-deoxyadenosine triphosphate (2CdATP). CdATP is the primary cytotoxic metabolite of 2CdA, and inhibits enzymes responsible for DNA replication and repair including ribonucleotide reductase and DNA polymerase (Bontemps et al. 2000). It is also incorporated into the DNA template resulting in inhibition of DNA elongation past this analogue (Plunkett and Saunders 1991). The aim of this study was to investigate whether cladribine was could also reduce the proliferation of human breast cancer MCF-7 cells.

The cytotoxic effects of 2CdA on MCF-7 cells were investigated using the MTS (3-(4,5-dimethylthiazol-2-yl)-5-(3-carboxymethoxyphenyl)-2-(4-sulfophenyl)-2H-tetrazolium inner salt) cell viability assay. Cells were seeded at 10,000 cells/well in 96 well plates and left to plate for 24 hours. Drugs were made up in RPMI media without phenol red and incubated with the cells for 96 hours before the MTS assay was performed according to manufacturer’s instructions. Results were analysed using one-way ANOVA. Drug treatments were tested in triplicate and experiments repeated at least 3 times.

2CdA (0.1-500μM) produced a dose-dependent reduction in cell viability with a maximum inhibition of 50.6±6.9% at 500μM compared to control. 1-500μM 2CdA were significantly different (p<0.05) compared to control. There was no significant difference between 1 and 500μM. The irreversible adenosine deaminase inhibitor, pentostatin (0.1-500μM), also used to treat hairy cell leukaemia (Bryson et al. 1993), produced a maximum inhibition of 28.3±6.2% at 500μM compared to control but this was not significant. Pre-incubation with the nucleoside transport inhibitors, dilazep, 10μM (alone 96.25±5.43%; +2CdA 55.9±4.0%) and 6-S-[(4-Nitrophenyl)methyl]-6-thioinosine (NBMPR), 100nM, (alone 101.3±3.54%; +2CdA 73.7±6.3%) had no significant effect alone or on the inhibition by 50μM 2CdA alone (64.08±2.45%). Incubation with 1μM 5-(3-Bromophenyl)-7-[6-(4-morpholinyl)-3-pyrido[2,3-d]pyrimidin-4-amine dihydrochloride (ABT 702), an adenosine kinase inhibitor, had no significant effect alone (89.69±5.73%) or on the inhibition by 50 μM 2CdA (70.06±10.06%)

These data suggest that 2CdA can significantly inhibit the growth of MCF-7 cells, whereas pentostatin cannot. The effect of 2CdA is likely to be intracellular as it was not inhibited by dilazep or NBMPR. The action of 2CdA is not dependant upon intracellular phosphorylation by adenosine kinase, however. These results highlight an interesting difference between the anti-proliferative effects of 2CdA and pentostatin on white blood cells where both drugs are effective and breast cancer cells where only 2CdA is active. As 2CdA is already used clinically as an anti-cancer drug, our data could be relevant for the treatment of breast cancer.

Bontemps F. et al. (2000) Biochemical Pharmacology 59:1237-1243

Bryson H.M. and Sorkin E.M. (1993) Drugs 46:872–894

Plunkett W. and Saunders P.P. (1991) Pharmacological Therapy 49:239-268