Mechanisms of apoptosis and cell-cycle arrest in subcutaneous breast tumours treated sequentially with doxorubicin followed by zoledronic acid

Breast cancer patients commonly receive a combination of different therapies; for patients with late-stage breast cancer involving metastasis to the bone, a chemotherapeutic agent is usually given in combination with the antiresorptive drug zoledronic acid (zol) (Novartis Pharma, Basel, Switzerland). We have previously reported that administration of doxorubicin (dox) (Pharachemie BV, Haarlem, The Netherlands) 24 hours prior to zol inhibits subcutaneous breast tumour growth, inhibits tumour cell proliferation and increases apoptosis in vivo. The aims of the present study were to determine the mechanisms by which dox and zol exert their synergistic antitumour effects.

MDA-MB-436-GFP (MDA-G8) cells (0.5 × 10⁸) were inoculated into the right flank of female MF1 nude mice (n = 3/array). Mice were treated once per week for 6 weeks with saline, 2 mg/kg dox, 100 μg/kg zol or dox followed 24 hours later by zol. Animals were sacrificed 24 hours following final treatment and one half of each tumour was stored in RNAlater and the other half in protein lysis buffer. RNA was extracted using a SuperArray ArrayGrade™ total RNA extraction kit (Tebu-bio, Peterborough, UK) and biotin-labelled riboprobes were subsequently produced using a SuperArray TrueLabelling-AMP™ 2.0 kit (Tebu-bio). Four micrograms of biotin-labelled RNA from each group was hybridised overnight at 60°C to separate GEArray cell-cycle and apoptosis pathway-specific microarrays (Superarray.com; Tebu-bio). Gene expression was analysed using GEAsuite software (Superarray.com; Tebu-bio) and gene maps were produced using Pathway Architecture software (Stratagene, CA, USA). Genes were considered relevant if they showed a twofold or greater change in gene expression compared with control and they showed direct interactions on the gene map. Expression of relevant genes was confirmed by quantitative PCR and protein expression assessed by western blot.

Molecular analysis of subcutaneous MDA-G8 tumours showed no effect on tumour cell cycle or apoptosis following administration of 100 μg/kg zol. Conversely, 2 mg/kg dox caused a cell-cycle block at G₁-S and G₂-M with a downregulation of cyclin E/CDK2 and cyclin B/CDC2; dox alone did not affect apoptosis. When dox was administered 24 hours prior to zol, however, the cell cycle was further suppressed, compared with dox alone, there was a downregulation of cyclin E1, cyclin B, cyclin D₁ and cyclin D₃ as well as their related cyclin-dependent kinases CDK2, CDC2, CDK4 and CDK7. Furthermore, tumours treated sequentially with dox then zol showed an induction in the apoptotic pathway with an upregulation in Bax, a downregulation in Bc12 and an increase in caspase 3 cleavage.

In subcutaneous MDA-G8 tumours: administration of zol does not effect the apoptotic cell cycle pathways, administration of zol disrupts the cell cycle but has no effect on apoptosis, and sequential administration of dox followed by zol results in cell-cycle inhibition and induction of apoptosis.