Breast cancer patients commonly receive a combination of different therapies; however, our understanding of how such combined treatments work is incomplete. We have previously shown that sequential administration of the cytotoxic agent doxorubicin (dox) (Pharmachemie BV, Haarlem, The Netherlands) followed by the antiresorptive agent zoledronic acid (zol) (Novartis Pharma, Basel, Switzerland) synergistically increased tumour cell apoptosis in vitro, and also increased tumour cell apoptosis, decreased tumour cell proliferation and reduced subcutaneous in breast tumour growth in vivo. In contrast, pretreating the cells with zol before dox or adding both drugs simultaneously did not cause synergy. The aim of the present study was to determine the mechanism by which sequential administration of dox followed by zol exerts the increased antitumour effects.
All experiments were carried out using MDA-MB-436 breast cancer cells, or MDA-MB-436 cells expressing green fluorescent protein (MDA-G8). Effects of dox on cell membrane integrity were monitored following propidium iodide (PI) or 7-amino-actinomycin D (7AAD) staining. Effects of single or sequential treatment with dox and zol were assessed using Annexin (apoptosis antibody), TMRE (mitochondrial membrane potential dye) and 7AAD (permeable membrane dye) staining by flow cytometry. Uptake of zol was assessed following western blotting using an antibody to the unprenylated form of Rap1a.
Following administration of 1 nM dox for 24 hours, 95% of the MDA-G8 showed uptake of both PI and 7AAD. The cells showed no sign of apoptosis and remained viable. Administration of 25 μM zol for 1 hour to cells pretreated with dox for 24 hours resulted in increased cell death compared with that caused by treatment with either dox or zol alone. Accumulation of unprenylated Rap1a was detected following treatment for 1 hour with lower doses of zol (8 μM) in the dox then zol treated cells, compared with cells treated with zol alone (12 μM). These data imply that pretreatment with dox facilitated the uptake of zol.
Treatment of MDA-MB-436 cells with 1 nM dox disrupts cell membrane integrity without reducing cell viability. Administration of dox 24 hours prior to zol facilitates the uptake of zol in MDA-MB-436 breast cancer cells.
Breast Cancer Campaign funded this work.