Combined effects of the bisphosphonate, zoledronic acid and the aromatase inhibitor letrozole on breast cancer cells in vitro: evidence of synergistic interaction

Abstract

Background: Aromatase inhibitors are widely used in the treatment of oestrogen receptor-positive post-menopausal breast cancer. These patients may also be receiving the bisphosphonate, zoledronic acid (ZA) to prevent bone loss or reduce skeletal morbidity in the setting of advanced disease. The potential biological interaction of these two drugs in breast cancer has not been assessed.

Methods: Aromatase-expressing breast cancer cells were treated with letrozole and ZA either simultaneously or in sequence, and the resulting apoptosis was assessed by staining with Hoechst 33342 and propidium iodide and examined using a fluorescent inverted Leica DMIRB microscope and a UV filter.

Results: We found that letrozole and ZA induce levels of apoptosis in breast cancer cells in vitro that are significantly greater compared with treatment with each drug alone. However, this potentially, synergistic relationship is drug-sequence dependent, occurring only when cells are treated with letrozole, followed by ZA. The converse sequence, or administering drugs simultaneously, induces levels of apoptosis no greater than each drug alone.

Conclusion: Owing to the enhanced anti-tumour efficacy of sequential drug administration, our findings may indicate that, for post-menopausal women who require treatment with letrozole, ZA should also be considered.

Figure 1

Experiment design for investigating the effects of sequential treatment of MCF7-Ca cells with letrozole (100 nM) and zoledronic acid (25 μM). Drug sequence 1: ‘letrozole followed by zoledronic acid’ drug sequence 2: ‘zoledronic acid followed by letrozole’ drug sequence 3: ‘zoledronic acid and letrozole together’).

Figure 2

Growth inhibition of MCF7-Ca cells treated with letrozole (0–100 nM) for 24–72 h (^*P=0.001 vs control).

Figure 3

Induction of apoptosis vs necrosis in MCF7-Ca cells after treatment with letrozole (0–100 nM) for 72 h continuously in oestrogen-free media (^*P<0.05 and ^**P<0.001 vs control).

Figure 4

Growth inhibition of MCF7-Ca cells treated with zoledronic acid (0–25 μM) for 1 h (^*P<0.05 and ^**P<0.01 compared with untreated controls).

Figure 5

(A) Induction of apoptosis vs necrosis in MCF7-Ca cells after treatment with zoledronic acid (0–25 μM) for 72 h continuously (^*P<0.05 and ^**P<0.01 vs control). (B) Induction of apoptosis vs necrosis in MCF7-Ca cells after treatment with zoledronic acid (0–25 μM) for 1 h (^*P<0.05, ^**P⩽0.01 and ^§P<0.001, compared with untreated controls).

Figure 6

The effect of drug sequencing with ‘letrozole followed by ZA’ (A), ‘ZA followed by letrozole’ (B) and ‘ZA and letrozole together’ (C) on resultant apoptosis when MCF7-Ca cells are treated with letrozole (100 nM) and zoledronic acid (10 μM), each for 24 h in oestrogen-free media plus AD (^*P<0.001, compared with the drugs in combination).

Figure 7

The effect of drug sequencing with letrozole followed by ZA on apoptosis when a clinically relevant concentration of ZA is used (^*P<0.01 for letrozole followed by ZA vs each drug alone).

Figure 8

The effect of the mevalonate pathway intermediary, geranylgeraniol (GGOH) (50 μM), on the apoptosis induced by treatment of MCF7-Ca cells with letrozole (100 nM, 24 h) followed by zoledronic acid (10 μM, for 1 h) (^*P=0.05 compared with ‘letrozole followed by ZA’).