Breast cancer has a prodigious capacity to metastasize to bone. In women with advanced
breast cancer and bone
metastases,
bisphosphonates reduce the incidence of hypercalcaemia and skeletal morbidity. Recent clinical findings suggest that some
bisphosphonates reduce the tumour burden in bone with a consequent increase in survival, raising the possibility that
bisphosphonates may have a direct effect on
breast cancer cells. We have investigated the in vitro effects of
bisphosphonates zoledronate,
pamidronate,
clodronate and
EB 1053 on growth, viability and induction of apoptosis in three human
breast cancer cell lines (MDA-MB-231, Hs 578T and MCF-7). Cell growth was monitored by
crystal violet dye assay, and cell viability was quantitated by MTS
dye reduction. Induction of apoptosis was determined by identification of morphological features of apoptosis using time-lapse videomicroscopy, identifying morphological changes in nucleis using Hoechst staining, quantitation of DNA fragmentation, level of expression of bcl-2 and bax
proteins and identification of the proteolytic cleavage of
Poly (ADP)-ribose polymerase (PARP). All four
bisphosphonates significantly reduced cell viability in all three cell lines.
Zoledronate was the most potent
bisphosphonate with IC50 values of 15, 20 and 3 microM respectively in MDA-MB-231, MCF-7 and Hs 578T cells. Corresponding values for
pamidronate were 40, 35 and 25 microM, whereas
clodronate and
EB 1053 were more than two orders of magnitude less potent. An increase in the proportion of cells having morphological features characteristic of apoptosis, characteristic apoptotic changes in the nucleus, time-dependent increase in the percentage of fragmented chromosomal
DNA, down-regulation in bcl-2
protein and proteolytic cleavage of PARP, all indicate that
bisphosphonates have direct anti-tumour effects on human
breast cancer cells.