Bisphosphonates induce apoptosis in human breast cancer cell lines

Breast cancer has a prodigious capacity to metastasize to bone. In women wi th advanced breast cancer and bone metastases, bisphosphonates reduce the i ncidence 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 bisphosphonat es may have a direct effect on breast cancer cells. We have investigated th e 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 m onitored 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, i dentifying morphological changes in nucleis using Hoechst staining, quantit ation of DNA fragmentation, level of expression of bcl-2 and bar proteins a nd identification of the proteolytic cleavage of Poly (ADP)-ribose polymera se (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 mu M respectively in MDA-MB-231, MCF-7 and Hs 5 78T cells. Corresponding values for pamidronate were 40, 35 and 25 mu M, wh ereas clodronate and EB 1053 were more than two orders of magnitude less po tent. An increase in the proportion of cells having morphological features characteristic of apoptosis, characteristic apoptotic changes in the nucleu s, time-dependent increase in the percentage of fragmented chromosomal DNA, down-regulation in bcl-2 protein and proteolytic cleavage of PARP, all ind icate that bisphosphonates have direct anti-tumour effects on human breast cancer cells. (C) 2000 Cancer Research Campaign.