Original articleInduction of cell death of human osteogenic sarcoma cells by zoledronic acid resembles anoikis
Introduction
Osteosarcoma is the most common primary malignant tumour of bone in children and adolescents. The first choice of treatment for osteosarcoma is chemotherapy, and multiple anticancer drugs such as doxorubicin, cisplatin, etoposide, and cyclophosphamide are commonly used, either alone or in combination [1]. However, despite significant improvements in patient survival and treatment of the primary tumour, some groups of patients who present with metastatic disease, particularly osteolytic bone metastases, as well as patients with tumours that recur after treatment, continue to have a poor prognosis. In addition, the frequent acquisition of drug-resistant phenotypes and the occurrence of “second malignancies” often associated with chemotherapy, remain as serious problems. Therefore, there is a pressing need to develop new and alternative approaches to the current treatment of osteosarcomas.
Bisphosphonates (BPs) are currently the most important class of inhibitors of osteoclast-mediated bone resorption and are used extensively for the treatment of skeletal diseases, such as Paget's disease, postmenopausal osteoporosis, and tumour-induced osteolysis [2], [3]. BPs have a high affinity for the hydroxyapatite mineral in bone and are taken up selectively and adsorbed to mineral surfaces at sites of increased bone turnover, where they inhibit osteoclast activity [4]. BPs can inhibit the recruitment, proliferation, and differentiation of osteoclast precursor cells [5], [6] as well as reducing the activity and viability of mature osteoclasts by the induction of apoptosis [7]. In addition, BPs may exert their actions indirectly via effects on osteoblasts [8].
BPs can be divided into two distinct pharmacological classes based on their molecular mechanisms of action [9]. Non-nitrogen-containing BPs, such as chlodronate and etidronate, are metabolized intracellularly to cytotoxic analogues of ATP that inhibit osteoclast activity [10]. In contrast, the newer nitrogen-containing BPs such as alendronate, residronate, pamidronate, and ZOL induce apoptosis in osteoclasts by inhibiting enzymes of the mevalonate pathway [11], [12], a biosynthetic pathway responsible for the production of cholesterol and isoprenoid lipids, particularly farnesyl- and geranyl-pyrophosphates. These are required for the posttranslational modification (prenylation) of small GTPases such as Ras, Rho, Rac, and Rab. Small GTPases are important signalling proteins that regulate a variety of cell processes that include cell attachment, cytoskeletal assembly, intracellular signalling, and apoptosis [13].
Recently, BPs have also been investigated as treatments for the hypercalcaemia of malignancy and bone pain [14], [15] that is due to focal tumour-induced osteolysis. Associated in vitro experiments have shown the potential for BPs to affect the proliferation and survival of tumour cells themselves, including the induction of apoptosis in human myeloma [16], [17], breast [18], [19], and prostate [20] cancer cells. Furthermore, BP treatment was shown to inhibit the progression and development of bone metastases of breast cancer by promoting apoptosis in both osteoclasts and metastatic breast cancer cells [21], [22]. The effect of BPs on osteosarcoma cells has not been extensively studied, although recently the antiproliferative effect of pamidronate in human and rat osteosarcoma cells in vitro has been demonstrated [39], [40]. There are no reports of the effects of the potent BP ZOL on human osteosarcoma cells.
Zoledronic acid (ZOL) is a new-generation nitrogen-containing BP and is the most potent inhibitor of bone resorption of the currently available bisphosphonates in clinical trials. Furthermore, recent phase I trials in patients with bone metastases or multiple myeloma demonstrated that ZOL was safe and well tolerated at all dose levels tested [23], [24], [25]. In this study we have investigated the cytotoxic effects of ZOL as a single agent, and in combination with clinically relevant anticancer drugs, in a panel of human osteogenic sarcoma cell lines. We found that ZOL was a potent inducer of apoptosis in a number of these cell lines, by a mechanism that involved geranylgeranyl-pyrophosphate and cell detachment, and which was independent of caspase activation. Although chemotherapeutic agents and ZOL separately reduced the proliferation of osteosarcoma cells, combination therapy was no more effective than either agent alone.
Section snippets
Cells and reagents
The human osteogenic sarcoma cell lines BTK-143, MG-63, G-292, SJSA-1, SaOS2, and HOS were obtained from the American Type Culture Collection (Rockville, MD) and cultured in Dulbecco's modified Eagle's medium (DMEM), supplemented with glutamine (2 mM), penicillin (100 IU/ml), streptomycin (100 μg/ml), gentamicin (160 μg/ml), and 10% fetal bovine serum (Biosciences, Sydney, Australia), in a humidified atmosphere containing 5% CO2.
ZOL was generously provided by Novartis Pharmaceuticals, Australia
Effects of ZOL on cell proliferation
The dose-dependent effects of ZOL on cell proliferation were tested against a panel of six human osteogenic sarcoma cell lines (HOS, BTK-143, G-292, SaOS2, SJSA-1, and MG-63). Cells were seeded in 96-well plates in serum-containing media and allowed to attach. Treatment with ZOL for 72 h dose-dependently resulted in fewer cells, compared with untreated control cultures, in all six osteogenic sarcoma cell lines. However, the efficacy of ZOL in reducing cell number varied considerably between
Discussion
BPs are potent inhibitors of bone resorption and are widely used for the treatment of tumour-induced osteolysis. However, recent studies have also shown the potential for BPs to affect directly the proliferation and survival of a variety of tumour cells themselves including those of breast [18], [19], [21], prostate [20], and multiple myeloma [16], [17], [30]. Osteogenic sarcomas may be good targets for BP treatment, since high concentrations of BP may be more easily achievable in the vicinity
Acknowledgements
This work was supported by grants from the Cancer Council of South Australia, and A. Evdokiou is a Fellow of the Council. Grants from the Adelaide Bone and Joint Research Foundation (ABJRF), the Royal Adelaide Hospital, Adelaide University, and the National Health and Medical Research Council of Australia (NHMRC) are gratefully acknowledged, and A. Labrinidis was supported from the above-mentioned funding bodies. A Reginald Walker Medical Research Scholarship from Adelaide University supported
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