Differential effects of bisphosphonates on breast cancer cell lines
Introduction
Bisphosphonates are analogues of endogenous pyrophosphate in which a carbon atom replaces the central atom of oxygen [1]. This carbon substitution makes these compounds resistant to hydrolysis and allows two additional side chains (R1 and R2) of variable structure. One of these side chains (R1) usually contains a hydroxyl moiety, which allows high affinity for calcium crystals and bone mineral. The differences at the other side chain (R2) produce marked differences in the anti-resorptive potency of different bisphosphonates and is the key factor for inhibition of bone resorbtion [2].
First generation bisphosphonates have either a single atom or alkyl chain at R2 (e.g. etidronate and clodronate). An up to 100-fold increase in potency was achieved by the introduction of a primary, secondary or tertiary amino group (–NH2) at the extremity of the R2 alkyl chain, to form the second generation amino-bisphosphonates (e.g. alendronate, pamidronate and ibandronate). Potency reaches the peak when the nitrogen atom is included within a ring structure in the R2 side chain (as in risedronate and zoledronic acid) [3].
Bisphosphonates have a high affinity for bone and are preferentially delivered to sites of increased bone formation or resorption. Once deposited on the bone surface they are ingested by osteoclasts. Bisphosphonates are not split by hydrolytic enzymes of osteoclasts and reduce the activity of osteoclasts by a variety of means. However, this anti-resorptive effect is the pharmacological basis of treating tumor osteolysis.
Tumor osteolysis is characterized by a self-sustaining cycle of interactions between metastases and the cell systems of the bones [4]. Metastatic tumor cells produce osteolytic factors and prostaglandins. These activate the osteoclasts to degradation of mineralized bone matrix. Growth factors and cytokines, that are stored in the bone, are released and may increase the proliferation rate of micro metastases.
The efficacy of bisphosphonates was thought to lie purely in the inhibition of bone resorption by osteoclasts. However, bone resorption is inhibited in a variety of ways, which include effects on osteoclast formation, resorptive activity and viability. Bisphosphonates have inhibitory effects on osteogenesis. Recent studies showed that bisphosphonates interact with surface receptors on precursor cells and block differentiation of mature osteoclasts [5].
Osteoclasts are the bone cells most likely to interact to high concentrations of bisphosphonates. After cellular uptake, bisphosphonate-treated osteoclasts show important changes in morphology.
These include the lack of the ruffled border demonstrating the disruption of cytoskeleton and the loss of actin rings. These structural alterations lead to a decreased osteoclast function [6].
It has also been reported that bisphosphonates can shorten the life span of osteoclasts by induction of programmed cell death (apoptosis) [7], [8]. Non-amino-bisphosphonates can be metabolized to methylene-containing analogs of ATP, which are extremely resistant to hydrolysis. These non-hydrolyzable ATP analogs accumulate intracellulary and inhibit metabolic enzymes that play important roles in cellular growth, differentiation and activity. In case of the aminobisphosphonates (such as alendronate, risedronate, pamidronate, ibandronate and zoledronate) apoptosis seems to be caused by the inhibition of important enzymes in the mevalonate pathway on which depends the synthesis of cholesterol and isoprenoid lipids, that are required for the prenylation of small GTP-ases, such as Ras, Rho and Rac, which are signaling proteins that regulate a variety of cellular processes. In this way, amino-bisphosphonates can deprive osteoclasts of important regulators of intracellular dynamics, leading to poor cell functioning and eventually programmed cell death.
There are several lines of evidence suggesting that the apoptotic and anti-proliferative effect of bisphosphonates on osteoclasts may exert directly on tumor cells [9].
Diel et al. found in 302 women with primary breast cancer, who received adjuvant therapy with bisphosphonates, a reduction in bone as well as visceral metastasis [10].
Furthermore, bisphosphonate treatment has been shown to inhibit the progression and development of bone metastases in a mouse model of breast cancer [11]. Such a beneficial effect of bisphosphonates on tumor burden in bone may result from a direct anti-tumor effect on breast cancer cells.
This in vitro study is performed to investigate the balance between apoptosis and proliferation of these bisphosphonates on breast cancer cells.
Section snippets
Drugs
Pamidronate (APD) was obtained from Fargron Pharmaceuticals BV (Nieuwerkerk a/d IJssel, The Netherlands), risedronate (Rise) from Proctor and Gamble (Rotterdam, The Netherlands), alendronate (Alen) from Merck Sharp and Dohme (Haarlem, The Netherlands). Clodronate (Clod) and ibandronate (Iban) came from Roche (Mannheim, Germany) and zoledronate (Zol) from Novartis Pharma (Basel, Switzerland).
Cell culture
Three human breast cancer cell lines were obtained from the American Type Culture Collection (ATCC,
Results
Bisphosphonates influence cell proliferation and programmed cell death in different breast cancer cell lines. However, not all bisphosphonates act equally on breast cancer cells in vitro.
The proliferation values in percentages versus controls with SD are given in Fig. 1, and the apoptosis values in percentages versus controls with SD are shown in Fig. 2.
Fig. 3 demonstrates the apoptosis/proliferation ratios. If a ratio is greater than one, overall cell growth can be considered reduced.
Discussion
Numerous clinical trials in patients with metastatic breast cancer have demonstrated that bisphosphonates have the ability to reduce the occurrence of pathologic fractures, bone pain, hypercalcaemic episodes and the need for radiation therapy and surgery [16], [17]. Also some clinical trials showed that patients who were treated with bisphosphonates suffered from less new metastases [18].
Recent studies suggest that the efficacy of bisphosphonates, besides the strong anti-osteoclastic activity,
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