Breast Cancer Research and Treatment 71: 257–268, 2002. © 2002 Kluwer Academic Publishers. Printed in the Netherlands.
Report
Distinct mechanisms of bisphosphonate action between osteoblasts and breast cancer cells: identity of a potent new bisphosphonate analogue Gregory G. Reinholz1 , Barbara Getz1 , Emily S. Sanders1 , Marat Ya. Karpeisky3,4 , Nelly Sh. Padyukova3, Sergey N. Mikhailov3, James N. Ingle2 , and Thomas C. Spelsberg 1 Department
of Biochemistry and Molecular Biology, 2 Department of Oncology, Mayo Clinic, Rochester, MN,
USA; 3 Engelhardt Institute of Molecular Biology, Russian Academy of Sciences, Moscow, Russia, 4 MBC Research, Inc., Boulder, CO, USA Key words: bisphosphonates, breast neoplasms, mevalonate pathway, osteoblasts Summary While the effects of bisphosphonates on bone-resorbing osteoclasts have been well documented, the effects of bisphosphonates on other cell types are not as well studied. Recently, we reported that bisphosphonates have direct effects on bone-forming human fetal osteoblast cells (hFOB) [1]. In this report, the role of the mevalonate pathway in the actions of bisphosphonates on hFOB, and MDA-MB-231 human breast cancer cells was examined. These studies included a novel bisphosphonate analog, the anhydride formed between arabinocytidine 5 phosphate and etidronate (Ara-CBP). Ara-CBP was the most potent inhibitor of hFOB and MDA-MB-231 cell proliferation, and stimulator of hFOB cell mineralization compared to etidronate, the anhydride formed between AMP and etidronate (ABP), pamidronate, and zoledronate. Inhibition of hFOB cell proliferation by Ara-CBP and zoledronate was partially reversed by mevalonate pathway intermediates, and stimulation of hFOB cell mineralization was completely reversed by mevalonate pathway intermediates. These results suggest that zoledronate and Ara-CBP act, at least in part, via inhibition of the mevalonate pathway in hFOB cells. In contrast, none of the mevalonate pathway intermediates reversed the inhibition of MDA-MB-231 cell proliferation by the bisphosphonates, or the effects of pamidronate on hFOB cells. As a positive control, the effects of mevastatin on hFOB and MDA-MB231 cells were completely reversed by mevalonate. In summary, these data suggest that zoledronate and Ara-CBP induce human osteoblast differentiation via inhibition of the mevalonate pathway. In contrast, the inhibition of MDA-MB-231 cell proliferation by the bisphosphonates appears to be through mechanisms other than inhibition of the mevalonate pathway.
Introduction Autopsy studies have revealed that the majority of patients who had died from breast cancer had metastases to bone [2–4]. Skeletal metastases are the cause of considerable complications in cancer patients including hypercalcemia, severe bone pain, spinal cord compression, and increased fracture frequency. Treatment of breast cancer patients with bisphosphonates reduces the occurrence of these complications by delaying the progression of bone metastases and reducing the number of new metastases in breast cancer patients [5–8]. However, the mechanism of action of these compounds is not completely understood.
The current dogma on bisphosphonate action in bone is that they act specifically on the bone resorbing osteoclast cells [9–20]. However, recent studies, from this and other laboratories, suggest that bisphosphonates can also directly affect osteoblast [1, 21–25], breast cancer and myeloma cells [26–27]. Studies in our laboratory have shown direct actions of bisphosphonates on hFOB cells [1]. hFOB cells were developed in this laboratory and are a unique normal human bone cell line that can produce bone in vitro [28]. hFOB cells also form bone in animals when injected into muscle tissue and form matrix detectable by electron microscopy (M. Subramaniam et al., manuscript in preparation). Treatment of hFOB cells with
258
G G Reinholz et al.
pamidronate and zoledronate enhanced the differentiation of these cells from the proliferation stage to the matrix maturation and mineralization stages [1]. Past studies suggest that the main mechanism of action of the nitrogen-containing bisphosphonates, such as pamidronate and zoledronate, in osteoclasts and other cell types, is via inhibition of the mevalonate pathway [9, 17, 20, 27, 29–32]. On the other hand, the non-nitrogen containing bisphosphonates, such as etidronate, are thought to act through the production of cytotoxic adenosine metabolites (e.g., ABP) [33, 34]. Because the mechanism of bisphosphonate action in osteoblasts and breast cancer cells has not been reported, we investigated the role of the mevalonate pathway in the bisphosphonate action on hFOB and MDA-MB-231 cells. We also compared the potency of Ara-CBP to etidronate, ABP (a reported metabolite of etidronate) [33], pamidronate, zoledronate, and mevastatin on hFOB cell proliferation and mineralization and MDA-MB-231 breast cancer cell proliferation. Lastly, the action of Ara-CBP [35] was compared to Ara-C and Ara-CMP to determine if both or one of these moieties of Ara-CBP are responsible for the significant effects of Ara-CBP on hFOB and MDA-MB-231 cells. Materials and methods Materials Pamidronate was produced by Novartis Pharma AG (Basel, Switzerland). Etidronate was produced by MGI Pharm Inc. (Minnetonka, MN). The Ara-CBP and ABP were synthesized at the Russian Academy of Sciences [35]. The zoledronate was provided by Novartis Pharma AG (Basel, Switzerland). DMEM: Ham’s F-12 medium (1:1), mevastatin, mevalonic acid lactone, geranylgeraniol, farnesol, Alizarin Red S stain, Ara-C and Ara-CMP were purchased from Sigma (St. Louis, MO). Fetal bovine serum was purchased from Summit Biotechnology (Ft. Collins, CO). MDA-MB-231 cells were purchased from ATCC (Manassas, VA). See Figure 1 for the structures of AraCBP, ABP, etidronate, Ara-C, Ara-CMP, pamidronate, zoledronate, and mevastatin. Cell culture The hFOB cells were previously developed and characterized in this laboratory [28]. Briefly, hFOB cells were derived from primary cultures of fetal tissue and are conditionally immortalized with a gene coding for
the temperature sensitive mutant (ts A58) of the SV40 large T-antigen. This cell line was isolated from the primary cultures based on its osteoblast phenotype. Incubation of hFOB cells at the permissive temperature (34◦C) results in rapid cell division, whereas little or no cell division occurs at the restrictive temperature (39◦C). hFOB cells were maintained at 34◦ C in DMEM:Ham’s F-12 medium (1:1) supplemented with 10% (v/v) fetal bovine serum and 300 µg/ml geneticin. MDA-MB-231 cells were maintained at 37◦ C in DMEM:Ham’s F-12 medium (1:1) supplemented with 10% (v/v) fetal bovine serum. Culture medium was removed and replaced with fresh medium (with or without the appropriate test compounds) every 3 or 4 days during experimentation. Cell proliferation hFOB or MDA-MB-231 cells were seeded at 20,000 cells/cm2 in 96-well plates and incubated for 24 h in normal culture medium. The medium was then replaced with 100 µl/well of fresh medium containing the compounds of interest. The relative number of viable cells in each well was then determined 7 days after treatment using the Cell Titer 96 AQueous One Solution Cell Proliferation Assay (Promega, Madison Wisconsin). Briefly, 20 µl of Cell Titer 96 AQueous One Solution was added to each well including three wells containing only medium for background subtraction. The cells were then incubated at 37◦ C for 30 min. The absorbance at 490 nm in each well was then determined using a SpectraMax 340, plate reader/spectrophotometer (Molecular Devices Corporation, Sunnyvale, CA). Mineralization hFOB cells were seeded at 20,000 cells/cm2 in 12well and 96-well plates and incubated at 34◦ C for 24 h in normal culture medium. The medium was then replaced with fresh medium containing the compounds of interest. After 7 days, the degree of mineralization was determined in the 12-well plates using alizarin red staining. Briefly, medium was aspirated from the wells and the cells were rinsed twice with PBS. The cells were fixed with ice-cold 70% (v/v) ethanol for 1 h. The ethanol was removed and the cells were rinsed twice with deionized water. The cells were then stained with 40 mM Alizarin Red S in deionized water adjusted to pH 4.2, for 10 min at room temperature. The Alizarin Red S solution was removed by aspiration and the cells were rinsed five times with
Mechanisms of bisphosphonate action
259
Figure 1. Chemical structures of Ara-CBP, ABP, etidronate, Ara-C, Ara-CMP, pamidronate, zoledronate and mevastatin.
deionized water. The water was removed by aspiration and the cells were incubated in PBS for 15 min at room temperature on an orbital rotator. The PBS was removed and the cells were rinsed once with fresh PBS. The cells were then destained for 15 min with 10% (w/v) cetylpyridinium chloride in 10 mM sodium phosphate, pH 7.0. The extracted stain was then transferred to a 96-well plate and the absorbance at 562 nm was measured using a SpectraMax 340, plate reader/spectrophotometer (Molecular Devices Corporation, Sunnyvale, CA). The concentration of Alizarin Red S staining in the samples was determined by comparing the absorbance values to those obtained from Alizarin Red S standards. The mineralization values were normalized to the relative number of viable cells
as determined directly in the 96-well plates using the above proliferation assay. Statistical analysis Significance was determined using the two-tailed Student’s t-test.
Results hFOB cell proliferation The effects of Ara-CBP, etidronate, ABP, pamidronate, zoledronate, and mevastatin on hFOB cell prolif-
260
G G Reinholz et al.
Figure 2. Schematic representation of the mevalonate pathway and the effects of nitrogen-containing bisphosphonates and statins [compiled from references 9, 17, 20, 29–32]. HMG-CoA, 3-hydroxy-3-methylglutaryl-coenzyme A, N-BPs, nitrogen-containing bisphosphonates, FOH, farnesol, GGOH, geranylgeraniol.
eration were compared, and the role of the mevalonate pathway was examined. See Figure 1 for chemical structures, and Figure 2 for a schematic of the mevalonate pathway. As shown in Figure 3(A), etidronate and ABP had little effect on hFOB cell proliferation between 10−8 to 10−5 M while pamidronate (10−5 M) and zoledronate (10−5 M) reduced hFOB cell proliferation to 57% and 35% of control, respectively. Mevastatin was more potent than pamidronate and zoledronate decreasing hFOB cell proliferation to 76% of control at 10−6 M and causing complete cell death at 10−5 M. Ara-CBP was the most potent, and inhibited hFOB cell proliferation at all concentrations tested (10−8 –10−5 M). In order to determine the role of the mevalonate pathway in the anti-proliferative effects of mevastatin, pamidronate, zoledronate, and Ara-CBP, the hFOB cells were treated simultaneously with the bisphosphonates or mevastatin, plus mevalonate, farnesol, or geranylgeraniol. Farnesol and geranylgeraniol are converted into the mevalonate pathway intermediates farnesyl diphosphate and geranylgeranyl diphosphate, respectively (Figure 1) via a salvage pathway [29]. As shown in Figure 3(B), treatment of the hFOB cells with mevalonate (2 × 10−4 M), farnesol (10−5 M), or gernylgeraniol (10−5 M) alone had little effect on
hFOB cell proliferation (103, 104, and 93% of control, respectively). As shown in Figure 3(B), treatment of hFOB cells with mevastatin (10−6 M) reduced hFOB cell proliferation to 68% of control. As expected, treatment of the hFOB cells simultaneously with mevalonate (2 × 10−4 M) and mevastatin (10−6 M) completely restored the hFOB cell proliferation to control levels (102% of control). These results suggest that mevastatin inhibits hFOB cell proliferation by inhibiting of 3-hydroxy-3-methylglutaryl-coenzyme A reductase activity. Treatment of the hFOB cells with pamidronate (10−5 M) reduced hFOB cell proliferation to 80% of control. The simultaneous addition of the mevalonate pathway intermediates with pamidronate did not reverse the action of pamidronate on hFOB cell proliferation. Thus, pamidronate appears to inhibit hFOB cell proliferation via mechanisms other than inhibition of the mevalonate pathway. Treatment of the hFOB cells with zoledronate (10−5 M) reduced hFOB cell proliferation to 60% of control. Neither mevalonate (2 × 10−4 M) nor farnesol (10−5 M) reversed the effect of zoledronate while geranylgeraniol (10−5 M) partially reversed the effect of zoledronate restoring hFOB cell proliferation to 80% of control. These results suggest that the inhibition of hFOB cell proliferation by zoledronate is due in part to inhibition
Mechanisms of bisphosphonate action
261
Figure 3. Comparative effects of bisphosphonates and mevastatin on hFOB cell proliferation: role of the mevalonate pathway. hFOB cells were seeded at 20,000 cell/cm2 in 96-well plates and incubated in normal culture medium at 37◦ C for 24 h. (A) The medium was then replaced with fresh culture medium containing various concentrations (10−8 –10−5 M) of bisphosphonates or mevastatin. The medium was then replaced with the appropriate medium every 3 or 4 days. After 10 days, the hFOB cell proliferation was assessed as described in ‘Materials and Methods’. (B) The medium was then replaced with fresh culture medium only or medium containing pamidronate (10−5 M), zoledronate (10−5 M), MBC-11 (10−7 M), mevastatin (10−6 M), mevalonate (2 × 10−4 M), farnesol (10−5 M), geranylgeraniol (10−5 M) or combinations of the above as shown. The medium was then replaced with the appropriate medium on day 4 of treatment. After 7 days, the hFOB cell proliferation was assessed as described in ‘Materials and Methods’. NM, normal medium; M, mevalonate; F, farnesol; G, geranylgeraniol; PAM, pamidronate; Zol, zoledronate The data represent the mean values (n = 4). Error bars, SDs from the mean values. ∗ p < 0.04, ∗∗ p < 0.01 compared to treatment with bisphosphonate or mevastatin alone.
of geranylgeranyl diphosphate synthesis. Treatment of hFOB cells with Ara-CBP (10−7 M) reduced hFOB cell proliferation to 30% of control. Each of the mevalonate pathway intermediates partially reversed the action of Ara-CBP on hFOB cell proliferation with geranylgeraniol (10−5 M) having the greatest effect restoring hFOB cell proliferation to 77% of control. These results suggest that Ara-CBP inhibits hFOB cell proliferation by inhibiting the mevalonate pathway and likely additional mechanisms.
hFOB cell mineralization The effects of Ara-CBP, etidronate, ABP, pamidronate, zoledronate, and mevastatin on hFOB cell mineralization were compared. As shown in Figure 4(A), etidronate and ABP had no effect on hFOB cell mineralization between 10−8 to 10−5 M while pamidronate (10−5 M) and zoledronate (10−5 M) increased hFOB cell mineralization to 162% and 195% of control, respectively. Again, Ara-CBP was the most potent
262
G G Reinholz et al.
Figure 4. Comparative effects of bisphosphonates and mevastatin on hFOB cell mineralization: role of the mevalonate pathway. hFOB cells were seeded at 20,000 cell/cm2 in 12 and 96-well plates and incubated in normal culture medium at 37 ◦ C for 24 h. (A) The medium was then replaced with fresh culture medium containing various concentrations (10−8 –10−5 M) of bisphosphonates or mevastatin. The medium was then replaced with the appropriate medium every 3 or 4 days. After 10 days, the hFOB cell mineralization was assessed as described in ‘Materials and Methods’. (B) The medium was then replaced with fresh culture medium only or medium containing pamidronate (10−5 M), zoledronate (10−5 M), ABP (10−7 M), mevastatin (10−6 M), mevalonate (2 × 10−4 M), farnesol (10−5 M), geranylgeraniol (10−5 M) or combinations of the above as shown. The medium was then replaced with the appropriate medium on day 4 of treatment. After 7 days, the hFOB cell mineralization was assessed as described in ‘Materials and Methods’. NM, normal medium; M, mevalonate; F, farnesol; G, geranylgeraniol; PAM, pamidronate; Zol, zoledronate. The data represent the mean values (n = 4). Error bars, SDs from the mean values. ∗ p < 0.02, ∗∗ p < 0.0002 compared to treatment with bisphosphonate or mevastatin alone.
of the compounds tested. Ara-CBP maximally induced hFOB cell mineralization to 427% of control at 10−6 M. Interestingly, treatment of hFOB cells with mevastatin (10−6 M) reduced hFOB cell mineralization to 24% of control. In order to determine the role of the mevalonate pathway in the effects of mevastatin, pamidronate, zoledronate, and Ara-CBP, on hFOB cell mineraliz-
ation, the hFOB cells were treated simultaneously with the bisphosphonates or mevastatin, plus the mevalonate pathway intermediates. As shown in Figure 4(B), treatment of the hFOB cells with mevalonate (2 × 10−4 M), farnesol (10−5 M) or geranylgeraniol (10−5 M) alone had little effect on hFOB cell mineralization (105, 94, and 87% of control, respectively).
Mechanisms of bisphosphonate action As shown in Figure 4(B), treatment of hFOB cells with mevastatin (10−6 M) reduced hFOB cell mineralization to 24% of control. As expected, this inhibitory action of mevastatin was completely reversed by mevalonate (2 × 10−4 M) to 107% of control and partially reversed by geranylgeraniol (10−5 M) to 86% of control. These results suggest that mevastatin decreases hFOB cell mineralization by inhibiting mevalonate synthesis. Treatment of hFOB cells with pamidronate (10−5 M) increased hFOB cell mineralization to 142% of control. Similar to the proliferation experiments, simultaneous treatment of hFOB cells with pamidronate and the mevalonate pathway intermediates did not reverse the action of pamidronate on hFOB cell mineralization. Therefore, it appears that pamidronate enhances hFOB cell mineralization via mechanisms other than inhibition of the mevalonate pathway. Treatment of hFOB cells with zoledronate (10−5 M) increased hFOB cell mineralization to 120% of control. Geranylgeraniol (10−5 M) reversed the effect of zoledronate to 96% of control. These results suggest that zoledronate enhances hFOB cell mineralization by inhibiting geranylgeranyl diphosphate synthesis. Treatment of hFOB cells with Ara-CBP (10−7 M) increased hFOB cell mineralization to 287% of control. Both mevalonate (2 × 10−4 M) and farnesol (10−5 M) partially reversed the action of Ara-CBP returning hFOB cell mineralization to 152% and 130% of control respectively, while geranylgeraniol (10−5 M) almost completely reversed the action of Ara-CBP to 107% of control. These results suggest that Ara-CBP enhances hFOB cell mineralization by inhibiting the mevalonate pathway.
263
most potent inhibitor of MDA-MB-231 cell proliferation reducing proliferation to 83%–6% of control between 10−8 M and 10−4 M. In order to determine the role of the mevalonate pathway in the effects of mevastatin, pamidronate, zoledronate, and Ara-CBP on MDA-MB-231 cell proliferation, the MDA-MB-231 cells were treated simultaneously with the bisphosphonates or mevastatin, plus the mevalonate pathway intermediates. As shown in Figure 5(B), treatment of the MDA-MB-231 cells with mevalonate (2 × 10−4 M), farnesol (2 × 10−5 M) or geranylgeraniol (2 × 10−5 M) alone had little effect on MDA-MB-231 cell proliferation (96, 100 and, 92% of control, respectively). As shown in Figure 5(B), treatment of the MDAMB-231 cells with mevastatin (10−6 M) reduced proliferation to 71% of control. As expected, addition of mevalonate (2 × 10−4 M) completely reversed the effect of mevastatin, while farnesol (2 × 10−5 M) further reduced proliferation and geranylgeraniol (2 × 10−5 M) partially reversed the action of mevastatin to 85% of control. Pamidronate (5 × 10−5 M) treatment decreased MDA-MB-231 cell proliferation to 49% of control. Similar to the results with the hFOB cells, addition of the mevalonate pathway intermediates had no effect on the action of pamidronate. Treatment of MDA-MB-231 cells with zoledronate (2 × 10−5 M) reduced proliferation to 50% of control. The mevalonate pathway intermediates had little effect on the action of zoledronate. Ara-CBP (10−6 M) treatment decreased the proliferation of MDA-MB231 cells to 25% of control. Again, addition of the mevalonate pathway intermediates had no effect on the action of Ara-CBP on MDA-MB-231 cell proliferation.
MDA-MB-231 breast cancer cell proliferation The effects of Ara-CBP, etidronate, ABP, pamidronate, zoledronate, and mevastatin on MDA-MB-231 breast cancer cell proliferation were compared. As shown in Figure 5(A), etidronate and ABP had no effect on MDA-MB-231 cell proliferation between 10−8 to 10−4 M while pamidronate and zoledronate inhibited MDA-MB-231 cell proliferation with similar potencies decreasing proliferation to 91% and 84% of control at 10−5 M and 12% and 7% of control at 10−4 M respectively. Mevastatin inhibited MDAMB-231 cell proliferation with greater potency than pamidronate and zoledronate reducing proliferation from 68% to 3% of control between 10−6 M and 10−4 M. As with the hFOB cells, Ara-CBP was the
Comparative effects of Ara-CBP, Ara-C, and Ara-CMP on hFOB and MDA-MB-231 breast cancer cells In order to determine if the effects of Ara-CBP on hFOB and MDA-MB-231 cells are due primarily to the arabinocytidine moiety, the effects of Ara-CBP, Ara-C and Ara-CMP on hFOB cell proliferation and mineralization, MDA-MB-231 cell proliferation were compared (see Figure 2 for structures). As shown in Figure 6(A), Ara-CBP, Ara-C and Ara-CMP all inhibited hFOB cell proliferation, however, Ara-CBP was approximately 10-fold more potent than AraC and Ara-CMP. As shown in Figure 6(B), AraC (10−7 M) increased hFOB cell mineralization to
264
G G Reinholz et al.
Figure 5. Comparative effects of bisphosphonates and mevastatin on MDA-MB-231 cell proliferation: role of the mevalonate pathway. MDA-MB-231 cells were seeded at 20,000 cell/cm2 in 96-well plates and incubated in normal culture medium at 37◦ C for 24 h. (A) The medium was then replaced with fresh culture medium containing various concentrations (10−8 –10−4 M) of bisphosphonates or mevastatin. The medium was then replaced with the appropriate medium every 3 or 4 days. After 7 days, the hFOB cell proliferation was assessed as described in ‘Materials and Methods’. (B) The medium was then replaced with fresh culture medium only or medium containing pamidronate (5 × 10−5 M), zoledronate (2 × 10−5 M), Ara-CBP (10−6 M), mevastatin (10−6 M), mevalonate (2 × 10−4 M), farnesol (2 × 10−5 M), geranylgeraniol (2 × 10−5 M) or combinations of the above as shown. The medium was then replaced with the appropriate medium on day 4 of treatment. After 7 days, the MDA-MB-231 cell proliferation was assessed as described in ‘Materials and Methods’. NM, normal medium; M, mevalonate; F, farnesol; G, geranylgeraniol; PAM, pamidronate; Zol, zoledronate. The data represent the mean values (n = 4). Error bars, SDs from the mean values. ∗ p < 0.001 compared to treatment with mevastatin alone.
119% of control and Ara-CBP (10−7 M) increased mineralization to 180% of control, whereas Ara-CMP did not increase hFOB cell mineralization at any of the concentrations tested. As shown in Figure 6(C), Ara-CBP, Ara-C and Ara-CMP all inhibited MDAMB-231 cell proliferation, however, Ara-CBP was approximately 100-fold more potent than either Ara-C or Ara-CMP.
Discussion Inhibition of the mevalonate pathway has been implicated in the mechanism of action of nitrogencontaining bisphosphonates in osteoclasts [9, 20, 32] and other cell types [27, 30, 31]. Therefore, the role of this pathway in the action of bisphosphonates in hFOB and MDA-MB-231 cells was examined. The
Mechanisms of bisphosphonate action
Figure 6. Comparative effects of Ara-CBP, Ara-C and Ara-CMP on hFOB and MDA-MB-231 cells. hFOB and MDA-MB-231 cells were seeded at 20,000 cell/cm2 in 96-well plates and incubated in normal culture medium at 37◦ C for 24 h. The medium was then replaced with fresh culture medium containing various concentrations (10−8 –10−5 M) of Ara-CBP, Ara-C or Ara-CMP. The medium was then replaced with the appropriate medium every 3 or 4 days. (A) After 10 days, the hFOB cell proliferation was assessed as described in ‘Materials and Methods’. (B) After 10 days, the hFOB cell mineralization was assessed as described in ‘Materials and Methods’. (C) After 7 days, the MDA-MB-231 cell proliferation was assessed as described in ‘Materials and Methods’. The data represent the mean values (n = 4). Error bars, SDs from the mean values.
265
3-hydroxy-3-methylglutaryl-coenzyme A reductase (EC 1.1.1.34) inhibitor, mevastatin, was used as a control. As expected, the effects of mevastatin on hFOB cells and MDA-MB-231 cells are completely reversed by mevalonate. These results suggest that mevastatin inhibits the mevalonate pathway in these cells as reported with other cell types [36]. Interestingly, mevastatin decreased the proliferation of the hFOB cells as did the bisphosphonates, and at similar concentrations. However, mevastatin decreased hFOB cell mineralization while pamidronate, zoledronate and Ara-CBP increased hFOB cell mineralization. Our results with mevastatin do not correlate with a recent study that reported increased bone formation in rodents after treatment with statins [37], nor with the clinical evidence linking use of oral statins to increased bone-mineral density and reduced fracture risk in postmenopausal women [38, 39]. The reported increase in bone formation induced by statins has been attributed to increased expression of BMP-2 [37, 40, 41]. We have observed that mevastatin treatment also induces BMP-2 mRNA levels in hFOB cells (data not shown). Thus, it is not clear at this time why mevastatin decreases hFOB cell mineralization. This observation is currently under investigation. The results presented herein suggest that both zoledronate and the novel bisphosphonate analogue, Ara-CBP, affect hFOB cells via inhibition of the mevalonate pathway. The inhibition of hFOB cell proliferation by zoledronate and Ara-CBP was partially reversed by mevalonate pathway intermediates. Whereas the induction of hFOB cell mineralization by zoledronate and Ara-CBP was completely reversed by the mevalonate pathway intermediate, geranylgeraniol. Thus, while the induction of hFOB cell mineralization by zoledronate and Ara-CBP may be completely due to inhibition of the mevalonate pathway, the inhibition of hFOB cell proliferation may involve additional unknown mechanisms. Interestingly, the effects of pamidronate on hFOB cells were not reversed by the mevalonate pathway intermediates, including squalene (data not shown), suggesting that the mechanism of action of pamidronate on these cells does not involve inhibition of the mevalonate pathway. Furthermore, the inhibition of MDA-MB-231 cell proliferation by the bisphosphonates was not reversed by mevalonate pathway intermediates. Thus, the mechanism of zoledronate and Ara-CBP action appears to be cell-type specific. In this manuscript, the effects of a novel arabinocytidine-bisphosphonate, Ara-CBP, on hFOB cells
266
G G Reinholz et al.
and MDA-MB-231 breast cancer cells were compared to etidronate, ABP, pamidronate, zoledronate, and mevastatin. Ara-CBP is similar in structure to the reported adenosine metabolite of etidronate, ABP, with the adenosine moiety replaced by the antineoplastic arabinocytidine moiety [33, 34]. Ara-CBP was more potent than either pamidronate or zoledronate in inhibiting hFOB cell proliferation and enhancing in vitro mineralization. Ara-CBP affected the hFOB cells in a wide range of doses (10−8–10−5 M). This is in contrast to the narrow effective range for pamidronate and zoledronate (10−6–10−5 M) observed in these studies. These results support the notion that in addition to the anti-resorptive effects of bisphosphonates on osteoclasts, bisphosphonates may affect bone formation. Furthermore, the results with Ara-CBP demonstrate that the basic structure of the bisphosphonates can be altered to enhance the effects of bisphosphonates on osteoblasts, at least in vitro. Because etidronate and ABP had no effect on hFOB or MDA-MB-231 cells at the concentrations tested, while Ara-CBP showed dramatic effects, one might expect that the effects of Ara-CBP are due solely to the arabinocytidine moiety. However, clearly, Ara-CBP was more potent than Ara-C and Ara-CMP. Therefore, the effects of Ara-CBP in these assays cannot be fully explained by the bisphosphonate or arabinocytidine moieties alone, and are likely due to intact Ara-CBP. Pamidronate, zoledronate, Ara-CBP and mevastatin also inhibited MDA-MB-231 breast cancer cells with the same order of potency observed in the hFOB cells. Similar results were also obtained with T47-D breast cancer cells (data not shown). These results support previous observations indicating that bisphosphonates can inhibit breast cancer cell proliferation [26]. These direct effects of bisphosphonates on breast cancer cells and osteoblasts may help explain the mechanism of bisphosphonate inhibition of breast cancer metastasis to bone. While it is difficult to predict what will occur in vivo, the potency of the known bisphosphonates reported in these assays does follow the reported rank potency of these compounds in vivo with zoledronate > pamidronate > etidronate [42]. Thus, it would be most interesting to test the in vivo effects of Ara-CBP, which displays much greater potency than zoledronate in our models, on animal models of bone metabolism and breast cancer metastasis. In summary, the bisphosphonates pamidronate, zoledronate, and Ara-CBP have direct effects on hFOB cells but act through distinct mechanisms. While pa-
midronate does not appear to act via inhibition of the mevalonate pathway, other bisphosphonates, zoledronate and Ara-CBP, do appear to inhibit this pathway in osteoblasts. To the best of our knowledge, this is the first report demonstrating that certain bisphosphonates can affect osteoblast cell activities through inhibition of the mevalonate pathway. Interestingly, while mevastatin also inhibits the mevalonate pathway in hFOB cells, mevastatin inhibits hFOB cell mineralization whereas zoledronate and Ara-CBP stimulate hFOB cell mineralization. Thus, inhibition of different steps in the mevalonate pathway appears to profoundly affect the resulting action on hFOB cell function. In MDA-MB-231 cells, pamidronate, zoledronate, AraCBP and mevastatin all inhibit proliferation, but only mevastatin acts via inhibition of the mevalonate pathway. Thus, the mechanisms of bisphosphonate action are compound and cell-type specific. Further understanding the mechanism of bisphosphonate action which appears to differ between the bone and breast cancer cells, should aid in the development of more effective compounds for the treatment of cancer metastasis to bone. The results with Ara-CBP presented herein are also encouraging and warrant further investigation of this compound in animal models of cancer metastasis to bone.
Acknowledgements This work was supported by the Mazza Foundation, the Mayo Clinic Comprehensive Cancer Center, and NIH training grant HD 07108-22 to Gregory G. Reinholz. We also wish to acknowledge Monica M. Reinholz for performing the BMP-2 gene expression analysis, and for her critical evaluation of this manuscript.
References 1. Reinholz GG, Getz B, Pederson L, Sanders ES, Subramaniam M, Ingle JN, Spelsberg TC: Bisphosphonates directly regulate cell proliferation, differentiation and gene expression in human osteoblasts. Cancer Res 60: 6001–6007, 2000 2. Cifuentes N, Pickren JW: Metastases from carcinoma of mammary gland: an autopsy study. J Surg Oncol 11: 193–205, 1979 3. Coleman RE, Rubens RD: The clinical course of bone metastases from breast cancer. Br J Cancer 55: 61–66, 1987 4. Weiss L: Comments on hematogenous metastatic patterns in humans as revealed by autopsy. Clin Exp Metastasis 10: 191– 199, 1992
Mechanisms of bisphosphonate action 5. Hortobagyi GN, Theriault RL, Porter L, Blayney D, Lipton A, Sinoff C, Wheeler H, Simeone JF, Seaman J, Knight RD, Heffernan M, Reitsma DJ: Efficacy of pamidronate in reducing skeletal complications in patients with breast cancer and lytic bone metastases. N Engl J Med 335: 1785–1791, 1996 6. Conte PF, Latreille J, Mauriac L, Calabresi F, Santos R, Campos D, Bonneterre J, Francini G, Ford JM: Delay in progression of bone metastases in breast cancer patients treated with intravenous pamidronate: results from a multinational randomized controlled trial. J Clin Oncol 14: 2552–2559, 1996 7. Hortobagyi GN, Theriault RL, Lipton A, Porter L, Blayney D, Sinoff C, Wheeler H, Simeone JF, Seaman JJ, Knight RD, Heffernan M, Mellars K, Reitsma DJ: Long-term prevention of skeletal complications of metastatic breast cancer with pamidronate. J Clin Oncol 16: 2038–2044, 1998 8. Diel IJ, Solomayer E, Costa SD, Gollan C, Goerner R, Wallwiener D, Kaufmann M, Bastert G: Reduction in new metastases in breast cancer with adjuvant clodronate treatment. N Engl J Med 339: 357–363, 1998 9. Russell RGG, Rogers MJ: Bisphosphonates: from the laboratory to the clinic and back again. Bone 25: 97–106, 1999 10. Hughes DE, MacDonald BR, Russell RGG, Gowen M: Inhibition of osteoclast-like cell formation by bisphosphonates in long-term cultures of human bone marrow. J Clin Invest 83 1930–1935, 1989 11. Cecchini MG, Felix R, Fleisch H, Cooper PH: Effect of bisphosphonates on proliferation and viability of mouse bone marrow-derived macrophages. J Bone Miner Res 2: 135–142, 1987 12. Löwik CWGM, van der Pluijm G, van der Wee-Pals LJA, Bloys van Treslong-de Groot H, Bijvoet OLM: Migration and phenotypic transformation of osteoclast precursors into mature osteoclasts: the effect of a bisphosphonate. J Bone Miner Res 3: 185–192, 1988 13. Evans CE, Braidman IP: Effects of two novel bisphosphonates on bone cells in vitro. Bone Miner 26: 95–107, 1994 14. Rogers MJ, Chilton, KM, Coxon FP, Lawry J, Smith MO, Suri S, Russell RGG: Bisphosphonates induce apoptosis in mouse macrophage-like cells in vitro by a nitric oxideindependent mechanism. J Bone Miner Res 11: 1482–1491, 1996 15. Hughes DE, Wright KR, Uy HL, Sasaki A, Yoneda T, Roodman GD, Mundy GR, Boyce BF: Bisphosphonates promote apoptosis in murine osteoclasts in vitro and in vivo. J Bone Miner Res 10: 1478–1487, 1995 16. Ito M, Amizuka N, Nakajima T, Ozawa H: Ultrastructural and cytochemical studies on cell death of osteoclasts induced by bisphosphonate treatment. Bone 25: 447–452, 1999 17. Reszka AA, Halasy-Nagy JM, Masarachia PJ, Rodan GA: Bisphosphonates act directly on the osteoclast to induce caspase cleavage of Mst1 kinase during apoptosis. J Biol Chem 274: 34967–34973, 1999 18. Carano A, Teitelbaum SL, Konsek JD, Schlesinger PH, Blair HC: Bisphosphonates directly inhibit the bone resorption activity of isolated avian osteoclasts in vitro. J Clin Invest 85: 456–461, 1990 19. Selander K, Lehenkari P, Väänänen HK: The effects of bisphosphonates on the resorption cycle of isolated osteoclasts. Calcif Tissue Int 55: 368–375, 1994 20. Fisher JE, Rogers MJ, Halasy JM, Luckman SP, Hughes DE, Masarachia PJ, Wesolowski G, Russell RGG, Rodan GA, Reszka AA: Alendronate mechanism of action: geranylgeraniol, an intermediate in the mevalonate pathway, prevents
21.
22.
23.
24.
25.
26.
27.
28.
29.
30.
31.
32.
33.
34.
35.
36.
267
inhibition of osteoclast formation, bone resorption, and kinase activation in vitro. Proc Natl Acad Sci USA 96: 133–138, 1999 Giuliani N, Pedrazzoni M, Negri G, Passeri G, Impicciatore M, Girasole G: Bisphosphonates stimulate formation of osteoblast precursors and mineralized nodules in murine and human bone marrow cultures in vitro and promote early osteoblastogenesis in young and aged mice in vivo. Bone 22: 455–461, 1998 Khokher MA, Dandona P: Diphosphonates inhibit human osteoblast secretion and proliferation. Metabolism 38: 184–187, 1989 Klein BY, Ben-Bassat H, Breuer E, Solomon V, Golomb G: Structurally different bisphosphonates exert opposing effects on alkaline phosphatase and mineralization in marrow osteoprogenitors. J Cell Biochem 68: 186–194, 1998 García-Moreno C, Serrano S, Nacher M, Farré M, Díez A, Mariñoso ML, Carbonell J, Mellibovsky L, Nogués X, Ballester J, Aubía J: Effect of alendronate on cultured normal human osteoblasts. Bone 22: 233–239, 1998 Plotkin LI, Weinstein RS, Parfitt AM, Roberson PK, Manolagas SC, Bellido T: Prevention of osteocyte and osteoblast apoptosis by bisphosphonates and calcitonin. J Clin Invest 104: 1363–1374, 1999 Fromigue O, Lagneaux L, Body JJ: Bisphosphonates induce breast cancer cell death in vitro. J Bone Miner Res 15: 2211– 2221, 2000 Shipman CM, Croucher PI, Russell RGG, Helfrich MH, Rogers MJ: The bisphosphonate incadronate (YM175) causes apoptosis of human myeloma cells in vitro by inhibiting the mevalonate pathway. Cancer Res 58: 5294–5297, 1998 Harris SA, Enger RJ, Riggs BL, Spelsberg TC: Development and characterization of a conditionally immortalized human fetal osteoblastic cell line. J Bone Miner Res 10: 178–186, 1995 Crick DC, Andres DA, Waechter CJ: Novel salvage pathway utilizing farnesol and geranylgeraniol for protein isoprenylation. Biochem Biophys Res Commun 237: 483–487, 1997 Amin D, Cornell SA, Gustafson SK, Needle SJ, Ullrich JW, Bilder GE, Perrone MH: Bisphosphonates used for the treatment of bone disorders inhibit squalene synthase and cholesterol biosynthesis. J Lipid Res 33: 1657–1663, 1992 van Beek E, Pieterman E, Cohen L, Löwik C, Papapoulos S: Farnesyl pyrophosphate synthase is the molecular target of nitrogen-containing bisphosphonates. Biochem Biophys Res Commun 264: 108–111, 1999 Fisher JE, Rodan GA, Reszka AA: In vivo effects of bisphosphonates on the osteoclast mevalonate pathway. Endocrinology 141: 4793–4796, 2000 Pelorgeas S, Martin J, Satre M: Cytotoxicity of dichloromethane diphosphonate and of 1-hydroxyethane-1,1-diphosphonate in the amoebae of the slime mould dictyostelium discoideum. Biochem Pharmocol 44: 2157–2163, 1992 Rogers MJ, Russell RGG, Blackburn GM, Williamson MP, Watts DJ: Metabolism of halogenated bisphosphonates by the cellular slime mould dictyostelium discoideum. Biochem and Biophys Res Commun 189: 414–423, 1992 Andreeva OI, Efimtseva EV, Padyukova NSh, Kochetkov SN, Mikhailov SN, Dixon HBF, Karpeisky MYa: Interaction of HIV-1 reverse transcriptase and T7 RNA polymerase with phosphonate analogs of NTP and inorganic pyrophosphate. Mol Biol 35: 717–729, 2001 Endo A: Chemistry, biochemistry, and phamacology of HMGCoA reductase inhibitors. Klin Wochenshr 66: 421–427, 1988
268 37.
38.
39.
40.
G G Reinholz et al. Mundy G, Garrett R, Harris S, Chan J, Chen D, Rossini G, Boyce B, Zhao M, Gutierrez G: Stimulation of bone formation in vitro and in rodents by statins. Science 286: 1946–1949, 1999 Edwards CJ, Hart DJ, Spector TD: Oral statins and increased bone-mineral density in postmenopausal women. Lancet 355: 2218–2219, 2000 Chan KA, Andrade SE, Boles M, Buist DSM, Chase GA, Donahue JG, Goodman MJ, Gurwitz JH, LaCroix AZ, Platt R: Inhibitors of hydroxymethylglutaryl-coenzyme A reductase and risk of fracture among older women. Lancet 355: 2185–2188, 2000 Sugiyama M, Kodama T, Konishi K, Abe K, Asami S, Oikawa S: Compactin and simvastatin, but not pravastatin, induce
bone morphogenetic protein-2 in human osteosarcoma cells. Biochem Biophys Res Commun. 271: 699–692, 2000 41. Garrett IR, Esparza J, Chen D, Zhao M, Gutierrez G, Escobedo A, Horn D, Mundy GR: Statins mediate their effects on osteoblasts by inhibition of HMG-CoA reductase and ultimately BMP-2. J Bone Min Res 15 (suppl 1): S225, 2000 (abstr) 42. Fleisch H: Bisphosphonates: mechanism of action. Endocr Rev 19: 80–100, 1998
Address for offprints and correspondence: Gregory G. Reinholz, Department of Orthopedics, Mayo Clinic, 200 First Street S.W., Rochester, MN 55905, USA; Tel.: 507-266-5237; Fax: 507-2845075; E-mail:
[email protected]
