Journal of the National Cancer Institute Advance Access originally published online on August 11, 2008
JNCI Journal of the National Cancer Institute 2008 100(16):1167-1178; doi:10.1093/jnci/djn240
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© The Author 2008. Published by Oxford University Press.
ARTICLES |
Antitumor Effects of Doxorubicin Followed by Zoledronic Acid in a Mouse Model of Breast Cancer
Affiliations of authors: Academic Unit of Clinical Oncology, School of Medicine and Biomedical Sciences (PDO, HM, DVL, REC, IH); Centre for Stem Cell Biology, Department of Biomedical Sciences, University of Sheffield, UK (MJ); Department of Pharmaceutics, University of Kuopio, Finland (HM)
Correspondence to: Ingunn Holen, PhD, Academic Unit of Clinical Oncology, School of Medicine and Biomedical Sciences, University of Sheffield, Beech Hill Road, Sheffield, S10 2RX, UK (e-mail: i.holen{at}sheffield.ac.uk).
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ABSTRACT |
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Background: The potent antiresorptive drug zoledronic acid (Zol) enhances the antitumor effects of chemotherapy agents in vitro. We investigated the effects of clinically achievable doses of doxorubicin (Dox) and Zol, given alone, in sequence, and in combination, on the growth of established breast tumors in vivo.
Methods: Female MF1 nude mice were inoculated subcutaneously with 5 x 105 human breast cancer MDA-MB-436 cells that stably expressed green fluorescent protein (ie, MDA-G8 cells). Beginning on day 7 after tumor cell injection, the mice were injected weekly for 6 weeks with saline, Dox (2 mg/kg body weight via intravenous injection), Zol (100 µg/kg body weight via intraperitoneal injection), Dox plus Zol, Zol followed 24 hours later by Dox, or Dox followed 24 hours later by Zol (n = 8–9 mice per group). The effects of treatment on tumor growth were determined by measuring tumor volume; on tumor cell apoptosis and proliferation by immunohistochemistry using antibodies for caspase-3 and Ki-67, respectively; and on bone by microcomputed tomography and bone histomorphometry. All P values are two-sided.
Results: Treatment with Dox or Zol alone or Zol followed 24 hours later by Dox did not statistically significantly decrease final tumor volume compared with saline. Mice treated with Dox plus Zol had statistically significantly smaller final tumor volumes than those treated with Dox alone (mean = 122 mm3 vs 328 mm3, difference = 206 mm3, 95% confidence interval [CI] = 78 to 335 mm3, P < .001), with Zol alone (122 mm3 vs 447 mm3, difference = 325 mm3, 95% CI = 197 to 454 mm3, P < .001), or with Zol followed 24 hours later by Dox (122 mm3 vs 418 mm3, difference = 296 mm3, 95% CI = 168 to 426 mm3, P < .001). Treatment with Dox followed 24 hours later by Zol almost completely abolished tumor growth. Tumors from mice that were treated with Dox followed by Zol had more caspase-3–positive cells than tumors from mice treated with saline (mean number of caspase-3–positive cells per square millimeter: 605.0 vs 82.19, difference = 522.8, 95% CI = 488.2 to 557.4, P < .001), with Zol alone (605.0 vs 98.44, difference = 506.6, 95% CI = 472.0 to 541.2, P < .001), or with Zol followed by Dox (605.0 vs 103.1, difference = 501.9, 95% CI = 467.3 to 536.5, P < .001). The treatment-induced increase in the number of caspase-3–positive cells was mirrored by a decrease in the number of tumor cells positive for the proliferation marker Ki-67. No evidence of bone disease was detected in any of the treatment groups following microcomputed tomography and histological analysis of bone.
Conclusion: Sequential treatment with Dox followed by Zol elicited substantial antitumor effects in subcutaneous breast tumors in vivo, in the absence of bone disease.
Prior knowledge Zoledronic acid is a potent inhibitor of osteoclastic bone resorption that also enhances the antitumor effects of chemotherapy agents both in vitro and in in vivo models with a high degree of tumor-induced bone disease. Study design An examination of the effects of clinically achievable doses of doxorubicin and zoledronic acid, given alone, in sequence, and in combination, on the growth of tumors derived from a human breast cell line that does not metastasize readily to bone in a mouse model. Contribution Sequential treatment with doxorubicin followed by zoledronic acid inhibited the growth of subcutaneous breast tumors in vivo, in the absence of bone disease. Implications There may be benefits of combining zoledronic acid with cytotoxic agents for the treatment of patients with early-stage breast cancer. Limitations Mice were treated with higher total doses of the drugs than those that breast cancer patients receive. The relevance of the mouse model to humans is not certain. From the Editors
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The potent third-generation nitrogen-containing bisphosphonate zoledronic acid is the only bisphosphonate that has been approved by the US Food and Drug Administration for the treatment of cancer-induced bone disease resulting from a range of solid tumors that affect bone as well as from multiple myeloma. Zoledronic acid inhibits osteoclastic bone resorption by inhibiting the activities of several enzymes of the mevalonate pathway (1), including farnesyl pyrophosphate synthase (2,3) and geranylgeranyl pyrophosphate synthase (4). Inhibition of the mevalonate pathway results in incomplete farnesylation and geranylgeranylation of signaling guanosine triphosphatases, including Ras, Rho, and Rac (5), which ultimately causes osteoclasts to undergo apoptosis (6). This effect is not limited to osteoclasts; the mevalonate pathway is present in all nucleated cells and constitutes an important part of the metabolic process that results in cholesterol synthesis.
Although the main target of nitrogen-containing bisphosphonates is the osteoclast, an increasing number of reports have described the potential direct antitumor effects of these compounds in in vitro model systems [reviewed in (7)]. These antitumor effects include inhibition of tumor cell growth and induction of cancer cell apoptosis (8–10), inhibition of tumor cell adhesion and invasion (11–13), and antiangiogenic activity (14,15). Antitumor effects of zoledronic acid have also been reported in in vivo model systems that mimic tumor-induced bone disease associated with a variety of cancer types, including multiple myeloma (16), osteosarcoma, (17), breast (18,19), prostate (20), and leukemia and lymphoma (21). Matsumoto et al. (22) reported that zoledronic acid (80 µg/kg body weight via subcutaneous injection three times per week for 2 weeks) inhibited the growth of human small-cell lung cancer cell–derived tumors that were subcutaneously implanted in nude mice. Li et al. (23) reported that a substantially lower dose of zoledronic acid (1 µg/kg body weight per week for 3 weeks) statistically significantly reduced tumor growth and increased survival among mice bearing subcutaneous tumors derived from murine non–small-cell lung cancer cells, which suggests that lung cancers may be particularly sensitive to the antitumor effects of this agent. In a transgenic mouse model of cervical cancer, zoledronic acid at a dose of 100 µg/kg body weight per day markedly inhibited the development of cervical tumors (24), which suggests that this compound has the potential to affect the growth of extraosseous soft tissue tumors. In addition to being the most potent of the bisphosphonates in relation to inhibition of bone resorption, zoledronic acid has shown synergistic induction of apoptosis in vitro when combined with paclitaxel, etoposide, cisplatinum, and irinotecan in lung cancer cells (22), with gemcitabine in colon cancer cells (25), with doxorubicin, paclitaxel, or tamoxifen in breast cancer cells (26,27), and with dexamethasone in myeloma cells (28).
There is also emerging evidence from a number of in vivo model systems of the beneficial effects of combined treatment with zoledronic acid and anticancer agents [reviewed in (29)]. For example, Melisi et al. (30) reported that treatment of mice with zoledronic acid and a cyclooxygenase-2 inhibitor (SC236) or a tyrosine kinase inhibitor (gefitinib) inhibited the growth of subcutaneous PC3 cell–derived prostate cancer xenografts more than treatment with the individual compounds. Heymann et al. (31) showed that zoledronic acid (100 µg/kg body weight via subcutaneous injection two times per week for 5 weeks) combined with ifosfamide almost complete inhibited tumor growth in a rat model of osteosarcoma. Hiraga et al. (32) reported that zoledronic acid (250 µg/kg body weight per day) in combination with tegafur–uracil (UFT) decreased the number of bone metastases compared with UFT alone in a mouse model of breast cancer. A recent study of bone metastases from neuroblastoma in a mouse model reported that administration of zoledronic acid (4 µg in 100 µL via subcutaneous injection five times per week) in combination with cyclophosphamide and topotecan reduced the number of osteolytic lesions and prolonged survival compared with the combination of cyclophosphamide and topotecan alone (33).
The majority of the studies that have investigated the effects of zoledronic acid on tumor growth in vivo have done so in models with a high degree of tumor-induced bone disease, whereas few studies, to our knowledge, have examined the effects of combined treatments on peripheral tumors that do not metastasize to bone. One of the few studies to investigate peripheral tumors that lack bone involvement was reported by Kuroda et al. (34), who examined the effects of combined treatment with zoledronic acid and imatinib mesylate in a mouse leukemia model. Combined treatment with zoledronic acid (80 µg/kg body weight via intravenous injection three times per week for 10 weeks) and imatinib mesylate caused a substantial increase in survival of mice engrafted with BV173 leukemia cells compared with the single agents.
Zoledronic acid is increasingly being used to control bone disease in breast cancer patients who are also being treated with anthracyclines. Anthracyclines, such as doxorubicin, are commonly used to manage both early and metastatic breast cancer. Doxorubicin has a complex mechanism of action that includes inhibition of topoisomerase II and that causes induction of DNA double-strand breaks (35), interference of DNA unwinding (36), induction of differentiation, and generation of reactive oxygen species (37). Doxorubicin induces growth arrest and nonapoptotic cell death accompanied by DNA damage in breast cancer cells in vitro but does not appear to induce apoptosis (38). An increasing number of breast cancer patients are treated with zoledronic acid to control their bone disease, in addition to chemotherapy regimes that often include doxorubicin. However, the cellular and molecular effects of treating breast cancer with doxorubicin and zoledronic acid are largely unknown. We have previously shown that sequential treatment of breast cancer cells in vitro with doxorubicin followed by zoledronic acid causes a synergistic increase in apoptotic cell death (26). In this study, we examined the effects of sequential treatment using these two agents on the subcutaneous growth of human breast cancer cells in a mouse model with no tumor-associated bone involvement.
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Materials and Methods |
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Cell Culture and Transfection
Human breast cancer MDA-MB-436 cells (obtained from the European Collection of Cell Cultures, Wiltshire, UK) were cultured in Dulbecco's modified Eagle medium (DMEM) supplemented with 4500 mg/L glucose, L-glucosamine, and pyruvate (Invitrogen–Gibco, Paisley, UK) and with 10% fetal calf serum (FCS). The cells were transfected with pMax-GFP (10 µg; Amaxa Biosystems, Cologne, Germany), an expression vector that contains the coding sequences for enhanced green fluorescent protein (eGFP) and geneticin resistance, with the use of a ProFection Mammalian Transfection System–Calcium Phosphate (Promega, Southampton, UK). eGFP–expressing MDA-MB-436 cells were selected on the basis of geneticin resistance by culturing in medium that contained 200 µg/mL geneticin (Melford Laboratories, Ipswitch, UK). Cells that expressed high levels of eGFP were selected and plated at a density of 1 cell per well into a 96-well tissue culture plate (Nunc, Rochester, NY) by single-cell disposition on a MoFlow High performance cell sorter (DakoCytomation, Cambridge, UK) with the 288-nm blue laser line from an Innova 190C ion laser (Coherent, Santa Clara, CA). eGFP fluorescence was detected by a 530/40-band pass filter. Cells were cultured in DMEM plus 4500 mg/L glucose, L-glucosamine, and pyruvate (Invitrogen-Gibco, Paisley, UK) supplemented with 10% FCS. MDA-G8 cells are a clone of eGFP–expressing cells that were derived from a single MDA-MB-436 cell. Use of eGFP-expressing tumor cells in this study allowed noninvasive in vivo monitoring of tumor take and subsequent growth. Tumor growth was monitored by visualization of eGFP fluorescence with the use of an Illumatool Lighting System equipped with a yellow filter (LightTools Research, Encinitas, CA).
Subcutaneous Growth of MDA-G8 Tumors In Vivo
MDA-G8 cells (0.5 x 106) were inoculated subcutaneously into the right flanks of 6-week-old-female MF1 nu/nu mice (n = 130; Charles River, Kent, UK). A total of 108 (83%) of the inoculated mice developed palpable tumors by day 7 after tumor cell injection; these mice were randomly assigned to the various treatment groups (n = 8 mice per group for dose response experiments and n = 8–9 mice per group for combination experiments).
An initial dose–response experiment was carried out to examine the effects of the individual agents on tumor growth; in this experiment, treatment commenced on day 7 after tumor cell injection, when the subcutaneous tumors had reached a palpable size. Mice were administered a single intravenous injection once per week for 6 weeks of doxorubicin (Pharmachemie B.V., Haarlem, The Netherlands) at either 2, 4, or 8 mg per kg body weight or a single intraperitoneal injection once per week for 6 weeks of zoledronic acid ([1-hydroxy-2- (1H-imidazoledronic acid-1-yl) ethylidene] bisphosphonic acid), supplied as the anhydrous disodium salt by Novartis Pharma AG, Basel, Switzerland) at 100 µg, 1.2 mg, or 6 mg per kg body weight (n = 8 mice per group).
For combination experiments, mice were injected intraperitoneally with 1) saline, 2) doxorubicin (2 mg/kg body weight; equivalent to a dose of 6.5 mg/m2 in patients [calculated using the conversion calculator available at http://www.fda.gov/cder/cancer/animalframe.htm]), 3) zoledronic acid (100 µg/kg body weight; equivalent to a dose of 0.32 mg/m2 in patients), 4) doxorubicin and zoledronic acid together (ie, administered within 30 seconds of each other), 5) zoledronic acid followed 24 hours later by doxorubicin, or 6) doxorubicin followed 24 hours later by zoledronic acid (n = 8–9 mice per group). All mice were treated once per week for 6 weeks.
All mice were killed by cerebral dislocation 24 hours after their final treatment and the subcutaneous tumors were excised. Half of each tumor was fixed in 4% paraformaldehyde in phosphate-buffered saline (PBS; pH 7.4) for 24 hours and used for immunohistochemical analysis; the other half was placed in the cell lysis buffer from an MCL1-1KT mammalian cell lysis kit (Sigma, Poole, UK) and protein was extracted according to the manufacturer's instructions. We simultaneously fixed and decalcified the tibia and femur from the right hind leg of each mouse in a solution of 1% paraformaldehyde and 0.5 M EDTA in PBS for 4 weeks and then embedded them in paraffin and cut 3-µm sections for histochemical and immunohistochemical staining. The tibia and femur from the left hind leg of each mouse were fixed in 10% formalin for 24 hours, and then transferred to 70% ethanol and used for microcomputed tomography analysis. Experiments were carried out in accordance with local guidelines and with Home Office approval under project license 40/2343 held by Professor N. J. Brown, University of Sheffield, UK.
Immunohistochemistry of Subcutaneous MDA-G8 Tumors from Treated Mice
Immunohistochemistry for caspase-3 was performed with the use of a rabbit polyclonal antibody that recognizes active mouse caspase-3 (AF835, 1:750 dilution; R&D Systems, Minneapolis, MN), followed by a biotin–conjugated anti-rabbit secondary antibody (1:200 dilution; Vector Laboratories, Peterborough, UK) as described by Marshman et al. (39). Immunohistochemistry for the cell proliferation antigen Ki-67 was carried out as previously described (40) using a mouse monoclonal antibody specific for human Ki-67 (MIB-1, 1:125 dilution; DakoCytomation, Cambridge, UK) followed by a biotin-conjugated anti-mouse secondary antibody (1:200; Vector Laboratories). The endothelial cell antigen CD34 was detected essentially as described for Ki-67 with the use of a rat monoclonal antibody specific for mouse CD34 (MCA1825GA, 1:50 dilution; Serotec, Oxford, UK), followed by a biotin-conjugated anti-rat secondary antibody (E0467, 1:200 dilution; DakoCytomation). The biotin-conjugated secondary antibody was detected by incubation with 3,3-diaminobenzidine (DakoCytomation), a chromogenic substrate for peroxidase that stains antigen–antibody complexes brown.
Four sections per tumor sample were stained with each primary antibody, and the numbers of active caspase-3–positive or Ki-67–positive cells in five randomly chosen 750-µm2 fields of view per section were scored with the use of an Leica BMRB upright microscope and OsteoMeasure software (OsteoMetrics Inc., Decatur, GA).
Osteoclast Histochemistry and Scoring
Osteoclasts were detected in sections of the right tibias from treated mice by toluidine blue and tartate-resistant acid phosphatase staining as previously described (41). Two 3-µm sections per tibia were scored; the numbers of osteoclasts per millimeter of cortical bone surface and the area of bone surface in contact with osteoclasts were quantified using a Leica RMRB upright microscope and OsteoMeasure software (Osteometrics Inc.) as previously described (42).
DNA Microarray Analysis of Gene Expression in Subcutaneous MDA-G3 Tumors from Treated Mice
Three tumors from mice in each of the following treatment groups were pooled and used to extract total RNA with the use of a SuperArray, ArrayGrade total RNA isolation kit (tebu-bio, Peterborough, UK) according to the standard protocol described in the manufacturer's instructions: saline control, doxorubicin (2 mg/kg body weight), zoledronic acid (100 µg/kg body weight), and doxorubicin followed 24 hours later by zoledronic acid. Tumor mRNA (2 µg) from each treatment group was used to produce biotin-labeled complementary RNA (cRNA) riboprobes with the use of a SuperArray TrueLabeling-AMP 2.0 kit (tebu-bio) and biotinylated-UTP (Perkin Elmer, Boston, MA). The cRNA riboprobes were purified with the use of a SuperArray ArrayGrade cRNA cleanup kit (tebu-bio), and riboprobe from each treatment group was hybridized separately to an Oligo GEArray Human Cell Cycle DNA microarray and to an apoptosis pathway–specific DNA microarray (OHS-020 and OHS-012, respectively; tebu-bio). Gene expression was analyzed using GEArray Expression Analysis Suite (version 2.0; SuperArray, Frederick MD [http://geasuite.superarray.com]).
Real-Time Polymerase Chain Reaction
Total RNA was extracted from subcutaneous MDA-G8 tumors with the use of Trizol reagent (Invitrogen AB, Stockholm, Sweden) according to the manufacturer's protocol. RNA was reverse transcribed using Superscript II (Invitrogen) in a total volume of 20 µL, and the resulting first-strand complementary DNA (cDNA) was used as a template for real-time quantitative polymerase chain reaction (PCR). Three randomly chosen tumors per treatment group were analyzed separately for relative mRNA expression levels of selected genes compared with the housekeeping gene glyceraldehyde-3-phosphate dehydrogenase (GAPDH; Hs99999905_m1, Applied Biosystems, Warrington, UK) using an ABI 7900 PCR System (Perkin Elmer, Applied Biosystems, Foster City, CA) and Taqman universal master mix (Applied Biosystems). Relative levels of apoptosis and cell cycle–related genes were assessed using the following Taqman gene expression assays (Applied Biosystems): caspase 2 (Hs00154240_m1), caspase 2 and RIPK1 domain–containing adaptor with death domain (CRADD) (Hs00388731_m1), tumor protein 53 (TP53) (Hs99999147_m1), B-cell CLL/lymphoma 2 (BCL-2) (Hs00236808_s1), cyclin-dependent kinase inhibitor 1A (CDKN1A) (Hs00355782_m1), cyclin-dependent kinase inhibitor 1B (CDKN1B) (Hs00153277_m1), cyclin D1 (Hs99999004_m1), cyclin D3 (Hs 01017690_g1), cell division cycle 2, G1 to S and G2 to M (CDC2) (Hs00364293_m1), cyclin-dependent kinase 7 (CDK7) (Hs00387062_m1), mitotic arrest deficient like-1 (MAD2L1) (Hs01554515_g1), and transcription factor DP1 (TFDP1) (Hs01104728).
Microcomputed Tomography Imaging
Microcomputed tomography analysis of tibiae and femurs from the left hind legs of treated mice was carried out with the use of a Skyscan 1172 X-ray–computed microtomograph (Skyscan, Aartselaar, Belgium) that was equipped with an X-ray tube (voltage, 49 kV; current, 200 µA) and a 0.5-mm aluminum filter. Pixel size was set to 4.37 µm, and scanning was initiated from the top of the proximal tibia or the distal femur. For each sample, 275 cross-sectional images were reconstructed with NRecon software (version 1.4.3, Skyscan). After reconstruction, the volume of interest was defined on the two-dimensional acquisition images using a hand-drawing tool. For measurements of trabecular bone, the volume of interest included cancellous bone and excluded the cortices. Trabecular bone volume fraction (BV/TV)—the ratio of the volume of bone present (BV) to the volume of the cancellous space (TV)—was calculated for 1 mm of the bone, starting 0.2 mm from the growth plate. For cortical bone measurement, the volume of interest was included only the cortices. Cortical volumes of the tibia and femur were calculated for 1.5 and 0.9 mm of bone, respectively. Three-dimensional modeling and analysis of the bone were performed with the use of CTAn (version 1.5.0.2) and CTvol (version 1.9.4.1) software (Skyscan).
Statistical Analysis
Statistical analysis of effects of treatments on mean bone volumes, number of osteoclasts, and percentage of cortical bone surface in contact with osteoclasts was carried out using analysis of variance followed by Dunnett's two-sided multiple comparisons test. Statistical analysis of all other data was carried out using a one-way Kruskal–Wallis (nonparametric) test followed by Dunn's multiple comparisons test. Statistical significance was defined as P less than or equal to .05. All P values are two-sided.
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Effect of Sequential Treatment with Doxorubicin and Zoledronic on the Growth of Human MDA-G8 Tumors in Immunodeficient Mice
The main aim of this study was to examine whether treatment of established subcutaneous tumors derived from human breast cancer cells with doxorubicin followed by zoledronic acid could inhibit tumor growth at doses at which the individual drugs alone could not. The breast cancer cells that we used in this study, a clone of MDA-MB-436 cells stably transfected with GFP-containing expression vector (MDA-G8 cells), were chosen because sequential treatment of this cell line in vitro with doxorubicin and zoledronic acid induced a synergistic increase in apoptotic cell death (26). Initial in vivo dose–response experiments were carried out to determine the dose of doxorubicin and of zoledronic acid that would be used in the combined and sequential treatment schedules; the doses we tested included clinically relevant doses of doxorubicin (2 mg/kg body weight via intravenous injection) and zoledronic acid (100 µg/kg body weight via intraperitoneal injection), which were administered once weekly for 6 weeks to mice bearing established subcutaneous MDA-G8 tumors (n = 8 mice per group) (Figure 1). Doses of doxorubicin and zoledronic acid that, when administered alone, had no statistically significant effect on tumor growth compared with saline were chosen for the subsequent combination experiments. Doxorubicin at the 4- and 8-mg/kg doses caused statistically significant inhibition of tumor growth compared with saline, whereas the 2-mg/kg dose did not. Zoledronic acid at the 100-µg/kg dose or the 1.2-mg/kg dose had no statistically significant effect on tumor growth compared with saline, whereas the highest dose tested (6 mg/kg) seemed, surprisingly, to stimulate tumor growth. Histological analysis revealed that tumors from mice treated with 6 mg zoledronic acid per kilogram body weight had an acellular core and were filled with liquid, which accounted for their larger size (data not shown).
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On the basis of the these results, in subsequent experiments tumor-bearing mice were treated with doxorubicin at 2 mg/kg body weight via intravenous injection (n = 8) and zoledronic acid at 100 µg/kg body weight via intraperitoneal injection (n = 8), alone or in combination according to the following treatment schedules: 1) doxorubicin and zoledronic acid together (n = 9), 2) zoledronic acid followed 24 hours later by doxorubicin (n = 9), or 3) doxorubicin followed 24 hours later by zoledronic acid (n = 9). Control mice were treated with saline (n = 9). All mice were treated once per week for 6 weeks and were sacrificed 24 hours after their final treatment. We assessed the effect of the various treatments on body weight twice per week during treatment. Analysis of body weight (assessed as a percentage of the initial body weight) showed that there was no statistically significant difference between the combination treatment groups and the control treatments (ie, saline, doxorubicin alone, or zoledronic acid alone), suggesting that the treatments were not overtly toxic for the mice (data not shown).
As shown in Figure 2, we observed moderate inhibition of subcutaneous tumor growth in mice that were treated with 2 mg/kg doxorubicin alone or with 100 µg/kg zoledronic acid alone compared to the saline control. Between days 32 and 42 following tumor cell inoculation, the rate of tumor growth did not differ among the groups that received saline, zoledronic acid alone, or doxorubicin alone (mean tumor doubling times of 6.4, 6.1, and 7.2 days, respectively), indicating that the single agents slowed tumor growth in the initial stages of tumor development, but had no effect in the later stages. Mice treated with zoledronic acid followed 24 hours later by doxorubicin displayed no statistically significant reduction in tumor size compared with mice treated with either drug alone. Mice treated with zoledronic acid and doxorubicin together had statistically significant smaller mean tumor volumes on day 42 than those treated with doxorubicin alone (122 mm3 vs 328 mm3, difference = 206 mm3, 95% CI = 78 to 335 mm3, P < .001), with zoledronic acid alone (122 mm3 vs 447 mm3, difference = 325 mm3, 95% CI = 197 to 454 mm3, P < .001), or with zoledronic acid followed 24 hours later by doxorubicin (122 mm3 vs 418 mm3, difference = 296 mm3, 95% CI = 168 to 426 mm3, P < .001). Tumor growth was almost completely abolished in mice treated with doxorubicin followed 24 hours later by zoledronic acid (mean tumor volume = 13 mm3, 95% CI = 7 to 19 mm3). The difference in mean tumor volume between mice treated with zoledronic acid and doxorubicin together and mice in each of the other treatment groups was statistically significant 32 days after tumor cell inoculation (P < .01 at day 32, P < .001 from day 35 until day 42), whereas the difference in mean tumor volume between mice treated with doxorubicin followed by zoledronic acid and each of the other treatment groups was statistically significant beginning on day 28 after tumor cell inoculation until the termination of the experiment on day 42 (P < .01 at day 28, P < .001 from day 32 until day 42). The mean tumor doubling times in the various treatment groups reflected the observed inhibition of tumor growth (6.4 days in the saline control group, 7.2 days in the doxorubicin alone group, 6.1 days in the zoledronic acid alone group, 5.6 days in the zoledronic acid followed 24 hours later by doxorubicin group, 9.6 days in the doxorubicin and zoledronic acid together group, and 253.6 days in the doxorubicin followed 24 hours later by zoledronic acid group).
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These results show that sequential treatment of tumor-bearing mice with doxorubicin followed by zoledronic acid caused a substantial decrease in subcutaneous breast tumor growth and that the order in which the drugs were administered was essential to achieve maximum growth inhibition. In addition, we found that sequential administration was superior to giving the drugs together.
Effect of Sequential Treatment with Doxorubicin and Zoledronic Acid on Tumor Cell Apoptosis and Proliferation
We next examined whether the reduction in tumor volume that we observed in mice that received the sequential treatment was due to tumor cell apoptosis by staining tumors sections with an antibody against active caspase-3, a marker of apoptosis, and scoring of the number of caspase-3–positive tumor cells per square millimeter (Figure 3, A). Tumors from mice that were sequentially treated with doxorubicin followed by zoledronic acid had a 7.5- fold increase in the number of apoptotic cells compared with tumors from the saline control group (mean number of caspase-3–positive cells per square millimeter, doxorubicin then zoledronic acid vs saline: 605.0 vs 82.19, difference = 522.8, 95% CI = 488.2 to 557.4, P < .001). Tumors from mice treated with doxorubicin followed by zoledronic acid also had statistically significantly more caspase-3–positive cells than tumors from mice treated with doxorubicin alone (605.0 vs 120.6, difference = 484.4, 95% CI = 449.8 to 519.0, P < .001), with zoledronic acid alone (605.0 vs 98.44, difference = 506.6, 95% CI = 472.0 to 541.2, P<.001), or with zoledronic acid followed by doxorubicin (605.0 vs 103.1, difference = 501.9, 95% CI = 467.3 to 536.5, P <. 001) (Figure 3, A). In addition, tumors from mice treated simultaneously with doxorubicin and zoledronic acid had statistically significantly more caspase-3–positive cells per square millimeter than tumors from mice treated with doxorubicin alone (232.5 vs 120.6, difference = 111.9, 95% CI = 77.3 to 146.5, P = .004) or zoledronic acid alone (232.5 vs 98.4, difference = 134.1, 9% CI = 99.4 to 168.7, P = .003). However, the mean number of caspase-3–positive cells per square millimeter in tumors from mice treated with doxorubicin and zoledronic acid simultaneously was 2.5-fold lower than that in tumors from mice treated with doxorubicin followed by zoledronic acid (232.5 vs 605.0, difference = 372.5, 95% CI = 337.9 to 407.1, P = .008). Treatment with the single agents alone did not induce statistically significant levels of apoptosis compared with saline (Figure 3, A).
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We also assessed the effects of the various treatments on tumor cell proliferation by analyzing the number of Ki-67–positive cells in the tumors from each treatment group (Figure 3, B). Weekly administration of either doxorubicin or zoledronic acid alone for 6 weeks did not cause a statistically significant decrease in the number of Ki-67–positive cells in MDA-G8 tumors compared with saline treatment (mean number of Ki-67–positive tumors cells per square millimeter for doxorubicin alone, zoledronic acid alone, and saline: 221.9 [95% CI = 209.3 to 234.5], 208.8 [95% CI = 196.6 to 220.9], and 231.4 [95% CI = 215.2 to 247.6], respectively). In addition, treatment with zoledronic acid followed by doxorubicin did not affect tumor cell proliferation compared with saline control (247.0 [95% CI = 229.3 to 264.7] vs 231.4 [95% CI = 215.2 to 247.6] Ki-67–positive tumor cells per square millimeter). However, tumors from mice that were sequentially treated with doxorubicin then zoledronic acid had fewer Ki-67–positive cells than tumors of mice treated with saline (mean number of Ki-67–positive cells per square millimeter: 17.9 vs 221.9, difference = 213.4, 95% CI = 185.6 to 241.3, P < .001) or with the two drugs together (mean number of Ki-67–positive cells per square millimeter: 17.9 vs 92.5, difference = 74.5, 95% CI = 46.7 to 102.4, P = .003). These data indicate that administration of zoledronic acid 24 hours after doxorubicin caused substantial inhibition of tumor cell proliferation in subcutaneous MDA-G8 tumors compared with all other treatment schedules tested.
To elucidate the potential mechanism by which the sequential treatment inhibited tumor growth, we used pathway-specific gene arrays to examine alterations in the expression of 224 key genes associated with cell proliferation and apoptosis in tumors from mice treated with saline, with 2 mg/kg doxorubicin, with 100 µg/kg zoledronic acid, or with doxorubicin followed 24 hour later by zoledronic acid. We identified a set of 30 genes that showed at least a twofold change in expression in tumors treated with doxorubicin followed by zoledronic acid compared with any of the controls (saline or the single agents). These genes were initially clustered into groups with the use of GEAsuite software; Pathway Architect software (Stratagene, Amsterdam, The Netherlands) was subsequently used to generate gene maps that linked the genes to specific pathways (data not shown). Genes that clustered into groups and showed potential direct interactions on the gene maps were further analyzed by real-time quantitative PCR to confirm the changes in gene expression that were identified by the arrays. Expression of the following genes was increased by at least twofold in tumors from mice treated with doxorubicin followed by zoledronic acid compared with tumors from mice treated with single drug alone (doxorubicin or zoledronic acid) or control (saline): caspase 2, cyclin-dependent kinase inhibitor 1A (CDKN1A), cyclin-dependent kinase inhibitor 1B (CDKN1B), caspase 2 and RIPK1 domain containing adaptor with death domain (CRADD), and tumor protein P53 (TP53). Expression of the genes for B-cell CLL/lymphoma 2 (Bcl2), cyclin D1 (CCND1), cyclin D3 (CCND3), cell division cycle 2, G1 to S and G2 to M (CDC2), cyclin-dependent kinase 7 (CDK7), mitotic arrest deficient like-1 (MAD2L1), and transcription factor DP1 (TFDP1) was decreased by at least twofold (data not shown). Our data show that sequential treatment with doxorubicin followed by zoledronic acid induced differential alterations in tumor gene expression compared with that caused by the single agents, which possibly contributed to its enhanced antitumor effect.
Effect of Sequential Treatment with Doxorubicin Followed by Zoledronic Acid on Tumor Neoangiogenesis
Because both zoledronic acid and doxorubicin have been reported to affect tumor angiogenesis (24), we next examined whether inhibition of tumor growth following sequential treatment was associated with a reduction in the vascularization level of the tumors. Tumors from mice in the five treatment groups were subjected to immunohistochemistry with an antibody that detects CD34, a marker of endothelial cells in blood vessels. Tumors from mice treated with saline displayed a high degree of vascularization, which was clearly visible following detection of CD34 expression (Figure 4, A). Tumors from mice treated with doxorubicin alone (Figure 4, B) or with zoledronic acid alone (Figure 4, C) displayed no obvious differences in the degree of vascularization compared with the saline control. By contrast, tumors from mice treated simultaneously with doxorubicin and zoledronic acid (Figure 4, D) displayed a marked reduction in the integrity of the tumor vasculature compared with those from saline-treated control mice or those from mice treated with either of the single agents; they had few clearly defined vessels and many dispersed endothelial cells. Tumors from mice treated with zoledronic acid followed by doxorubicin (Figure 4, E) had no obvious change in tumor vascularization compared with the saline control. By contrast, tumors from mice treated with doxorubicin followed by zoledronic acid (Figure 4, F) exhibited very low levels of vascularization compared with all other groups; these tumors appeared to contain few vessels and displayed a low number of CD34-positive endothelial cells. These observations suggest that doxorubicin and zoledronic acid do not affect blood vessel formation when administered individually and at low concentrations to mice whereas combination treatment with doxorubicin followed 24 hours later by zoledronic acid results in a substantial reduction of new tumor blood vessel formation. The inhibition of tumor growth observed in the mice treated either simultaneously or sequentially with doxorubicin and zoledronic acid may have been caused in part by reduced vascularization and is consistent with the antiangiogenic effects of high-dose zoledronic acid monotherapy reported by Giraudo et al. (24).
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Effect of Doxorubicin and Zoledronic Acid on Bone
The majority of reports of the antitumor effects of zoledronic acid have focused on the effects of this drug either on bone itself or on tumors growing within the bone microenvironment. The substantial inhibition of subcutaneous tumor growth shown in this study suggests that the combined therapies also have a direct effect on tumors at extraosseous sites. Histological evaluation of sections of bones from tumor-bearing mice showed no evidence of bone involvement by tumor cells (data not shown). We carried out a comprehensive analysis of bone in mice from all treatment groups to examine whether the treatments affected bone structure and/or integrity and to confirm that the antiresorptive effect of zoledronic acid was not affected by doxorubicin treatment. The trabecular and cortical bone volumes were determined because they are structurally distinct areas of bone. Next, the number of osteoclasts per square millimeter of cortical bone and the percentage of bone surface in contact with osteoclasts were determined to allow us to distinguish between changes in the number of bone-resorbing cells and in the area of bone in contact with these.
The antiresorptive capacity of zoledronic acid is well established, and the dose used in this study (100 µg/kg body weight once per week) did reduce osteoclast activity. As shown in Table 1, weekly treatment with zoledronic acid for 6 weeks, either alone or in combination or sequence with doxorubicin, resulted in a statistically significant decrease in percentage of cortical bone surface that was in contact with osteoclasts compared with saline (mean percentage of cortical bone surface in contact with osteoclasts: zoledronic acid vs saline, 2.1 vs 8.3, difference = 6.2, 95% CI = 4.0 to 8.4, P < .001; zoledronic acid and doxorubicin vs saline, 1.7 vs 8.3, difference = 6.6, 95% CI = 4.3 to 8.8, P < .001; zoledronic acid then doxorubicin vs saline, 2.2 vs 8.3, difference = 6.1, 95% CI = 3.9 to 8.3, P < .001; doxorubicin then zoledronic acid vs saline, 2.1 vs 8.3, difference = 6.1, 95% CI = 3.9 to 8.4, P < .001). Treatment with doxorubicin alone had no statistically significant effect on the number of osteoclasts or on the surface area of cortical bone in contact with osteoclasts compared with the saline control.
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Mice treated with zoledronic acid, either alone or in sequence (or combination) with doxorubicin, had greater mean trabecular bone volume (both in the tibia [data not shown] and in the femur) than mice treated with saline (for the femur, zoledronic acid alone vs saline: 65.7 mm3 vs 19.7 mm3, difference = 46.0 mm3, 95% CI = 33.1 to 58.9 mm3, P < .001; zoledronic acid with doxorubicin vs saline: 66.2 mm3 vs 19.7 mm3, difference = 46.6 mm3, 95% CI = 33.7 to 59.4 mm3, P < .001; zoledronic acid then doxorubicin vs saline: 55.8 mm3 vs 19.7 mm3, difference = 36.1 mm3, 95% CI = 23.2 to 49.0 mm3, P < .001; doxorubicin then zoledronic acid vs saline: 57.1 mm3 vs 19.7 mm3, difference = 37.4 mm3, 95% CI = 24.5 to 50.3 mm3, P < .001) (Table 1). There were no statistically significant effects on bone structure or integrity in the mice treated with zoledronic acid in combination or sequence with doxorubicin, compared to zoledronic acid alone, showing that doxorubicin treatment did not affect the antiresorptive capacity of zoledronic acid (Table 1).
Microcomputed tomography analysis of tibiae confirmed that there was no evidence of tumor-induced bone loss in any of the treatment groups (Figure 5). Zoledronic acid, either alone or in combination or sequence with doxorubicin, caused a statistically significant increase in both trabecular and cortical bone volume compared with saline control, whereas treatment with doxorubicin alone did not.
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In this study, we provide evidence supporting the potential ability of zoledronic acid to elicit antitumor effects outside bone. Several groups (43–45) have previously reported anticancer effects of bisphosphonates in mouse models of tumor-induced bone disease. However, when these drugs were used to treat tumors associated with extensive bone destruction, their growth inhibitory effects were attributed to their ability to inhibit osteoclast activity, which resulted in decreases in bone resorption and in the subsequent release of growth factors from the bone matrix (43–45). This study was carried out using a cell line that does not metastasize readily to bone, which allowed us to study the effects of bisphosphonates in an animal model of peripheral breast tumors that had no direct bone involvement. Direct anticancer effects of bisphosphonates have been demonstrated in vitro. In vivo, however, the bioavailability of bisphosphonates in soft tissue is poor. In clinical practice, administration of zoledronic acid (4 mg in 100 mL over 15 minutes) results in a peak systemic concentration of 1–2 µM, and the drug is rapidly cleared from the circulation within 1–2 hours (46). It is therefore likely that peripheral tumors are exposed to low concentrations of zoledronic acid for only a few hours. The zoledronic acid dose of 100 µg/kg that we used in this study is equivalent to the human 4-mg dose after correction for the higher molecular weight of the research-grade form of the drug (ie, dinatrium hydrate). In addition, we treated mice weekly (rather than monthly) and found no reduction in the volume of subcutaneous MDA-G8 tumors in mice that were treated with zoledronic acid alone compared with saline treatment. Zoledronic acid had no effect on the levels of tumor cell apoptosis or proliferation; nor did it reduce tumor vascularization compared with that in tumors from saline-treated mice. We conclude that treating mice in this model with a dose of zoledronic acid equivalent to the one used in the clinical treatment of cancer-induced bone disease had no effect on tumors at peripheral sites.
We next investigated the potential antitumor effects of zoledronic acid combined with the commonly used chemotherapy agent doxorubicin. Doxorubicin has well-characterized properties as an anticancer agent (35–37). Consistent with previously published data (47), we found that treatment of mice with doxorubicin resulted in an apoptosis-independent (no evidence of increased levels of active caspase-3 in the tumors [Figure 3, A]), dose-dependent decrease in tumor burden. Cancer patients are often treated with a combination of therapies, and in some cases combining some drugs synergistically increases their anticancer effects [reviewed by Miles et al. (48)]. Indeed, evidence from in vitro and in vivo models indicates that bisphosphonates have synergistic antitumor activity when used in combination with cytotoxic drugs, targeted molecular therapies, and radiotherapy [reviewed by Santini et al. (29)]. The demonstration of these synergistic effects suggests that low serum concentrations of bisphosphonates in vivo can be sufficient to exert antitumor effects in peripheral tissues if bisphosphonates are combined with other drugs. Doxorubicin is used routinely in the treatment of breast cancer patients, and patients with late-stage breast cancer that has metastasized to the bone may be treated with this drug along with zoledronic acid. Studies in our laboratory have shown that doxorubicin and zoledronic acid can synergistically increase apoptosis in (26) and reduce the invasiveness of (49) breast cancer cell lines in vitro depending on the order in which they are applied to the cells. The increased level of apoptosis was not accompanied by a corresponding increase in the level of unprenylated Rap1a in vitro (P. D. Ottewell, D. V. Lefley, I. Holen, unpublished observations), suggesting that pretreatment with doxorubicin does not increase the ability of subsequently administered zoledronic acid to inhibit protein prenylation. Therefore, we speculate that the mechanism of action underlying the antitumor effect caused by sequential treatment with doxorubicin followed by zoledronic acid may not be due to the accumulation of unprenylated proteins, but this remains to be established.
Here we have shown in an in vivo mouse model that doses of doxorubicin and zoledronic acid that had no major effects on tumor growth when administered individually reduced tumor burden when doxorubicin was administered 24 hours before zoledronic acid but not when the drugs were administered in the reverse order. This finding suggests that an initial "priming" of the cells by doxorubicin renders them more sensitive to subsequent exposure to zoledronic acid. In addition, although simultaneous administration of doxorubicin and zoledronic acid reduced tumor growth, increased tumor cell apoptosis, and decreased tumor cell proliferation and vascularization compared with saline, these effects were statistically significantly more pronounced when the drugs were administered 24 hours apart. These data support the notion that the effects of doxorubicin on the tumor cells manifest themselves over a few hours. Furthermore, the reduction in tumor growth seen following sequential treatment with doxorubicin followed by zoledronic acid appears to be a result of multiple antitumor effects exerted by these drugs, including increased tumor cell apoptosis, reduced tumor cell proliferation, and reduced tumor angiogenesis. These results, taken together with the previously published in vitro data (26), strongly support the hypothesis that doxorubicin and zoledronic acid exert statistically significantly increased antitumor effects when given in sequence.
The mechanism of action of the sequential doxorubicin and zoledronic acid treatment remains to be identified. In vitro studies have shown that the synergistic induction of tumor cell apoptosis can be completely abolished by adding geranylgeraniol (GGOH), an intermediate of the mevalonate pathway downstream of farnesyl diphosphate synthase, the enzyme that is targeted by zoledronic acid, at the same time as zoledronic acid (26). These data indicate that, at least in vitro, the increased levels of tumor cell apoptosis caused by the combined treatments depend on inhibition of prenylation of one or more molecular targets. However, coadministration of GGOH is not feasible in in vivo model systems, which prevents a direct confirmation that the effects of sequential treatment are due to disruption of prenylation caused by zoledronic acid. Other potential mechanisms of action may also be responsible for the observed effects on tumor size. Zoledronic acid is a highly charged hydrophilic molecule that does not readily cross the plasma cell membrane. It reaches pharmacologically active concentrations only in cells that exhibit marked fluid-phase endocytosis, such as osteoclasts and macrophages (50). One hypothesis that we are currently testing to explain the increased antitumor effect observed with sequential doxorubicin and zoledronic acid treatment is that zoledronic acid uptake by tumor cells is enhanced by prior exposure of the cells to doxorubicin. In support of this hypothesis, we have found that pretreatment of breast cancer cells with 10 nM doxorubicin for 24 hours in vitro increases subsequent uptake of 7-amino-actinomycin D, a fluorescent DNA stain, suggesting that doxorubicin treatment may facilitate uptake of subsequently administered compounds, including zoledronic acid (P. D. Ottewell, D. V. Lefley, I. Holen, unpublished observations). In addition, we established that tumor expression of several genes associated with cell cycle progression and apoptosis was altered following sequential treatment of mice with doxorubicin and zoledronic acid compared with expression in tumors from mice given the single agents. Although these data are preliminary, they support the hypothesis that administration of the drugs in a particular treatment schedule initiates specific changes in gene expression in subcutaneous tumors. This is an intriguing observation because no other studies have, to our knowledge, identified similar effects on gene expression in tumors not associated with bone following treatment with zoledronic acid.
Following in vivo administration, zoledronic acid binds rapidly to bone, causing inhibition of osteoclast function and bone resorption (51). As expected, we found that administration of 100 µg zoledronic acid per kilogram body weight once per week for 6 weeks resulted in increases in the thickness of cortical and trabecular bone and decreases in the number of osteoclasts and in the surface area of bone in contact with osteoclasts. Although the dose of zoledronic acid used was able to inhibit bone resorption, it was unable to reduce peripheral tumor growth, suggesting that the reduced release of bone-associated growth factors and cytokines caused by zoledronic acid alone is not sufficient to reduce the growth of tumors outside the bone microenvironment.
We also investigated whether mice bearing subcutaneous MDA-G8 tumors had developed secondary tumors in bone. Histological assessment of bones from mice in all treatment groups showed that there were no tumor cells present in any of the bones, confirming that sequential administration with doxorubicin then zoledronic acid exhibits direct antitumor effects in soft tissue.
Our study has several limitations. First, although the treatment schedule we used consisted of clinically achievable doses of both doxorubicin (2 mg/kg body weight, equivalent to 6.5 mg/m2 in a patient) and zoledronic acid (100 µg/kg body weight, equivalent to a 4-mg clinical dose), the mice were treated weekly for 6 weeks. By contrast, breast cancer patients are treated with a standard infusion of 4 mg zoledronic acid once every 3–4 weeks and one cycle of doxorubicin every 3 weeks. Thus, the mice received higher total doses of the drugs than those that breast cancer patients receive. Second, because zoledronic acid is rapidly cleared from extraskeletal sites following administration, it is unclear how doses of this drug that are sufficient to elicit the observed combined effect reach peripheral tumors. Finally, the precise molecular mechanism of action underlying the observed antitumor effects, as well as the relevance of this mouse study for human breast cancer, remains to be established.
To our knowledge, this is the first report to show that doxorubicin and zoledronic acid inhibit subcutaneous breast tumor growth in vivo in a treatment sequence–specific fashion and in the absence of tumor-induced bone disease. Our data suggest that there may be benefits of combining zoledronic acid with cytotoxic agents for the treatment of patients with early-stage breast cancer.
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Funding |
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H.M. is supported by the Academy of Finland, Finnish Cultural Foundation, and Saastamoinen Foundation. M.J. is supported by the Medical Research Council, UK. The study was sponsored by a grant from Breast Cancer Campaign, UK (to I.H.). The study sponsors had no role in the design of the study; the collection, analysis, or interpretation of the data; the writing of the manuscript; or the decision to submit the manuscript for publication.
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NOTES |
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We wish to thank Professor N. J. Brown (University of Sheffield, UK) who holds the Home Office project license and Dr Jonathan Green (Novartis Pharma, Switzerland) for the kind gift of zoledronic acid.
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Manuscript received December 17, 2008; revised June 2, 2008; accepted June 16, 2008.
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