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Experimental Therapeutics |
Division of Endocrinology and Metabolism, Department of Medicine, The University of Texas Health Science Center at San Antonio, San Antonio, Texas 78229-3900
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ABSTRACT |
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 Top ABSTRACT INTRODUCTIONMATERIALS AND METHODSRESULTSDISCUSSIONREFERENCES |
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INTRODUCTION |
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TopABSTRACTINTRODUCTIONMATERIALS AND METHODSRESULTSDISCUSSIONREFERENCES |
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On the other hand, it is unclear why tumor burden in bone is decreased by BPs. Recent in vitro data have shown that BPs inhibit the attachment of breast and prostate cancer cells to bone matrix (9 , 10) and enhance apoptosis in myeloma cells (11, 12, 13) and breast cancer cells (14 , 15) . Moreover, a clinical study has shown that the BP clodronate suppresses distant metastases in both bone and visceral organs in breast cancer patients (16) . These results suggest that BPs may have some anticancer effects, which in turn reduce metastatic tumor burden in bone. However, the effects of BP on cancer cells colonized in bone are yet to be elucidated.
In the present study, to further elucidate the mechanism by which BP inhibits bone metastases, we examined the effects of the BP ibandronate on MDA-231 human breast cancer cells metastasized in bone, using a well-characterized animal model of bone metastasis. Our results show that ibandronate inhibits bone metastases of MDA-231 breast cancer by promoting apoptosis not only in osteoclasts but also metastatic MDA-231 cells.
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MATERIALS AND METHODS |
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TopABSTRACTINTRODUCTIONMATERIALS AND METHODSRESULTSDISCUSSIONREFERENCES |
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Animal Model
Animals.
Four-week-old female BALB/c-nu/nu mice (Harlan Industries, Houston, TX) were used for the experiments. Mice were kept in laminar flow isolators in our animal facilities.
Intracardiac Inoculation.
Intracardiac injection was performed as described previously (8
, 18)
. Subconfluent MDA-231 cells were refed with fresh medium 24 h before injection. Cells (1 x 105) suspended in 0.1 ml of PBS were injected with a 27-gauge needle into the left cardiac ventricles of mice under anesthesia (pentobarbital, 0.05 mg/g of body weight).
Orthotopic Inoculation.
MDA-231 cells (5 x 106) were prepared in a manner similar to the intracardiac injection, suspended in 0.1 ml of PBS, and injected into the mammary fat pads of mice under anesthesia (pentobarbital, 0.05 mg/g of body weight).
BP
The BP ibandronate [1-hydroxy-3-(methylpentylamino)propylidine bisphosphonate; BM 21.0955; Ref. 19
], which was kindly provided by Boehringer Mannheim GmbH (Mannheim, Germany), was used after dilution in PBS.
Experimental Protocol
The administration protocol for ibandronate is depicted in Fig. 1
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In orthotopic tumor formation experiments, MDA-231 cells were inoculated in the mammary fat pad on day 0 and ibandronate (4 µg/mouse) was administrated s.c. daily from day 7 to day 28. Control mice received PBS. The experiments were carried out twice.
Radiographic Measurement of Osteolytic Lesion Area
Areas of osteolytic lesions were determined on radiographs as described previously (8)
. Animals were anesthetized deeply with pentobarbital (0.05 mg/g of body weight), laid in prone position against the films (22 x 27 cm; X-OMAT AR; Eastman Kodak Co., Rochester, NY), and exposed to an X-ray at 35 kV for 6 s using a Faxitron radiographic inspection unit (43855A; Faxitron X-ray Corporation, Buffalo Grove, IL). Films were developed using a RP X-OMAT processor (M6b; Eastman Kodak Co.). Radiographs were analyzed extensively and carefully by three different individuals who had no prior knowledge of the experimental protocol. The areas of osteolytic bone metastases, which were observed as demarcated radiolucent lesions in the hindlimbs, were quantitatively assessed using a computer-assisted JAVA image analysis system (Jandel Scientific, Corte Madera, CA). Changes in size of osteolytic lesions were analyzed by comparing the radiographs of each individual mouse taken at day 21 with those taken at day 28. Data are shown as the percentage of increase in osteolytic lesion: (size of osteolytic lesion at day 28 - size of osteolytic lesion at day 21)/size of osteolytic lesion at day 21.
Histological and Histochemical Examination
Mice anesthetized with pentobarbital (0.05 mg/g of body weight) were fixed by perfusion first with saline and then with 4% paraformaldehyde buffered with 0.1 M phosphate buffer (pH 7.4) through the left cardiac ventricle as described previously (18)
. In bone metastasis experiments, the femora and tibiae were dissected, immersed in the same fixative overnight, and decalcified in 4.13% EDTA at room temperature for 1 week. In orthotopic tumor formation experiments, tumors in mammary fat pads were dissected, weighed, and immersed in the same fixative overnight. Paraffin sections were made following conventional methods and stained with H&E. Histochemical examination for TRAP was also performed as described previously (18)
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Histomorphometrical Examination
Tumor Burden.
We performed histomorphometrical analysis of tumor burden in the metastatic tumors in the distal femoral and proximal tibial metaphyses of both hindlimbs, using longitudinal sections stained with H&E (x200 magnification) and the OsteoMeasure System (Osteometrics, Atlanta, GA). To determine the tumor area, five fields were randomly selected in each section. Data are shown as tumor area (mm2)/total area of the field (mm2).
Osteoclast Number.
The number of TRAP-positive multinucleated osteoclasts at the interface between tumor and bone was counted in five fields of each section (x400 magnification) and expressed per millimeter of this interface distance as described previously (8)
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Apoptosis in Osteoclasts.
Apoptotic osteoclasts were identified by the presence of chromatin condensation and/or nuclear fragmentation in osteoclasts showing cytoplasmic contraction and loss of adhesion to the underlying bones, using TRAP-stained sections (7)
. The number of apoptotic osteoclasts at the tumor-bone interface was counted in five fields of each section (x400 magnification), and data are shown as the number of apoptotic osteoclasts/total number of osteoclasts counted.
Apoptosis and Mitosis of MDA-231 Cells.
Apoptosis in MDA-231 cells was determined with TUNEL-stained sections (x400 magnification). TUNEL staining was performed with the ApopTag Plus In Situ Apoptosis Detection Kit (Oncor, Inc., Gaithersburg, MD) according to the manufacturer’s protocol. Mitotic cells were quantified using H&E-stained sections at x400 magnification. To determine the number of apoptotic and mitotic cells, we randomly selected and counted five fields of nonnecrotic areas of metastatic tumors in bone or mammary tumors in each specimen. Data are expressed as number of cells/mm2 tumor area.
In Vitro Analysis of Apoptosis
DNA Fragmentation Assay.
MDA-231 cells (5 x 105) were cultured in 10-cm plates for 24 h and treated with 1, 10, or 100 µM ibandronate for an additional 72 h. Intranucleosomal DNA fragmentation was detected by agarose gel electrophoresis according to a method described previously (20)
. In some experiments, DNA fragmentation of MDA-231 cells treated with ibandronate in the presence or absence of a caspase inhibitor, Z-VAD-FMK (50 µM; Promega Co., Madison, WI), was also examined.
Caspase Assay.
MDA-231 cells (2 x 105) were cultured in 6-well plates for 24 h and treated with 100 µM ibandronate for an additional 72 h in the presence or absence of 50 µM Z-VAD-FMK. Caspase-3 activity was measured by proteolytic cleavage of the caspase-3 substrate Ac-DEVD-pNA, using the CaspACE Assay System (Promega) according to manufacturer’s instruction.
Statistical Analysis
All data were analyzed by Student’s t test or Welch’s t test for unpaired samples. All data were presented as the mean ± SE.
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RESULTS |
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TopABSTRACTINTRODUCTIONMATERIALS AND METHODSRESULTSDISCUSSIONREFERENCES |
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. These osteolytic lesions markedly enlarged at 7 days in the untreated group (Fig. 2A-b)
. In contrast, treatment with ibandronate profoundly impaired the enlargement of these osteolytic lesions (Fig. 2A-d)
. Quantitative radiological assessment showed that ibandronate significantly inhibited the progression of the osteolytic bone metastases (Fig. 2B)
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. In contrast, in the ibandronate-treated bone, colonization of metastatic MDA-231 breast cancer cells in the bone marrow cavity was markedly decreased and the majority of trabecular bones remained undestroyed (Fig. 3A-b)
. Consistent with these histological observations, histomorphometrical analysis demonstrated that ibandronate significantly decreased the metastatic MDA-231 tumor burden in bone (Fig. 3B)
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. Nuclear chromatin condensation and/or fragmentation of the nucleus were also observed in these cancer cells. These histological findings are characteristic features of apoptotic cancer cells (21)
, which were clearly distinguishable from mitotic cancer cells (Fig. 4A)
. To quantify the number of apoptotic cancer cells in bone metastases, we used the TUNEL intranucleosomal DNA fragment end-labeling technique. TUNEL-positive cells were clearly identified as brown-stained cells (Fig. 4B)
. We found that ibandronate significantly increased the number of apoptotic MDA-231 breast cancer cells in bone metastases (Fig. 4C-a)
. However, mitosis in metastatic MDA-231 breast cancer cells was not affected by ibandronate (Fig. 4C-b)
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and mitosis (Fig. 5b)
in MDA-231 breast cancer cells in these tumors between the untreated and the ibandronate-treated group.
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Effects of Ibandronate on Apoptosis in MDA-231 Cells in Vitro.
To determine the effects of ibandronate on the apoptosis in MDA-231 cells in vitro, we assessed intranucleosomal DNA fragmentation using agarose gel electrophoresis. Ibandronate at 1 and 10 µM had no effects on MDA-231 cell apoptosis, whereas ibandronate at 100 µM induced evident apoptosis in MDA-231 cells (Fig. 6A)
. The DNA fragmentation induced by ibandronate was almost completely inhibited by the caspase inhibitor Z-VAD-FMK (Fig. 6B)
. To examine the mechanism of apoptosis induced by ibandronate, we measured caspase-3 activity (Fig. 6C)
. Ibandronate (100 µM) significantly increased caspase-3 activity in MDA-231 cells, and the caspase inhibitor Z-VAD-FMK reduced this increase to a level similar to that of the control (Fig. 6C)
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. In contrast, the number of osteoclasts was dramatically decreased in ibandronate-treated bone (Fig. 7A-b)
. Consistent with these histochemical observations, histomorphometrical analysis demonstrated a marked decrease in osteoclast number in ibandronate-treated bone (Fig. 7B)
. Furthermore, ibandronate also increased the number of apoptotic osteoclasts in bone metastases. Apoptotic TRAP-positive osteoclasts were readily recognizable by their unique morphological appearance. They were detached from bone surfaces and exhibited typical nuclear chromatin condensation (Fig. 8A)
. Quantitative analysis demonstrated that there was a marked increase in the number of apoptotic osteoclasts in ibandronate-treated bone as measured by the percentage of apoptosis at the tumor bone interface (Fig. 8B)
. These results are consistent with our previous results (7
, 8)
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DISCUSSION |
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TopABSTRACTINTRODUCTIONMATERIALS AND METHODSRESULTSDISCUSSIONREFERENCES |
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Whether the enhancement of MDA-231 cell apoptosis in bone following ibandronate treatment in bone is caused by direct anticancer actions on MDA-231 breast cancer cells or indirect mechanisms associated with the uniqueness of the bone microenvironment needs to be elucidated. Bone is a storehouse of varieties of growth factors, such as insulin-like growth factor and transforming growth factor ß (23) . These growth factors are released into the bone microenvironment in active forms as a consequence of osteoclastic bone resorption (24) . BP probably limits the supply of these bone-derived growth factors that facilitate the proliferation and survival of breast cancer cells colonizing bone through inhibition of osteoclastic bone resorption. This growth factor-deficient condition may, in turn, cause increased apoptosis in these bone-colonizing breast cancer cells. In support of this notion, Kostenuik et al. (25) have demonstrated that stimulation of bone resorption increases the growth of metastatic Walker 256 tumor in bone. Moreover, we recently observed that the conditioned medium of resorbed bone promoted the proliferation of MDA-231 breast cancer cells in culture in a preliminary experiment. Of note, the conditioned medium of ibandronate-treated bones in which bone resorption was inhibited failed to stimulate the proliferation of MDA-231 breast cancer cells in the same experiment.5 When these results are taken together, it seems likely that ibandronate, at least in part, promotes apoptosis in metastatic MDA-231 breast cancer cells in bone through inhibition of osteoclastic bone resorption.
Several previous reports have shown that BPs induce apoptosis in cancer cells through inhibition of the mevalonate pathway (13) , reduction of bcl-2 expression (14) , and activation of caspase (14 , 15) . Our in vitro data showed that ibandronate (100 µM) elevated caspase-3 activity and induced apoptosis in MDA-231 breast cancer cells as determined by DNA fragmentation. These effects of ibandronate were reversed in the presence of the caspase inhibitor Z-VAD-FMK. Thus, ibandronate likely enhances apoptosis in MDA-231 breast cancer cells, at least in part, through an activation of caspase-3. Our results are consistent with those obtained in previous studies (9, 10, 11, 12, 13, 14, 15) and support the notion that BPs have direct anticancer actions in vitro. However, our in vivo data demonstrated that ibandronate reduced MDA-231 tumor burden in bone but failed to inhibit MDA-231 tumor formation in the mammary fat pad, suggesting that the anticancer effects of ibandronate may be bone specific. Furthermore, contrary to the results reported by Diel et al. (16) , more recent studies showed that the bisphosphonate clodronate had no effects on visceral organ metastases in breast cancer patients (26 , 27) . Thus, whether BPs possess direct anticancer effects in vivo is still an open question, and further study is needed.
Recent studies have shown that BPs promote apoptosis in osteoclasts (28 , 29) and cancer cells (13) through the same mechanism that involves mevalonate pathway. However, it should be noted that the concentrations required to promote apoptosis in MDA-231 breast cancer cells in vitro are much higher than those required in osteoclasts. We previously have shown that BPs, including pamidronate, clodronate, and risedronate, at 1 µM markedly increased apoptosis in osteoclasts (7) . On the other hand, we show here that ibandronate at 1 and 10 µM fails to induce apoptosis in MDA-231 breast cancer cells and that apoptosis is observed only when MDA-231 cells are treated with 100 µM ibandronate. Consistent with our results, other groups have also reported that relatively high concentrations of BPs (>10 µM) are required to promote apoptosis in tumor cells (11, 12, 13, 14, 15) . Thus, it is likely that higher concentrations of BP are necessary to promote apoptosis in cancer cells than in osteoclasts in bone metastases. Regarding the concentration of BP in bone, the authors of one study suggested that the concentration of the BP alendronate reaches as high as 800 µM at the osteoclast-bone interface (30) , whereas the authors of another study reported that an estimated concentration of the BP incadronate is as low as 0.7 µg/g (<1 µM) in the metastatic tumor nests in bone (31) . If the former is the case, it is possible that BPs directly promote apoptosis in tumor cells in bone. On the other hand, if the latter is the case, BPs seem likely to promote apoptosis in tumor cells in bone indirectly through inhibition of osteoclastic bone resorption. Whichever is the case, it is suggested that BP concentrations in bone need to be maintained at sufficiently high levels to enhance apoptosis in metastatic cancer cells.
In conclusion, our results suggest that BPs inhibit bone metastasis of breast cancer by promoting apoptosis in both osteoclasts and metastatic breast cancer cells. These two cellular mechanisms may be cumulatively responsible for the suppression of osteolytic bone metastases by BPs in breast cancer.
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FOOTNOTES |
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1 This work was supported by NIH Grants PO1-CA40035, RO1-AR28149, and RO1-DK45229. 
2 Present address: Department of Biochemistry, Osaka University Faculty of Dentistry, 1-8 Yamadaoka, Suita, Osaka 565-0871, Japan.
3 To whom requests for reprints should be addressed, at Division of Endocrinology and Metabolism, Department of Medicine, The University of Texas Health Science Center at San Antonio, Mail Code 7877, 7703 Floyd Curl Drive, San Antonio, TX 78229-3900. Phone: (210) 567-4901; Fax: (210) 567-6693; E-mail: yoneda{at}uthscsa.edu
4 The abbreviations used are: BP, bisphosphonate; TRAP, tartrate-resistant acid phosphatase; TUNEL, terminal deoxynucleotidyl transferase-mediated dUTP nick end labeling; Z-VAD-FMK, z-Val-Ala-Asp-fluoromethyl ketone; Ac-DEVD-pNA, N-acetyl-Asp-Glu-Val-Asp-p-nitroaniline.
5 T. Yoneda, A. Myoui, A. Sasaki, P. J. Williams, N. Hashimoto, G. R. Mundy, manuscript in preparation.
Received 3/27/00. Accepted 3/27/01.
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