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Use of bisphosphonates for the treatment of bone metastasis in experimental animal models
C ANCER TREATMENT REVIEWS 1999; 25: 293–299 Ar ticle No. ctr v. 1999.0133, available online at http://www.idealibrar y.com on
LABORATORY–CLINIC INTERFACE
Use of bisphosphonates for the treatment of bone metastasis in experimental animal models T. Yoneda*†,T. Michigami*‡, B. Yi*, P. J.Williams*, M. Niewolna*, T. Hiraga* *Division of Endocrinology and Metabolism, Department of Medicine,The University of Texas Health Science Center at San Antonio, San Antonio,Texas, USA; †Department of Biochemistry, Osaka University Faculty of Dentistry, Suita, Osaka 565, Japan Therapeutic effectiveness of bisphosphonates (BP) on bone metastases in patients with cancers including those of the breast and prostate has been well documented. However, there are still many important questions that remain unsolved or controversial. To obtain answers for these questions that are not readily addressed in a well-controlled manner in clinical studies, we have developed two animal models of bone metastasis (orthotopic and experimental). Using these models, we studied the effects of BP alone or in combination with anti-cancer agents on the metastasis of breast cancer to bone and visceral organs. In addition, we also determined the effects of BP on osteosclerotic metastases. We found that BP impaired the progression of bone metastases primarily through enhancing apoptosis in osteoclasts and breast cancer cells colonized in bone. In some situations, however, BP alone increased metastases in visceral organs including liver and adrenal glands. However, combination of BP with anti-cancer agents enhanced the suppression of tumour in both bone and visceral organs, leading to prolonged survival of tumour-bearing animals. Of potential importance, preventative administration of BP inhibited the development of eventual osteosclerotic bone metastases.These results suggest that BP exhibits diverse beneficial effects on osteolytic and osteoblastic bone metastasis and non-bone organ metastasis in breast cancer when administered appropriately. They also suggest that the animal models of bone metastasis described here allow us to produce clinicallyrelevant information that is useful for the design of optimal regimens of BP for the treatment of breast cancer patients with bone and visceral metastases. © 1999 Harcourt Publishers Ltd Key words: Osteolytic bone metastasis; osteoclasts; bisphosphonates; breast cancer; prostate cancer; osteosclerotic bone metastasis.
Large bodies of clinical evidence have been accumulated demonstrating that bisphosphonates (BP) effectively suppress bone metastasis and its complications, particularly bone pain, in patients with breast cancer (1, 2), prostate cancer (3) and multiple myeloma (4–7). Because of its characteristic chemical structure, BP selectively accumulates in bone (8). Corresponding author: Toshiyuki Yoneda, Division of Endocrinology and Metabolism, Department of Medicine, UTHSCSA, 7703 Floyd Curl Drive, San Antonio, Texas 78284–7877, USA. †Current address: Research Institute, Osaka Medical Center for Maternal and Child Health, Izunci, Osaka, Japan. 0305-7372/99/050293 + 07 $12.00/0
BP then specifically inhibits osteoclasts through yetunclear mechanisms (8). These properties of BP provide the theoretical basis for the effectiveness of BP in the treatment of bone metastasis in which osteoclasts play a key role (9–12). On the other hand, effects of BP in vivo on metastatic cancer cells in the development of bone metastasis remain unclear, although several recent in-vitro studies suggest that BP promotes apoptosis in certain cancer cells (13–15). Thus, determination of the effects of BP on cancer cells colonizing bone is critical to increase our understanding of the mode of BP action and, more importantly, therapeutic efficacy of BP for the treatment of bone metastasis. © 1999 HARCOURT PUBLISHERS LTD
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Cancer patients who develop bone metastases, in almost all cases, also have metastases in visceral organs which are the primary direct cause of death (16–18). Accordingly, it is important to determine the effects of BP on metastases in visceral organs. In this context, it should be noted that clinical studies reported to date show that BP fails to increase the survival of cancer patients, despite the fact that BP markedly suppresses bone metastases (1, 19, 20). These results indicate that suppression of bone metastasis does not improve the survival of cancer patients and suggest that BP has little effects on visceral organ metastases. On the other hand, a recent study described by Diel et al., (18) reported that the BP clodronate in combination with conventional anti-cancer therapies enhanced inhibition of visceral organ metastases and prolonged the survival compared with anti-cancer therapy alone in breast cancer patients. The data suggest that clodronate may possess direct anti-cancer and/or adjuvant effects on metastases in non-bone organs. However, a subsequent analogous clinical study showed clodronate did not suppress visceral organ metastases nor prolong the survival in breast cancer patients (21). Thus, the effects of BP on visceral organ metastasis are still controversial and need to be elucidated. In most cases of prostate cancer (3, 22) and often breast cancer (23), osteosclerotic bone metastases develop. Although very little is known about the mechanisms underlying osteosclerotic bone metastasis, there has been a long-standing notion that the precedence of bone resorption is essential for the subsequent development of osteosclerotic bone metastases (24, 25). It has been proposed that bone resorption releases growth factors and calcium stored in bone, which in turn facilitates osteoblasts to proliferate and mineralize. Consistent with this notion, it is frequently observed that blood or urinary levels of biochemical markers of bone resorption are elevated during the advancement of osteosclerotic bone metastases in prostate cancer patients (26). BP, therefore, could inhibit the development of osteosclerotic bone metastases through inhibiting preceding bone resorption. However, this intriguing possibility has not been extensively explored yet. Here, we describe the results of the studies in which we addressed these issues using two animal models of bone metastasis.
ANIMAL MODELS OF BONE METASTASIS We have developed two animal models of bone metastasis of human and mouse breast cancer in our laboratory over the last several years.
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Experimental bone metastasis model (heart injection model) Characteristics of this model have been described in detail elsewhere (27, 28). In this model, cancer cells are directly introduced into the arterial circulation through the left ventricle of the heart in young mice. Thus, this model represents an experimental bone metastasis model. The most notable feature of this model is that the development of metastases is preferentially observed in bone through yet-unknown mechanisms. Metastasis to non-bone organs including adrenal glands, ovary and brain is rare and few metastases are detected in lung or liver at a histological level. As such, this model is suitable specifically to study the events involved in bone metastasis. Another feature of this model is that the capacity of human cancers including breast, prostate and neuroblastoma to develop bone metastases can be studied by using nude mice. In most of our studies, we used a human oestrogen-independent breast cancer cell line MDA-MB-231 (MDA-231). These cancer cells develop radiologically distinctive osteolytic bone metastases that histologically demonstrate numerous osteoclasts and aggressive tumour colonization in bone 3 to 4 weeks after cell inoculation.
Spontaneous (orthotopic) bone metastasis model of mouse mammary tumour 4T1 Experimental metastasis models such as the heart injection model described above lack critical early steps occurring between tumour growth at the primary site and entry into the circulation (intravasation). Moreover, the heart injection model described above rarely forms visceral organ metastases, which many cancer patients already have developed at the time of detection of bone metastases. Thus, the results of the experiments in which the effectiveness of a novel therapeutic approach is examined in experimental bone metastasis models do not allow us confidently to extrapolate the data to cancer patients with bone and visceral organ metastases. To overcome these problems, we have established an orthotopic metastasis model of mouse mammary tumour called 4T1 (29, 30). This mammary tumour cells form tumour 7 to 10 days after subcutaneous inoculation into the orthotopic mammary fat pad and subsequently develop bone and visceral organ metastases 3 to 5 weeks after inoculation in female mice. From technical points of view, this model produces bone metastases in 100% of animals, whereas the heart injection model does not because of its dependence on technical skill. Another advantageous feature is
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Figure 1 Apoptosis in osteoclast and metastatic cancer cells in bone in ibandronate-treated mice in the heart injection model. A tartrateresistant acid phosphatase (TRAP)-positive osteoclast (thick arrow) exhibits representative apoptosis with nuclear condensation and is detached from bone surface. In addition, metastatic MDA-231 human breast cancer cells (thin arrow) also show histologically representative apoptotic morphology.
that this model allows us to use immunocompetent syngeneic female mice (Balb/c). Overall, this model more closely represents the situation occurring in the metastasis in breast cancer patients than heart injection model, except that 4T1 tumour is not of human origin.
EFFECTS OF BP ON BREAST CANCER CELLS COLONIZED IN BONE We studied the effects of the BP ibandronate on bone metastasis of MDA-231 human breast cancer using the heart injection model. Ibandronate was given after osteolytic bone metastases were established. Progression of osteolytic bone metastases was monitored weekly by radiographs. Bones with radiologically-evident lesions were processed for histological and histomorphometrical examination. Radiographical examination showed that ibandronate significantly inhibited the progression of established bone metastases, as we have previously reported using risedronate (31). Histomorphometrical analysis of these osteolytic lesions demonstrated that ibandronate decreased osteoclast number and metastatic tumour burden in bone. Moreover, histological examination revealed that ibandronate markedly increased apoptosis in osteoclasts (Figure 1), which is consistent with our previous in-vivo observation (32). Induction of apoptosis in osteoclasts in culture by BP through inhibition of
the mevalonate pathway which is critical for the prenylation of small GTP proteins such as Ras, Rho and Rab has been reported (33, 34). Subsequently, we also determined the effects of ibandronate on apoptosis and mitosis in metastatic MDA-231 breast cancer cells in bone. We found that apoptosis in these cancer cells was also increased by the treatment with ibandronate, whereas mitosis was not changed, suggesting that increased apoptosis is not due to the cytotoxic effect of ibandronate. To determine whether increased apoptosis in cancer cells colonized in bone is specific for bone, the effect of ibandronate on apoptosis in MDA-231 tumour formed at the mammary fat pad was next examined. Our data showed that ibandronate did not increase apoptosis in MDA-231 tumour in the mammary fat pad. The data suggest that ibandronate enhances apoptosis in cancer cells specifically in bone. The result also appears to suggest that ibandronate does not have direct anti-cancer effect. It seems likely that apoptotic effects of ibandronate are attributable to the restriction of bone-derived growth factor supply by inhibiting bone resorption. There could be, however, an alternative interpretation of this result. Since BP predominantly accumulates in bone after systemic administration, the local concentration of ibandronate in the mammary fat pad was most unlikely to be sufficient to increase apoptosis in cancer cells. In support of this interpretation, it has been shown that considerably higher concentrations (>10–4 M) of BP are required to induce apoptosis in some cancer
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cells in culture (13,15). More recently, BP at high concentrations has been found to induce apoptosis through inhibiting the mevalonate pathway in myeloma cells in culture (14). Thus, whether BP increases apoptosis in metastatic breast cancer cells in bone through direct anti-cancer action or not is still an open question.
EFFECTS OF BP ON VISCERAL ORGAN METASTASIS Heart injection model of bone metastasis Single Use of BP MDA-231 human breast cancer cells occasionally spread to adrenal glands after intracardiac inoculation. By repeated passages in the metastases in adrenal glands, we established a subclone that selectively and consistently metastasizes to both bone and adrenal glands. We then stably transfected the reporter gene luciferase into this subclone (MDA231F9AD/Luc). Using this subclone, we determined the effects of ibandronate on metastases in bone and adrenal glands. In the first set of experiments, ibandronate was administered in a preventative manner in which mice received daily subcutaneous injections of ibandronate from the time of intracardiac inoculation of cells to the end of experiments. Radiographical and histological examination demonstrated that ibandronate administered according to this protocol profoundly decreased osteolytic bone metastases. In contrast, metastasis to adrenal glands determined by luciferase activity was significantly increased in ibandronate-treated animals. Similar experimental observations have been reported using risedronate (31), pamidronate (35), and alendronate (36). The mechanism of increased soft organ metastasis by BP administration is not known. It is possible that this observation is specific for experimental bone metastasis model, which does not simulate the situation in cancer patients. In the second set of experiments, ibandronate was administered in a therapeutic fashion in which mice received daily subcutaneous injections of ibandronate after bone metastases were established until the end of experiments. Thus, in these experiments, the total amount of ibandronate was less and the period of administration was shorter than the preventative experiments. Our results demonstrate that ibandronate significantly impaired the progression of bone metastases. Of note, ibandronate administered according to this protocol did not increase adrenal metastases. It is, therefore, probable that
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administration of BP subsequent to the detection of bone metastases does not cause an increase in soft organ metastases in breast cancer patients.
Combined Use of BP with anti-cancer agent In the experiments described above, mice were treated with only BP and no anti-cancer agents. On the other hand, breast cancer patients with bone metastases are primarily treated with anti-cancer agents and given BP as an adjuvant therapy only when necessary. In fact, most clinical studies have been performed using BP in combination with conventional anti-cancer therapies (1,18,20). We therefore determined the effects of ibandronate combined with an anti-cancer agent doxorubicin. We found that combined treatment with doxorubicin and ibandronate in a preventative manner suppressed both bone and adrenal metastases. Doxorubicin alone moderately decreased adrenal metastases but failed to inhibit bone metastases in these experiments. Thus, combination of BP and anti-cancer agents probably produces synergistic effects on both bone and soft organ metastases.
Orthotopic model of bone metastasis As described above, 4T1 mammary tumour cells begin to show tumour formation at the orthotopic site 1 week after cell inoculation and develop both bone and visceral organ metastases 3 to 4 weeks after the inoculation. Thus, this model is most suitable to determine the therapeutic effects of BP and also compare the effects of BP on metastases between bone and non-bone organs. To facilitate quantitative analysis, 4T1 cells were stably transfected with the reporter gene luciferase (4T1/Luc). In addition, metastases in bone and non-bone organs were analysed by histology and histomorphometry. Since osteolytic bone metastases of 4T1 mouse mammary tumour are not as distinctive on radiographs as those of MDA-231 human breast cancer, weekly radiological monitoring was not conducted and radiographs were taken only at the end of experiments.
Single use of BP We tested the newest and most potent BP zoledronate in this model. Zoledronate was administered in a therapeutic manner in which single bolus intravenous injection from tail vein was given when tumour formation at the orthotopic inoculation site became visible 7 to 10 days after cell inoculation. Similar administration protocol of BP to this has been most frequently used for breast cancer patients with
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bone metastases. Histomorphometrical and histological examination showed that zoledronate suppressed bone metastases in a dose-dependent fashion. Moreover, zoledronate decreased osteoclast number, increased apoptosis in osteoclasts and metastatic cancer cells and diminished metastatic tumour burden in bone. Metastases in lung and liver determined by luciferase activity were not increased by single bolus iv injection of zoledronate.
Combined use of BP with anti-cancer agent We tested incadronate or zoledronate in combination with an anti-cancer agent UFT. UFT is an oral anticancer agent consisting of tegafur, a prodrug of fluorouracil, and uracil (with a fluorouracil degradation-inhibitory effect) (37) and has been shown to have therapeutic effects in breast cancer patients (38). In these experiments, incadronate or zoledronate was administered by single bolus iv injection and UFT was given orally once a day from 7 days after tumour inoculation to the end of experiments. Incadronate or zoledronate combined with UFT inhibited not only bone metastases but also lung or liver metastases in an additive fashion. More importantly, combined treatment with BP and UFT increased the survival of tumour-bearing animals. These results are consistent with those of a previous clinical study reported by Diel et al. (18). UFT alone marginally but significantly suppressed tumour formation at the orthotopic inoculation site and moderately decreased lung and liver metastases. UFT alone also inhibited bone metastases. In summary, our results suggest that BP may influence visceral organ metastases of breast cancer in some situations. However, our experimental results suggest that as long as BP is administered according to currently used therapeutic manners including single bolus i.v. injection and in combination with anti-cancer therapies, it is most unlikely that BP affects visceral organ metastases in breast cancer patients.
EFFECTS OF BP ON OSTEOSCLEROTIC BONE METASTASIS To study the effects of BP on osteosclerotic bone metastasis, prostate cancer is obviously the most appropriate model. Unfortunately, however, there is currently no prostate cancer that consistently develops osteosclerotic bone metastases in experimental animals. This is a major reason why very little information is available on the effects of BP on osteosclerotic bone metastases at the present time. We have
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recently observed that intracardiac inoculation of human estrogen-dependent breast cancer cells MCF7 over-expressing the Neu/HER/ErbB-2 oncogene develop osteosclerotic bone metastases 8 to 10 weeks after the inoculation. It has been clinically recognized that approximately 30% of breast cancer patients manifest predominant osteosclerotic bone metastases (23). Close histological examination as a function of time revealed that MCF-7/Neu cells formed osteosclerotic bone metastases following osteolysis which takes place around 4 to 6 weeks of the inoculation. The observation is consistent with the longstanding notion that bone resorption is a prerequisite for the consequent development of osteosclerotic bone metastases. Using this newly-established model of osteosclerotic bone metastasis of breast cancer, we examined the effects of the BP ibandronate. One group of mice received daily subcutaneous injections of ibandronate from 7 days before cell inoculation to 4 weeks after the inoculation to inhibit initial osteolysis, left untreated thereafter and killed 10 weeks after inoculation (early treatment). Another group of mice received ibandronate from 6 to 10 weeks after cell inoculation during which period osteosclerosis predominantly takes place and killed at week 10 (late treatment). Our data showed that early treatment inhibited the development of osteosclerotic bone metastases, whereas late treatment failed to inhibit them. These results suggest that bone resorption is necessary for the eventual development of osteosclerotic bone metastases and that inhibition of initial osteoclastic bone resorption by the administration of ibandronate inhibits the following development of osteosclerotic bone metastases. They also suggest that BP may have therapeutic effects on osteosclerotic bone metastases in prostate cancer when administered at appropriate stage of the advancement of the disease.
CONCLUDING REMARKS BP has been successfully used for the treatment of cancer patients with bone metastases for almost three decades. Nonetheless, a number of important questions still remain unsolved. These questions are not readily addressed in a well-controlled manner in cancer patients with bone metastases. In this article, the authors have introduced two different types (experimental and orthotopic) of animal models of bone metastasis and shown that these animal models are useful to obtain relevant information for these yet-unanswered clinical questions. Figure 2 summarizes the results described here. We expect that optimal utilization of these animal models produces
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Cancer Cell Visceral Organs
Mevalonate Pathway
BP
Apoptosis Osteoclast
BP Osteoblasts
Mevalonate Pathway
BP Bone • Growth Factors (TGFb, IGFs) • Calcium
Figure 2 BP effects on osteoclasts and metastatic cancer cells in bone. BP preferentially accumulates in bone, directly enters osteoclast and inhibits mevalonate pathway that is critical to the prenylation of small GTP proteins such as Ras, Rho and Rab, leading to induction of apoptosis and inhibition of bone resorption. Due to an inhibition of osteoclastic bone resorption, supply of bone-derived growth factors such as TGFβ and IGFs to metastatic cancer cells is largely restricted, which in turn cause apoptosis in these cancer cells. Moreover, BP released into the marrow cavity as a consequence of osteoclastic bone resorption might directly induce apoptosis in metastatic cancer cells through inhibiting mevalonate pathway, although this is yet to be proved. In case of osteosclerotic metastasis of prostate cancer, inhibition of bone resorption by BP may limit the supply of bone-stored growth factors and calcium that are necessary for osteoblasts to proliferate and mineralize. Effects of BP on visceral organ metastasis are still unclear. Dotted lines indicate inhibition.
further information regarding the issues such as mode of BP action on bone and non-bone organ metastases, most efficacious protocol of BP administration to cancer patients with bone and visceral organ metastases and effectiveness of novel therapeutic approaches combined with BP for the treatment of bone metastasis. Finally, these study models should provide us with the opportunity to determine the cellular and molecular mechanisms underlying the predilection of breast and prostate cancer, neuroblastoma and myeloma for colonizing skeletons.
ACKNOWLEDGEMENT The authors thank Roche, Novartis, and Taiho Pharmaceutical Company for providing us with ibandronate, zoledronate and UFT, respectively. The authors also thank Miss Mie Masuda for her secretarial assistance. This work was supported by NIH grants PO1CA40035, RO1-AR28149 and RO1-DK45229.
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EFFECTS OF BISPHOSPHONATES ON BONE METASTASIS 8. Sato M, Grasser W, Endo N, Akins R, Simmons H, Thompson DD, Golub E, Rodan GA. Bisphosphonate action. Alendronate localization in rat bone and effects on osteoclast ultrastructure. J Clin Invest 1991; 88: 2095–2105. 9. Mundy GR, Yoneda T. Facilitation and suppression of bone metastasis. Clin Orthop 1995; 312: 34–44. 10. Mundy GR, Yoneda T. Bisphosphonates as anticancer drug. N Engl J Med 1998; 339: 357–363. 11. Yoneda T. Mechanisms of preferential metastasis of breast cancer to bone. Int J Oncol. 1996; 9: 103–109. 12. Yoneda T. Cellular and molecular mechanisms of breast and prostate cancer metastasis to bone. Eur J Cancer 1998; 34: 240–245. 13. Shipman CM, Rogers MJ, Apperley JF, Russell RGG, Croucher P I. Bisphosphonate induce apoptosis in human myeloma cell lines: a novel anti-tumour activity. Br J Haematol 1997; 98: 665–672. 14. 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 1998; 58: 5294–5297. 15. Aparicio A, Gardner A, Tu Y, Savage A, Berenson J, Lichtenstein A. In vitro cytoreductive effects on multiple myeloma cells induced by bisphosphonates. Leukemia 1998; 12: 220–229. 16. Coleman RE, Rubens RD. The clinical course of bone metastases from breast cancer. Br J Cancer 1987; 55: 61–66. 17. Koenders PG, Beex LVAM, Langens R, Kloppenborg PWC, Smals AGH, Benraad ThJ, Breast cancer study group. Breast Cancer Res Treat 1991; 18: 27–32. 18. Diel IJ, Solomayer E-F, 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 1998; 339: 357–363. 19. Body JJ, Bartl R, Burckhardt P, Delmas PD, Diel IJ, Fleish H, Kanis JA, Kyle RA, Mundy GR, Paterson A H G Rubens RD. Current use of bisphosphonates in oncology. J Clin Oncol 1998; 16: 3890–3899. 20. Theriault RL, Hortobagyi GN, Leff R, Gluck S, Stewart JF, Costello S, Kennedy I, Simeone J, Seaman JJ, Knight RD, Mellars K, Heffernan M, Reitsman DJ. Pamidronate reduces skeletal morbidity in women with advanced breast cancer and lytic bone lesions: A randomized, placebo-controlled trial. J Clin Oncol 1999; 17: 846–854. 21. McCloskey EV, Powles T, Paterson AHG, Ashley S, Kanis JA. Clodronate reduces incidence of skeletal metastases in women with primary breast cancer. Bone 1998; 23 (Suppl): S189 22. Milch RA, Changus GW. Response of bone to tumor invasion. Cancer 1956; 9: 340–351. 23. Kamby C, Andersen J, Ejlertsen B, Birkler NE, Rytter L, Zedeler K, Thorpe SM, Norgaard T, Rose C. Histological grade and steroid receptor content of primary breast cancer – impact on prognosis and possible modes of action. Br J Cancer 1988; 58: 480–486.
299 24. Urwin GH, Percival RC, Harris S, Beneton MNC, Williams JL, Kanis SA. Generalized increase in bone resorption in carcinoma of the prostate. Eur J Urol 1985; 57: 721–723. 25. Clarke NW, McClure J, George NJR. Morphometric evidence for bone resorption and replacement in prostate cancer. Br J Urol 1991; 68: 74–80. 26. Pecherstorfer M, Ludwig H, Zimmer-Roth H, Schiling T, Woitge HW, Schmidt H, Baumgartner G, Thiebaud D, Ludwig H, Seibel MJ. The diagnostic value of urinary pyridinium cross-links of collagen, alkaline phosphatase and urinary calcium excretion in neoplastic bone disease. J Clin Endocrinol Metab 1995; 121: 542–548. 27. Yoneda T, Sasaki A, Mundy GR. Osteolytic bone disease in breast cancer. Breast Cancer Res Treat 1994; 32: 73–84. 28. Yoneda T. Arterial microvascularization and breast cancer colonization in bone. Histol Histopathol 1997; 12: 1145–1149. 29. Aslakson CJ, Miller FR. Selective events in the metastatic process defined by analysis of the sequential dissemination of subpopulations of a mouse mammary tumor. Cancer Res 1992; 52: 1399–1405. 30. Lelekakis M, Moseley JM, Martin JM, Hards D, Williams E, Ho P, Lowen D, Javni J, Miller FR, Slavin J, Anderson RL. A novel orthotopic model of breast cancer metastasis to bone. Clin Exp Metastasis 1999; 17: 163–170. 31. Sasaki A, Boyce BF, Story B, Wright KR, Chapman M, Boyce R, Mundy GR, Yoneda T. Bisphosphonate risedronate reduces metastatic human breast cancer burden in bone in nude mice. Cancer Res 1995; 55: 3551–3557. 32. 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 1995; 10: 1478–1487. 33. Fisher JE, Rogers MJ, Halasy JM, Luckman SP, Hughes DE, Masarachia PJ, Wesolowski G, Russel RGG, Rodan GA, Reszka AA. Alendronate mechanism of action: geranylgeraniol, an intermediate in the mevalonate pathway, prevents inhibition of osteoclast formation, bone resorption, and kinase activation in vitro. Proc Natl Acad Sci USA 1999; 96: 133–138. 34. Luckman SP, Hughes DE, Coxon FP, Russell RGG, Rogers MJ. Nitrogen-containing bisphosphonates inhibit the mevalonate pathway and prevent post-transitional prenylation of GTPbinding proteins, including Ras. J Bone Miner. Res 1998; 13: 581–589. 35. Kostenuik PJ, Orr FW, Suyama K, Singh G. Increased growth rate and tumor burden of spontaneously metastatic Walker 256 cancer cells in the skeleton of bisphosphonates-treated rats. Cancer Res 1993; 53: 5452–5457. 36. Stearns ME, Wang M. Effects of alendronate and taxol on PC3ML cell bone metastases in SCID mice. Invasion Metastasis 1996; 16: 116–131. 37. Takiuchi H, Ajani JA. Uracil-tegafur in gastric carcinoma: A comprehensive review. J Clin Oncol 1998; 16: 2877–2885. 38. Tashiro H, Nomura Y, Ohsaki A. A double blind comparative study of tegafur (FT) and UFT (a comibantion of tegafur and yracil) in advanced breast cancer. Jpn J Clin Oncol 1994; 24: 212–217.
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Use of bisphosphonates for the treatment of bone metastasis in experimental animal models T. Yoneda*†,T. Michigami*‡, B. Yi*, P. J.Williams*, M. Niewolna*, T. Hiraga* *Division of Endocrinology and Metabolism, Department of Medicine,The University of Texas Health Science Center at San Antonio, San Antonio,Texas, USA; †Department of Biochemistry, Osaka University Faculty of Dentistry, Suita, Osaka 565, Japan Therapeutic effectiveness of bisphosphonates (BP) on bone metastases in patients with cancers including those of the breast and prostate has been well documented. However, there are still many important questions that remain unsolved or controversial. To obtain answers for these questions that are not readily addressed in a well-controlled manner in clinical studies, we have developed two animal models of bone metastasis (orthotopic and experimental). Using these models, we studied the effects of BP alone or in combination with anti-cancer agents on the metastasis of breast cancer to bone and visceral organs. In addition, we also determined the effects of BP on osteosclerotic metastases. We found that BP impaired the progression of bone metastases primarily through enhancing apoptosis in osteoclasts and breast cancer cells colonized in bone. In some situations, however, BP alone increased metastases in visceral organs including liver and adrenal glands. However, combination of BP with anti-cancer agents enhanced the suppression of tumour in both bone and visceral organs, leading to prolonged survival of tumour-bearing animals. Of potential importance, preventative administration of BP inhibited the development of eventual osteosclerotic bone metastases.These results suggest that BP exhibits diverse beneficial effects on osteolytic and osteoblastic bone metastasis and non-bone organ metastasis in breast cancer when administered appropriately. They also suggest that the animal models of bone metastasis described here allow us to produce clinicallyrelevant information that is useful for the design of optimal regimens of BP for the treatment of breast cancer patients with bone and visceral metastases. © 1999 Harcourt Publishers Ltd Key words: Osteolytic bone metastasis; osteoclasts; bisphosphonates; breast cancer; prostate cancer; osteosclerotic bone metastasis.
Large bodies of clinical evidence have been accumulated demonstrating that bisphosphonates (BP) effectively suppress bone metastasis and its complications, particularly bone pain, in patients with breast cancer (1, 2), prostate cancer (3) and multiple myeloma (4–7). Because of its characteristic chemical structure, BP selectively accumulates in bone (8). Corresponding author: Toshiyuki Yoneda, Division of Endocrinology and Metabolism, Department of Medicine, UTHSCSA, 7703 Floyd Curl Drive, San Antonio, Texas 78284–7877, USA. †Current address: Research Institute, Osaka Medical Center for Maternal and Child Health, Izunci, Osaka, Japan. 0305-7372/99/050293 + 07 $12.00/0
BP then specifically inhibits osteoclasts through yetunclear mechanisms (8). These properties of BP provide the theoretical basis for the effectiveness of BP in the treatment of bone metastasis in which osteoclasts play a key role (9–12). On the other hand, effects of BP in vivo on metastatic cancer cells in the development of bone metastasis remain unclear, although several recent in-vitro studies suggest that BP promotes apoptosis in certain cancer cells (13–15). Thus, determination of the effects of BP on cancer cells colonizing bone is critical to increase our understanding of the mode of BP action and, more importantly, therapeutic efficacy of BP for the treatment of bone metastasis. © 1999 HARCOURT PUBLISHERS LTD
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Cancer patients who develop bone metastases, in almost all cases, also have metastases in visceral organs which are the primary direct cause of death (16–18). Accordingly, it is important to determine the effects of BP on metastases in visceral organs. In this context, it should be noted that clinical studies reported to date show that BP fails to increase the survival of cancer patients, despite the fact that BP markedly suppresses bone metastases (1, 19, 20). These results indicate that suppression of bone metastasis does not improve the survival of cancer patients and suggest that BP has little effects on visceral organ metastases. On the other hand, a recent study described by Diel et al., (18) reported that the BP clodronate in combination with conventional anti-cancer therapies enhanced inhibition of visceral organ metastases and prolonged the survival compared with anti-cancer therapy alone in breast cancer patients. The data suggest that clodronate may possess direct anti-cancer and/or adjuvant effects on metastases in non-bone organs. However, a subsequent analogous clinical study showed clodronate did not suppress visceral organ metastases nor prolong the survival in breast cancer patients (21). Thus, the effects of BP on visceral organ metastasis are still controversial and need to be elucidated. In most cases of prostate cancer (3, 22) and often breast cancer (23), osteosclerotic bone metastases develop. Although very little is known about the mechanisms underlying osteosclerotic bone metastasis, there has been a long-standing notion that the precedence of bone resorption is essential for the subsequent development of osteosclerotic bone metastases (24, 25). It has been proposed that bone resorption releases growth factors and calcium stored in bone, which in turn facilitates osteoblasts to proliferate and mineralize. Consistent with this notion, it is frequently observed that blood or urinary levels of biochemical markers of bone resorption are elevated during the advancement of osteosclerotic bone metastases in prostate cancer patients (26). BP, therefore, could inhibit the development of osteosclerotic bone metastases through inhibiting preceding bone resorption. However, this intriguing possibility has not been extensively explored yet. Here, we describe the results of the studies in which we addressed these issues using two animal models of bone metastasis.
ANIMAL MODELS OF BONE METASTASIS We have developed two animal models of bone metastasis of human and mouse breast cancer in our laboratory over the last several years.
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Experimental bone metastasis model (heart injection model) Characteristics of this model have been described in detail elsewhere (27, 28). In this model, cancer cells are directly introduced into the arterial circulation through the left ventricle of the heart in young mice. Thus, this model represents an experimental bone metastasis model. The most notable feature of this model is that the development of metastases is preferentially observed in bone through yet-unknown mechanisms. Metastasis to non-bone organs including adrenal glands, ovary and brain is rare and few metastases are detected in lung or liver at a histological level. As such, this model is suitable specifically to study the events involved in bone metastasis. Another feature of this model is that the capacity of human cancers including breast, prostate and neuroblastoma to develop bone metastases can be studied by using nude mice. In most of our studies, we used a human oestrogen-independent breast cancer cell line MDA-MB-231 (MDA-231). These cancer cells develop radiologically distinctive osteolytic bone metastases that histologically demonstrate numerous osteoclasts and aggressive tumour colonization in bone 3 to 4 weeks after cell inoculation.
Spontaneous (orthotopic) bone metastasis model of mouse mammary tumour 4T1 Experimental metastasis models such as the heart injection model described above lack critical early steps occurring between tumour growth at the primary site and entry into the circulation (intravasation). Moreover, the heart injection model described above rarely forms visceral organ metastases, which many cancer patients already have developed at the time of detection of bone metastases. Thus, the results of the experiments in which the effectiveness of a novel therapeutic approach is examined in experimental bone metastasis models do not allow us confidently to extrapolate the data to cancer patients with bone and visceral organ metastases. To overcome these problems, we have established an orthotopic metastasis model of mouse mammary tumour called 4T1 (29, 30). This mammary tumour cells form tumour 7 to 10 days after subcutaneous inoculation into the orthotopic mammary fat pad and subsequently develop bone and visceral organ metastases 3 to 5 weeks after inoculation in female mice. From technical points of view, this model produces bone metastases in 100% of animals, whereas the heart injection model does not because of its dependence on technical skill. Another advantageous feature is
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Figure 1 Apoptosis in osteoclast and metastatic cancer cells in bone in ibandronate-treated mice in the heart injection model. A tartrateresistant acid phosphatase (TRAP)-positive osteoclast (thick arrow) exhibits representative apoptosis with nuclear condensation and is detached from bone surface. In addition, metastatic MDA-231 human breast cancer cells (thin arrow) also show histologically representative apoptotic morphology.
that this model allows us to use immunocompetent syngeneic female mice (Balb/c). Overall, this model more closely represents the situation occurring in the metastasis in breast cancer patients than heart injection model, except that 4T1 tumour is not of human origin.
EFFECTS OF BP ON BREAST CANCER CELLS COLONIZED IN BONE We studied the effects of the BP ibandronate on bone metastasis of MDA-231 human breast cancer using the heart injection model. Ibandronate was given after osteolytic bone metastases were established. Progression of osteolytic bone metastases was monitored weekly by radiographs. Bones with radiologically-evident lesions were processed for histological and histomorphometrical examination. Radiographical examination showed that ibandronate significantly inhibited the progression of established bone metastases, as we have previously reported using risedronate (31). Histomorphometrical analysis of these osteolytic lesions demonstrated that ibandronate decreased osteoclast number and metastatic tumour burden in bone. Moreover, histological examination revealed that ibandronate markedly increased apoptosis in osteoclasts (Figure 1), which is consistent with our previous in-vivo observation (32). Induction of apoptosis in osteoclasts in culture by BP through inhibition of
the mevalonate pathway which is critical for the prenylation of small GTP proteins such as Ras, Rho and Rab has been reported (33, 34). Subsequently, we also determined the effects of ibandronate on apoptosis and mitosis in metastatic MDA-231 breast cancer cells in bone. We found that apoptosis in these cancer cells was also increased by the treatment with ibandronate, whereas mitosis was not changed, suggesting that increased apoptosis is not due to the cytotoxic effect of ibandronate. To determine whether increased apoptosis in cancer cells colonized in bone is specific for bone, the effect of ibandronate on apoptosis in MDA-231 tumour formed at the mammary fat pad was next examined. Our data showed that ibandronate did not increase apoptosis in MDA-231 tumour in the mammary fat pad. The data suggest that ibandronate enhances apoptosis in cancer cells specifically in bone. The result also appears to suggest that ibandronate does not have direct anti-cancer effect. It seems likely that apoptotic effects of ibandronate are attributable to the restriction of bone-derived growth factor supply by inhibiting bone resorption. There could be, however, an alternative interpretation of this result. Since BP predominantly accumulates in bone after systemic administration, the local concentration of ibandronate in the mammary fat pad was most unlikely to be sufficient to increase apoptosis in cancer cells. In support of this interpretation, it has been shown that considerably higher concentrations (>10–4 M) of BP are required to induce apoptosis in some cancer
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cells in culture (13,15). More recently, BP at high concentrations has been found to induce apoptosis through inhibiting the mevalonate pathway in myeloma cells in culture (14). Thus, whether BP increases apoptosis in metastatic breast cancer cells in bone through direct anti-cancer action or not is still an open question.
EFFECTS OF BP ON VISCERAL ORGAN METASTASIS Heart injection model of bone metastasis Single Use of BP MDA-231 human breast cancer cells occasionally spread to adrenal glands after intracardiac inoculation. By repeated passages in the metastases in adrenal glands, we established a subclone that selectively and consistently metastasizes to both bone and adrenal glands. We then stably transfected the reporter gene luciferase into this subclone (MDA231F9AD/Luc). Using this subclone, we determined the effects of ibandronate on metastases in bone and adrenal glands. In the first set of experiments, ibandronate was administered in a preventative manner in which mice received daily subcutaneous injections of ibandronate from the time of intracardiac inoculation of cells to the end of experiments. Radiographical and histological examination demonstrated that ibandronate administered according to this protocol profoundly decreased osteolytic bone metastases. In contrast, metastasis to adrenal glands determined by luciferase activity was significantly increased in ibandronate-treated animals. Similar experimental observations have been reported using risedronate (31), pamidronate (35), and alendronate (36). The mechanism of increased soft organ metastasis by BP administration is not known. It is possible that this observation is specific for experimental bone metastasis model, which does not simulate the situation in cancer patients. In the second set of experiments, ibandronate was administered in a therapeutic fashion in which mice received daily subcutaneous injections of ibandronate after bone metastases were established until the end of experiments. Thus, in these experiments, the total amount of ibandronate was less and the period of administration was shorter than the preventative experiments. Our results demonstrate that ibandronate significantly impaired the progression of bone metastases. Of note, ibandronate administered according to this protocol did not increase adrenal metastases. It is, therefore, probable that
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administration of BP subsequent to the detection of bone metastases does not cause an increase in soft organ metastases in breast cancer patients.
Combined Use of BP with anti-cancer agent In the experiments described above, mice were treated with only BP and no anti-cancer agents. On the other hand, breast cancer patients with bone metastases are primarily treated with anti-cancer agents and given BP as an adjuvant therapy only when necessary. In fact, most clinical studies have been performed using BP in combination with conventional anti-cancer therapies (1,18,20). We therefore determined the effects of ibandronate combined with an anti-cancer agent doxorubicin. We found that combined treatment with doxorubicin and ibandronate in a preventative manner suppressed both bone and adrenal metastases. Doxorubicin alone moderately decreased adrenal metastases but failed to inhibit bone metastases in these experiments. Thus, combination of BP and anti-cancer agents probably produces synergistic effects on both bone and soft organ metastases.
Orthotopic model of bone metastasis As described above, 4T1 mammary tumour cells begin to show tumour formation at the orthotopic site 1 week after cell inoculation and develop both bone and visceral organ metastases 3 to 4 weeks after the inoculation. Thus, this model is most suitable to determine the therapeutic effects of BP and also compare the effects of BP on metastases between bone and non-bone organs. To facilitate quantitative analysis, 4T1 cells were stably transfected with the reporter gene luciferase (4T1/Luc). In addition, metastases in bone and non-bone organs were analysed by histology and histomorphometry. Since osteolytic bone metastases of 4T1 mouse mammary tumour are not as distinctive on radiographs as those of MDA-231 human breast cancer, weekly radiological monitoring was not conducted and radiographs were taken only at the end of experiments.
Single use of BP We tested the newest and most potent BP zoledronate in this model. Zoledronate was administered in a therapeutic manner in which single bolus intravenous injection from tail vein was given when tumour formation at the orthotopic inoculation site became visible 7 to 10 days after cell inoculation. Similar administration protocol of BP to this has been most frequently used for breast cancer patients with
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bone metastases. Histomorphometrical and histological examination showed that zoledronate suppressed bone metastases in a dose-dependent fashion. Moreover, zoledronate decreased osteoclast number, increased apoptosis in osteoclasts and metastatic cancer cells and diminished metastatic tumour burden in bone. Metastases in lung and liver determined by luciferase activity were not increased by single bolus iv injection of zoledronate.
Combined use of BP with anti-cancer agent We tested incadronate or zoledronate in combination with an anti-cancer agent UFT. UFT is an oral anticancer agent consisting of tegafur, a prodrug of fluorouracil, and uracil (with a fluorouracil degradation-inhibitory effect) (37) and has been shown to have therapeutic effects in breast cancer patients (38). In these experiments, incadronate or zoledronate was administered by single bolus iv injection and UFT was given orally once a day from 7 days after tumour inoculation to the end of experiments. Incadronate or zoledronate combined with UFT inhibited not only bone metastases but also lung or liver metastases in an additive fashion. More importantly, combined treatment with BP and UFT increased the survival of tumour-bearing animals. These results are consistent with those of a previous clinical study reported by Diel et al. (18). UFT alone marginally but significantly suppressed tumour formation at the orthotopic inoculation site and moderately decreased lung and liver metastases. UFT alone also inhibited bone metastases. In summary, our results suggest that BP may influence visceral organ metastases of breast cancer in some situations. However, our experimental results suggest that as long as BP is administered according to currently used therapeutic manners including single bolus i.v. injection and in combination with anti-cancer therapies, it is most unlikely that BP affects visceral organ metastases in breast cancer patients.
EFFECTS OF BP ON OSTEOSCLEROTIC BONE METASTASIS To study the effects of BP on osteosclerotic bone metastasis, prostate cancer is obviously the most appropriate model. Unfortunately, however, there is currently no prostate cancer that consistently develops osteosclerotic bone metastases in experimental animals. This is a major reason why very little information is available on the effects of BP on osteosclerotic bone metastases at the present time. We have
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recently observed that intracardiac inoculation of human estrogen-dependent breast cancer cells MCF7 over-expressing the Neu/HER/ErbB-2 oncogene develop osteosclerotic bone metastases 8 to 10 weeks after the inoculation. It has been clinically recognized that approximately 30% of breast cancer patients manifest predominant osteosclerotic bone metastases (23). Close histological examination as a function of time revealed that MCF-7/Neu cells formed osteosclerotic bone metastases following osteolysis which takes place around 4 to 6 weeks of the inoculation. The observation is consistent with the longstanding notion that bone resorption is a prerequisite for the consequent development of osteosclerotic bone metastases. Using this newly-established model of osteosclerotic bone metastasis of breast cancer, we examined the effects of the BP ibandronate. One group of mice received daily subcutaneous injections of ibandronate from 7 days before cell inoculation to 4 weeks after the inoculation to inhibit initial osteolysis, left untreated thereafter and killed 10 weeks after inoculation (early treatment). Another group of mice received ibandronate from 6 to 10 weeks after cell inoculation during which period osteosclerosis predominantly takes place and killed at week 10 (late treatment). Our data showed that early treatment inhibited the development of osteosclerotic bone metastases, whereas late treatment failed to inhibit them. These results suggest that bone resorption is necessary for the eventual development of osteosclerotic bone metastases and that inhibition of initial osteoclastic bone resorption by the administration of ibandronate inhibits the following development of osteosclerotic bone metastases. They also suggest that BP may have therapeutic effects on osteosclerotic bone metastases in prostate cancer when administered at appropriate stage of the advancement of the disease.
CONCLUDING REMARKS BP has been successfully used for the treatment of cancer patients with bone metastases for almost three decades. Nonetheless, a number of important questions still remain unsolved. These questions are not readily addressed in a well-controlled manner in cancer patients with bone metastases. In this article, the authors have introduced two different types (experimental and orthotopic) of animal models of bone metastasis and shown that these animal models are useful to obtain relevant information for these yet-unanswered clinical questions. Figure 2 summarizes the results described here. We expect that optimal utilization of these animal models produces
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Cancer Cell Visceral Organs
Mevalonate Pathway
BP
Apoptosis Osteoclast
BP Osteoblasts
Mevalonate Pathway
BP Bone • Growth Factors (TGFb, IGFs) • Calcium
Figure 2 BP effects on osteoclasts and metastatic cancer cells in bone. BP preferentially accumulates in bone, directly enters osteoclast and inhibits mevalonate pathway that is critical to the prenylation of small GTP proteins such as Ras, Rho and Rab, leading to induction of apoptosis and inhibition of bone resorption. Due to an inhibition of osteoclastic bone resorption, supply of bone-derived growth factors such as TGFβ and IGFs to metastatic cancer cells is largely restricted, which in turn cause apoptosis in these cancer cells. Moreover, BP released into the marrow cavity as a consequence of osteoclastic bone resorption might directly induce apoptosis in metastatic cancer cells through inhibiting mevalonate pathway, although this is yet to be proved. In case of osteosclerotic metastasis of prostate cancer, inhibition of bone resorption by BP may limit the supply of bone-stored growth factors and calcium that are necessary for osteoblasts to proliferate and mineralize. Effects of BP on visceral organ metastasis are still unclear. Dotted lines indicate inhibition.
further information regarding the issues such as mode of BP action on bone and non-bone organ metastases, most efficacious protocol of BP administration to cancer patients with bone and visceral organ metastases and effectiveness of novel therapeutic approaches combined with BP for the treatment of bone metastasis. Finally, these study models should provide us with the opportunity to determine the cellular and molecular mechanisms underlying the predilection of breast and prostate cancer, neuroblastoma and myeloma for colonizing skeletons.
ACKNOWLEDGEMENT The authors thank Roche, Novartis, and Taiho Pharmaceutical Company for providing us with ibandronate, zoledronate and UFT, respectively. The authors also thank Miss Mie Masuda for her secretarial assistance. This work was supported by NIH grants PO1CA40035, RO1-AR28149 and RO1-DK45229.
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