Combined Therapy with a New Bisphosphonate, Minodronate

5380 Vol. 9, 5380–5385, November 1, 2003 Clinical Cancer Research

Combined Therapy with a New Bisphosphonate, Minodronate (YM529), and Chemotherapy for Multiple Organ Metastases of Small Cell Lung Cancer Cells in Severe Combined Immunodeficient Mice

Seiji Yano,1,2 Helong Zhang,1 Masaki Hanibuchi, visceral (lung and liver) metastasis. However, neither Toyokazu Miki, Hisatsugu Goto, Hisanori Uehara, YM529 alone nor VP-16 alone significantly prolonged the and Saburo Sone survival of SBC-5-bearing mice. Combined use of YM529 and VP-16 further inhibited the production of bone metas- Departments of Internal Medicine and Molecular Therapeutics [S. Y., tasis and significantly prolonged survival. H. Z., M. H., T. M., H. G., S. S.] and Environmental and Molecular Pathology [H. U.], Course of Bioregulation and Medical Oncology, Conclusions: Combined therapy with bisphosphonate University of Tokushima School of Medicine, Tokushima, Japan and chemotherapy may be useful for small cell lung cancer patients with multiple organ metastases including bone metastasis. ABSTRACT Purpose: Lung cancer in the advanced stage frequently INTRODUCTION metastasizes to multiple organs, including the liver, lungs, Lung cancer is the most common cause of cancer deaths in lymph nodes, and bone. Bisphosphonates have been widely the world. Lung cancer frequently metastasizes to the systemic used to treat osteolytic bone metastasis in the past years; lymph nodes and distant organs, including the liver, lung, kid- however, many studies have implicated that a single use of ney, and bone, and Ͼ90% of deaths from lung cancer can be bisphosphonates could not prolong the survival of patients. attributed to metastases (1). Therefore, metastasis is a critical In the present study, using a multiple-organ metastasis problem in the treatment of lung cancer patients. More than model of human lung cancer cells, we examined the effect of one-third of patients with advanced lung cancer manifest osteo- combined therapy with a new bisphosphonate (YM529) and lytic bone metastases that can cause bone pain, hypercalcemia, etoposide (VP-16). nerve compression syndromes, and even fractures (2). These Experimental Design: Human small cell lung cancer events might be the main causes of the decrease in quality of life (SBC-5) cells i.v. inoculated into natural killer cell-depleted in lung cancer patients with bone metastases. Therefore, the severe combined immunodeficient mice metastasized to mul- prevention and treatment of osteolytic bone metastases are clin- tiple organs, including the lungs, liver, kidneys, lymph ically important. nodes, and bone. SBC-5-bearing mice were treated with Bisphosphonates are potent inhibitors of osteoclastic bone YM529 and/or VP-16 and sacrificed 5 weeks after tumor cell resorption and have been widely used in the treatment of hy- inoculation. Bone metastasis was assessed by X-ray photo- percalcemia (3). In addition, several bisphosphonate products, graphs, and visceral metastasis was evaluated macroscopi- for example, clodronate (4), etidronate (5), alendronate (6), cally. The number of osteoclasts in the bone lesions was ibandronate (7), pamidronate (8), and zoledronic acid (9), have examined by tartrate-resistant acid phosphatase staining. been developed and used in the treatment of cancer patients with Results: Monotherapy with YM529 suppressed the pro- bone metastases. These bisphosphonates, however, could im- duction of bone metastases, but not visceral metastasis. His- prove the quality of life but could not improve the survival of tological analyses revealed that the number of osteoclasts in advanced cancer patients with bone metastases (10). bone lesions was lower in YM526-treated mice, compared We recently established a model of multiple-organ metas- with control mice. VP-16 inhibited both bone metastasis and tasis with a small cell lung cancer cell line, SBC-5, in NK3 cell-depleted SCID mice (11). In this model, SBC-5 cells me- tastasize into multiple organs, such as the lung, liver, kidneys, systemic lymph nodes, and bone, resembling characteristics of Received 4/16/03; revised 7/18/03; accepted 7/22/03. small cell lung cancer in humans. We found that a new bisphos- The costs of publication of this article were defrayed in part by the payment of page charges. This article must therefore be hereby marked phonate, minodronate (YM529), inhibited osteolytic bone me- advertisement in accordance with 18 U.S.C. Section 1734 solely to tastasis via the inhibition of bone resorption; however, it could indicate this fact. not prolong the survival of tumor-bearing mice because of This work was supported in part by a Grant-in-aid for Cancer Research visceral metastasis (12). from the Ministry of Education, Science, Sports and Culture of Japan. 1 Both authors contributed equally to this work. 2 To whom requests for reprints should be addressed, at Department of Internal Medicine and Molecular Therapeutics, Course of Bioregulation and Medical Oncology, University of Tokushima School of Medicine, 3 The abbreviations used are: NK, natural killer; SCID, severe combined Kuramoto-cho 3-18-15, Tokushima 770-8503, Japan. Phone: 81-88- immunodeficient; PTHrP, parathyroid hormone-related protein; VEGF, 633-9274; Fax: 81-88-633-2134; E-mail: [email protected] vascular endothelial growth factor; TRAP, tartrate-resistant acid phos- u.ac.jp. phatase; IL, interleukin.

In the present study, we examined the therapeutic efficacy treated with i.v. administration of YM529 (0.2 ␮g) on day 7 of YM529 combined with etoposide (VP-16), which is com- and/or VP-16 (200 ␮g) on days 2, 3, 9, and 10, based on the monly used for small cell lung cancer patients, in terms of previous report (17). metastasis formation and survival. Five weeks after the tumor cell inoculation, the mice were anesthetized by i.p. injection of pentobarbital (0.5 mg/body), and X-ray photographs of the mice were taken to evaluate bone MATERIALS AND METHODS metastasis. Then, mice were sacrificed by cutting the subclavian Cell Culture. The human small cell lung cancer cell line artery, and all major organs were removed. The lungs were fixed SBC-5 was provided by Dr. K. Hiraki (Okayama University, in Bouin’s solution for 24 h. The number of metastatic lesions Okayama, Japan; Ref. 11). The SBC-5 cells were maintained in larger than 0.5 mm in diameter in the major organs was counted RPMI 1640 supplemented with 10% heat-inactivated fetal bo- macroscopically. vine serum (CRPMI-1640) and 1% gentamicin. The cells were For evaluatation of survival, tumor-bearing mice were sac- incubated at 37°C in a humidified atmosphere of 5% CO2 in air. rificed when the mice became moribund. Reagents. Antimouse IL-2 receptor ␤ chain monoclonal Histology and Immunohistochemistry. The hind limbs antibody, TM-␤1 (IgG2b), was supplied by Drs. M. Miyasaka of the mice were taken and fixed in 10% formalin. The bone and T. Tanaka (Osaka University, Osaka, Japan; Ref. 13). Mi- specimens were decalcified in 10% EDTA solution for 1 week nodronate (YM529) was provided by Yamanouchi Pharmaceu- and then embedded in paraffin. Tissue sections (4-␮m thick) tical Co. (Ibaragi, Japan; Ref. 14). Etoposide (VP-16) was were processed. For detection of osteoclasts, TRAP staining was obtained from Nippon Kayaku Co. (Tokyo, Japan). performed using a Sigma Diagnostics Acid Phosphatase Kit Animals. Male S EB-17/Icr-scid mice, age 6–8 weeks, (Sigma Diagnostics, St. Louis, MO). The number of TRAP- were obtained from CLEA (Osaka, Japan) and maintained under positive osteoclasts at the tumor-bone interface was counted specific pathogen-free conditions throughout the experiment. under a microscope in five random fields at ϫ200 magnifica- Experiments were performed according to the guidelines of our tion. Sections (4-␮m thick) of formalin-fixed, paraffin-embed- university. ded tumors were also stained with H&E for routine histological In Vitro Effect of YM529 on Proliferation of SBC-5 examination. Cells. SBC-5 cells at 80% confluence were harvested, plated Statistical Analysis. The Mann-Whitney U test was used into 96-well tissue culture plates (5000 cells/100 ␮l/well), and to determine the significance of difference in the number of incubated for 24 h at 37°C in 5% CO2. Then, various concen- multiorgan (bone, liver, lungs, kidneys, and lymph nodes) me- trations of YM529 were added to the cultures. After a 72-h tastases between YM529 and/or VP-16-treated groups and the incubation at 37°C, 50 ␮l of the 3-(4,5-dimethylthiazol-2-yl)- untreated group. The significance of differences in the number 2,5-diphenyltetrazolium bromide stock solution (2 mg/ml) were of TRAP-positive cells and the survival rate of the mice were added to each well, and the cells were further incubated for 2 h analyzed by Student’s t test (two-tailed) and the log-rank test, at 37°C (15). Then, the culture media were removed, and 100 ␮l respectively. P Ͻ 0.05 was considered significant in all exper- of DMSO were added to dissolve the dark blue crystals. Ab- iments. sorbance was measured with a MTP-32 Microplate Reader (Corona Electric, Ibaragi, Japan) at test and reference wave- lengths of 550 and 630 nm, respectively. RESULTS Effect of YM529 on Production of PTHrP and VEGF of In Vitro Effects of YM529 and VP-16 on Proliferation of SBC-5 Cells. SBC-5 cells at 80% confluence were harvested, SBC-5 Cells. YM529 at Ͻ1 ␮g/ml did not inhibit prolifera- plated into 6-well tissue culture plates (1 ϫ 105 cells/2 ml/well), tion of SBC-5 cells, whereas it did inhibit proliferation of SBC-5 Ͼ and incubated for 24 h at 37°C and 5% CO2. Then, the cultures cells at higher concentrations. VP-16 at 100 ng/ml suppressed were washed, and various concentrations of YM529 were proliferation of SBC-5 cells. YM529 at 1 ␮g/ml did not affect added. After a 72-h incubation at 37°C, culture supernatants the susceptibility of SBC-5 cells to VP-16 (Fig. 1). were collected, and the concentrations of PTHrP and VEGF Effect of YM529 and VP-16 on Production of PTHrP were determined using radioimmunoassay (Otsuka Assay, To- and VEGF by SBC-5 Cells. We reported previously (11) that kushima, Japan) and ELISA (R&D Systems, Minneapolis, MN), SBC-5 cells produced PTHrP and VEGF, which are thought to respectively. be crucial regulatory molecules for bone resorption and angio- Model of Multiple-Organ Metastasis by SBC-5 Cells genesis, respectively. We next examined the effect of YM529 and Antimetastatic Effect of YM529. To facilitate the me- and VP-16 on the production of these two molecules by SBC-5 tastasis of SBC-5 small cell lung cancer cells, NK cells were cells. Noncytotoxic concentrations of YM529 or VP-16 did not depleted in SCID mice (16). For NK cell depletion, TM-␤1 affect the production of these two molecules (Fig. 2). In addi- monoclonal antibody (300 ␮g/300 ␮l PBS/mouse) was injected tion, combined treatment with YM-529 and VP-16 had no effect i.p. into SCID mice 2 days before tumor cell inoculation. SBC-5 on the production of PTHrP or VEGF (data not shown). cells at the subconfluent condition were harvested and washed Effects of YM529 and VP-16 Treatments on Multiorgan with Ca2ϩ- and Mg2ϩ-free PBS (CMF-PBS). Cell viability was Metastases in SCID Mice. SBC-5 cells injected into NK determined by the trypan blue exclusion test, and only single cell-depleted SCID mice developed osteolytic bone metastatic cell suspensions of Ͼ90% viability were used. SBC-5 cells (1 ϫ lesions in the limbs, vertebral bone, pelvis scapulae, and the 106 cells/300 ␮l) were injected into the lateral tail vein of mice hind limbs. These lesions were detected on day 28 by radiog- on day 0. At the indicated periods, tumor-bearing mice were raphy (11). Several mice experienced paralysis (probably asso-

Fig. 1 Effects of YM529 and VP-16 on proliferation of SBC-5 cells. Fig. 2 Effect of YM529 and VP-16 on production of PTHrP and ϫ 4 SBC-5 cells were plated into 96-well tissue culture plates (5 10 VEGF by SBC-5 cells. SBC-5 cells (1 ϫ 105 cells/2 ml/well) were cells/well) and incubated for 24 h. Various concentrations of YM529 incubated for 24 h. Then, the cultures were washed, and various con- (A), VP-16 (A), or both (B) were added to the culture and incubated for centrations of YM529 (A) or VP-16 (B) were added. After 72 h of 70 h. Then, cell proliferation was determined by 3-(4,5-dimethylthiazol- incubation, culture supernatants were collected, and the concentrations 2-yl)-2,5-diphenyltetrazolium bromide assay. Data shown are represent- of PTHrP (Ⅺ) and VEGF (f) were determined using radioimmunoassay ative of three experiments with similar results. and ELISA, respectively. Values shown are representative of two independent experiments with similar results. Bars represent SDs.

ciated with spinal cord compression and bone metastases in the hind limbs) 4 weeks after SBC-5 cell inoculation, and the incidence of mice with paralysis became 30–50% five weeks of YM529 and VP-16 tended to decrease the number of oste- after inoculation. In addition, SBC-5 cells produced macroscop- oclasts, compared with single use of YM529 or VP-16, although ically detectable metastasis in the visceral organs, such as the the difference was not significant. liver, lungs, kidneys, and lymph nodes by day 28. A single Effect of YM529 Administration on Survival of Mice injection of 0.2 ␮g of YM529 significantly reduced the forma- Inoculated with SBC-5 Cells. We finally evaluated the ther- tion of bone metastasis; however, it had no effect on the devel- apeutic effect of YM529 on survival. The SCID mice inoculated opment of metastasis to other organs, such as the liver, lungs, with SBC-5 cells became moribund on days 35–42 (Fig. 5). kidneys, or systemic lymph nodes (Fig. 3; Table 1). Chemother- Treatment with YM529 or VP-16 marginally prolonged the apy with VP-16 significantly inhibited the development of me- survival of SBC-5-bearing SCID mice, but the difference was tastasis to the liver, lungs, and bone, but not to the kidneys or not significant. However, combined treatment significantly pro- Ͻ lymph nodes. The combined use of YM529 and VP-16 further longed the survival of these mice (P 0.05). inhibited bone metastasis, compared with treatment with YM529 alone or VP-16 alone. These results suggested that both DISCUSSION YM529 and VP-16 show promise in treating bone metastasis, Metastasis to multiple organs is a critical problem for lung and their combined use is necessary for control of multiorgan cancer patients. In particular, small cell lung cancer metastasizes metastasis. to various distant organs, including the liver, systemic lymph Effect of YM529 and VP-16 on the Number of Oste- nodes, and bone. In the present study, we demonstrated that a oclasts in Bone Lesions. Histological analysis of the un- new nitrogen-containing bisphosphonate (YM529) had a thera- treated mice revealed that osteolytic bone lesions consisted of peutic effect against osteolytic bone metastasis, but not visceral cancer cells. Numerous osteoclasts stained by TRAP staining metastasis, produced by SBC-5 cells. On the other hand, VP-16 were observed along the trabecular bone surface surrounded by suppressed the metastasis to the liver, lungs, and bone, but not SBC-5 cells. The number of osteoclasts was significantly lower to the kidneys or lymph nodes. Combined use of these two in bone lesions of mice treated with either YM529 or VP-16, agents could produce a better effect in terms of survival and compared with control mice (Fig. 4). In addition, combined use development of metastasis.

Fig. 3 Inhibition of osteolytic bone metastasis by combined therapy with YM529 and VP-16. SBC-5 cells (1 ϫ 106 cells/mouse) were i.v. injected into the NK cell-depleted SCID mice on day 0. The mice were treated with i.v. administration of YM529 (0.2 ␮g) on day 7 and/or VP-16 (200 ␮g) on days 2, 3, 9, and 10. Bone metastasis was assessed by X-ray photograph on day 35. A, control mouse; B, mouse treated with YM529 and VP-16. Arrows indicate osteolytic bone metastasis.

Table 1 Effect of YM529 and/or VP-16 on multiorgan metastasis of SBC-5 cells in NK cell-depleted SCID mice SBC-5 cells (1 ϫ 106 cells/mouse) were i.v. injected into the NK cell-depleted SCID mice on day 0. The mice were treated with i.v. administration of YM529 (0.2 ␮g) on day 7 and/or VP-16 (200 ␮g) on days 2, 3, 9, and 10. Metastasis was evaluated on day 35, as described in “Materials and Methods.” Data shown are representative of two independent experiments with similar results. Bone Liver Lung Kidney Lymph node Treatment Incidence No.a Incidence No. Incidence No. Incidence No. Incidence No. Control 5/5 14 (11–18) 5/5 72 (61–84) 5/5 27 (19–41) 5/5 5 (3–7) 5/5 5 (3–11) YM529 4/5 3 (0–7)b 5/5 66 (58–88) 5/5 23 (12–31) 5/5 4 (3–4) 5/5 3 (2–4) VP-16 5/5 7 (3–11)b 5/5 41 (14–83)c 5/5 13 (8–21)c 5/5 4 (2–5) 5/5 4 (2–5) YM529 ϩ VP-16 3/5 1 (0–3)b,c,d 5/5 43 (25–73)c 5/5 12 (9–23)c 5/5 3 (2–7) 5/5 3 (2–4) a Number of metastatic nodules [median (range)]. b P Ͻ 0.01 compared with control. c P Ͻ 0.05 compared with control. d P Ͻ 0.05 compared with VP-16 alone.

A large number of studies have reported the inhibitory life because of bone metastasis. Collectively, the control of effects of bisphosphonates against bone metastasis; however, both bone metastasis and visceral metastases by multiple the survival benefit of bisphosphonates is still controversial modalities may be necessary to prolong the survival of cancer in experimental animal models (18–20). In the present study, patients with multiple organ metastases. To further prolong treatment with YM529 alone could not significantly prolong survival, experiments to evaluate the effect of continuing survival, although it inhibited bone metastasis remarkably, treatment with YM529 and VP-16 are ongoing. suggesting that selective inhibition of bone metastasis is not It is well accepted that bone destruction caused by bone sufficient for prolonging survival. Our results are consistent metastasis is mediated by various factors produced or induced with most of the clinical studies showing that bisphospho- by tumor cells and/or microenvironments that stimulate the nates can improve the quality of the life but not the survival formation and activation of osteoclasts, the normal bone-resorb- of advanced cancer patients with bone metastases (21, 22). ing cells (23, 24). Several factors, including IL-1, IL-6, receptor On the other hand, a chemotherapeutic agent, VP-16, sup- activator of nuclear factor ␬B (RANK) ligand, macrophage pressed metastasis to some visceral organs (the liver and inflammatory protein-1␣, and PTHrP, have been implicated as lungs). It also suppressed bone metastasis in our protocol, but factors that enhance osteoclast formation and bone destruction with much less efficiency than YM529. In addition, VP-16 in malignant diseases (23). Of these factors, PTHrP has been alone could not prolong survival. Because the mice with reported to indirectly activate osteoclasts by induction or stim- severe bone metastases became paralyzed and could not ulation of RANK ligand expression by osteoblasts and hence obtain enough food or water, the poor prognosis of the mice augment bone resorption (23). We demonstrated previously that treated with VP-16 alone may be due not only to the pro- SBC-5 cells overexpressed PTHrP, and treatment with anti- gression of visceral metastasis but also to restricted quality of PTHrP neutralizing antibody inhibited the development of bone

Fig. 5 The survival of mice treated with or without YM529 and/or VP-16. SBC-5 cells (1 ϫ 106) were i.v. inoculated into NK cell-depleted SCID mice (10 mice/group) on day 0. The mice were treated with i.v. administration of YM529 (0.2 ␮g) on day 7 and/or VP-16 (200 ␮g) on days 2, 3, 9, and 10, and survival of the mice was evaluated as described in “Materials and Methods.”

VP-16 against bone metastasis may be predominantly due to inhibition of osteoclast generation and/or function by YM529 plus suppression of cell proliferation by VP-16 in the bone lesions. Fig. 4 Effect of YM529 on the number of osteoclasts in the bone lesions. SBC-5 cells (1 ϫ 106 cells/mouse) were i.v. injected into NK cell-depleted In conclusion, use of bisphosphonate (YM529) alone in- SCID mice on day 0. The mice were treated with i.v. administration of hibited bone metastasis but was not sufficient to prolong the YM529 (0.2 ␮g) on day 7 and/or VP-16 (200 ␮g) on days 2, 3, 9, and 10. survival of mice with bone metastasis and visceral dissemina- On day 35, bone lesions were harvested, and osteoclasts were stained by tion. Combined use of YM529 with VP-16 to suppress visceral TRAP staining, as described in “Materials and Methods.” A, bone lesions of metastasis successfully suppressed multiorgan metastases and control mouse; B, bone lesions of mouse treated with YM529 and VP-16; C, number of TRAP-positive-stained cells in the bone lesions of mice prolonged survival. Therefore, a combined modality with treated with or without YM529 and/or VP-16. Bars represent SD. T, tumor; bisphosphonate and chemotherapy may be useful for lung can- -P Ͻ 0.01. cer patients who have bone metastasis and visceral dissemina ,ء .O, bone; HPF, high power field tion.

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Seiji Yano, Helong Zhang, Masaki Hanibuchi, et al.

Clin Cancer Res 2003;9:5380-5385.

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