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.

PURPOSE: Lung cancer in the advanced stage frequently metastasizes to multiple organs, including the liver, lungs, lymph nodes, and bone. Bisphosphonates have been widely used to treat osteolytic bone metastasis in the past years; however, many studies have implicated that a single use of bisphosphonates could not prolong the survival of patients. In the present study, using a multiple-organ metastasis model of human lung cancer cells, we examined the effect of combined therapy with a new bisphosphonate (YM529) and etoposide (VP-16). EXPERIMENTAL DESIGN: Human small cell lung cancer (SBC-5) cells i.v. inoculated into natural killer cell-depleted severe combined immunodeficient mice metastasized to multiple organs, including the lungs, liver, kidneys, lymph nodes, and bone. SBC-5-bearing mice were treated with YM529 and/or VP-16 and sacrificed 5 weeks after tumor cell inoculation. Bone metastasis was assessed by X-ray photographs, and visceral metastasis was evaluated macroscopically. The number of osteoclasts in the bone lesions was examined by tartrate-resistant acid phosphatase staining. RESULTS: Monotherapy with YM529 suppressed the production of bone metastases, but not visceral metastasis. Histological analyses revealed that the number of osteoclasts in bone lesions was lower in YM526-treated mice, compared with control mice. VP-16 inhibited both bone metastasis and visceral (lung and liver) metastasis. However, neither YM529 alone nor VP-16 alone significantly prolonged the survival of SBC-5-bearing mice. Combined use of YM529 and VP-16 further inhibited the production of bone metastasis and significantly prolonged survival. CONCLUSIONS: Combined therapy with bisphosphonate and chemotherapy may be useful for small cell lung cancer patients with multiple organ metastases including bone metastasis.     

Effect of YM529 on a model of mandibular invasion by oral squamous cell carcinoma in mice.

PURPOSE: This study examined the mechanisms of osteoclast-mediated bone invasion in a model of oral squamous cell carcinoma (OSCC). C3H/HeN mice were inoculated with SCC VII cells into the masseter region to establish an animal model of mandibular invasion by OSCC. EXPERIMENTAL DESIGN: The mice were divided into three groups: a control group, given daily s.c. injections of saline; group 1, given 2 microg per mouse per day of the bisphosphonate YM529; and group 2, given 10 microg per mouse per day of YM529. After 3 weeks of treatment, the lesions were studied by micro-computed tomography. After tartrate-resistant acid phosphatase (TRAP) staining, the osteoclasts were easily identified, and the percentages of the area occupied by osteoclasts were calculated by computer for each sample. The tumors were analyzed by RT-PCR to determine the mRNA expression of interleukin-6 (IL-6), parathyroid hormone-related protein (PTHrP), tumor necrosis factor-alpha (TNF-alpha), receptor activator of nuclear factor-kappaB (RANK), RANK ligand (RANKL), and osteoprotegerin. RESULTS: SCC VII cells rapidly multiplied in the masseter muscle of the mice. Bone invasion was evident only in the control group on micro-computed tomography. On TRAP-stained slices, the percentages of osteoclasts in groups 1 and 2 were significantly lower than that in the control group. The mRNA expressions of IL-6, PTHrP, THF-alpha, and RANK decreased as the concentration of YM529 increased. CONCLUSIONS: We conclude that various cancer-derived cytokines play important roles in the invasion of bone by OSCC. YM529, a third-generation bisphosphonate, can suppress osteoclast-mediated bone invasion by OSCC. The mechanism of this effect might involve inhibition of cytokines such as IL-6, PTHrP, TNF-alpha, and RANK by YM529.     

Anti-tumor effect of bisphosphonate (YM529) on non-small cell lung cancer cell lines

Background  

YM529 is a newly developed nitrogen-containing bisphosphonate (BP) classified as a third-generation BP that shows a 100-fold greater potency against bone resorption than pamidronate, a second-generation BP. This agent is, therefore expected to be extremely useful clinically for the treatment of osteoporosis and hypercalcemia. Recently, YM529 as well as other third-generation BPs have also been shown to exert anti-tumor effects against various types of cancer cells both in vitro or/and in vivo. In this study, we investigate the anti-tumor effect of YM529 on non-small cell lung cancer (NSCLC).     

A newly developed bisphosphonate, YM529, is a potent apoptosis inducer of human myeloma cells

We examined the effect of YM529, a newly developed third-generation bisphosphonate (BP), on the growth of human myeloma cell lines using the trypan blue dye exclusion test and Alamar blue assay. BPs induced inhibition of proliferation in all cell lines dose-dependently, and YM529 had a most potent growth inhibitory effect, followed by incadronate and pamidronate. Flow cytometric analysis using annexinV and 7AAD showed that YM529 most significantly induced apoptosis of all myeloma cell lines. These observations suggested that YM529 is a potent apoptosis inducer of myeloma cells, and might have some benefit not only on the improvement of bone lesions but also on survival in some myeloma patients.     

Combination with third‐generation bisphosphonate (YM529) and interferon‐alpha can inhibit the progression of established bone renal cell carcinoma

The aim of this study was to investigate whether the third‐generation nitrogen‐containing bisphosphonate (YM529) can inhibit the progression of established bone renal cell carcinoma (RCC) and to elucidate its mechanism. Antiproliferative effect and apoptosis induction of RCC cells and mouse osteoclasts by YM529 and/or interferon‐alpha (IFN‐α) were evaluated in vitro using cell counting and in vivo using soft X‐ray, the TUNEL method and tartrate‐resistant acid phosphatase stain. For the in vivo study, male athymic BALB/cA Jc1‐nu nude mice bearing human RCC cell line RBM1‐IT4 cells were treated with YM529 and/or IFN‐α. The biological activity of osteoclasts was evaluated using the pit formation assay. The antiangiogenetic effect by YM529 and/or IFN‐α was analyzed using micro‐vessel density and in situ mRNA hybridization. Osteoclast number in bone tumors was decreased in YM529‐treated mouse. YM529 also inhibited osteoclast activity and proliferation in vitro, whereas basic fibroblast growth factor expressions and micro‐vessel density within tumors were inhibited by IFN‐α. Neither YM529 nor IFN‐α alone significantly inhibited the growth of established bone metastatic tumors. Combined treatment with YM529 and IFN‐α may be beneficial in patients with human RCC bone metastasis. Their effects are mediated by osteoclast recruitment inhibition and inactivation by YM529 and antiangiogenesis by IFN‐α.     

A third-generation bisphosphonate, minodronic acid (YM529), successfully prevented the growth of bladder cancer in vitro and in vivo

Minodronic acid (YM529) is a third-generation bisphosphonate (BP) that has been shown to directly and indirectly prevent proliferation, induce apoptosis, and inhibit metastasis of various types of cancer cells. In this study, we have investigated the therapeutic efficacy of YM529 against bladder cancer, both in vitro and in vivo. YM529 inhibited geranylgeranylation as well as farnesylation and reduced the growth of all seven bladder cancer cell lines in a dose- and time-dependent manner in vitro. YM529 demonstrated a good synergistic or additive antiproliferative effect when administered in combination with cisplatin or paclitaxel. Immunohistochemical study revealed YM529 inhibited the prenylation of Rap1A in vivo. YM529 administered systemically did not markedly inhibit the growth of visceral metastases but it showed a significant anticancer effect on bone metastases monitored by an in vivo imaging system. Moreover, intravesical YM529 demonstrated significant growth inhibition in a bladder cancer orthotopic model. No adverse effects were associated with the systemic as well as the intravesical treatment regimens. In conclusion, our study suggests that YM529 may be a potent anticancer agent for bladder cancer. The efficacy and safety of this BP as an agent for combination chemotherapies against bladder cancer should be verified by early-phase clinical trials.     

Effect of a newly developed bisphosphonate,YH529, on osteolytic bone metastases in nude mice

YH529, [1-hydroxy-2-(imidazo [1,2-a] pyridin-3-yl) ethylidene]-bisphosphonic acid monohydrate, is a newly developed third-generation bisphosphonate with a potent inhibitory activity toward osteoclastic bone resorption. The primary cellular mechanism of osteolysis associated with metastatic cancer is osteoclast-mediated. It is likely that bisphosphonates would be efficacious in this situation. In the present study, we examined the effect of YH529 in a nude mice bone metastasis model, in which the intracardiac injection of a human breast cancer cell line, MDA-MB-231(MDA-231), leads to osteolytic bone metastases. To examine whether YH529 would prevent such bone metastasis, we administered YH529 s.c. to nude mice simultaneously with cancer cell inoculation through the entire experimental period (protocol 1) or performed short-term prophylactic administration before inoculation of the MDA-231 cells (protocol 2). In addition, to examine the possible therapeutic effects of the drug on established bone metastases, we injected YH529 after radiographically small but distinct osteolytic bone metastases had been detected (protocol 3). In all protocols, YH529 (2 μg/mouse/day) markedly inhibited bone metastases as well as the progression of established metastatic foci that were quantified on the radiographs. Histological examination and histomorphometrical analysis revealed that YH529 markedly reduced the number of osteoclasts and the size of the tumor at the metastatic bone sites. Our results suggest that YH529 may suppress metastasis formation and tumor growth in bone through inhibition of osteoclastic bone resorption. Int. J. Cancer 77:279–285, 1998.© 1998 Wiley-Liss, Inc.     

Induction of mitochondria-dependent apoptosis through the inhibition of mevalonate pathway in human breast cancer cells by YM529, a new third generation bisphosphonate

YM529, a new third generation bisphosphonate, induced apoptosis of a human breast cancer cell line, MX-1. Cytotoxic activity of YM529 was more potent than that of incadronate. YM529 activated caspase-9, but not caspase-8, and induced the release of cytochrome c into cytosol. YM529 increased Bax expression and decreased Bcl-2 expression, while it did not induce caspase-8-dependent Bid truncation. Farnesyl pyrophosphate prevented YM529-mediated cytotoxicity. These results suggest that YM529 is a potent therapeutic agent for human breast cancers, activating the mitochondria-dependent apoptotic pathway through the inhibition of protein farnesylation.     

Severe pulmonary toxicity in patients treated with a combination of docetaxel and gemcitabine for metastatic transitional cell carcinoma

Background: Both gemcitabine and docetaxel have been associated with pulmonary toxicity when used as single agents. We report a study in which three of five cases developed pulmonary toxicity (which proved fatal in one case) when these drugs were used in combination to treat metastatic transitional cell cancer.Patients and methods: Three patients developed dyspnoea, in two cases associated with pulmonary infiltrates, whilst receiving the combination of gemcitabine and docetaxel in a phase I trial. The case notes of all five patients entered into this trial were studied. A literature review was undertaken to gain information on reported pulmonary toxicity with the deoxy-cytidine analogues and taxanes given alone or in combination with or without radiotherapy.Results: Three patients developed delayed dyspnoea whilst receiving gemcitabine/docetaxel in combination. This settled with cessation of treatment in one patient, however in the remaining two cases significant hypoxia developed, associated radiologically with evidence of progressive pulmonary infiltrates. One of these patients developed respiratory failure after bronchoscopy and biopsy and died. His chest X-ray changes were consistent with adult respiratory distress syndrome. The transbronchial biopsy and post mortem lung histology in this patient showed diffuse alveolar damage. The remaining patient settled with high dose prednisolone but died subsequently of progressive metastatic disease.Conclusion: The combination of gemcitabine and docetaxel showed promising activity in this small study. The development of pulmonary symptoms in three cases with radiological lung infiltrates in two other cases was cause for concern. Patients receiving this drug combination should be closely monitored for similar problems.     

Inhibition by a new bisphosphonate (YM175) of bone resorption induced by the MBT-2 tumour of mice.

A new bisphosphonate, disodium dihydrogen (cycloheptylamino) methylene bisphosphonate monohydrate (YM175), was compared with 3-amino-1-hydroxypropylidene-1, 1-bisphosphonate (AHPrBP) and 1-hydroxyethylidene-1,1-bisphosphonate (HEBP) in terms of its effect on tumour induced osteolysis using a bladder tumour in mice (MBT-2). The method consisted of inoculating tumour cells subcutaneously (SC) over the calvaria in mice, resulting in a local tumour causing fragmentation of the bone. The compounds were active not only when administered preventively before establishment of bone resorption, but also in an inhibitory fashion once the variables were already under the influence of the tumour. This osteolysis was evaluated by measuring the increased area of bone resorption in reduced opacity to radiograph and histology. The results showed the following sequence of potency: YM175 > AHPrBP = HEBP. This inhibition was obtained with no apparent effect on the growth of the MBT-2 tumour. YM175 appears to be an interesting new bisphosphonate with possible clinical application.     

Complete response in a patient with liver metastasis of esophageal squamous cell carcinoma treated with docetaxel

On routine endoscopy a Type 2 tumor was found in the esophagogastric junction of a 74-year-old man. A histological diagnosis of squamous cell carcinoma was made based on a biopsy specimen, and lower esophagectomy and proximal gastrectomy were performed. The pathological diagnosis was pT3, N2, M0, pStage III. A low-dose FP treatment as adjuvant chemotherapy was given for only three weeks due to severe anorexia. A liver metastases measuring 22×24 mm in diameter at the s6 lesion was found with a CT examination a year and a half after the operation. A dose of 70 mg/m2 of docetaxel was given by intervenous infusion, and repeated every four weeks. Toxicities, grade 4 neutropenia and mild pneumonia associated with this chemotherapy regimen, were observed after five cycles. Therefore this treatment was discontinued. CT performed at that time showed a complete response (CR) and no more recurrences for six months. Docetaxel treatment is considered to be safe for outpatients and is one of the cures for metastatic esophageal cancer.     

荷人膀胱癌原位移植瘤裸小鼠动物模型的建立

目的建立模拟人的原位裸小鼠模型,评价抗人膀胱癌免疫毒素临床前的疗效。方法将体外培养的人移行细胞癌细胞系（BIU-87）原位移植于经酸处理的裸小鼠膀胱内,定期用核磁共振成像（MRI）检查,并进行解剖、组织学检查和免疫细胞化学的检测。结果在第7～36天内,成瘤率为92．9％（52／56）;其中第12～13天处死的裸小鼠中,95．2％（40／42）发展成移行细胞癌,以浅表性为主。组织学检查证实为Ⅱ～Ⅲ级;免疫细胞化学证实,该模型具有BIU-87细胞的特征。在肿瘤的发生发展中,MRI检查的结果与组织学检查的结果密切相关。接种后第7—9天可以检测出原位癌（CIS）,其后发展成乳头状瘤或浸润性疾病。结论模拟人的原位裸小鼠模型重复性好,是临床前研究膀胱灌注治疗的理想模型。     

Effect of photodynamic therapy in combination with ionizing radiation on human squamous cell carcinoma cell lines of the head and neck

Photodynamic therapy (PDT) is a promising treatment modality for head and neck, and other tumours, using drugs activated by light. A second generation drug, 5-aminolaevulinic acid (5-ALA), is a precursor of the active photosensitizer protoporphyrin IX (PpIX) and has fewer side-effects and much more transient phototoxicity than previous photosensitizers. We have investigated the effect of 5-ALA mediated PDT in combination with gamma-irradiation on the colony forming ability of several human head and neck tumour cell lines. The effect of treatments on the DNA cell cycle kinetics was also investigated. Our results indicate that the combination of 5-ALA mediated PDT and gamma-irradiation results in a level of cytotoxicity which is additive and not synergistic. 5-ALA mediated PDT had no discernible effect on DNA cell cycle distributions. gamma-irradiation-induced cell cycle arrest in G2 did not enhance the phototoxicity of 5-ALA.     

Efficacy of a nitrogen-containing bisphosphonate, minodronate, in conjunction with a p38 mitogen activated protein kinase inhibitor or doxorubicin against malignant bone tumor cells

Purpose We recently reported the sarcoma-selective antitumor effects of a newly developed nitrogen-containing bisphosphonate, minodronate (MIN), on malignant bone tumors. The aim of this study was to develop efficient combination MIN therapy in malignant bone tumors. Methods We examined downstream molecular events of MIN in osteosarcoma and Ewing’s sarcoma cells to search for a partner to combine with MIN. Furthermore, we evaluated the combined effects of MIN and clinically available Doxorubicin (DOX). Results We found that MIN inhibited Rap 1A prenylation, and extracellular signal-regulated kinase (ERK) or Akt phosphorylation in osteosarcoma (Saos-2) and Ewing’s sarcoma (SK-ES-1) cells. Interestingly, MIN activated p38 mitogen activated protein kinase (MAPK) only in SK-ES-1 cells and a p38 MAPK inhibitor augmented MIN-induced growth inhibition in SK-ES-1 cells. Doxorubicin (DOX) exerted synergistic effects on Saos-2 and SK-ES-1 cell lines. Daily injection of MIN enhanced the growth inhibition of SK-ES-1 xenograft sarcoma treated by DOX in nude mice. Conclusions These findings suggest that the inhibition of the p38 MAPK pathway may be attractive in overcoming cellular resistance against MIN. In the light of clinical settings, MIN may have a beneficial adjuvant role in the DOX treatment.     

Inhibition of growth and metastasis of orthotopic human prostate cancer in athymic mice by combination therapy with pegylated interferon-alpha-2b and docetaxel

We evaluated whether treatment of orthotopic human prostate cancer in nude mice with pegylated IFN-alpha-2b (PEG-IFN-alpha-2b) and docetaxel could represent a two-compartment targeting of primary tumor (tumor cells and tumor-associated endothelial cells) and inhibition of regional lymph node metastasis. The antiangiogenic properties of IFN were combined with the cytotoxic properties of docetaxel, resulting in apoptosis of both tumor cells and endothelium and hence significant inhibition of primary tumor growth. We first determined the optimal biological dose of PEG-IFN-alpha-2b (70,000 IU/week) necessary to down-regulate the expression of basic fibroblast growth factor, matrix metalloprotease-9, and matrix metalloprotease-2. The therapeutic dose of docetaxel (10 mg/kg/week) was determined by efficacy and minimal body weight loss. Therapy beginning 3 days after orthotopic implantation of PC3-MM2 prostate cancer cells reduced tumor weight by 37% in mice treated with PEG-IFN-alpha-2b, by 60% in mice treated with docetaxel, and by 83% in those given both drugs. PEG-IFN-alpha-2b also induced apoptosis of tumor-associated endothelial cells and hence a significant decrease in microvessel density. Our data indicate that the combination of PEG-IFN-alpha and docetaxel inhibits neoplastic angiogenesis by inducing a decrease in the local production of proangiogenic molecules by tumor cells, resulting in increased apoptosis of tumor-associated endothelial cells.     

Additive antitumor activities of taxoids in combination with the bisphosphonate ibandronate against invasion and adhesion of human breast carcinoma cells to bone.

A very common metastatic site for breast carcinoma is bone. Metastatic breast carcinoma cells stimulate osteoclast-mediated bone resorption leading to osteolysis. Bisphosphonates are powerful inhibitors of osteoclast activity, and are therefore used in combination with standard chemotherapy or hormonal therapy for the treatment of cancer-associated osteolytic metastases. However, there may be an added beneficial effect of the bisphosphonates, that is, additive or synergistic activities with cytotoxic agents. Here, we investigated the effects of the bisphosphonate ibandronate in combination with taxoids (taxol and taxotere) on induction of apoptosis, invasion and adhesion of breast carcinoma cells to bone. Ibandronate did not induce apoptosis of human MDA-MB-231 breast carcinoma cells, nor did it enhance the effectiveness of taxoid-induced apoptosis in MDA-MB-231 cells. In contrast, ibandronate enhanced the antitumor activity of taxoids against invasion and cell adhesion to bone. Our findings raise the interesting possibility that the combination of bisphosphonates and taxoids may be useful for the treatment of patients with cancer types that are known to metastasize preferentially to bone.