Immunohistochemical and functional studies for M3 muscarinic receptors and cyclo-oxygenase-2 expressed in the mouse atrium.

In mouse atrium, M2 and M3 muscarinic receptors (M2R and M3R) are involved in biphasic (negative and positive) inotropic actions of muscarinic agonists, and the positive inotropic action is reduced by indomethacin. The aim of our study was to determine the localization of M2R, M3R and cyclo-oxygenase (COX) in mouse atrium and to characterize muscarinic receptor-mediated positive inotropy. M2R immunoreactivity was found only on atrial myocardium, but M3R immunoreactivity was localized on both the myocardium and endocardial endothelium. COX-1 and COX-2 immunoreactivities were identified in both myocardial and endocardial endothelium. In electrically stimulated left atria, carbachol caused M2R-mediated negative inotropy followed by M3R-mediated positive inotropy. Removal of atrial endothelium reduced the positive inotropy without affecting the negative inotropy, suggesting that stimulation of endothelial M3R mediates the positive inotropy. N-[2-(cyclohexyloxy)-4-nitrophenyl]-methanesulfonamide (NS398, COX-2 inhibitor) decreased the carbachol-induced positive inotropy; however, 5-(4-chlorophenyl)-1-(4-methoxyphenyl)-3-trifluoromethylpyrazole (SC560, COX-1 inhibitor), 1-[[4,5-bis(4-methoxyphenyl)-2-thiazolyl]carbonyl]-4-methylpiperazine (FR122047, COX-1 inhibitor) and L-nitroarginine methylester did not affect the inotropic response. M3R activation caused positive chronotropy in spontaneously beating right atria when M2R-mediated negative chronotropy was suppressed and rate of contraction was low, <350 beats min⁻¹. Our results indicate that although M3Rs are located on both myocardial cells and endocardial endothelial cells, only endothelial M3Rs mediate positive inotropy in response to muscarinic agonists via activation of COX-2 in the mouse atrium. M3R-mediated positive chronotropy counteracting M2R-mediated negative chronotropy was also demonstrated.

Serological test and chest computed tomography findings in patients with Mycobacterium avium complex lung disease.

The present authors have previously reported the usefulness of a serodiagnostic test to detect serum glycopeptidolipid (GPL) core antibody in diagnosing Mycobacterium avium complex (MAC) lung disease in immunocompetent patients. The aim of the present study was to investigate correlations between the levels of antibody against GPL core and chest computed tomography (CCT) findings in patients with MAC lung disease. A total of 47 patients with MAC-positive culture from their sputum and who had radiographic abnormalities were investigated. Thirty-three patients met the American Thoracic Society criteria for MAC disease; 14 did not. All patients underwent both CCT examination and the serodiagnostic test for MAC at the same time. Small nodular shadows were seen on CCT in all 47 patients and bronchiectasis shadows were seen in 39 (83%) of them. There was a significant positive correlation between the extent of the disease and the level of GPL core immunoglobulin (Ig)A antibody. The levels of GPL core IgA antibody were significantly elevated in patients who had nodular shadows (10-30 mm) compared with patients who had small nodular shadows (<10 mm). The present results document that the levels of immunoglobulin A antibody against glycopeptidolipid core correlate with the chest computed tomography findings of Mycobacterium avium complex lung disease.

Hedgehog and Fgf signaling pathways regulate the development of tphR-expressing serotonergic raphe neurons in zebrafish embryos.

Serotonin (5HT) plays major roles in the physiological regulation of many behavioral processes, including sleep, feeding, and mood, but the genetic mechanisms by which serotonergic neurons arise during development are poorly understood. In the present study, we have investigated the development of serotonergic neurons in the zebrafish. Neurons exhibiting 5HT-immunoreactivity (5HT-IR) are detected from 45 h postfertilization (hpf) in the ventral hindbrain raphe, the hypothalamus, pineal organ, and pretectal area. Tryptophan hydroxylases encode rate-limiting enzymes that function in the synthesis of 5HT. As part of this study, we cloned and analyzed a novel zebrafish tph gene named tphR. Unlike two other zebrafish tph genes (tphD1 and tphD2), tphR is expressed in serotonergic raphe neurons, similar to tph genes in mammalian species. tphR is also expressed in the pineal organ where it is likely to be involved in the pathway leading to synthesis of melatonin. To better understand the signaling pathways involved in the induction of the serotonergic phenotype, we analyzed tphR expression and 5HT-IR in embryos in which either Hh or Fgf signals are abrogated. Hindbrain 5HT neurons are severely reduced in mutants lacking activity of either Ace/Fgf8 or the transcription factor Noi/Pax2.1, which regulates expression of ace/fgf8, and probably other genes encoding signaling proteins. Similarly, serotonergic raphe neurons are absent in embryos lacking Hh activity confirming a conserved role for Hh signals in the induction of these cells. Conversely, over-activation of the Hh pathway increases the number of serotonergic neurons. As in mammals, our results are consistent with the transcription factors Nk2.2 and Gata3 acting downstream of Hh activity in the development of serotonergic raphe neurons. Our results show that the pathways involved in induction of hindbrain serotonergic neurons are likely to be conserved in all vertebrates and help establish the zebrafish as a model system to study this important neuronal class.

Near-field optical microscope observation of dye-containing nano-domains.

A novel method for forming dye-containing nano-domains in thin films using a polymer alloy system has been developed. The polymer alloy system (PS-b-PMMA), which consists of polystyrene (PS) and polymethyl methacrylate (PMMA), forms microphase separation in thin films. The film was treated using a previously reported technique under vacuum conditions, and an organic dye was selectively dispersed into the PS. Selective association of the dye (diarylethene; cis-1, 2-dicyano-1, 2-bis (2,4,5-trimethyl-3-thienyl) ethene) with the PS nano-domains was then observed, with both transmission electron microscopy and scanning near-field optical microscopy with an absorption spectrum.

Melt segregation and strain partitioning: implications for seismic anisotropy and mantle flow.

One of the principal means of understanding upper mantle dynamics involves inferring mantle flow directions from seismic anisotropy under the assumption that the seismic fast direction (olivine a axis) parallels the regional flow direction. We demonstrate that (i) the presence of melt weakens the alignment of a axes and (ii) when melt segregates and forms networks of weak shear zones, strain partitions between weak and strong zones, resulting in an alignment of a axes 90 degrees from the shear direction in three-dimensional deformation. This orientation of a axes provides a new means of interpreting mantle flow from seismic anisotropy in partially molten deforming regions of Earth.

Bisphosphonate actions on cancer.

Bisphosphonates (BPs) suppress cancer cell colonization in bone associated with cancers such as breast cancer and multiple myeloma. The mechanism of the suppressive action of BPs is thought to be due to an inhibition of osteoclastic bone resorption which releases bone-stored growth factors that feed cancer cells colonizing bone. Recently, data are accumulating that BP suppresses growth and induces apoptosis in cancer cells in culture, suggesting that BP directly influences survival of cancer cells in an osteoclast-independent manner. These results raise the possibility that BP inhibits cancer growth in organs other than bone. However, evidence is limited that BP reduces tumor growth in non-bone sites in cancer patients. In this review, we discuss the effectiveness of BP on breast cancer colonization in non-bone sites and our results in animal models with metastases. With currently available clinical and in vivo experimental data, BPs are definitely beneficial for the treatment of cancer patients who manifest clinically detectable bone metastases. However, it is not recommended that BP be given as a preventative to patients with visceral metastases and of no evidence of bone metastases. Whether individual BP with different chemical structure has unique biological or biochemical action is an intriguing question but open at the moment.

Impairments and prognostic factors for survival in patients with idiopathic pulmonary fibrosis.

The aim of the present study was to evaluate exercise limiting factors using cardiopulmonary exercise testing (CPET) in patients with idiopathic pulmonary fibrosis (IPF), and to investigate whether these parameters are related to survival after CPET. We evaluated 41 patients with IPF (mean 68.2 years, 27 male) who performed CPET. The exercise capacity in patients with IPF was limited more strongly by gas exchange and/or ventilatory impairments, compared with cardiac impairment. Using univariate analysis, the severity of exercise-induced hypoxemia (EIH) evaluated by deltaPaO2/deltaVO2 (PaO2-slope), oxygen uptake at maximum exercise, oxygen pulse at maximum exercise, ventilatory equivalent for carbon dioxide at maximum exercise and age were significantly related to the survival rate. Interestingly, the PaO2-slope was most closely correlated with the survival rate using multiple analysis with a stepwise evaluation. Nevertheless, PaO2 at rest and at maximum exercise were not factors influencing survival. In patients with IPF, CPET can simultaneously evaluate the ability of both the cardiovascular and respiratory systems, and should be available so that parameters can be derived to make the necessary prognostic estimations, with the most useful parameter being the degree of EIH as represented by the PaO2-slope.

2,3,7,8-tetrachlorodibenzo-p-dioxin toxicity in the zebrafish embryo: local circulation failure in the dorsal midbrain is associated with increased apoptosis.

Effects of 2,3,7,8-tetrachlorodibenzo-p-dioxin (TCDD) on local circulation and apoptosis in the midbrain were investigated in zebrafish (Danio rerio) embryos during early development. Embryos were exposed to TCDD from 24 h post fertilization (hpf) until observation, in water maintained at 28.5 degrees C. TCDD decreased blood flow in the mesencephalic vein, the only vessel perfusing the dorsal midbrain of the embryo. At 50 hpf, blood flow was maximally reduced in this vessel and gradually returned to the control level at 60 hpf. In contrast, blood flows in the trunk and in other vessels of the head of the embryo did not significantly change until 72 hpf. Furthermore, TCDD exposure caused apoptosis in the midbrain at 60 hpf, and the TCDD dose response relationship for this effect was similar to that for reduced blood flow in the mesencephalic vein at 50 hpf. The effects of TCDD on apoptosis in the midbrain, but not on blood flow, were abolished by Z-VAD-FMK, a general caspase inhibitor. TCDD effects on both endpoints were mimicked by beta-naphthoflavone (BNF), an aryl hydrocarbon receptor (AHR) agonist, and almost abolished by concomitant exposure to TCDD and alpha-naphthoflavone (ANF), an AHR antagonist. Concomitant exposure to TCDD and either an inhibitor of cytochrome P450 (CYP) (SKF525A or miconazole) or an antioxidant (N-acetylcysteine or ascorbic acid) inhibited these effects of TCDD. The incidence of apoptosis in the midbrain was inversely related to blood flow in this brain region following these various treatments and graded TCDD exposure concentrations (r = -0.91). The same range of TCDD exposure concentrations that reduced blood flow and increased apoptosis in the midbrain greatly enhanced CYP1A mRNA expression and immunoreactivity at 50 hpf in endothelial cells of blood vessels including the mesencephalic vein and the heart, but not the brain parenchyma. Taken together, these results suggest that TCDD induces apoptosis in the midbrain of the zebrafish embryo secondary to local circulation failure, which could be related to AHR activation, induction of CYP1A, and oxidative stress.

An endocrinopathy characterized by dysfunction of the pituitary-adrenal axis and alopecia universalis: supporting the entity of a triple H syndrome.

We demonstrate the rare disorder of triple H syndrome in a 25-year-old man. He was pointed out as having short stature, at -5.9 s.d., and diagnosed as GH deficient at 6 years old. Approximately a year ago, he noticed systematic hair loss. He lost body weight by 7 kg during the last half year. He was admitted to Jichi Medical School Hospital because of unconsciousness. Physical findings showed disturbance of consciousness with Japan Coma Scale I-3. He had emaciation and alopecia universalis. Laboratory findings showed plasma glucose was as low as 1.11 mmol/l. GH and ACTH deficiency with hypoadrenocorticism were clarified. His intelligence was in the low normal range with a WAIS IQ of 70, and anterograde amnesia was suggested in the presence of a little, but not significant, morphological change in the hippocampus on a magnetic resonance imaging scan. Replacement by a physiological dose of hydrocortisone normalized plasma glucose, and restored body weight and growth of hair during the 7 month therapeutic period. The present finding strongly supports a clinical entity of triple H syndrome, including ACTH deficiency, alopecia universalis and anterograde amnesia, and that there may be some variation of the triad among the subjects.

Clinical evaluation of anti-tuberculous glycolipid immunoglobulin G antibody assay for rapid serodiagnosis of pulmonary tuberculosis.

Previously we reported the development of a highly sensitive enzyme-linked immunosorbent assay specific for anti-tuberculous glycolipid (anti-TBGL) for the rapid serodiagnosis of tuberculosis. In this study, the usefulness of an anti-TBGL antibody assay kit for rapid serodiagnosis was evaluated in a controlled multicenter study. Antibody titers in sera from 318 patients with active pulmonary tuberculosis (216 positive for Mycobacterium tuberculosis in smear and/or culture tests and 102 smear and culture negative and clinically diagnosed), 58 patients with old tuberculosis, 177 patients with other respiratory diseases, 156 patients with nonrespiratory diseases, and 454 healthy subjects were examined. Sera from 256 younger healthy subjects from among the 454 healthy subjects were examined as a control. When the cutoff point of anti-TBGL antibody titer was determined as 2.0 U/ml, the sensitivity for active tuberculosis patients was 81.1% and the specificity was 95.7%. Sensitivity in patients with smear-negative and culture-negative active pulmonary tuberculosis was 73.5%. Even in patients with noncavitary minimally advanced lesions, the positivity rate (60.0%) and the antibody titer (4.6 +/- 9.4 U/ml) were significantly higher than those in the healthy group. These results indicate that this assay using anti-TBGL antibody is useful for the rapid serodiagnosis of active pulmonary tuberculosis.

A bone-seeking clone exhibits different biological properties from the MDA-MB-231 parental human breast cancer cells and a brain-seeking clone in vivo and in vitro.

Breast cancer has a predilection for spreading to bone. The mechanism of preferential metastasis of breast cancer to bone is unknown. We hypothesize that breast cancer cells that develop bone metastases have the capacity to facilitate their colonization in bone. To examine this hypothesis, we established bone-seeking (MDA-231BO) and brain-seeking (MDA-231BR) clones of the human breast cancer cell line MDA-MB-231 by repeated sequential passages in nude mice and in vitro of metastatic cells obtained from bone and brain metastases, respectively. These clones were examined for distinguishing biological characteristics and compared with the MDA-231 parental cells (MDA-231P) in vivo and in vitro. Both the MDA-231BR and the MDA-231BO showed identical tumorigenicity to MDA-231P at the orthotopic site. MDA-231P that was inoculated into the heart developed metastases in bone, brain, ovary, and adrenal glands. On the other hand, MDA-231BO exclusively metastasized to bone with larger osteolytic lesions than MDA-231P. MDA-231BR exclusively disseminated to brain and failed to develop bone metastases. In culture, MDA-231BO produced greater amounts of parathyroid hormone-related protein (PTH-rP) than MDA-231BR and MDA-231P in the absence or presence of transforming growth factor beta (TGF-beta). Furthermore, the anchorage-independent growth of MDA- 231BO in soft agar was not inhibited by TGF-beta, whereas TGF-beta profoundly inhibited the growth of MDA-231P and MDA-231BR. Insulin-like growth factor I (IGF-I) markedly promoted the anchorage-independent growth of MDA-231BO, whereas marginal or no stimulation was observed in MDA-231BR or MDA-231P, respectively. Our data suggest that these phenotypic changes allow breast cancer cells to promote osteoclastic bone resorption, survive, and proliferate in bone, which consequently leads to the establishment of bone metastases.

The bisphosphonate ibandronate promotes apoptosis in MDA-MB-231 human breast cancer cells in bone metastases.

Bisphosphonate (BP), a specific inhibitor of osteoclasts, has been widely used as a beneficial agent for the treatment of bone metastases in patients with breast cancer. It is well recognized that BP reduces osteolysis by promoting apoptosis in osteoclasts. However, recent animal and human data suggest that BPs not only reduce osteolysis associated with metastatic breast cancer, but also decrease tumor burden in bone. The mechanisms by which tumor burden is decreased following BP administration are unknown. Here we examined the effects of the BP ibandronate on MDA-231 human breast cancer cells in bone metastases in a well-characterized animal model of bone metastasis. Ibandronate, which was administered (s.c. daily; 4 microg/mouse/day) after bone metastases were established, inhibited the progression of established osteolytic bone metastases as assessed by radiographic analysis. Histological and histomorphometrical examination revealed that ibandronate reduced osteoclastic bone resorption, with increased apoptosis in osteoclasts. Furthermore, ibandronate also significantly decreased the MDA-231 tumor burden, with increased apoptosis in MDA-231 breast cancer cells in bone metastases. In contrast, ibandronate failed to inhibit MDA-231 tumor formation with no effects on apoptosis in MDA-231 breast cancer cells in the orthotopic mammary fat pads. These data suggest that the effects of ibandronate on apoptosis in MDA-231 breast cancer cells are restricted in bone in which ibandronate selectively deposits. Consistent with these in vivo results, a relatively high concentration of ibandronate (100 microM) increased caspase-3 activity and induced DNA fragmentation in MDA-231 breast cancer cells in culture. Moreover, a caspase inhibitor, z-Val-Ala-Asp-fluoromethyl ketone, blocked ibandronate-induced DNA fragmentation in MDA-231 cells, suggesting an involvement of caspase-3 in ibandronate-induced apoptosis. Our results suggest that BP suppresses bone metastases through promotion of apoptosis in metastatic cancer cells as well as in osteoclasts. However, it still remains open whether BP has direct anticancer actions in vivo.

Relaxation behavior of laser-polarized (129)Xe gas: size dependency and wall effect of the T(1) relaxation time in glass and gelatin bulbs.

Size dependency of the relaxation time T(1) was measured for laser-polarized (129)Xe gas encapsulated in different sized cavities made by glass bulbs or gelatin capsules. The use of laser-polarized gas enhances the sensitivity a great deal, making it possible to measure the longer (129)Xe relaxation time in quite a short time. The size dependency is analyzed on the basis of the kinetic theory of gases and a relationship is derived in which the relaxation rate is connected with the square inverse of the diameter of the cavity. Such an analysis provides a novel parameter which denotes the wall effect on the relaxation rate when a gas molecule collides with the surface once in a second. The relaxation time of (129)Xe gas is also dependent on the material which forms the cavity. This dependency is large and the relaxation study using polarized (129)Xe gas is expected to offer important information about the state of the matter of the cavity wall.

Preclinical studies with zoledronic acid and other bisphosphonates: impact on the bone microenvironment.

The propensity for breast cancer cells to metastasize to bone and to induce osteolysis has long been recognized. Characteristics of both the tumor cells and the bone microenvironment contribute to this phenomenon. The presence of tumor in bone is associated with activation of osteoclasts, resulting in excessive bone resorption and subsequent osteolysis. Breast cancer cells and other tumor types influence osteoclastic bone resorption by increasing the number of osteoclasts and enhancing their resorptive activity. Parathyroid hormone-related peptide, in addition to its role in humorally mediated hypercalcemia, is secreted by metastatic breast cancer cells in bone in which it acts as a paracrine factor to stimulate osteoclasts. As bone matrix is broken down by activated osteoclasts, a rich supply of mitogenic factors is released, including insulin-like growth factors, bone morphogenetic proteins, and fibroblast growth factors. Transforming growth factor (TGF)-beta, one of the most abundant of the bone-derived factors, promotes increased production of parathyroid hormone-related peptide by tumor cells, establishing a "vicious cycle" leading to progressive tumor growth and bone destruction. Bisphosphonates interrupt this cycle by inhibiting osteoclasts, in part by inducing osteoclast apoptosis. In several animal models of breast cancer metastasis to bone, bisphosphonates decrease the number of new bone metastases and inhibit progression of existing lesions. A single 3 microg intravenous injection of zoledronic acid (Zometa; Novartis Pharmaceuticals Corp, East Hanover, NJ), a new highly potent bisphosphonate, prevented destruction of trabecular bone in an orthotopic mouse mammary tumor model. Tumor volume in bone was decreased by zoledronic acid in a dose-dependent manner in the same model, and tumor cell apoptosis was increased by zoledronic acid in bone metastases in the 4T1 murine model of mammary carcinoma metastasis. Zoledronic acid at a dose of 1.0 microg/d for 10 days also reduced bone lesion area in a nude mouse model with existing bone metastases. Although bisphosphonates, including zoledronic acid, are able to induce apoptosis in tumor cells in vitro, studies in animal models to date have generally not shown a reduction in nonosseous tumor. Therefore, bisphosphonate-associated tumor reduction in bone is most likely mediated by osteoclast inhibition or is related to high local concentrations of bisphosphonates in the bone compartment.

2, 3, 7, 8-tetrachlorodibenzo-p-dioxin induces apoptosis in the dorsal midbrain of zebrafish embryos by activation of arylhydrocarbon receptor.

Neurotoxic effects of 2, 3, 7, 8-tetrachlorodibenzo-p-dioxin (TCDD) has not been fully elucidated, despite the known potent agonist of arylhydrocarbon receptor (AhR), which activation induces cytochrome P450 1A and several representative toxicities of halogenated aromatic hydrocarbons. In the present study, the effects of TCDD on cell death in zebrafish embryos (Danio rerio) during the early stage of development were investigated. As shown by terminal transferase-mediated nick-end-labeling staining, TCDD exposure significantly increased the occurrence of pycnotic cell death (PCD), especially in the dorsal midbrain (optic tectum). The ultrastructures of these pycnotic cells showed apoptotic features such as condensation and cleavage of chromatin. TCDD-induced PCD was mimicked by beta-naphthoflavone (AhR agonist), and inhibited by alpha-naphthoflavone (AhR antagonist). These results suggest that AhR activation can induce apoptosis in the central nervous system during development.

Ca(2+) signaling in porcine duodenal glands by muscarinic receptor activation.

The duodenal glands have been thought to play an important role in defense against proximal duodenal ulcer; however, the secretory mechanisms of these glands remain to be determined. In isolated duodenal acinar cells of the pig, we investigated the effects of ACh on intracellular Ca(2+) concentration ([Ca(2+)](i)) and on membrane currents with fura 2 fluorometry and the patch clamp technique. ACh caused a transient increase in [Ca(2+)](i), and the increase was markedly inhibited by atropine or 4-diphenylacetoxy-N-methylpiperidine methiodide but not by hexamethonium, pirenzepine, or methoctramine. The expression of mRNA for the M(3) subtype far exceeded that for either M(1) or M(2) as revealed by real-time quantitative PCR and in situ hybridization. The rise in [Ca(2+)](i) evoked by ACh was largely inhibited by thapsigargin but slightly affected by extracellular Ca(2+) deprivation. Caffeine had no effect on [Ca(2+)](i). ACh elicited Ca(2+)-dependent K(+) currents, a finding similar to the response to inositol 1,4,5,-trisphosphate applied intracellularly. These results suggest the presence of M(3) receptors linked to Ca(2+) release in porcine duodenal glands.

[Detection of rifampin-resistant Mycobacterium tuberculosis by line probe assay (LiPA)].

A recently described reverse hybridization-based line probe assay is used for the rapid detection of the mutations in the rpoB genes of rifampin-resistant Mycobacterium tuberculosis and for the identification of the M. tuberculosis complex. A multicenter study that included 5 laboratories was performed to evaluate the line probe assay in comparison with the in vitro susceptibility test. A total of 406 mycobacteria isolates which were composed of 103 rifampin-resistant and 230 rifampin-susceptible M. tuberculosis isolates, and 73 mycobacteria other than tubercle bacilli (MOTT), were subjected to this study. All 333 M. tuberculosis isolates were discriminated correctly from MOTT bacilli by a line probe assay. Concordance rates with sequencing results for five wild-type probes (S probes) and four specific mutations (R probes) for detecting the mutations in the rpoB genes were both 100%. The overall concordance rate with the in vitro susceptibility testing results was 98.5% (328 of 333 isolates). These results indicate that a line probe assay kit may be useful for the rapid diagnosis of rifampin-resistant tuberculosis.

Actions of bisphosphonate on bone metastasis in animal models of breast carcinoma.

BACKGROUND: Bone, which abundantly stores a variety of growth factors, provides a fertile soil for cancer cells to develop metastases by supplying these growth factors as a consequence of osteoclastic bone resorption. Accordingly, suppression of osteoclast activity is a primary approach to inhibit bone metastasis, and bisphosphonate (BP), a specific inhibitor of osteoclasts, has been widely used for the treatment of bone metastases in cancer patients. To obtain further insights into the therapeutic usefulness of BP, the authors studied the effects of BP on bone and visceral metastases in animal models of metastasis. METHODS: The authors used two animal models of breast carcinoma metastasis that they had developed in their laboratory over the last several years. One model uses female young nude mice in which inoculation of the MDA-MB-231 or MCF-7 human breast carcinoma cells into the left cardiac ventricle selectively develops osteolytic or osteosclerotic bone metastases, respectively. Another model uses syngeneic female mice (Balb/c) in which orthotopic inoculation of the 4T1 murine mammary carcinoma cells develops metastases in bone and visceral organs including lung, liver, and kidney. RESULTS: BP inhibited the development and progression of osteolytic bone metastases of MDA-MB-231 breast carcinoma through increased apoptosis in osteoclasts and breast carcinoma cells colonized in bone. In a preventative administration, however, BP alone increased the metastases to visceral organs with profound inhibition of bone metastases. However, combination of BP with anticancer agents such as uracil and tegafur or doxorubicin suppressed the metastases not only in bone but also visceral organs and prolonged the survival in 4T1 mammary tumor-bearing animals. Of interest, inhibition of early osteolysis by BP inhibited the subsequent development of osteosclerotic bone metastases of MCF-7 breast carcinoma. CONCLUSIONS: These results suggest that BP has beneficial effects on bone metastasis of breast carcinoma and is more effective when combined with anticancer agents. They also suggest that the animal models of bone metastasis described here allow us to design optimized regimen of BP administration for the treatment of breast carcinoma patients with bone and visceral metastases.

Use of bisphosphonates for the treatment of bone metastasis in experimental animal models.

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 clinically- relevant information that is useful for the design of optimal regimens of BP for the treatment of breast cancer patients with bone and visceral metastases.