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1.
Neurosci Lett ; 469(1): 84-7, 2010 Jan 18.
Article in English | MEDLINE | ID: mdl-19944131

ABSTRACT

Caloric and food restriction attenuate oxidative stress. The effect of aging and every-other-day (EOD) feeding on oxygen radical-dependent chemiluminescent intensity was examined in ex vivo brain slices from Fischer rats during oxygenation and hypoxia-reoxygenation with lucigenin, a chemilumigenic probe used for detecting superoxide anion radicals. The chemiluminescent intensity increased during reoxygenation after hypoxic treatment, and the chemiluminescence in the brain slices at the baseline and during reoxygenation increased with age. However, no difference was observed in the superoxide-dependent chemiluminescence between brain slices prepared from the aged rats fed EOD and those fed ad libitum. Our results indicated that age-dependent increases in superoxide production might be associated with enhanced oxidative stress in aged Fischer rat brains. However, the present study newly indicated that decreased superoxide production might not be a major causal factor in caloric and food restriction attenuated oxidative stress.


Subject(s)
Aging/metabolism , Brain/metabolism , Caloric Restriction , Reactive Oxygen Species/metabolism , Animals , Eating , Hypoxia/metabolism , In Vitro Techniques , Luminescence , Oxidative Stress , Rats , Rats, Inbred F344 , Superoxides/metabolism
2.
Article in English | MEDLINE | ID: mdl-19342773

ABSTRACT

Parathyroid hormone-related protein (PTHrP) plays an important role in regulating embryonic skeletal development and is abnormally regulated in the pathogenesis of skeletal complications observed with many cancers and osteoporosis. It exerts its action through binding to a G-protein-coupled seven-transmembrane cell-surface receptor (GPCR). Structurally, GPCRs are very difficult to study by X-ray crystallography. In this study, a monoclonal antibody Fab fragment which recognizes the same region of PTHrP as its receptor, PTH1R, was used to aid in the crystallization of PTHrP. The resultant protein complex was crystallized using the hanging-drop vapour-diffusion method with polyethylene glycol as a precipitant. The crystals belonged to the orthorhombic space group P2(1)2(1)2, with unit-cell parameters a = 72.6, b = 96.3, c = 88.5 A, and diffracted to 2.0 A resolution using synchrotron radiation. The crystal structure will shed light on the nature of the key residues of PTHrP that interact with the antibody and will provide insights into how the antibody is able to discriminate between PTHrP and the related molecule parathyroid homone.


Subject(s)
Antibodies, Monoclonal/chemistry , Immunoglobulin Fab Fragments/chemistry , Parathyroid Hormone-Related Protein/chemistry , Receptors, G-Protein-Coupled/chemistry , Crystallography, X-Ray , Electrophoresis, Polyacrylamide Gel , Humans , Neutralization Tests , Protein Structure, Tertiary
3.
J Biol Chem ; 284(23): 15557-63, 2009 Jun 05.
Article in English | MEDLINE | ID: mdl-19346515

ABSTRACT

Parathyroid hormone-related protein (PTHrP) plays a vital role in the embryonic development of the skeleton and other tissues. When it is produced in excess by cancers it can cause hypercalcemia, and its local production by breast cancer cells has been implicated in the pathogenesis of bone metastasis formation in that disease. Antibodies have been developed that neutralize the action of PTHrP through its receptor, parathyroid hormone receptor 1, without influencing parathyroid hormone action through the same receptor. Such neutralizing antibodies against PTHrP are therapeutically effective in animal models of the humoral hypercalcemia of malignancy and of bone metastasis formation. We have determined the crystal structure of the complex between PTHrP (residues 1-108) and a neutralizing monoclonal anti-PTHrP antibody that reveals the only point of contact is an alpha-helical structure extending from residues 14-29. Another striking feature is that the same residues that interact with the antibody also interact with parathyroid hormone receptor 1, showing that the antibody and the receptor binding site on the hormone closely overlap. The structure explains how the antibody discriminates between the two hormones and provides information that could be used in the development of novel agonists and antagonists of their common receptor.


Subject(s)
Antibody Specificity , Cyclic AMP/metabolism , Receptor, Parathyroid Hormone, Type 1/immunology , Animals , Binding Sites , Bone Neoplasms , Cell Line, Tumor , Crystallography, X-Ray , Humans , Ligands , Models, Molecular , Neutralization Tests , Osteosarcoma , Protein Conformation , RNA, Messenger/genetics , Rats , Receptor, Parathyroid Hormone, Type 1/chemistry , Receptor, Parathyroid Hormone, Type 1/genetics , Receptor, Parathyroid Hormone, Type 1/metabolism , Surface Properties , X-Ray Diffraction
4.
Cancer Sci ; 98(10): 1563-9, 2007 Oct.
Article in English | MEDLINE | ID: mdl-17645771

ABSTRACT

It has been shown that in the mouse colon 26 tumor model, tumors grown in the subcutis (subcutis colon 26) caused early onset of cachectic syndromes, whereas those in the liver (liver colon 26) did not. Both interleukin (IL)-6 and parathyroid hormone-related protein (PTHrP) were involved in the development of cachectic syndromes in this tumor model. However, whether expression of PTHrP and IL-6 is differently regulated in the tumor microenvironment is unclear. In the present study, culturing the colon 26 cells under different conditions in vitro revealed that IL-6 production was increased by monolayer culture under a low-glucose condition but not by spheroid culture. In contrast, PTHrP production was increased by spheroid culture but not by monolayer culture, even under a low-glucose condition. Gene expression profiling revealed that the expression of cyclooxygenase (COX)-2 was up-regulated in both subcutis colon 26 and spheroid cultures, and that COX-2 inhibitor NS-398 suppressed PTHrP production in spheroid cultures. Furthermore, administration of NS-398 decreased the PTHrP level without affecting the tumor growth in mice bearing subcutis colon 26. These results demonstrate that production of PTHrP and IL-6 largely depends on the microenvironments in which tumors are developed or metastasized and that up-regulation of COX-2 in a necrobiotic environment leads to PTHrP production, thereby causing cachectic syndromes.


Subject(s)
Biomarkers, Tumor/metabolism , Colonic Neoplasms/metabolism , Cyclooxygenase 2/metabolism , Parathyroid Hormone-Related Protein/metabolism , Animals , Becaplermin , Cachexia/etiology , Calcium/metabolism , Colonic Neoplasms/pathology , Cyclooxygenase Inhibitors/pharmacology , Gene Expression Profiling , Glucose/metabolism , Humans , Interleukin-6/metabolism , Male , Mice , Necrosis , Nitrobenzenes/pharmacology , Oligonucleotide Array Sequence Analysis , Platelet-Derived Growth Factor/metabolism , Proto-Oncogene Proteins c-sis , Reverse Transcriptase Polymerase Chain Reaction , Sulfonamides/pharmacology , Tumor Cells, Cultured
5.
Anticancer Res ; 25(6B): 3817-23, 2005.
Article in English | MEDLINE | ID: mdl-16309168

ABSTRACT

BACKGROUND: Parathyroid hormone-related protein (PTHrP) has been implicated in bone metastasis. However, the effects on bone metastasis of blocking the PTHrP function have not been tested in the clinic. Here, the effects of a humanized anti-PTHrP monoclonal antibody (mAb) on bone metastasis in a human xenograft model are shown. MATERIALS AND METHODS: Subline MDA-5a, with high bone metastatic activity, was established from the human breast cancer cell line MDA-MB-231. Mice were injected with MDA-5a and an anti-PTHrP monoclonal antibody (mAb) raised against human PTHrP (1-34); bone metastasis was evaluated by X-ray photography. RESULTS: MDA-5a produced elevated levels of PTHrP, Interleukin 8 (IL-8), IL-6 and matrix metalloproteinase 1 (MMP-1) and frequently metastasized to the bone. Administration of the humanized anti-PTHrP mAb significantly suppressed osteolytic bone metastasis of MDA-5a and caused osteogenesis at the sites of metastasis. CONCLUSION: The humanized anti-PTHrP mAb was effective against bone metastasis by inducing osteogenesis and, therefore, will provide a new treatment option for bone metastasis in breast cancer.


Subject(s)
Antibodies, Monoclonal/pharmacology , Bone Neoplasms/prevention & control , Breast Neoplasms/drug therapy , Parathyroid Hormone-Related Protein/immunology , Animals , Antibodies, Monoclonal/immunology , Bone Neoplasms/genetics , Bone Neoplasms/metabolism , Bone Neoplasms/secondary , Breast Neoplasms/genetics , Breast Neoplasms/metabolism , Breast Neoplasms/pathology , Cell Line, Tumor , Female , Gene Expression Profiling , Humans , Interleukins/biosynthesis , Interleukins/genetics , Matrix Metalloproteinase 1/biosynthesis , Matrix Metalloproteinase 1/genetics , Mice , Mice, Inbred BALB C , Mice, Nude , Oligonucleotide Array Sequence Analysis , Parathyroid Hormone-Related Protein/biosynthesis , Parathyroid Hormone-Related Protein/genetics , Xenograft Model Antitumor Assays
6.
Clin Cancer Res ; 11(11): 4198-203, 2005 Jun 01.
Article in English | MEDLINE | ID: mdl-15930357

ABSTRACT

PURPOSE: Bisphosphonate and calcitonin lower blood calcium in humoral hypercalcemia of malignancy (HHM) by suppressing osteoclastic bone resorption, but repeated administration of these drugs often leads to relapse. In this study, we examined the roles of parathyroid hormone-related protein (PTHrP) in the development of bisphosphonate- and calcitonin-refractory HHM. EXPERIMENTAL DESIGN: Nude rats bearing the LC-6 JCK tumor xenograft (LC-6 rats) exhibited high bone turnover and HHM. Repeated administration of alendronate induced a sustained suppression of the bone resorption, but it caused only early and transient reduction of the blood calcium levels, leading to unresponsiveness to the drug. Because high blood levels of PTHrP were detected in the LC-6 rats, those that developed alendronate-refractory HHM were treated with an anti-PTHrP antibody. RESULTS: Administration of anti-PTHrP antibody to animals that received repeated administration of alendronate, thereby developing alendronate-refractory HHM, resulted in an increase in fractional excretion of calcium and a marked decrease of blood calcium level. Drug-refractory HHM was also observed in animals that received another osteoclast inhibitor, an eel calcitonin analogue elcatonin. The blood calcium level decreased after the initial administration of elcatonin, but it eventually became elevated during repeated administration. Administration of the anti-PTHrP antibody, but not of alendronate, effectively reduced the blood calcium of the animals that developed elcatonin-refractory HHM. CONCLUSION: High levels of circulating PTHrP and the resulting augmentation of renal calcium reabsorption is one of the major causes of the emergence of osteoclast inhibitor-refractory HHM. Thus, blockage of PTHrP functions by a neutralizing antibody against PTHrP would benefit patients who develop bisphosphonate- or calcitonin-refractory HHM.


Subject(s)
Alendronate/therapeutic use , Calcium/metabolism , Hypercalcemia/etiology , Kidney/metabolism , Lung Neoplasms/complications , Parathyroid Hormone-Related Protein/physiology , Alendronate/administration & dosage , Animals , Antibodies, Monoclonal/immunology , Antibodies, Monoclonal/pharmacology , Bone Resorption/prevention & control , Calcitonin/administration & dosage , Calcitonin/analogs & derivatives , Calcitonin/therapeutic use , Calcium/blood , Calcium/pharmacokinetics , Cell Line, Tumor , Diphosphonates/administration & dosage , Diphosphonates/therapeutic use , Drug Resistance , Humans , Hypercalcemia/drug therapy , Kidney/drug effects , Lung Neoplasms/metabolism , Lung Neoplasms/pathology , Mice , Mice, Inbred BALB C , Mice, Nude , Parathyroid Hormone/blood , Parathyroid Hormone-Related Protein/blood , Parathyroid Hormone-Related Protein/immunology , Rats , Rats, Inbred F344 , Rats, Nude , Time Factors , Xenograft Model Antitumor Assays
7.
Int J Cancer ; 116(3): 471-8, 2005 Sep 01.
Article in English | MEDLINE | ID: mdl-15800941

ABSTRACT

Nude rats bearing the LC-6-JCK human lung cancer xenograft displayed cancer-associated wasting syndrome in addition to humoral hypercalcemia of malignancy. In these rats, not only PTHrP but also several other human proinflammatory cytokines, such as IL-6, leukemia-inducing factor, IL-8, IL-5 and IL-11, were secreted to the bloodstream. Proinflammatory cytokines induce acute-phase reactions, as evidenced by a decrease of serum albumin and an increase in alpha1-acid glycoprotein. Tumor resection abolished the production of proinflammatory cytokines and improved acute-phase reactions, whereas anti-PTHrP antibody affected neither proinflammatory cytokine production nor acute-phase reactions. Nevertheless, tumor resection and administration of anti-PTHrP antibody similarly and markedly attenuated not only hypercalcemia but also loss of fat, muscle and body weight. Body weight gain by anti-PTHrP antibody was associated with increased food consumption; increased body weight from anti-PTHrP antibody was observed when animals were freely fed but not when they were given the same feeding as those that received only vehicle. Furthermore, nude rats bearing LC-6-JCK showed reduced locomotor activity, less eating and drinking and low blood phosphorus; and anti-PTHrP antibody restored them. Although alendronate, a bisphosphonate drug, decreased blood calcium, it affected neither locomotor activity nor serum phosphorus level. These results indicate that PTHrP represses physical activity and energy metabolism independently of hypercalcemia and proinflammatory cytokine actions and that deregulation of such physiologic activities and functions by PTHrP is at least in part involved in PTHrP-induced wasting syndrome.


Subject(s)
Adenocarcinoma/pathology , Carcinoma, Squamous Cell/pathology , Energy Metabolism , Kidney Neoplasms/pathology , Laryngeal Neoplasms/pathology , Lung Neoplasms/pathology , Motor Activity , Pancreatic Neoplasms/pathology , Parathyroid Hormone-Related Protein/physiology , Wasting Syndrome/physiopathology , Adenocarcinoma/veterinary , Animals , Body Weight , Carcinoma, Squamous Cell/veterinary , Cytokines/blood , Cytokines/metabolism , Disease Models, Animal , Feeding Behavior , Humans , Hypercalcemia/complications , Hypercalcemia/physiopathology , Hypercalcemia/veterinary , Kidney Neoplasms/veterinary , Laryngeal Neoplasms/veterinary , Lung Neoplasms/veterinary , Pancreatic Neoplasms/veterinary , Rats , Rats, Nude , Transplantation, Heterologous , Wasting Syndrome/veterinary
8.
Anticancer Res ; 24(5A): 2665-73, 2004.
Article in English | MEDLINE | ID: mdl-15517871

ABSTRACT

A humanized monoclonal antibody against parathyroid hormone-related protein (PTHrP) was generated from the mouse monoclonal antibody raised against the peptide corresponding to the N-terminal 34 amino acids of the human PTHrP [(PTHrP(1-34)]. The humanized antibody interacted with the PTHrP(1-34) with a kD value of 1.90 x 10(-10) M, and the epitope resides between the amino acids 20 and 30 of the PTHrP. PTHrP(1-34) significantly increased the intracellular cAMP levels in the rat osteosarcoma cells that expressed PTHR1, and the 5 microg/mL or higher concentrations of the humanized antibody almost completely blocked the PTHrP-induced cAMP production even in the presence of 2 microg/mL PTHrP(1-34), demonstrating its ability to fully neutralize PTHrP function. There was no significant difference in the potency of the mouse, chimera, or the humanized antibodies to suppress the PTHrP-induced increase in the intracellular cAMP in ROS cells. Furthermore, at the same doses, the administration of the chimera or the humanized antibody was equally effective in reducing the blood ionized calcium levels of hypercalcemic mice bearing the PAN-7-JCK human pancreatic cancer xenograft or the LC-6-JCK human lung cancer xenograft that secreted PTHrP. Thus, humanized anti-PTHrP may be useful for the treatment of the humoral hypercalcemia of malignancy in humans.


Subject(s)
Antibodies, Monoclonal/immunology , Antibodies, Monoclonal/pharmacology , Hypercalcemia/therapy , Parathyroid Hormone-Related Protein/antagonists & inhibitors , Parathyroid Hormone-Related Protein/immunology , Peptide Fragments/immunology , Amino Acid Sequence , Animals , Antibodies, Monoclonal/biosynthesis , Antibodies, Monoclonal/genetics , Cell Line, Tumor , Humans , Hypercalcemia/blood , Hypercalcemia/etiology , Immunoglobulin Heavy Chains/genetics , Immunoglobulin Light Chains/genetics , Lung Neoplasms/blood , Lung Neoplasms/complications , Male , Mice , Mice, Inbred BALB C , Mice, Nude , Molecular Sequence Data , Pancreatic Neoplasms/blood , Pancreatic Neoplasms/complications , Rats , Rats, Inbred F344 , Rats, Nude , Recombinant Fusion Proteins/biosynthesis , Recombinant Fusion Proteins/genetics , Recombinant Fusion Proteins/immunology , Recombinant Fusion Proteins/pharmacology , Xenograft Model Antitumor Assays
9.
Semin Oncol ; 30(5 Suppl 16): 167-73, 2003 Oct.
Article in English | MEDLINE | ID: mdl-14613038

ABSTRACT

Parathyroid hormone-related protein (PTHrP) plays a central role in humoral hypercalcemia of malignancy (HHM), which is one of the most frequent paraneoplastic syndromes. PTHrP produced by the tumor acts through a common PTH/PTHrP receptor to promote bone resorption, inhibit calcium excretion from the kidney, and induce hypercalcemia. Patients with HHM often develop cachexia associated with typical symptoms such as anorexia, malaise, nausea, constipation, polyuria, polydipsia, and confusion. The etiology of the cachexia is not fully understood but is thought to be caused by hypercalcemia and various cytokines such as interleukin-6, tumor necrosis factor-alpha, leukemia inhibitory factor, and others. In this study, we investigated the role of PTHrP in hypercalcemia and cachexia in HHM by using humanized anti-PTHrP antibody. A mouse monoclonal antibody that binds to PTHrP amino acid sequence 1-34 and inhibits PTHrP function has been humanized to create a specific and potent agent for the treatment of patients with HHM. The mouse monoclonal antibody has been shown to have antihypercalcemic activity against nude mice bearing human tumors. Because a mouse antibody is highly immunogenic in human patients, the complementarity-determining regions from the mouse antibody were grafted into a human antibody. The resulting humanized antibody specifically recognizes PTHrP(1-34) and neutralizes PTHrP functions in vitro and in vivo. The humanized anti-PTHrP antibody was administered intravenously to HHM model animals bearing tumors such as LC-6 human lung carcinoma. These animals showed symptoms similar to those of patients with HHM (eg, hypercalcemia and cachexia). The humanized anti-PTHrP antibody-treated animals responded with normalization of blood ionized calcium level through an improvement of bone metabolism and calcium excretion. Moreover, the treated animals also showed an improvement in body weight, ultromotivity, metabolic alkalosis, food consumption, water intake, serum phosphorus, and renal function. Consequently, the humanized antibody-treated animals experienced complete resolution of hypercalcemia and cachexia. These results suggest that the humanized antibody would be an effective and beneficial agent for patients with HHM, and that PTHrP is a major pathogenetic factor of hypercalcemia and cachexia in patients with HHM.


Subject(s)
Antibodies, Monoclonal/therapeutic use , Cachexia/drug therapy , Hypercalcemia/drug therapy , Neoplasms/metabolism , Paraneoplastic Syndromes/drug therapy , Parathyroid Hormone-Related Protein/metabolism , Animals , Cachexia/etiology , Cachexia/metabolism , Humans , Hypercalcemia/etiology , Hypercalcemia/metabolism , Mice , Models, Animal , Neoplasms/complications , Paraneoplastic Syndromes/metabolism , Parathyroid Hormone-Related Protein/immunology
10.
Cancer Lett ; 199(2): 147-55, 2003 Sep 25.
Article in English | MEDLINE | ID: mdl-12969787

ABSTRACT

We investigated the effects of treatment with anti-parathyroid hormone-related protein (1-34) monoclonal murine antibody (anti-PTHrP MoAb) on apoptosis and the differentiation of chondrosarcoma HTB-94 cells. Treatment with anti-PTHrP MoAb accelerated apoptosis of HTB-94 cells in a dose-dependent manner, and anti-PTHrP MoAb also promoted the chondrogenic differentiation of HTB-94 cells. The induction of apoptosis by anti-PTHrP MoAb via imbalance of Bcl-2/Bax ratio and activation of caspase-3 may provide a mechanistic explanation for its potential antitumor effects. Our results suggest the possibility that anti-PTHrP MoAb may be beneficial as a new treatment for chondrosarcoma.


Subject(s)
Antibodies, Monoclonal/pharmacology , Apoptosis/drug effects , Bone Neoplasms/pathology , Cell Differentiation/drug effects , Chondrosarcoma/pathology , Parathyroid Hormone-Related Protein/immunology , Bone Neoplasms/immunology , Bone Neoplasms/metabolism , Caspase 3 , Caspases/metabolism , Cell Division/drug effects , Chondrosarcoma/immunology , Chondrosarcoma/metabolism , Collagen Type II/genetics , Collagen Type II/metabolism , Collagen Type X/genetics , Collagen Type X/metabolism , DNA Primers/chemistry , Humans , Immunoenzyme Techniques , Peptide Fragments/immunology , Proto-Oncogene Proteins/genetics , Proto-Oncogene Proteins/metabolism , Proto-Oncogene Proteins c-bcl-2/genetics , Proto-Oncogene Proteins c-bcl-2/metabolism , Reverse Transcriptase Polymerase Chain Reaction , Tumor Cells, Cultured , bcl-2-Associated X Protein
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