herbimycin and Hypercalcemia

Anti-bone resorption properties of the Korean herbal formulation, Honghwain (HHI; Carthamus tinctorius L. seed) was biochemically investigated. On processing bone metabolism, PGE2 accelerated production of IL-1beta in fetal mouse osteoblast and stimulated physiological activation substance, IL-1beta. The novel class of Src tyrosine kinase inhibitors, Herbimycin A (HERB) and HHI reduced COX-2 mRNA levels as well as PGE2 production induced by IL-1beta, TNF-alpha and IL-6. HHI inhibited in vitro and in vivo bone resorption by inhibition of phosphorylation of peptide substrates. HHI dose-dependently reduced the hypercalcemia induced in mice by IL-1beta and partly prevented bone loss and microarchitectural changes in young ovariectomized rats, showing that the protective effect on bone was exerted via the inhibition of bone resorption. These results indicate that the synergy between IL-beta, TNF-alpha, IL-6 on PGE2 production is due to an enhanced gene expression of COX-2 and that tyrosine kinase (s) are involved in the signal transduction of COX-2 in mouse calvarial osteoblasts. Thus, HHI as a possible Src family kinase inhibitor may be useful for the treatment of diseases associated with elevated bone loss.

Topics: Animals; Benzoquinones; Bone and Bones; Bone Resorption; Carthamus; Cell Separation; Cell Survival; Cells, Cultured; Cyclooxygenase 1; Cyclooxygenase 2; Cyclooxygenase 2 Inhibitors; Cyclooxygenase Inhibitors; Cytokines; Dinoprostone; Female; Hypercalcemia; Isoenzymes; Lactams, Macrocyclic; Male; Membrane Proteins; Mice; Osteoblasts; Osteoporosis; Ovariectomy; Parathyroid Hormone; Plant Extracts; Prostaglandin Antagonists; Prostaglandin-Endoperoxide Synthases; Protein-Tyrosine Kinases; Quinones; Rifabutin; RNA, Messenger; Seeds

Interleukin-6 (IL-6) is a multifunctional cytokine that is produced not only by a variety of normal cells but also by cancer cells. IL-6 produced by cancer cells stimulates the proliferation of these cancer cells in an autocrine/ paracrine manner and causes paraneoplastic syndromes including hypercalcemia, cachexia, and leukocytosis. We have reported previously that a human oral squamous cancer associated with hypercalcemia produces large amounts of IL-6, that animals bearing this cancer exhibit elevated levels of plasma IL-6, and that neutralizing antibodies to human IL-6 reverse hypercalcemia in tumor-bearing animals, indicating an important role of IL-6 in the hypercalcemia in this model. Because these cancer cells overexpress epidermal growth factor receptors (EGFR) with intrinsic tyrosine kinase (TK) activity similar to many other squamous cancers, we examined the effects of herbimycin A, a tyrosine kinase inhibitor, on IL-6 production and hypercalcemia in animals bearing this cancer to develop a new approach to treat the hypercalcemia associated with malignancy. Intraperitoneal administration (once a day for 2 days) of herbimycin A to cancer-bearing hypercalcemic mice reduced the plasma levels of human IL-6 and impaired the hypercalcemia. During 2-day treatment with herbimycin A, no changes were observed in tumor size. Of interest, plasma levels of mouse, but not human, soluble IL-6 receptors were also elevated. However, herbimycin A showed no effects on plasma levels of mouse soluble IL-6 receptors. Herbimycin A suppressed the tyrosine autophosphorylation of EGFR and IL-6 mRNA expression and production, all of which were stimulated by EGF. The data raise the possibility that TK inhibitors may be potential mechanism-based therapeutic agents for the treatment of hypercalcemia associated with squamous cancers which overexpress EGFR.

Topics: Animals; Antibiotics, Antineoplastic; Antigens, CD; Benzoquinones; Carcinoma, Squamous Cell; Enzyme Inhibitors; Epidermal Growth Factor; ErbB Receptors; Humans; Hypercalcemia; Interleukin-6; Lactams, Macrocyclic; Male; Mice; Mice, Nude; Neoplasm Transplantation; Protein-Tyrosine Kinases; Quinones; Receptors, Interleukin; Receptors, Interleukin-6; Rifabutin; Solubility; Tumor Cells, Cultured

Since absence of expression of the c-src gene product in mice indicates that the pp60c-src tyrosine kinase is required and essential for osteoclastic bone resorption, we tested the effects of the antibiotic herbimycin A, which is an inhibitor of pp60c-src on osteoclastic bone resorption in vitro and on hypercalcemia in vivo. We examined the effects of herbimycin A on the formation of bone resorbing osteoclasts in mouse long-term marrow cultures, on isolated rodent osteoclasts and on bone resorption in organ cultures of fetal rat long bones stimulated by parathyroid hormone. We found that herbimycin A in concentrations of 1-100 ng/ml inhibited bone resorption in each of these systems. We determined the effects of herbimycin A (100 ng/ml) on src tyrosine kinase activity in mouse marrow cultures and found that it was decreased. Herbimycin A also decreased elevated blood calcium levels that were induced either by repeated subcutaneous injections of recombinant human interleukin-1 alpha or by a human tumor. There was no evidence for toxicity in any of these culture systems or in mice treated with herbimycin A. A different tyrosine kinase inhibitor that does not inhibit pp60c-src was used as a control and caused none of these effects. These data suggest that pp60c-src tyrosine kinase inhibitors may be useful pharmacologic inhibitors of osteoclastic bone resorption and hypercalcemia.

Topics: Animals; Benzoquinones; Bone and Bones; Bone Marrow; Bone Resorption; Cells, Cultured; CSK Tyrosine-Protein Kinase; Hypercalcemia; Lactams, Macrocyclic; Mice; Osteoclasts; Protein-Tyrosine Kinases; Quinones; Rats; Rifabutin; src-Family Kinases