Although (-)-epigallocatechin gallate (EGCG) has been reported to induce apoptosis in a variety of tumor cells, detailed mechanisms remain to be explored. In the present study, we investigated the antitumor mechanism of EGCG by using human T-cell acute lymphoblastic leukemia Jurkat cells. We focused on the involvement of reactive oxygen species, as we found previously that EGCG caused apoptotic cell death in osteoclastic cells due mainly to promotion of the reduction of Fe(III) to Fe(II) to trigger Fenton reaction, which affords hydroxyl radical from hydrogen peroxide [H(2)O(2) + Fe(II) --> (*)OH + OH(-) + Fe(III)]. EGCG (12.5-50 micro M) decreased the viability of Jurkat cells and caused concomitant increase in cellular caspase-3 activity. Catalase and the Fe(II)-chelating reagent o-phenanthroline suppressed the EGCG effects, indicating involvements of both H(2)O(2) and Fe(II) in the mechanism. Unexpectedly, epicatechin gallate (ECG), which has Fe(III)-reducing potency comparable with EGCG, failed to decrease the viability of Jurkat cells, while epigallocatechin (EGC), which has low capacity to reduce Fe(III), showed cytotoxic effects similar to EGCG. These results suggest that, unlike in osteoclastic cells, a mechanism other than Fe(III) reduction plays a role in catechin-mediated Jurkat cell death. We found that EGCG causes an elevation of H(2)O(2) levels in Jurkat cell culture, in cell-free culture medium and sodium phosphate buffer. Catechins with a higher ability to produce H(2)O(2) were more cytotoxic to Jurkat cells. Hydrogen peroxide itself exerted Fe(II)-dependent cytotoxicity. Amongst tumor and normal cell lines tested, cells exhibiting lower H(2)O(2)-eliminating activity were more sensitive to EGCG. From these findings, we propose the mechanism that make catechins cytotoxic in certain tumor cells is due to their ability to produce H(2)O(2) and that the resulting increase in H(2)O(2) levels triggers Fe(II)-dependent formation of highly toxic hydroxyl radical, which in turn induces apoptotic cell death.

LPS is a potent stimulator of bone resorption in inflammatory diseases. The mechanism by which LPS induces osteoclastogenesis was studied in cocultures of mouse osteoblasts and bone marrow cells. LPS stimulated osteoclast formation and PGE2 production in cocultures of mouse osteoblasts and bone marrow cells, and the stimulation was completely inhibited by NS398, a cyclooxygenase-2 inhibitor. Osteoblasts, but not bone marrow cells, produced PGE2 in response to LPS. LPS-induced osteoclast formation was also inhibited by osteoprotegerin (OPG), a decoy receptor of receptor activator of NF-κB ligand (RANKL), but not by anti-mouse TNFR1 Ab or IL-1 receptor antagonist. LPS induced both stimulation of RANKL mRNA expression and inhibition of OPG mRNA expression in osteoblasts. NS398 blocked LPS-induced down-regulation of OPG mRNA expression, but not LPS-induced up-regulation of RANKL mRNA expression, suggesting that down-regulation of OPG expression by PGE2 is involved in LPS-induced osteoclast formation in the cocultures. NS398 failed to inhibit LPS-induced osteoclastogenesis in cocultures containing OPG knockout mouse-derived osteoblasts. IL-1 also stimulated PGE2 production in osteoblasts and osteoclast formation in the cocultures, and the stimulation was inhibited by NS398. As seen with LPS, NS398 failed to inhibit IL-1-induced osteoclast formation in cocultures with OPG-deficient osteoblasts. These results suggest that IL-1 as well as LPS stimulates osteoclastogenesis through two parallel events: direct enhancement of RANKL expression and suppression of OPG expression, which is mediated by PGE2 production.

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Lipopolysaccharide (LPS), a cell component of Gram-negative bacteria, is a pathogen of inflammatory bone loss. To examine the effects of LPS on the survival and fusion of osteoclasts, mononuclear osteoclasts (preosteoclasts, pOCs) were collected from a mouse co-culture system and cultured in the presence or absence of LPS. Most pOCs died within 24 h in the absence of any stimulus. LPS as well as receptor activator of NF-kappaB ligand (RANKL) supported the survival of pOCs, and induced their fusion to form multinucleated cells (MNCs). Like authentic osteoclasts, MNCs induced by LPS expressed calcitonin receptors, and formed actin rings on culture plates. LPS-induced MNC formation in pOC cultures was observed even in the presence of osteoprotegerin and interleukin (IL)-1-receptor antagonists. MNC formation was also stimulated by LPS in pOC cultures prepared from tumor necrosis factor (TNF)-receptor-I or TNF-receptor-II deficient mice. LPS induced the degradation of IkappaB in pOCs within 20 min. Lactacystin, an inhibitor of NF-kappaB activation, and wortmannin, an inhibitor of phosphatidylinositol-3 kinase, strongly inhibited LPS-induced MNC formation in pOC cultures. LPS induced pit-forming activity of pOCs in the presence of macrophage-colony stimulating factor (M-CSF). These findings suggest that LPS stimulates the survival and fusion of pOCs, independent of RANKL, IL-1 or TNF-alpha action. Activation of NF-kappaB and phosphatidylinositol-3 kinase appeared to be involved in LPS-induced effects on pOCs. These observations suggest that LPS is involved directly in inflammatory bone loss, and also indirectly through the production of LPS-induced host factors such as IL-1 and TNF-alpha.

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Four new analogues of concanamycin family, designated concanamycins D, E, F and G, were isolated from the mycelium of Streptomyces sp. A1509by solvent extraction, silica gel column chromatography and HPLC.Structures of these compoundswere identified by the combination of spectroscopic analyses. All of these compoundswere structurally related to concanamycinsA, B and C, which had been isolated previously, and inhibited the acidification of rat liver lysosomes at 10~1 2^-10~9 mconcentration. The structure-activity study showedthat the 18-memberedmacrolide ring and the 6-membered hemiketal ring portions of the molecules of concanamycin family are responsible for potent inhibitory activity.