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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