Transforming growth factor-β (TGF-β) has been shown to induce apoptosis in normal hepatocytes and hepatoma cells both in vivo and in vitro. However, the mechanism by which TGF-β induces apoptosis is not clear. The antiapoptotic activity of antioxidants including N-acetyl-L-cysteine (Ac-Cys), ascorbic acid and a novel free radical scavenger, carboxyfullerene (C 60 ) on TGF-β-treated human hepatoma Hep3B cells was examined. Only the water-soluble hexacarboxylic acid derivative of C 60 was found to prevent TGF-β-induced apoptosis. Antiapoptotic activity of C 60 correlated its ability to eliminate TGF-β-generated reactive oxygen species (ROSs). However, C 60 did not interfere with TGF-β-activated PAI-1 promoter activity in the Hep3B cells. These results indicate that the signaling pathway of TGF-β-induced apoptosis may be related to the generation of ROSs and may be uncoupled from the TGF-β-activated gene promoter activity. Furthermore, the regioisomer of C 60 with a C 3 symmetry was more potent in protecting cells from apoptosis than that with a D 3 symmetry, and the C3 isomer had stronger interactions with lipid bilayers than the D 3 isomer. The spectroscopic analysis revealed that the C 3 isomer had stronger interactions with artificial lipid bilayers than the D 3 isomer. Therefore, our study indicates that C 60 may interact with membrane to eliminate TGF-β-induced ROSs and to prevent apoptosis occur in human hepatoma cells.Keywords : carboxyfullerene ; transforming growth factor-β ; apoptosis ; reactive oxygen species.Since its discovery, the pure carbon spheres of buckminster-inhibits cell proliferation, depending on the cell types and the presence of other growth factors [7Ϫ11]. Recent studies have fullerene (C 60 ) have generated great interest from many different branches of science and engineering. To investigate the chemical shown that TGF-β induces apoptosis in liver cells in vitro [12Ϫ 17], and transgenic mice overexpressing TGF-β develop continand physical characteristics of C 60 (and its larger fullerenes), many novel properties of C 60 were observed including its avid uing apoptotic death of hepatocytes as well as hepatic fibrosis in vivo [18]. Exogenous administration of TGF-β to rodents also reactivity with free radicals [1]. Buckminsterfullerenes, for example, are capable of adding multiple radicals to each molecule. results in a significant increase in hepatic cell death [13,15,19].These data strongly suggest that apoptosis induced by TGF-β The addition of as many as 34 methyl radicals to a single C 60 sphere has been reported, leading Krusic et al.[1] to characterize may be involved in various hepatic lesions. However, despite the identification of TGF-β receptors and the genes involved in C 60 as a 'radical sponge'. However, native C 60 is soluble only in organic solvents. Dugan et al. [2] have evaluated the possibility its downstream signaling pathway [20Ϫ27], the mechanism of TGF-β-induced apoptosis remains largely unknown. that the potent innate anti-oxidant properties of C 60 could be harn...

Ceramide has been suggested as the secondary messenger mediating the apoptotic signal for Fas engagement. By using different inhibitors, we demonstrated here that ceramide is unlikely a mediator of Fas-initiated apoptosis. First, cAMP prevented cell death induced by ceramide but not by Fas. Second, ceramide-triggered, but not Fas-triggered, apoptosis was antagonized by the free radical scavenger C60. Third, the metal chelator pyrrolidinedithiocarbamate suppressed ceramide-initiated DNA fragmentation but had no effect on the Fas-induced cell death. Fourth, the SAPK/ERK kinase dominant negative mutant, which attenuated ceramide-induced cell death, did not prevent Fas-induced apoptosis. Finally, activation of NF-κB inhibited ceramide-induced but not Fas-initiated apoptosis. The fact that many antagonists of ceramide-induced apoptosis could not suppress Fas-mediated cell death clearly indicates that ceramide is not the mediator for Fas-initiated apoptotic signal.

No abstract