In the present study, we studied the molecular mechanism underlying cell death induced by a cancer chemoprotective compound benzyl isothiocyanate (BITC). The cytotoxic effect of BITC was examined in rat liver epithelial RL34 cells. Apoptosis was induced when the cells were treated with 20 M BITC, characterized by the appearance of phosphatidylserine on the outer surface of the plasma membrane and caspase-3 activation, whereas no caspase activation and propidium iodide incorporation into cell were detected with 50 M BITC that induced necrosis. The mitochondrial death pathway was suggested to be involved in BITC-induced apoptosis because the treatment of cells with BITC-induced caspase-9-dependent apoptosis and mitochondrial transmembrane potential (⌬⌿m) alteration. We demonstrated here for the first time that BITC directly modifies mitochondrial functions, including inhibition of respiration, mitochondrial swelling, and release of cytochrome c. Moreover, glutathione depletion by diethyl maleate significantly accelerated BITC-triggered apoptosis, suggesting the involvement of a redox-dependent mechanism. This was also implicated by the observations that intracellular accumulation of reactive oxygen species, including superoxide (O 2 . ) and hydroperoxides (HPOs), was indeed detected in the cells treated with BITC and that the intracellular HPO level was significantly attenuated by pretreatment with N-acetylcysteine. The treatment with a pharmacological scavenger of O 2 . , Tiron, also diminished the HPO formation by ϳ80%, suggesting that most of the HPOs were H 2 O 2 derived from the dismutation of O 2 . . These results suggest that BITC induces apoptosis through a mitochondial redox-sensitive mechanism.
Cell death plays a pivotal role in the body to maintain homeostasis during aging. Studies have shown that damaged cells, which must be removed from the body, accumulate during aging. Decay of the capacity and͞or control of cell death during aging is widely considered to be involved in some age-dependent diseases. We investigated the accumulation of protein carbonyls and the role of cell death induced by hydrogen peroxide in human fibroblasts from individuals of various ages (17-80 years). The results showed that levels of oxidatively modified proteins increased with age, not only in whole-cell lysates but also in mitochondrial fractions, and this change correlates with a decline in the intracellular ATP level. Exposure of fibroblasts to hydrogen peroxide led to cell death by apoptosis and necrosis. Younger (<60 years old) cells were more resistant to necrosis induced by hydrogen peroxide than were older cells (>60 years old), which contained lower levels of free ATP than did younger cells. Treatment of cells of all ages with inhibitors of ATP synthesis (oligomycin, 2,4-dinitrophenol, or 2-deoxyglucose) made them more susceptible to cell death but also led to a switch in the death mode from apoptosis to necrosis. Furthermore, hydrogen peroxide treatment led to a greater accumulation of several inflammatory cytokines (IL-6, IL-7, IL-16, and IL-17) and increased necrosis in older cells. These results suggest that agerelated decline in the ATP level reduces the capacity to induce apoptosis and promotes necrotic inflammation. This switch may trigger a number of age-dependent disorders.protein carbonyls ͉ cytokines ͉ oxidative stress ͉ mitochondria A ging is characterized by a general decline in physiological function that leads to morbidity and mortality. Although specific causes of this decline are not well known, there is some evidence that sustained damage inflicted by endogenously produced oxidants contribute to the development of the age-related deficits (1). These oxidants include superoxide (O 2•Ϫ ), hydrogen peroxide, and hydroxyl radicals (HO Ϫ ) and possibly singlet oxygen ( 1 O 2 ). They damage cellular macromolecules, including DNA (2), proteins (3), and lipids (4). The continuous threat of oxidative damage to the cell, tissue, and organism as a whole is underscored by the existence of an impressive array of cellular defenses that have evolved to battle these reactive oxidants. However, these defenses are not perfect and, consequently, cellular macromolecules become oxidatively damaged. If a cell becomes damaged and nonfunctional, apoptosis provides a mechanism to eliminate it to make room for its replacement with a new cell. If the apoptotic process does not work well, the accumulation of damaged cells will contribute significantly to aging (5) and age-associated degenerative diseases such as cancer, cardiovascular disease, immune system decline, brain dysfunction, and cataracts.Mitochondria constitute the greatest source of oxidants on the basis of the following evidence: (i) the mitochondrial electron ...
In the present study, we clarified the molecular mechanism underlying the relationship between benzyl isothiocyanate (BITC)-induced cell cycle arrest and apoptosis and the involvement of mitogen-activated protein kinases (MAPKs). The exposure of Jurkat human T-cell leukemia cells to BITC resulted in the inhibition of the G 2 -M progression that coincided with the apoptosis induction. The experiment using the phase-specific synchronized cells demonstrated that the G 2 -M phase-arrested cells are more sensitive to undergoing apoptotic stimulation by BITC than the cells in other phases. We also confirmed that BITC activated c-Jun N-terminal kinase (JNK) and p38 MAPK, but not extracellular signal-regulated kinase, at the concentration required for apoptosis induction. An experiment using a JNK-specific inhibitor SP600125 or a p38 MAPK inhibitor SB202190 indicated that BITC-induced apoptosis might be regulated by the activation of these two kinases. Conversely, BITC is likely to confine the Jurkat cells in the G 2 -M phase mainly through the p38 MAPK pathway because only the p38 MAPK inhibitor significantly attenuated the accumulation of inactive phosphorylated Cdc2 protein and the G 2 -Marrested cell numbers. We reported here for the first time that the antiapoptotic Bcl-2 protein was phosphorylated by the BITC treatment without significant alteration of the Bcl-2 total protein amount. This was abrogated by a JNK specific inhibitor SP600125 at the concentration required for specific inhibition of the c-Jun phosphorylation. Moreover, the spontaneous phosphorylation of antiapoptotic Bcl-2 in the G 2 -M synchronized cells was enhanced synergistically by the BITC treatment. Involvement of the MAPK activation in the Bcl-2 phosphorylation and apoptosis induction also was observed in HL-60 and HeLa cells. Thus, we identified the phosphorylated Bcl-2 as a key molecule linking the p38 MAPK-dependent cell cycle arrest with the JNK activation by BITC.
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