Abstract
L-amino-acid oxidases (LAO) purified from fungi induce cell death in various mammalian cells including human tumor cell lines. The mechanism, however, remains poorly understood. In this study, we aimed to define a precise mechanism of cell death induced in Jurkat and MCF7 cancer cell lines by ApLAO and CgLAO, LAOs isolated from Amanita phalloides and Clitocybe geotropa, respectively. Cell death induced by both LAOs is shown to be concentration- and time-dependent, with higher toxic effects in Jurkat cells. LAO activity is required for the cytotoxicity. Detailed study on Jurkat cells further demonstrated that ApLAO and CgLAO both induce the intrinsic mitochondrial pathway of apoptosis, accompanied by a time-dependent depolarization of the mitochondrial membrane through the generation of reactive oxygen species. Treatment with the LAOs resulted in an increased ratio of the expression of proapoptotic Bax to that of antiapoptotic Bcl-2, subsequently leading to the activation of caspase-9 and -3. However, the pancaspase inhibitor, Z-VAD-FMK, did not completely abolish the cell death induced by either ApLAO or CgLAO, suggesting an alternative pathway for LAO-induced apoptosis. Indeed, caspase-8 activity in ApLAO- and CgLAO-treated cells was increased. Further, Fas/FasL (Fas ligand) antagonist caused a slight reduction in toxin-induced cell death, supporting the involvement of ApLAO and CgLAO in death-receptor-mediated apoptosis. These results thus provide new evidence that ApLAO and CgLAO induce apoptosis in Jurkat cells via both the intrinsic and extrinsic pathways, although the significantly higher increase of caspase-9 over caspase-8 activity suggests that it is the intrinsic pathway that is the predominant mode of ApLAO- and CgLAO-induced apoptosis.
Highlights
Apoptosis is a controlled form of cell death that has an important role in the development and maintenance of higher organisms
Jurkat cells were pretreated with Fas/Fas ligand (FasL) antagonist Kp7-6 (0.5 mM) for 1 h and treated with ApLAO (0.5 μg/ml) or L-amino-acid oxidases (LAO) from Clitocybe geotropa (CgLAO) (5 μ/ml) for an additional 24 h
The toxic effects of a toxogein isolated from the fruiting bodies of the edible trooping funnel C. geotropa (CgLAO) have been studied in comparison with those of toxophallin isolated from the death cap A. phalloides (ApLAO), using Jurkat and MCF7 cancer cell lines
Summary
Apoptosis is a controlled form of cell death that has an important role in the development and maintenance of higher organisms. Deregulation of apoptosis disrupts the fine balance between cell proliferation and cell death leading to diseases such as cancer.[1] Apoptosis is defined by several morphological and biochemical hallmarks such as cell shrinkage, membrane blebbing, chromatin condensation and exposure of phosphatidylserine at the plasma membrane.[2] It is triggered by two main pathways, extrinsic and/or intrinsic The former, known as the death receptor pathway, involves the ligation of death receptors such as the tumor necrosis factor receptor and Fas receptor, and results in activation of the initiator caspase-8. LAO activity induces edema and causes hemolytic and hemorrhagic effects; it promotes antimicrobial, antiviral and antiparasitic effects, induces apoptosis of tumor cells and inhibits platelet aggregation These effects caused by LAO activity are mediated mainly by the production of H2O2,17,18 leading to oxidative stress.[14] the apoptosis-inducing effects of several snake venom LAOs have been studied, with a view to their possible use in cancer therapy, the exact mechanism is still not known. Detailed study on Jurkat cells has demonstrated that ApLAO and CgLAO are involved in the multiple signaling pathways triggering apoptosis
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