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Cytotoxic L -amino-acid oxidases from Amanita phalloides and Clitocybe geotropa induce caspase-dependent apoptosis

View Article: PubMed Central - PubMed

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.

No MeSH data available.


Effects of ApLAO and CgLAO in death receptor-mediated apoptosis of Jurkat cells. (a) Representative images of the localization of ApLAO conjugated with FITC (ApLAO-FITC; green fluorescence) in control Jurkat cells and in cells treated with ApLAO-FITC for 1, 10 and 60 min (white arrows), as analyzed by fluorescence microscopy. Scale bars=20 μm (b and c) Jurkat cells were pretreated with Fas/FasL inhibitor Kp7-6 (0.1–0.5 mM) for 1 h, followed by ApLAO (0.5 μg/ml) or CgLAO (5 μg/ml) treatment. After 24 h, cell viability was assessed by MTS assay (b). Cells were treated in quadruplicate. Results are the means±S.D. of three independent assays. *P<0.05. Cytotoxicity was assessed by staining cells with PI and the percentage of PIpos cells was determined by flow cytometry (c). Cells were treated in duplicate. Results are the means±S.D. of two independent assays. *P<0.05.
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fig6: Effects of ApLAO and CgLAO in death receptor-mediated apoptosis of Jurkat cells. (a) Representative images of the localization of ApLAO conjugated with FITC (ApLAO-FITC; green fluorescence) in control Jurkat cells and in cells treated with ApLAO-FITC for 1, 10 and 60 min (white arrows), as analyzed by fluorescence microscopy. Scale bars=20 μm (b and c) Jurkat cells were pretreated with Fas/FasL inhibitor Kp7-6 (0.1–0.5 mM) for 1 h, followed by ApLAO (0.5 μg/ml) or CgLAO (5 μg/ml) treatment. After 24 h, cell viability was assessed by MTS assay (b). Cells were treated in quadruplicate. Results are the means±S.D. of three independent assays. *P<0.05. Cytotoxicity was assessed by staining cells with PI and the percentage of PIpos cells was determined by flow cytometry (c). Cells were treated in duplicate. Results are the means±S.D. of two independent assays. *P<0.05.

Mentions: The Fas receptor is a death receptor on the surface of cells that leads to the extrinsic apoptotic pathway, through caspase-8 activation.26 As an increase in caspase-8 activation was observed in cells treated with LAOs, we further investigated whether the extrinsic pathway of apoptosis is triggered. To address this question, ApLAO protein was conjugated with a fluorescent molecule FITC (ApLAO-FITC). Exposure of Jurkat cells to ApLAO-FITC showed the periplasmic membrane localization of ApLAO after 1 and 10 min of treatment, whereas 1 h treatment resulted in morphological changes and reduced fluorescence of the conjugated protein (Figure 6a). Further, the ability of LAO to induce cell death by association with death receptor was investigated. Jurkat cells were pretreated with Fas/FasL antagonist Kp7-6 (0.5 mM) for 1 h and then treated with ApLAO (0.5 μg/ml) or CgLAO (5 μ/ml) for an additional 24 h. Fas/FasL antagonist had a small impact on the viability of ApLAO- and CgLAO-reduced cells (Figure 6b). Furthermore, ApLAO- and CgLAO-induced damage of cell membrane integrity, as assessed by flow cytometry, was reduced in the presence of 0.5 mM Kp7-6 (Figure 6c), suggesting that Fas receptor has some role in LAO-induced cell death.


Cytotoxic L -amino-acid oxidases from Amanita phalloides and Clitocybe geotropa induce caspase-dependent apoptosis
Effects of ApLAO and CgLAO in death receptor-mediated apoptosis of Jurkat cells. (a) Representative images of the localization of ApLAO conjugated with FITC (ApLAO-FITC; green fluorescence) in control Jurkat cells and in cells treated with ApLAO-FITC for 1, 10 and 60 min (white arrows), as analyzed by fluorescence microscopy. Scale bars=20 μm (b and c) Jurkat cells were pretreated with Fas/FasL inhibitor Kp7-6 (0.1–0.5 mM) for 1 h, followed by ApLAO (0.5 μg/ml) or CgLAO (5 μg/ml) treatment. After 24 h, cell viability was assessed by MTS assay (b). Cells were treated in quadruplicate. Results are the means±S.D. of three independent assays. *P<0.05. Cytotoxicity was assessed by staining cells with PI and the percentage of PIpos cells was determined by flow cytometry (c). Cells were treated in duplicate. Results are the means±S.D. of two independent assays. *P<0.05.
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fig6: Effects of ApLAO and CgLAO in death receptor-mediated apoptosis of Jurkat cells. (a) Representative images of the localization of ApLAO conjugated with FITC (ApLAO-FITC; green fluorescence) in control Jurkat cells and in cells treated with ApLAO-FITC for 1, 10 and 60 min (white arrows), as analyzed by fluorescence microscopy. Scale bars=20 μm (b and c) Jurkat cells were pretreated with Fas/FasL inhibitor Kp7-6 (0.1–0.5 mM) for 1 h, followed by ApLAO (0.5 μg/ml) or CgLAO (5 μg/ml) treatment. After 24 h, cell viability was assessed by MTS assay (b). Cells were treated in quadruplicate. Results are the means±S.D. of three independent assays. *P<0.05. Cytotoxicity was assessed by staining cells with PI and the percentage of PIpos cells was determined by flow cytometry (c). Cells were treated in duplicate. Results are the means±S.D. of two independent assays. *P<0.05.
Mentions: The Fas receptor is a death receptor on the surface of cells that leads to the extrinsic apoptotic pathway, through caspase-8 activation.26 As an increase in caspase-8 activation was observed in cells treated with LAOs, we further investigated whether the extrinsic pathway of apoptosis is triggered. To address this question, ApLAO protein was conjugated with a fluorescent molecule FITC (ApLAO-FITC). Exposure of Jurkat cells to ApLAO-FITC showed the periplasmic membrane localization of ApLAO after 1 and 10 min of treatment, whereas 1 h treatment resulted in morphological changes and reduced fluorescence of the conjugated protein (Figure 6a). Further, the ability of LAO to induce cell death by association with death receptor was investigated. Jurkat cells were pretreated with Fas/FasL antagonist Kp7-6 (0.5 mM) for 1 h and then treated with ApLAO (0.5 μg/ml) or CgLAO (5 μ/ml) for an additional 24 h. Fas/FasL antagonist had a small impact on the viability of ApLAO- and CgLAO-reduced cells (Figure 6b). Furthermore, ApLAO- and CgLAO-induced damage of cell membrane integrity, as assessed by flow cytometry, was reduced in the presence of 0.5 mM Kp7-6 (Figure 6c), suggesting that Fas receptor has some role in LAO-induced cell death.

View Article: PubMed Central - PubMed

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.

No MeSH data available.