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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.

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ApLAO- and CgLAO-induced loss of mitochondrial function in Jurkat cells. (a and b) Cells were exposed to ApLAO (0.5 μg/ml) (a) or CgLAO (5 μg/ml) (b) in the absence or presence of catalase (1000 U/ml) and/or L-Leu (790 μg/ml) for the time period indicated. Mitochondrial transmembrane potential (Δψm) was then measured by flow cytometry using mitochondria-sensitive CMXRos dye. Cells were treated in duplicate. Results are the means±S.D. of two independent assays. *P<0.05. (c and d) Western blots showing the effect of ApLAO (0.5 μg/ml) (c) and CgLAO (5 μg/ml) (d) treatment at the time period indicated on the protein levels of Bax (upper panel) and Bcl-2 (lower panel) in Jurkat cells. The bar plot shows densitometric analysis of protein expression and represents the ratio of Bax to Bcl-2 expression (Bax/Bcl-2) relative to that in control cells. Results are the means±S.D. of two independent assays. *P<0.05.
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fig5: ApLAO- and CgLAO-induced loss of mitochondrial function in Jurkat cells. (a and b) Cells were exposed to ApLAO (0.5 μg/ml) (a) or CgLAO (5 μg/ml) (b) in the absence or presence of catalase (1000 U/ml) and/or L-Leu (790 μg/ml) for the time period indicated. Mitochondrial transmembrane potential (Δψm) was then measured by flow cytometry using mitochondria-sensitive CMXRos dye. Cells were treated in duplicate. Results are the means±S.D. of two independent assays. *P<0.05. (c and d) Western blots showing the effect of ApLAO (0.5 μg/ml) (c) and CgLAO (5 μg/ml) (d) treatment at the time period indicated on the protein levels of Bax (upper panel) and Bcl-2 (lower panel) in Jurkat cells. The bar plot shows densitometric analysis of protein expression and represents the ratio of Bax to Bcl-2 expression (Bax/Bcl-2) relative to that in control cells. Results are the means±S.D. of two independent assays. *P<0.05.

Mentions: It is well known that damage of mitochondria has a very important role in the intrinsic pathway of apoptosis.25 Alteration in the Δψm was therefore evaluated by flow cytometric analysis, using the mitochondria-sensitive MitoTracker Red CMXROS dye, in cells treated with ApLAO (0.5 μg/ml) or CgLAO (5 μg/ml) in the absence or presence of catalase (1000 U/ml). Significant dissipation Δψm appeared after 6 h treatment with ApLAO (Figure 5a) and with CgLAO (Figure 5b), increasing further with time of incubation. After 24 h, in the case of ApLAO treatment, Δψm was reduced to 49.2±6.2% of the value for control cells. Following CgLAO treatment, Δψm was reduced to 55.7±8.3% that of control cells. This reduction, however, was reversed in the presence of catalase, when mitochondrial function after 24 h of incubation with ApLAO recovered to 71.4±5.1% of the control value and, with CgLAO, to 76.7±0.9% (Figures 5a and b). When an amino-acid, L-Leu, was used, no significant changes in the ApLAO- or CgLAO-increased loss of vΔψm were observed (Figures 5a and b).


Cytotoxic L -amino-acid oxidases from Amanita phalloides and Clitocybe geotropa induce caspase-dependent apoptosis
ApLAO- and CgLAO-induced loss of mitochondrial function in Jurkat cells. (a and b) Cells were exposed to ApLAO (0.5 μg/ml) (a) or CgLAO (5 μg/ml) (b) in the absence or presence of catalase (1000 U/ml) and/or L-Leu (790 μg/ml) for the time period indicated. Mitochondrial transmembrane potential (Δψm) was then measured by flow cytometry using mitochondria-sensitive CMXRos dye. Cells were treated in duplicate. Results are the means±S.D. of two independent assays. *P<0.05. (c and d) Western blots showing the effect of ApLAO (0.5 μg/ml) (c) and CgLAO (5 μg/ml) (d) treatment at the time period indicated on the protein levels of Bax (upper panel) and Bcl-2 (lower panel) in Jurkat cells. The bar plot shows densitometric analysis of protein expression and represents the ratio of Bax to Bcl-2 expression (Bax/Bcl-2) relative to that in control cells. Results are the means±S.D. of two independent assays. *P<0.05.
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fig5: ApLAO- and CgLAO-induced loss of mitochondrial function in Jurkat cells. (a and b) Cells were exposed to ApLAO (0.5 μg/ml) (a) or CgLAO (5 μg/ml) (b) in the absence or presence of catalase (1000 U/ml) and/or L-Leu (790 μg/ml) for the time period indicated. Mitochondrial transmembrane potential (Δψm) was then measured by flow cytometry using mitochondria-sensitive CMXRos dye. Cells were treated in duplicate. Results are the means±S.D. of two independent assays. *P<0.05. (c and d) Western blots showing the effect of ApLAO (0.5 μg/ml) (c) and CgLAO (5 μg/ml) (d) treatment at the time period indicated on the protein levels of Bax (upper panel) and Bcl-2 (lower panel) in Jurkat cells. The bar plot shows densitometric analysis of protein expression and represents the ratio of Bax to Bcl-2 expression (Bax/Bcl-2) relative to that in control cells. Results are the means±S.D. of two independent assays. *P<0.05.
Mentions: It is well known that damage of mitochondria has a very important role in the intrinsic pathway of apoptosis.25 Alteration in the Δψm was therefore evaluated by flow cytometric analysis, using the mitochondria-sensitive MitoTracker Red CMXROS dye, in cells treated with ApLAO (0.5 μg/ml) or CgLAO (5 μg/ml) in the absence or presence of catalase (1000 U/ml). Significant dissipation Δψm appeared after 6 h treatment with ApLAO (Figure 5a) and with CgLAO (Figure 5b), increasing further with time of incubation. After 24 h, in the case of ApLAO treatment, Δψm was reduced to 49.2±6.2% of the value for control cells. Following CgLAO treatment, Δψm was reduced to 55.7±8.3% that of control cells. This reduction, however, was reversed in the presence of catalase, when mitochondrial function after 24 h of incubation with ApLAO recovered to 71.4±5.1% of the control value and, with CgLAO, to 76.7±0.9% (Figures 5a and b). When an amino-acid, L-Leu, was used, no significant changes in the ApLAO- or CgLAO-increased loss of vΔψm were observed (Figures 5a and b).

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.


Related in: MedlinePlus