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Brevinin-2R(1) semi-selectively kills cancer cells by a distinct mechanism, which involves the lysosomal-mitochondrial death pathway.

Ghavami S, Asoodeh A, Klonisch T, Halayko AJ, Kadkhoda K, Kroczak TJ, Gibson SB, Booy EP, Naderi-Manesh H, Los M - J. Cell. Mol. Med. (2008)

Bottom Line: The decrease in mitochondrial membrane potential (DeltaPsim), or total cellular ATP levels, and increased reactive oxygen species (ROS) production, but not caspase activation or the release of apoptosis-inducing factor (AIF) or endonuclease G (Endo G), were early indicators of Brevinin-2R-triggered death.Autophagosomes have been detected upon Brevinin-2R treatment.Our results show that Brevinin-2R activates the lysosomalmitochondrial death pathway, and involves autophagy-like cell death.

View Article: PubMed Central - PubMed

Affiliation: Department of Biochemistry and Medical Genetics, Manitoba Institute of Cell Biology, Cancer Care Manitoba, Winnipeg, Manitoba, Canada.

ABSTRACT
Brevinin-2R is a novel non-hemolytic defensin that was isolated from the skin of the frog Rana ridibunda. It exhibits preferential cytotoxicity towards malignant cells, including Jurkat (T-cell leukemia), BJAB (B-cell lymphoma), HT29/219, SW742 (colon carcinomas), L929 (fibrosarcoma), MCF-7 (breast adenocarcinoma), A549 (lung carcinoma), as compared to primary cells including peripheral blood mononuclear cells (PBMC), T cells and human lung fibroblasts. Jurkat and MCF-7 cells overexpressing Bcl2, and L929 and MCF-7 over-expressing a dominant-negative mutant of a pro-apoptotic BNIP3 (DeltaTM-BNIP3) were largely resistant towards Brevinin-2R treatment. The decrease in mitochondrial membrane potential (DeltaPsim), or total cellular ATP levels, and increased reactive oxygen species (ROS) production, but not caspase activation or the release of apoptosis-inducing factor (AIF) or endonuclease G (Endo G), were early indicators of Brevinin-2R-triggered death. Brevinin-2R interacts with both early and late endosomes. Lysosomal membrane permeabilization inhibitors and inhibitors of cathepsin-B and cathepsin-L prevented Brevinin-2R-induced cell death. Autophagosomes have been detected upon Brevinin-2R treatment. Our results show that Brevinin-2R activates the lysosomalmitochondrial death pathway, and involves autophagy-like cell death.

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Brevinin kills cancer cells by a mechanism not relying on caspases. (A) BJAB and MCF-7 cells were treated with Brevinin-2R (10 μg/ml) for indicated times. Some samples were cotreated with zVAD-fmk (60 μm) broad spectrum caspase inhibitor. Cell viability was assessed by MTT assay. Data represent average values obtained from three independent experiments. (B) Jurkat cells were treated with Brevinin-2R (10 μg/ml) and with anti-CD95 (0.5 μg/ml) for indicated time periods which were chosen based on the assumption that caspase activation should become detectable about 1–2 hrs prior to morphologic manifestation of cell death. Total cell extracts were harvested, resolved on SDS-PAGE and active subunits of caspase-3, -8 and -9 were detected by Western blot. (C) In an experiment parallel to the one depicted in (B), caspase activity in Jurkat cells was measured by a Caspase-Glo® luminometric assay. The caspase activity is represented as a ‘-fold increase’ as compared to the control. The data represent triplicates of three independent experiments. (D) MCF-7 cells treated with Brevinin-2R for 6 hrs were photographed to indicate the morphology of dying cells.
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fig02: Brevinin kills cancer cells by a mechanism not relying on caspases. (A) BJAB and MCF-7 cells were treated with Brevinin-2R (10 μg/ml) for indicated times. Some samples were cotreated with zVAD-fmk (60 μm) broad spectrum caspase inhibitor. Cell viability was assessed by MTT assay. Data represent average values obtained from three independent experiments. (B) Jurkat cells were treated with Brevinin-2R (10 μg/ml) and with anti-CD95 (0.5 μg/ml) for indicated time periods which were chosen based on the assumption that caspase activation should become detectable about 1–2 hrs prior to morphologic manifestation of cell death. Total cell extracts were harvested, resolved on SDS-PAGE and active subunits of caspase-3, -8 and -9 were detected by Western blot. (C) In an experiment parallel to the one depicted in (B), caspase activity in Jurkat cells was measured by a Caspase-Glo® luminometric assay. The caspase activity is represented as a ‘-fold increase’ as compared to the control. The data represent triplicates of three independent experiments. (D) MCF-7 cells treated with Brevinin-2R for 6 hrs were photographed to indicate the morphology of dying cells.

Mentions: We examined the time kinetics of Brevinin-2R-induced cell death and its dependence on the cas-pase family of proteases. Brevinin-2R applied at a concentration of 10 μg/ml induced cell death within the first 2–4 hrs (Fig. 2A). Brevinin-2R-triggered toxicity could not be efficiently blocked by the broad-spectrum caspase inhibitor zVAD-fmk. Processing and activation of caspase-3 -9 and -8 were not detected in Brevinin-2R-treated Jurkat cells (Fig. 2B). This was confirmed by our inability to detect a significant increase in the corresponding enzymatic activities (DEVD-ase, LEHD-ase, IETD-ase; Fig. 2C). DEVD-ase (Caspase-3/7) activity was increased ∼1-fold and these traces of DEVD-ase activity (Fig. 2C) may explain some protection from Brevinin-2R-induced cell death observed upon treatment with zVAD-fmk pan-caspase inhibitor. The morphology of dying cells resembled apoptosis to some degree, with cells rapidly shrinking but rarely exhibiting other hallmarks of apoptosis, including membrane bleb-bing or cell detachment (Fig. 2D).


Brevinin-2R(1) semi-selectively kills cancer cells by a distinct mechanism, which involves the lysosomal-mitochondrial death pathway.

Ghavami S, Asoodeh A, Klonisch T, Halayko AJ, Kadkhoda K, Kroczak TJ, Gibson SB, Booy EP, Naderi-Manesh H, Los M - J. Cell. Mol. Med. (2008)

Brevinin kills cancer cells by a mechanism not relying on caspases. (A) BJAB and MCF-7 cells were treated with Brevinin-2R (10 μg/ml) for indicated times. Some samples were cotreated with zVAD-fmk (60 μm) broad spectrum caspase inhibitor. Cell viability was assessed by MTT assay. Data represent average values obtained from three independent experiments. (B) Jurkat cells were treated with Brevinin-2R (10 μg/ml) and with anti-CD95 (0.5 μg/ml) for indicated time periods which were chosen based on the assumption that caspase activation should become detectable about 1–2 hrs prior to morphologic manifestation of cell death. Total cell extracts were harvested, resolved on SDS-PAGE and active subunits of caspase-3, -8 and -9 were detected by Western blot. (C) In an experiment parallel to the one depicted in (B), caspase activity in Jurkat cells was measured by a Caspase-Glo® luminometric assay. The caspase activity is represented as a ‘-fold increase’ as compared to the control. The data represent triplicates of three independent experiments. (D) MCF-7 cells treated with Brevinin-2R for 6 hrs were photographed to indicate the morphology of dying cells.
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Related In: Results  -  Collection

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fig02: Brevinin kills cancer cells by a mechanism not relying on caspases. (A) BJAB and MCF-7 cells were treated with Brevinin-2R (10 μg/ml) for indicated times. Some samples were cotreated with zVAD-fmk (60 μm) broad spectrum caspase inhibitor. Cell viability was assessed by MTT assay. Data represent average values obtained from three independent experiments. (B) Jurkat cells were treated with Brevinin-2R (10 μg/ml) and with anti-CD95 (0.5 μg/ml) for indicated time periods which were chosen based on the assumption that caspase activation should become detectable about 1–2 hrs prior to morphologic manifestation of cell death. Total cell extracts were harvested, resolved on SDS-PAGE and active subunits of caspase-3, -8 and -9 were detected by Western blot. (C) In an experiment parallel to the one depicted in (B), caspase activity in Jurkat cells was measured by a Caspase-Glo® luminometric assay. The caspase activity is represented as a ‘-fold increase’ as compared to the control. The data represent triplicates of three independent experiments. (D) MCF-7 cells treated with Brevinin-2R for 6 hrs were photographed to indicate the morphology of dying cells.
Mentions: We examined the time kinetics of Brevinin-2R-induced cell death and its dependence on the cas-pase family of proteases. Brevinin-2R applied at a concentration of 10 μg/ml induced cell death within the first 2–4 hrs (Fig. 2A). Brevinin-2R-triggered toxicity could not be efficiently blocked by the broad-spectrum caspase inhibitor zVAD-fmk. Processing and activation of caspase-3 -9 and -8 were not detected in Brevinin-2R-treated Jurkat cells (Fig. 2B). This was confirmed by our inability to detect a significant increase in the corresponding enzymatic activities (DEVD-ase, LEHD-ase, IETD-ase; Fig. 2C). DEVD-ase (Caspase-3/7) activity was increased ∼1-fold and these traces of DEVD-ase activity (Fig. 2C) may explain some protection from Brevinin-2R-induced cell death observed upon treatment with zVAD-fmk pan-caspase inhibitor. The morphology of dying cells resembled apoptosis to some degree, with cells rapidly shrinking but rarely exhibiting other hallmarks of apoptosis, including membrane bleb-bing or cell detachment (Fig. 2D).

Bottom Line: The decrease in mitochondrial membrane potential (DeltaPsim), or total cellular ATP levels, and increased reactive oxygen species (ROS) production, but not caspase activation or the release of apoptosis-inducing factor (AIF) or endonuclease G (Endo G), were early indicators of Brevinin-2R-triggered death.Autophagosomes have been detected upon Brevinin-2R treatment.Our results show that Brevinin-2R activates the lysosomalmitochondrial death pathway, and involves autophagy-like cell death.

View Article: PubMed Central - PubMed

Affiliation: Department of Biochemistry and Medical Genetics, Manitoba Institute of Cell Biology, Cancer Care Manitoba, Winnipeg, Manitoba, Canada.

ABSTRACT
Brevinin-2R is a novel non-hemolytic defensin that was isolated from the skin of the frog Rana ridibunda. It exhibits preferential cytotoxicity towards malignant cells, including Jurkat (T-cell leukemia), BJAB (B-cell lymphoma), HT29/219, SW742 (colon carcinomas), L929 (fibrosarcoma), MCF-7 (breast adenocarcinoma), A549 (lung carcinoma), as compared to primary cells including peripheral blood mononuclear cells (PBMC), T cells and human lung fibroblasts. Jurkat and MCF-7 cells overexpressing Bcl2, and L929 and MCF-7 over-expressing a dominant-negative mutant of a pro-apoptotic BNIP3 (DeltaTM-BNIP3) were largely resistant towards Brevinin-2R treatment. The decrease in mitochondrial membrane potential (DeltaPsim), or total cellular ATP levels, and increased reactive oxygen species (ROS) production, but not caspase activation or the release of apoptosis-inducing factor (AIF) or endonuclease G (Endo G), were early indicators of Brevinin-2R-triggered death. Brevinin-2R interacts with both early and late endosomes. Lysosomal membrane permeabilization inhibitors and inhibitors of cathepsin-B and cathepsin-L prevented Brevinin-2R-induced cell death. Autophagosomes have been detected upon Brevinin-2R treatment. Our results show that Brevinin-2R activates the lysosomalmitochondrial death pathway, and involves autophagy-like cell death.

Show MeSH
Related in: MedlinePlus