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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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Brevenin-2R-induced cell death involves lysosomal activation and is inhibited by specific inhibitors of the lyso-somal proton pump and cathepsin B/L. Pretreatment for 1 hr with Baf-A1 (0.05 μm) (A), z-FF-fmk (100 μM)(B, D) and CA-074-Me (20 μm) (C, E) reduced the cytotoxic effect of Brevinin-2R (5 and 10 μg/ml for 2 hrs) on L929 and MCF-7 as assessed by MTT assay. Data represent average values from triplicates of three independent experiments.(F): Early increase in cytoplasmic lysotracker red-stained granules upon Brevinin-2R treatment is inhibited by Baf A1 pre-treatment. L929 were treated with Brevinin-2R (10 μg/ml for 2 hrs) and stained with the acidophilic lysosomal probe LysoTracker Red (LTR). Brevinin-2R caused an increase in volume and frequency of cytoplasmic granules staining with LTR. Nuclei were visualized by DAPI.(G): L929 pretreated with Baf-A1 (0.05 μM) failed to show cytoplasmic granular pattern of LTR fluorescence after Brevinin-2R-treatment. Nuclei were visualized by DAPI.(H): MCF-7 were treated with Brevinin-2R (10 μg/ml for 2 hrs) and immunostained with an anti-LAMP-Cy5-conjugated (magenta) antibody and an anti-cathepsin-B-antibody followed by corresponding Cy-3 conjugated secondary antibody (red). Cell nuclei were counterstained with DAPI. In control cells cathepsin-B colocalized with LAMP. Upon treatment with Brevinin-2R, cathepsin-B translocated to the cytosol and the nucleus.(I) Brevinin-2R shows very high interaction with lysosomes. The precise interaction of Brevinin-2R with lysosomes was determined by confocal microscopy. L929 cells were treated either with FITC-labeled scrambled Brevinin-2R-like peptide and Brevinin-2R (both 10 μg/ml for 2 hrs). Brevinin-2R, but not, scrambled Brevinin-2R-like peptide showed strong direct interaction with lysosomes. Cells were counterstained with DAPI and LTR to visualize nuclei and lysosomes, respectively.(J, K) Brevinin-2R activates early and late endosomes. MCF-7 were treated with Brevinin-2R (10 μg/ml for 2 hrs) prior to immunostaining with an anti-early endosome marker (EEM1) (J) and anti-mannose 6-phosphate receptor (late endosome marker, LEM) (K) followed by corresponding FITC-conjugated secondary antibody (green). Cell nuclei were counterstained with DAPI (L, M, N, O). Brevinin-2R induces morphological hallmarks of autophagic cell death. Untreated (L) or treated (M, N, O; Brevinin-2R:10 μg/ml for 2 hrs) L929 cells were investigated by Electron Microscopy. We observed (M) disintegration of mitochondria and ER, (N) autophagosomes and (O) strongly increased cytoplasmic vacuolization and autophagic sequestration of organelles. Magnification: 8.4 × 103 (L, M), 4.6 × 103 (N) and 11.5 × 103 (M).
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fig04: Brevenin-2R-induced cell death involves lysosomal activation and is inhibited by specific inhibitors of the lyso-somal proton pump and cathepsin B/L. Pretreatment for 1 hr with Baf-A1 (0.05 μm) (A), z-FF-fmk (100 μM)(B, D) and CA-074-Me (20 μm) (C, E) reduced the cytotoxic effect of Brevinin-2R (5 and 10 μg/ml for 2 hrs) on L929 and MCF-7 as assessed by MTT assay. Data represent average values from triplicates of three independent experiments.(F): Early increase in cytoplasmic lysotracker red-stained granules upon Brevinin-2R treatment is inhibited by Baf A1 pre-treatment. L929 were treated with Brevinin-2R (10 μg/ml for 2 hrs) and stained with the acidophilic lysosomal probe LysoTracker Red (LTR). Brevinin-2R caused an increase in volume and frequency of cytoplasmic granules staining with LTR. Nuclei were visualized by DAPI.(G): L929 pretreated with Baf-A1 (0.05 μM) failed to show cytoplasmic granular pattern of LTR fluorescence after Brevinin-2R-treatment. Nuclei were visualized by DAPI.(H): MCF-7 were treated with Brevinin-2R (10 μg/ml for 2 hrs) and immunostained with an anti-LAMP-Cy5-conjugated (magenta) antibody and an anti-cathepsin-B-antibody followed by corresponding Cy-3 conjugated secondary antibody (red). Cell nuclei were counterstained with DAPI. In control cells cathepsin-B colocalized with LAMP. Upon treatment with Brevinin-2R, cathepsin-B translocated to the cytosol and the nucleus.(I) Brevinin-2R shows very high interaction with lysosomes. The precise interaction of Brevinin-2R with lysosomes was determined by confocal microscopy. L929 cells were treated either with FITC-labeled scrambled Brevinin-2R-like peptide and Brevinin-2R (both 10 μg/ml for 2 hrs). Brevinin-2R, but not, scrambled Brevinin-2R-like peptide showed strong direct interaction with lysosomes. Cells were counterstained with DAPI and LTR to visualize nuclei and lysosomes, respectively.(J, K) Brevinin-2R activates early and late endosomes. MCF-7 were treated with Brevinin-2R (10 μg/ml for 2 hrs) prior to immunostaining with an anti-early endosome marker (EEM1) (J) and anti-mannose 6-phosphate receptor (late endosome marker, LEM) (K) followed by corresponding FITC-conjugated secondary antibody (green). Cell nuclei were counterstained with DAPI (L, M, N, O). Brevinin-2R induces morphological hallmarks of autophagic cell death. Untreated (L) or treated (M, N, O; Brevinin-2R:10 μg/ml for 2 hrs) L929 cells were investigated by Electron Microscopy. We observed (M) disintegration of mitochondria and ER, (N) autophagosomes and (O) strongly increased cytoplasmic vacuolization and autophagic sequestration of organelles. Magnification: 8.4 × 103 (L, M), 4.6 × 103 (N) and 11.5 × 103 (M).

Mentions: The vacuolar H+ -ATPase inhibitor Baf A1 (0.05 μm), the irreversible inhibitor of cathepsin-B and cathepsin-L zFF-fmk (100 μm) and the cathepsin-B inhibitor CA-074-Me (10 μm) partially inhibited Brevinin-2R cytotoxicity in MCF-7 and L929 (Fig. 4A–E). Staining of cells with the acidophilic lyso-somal probe LTR revealed that Brevinin-2R caused an increase in lysosomal volume (Fig. 4F) and this effect was not abolished in the presence of Baf A1 (Fig. 4G). We observed co-localization of cathepsin-B with the specific lysosomal marker LAMP-1 indicating the presence of cathepsin-B in lysosomes. In Brevinin-2R-treated cells, cathepsin-B translocated from the lysosome to the cytosol (Fig. 4H) but this lysosomal release of cathepsin-B was greatly reduced in the presence of Baf A1 (data not shown). Thus, Brevinin-2R caused a selective increase in lysosomal membrane permeabilization (LMP), which involved the activity of lysosomal vacuolar H+ ATPase. Furthermore, we obtained evidence for a direct interaction between Brevinin-2R and lysosomes, which may account for the observed lysosomal damage (Fig. 4I). Moreover, cells treated with this defensin showed early and late endosomal activation (Fig. 4J–K), and Brevinin-2R co-localized with markers for early (EEA1) and late endosomes (mannose-6-phosphate receptor, M-6-PR) (data was not shown). These observations indicate interaction of Brevinin-2R with the lysosomal compartment, which initiates a sequence of fatal events including lysoso-mal damage, cathepsin leakage into the cytosol and overall cell damage and death.


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)

Brevenin-2R-induced cell death involves lysosomal activation and is inhibited by specific inhibitors of the lyso-somal proton pump and cathepsin B/L. Pretreatment for 1 hr with Baf-A1 (0.05 μm) (A), z-FF-fmk (100 μM)(B, D) and CA-074-Me (20 μm) (C, E) reduced the cytotoxic effect of Brevinin-2R (5 and 10 μg/ml for 2 hrs) on L929 and MCF-7 as assessed by MTT assay. Data represent average values from triplicates of three independent experiments.(F): Early increase in cytoplasmic lysotracker red-stained granules upon Brevinin-2R treatment is inhibited by Baf A1 pre-treatment. L929 were treated with Brevinin-2R (10 μg/ml for 2 hrs) and stained with the acidophilic lysosomal probe LysoTracker Red (LTR). Brevinin-2R caused an increase in volume and frequency of cytoplasmic granules staining with LTR. Nuclei were visualized by DAPI.(G): L929 pretreated with Baf-A1 (0.05 μM) failed to show cytoplasmic granular pattern of LTR fluorescence after Brevinin-2R-treatment. Nuclei were visualized by DAPI.(H): MCF-7 were treated with Brevinin-2R (10 μg/ml for 2 hrs) and immunostained with an anti-LAMP-Cy5-conjugated (magenta) antibody and an anti-cathepsin-B-antibody followed by corresponding Cy-3 conjugated secondary antibody (red). Cell nuclei were counterstained with DAPI. In control cells cathepsin-B colocalized with LAMP. Upon treatment with Brevinin-2R, cathepsin-B translocated to the cytosol and the nucleus.(I) Brevinin-2R shows very high interaction with lysosomes. The precise interaction of Brevinin-2R with lysosomes was determined by confocal microscopy. L929 cells were treated either with FITC-labeled scrambled Brevinin-2R-like peptide and Brevinin-2R (both 10 μg/ml for 2 hrs). Brevinin-2R, but not, scrambled Brevinin-2R-like peptide showed strong direct interaction with lysosomes. Cells were counterstained with DAPI and LTR to visualize nuclei and lysosomes, respectively.(J, K) Brevinin-2R activates early and late endosomes. MCF-7 were treated with Brevinin-2R (10 μg/ml for 2 hrs) prior to immunostaining with an anti-early endosome marker (EEM1) (J) and anti-mannose 6-phosphate receptor (late endosome marker, LEM) (K) followed by corresponding FITC-conjugated secondary antibody (green). Cell nuclei were counterstained with DAPI (L, M, N, O). Brevinin-2R induces morphological hallmarks of autophagic cell death. Untreated (L) or treated (M, N, O; Brevinin-2R:10 μg/ml for 2 hrs) L929 cells were investigated by Electron Microscopy. We observed (M) disintegration of mitochondria and ER, (N) autophagosomes and (O) strongly increased cytoplasmic vacuolization and autophagic sequestration of organelles. Magnification: 8.4 × 103 (L, M), 4.6 × 103 (N) and 11.5 × 103 (M).
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fig04: Brevenin-2R-induced cell death involves lysosomal activation and is inhibited by specific inhibitors of the lyso-somal proton pump and cathepsin B/L. Pretreatment for 1 hr with Baf-A1 (0.05 μm) (A), z-FF-fmk (100 μM)(B, D) and CA-074-Me (20 μm) (C, E) reduced the cytotoxic effect of Brevinin-2R (5 and 10 μg/ml for 2 hrs) on L929 and MCF-7 as assessed by MTT assay. Data represent average values from triplicates of three independent experiments.(F): Early increase in cytoplasmic lysotracker red-stained granules upon Brevinin-2R treatment is inhibited by Baf A1 pre-treatment. L929 were treated with Brevinin-2R (10 μg/ml for 2 hrs) and stained with the acidophilic lysosomal probe LysoTracker Red (LTR). Brevinin-2R caused an increase in volume and frequency of cytoplasmic granules staining with LTR. Nuclei were visualized by DAPI.(G): L929 pretreated with Baf-A1 (0.05 μM) failed to show cytoplasmic granular pattern of LTR fluorescence after Brevinin-2R-treatment. Nuclei were visualized by DAPI.(H): MCF-7 were treated with Brevinin-2R (10 μg/ml for 2 hrs) and immunostained with an anti-LAMP-Cy5-conjugated (magenta) antibody and an anti-cathepsin-B-antibody followed by corresponding Cy-3 conjugated secondary antibody (red). Cell nuclei were counterstained with DAPI. In control cells cathepsin-B colocalized with LAMP. Upon treatment with Brevinin-2R, cathepsin-B translocated to the cytosol and the nucleus.(I) Brevinin-2R shows very high interaction with lysosomes. The precise interaction of Brevinin-2R with lysosomes was determined by confocal microscopy. L929 cells were treated either with FITC-labeled scrambled Brevinin-2R-like peptide and Brevinin-2R (both 10 μg/ml for 2 hrs). Brevinin-2R, but not, scrambled Brevinin-2R-like peptide showed strong direct interaction with lysosomes. Cells were counterstained with DAPI and LTR to visualize nuclei and lysosomes, respectively.(J, K) Brevinin-2R activates early and late endosomes. MCF-7 were treated with Brevinin-2R (10 μg/ml for 2 hrs) prior to immunostaining with an anti-early endosome marker (EEM1) (J) and anti-mannose 6-phosphate receptor (late endosome marker, LEM) (K) followed by corresponding FITC-conjugated secondary antibody (green). Cell nuclei were counterstained with DAPI (L, M, N, O). Brevinin-2R induces morphological hallmarks of autophagic cell death. Untreated (L) or treated (M, N, O; Brevinin-2R:10 μg/ml for 2 hrs) L929 cells were investigated by Electron Microscopy. We observed (M) disintegration of mitochondria and ER, (N) autophagosomes and (O) strongly increased cytoplasmic vacuolization and autophagic sequestration of organelles. Magnification: 8.4 × 103 (L, M), 4.6 × 103 (N) and 11.5 × 103 (M).
Mentions: The vacuolar H+ -ATPase inhibitor Baf A1 (0.05 μm), the irreversible inhibitor of cathepsin-B and cathepsin-L zFF-fmk (100 μm) and the cathepsin-B inhibitor CA-074-Me (10 μm) partially inhibited Brevinin-2R cytotoxicity in MCF-7 and L929 (Fig. 4A–E). Staining of cells with the acidophilic lyso-somal probe LTR revealed that Brevinin-2R caused an increase in lysosomal volume (Fig. 4F) and this effect was not abolished in the presence of Baf A1 (Fig. 4G). We observed co-localization of cathepsin-B with the specific lysosomal marker LAMP-1 indicating the presence of cathepsin-B in lysosomes. In Brevinin-2R-treated cells, cathepsin-B translocated from the lysosome to the cytosol (Fig. 4H) but this lysosomal release of cathepsin-B was greatly reduced in the presence of Baf A1 (data not shown). Thus, Brevinin-2R caused a selective increase in lysosomal membrane permeabilization (LMP), which involved the activity of lysosomal vacuolar H+ ATPase. Furthermore, we obtained evidence for a direct interaction between Brevinin-2R and lysosomes, which may account for the observed lysosomal damage (Fig. 4I). Moreover, cells treated with this defensin showed early and late endosomal activation (Fig. 4J–K), and Brevinin-2R co-localized with markers for early (EEA1) and late endosomes (mannose-6-phosphate receptor, M-6-PR) (data was not shown). These observations indicate interaction of Brevinin-2R with the lysosomal compartment, which initiates a sequence of fatal events including lysoso-mal damage, cathepsin leakage into the cytosol and overall cell damage and death.

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