Brevinin-2R(1) semi-selectively kills cancer cells by a distinct mechanism, which involves the lysosomal-mitochondrial death pathway.
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.
Affiliation: Department of Biochemistry and Medical Genetics, Manitoba Institute of Cell Biology, Cancer Care Manitoba, Winnipeg, Manitoba, Canada.
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.
- Amphibian Proteins/pharmacology*/toxicity
- Antimicrobial Cationic Peptides/pharmacology*/toxicity
- Mitochondria/drug effects*/metabolism/ultrastructure
- Adenosine Triphosphate/analysis/metabolism
- Cell Death/drug effects
- Cell Line, Tumor
- Cell Survival/drug effects
- Endosomes/drug effects/ultrastructure
- Fluorescent Dyes/metabolism
- HT29 Cells
- Jurkat Cells
- L Cells (Cell Line)
- Membrane Potential, Mitochondrial/drug effects
- Reactive Oxygen Species/metabolism
- Tetrazolium Salts/metabolism
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fig03: Brevinin-2R kills cancer cells by a novel pathway that damages mitochondria, involves BNIP3, and it is sensitive to the inhibition by Bcl2. (A) Bcl2 overexpression significantly protected from Brevinin-2R-induced cell death. Jurkat and MCF-7, and stable transfectants overexpressing Bcl2 (Jurkat-Bcl2, MCF-7-Bcl2) were treated with Brevinin-2R (10 μg/ml) for indicated time periods and cell viability was assessed by MTT assay. (B) Effect of Brevinin-2R on the growth of MCF-7, L929 and clones stably transfected with dominant negative mutant of BNIP3 (MCF-7- TM-BNIP3, L929-DTM-BNIP3). Cells were treated with Brevinin-2R (10 μg/ml) for the indicated times and cell viability was assessed by MTT assay.(C) Brevinin-2R changes mitochondrial membrane potential. Cytofluorimetric analysis of mitochondrial transmembrane potential (ΔΦ m) in Jurkat (left panel) and a clone that overexpresses Bcl2 (Jurkat-Bcl2, right panel). Cells were treated for 30 min with medium alone (upper diagrams), or with Brevinin-2R (10 μg/ml). Brevinin-2R treatment showed obvious changes in mitochondrial membrane potential and overexpression of Bcl2 resulted in significantly increased resistance toward changes in ΔΨm. (D, E, F) Increase in cellular ROS and decrease in total cell ATP content are early indicators of Brevinin-2R cell death. Brevinin-2R increased ROS production in L929 (D), Jurkat (E) and MCF-7 (F) cells. Cells were treated with Brevinin-2R (5 and 10 μg/ml) for the indicated time points and then ROS was measured using DHR123. The experiment was repeated four times and the average ROS values are indicated. DTM-BNIP3 and Bcl2 overexpression increased resistance against ROS production.(G, H, I) The effect of Brevinin-2R (5 and 10 μg/ml) on total ATP content of treated Jurkat (G), L929 (H) and MCF-7 (I) cells at indicated time points is shown. Overexpression of the dominant negative ΔTM-BNIP3-increased resistance toward ATP decrease as determined by a luciferase-based method. Data represent the average values from triplicates of three independent experiments.(J, K) Brevinin-2R does not trigger the release of pro-apoptotic mitochondrial proteins. The cellular localization of AIF (J) and Endo G (K) was determined by confocal microscopy. MCF-7 cells were treated with Brevinin-2R (10 μg/ml) for 3 hrs prior to immunostaining with anti-AIF and ENDO-G antibodies followed by detection with Cy-5-conjugated secondary antibody (magenta). Cells were counterstained with DAPI and mitotracker to visualize nuclei and mitochondria.(L) Brevinin-2R does not change expression of anti- and pro-apoptotic Bcl2 proteins. Western blot analysis of cell lysates from MCF-7 treated with Brevinin-2R (10 μg/ml) for 0, 2, 4 and 6 hrs revealed no changes in the production of Bcl2, Bcl-XL, Mcl-1, Bax, Bak. Glyceraldehyde 3-phosphate dehydrogenase (GAPDH) was included as a loading control. (M) Brevinin-2R shows very low interaction with mitochondria. Confocal microscopy was used to determine the interaction of Brevinin-2R with mitochondria. L929 cells were treated for 2 hrs with FITC-labeled scrambled Brevinin-2R-like peptide and Brevinin-2R. Both peptides showed very low direct interaction with mitochondria. Cells were counterstained with DAPI and mitotracker to visualize nuclei and mitochondria.(N, O) Brevinin-2R induces mitochondrial damage. TEM ultrastructural analysis of control L929 cells before (N) and after a 2 hrs incubation with Brevinin-2R (O) showed structural damage to mitochondria. Magnification:21.5 × 103.
ΔΨm was measured by flow cytometry using the fluorescent probe JC-1 (5,5, 6,6-tetrachloro-1,1, 3,3-tetraethyl-benzimidazole carbocyanide iodide) as previously described . In each experiment, at least 15,000 events were analyzed. The m was visualized as 3-D diagrams with FL2, FL1 and cell counts being the x/y/z axis, respectively (Fig. 3C). The measurement of ROS production was performed by flow cytometry using DHR123. Jurkat, Jurkat-Bcl2, MCF-7, L929, L929-Δ TM-BNIP3 cell lines (1.5 104) were treated with TNF at indicated concentrations for different time points. DHR 123 (1 μm) was added to treated cells at 37° C for 15 min before cells were harvested and washed three times with ice-cold PBS. Cells were left on ice for 15 min to stabilize fluorescence. The fluorescence intensity (FL-1 and FL-2 channels) was then measured by flow cytometry (FACS-Calibur, Becto-Dickinson).