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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-2R rapidly kills cancer cells from different histological origin. Jurkat, BJAB, MCF-7 A549 and L929 cells were treated for 4 hrs with the indicated concentrations of Brevinin-2R. Cell viability was assessed by MTT assay. The experiment was repeated 4 times and the average viability values are shown. (B–D) Brevinin-2R shows higher toxicity towards cancer cells than normal cells. PBMS and Jurkat (B), human T cell and Jurkat (C), human lung fibroblast and A549 (D) cells were treated with 2.5 μg/ml, 5 μg/ml or 10 μg/ml of Brevinin-2R and after the indicated time points their viability was assessed by MTT assay. The respective Brevinin-2R concentrations are indicated along with cell types used in the assay. Data represent the average values of quadruplicates from three independent experiments. (E) Brevinin-2R displays lower interaction with normal cells than with cancer cells. A549 and normal lung fibroblast at 2 × 106 cells were harvested. After blocking with 3% BSA, cells were incubated with 10 μg/ml FITC-conjugated Brevinin-2R. The stained cells were analyzed by FACS at 488 nm excitation. Automated analyses were performed using Cell Quest Pro software. Red histogram shows Brevinin-2R interaction with normal lung fibroblasts; blue histogram shows interaction with A549 tumor cells.(F) Low hemolytic activity of Brevinin-2R against sheep erythrocytes. Brevinin-2R at concentrations as high as 200 μg/ml resulted in only 2.5% hemolytic activity. Sheep erythrocytes hemolysed with 0.2% Triton-X100 served as a positive control and sheep erythrocytes treated with PBS served as a negative control. Hemolysis was evaluated by triplicate measurements of three independent experiments. (G) Brevinin-2R cytotoxic effect is dependent on its primary structure and sequence specific. To confirm the specific cytotoxicity of Brevinin-2R, Jurkat, MCF-7 and L929 were treated with a scrambled Brevinin-2R peptide (sequence see Materials and Methods) at 50 μg/ml for up to 48 hrs. Cell viability was assessed by MTT assay. The experiment was repeated four times and the average viability values are shown.
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fig01: Brevinin-2R rapidly kills cancer cells from different histological origin. Jurkat, BJAB, MCF-7 A549 and L929 cells were treated for 4 hrs with the indicated concentrations of Brevinin-2R. Cell viability was assessed by MTT assay. The experiment was repeated 4 times and the average viability values are shown. (B–D) Brevinin-2R shows higher toxicity towards cancer cells than normal cells. PBMS and Jurkat (B), human T cell and Jurkat (C), human lung fibroblast and A549 (D) cells were treated with 2.5 μg/ml, 5 μg/ml or 10 μg/ml of Brevinin-2R and after the indicated time points their viability was assessed by MTT assay. The respective Brevinin-2R concentrations are indicated along with cell types used in the assay. Data represent the average values of quadruplicates from three independent experiments. (E) Brevinin-2R displays lower interaction with normal cells than with cancer cells. A549 and normal lung fibroblast at 2 × 106 cells were harvested. After blocking with 3% BSA, cells were incubated with 10 μg/ml FITC-conjugated Brevinin-2R. The stained cells were analyzed by FACS at 488 nm excitation. Automated analyses were performed using Cell Quest Pro software. Red histogram shows Brevinin-2R interaction with normal lung fibroblasts; blue histogram shows interaction with A549 tumor cells.(F) Low hemolytic activity of Brevinin-2R against sheep erythrocytes. Brevinin-2R at concentrations as high as 200 μg/ml resulted in only 2.5% hemolytic activity. Sheep erythrocytes hemolysed with 0.2% Triton-X100 served as a positive control and sheep erythrocytes treated with PBS served as a negative control. Hemolysis was evaluated by triplicate measurements of three independent experiments. (G) Brevinin-2R cytotoxic effect is dependent on its primary structure and sequence specific. To confirm the specific cytotoxicity of Brevinin-2R, Jurkat, MCF-7 and L929 were treated with a scrambled Brevinin-2R peptide (sequence see Materials and Methods) at 50 μg/ml for up to 48 hrs. Cell viability was assessed by MTT assay. The experiment was repeated four times and the average viability values are shown.

Mentions: Brevinin-2R was the main component of fraction IV of the crude skin peptide extract (Supplementary Fig. S2A) and effective in killing various tumor cell lines (Jurkat, BJAB, MCF-7, L929, A549) at concentrations 2–4 times lower than required for R. ridibunda crude skin extract used in the previous experiments (compare Supplementary Fig. S1A–D with Fig. 1A). Similar to the crude skin extracts, the breast cancer adenocarcinoma cell line MCF-7 was most sensitive (Fig. 1A). These data indicate that Brevinin-2R, applied at clinically achievable low-micromolar concentrations, can rapidly kill cancer cells of different origin and species. Next, we compared the cytotoxic effect of Brevinin-2R with two anticancer agents doxorubicin and cisplatin (both tested at concentrations up to 50 μg/ml) on Jurkat and MCF-7 cells. The results showed that at 10 μg/ml (4 hrs) Brevinin-2R killed over 70% of MCF-7 and Jurkat cells (Fig. 1A), while the cytotoxicity of doxorubicin and cisplatin towards these cells after 4 hrs was about 30% (dox-orubicin), and 0% (cisplatin) for MCF-7 and 30% (doxorubicin), 5% (cisplatin) for Jurkat cells (Supplementary Fig. S3A–D). Thus, under the assayed conditions, Brevinin-2R was significantly more toxic toward these cells than doxorubicin and cisplatin (P < 0.0001) (compare Fig. 1A with Supplementary Fig. S3A–D).


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-2R rapidly kills cancer cells from different histological origin. Jurkat, BJAB, MCF-7 A549 and L929 cells were treated for 4 hrs with the indicated concentrations of Brevinin-2R. Cell viability was assessed by MTT assay. The experiment was repeated 4 times and the average viability values are shown. (B–D) Brevinin-2R shows higher toxicity towards cancer cells than normal cells. PBMS and Jurkat (B), human T cell and Jurkat (C), human lung fibroblast and A549 (D) cells were treated with 2.5 μg/ml, 5 μg/ml or 10 μg/ml of Brevinin-2R and after the indicated time points their viability was assessed by MTT assay. The respective Brevinin-2R concentrations are indicated along with cell types used in the assay. Data represent the average values of quadruplicates from three independent experiments. (E) Brevinin-2R displays lower interaction with normal cells than with cancer cells. A549 and normal lung fibroblast at 2 × 106 cells were harvested. After blocking with 3% BSA, cells were incubated with 10 μg/ml FITC-conjugated Brevinin-2R. The stained cells were analyzed by FACS at 488 nm excitation. Automated analyses were performed using Cell Quest Pro software. Red histogram shows Brevinin-2R interaction with normal lung fibroblasts; blue histogram shows interaction with A549 tumor cells.(F) Low hemolytic activity of Brevinin-2R against sheep erythrocytes. Brevinin-2R at concentrations as high as 200 μg/ml resulted in only 2.5% hemolytic activity. Sheep erythrocytes hemolysed with 0.2% Triton-X100 served as a positive control and sheep erythrocytes treated with PBS served as a negative control. Hemolysis was evaluated by triplicate measurements of three independent experiments. (G) Brevinin-2R cytotoxic effect is dependent on its primary structure and sequence specific. To confirm the specific cytotoxicity of Brevinin-2R, Jurkat, MCF-7 and L929 were treated with a scrambled Brevinin-2R peptide (sequence see Materials and Methods) at 50 μg/ml for up to 48 hrs. Cell viability was assessed by MTT assay. The experiment was repeated four times and the average viability values are shown.
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fig01: Brevinin-2R rapidly kills cancer cells from different histological origin. Jurkat, BJAB, MCF-7 A549 and L929 cells were treated for 4 hrs with the indicated concentrations of Brevinin-2R. Cell viability was assessed by MTT assay. The experiment was repeated 4 times and the average viability values are shown. (B–D) Brevinin-2R shows higher toxicity towards cancer cells than normal cells. PBMS and Jurkat (B), human T cell and Jurkat (C), human lung fibroblast and A549 (D) cells were treated with 2.5 μg/ml, 5 μg/ml or 10 μg/ml of Brevinin-2R and after the indicated time points their viability was assessed by MTT assay. The respective Brevinin-2R concentrations are indicated along with cell types used in the assay. Data represent the average values of quadruplicates from three independent experiments. (E) Brevinin-2R displays lower interaction with normal cells than with cancer cells. A549 and normal lung fibroblast at 2 × 106 cells were harvested. After blocking with 3% BSA, cells were incubated with 10 μg/ml FITC-conjugated Brevinin-2R. The stained cells were analyzed by FACS at 488 nm excitation. Automated analyses were performed using Cell Quest Pro software. Red histogram shows Brevinin-2R interaction with normal lung fibroblasts; blue histogram shows interaction with A549 tumor cells.(F) Low hemolytic activity of Brevinin-2R against sheep erythrocytes. Brevinin-2R at concentrations as high as 200 μg/ml resulted in only 2.5% hemolytic activity. Sheep erythrocytes hemolysed with 0.2% Triton-X100 served as a positive control and sheep erythrocytes treated with PBS served as a negative control. Hemolysis was evaluated by triplicate measurements of three independent experiments. (G) Brevinin-2R cytotoxic effect is dependent on its primary structure and sequence specific. To confirm the specific cytotoxicity of Brevinin-2R, Jurkat, MCF-7 and L929 were treated with a scrambled Brevinin-2R peptide (sequence see Materials and Methods) at 50 μg/ml for up to 48 hrs. Cell viability was assessed by MTT assay. The experiment was repeated four times and the average viability values are shown.
Mentions: Brevinin-2R was the main component of fraction IV of the crude skin peptide extract (Supplementary Fig. S2A) and effective in killing various tumor cell lines (Jurkat, BJAB, MCF-7, L929, A549) at concentrations 2–4 times lower than required for R. ridibunda crude skin extract used in the previous experiments (compare Supplementary Fig. S1A–D with Fig. 1A). Similar to the crude skin extracts, the breast cancer adenocarcinoma cell line MCF-7 was most sensitive (Fig. 1A). These data indicate that Brevinin-2R, applied at clinically achievable low-micromolar concentrations, can rapidly kill cancer cells of different origin and species. Next, we compared the cytotoxic effect of Brevinin-2R with two anticancer agents doxorubicin and cisplatin (both tested at concentrations up to 50 μg/ml) on Jurkat and MCF-7 cells. The results showed that at 10 μg/ml (4 hrs) Brevinin-2R killed over 70% of MCF-7 and Jurkat cells (Fig. 1A), while the cytotoxicity of doxorubicin and cisplatin towards these cells after 4 hrs was about 30% (dox-orubicin), and 0% (cisplatin) for MCF-7 and 30% (doxorubicin), 5% (cisplatin) for Jurkat cells (Supplementary Fig. S3A–D). Thus, under the assayed conditions, Brevinin-2R was significantly more toxic toward these cells than doxorubicin and cisplatin (P < 0.0001) (compare Fig. 1A with Supplementary Fig. S3A–D).

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