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Sensitivity of cancer cells to truncated diphtheria toxin.

Zhang Y, Schulte W, Pink D, Phipps K, Zijlstra A, Lewis JD, Waisman DM - PLoS ONE (2010)

Bottom Line: For in vivo studies, the chick chorioallantoic membrane (CAM) system was used to evaluate the effect of DT385 on angiogenesis.The CAM and mouse model system was used to evaluate the effect of DT385 on HEp3 and Lewis lung carcinoma (LLC) tumor growth, respectively.Furthermore, high concentrations of DT385 failed to affect growth arrested cells.

View Article: PubMed Central - PubMed

Affiliation: Departments of Biochemistry and Molecular Biology and Pathology, Dalhousie University, Halifax, Nova Scotia, Canada.

ABSTRACT

Background: Diphtheria toxin (DT) has been utilized as a prospective anti-cancer agent for the targeted delivery of cytotoxic therapy to otherwise untreatable neoplasia. DT is an extremely potent toxin for which the entry of a single molecule into a cell can be lethal. DT has been targeted to cancer cells by deleting the cell receptor-binding domain and combining the remaining catalytic portion with targeting proteins that selectively bind to the surface of cancer cells. It has been assumed that "receptorless" DT cannot bind to and kill cells. In the present study, we report that "receptorless" recombinant DT385 is in fact cytotoxic to a variety of cancer cell lines.

Methods: In vitro cytotoxicity of DT385 was measured by cell proliferation, cell staining and apoptosis assays. For in vivo studies, the chick chorioallantoic membrane (CAM) system was used to evaluate the effect of DT385 on angiogenesis. The CAM and mouse model system was used to evaluate the effect of DT385 on HEp3 and Lewis lung carcinoma (LLC) tumor growth, respectively.

Results: Of 18 human cancer cell lines tested, 15 were affected by DT385 with IC(50) ranging from 0.12-2.8 microM. Furthermore, high concentrations of DT385 failed to affect growth arrested cells. The cellular toxicity of DT385 was due to the inhibition of protein synthesis and induction of apoptosis. In vivo, DT385 diminished angiogenesis and decreased tumor growth in the CAM system, and inhibited the subcutaneous growth of LLC tumors in mice.

Conclusion: DT385 possesses anti-angiogenic and anti-tumor activity and may have potential as a therapeutic agent.

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Related in: MedlinePlus

Inhibition of protein synthesis by DT385.(A), U-87 MG cells were treated with 1 µM DT385 or control (either rP22, SUMO or PBS) for 24, 36 h and 48 h, respectively, and then labeled with [35S-]methionine for 15 minutes. Cell lysates (10 µg) were separated by SDS-PAGE (12% gel) and gels were stained with Coomassie blue (left), dried on Whatman paper and visualized by radiography using a phosphorimager (right). Representative images for 36 h treatment are shown. (B), quantification of (A). Radioactivity of cell lysates was determined by liquid scintillation counting. Data are expressed as percent of control. The average of CPM from irrelevant protein, rP22 or recombinant SUMO or PBS treatment was considered as the control CPM value. Results are the mean ± S.D. (n = 6, 2 independent experiments). Cell viability was also measured as described in the legend to Figure 1. Results are the mean ± S.D. of three independent experiments performed in triplicate.
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pone-0010498-g005: Inhibition of protein synthesis by DT385.(A), U-87 MG cells were treated with 1 µM DT385 or control (either rP22, SUMO or PBS) for 24, 36 h and 48 h, respectively, and then labeled with [35S-]methionine for 15 minutes. Cell lysates (10 µg) were separated by SDS-PAGE (12% gel) and gels were stained with Coomassie blue (left), dried on Whatman paper and visualized by radiography using a phosphorimager (right). Representative images for 36 h treatment are shown. (B), quantification of (A). Radioactivity of cell lysates was determined by liquid scintillation counting. Data are expressed as percent of control. The average of CPM from irrelevant protein, rP22 or recombinant SUMO or PBS treatment was considered as the control CPM value. Results are the mean ± S.D. (n = 6, 2 independent experiments). Cell viability was also measured as described in the legend to Figure 1. Results are the mean ± S.D. of three independent experiments performed in triplicate.

Mentions: Diphtheria toxin kills cells by catalysing the ADP-ribosylation of EF-2, leading to inhibition of protein synthesis [5], [28]. To investigate if the DT385 decreased cell viability by inhibiting protein synthesis, glioma U-87 MG cells were treated with 1 µM DT385 for 36 hours followed by labeling with [35S-] methionine for 15 minutes. As shown in Figure 5A, SDS-PAGE analysis of cell lysate showed that protein synthesis was severely reduced in DT385 treated cells. This inhibition occurred within 24 hrs of treatment and persisted during the duration of the assay (48 hr) (Figure 5B). The 36-h treatment gave the greatest decrease in protein labeling. These data suggest that, mechanistically, DT385 decreases cell viability by blocking protein synthesis. Similar results were obtained for Hela cells (data not shown).


Sensitivity of cancer cells to truncated diphtheria toxin.

Zhang Y, Schulte W, Pink D, Phipps K, Zijlstra A, Lewis JD, Waisman DM - PLoS ONE (2010)

Inhibition of protein synthesis by DT385.(A), U-87 MG cells were treated with 1 µM DT385 or control (either rP22, SUMO or PBS) for 24, 36 h and 48 h, respectively, and then labeled with [35S-]methionine for 15 minutes. Cell lysates (10 µg) were separated by SDS-PAGE (12% gel) and gels were stained with Coomassie blue (left), dried on Whatman paper and visualized by radiography using a phosphorimager (right). Representative images for 36 h treatment are shown. (B), quantification of (A). Radioactivity of cell lysates was determined by liquid scintillation counting. Data are expressed as percent of control. The average of CPM from irrelevant protein, rP22 or recombinant SUMO or PBS treatment was considered as the control CPM value. Results are the mean ± S.D. (n = 6, 2 independent experiments). Cell viability was also measured as described in the legend to Figure 1. Results are the mean ± S.D. of three independent experiments performed in triplicate.
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Related In: Results  -  Collection

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getmorefigures.php?uid=PMC2864767&req=5

pone-0010498-g005: Inhibition of protein synthesis by DT385.(A), U-87 MG cells were treated with 1 µM DT385 or control (either rP22, SUMO or PBS) for 24, 36 h and 48 h, respectively, and then labeled with [35S-]methionine for 15 minutes. Cell lysates (10 µg) were separated by SDS-PAGE (12% gel) and gels were stained with Coomassie blue (left), dried on Whatman paper and visualized by radiography using a phosphorimager (right). Representative images for 36 h treatment are shown. (B), quantification of (A). Radioactivity of cell lysates was determined by liquid scintillation counting. Data are expressed as percent of control. The average of CPM from irrelevant protein, rP22 or recombinant SUMO or PBS treatment was considered as the control CPM value. Results are the mean ± S.D. (n = 6, 2 independent experiments). Cell viability was also measured as described in the legend to Figure 1. Results are the mean ± S.D. of three independent experiments performed in triplicate.
Mentions: Diphtheria toxin kills cells by catalysing the ADP-ribosylation of EF-2, leading to inhibition of protein synthesis [5], [28]. To investigate if the DT385 decreased cell viability by inhibiting protein synthesis, glioma U-87 MG cells were treated with 1 µM DT385 for 36 hours followed by labeling with [35S-] methionine for 15 minutes. As shown in Figure 5A, SDS-PAGE analysis of cell lysate showed that protein synthesis was severely reduced in DT385 treated cells. This inhibition occurred within 24 hrs of treatment and persisted during the duration of the assay (48 hr) (Figure 5B). The 36-h treatment gave the greatest decrease in protein labeling. These data suggest that, mechanistically, DT385 decreases cell viability by blocking protein synthesis. Similar results were obtained for Hela cells (data not shown).

Bottom Line: For in vivo studies, the chick chorioallantoic membrane (CAM) system was used to evaluate the effect of DT385 on angiogenesis.The CAM and mouse model system was used to evaluate the effect of DT385 on HEp3 and Lewis lung carcinoma (LLC) tumor growth, respectively.Furthermore, high concentrations of DT385 failed to affect growth arrested cells.

View Article: PubMed Central - PubMed

Affiliation: Departments of Biochemistry and Molecular Biology and Pathology, Dalhousie University, Halifax, Nova Scotia, Canada.

ABSTRACT

Background: Diphtheria toxin (DT) has been utilized as a prospective anti-cancer agent for the targeted delivery of cytotoxic therapy to otherwise untreatable neoplasia. DT is an extremely potent toxin for which the entry of a single molecule into a cell can be lethal. DT has been targeted to cancer cells by deleting the cell receptor-binding domain and combining the remaining catalytic portion with targeting proteins that selectively bind to the surface of cancer cells. It has been assumed that "receptorless" DT cannot bind to and kill cells. In the present study, we report that "receptorless" recombinant DT385 is in fact cytotoxic to a variety of cancer cell lines.

Methods: In vitro cytotoxicity of DT385 was measured by cell proliferation, cell staining and apoptosis assays. For in vivo studies, the chick chorioallantoic membrane (CAM) system was used to evaluate the effect of DT385 on angiogenesis. The CAM and mouse model system was used to evaluate the effect of DT385 on HEp3 and Lewis lung carcinoma (LLC) tumor growth, respectively.

Results: Of 18 human cancer cell lines tested, 15 were affected by DT385 with IC(50) ranging from 0.12-2.8 microM. Furthermore, high concentrations of DT385 failed to affect growth arrested cells. The cellular toxicity of DT385 was due to the inhibition of protein synthesis and induction of apoptosis. In vivo, DT385 diminished angiogenesis and decreased tumor growth in the CAM system, and inhibited the subcutaneous growth of LLC tumors in mice.

Conclusion: DT385 possesses anti-angiogenic and anti-tumor activity and may have potential as a therapeutic agent.

Show MeSH
Related in: MedlinePlus