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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

DT385 causes apoptosis and cell death in U-87 MG cells.(A), U-87 MG cells growing in an 8- well chamber slide were treated with 1.2 µM DT385, or control (PBS) respectively for 3 days. Following treatments, cells were stained for apoptosis with FITC-labeled annexin V. Cell membrane integrity was evaluated with ethidium homodimer III (EtD-III). Representative images (100× magnifications) are shown. The arrow indicates cells that were labeled with both fluorescent dyes. (B), Graphical representation of (A). The cell number per high-power field (HPF, ×400) was the mean of the cell number obtained from 5 random HPF. Detached cells, which were stained with both annexin V and EtD-III positive, were also included in the cell number determination. Results are the mean ± S.D. of 3 experiments.
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pone-0010498-g003: DT385 causes apoptosis and cell death in U-87 MG cells.(A), U-87 MG cells growing in an 8- well chamber slide were treated with 1.2 µM DT385, or control (PBS) respectively for 3 days. Following treatments, cells were stained for apoptosis with FITC-labeled annexin V. Cell membrane integrity was evaluated with ethidium homodimer III (EtD-III). Representative images (100× magnifications) are shown. The arrow indicates cells that were labeled with both fluorescent dyes. (B), Graphical representation of (A). The cell number per high-power field (HPF, ×400) was the mean of the cell number obtained from 5 random HPF. Detached cells, which were stained with both annexin V and EtD-III positive, were also included in the cell number determination. Results are the mean ± S.D. of 3 experiments.

Mentions: DT kills cells by a mechanism involving cellular apoptosis. We also observed increased apoptosis in DT385 treated cells (Figure 3). The incubation of cultured U-87 with DT385 resulted in a dramatic increase in cellular apoptosis as measured by an increase in annexin V staining (85%) and the uptake of ethidium homodimer (87%).


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)

DT385 causes apoptosis and cell death in U-87 MG cells.(A), U-87 MG cells growing in an 8- well chamber slide were treated with 1.2 µM DT385, or control (PBS) respectively for 3 days. Following treatments, cells were stained for apoptosis with FITC-labeled annexin V. Cell membrane integrity was evaluated with ethidium homodimer III (EtD-III). Representative images (100× magnifications) are shown. The arrow indicates cells that were labeled with both fluorescent dyes. (B), Graphical representation of (A). The cell number per high-power field (HPF, ×400) was the mean of the cell number obtained from 5 random HPF. Detached cells, which were stained with both annexin V and EtD-III positive, were also included in the cell number determination. Results are the mean ± S.D. of 3 experiments.
© Copyright Policy
Related In: Results  -  Collection

Show All Figures
getmorefigures.php?uid=PMC2864767&req=5

pone-0010498-g003: DT385 causes apoptosis and cell death in U-87 MG cells.(A), U-87 MG cells growing in an 8- well chamber slide were treated with 1.2 µM DT385, or control (PBS) respectively for 3 days. Following treatments, cells were stained for apoptosis with FITC-labeled annexin V. Cell membrane integrity was evaluated with ethidium homodimer III (EtD-III). Representative images (100× magnifications) are shown. The arrow indicates cells that were labeled with both fluorescent dyes. (B), Graphical representation of (A). The cell number per high-power field (HPF, ×400) was the mean of the cell number obtained from 5 random HPF. Detached cells, which were stained with both annexin V and EtD-III positive, were also included in the cell number determination. Results are the mean ± S.D. of 3 experiments.
Mentions: DT kills cells by a mechanism involving cellular apoptosis. We also observed increased apoptosis in DT385 treated cells (Figure 3). The incubation of cultured U-87 with DT385 resulted in a dramatic increase in cellular apoptosis as measured by an increase in annexin V staining (85%) and the uptake of ethidium homodimer (87%).

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