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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

Internalization of DT385 by Cancer Cells.(A), Cancer cells were grown in an 8-well chamber slide. FITC-labeled DT385 (1 µM) was added to culture. The cells were observed and photographed under a Zeiss LSM 510 fluorescence confocal microscope (Carl Zeiss, Germany) after 36 h. Top and right sides of images demonstrates the orthogonal view of the confocal dataset shown in the x and y-axis of images. Magnifications are indicated below images. (B), U87 cells were incubated with 2 µM DT385 alone or in combination with 10 mM NH4Cl for the indicated times. The media was then replaced and cells were cultured for a total time of 36 hours after which cell viability was accessed with the MTS assay. The PBS vehicle control was considered as 100% viable. Results are expressed as percentages of PBS-treated cells. Results shown are representative of 2 independent experiments performed in triplicate.
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pone-0010498-g004: Internalization of DT385 by Cancer Cells.(A), Cancer cells were grown in an 8-well chamber slide. FITC-labeled DT385 (1 µM) was added to culture. The cells were observed and photographed under a Zeiss LSM 510 fluorescence confocal microscope (Carl Zeiss, Germany) after 36 h. Top and right sides of images demonstrates the orthogonal view of the confocal dataset shown in the x and y-axis of images. Magnifications are indicated below images. (B), U87 cells were incubated with 2 µM DT385 alone or in combination with 10 mM NH4Cl for the indicated times. The media was then replaced and cells were cultured for a total time of 36 hours after which cell viability was accessed with the MTS assay. The PBS vehicle control was considered as 100% viable. Results are expressed as percentages of PBS-treated cells. Results shown are representative of 2 independent experiments performed in triplicate.

Mentions: DT has been shown to enter toxin-sensitive mammalian cells by receptor-mediated endocytosis which involves the interaction of the receptor-binding domain of the protein with its extracellular receptor. Since DT385 does not possess a receptor-binding domain, it should be incapable of binding to cells and becoming internalized. To investigate if DT385 was internalized, we tracked fluorescently labeled DT385 with confocal microscopy. Tumor cells sensitive to DT385 were incubated with FITC-DT385 and imaged with confocal microscopy. As shown in Figure 4A, incubation of cells with DT385 resulted in the internalization of the toxin which was detectable in perinuclear vesicles. In contrast, incubation of cells with similar concentrations of FITC-bovine serum albumin did not result in internalization of the protein (Figure S4). This experiment suggested that the uptake of DT385 by cells was not due to non specific cellular uptake of the protein.


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)

Internalization of DT385 by Cancer Cells.(A), Cancer cells were grown in an 8-well chamber slide. FITC-labeled DT385 (1 µM) was added to culture. The cells were observed and photographed under a Zeiss LSM 510 fluorescence confocal microscope (Carl Zeiss, Germany) after 36 h. Top and right sides of images demonstrates the orthogonal view of the confocal dataset shown in the x and y-axis of images. Magnifications are indicated below images. (B), U87 cells were incubated with 2 µM DT385 alone or in combination with 10 mM NH4Cl for the indicated times. The media was then replaced and cells were cultured for a total time of 36 hours after which cell viability was accessed with the MTS assay. The PBS vehicle control was considered as 100% viable. Results are expressed as percentages of PBS-treated cells. Results shown are representative of 2 independent experiments performed in triplicate.
© Copyright Policy
Related In: Results  -  Collection

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getmorefigures.php?uid=PMC2864767&req=5

pone-0010498-g004: Internalization of DT385 by Cancer Cells.(A), Cancer cells were grown in an 8-well chamber slide. FITC-labeled DT385 (1 µM) was added to culture. The cells were observed and photographed under a Zeiss LSM 510 fluorescence confocal microscope (Carl Zeiss, Germany) after 36 h. Top and right sides of images demonstrates the orthogonal view of the confocal dataset shown in the x and y-axis of images. Magnifications are indicated below images. (B), U87 cells were incubated with 2 µM DT385 alone or in combination with 10 mM NH4Cl for the indicated times. The media was then replaced and cells were cultured for a total time of 36 hours after which cell viability was accessed with the MTS assay. The PBS vehicle control was considered as 100% viable. Results are expressed as percentages of PBS-treated cells. Results shown are representative of 2 independent experiments performed in triplicate.
Mentions: DT has been shown to enter toxin-sensitive mammalian cells by receptor-mediated endocytosis which involves the interaction of the receptor-binding domain of the protein with its extracellular receptor. Since DT385 does not possess a receptor-binding domain, it should be incapable of binding to cells and becoming internalized. To investigate if DT385 was internalized, we tracked fluorescently labeled DT385 with confocal microscopy. Tumor cells sensitive to DT385 were incubated with FITC-DT385 and imaged with confocal microscopy. As shown in Figure 4A, incubation of cells with DT385 resulted in the internalization of the toxin which was detectable in perinuclear vesicles. In contrast, incubation of cells with similar concentrations of FITC-bovine serum albumin did not result in internalization of the protein (Figure S4). This experiment suggested that the uptake of DT385 by cells was not due to non specific cellular uptake of the protein.

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