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Efficacy of adenovirally expressed soluble TRAIL in human glioma organotypic slice culture and glioma xenografts.

Liu Y, Lang F, Xie X, Prabhu S, Xu J, Sampath D, Aldape K, Fuller G, Puduvalli VK - Cell Death Dis (2011)

Bottom Line: It inhibited anchorage-independent glioma growth and exerted a bystander effect in transwell assays.Intratumoral injections of AdsTRAIL in a rodent intracranial glioma model resulted in reduced tumor growth and improved survival compared with Ad-enhanced green fluorescent protein (EGFP)- or vehicle-treated controls without toxicity.Human glioma organotypic slices treated with AdsTRAIL demonstrated apoptosis induction and caspase activation.

View Article: PubMed Central - PubMed

Affiliation: Department of Neuro-oncology, University of Texas MD Anderson Cancer Center, Houston, TX 77035, USA.

ABSTRACT
Tumor necrosis factor (TNF)-related apoptosis-inducing ligand (TRAIL) selectively induces apoptosis in malignant cells, including gliomas, and is currently in anticancer clinical trials. However, the full-length and tagged forms of TRAIL, unlike the untagged ligand (soluble TRAIL (sTRAIL)), exhibits toxicity against normal cells. Here, we report the generation and testing of an adenovirus (AdsTRAIL) that expresses untagged sTRAIL in an intracranial xenograft model and a human glioma organotypic slice culture model. AdsTRAIL efficiently induced apoptosis in glioma cell lines, including those resistant to sTRAIL, but not in normal human astrocytes (NHAs). It inhibited anchorage-independent glioma growth and exerted a bystander effect in transwell assays. Intratumoral injections of AdsTRAIL in a rodent intracranial glioma model resulted in reduced tumor growth and improved survival compared with Ad-enhanced green fluorescent protein (EGFP)- or vehicle-treated controls without toxicity. Human glioma organotypic slices treated with AdsTRAIL demonstrated apoptosis induction and caspase activation.

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Bystander effect of AdsTRAIL in gliomas cells. (a) TRAIL-resistant hMSCs (2 × 105 cells) were plated in the upper chamber of a six-well transwell plate and transduced with AdEGFP50MOI or AdsTRAIL* (AdsTRAIL50MOI+AdEGFP5MOI). After 24 h, U251HF (2 × 105 cells) were plated in the lower chamber. Changes in cell morphology in the lower chambers were examined at 48 h to identify apoptotic cells. (b) U251 cells from the lower chamber of the transwell experiment were harvested at 48 h and analyzed by flow cytometry for the sub-G1 (apoptotic) fraction
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fig4: Bystander effect of AdsTRAIL in gliomas cells. (a) TRAIL-resistant hMSCs (2 × 105 cells) were plated in the upper chamber of a six-well transwell plate and transduced with AdEGFP50MOI or AdsTRAIL* (AdsTRAIL50MOI+AdEGFP5MOI). After 24 h, U251HF (2 × 105 cells) were plated in the lower chamber. Changes in cell morphology in the lower chambers were examined at 48 h to identify apoptotic cells. (b) U251 cells from the lower chamber of the transwell experiment were harvested at 48 h and analyzed by flow cytometry for the sub-G1 (apoptotic) fraction

Mentions: Non-replicating adenoviruses engineered to express non-secreted proteins will cause an antitumor effect only in the cells transduced by the virus; adjacent cells that escape this effect could repopulate the tumor. In contrast, an adenoviral construct expressing a soluble ligand can exert a bystander effect on the non-transduced cells, thus contributing to a field antitumor effect. To test this concept, we transduced TRAIL-resistant human mesenchymal stem cells (hMSCs) with AdsTRAIL in the upper chamber of a two-chamber transwell experiment and plated TRAIL-sensitive U251HF cells in the lower chamber. To control for the possibility that the virus released from the hMSCs may cross the transwell and directly affect U251HF cells in the lower chamber, we transduced the hMSCs in the upper chamber with AdEGFP and monitored the lower chamber for green fluorescent cells. AdsTRAIL-transduced hMSCs in the upper chamber did not show any evidence of apoptosis either by morphological appearance or by flow-cytometric analysis for a sub-G1 fraction (Figure 4a). In contrast, the U251HF cells in the lower chamber efficiently underwent apoptosis; the cells, however, did not exhibit green fluorescence, indicating that the effects seen were not due to direct transfer of the adenoviral construct across the transwell membrane. These results were also confirmed by flow-cytometric analysis of the cells in both chambers (Figure 4b) and strongly support the possibility that AdsTRAIL induced a bystander effect by diffusion of the soluble protein.


Efficacy of adenovirally expressed soluble TRAIL in human glioma organotypic slice culture and glioma xenografts.

Liu Y, Lang F, Xie X, Prabhu S, Xu J, Sampath D, Aldape K, Fuller G, Puduvalli VK - Cell Death Dis (2011)

Bystander effect of AdsTRAIL in gliomas cells. (a) TRAIL-resistant hMSCs (2 × 105 cells) were plated in the upper chamber of a six-well transwell plate and transduced with AdEGFP50MOI or AdsTRAIL* (AdsTRAIL50MOI+AdEGFP5MOI). After 24 h, U251HF (2 × 105 cells) were plated in the lower chamber. Changes in cell morphology in the lower chambers were examined at 48 h to identify apoptotic cells. (b) U251 cells from the lower chamber of the transwell experiment were harvested at 48 h and analyzed by flow cytometry for the sub-G1 (apoptotic) fraction
© Copyright Policy - open-access
Related In: Results  -  Collection

License
Show All Figures
getmorefigures.php?uid=PMC3101700&req=5

fig4: Bystander effect of AdsTRAIL in gliomas cells. (a) TRAIL-resistant hMSCs (2 × 105 cells) were plated in the upper chamber of a six-well transwell plate and transduced with AdEGFP50MOI or AdsTRAIL* (AdsTRAIL50MOI+AdEGFP5MOI). After 24 h, U251HF (2 × 105 cells) were plated in the lower chamber. Changes in cell morphology in the lower chambers were examined at 48 h to identify apoptotic cells. (b) U251 cells from the lower chamber of the transwell experiment were harvested at 48 h and analyzed by flow cytometry for the sub-G1 (apoptotic) fraction
Mentions: Non-replicating adenoviruses engineered to express non-secreted proteins will cause an antitumor effect only in the cells transduced by the virus; adjacent cells that escape this effect could repopulate the tumor. In contrast, an adenoviral construct expressing a soluble ligand can exert a bystander effect on the non-transduced cells, thus contributing to a field antitumor effect. To test this concept, we transduced TRAIL-resistant human mesenchymal stem cells (hMSCs) with AdsTRAIL in the upper chamber of a two-chamber transwell experiment and plated TRAIL-sensitive U251HF cells in the lower chamber. To control for the possibility that the virus released from the hMSCs may cross the transwell and directly affect U251HF cells in the lower chamber, we transduced the hMSCs in the upper chamber with AdEGFP and monitored the lower chamber for green fluorescent cells. AdsTRAIL-transduced hMSCs in the upper chamber did not show any evidence of apoptosis either by morphological appearance or by flow-cytometric analysis for a sub-G1 fraction (Figure 4a). In contrast, the U251HF cells in the lower chamber efficiently underwent apoptosis; the cells, however, did not exhibit green fluorescence, indicating that the effects seen were not due to direct transfer of the adenoviral construct across the transwell membrane. These results were also confirmed by flow-cytometric analysis of the cells in both chambers (Figure 4b) and strongly support the possibility that AdsTRAIL induced a bystander effect by diffusion of the soluble protein.

Bottom Line: It inhibited anchorage-independent glioma growth and exerted a bystander effect in transwell assays.Intratumoral injections of AdsTRAIL in a rodent intracranial glioma model resulted in reduced tumor growth and improved survival compared with Ad-enhanced green fluorescent protein (EGFP)- or vehicle-treated controls without toxicity.Human glioma organotypic slices treated with AdsTRAIL demonstrated apoptosis induction and caspase activation.

View Article: PubMed Central - PubMed

Affiliation: Department of Neuro-oncology, University of Texas MD Anderson Cancer Center, Houston, TX 77035, USA.

ABSTRACT
Tumor necrosis factor (TNF)-related apoptosis-inducing ligand (TRAIL) selectively induces apoptosis in malignant cells, including gliomas, and is currently in anticancer clinical trials. However, the full-length and tagged forms of TRAIL, unlike the untagged ligand (soluble TRAIL (sTRAIL)), exhibits toxicity against normal cells. Here, we report the generation and testing of an adenovirus (AdsTRAIL) that expresses untagged sTRAIL in an intracranial xenograft model and a human glioma organotypic slice culture model. AdsTRAIL efficiently induced apoptosis in glioma cell lines, including those resistant to sTRAIL, but not in normal human astrocytes (NHAs). It inhibited anchorage-independent glioma growth and exerted a bystander effect in transwell assays. Intratumoral injections of AdsTRAIL in a rodent intracranial glioma model resulted in reduced tumor growth and improved survival compared with Ad-enhanced green fluorescent protein (EGFP)- or vehicle-treated controls without toxicity. Human glioma organotypic slices treated with AdsTRAIL demonstrated apoptosis induction and caspase activation.

Show MeSH
Related in: MedlinePlus