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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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(a) Various glioma cell lines were treated with 100 MOI AdsTRAIL or AdEGFP for the period indicated, and the number of viable cells was determined by a WST1 assay. The effects of AdsTRAIL were also assessed in NHAs. (b) Morphological changes after treatment with AdsTRAIL in glioma cells sensitive (D54 and U251HF) or resistant (U87 and SNB19) to sTRAIL. (c) U251HF cells were transduced with AdsTRAIL (100 MOI) and harvested at the periods indicated. The cells were stained with Annexin-V (0.6 μg/ml) and PI (50 μg/ml) and analyzed by flow cytometry. (d) U87 and SNB19 cells transduced with AdEGFP (100 MOI) were treated with sTRAIL (100 ng/ml) and analyzed 48 h later for the apoptotic cells (sub-G1 fraction) using flow cytometry. Untreated cells, cells treated with sTRAIL, and those transduced with AdsTRAIL were used as controls (data shown as mean percent sub-G1 from two independent experiments; error bars indicate standard error of mean)
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fig1: (a) Various glioma cell lines were treated with 100 MOI AdsTRAIL or AdEGFP for the period indicated, and the number of viable cells was determined by a WST1 assay. The effects of AdsTRAIL were also assessed in NHAs. (b) Morphological changes after treatment with AdsTRAIL in glioma cells sensitive (D54 and U251HF) or resistant (U87 and SNB19) to sTRAIL. (c) U251HF cells were transduced with AdsTRAIL (100 MOI) and harvested at the periods indicated. The cells were stained with Annexin-V (0.6 μg/ml) and PI (50 μg/ml) and analyzed by flow cytometry. (d) U87 and SNB19 cells transduced with AdEGFP (100 MOI) were treated with sTRAIL (100 ng/ml) and analyzed 48 h later for the apoptotic cells (sub-G1 fraction) using flow cytometry. Untreated cells, cells treated with sTRAIL, and those transduced with AdsTRAIL were used as controls (data shown as mean percent sub-G1 from two independent experiments; error bars indicate standard error of mean)

Mentions: Sensitivity to sTRAIL varies among cell types and several glioma cell lines are resistant to purified exogenous sTRAIL. Among the variety of factors that can govern such resistance, the short half-life of exogenous sTRAIL can limit the exposure and consequently the efficacy of the agent. We postulated that sustained expression of the ligand by AdsTRAIL would overcome this limitation. AdsTRAIL treatment caused reduced viability of several glioma cell lines, but not of normal human astrocytes (NHAs); these changes were most evident in D54, U251HF, LN229, and U373 cells and less so in U87 and SNB19 cells (Figure 1a). The sensitive cells demonstrated apoptotic morphology in response to both purified sTRAIL and AdsTRAIL; in contrast, U87 and SNB19 cells were resistant to sTRAIL but sensitive to AdsTRAIL (Figure 1b). Annexin V staining and flow-cytometric analysis showed a time-dependent increase in early apoptosis, which peaked by 12 h, followed by an increase in late apoptosis (Figure 1c). We confirmed that the increased cell death seen with AdsTRAIL was not due to a nonspecific increase in cellular sensitivity to sTRAIL in response to adenoviral transduction of glioma cells; TRAIL-resistant glioma cell lines, U87 and SNB19, transduced with Ad-enhanced green fluorescent protein (EGFP) remained resistant to sTRAIL, whereas when transduced with AdsTRAIL they efficiently underwent apoptosis, confirming that it was a direct effect of AdsTRAIL (Figure 1d and Supplementary Figure 1).


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)

(a) Various glioma cell lines were treated with 100 MOI AdsTRAIL or AdEGFP for the period indicated, and the number of viable cells was determined by a WST1 assay. The effects of AdsTRAIL were also assessed in NHAs. (b) Morphological changes after treatment with AdsTRAIL in glioma cells sensitive (D54 and U251HF) or resistant (U87 and SNB19) to sTRAIL. (c) U251HF cells were transduced with AdsTRAIL (100 MOI) and harvested at the periods indicated. The cells were stained with Annexin-V (0.6 μg/ml) and PI (50 μg/ml) and analyzed by flow cytometry. (d) U87 and SNB19 cells transduced with AdEGFP (100 MOI) were treated with sTRAIL (100 ng/ml) and analyzed 48 h later for the apoptotic cells (sub-G1 fraction) using flow cytometry. Untreated cells, cells treated with sTRAIL, and those transduced with AdsTRAIL were used as controls (data shown as mean percent sub-G1 from two independent experiments; error bars indicate standard error of mean)
© Copyright Policy - open-access
Related In: Results  -  Collection

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

fig1: (a) Various glioma cell lines were treated with 100 MOI AdsTRAIL or AdEGFP for the period indicated, and the number of viable cells was determined by a WST1 assay. The effects of AdsTRAIL were also assessed in NHAs. (b) Morphological changes after treatment with AdsTRAIL in glioma cells sensitive (D54 and U251HF) or resistant (U87 and SNB19) to sTRAIL. (c) U251HF cells were transduced with AdsTRAIL (100 MOI) and harvested at the periods indicated. The cells were stained with Annexin-V (0.6 μg/ml) and PI (50 μg/ml) and analyzed by flow cytometry. (d) U87 and SNB19 cells transduced with AdEGFP (100 MOI) were treated with sTRAIL (100 ng/ml) and analyzed 48 h later for the apoptotic cells (sub-G1 fraction) using flow cytometry. Untreated cells, cells treated with sTRAIL, and those transduced with AdsTRAIL were used as controls (data shown as mean percent sub-G1 from two independent experiments; error bars indicate standard error of mean)
Mentions: Sensitivity to sTRAIL varies among cell types and several glioma cell lines are resistant to purified exogenous sTRAIL. Among the variety of factors that can govern such resistance, the short half-life of exogenous sTRAIL can limit the exposure and consequently the efficacy of the agent. We postulated that sustained expression of the ligand by AdsTRAIL would overcome this limitation. AdsTRAIL treatment caused reduced viability of several glioma cell lines, but not of normal human astrocytes (NHAs); these changes were most evident in D54, U251HF, LN229, and U373 cells and less so in U87 and SNB19 cells (Figure 1a). The sensitive cells demonstrated apoptotic morphology in response to both purified sTRAIL and AdsTRAIL; in contrast, U87 and SNB19 cells were resistant to sTRAIL but sensitive to AdsTRAIL (Figure 1b). Annexin V staining and flow-cytometric analysis showed a time-dependent increase in early apoptosis, which peaked by 12 h, followed by an increase in late apoptosis (Figure 1c). We confirmed that the increased cell death seen with AdsTRAIL was not due to a nonspecific increase in cellular sensitivity to sTRAIL in response to adenoviral transduction of glioma cells; TRAIL-resistant glioma cell lines, U87 and SNB19, transduced with Ad-enhanced green fluorescent protein (EGFP) remained resistant to sTRAIL, whereas when transduced with AdsTRAIL they efficiently underwent apoptosis, confirming that it was a direct effect of AdsTRAIL (Figure 1d and Supplementary Figure 1).

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