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Enhancing the bystander killing effect of an oncolytic HSV by arming it with a secretable apoptosis activator.

Loya SM, Zhang X - Gene Ther. (2015)

Bottom Line: The rationale is that, due to its much smaller size, this apoptosis activator can overcome obstacles such as the dense collagen in the tumor tissues to spread more freely than the viral particles.When measured in vitro, Her2-COL-sFasL was found to efficiently induce caspase cleavage, resulting in an 80% reduction in cell viability.Once incorporated into the genome of an oncolytic type 2 herpes simplex virus, FusOn-H3, Her2-COL-sFasL potentiates the therapeutic efficacy of the virus in an aggressive syngeneic mammary tumor model.

View Article: PubMed Central - PubMed

Affiliation: 1] Department of Biology and Biochemistry, Center for Nuclear Receptors and Cell Signaling, University of Houston, Houston, TX, USA [2] Department of Molecular Virology and Microbiology, Baylor College of Medicine, Houston, TX, USA.

ABSTRACT
Although oncolytic viruses have shown great promise as cancer therapeutics, results from a recent phase III clinical trial indicate that their potency may need further improvement for a clear clinical benefit. Here, we report a novel strategy to increase the bystander effect of virotherapy by arming an oncolytic virus with a secreted form of a Her2 single chain antibody linked to a self-multimerizing Fas ligand extracellular domain (Her2-COL-sFasL). The rationale is that, due to its much smaller size, this apoptosis activator can overcome obstacles such as the dense collagen in the tumor tissues to spread more freely than the viral particles. When measured in vitro, Her2-COL-sFasL was found to efficiently induce caspase cleavage, resulting in an 80% reduction in cell viability. Once incorporated into the genome of an oncolytic type 2 herpes simplex virus, FusOn-H3, Her2-COL-sFasL potentiates the therapeutic efficacy of the virus in an aggressive syngeneic mammary tumor model. Our data suggest that arming an oncolytic virus with a secretable and self-multimerizing apoptosis inducer is a feasible strategy to improve the potency of virotherapy.

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Chimeric sFasL molecules induce caspase activation and cell death in vitroHCT116 cells were transfected with respective molecules. Media was collected after 48 hours and cellular debris removed. (a) Caspase-8 and −3 cleavage following treatment with chimeric sFasL molecules. Western blot analysis of equal amounts of HCT116 cellular protein after 24 hours of specified media treatment. Caspase-8 and caspase-3 antibodies were used to detect active cleavage products specified (p18 and p19, respectively). GAPDH was used as loading control. In (b and c) viable HCT116 cells counted using trypan blue exclusion assay 48 hours after specified media treatment. Then triplicate values were averaged and percent viable cells calculated by normalization to GFP control media treated cells. (b) Reduced HCT116 cell viability following supernatant transfer. Data shown is the average of three independent experiments. ** p<0.001 as compared with GFP transfected cell media treatment controls using student’s T test. (c) HCT116 cells treated in triplicate with equal amounts of COL-sFasL and Her2-COL-sFasL. Data are representative of two independent experiments. ns, not significant; ** p<0.001 using student’s T test as compared to cells treated with GFP media. Graphs show mean ±SEM.
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Figure 2: Chimeric sFasL molecules induce caspase activation and cell death in vitroHCT116 cells were transfected with respective molecules. Media was collected after 48 hours and cellular debris removed. (a) Caspase-8 and −3 cleavage following treatment with chimeric sFasL molecules. Western blot analysis of equal amounts of HCT116 cellular protein after 24 hours of specified media treatment. Caspase-8 and caspase-3 antibodies were used to detect active cleavage products specified (p18 and p19, respectively). GAPDH was used as loading control. In (b and c) viable HCT116 cells counted using trypan blue exclusion assay 48 hours after specified media treatment. Then triplicate values were averaged and percent viable cells calculated by normalization to GFP control media treated cells. (b) Reduced HCT116 cell viability following supernatant transfer. Data shown is the average of three independent experiments. ** p<0.001 as compared with GFP transfected cell media treatment controls using student’s T test. (c) HCT116 cells treated in triplicate with equal amounts of COL-sFasL and Her2-COL-sFasL. Data are representative of two independent experiments. ns, not significant; ** p<0.001 using student’s T test as compared to cells treated with GFP media. Graphs show mean ±SEM.

Mentions: Caspase-8 is an initiator caspase specific to the extrinsic pathway, whereas caspase-3 is an executioner caspase that activates the downstream events resulting in cell death through apoptosis.29 Therefore, we tested the ability of the secreted molecules to induce caspase cleavage to activate apoptosis by measuring the cleavage of caspase-8 and caspase-3 into their respective active cleaved forms (p18 and p19 respectively). An equal volume of the 48 hour HCT116 cell media used for Figures 1b and c was added to freshly seeded HCT116 cells, which were then incubated for 24 hours. Afterwards, cells were collected and samples with equal amounts of protein were loaded for Western blot analysis. HCT116 cells treated with media containing control sFasL molecules that lack a multimerization domain or control molecules lacking the sFasL domain show negligible caspase-8 or caspase-3 cleavage (Figure 2a). Whereas, the transfer of media containing Her2-sFasL, COL-sFasL, or Her2-COL-sFasL to HCT116 cells led to significant cleavage of caspase-8 and caspase-3 into their respective active products. These results thus indicate that the ability of these sFasL containing chimeric molecules to self multimerize into an active form is crucial for them to activate caspase cleavage.


Enhancing the bystander killing effect of an oncolytic HSV by arming it with a secretable apoptosis activator.

Loya SM, Zhang X - Gene Ther. (2015)

Chimeric sFasL molecules induce caspase activation and cell death in vitroHCT116 cells were transfected with respective molecules. Media was collected after 48 hours and cellular debris removed. (a) Caspase-8 and −3 cleavage following treatment with chimeric sFasL molecules. Western blot analysis of equal amounts of HCT116 cellular protein after 24 hours of specified media treatment. Caspase-8 and caspase-3 antibodies were used to detect active cleavage products specified (p18 and p19, respectively). GAPDH was used as loading control. In (b and c) viable HCT116 cells counted using trypan blue exclusion assay 48 hours after specified media treatment. Then triplicate values were averaged and percent viable cells calculated by normalization to GFP control media treated cells. (b) Reduced HCT116 cell viability following supernatant transfer. Data shown is the average of three independent experiments. ** p<0.001 as compared with GFP transfected cell media treatment controls using student’s T test. (c) HCT116 cells treated in triplicate with equal amounts of COL-sFasL and Her2-COL-sFasL. Data are representative of two independent experiments. ns, not significant; ** p<0.001 using student’s T test as compared to cells treated with GFP media. Graphs show mean ±SEM.
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Figure 2: Chimeric sFasL molecules induce caspase activation and cell death in vitroHCT116 cells were transfected with respective molecules. Media was collected after 48 hours and cellular debris removed. (a) Caspase-8 and −3 cleavage following treatment with chimeric sFasL molecules. Western blot analysis of equal amounts of HCT116 cellular protein after 24 hours of specified media treatment. Caspase-8 and caspase-3 antibodies were used to detect active cleavage products specified (p18 and p19, respectively). GAPDH was used as loading control. In (b and c) viable HCT116 cells counted using trypan blue exclusion assay 48 hours after specified media treatment. Then triplicate values were averaged and percent viable cells calculated by normalization to GFP control media treated cells. (b) Reduced HCT116 cell viability following supernatant transfer. Data shown is the average of three independent experiments. ** p<0.001 as compared with GFP transfected cell media treatment controls using student’s T test. (c) HCT116 cells treated in triplicate with equal amounts of COL-sFasL and Her2-COL-sFasL. Data are representative of two independent experiments. ns, not significant; ** p<0.001 using student’s T test as compared to cells treated with GFP media. Graphs show mean ±SEM.
Mentions: Caspase-8 is an initiator caspase specific to the extrinsic pathway, whereas caspase-3 is an executioner caspase that activates the downstream events resulting in cell death through apoptosis.29 Therefore, we tested the ability of the secreted molecules to induce caspase cleavage to activate apoptosis by measuring the cleavage of caspase-8 and caspase-3 into their respective active cleaved forms (p18 and p19 respectively). An equal volume of the 48 hour HCT116 cell media used for Figures 1b and c was added to freshly seeded HCT116 cells, which were then incubated for 24 hours. Afterwards, cells were collected and samples with equal amounts of protein were loaded for Western blot analysis. HCT116 cells treated with media containing control sFasL molecules that lack a multimerization domain or control molecules lacking the sFasL domain show negligible caspase-8 or caspase-3 cleavage (Figure 2a). Whereas, the transfer of media containing Her2-sFasL, COL-sFasL, or Her2-COL-sFasL to HCT116 cells led to significant cleavage of caspase-8 and caspase-3 into their respective active products. These results thus indicate that the ability of these sFasL containing chimeric molecules to self multimerize into an active form is crucial for them to activate caspase cleavage.

Bottom Line: The rationale is that, due to its much smaller size, this apoptosis activator can overcome obstacles such as the dense collagen in the tumor tissues to spread more freely than the viral particles.When measured in vitro, Her2-COL-sFasL was found to efficiently induce caspase cleavage, resulting in an 80% reduction in cell viability.Once incorporated into the genome of an oncolytic type 2 herpes simplex virus, FusOn-H3, Her2-COL-sFasL potentiates the therapeutic efficacy of the virus in an aggressive syngeneic mammary tumor model.

View Article: PubMed Central - PubMed

Affiliation: 1] Department of Biology and Biochemistry, Center for Nuclear Receptors and Cell Signaling, University of Houston, Houston, TX, USA [2] Department of Molecular Virology and Microbiology, Baylor College of Medicine, Houston, TX, USA.

ABSTRACT
Although oncolytic viruses have shown great promise as cancer therapeutics, results from a recent phase III clinical trial indicate that their potency may need further improvement for a clear clinical benefit. Here, we report a novel strategy to increase the bystander effect of virotherapy by arming an oncolytic virus with a secreted form of a Her2 single chain antibody linked to a self-multimerizing Fas ligand extracellular domain (Her2-COL-sFasL). The rationale is that, due to its much smaller size, this apoptosis activator can overcome obstacles such as the dense collagen in the tumor tissues to spread more freely than the viral particles. When measured in vitro, Her2-COL-sFasL was found to efficiently induce caspase cleavage, resulting in an 80% reduction in cell viability. Once incorporated into the genome of an oncolytic type 2 herpes simplex virus, FusOn-H3, Her2-COL-sFasL potentiates the therapeutic efficacy of the virus in an aggressive syngeneic mammary tumor model. Our data suggest that arming an oncolytic virus with a secretable and self-multimerizing apoptosis inducer is a feasible strategy to improve the potency of virotherapy.

Show MeSH
Related in: MedlinePlus