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Antitumor activity of Ad-IU2, a prostate-specific replication-competent adenovirus encoding the apoptosis inducer, TRAIL.

Jiménez JA, Li X, Zhang YP, Bae KH, Mohammadi Y, Pandya P, Kao C, Gardner TA - Cancer Gene Ther. (2009)

Bottom Line: Ad-IU2 induced fivefold greater apoptosis selectively in PSA/PSMA-positive CWR22rv and C4-2 cells than an oncolytic adenoviral control.Furthermore, prolonged infection with Ad-IU2 reversed TRAIL resistance in LNCaP cells.This study shows the potential clinical utility of a PSRCA armed with an apoptosis-inducing ligand.

View Article: PubMed Central - PubMed

Affiliation: Department of Urology, Indiana University School of Medicine, Indianapolis, IN, USA.

ABSTRACT
In this study, we analyzed the preclinical utility and antitumor efficacy of tumor necrosis factor-related apoptosis-inducing ligand (TRAIL) delivered by Ad-IU2, a prostate-specific replication-competent adenovirus (PSRCA), against androgen-independent prostate cancer. Through transcriptional control of adenoviral early genes E1a, E1b and E4, as well as TRAIL by two bidirectional prostate-specific enhancing sequences (PSES), the expression of TRAIL and adenoviral replication was limited to prostate-specific antigen (PSA) and prostate-specific membrane antigen (PSMA)-positive cells. Ad-IU2 induced fivefold greater apoptosis selectively in PSA/PSMA-positive CWR22rv and C4-2 cells than an oncolytic adenoviral control. Furthermore, prolonged infection with Ad-IU2 reversed TRAIL resistance in LNCaP cells. Ad-IU2 showed superior killing efficiency in PSA/PSMA-positive prostate cancer cells at doses five- to eight-fold lower than required by a PSRCA to produce a similar effect; however, this cytotoxic effect was not observed in non-prostatic cells. As an enhancement of its therapeutic efficacy, Ad-IU2 exerted a TRAIL-mediated bystander effect through direct cell-to-cell contact and soluble factors such as apoptotic bodies. In vivo, Ad-IU2 markedly suppressed the growth of subcutaneous androgen-independent CWR22rv xenografts compared with a PSRCA at 6 weeks after treatment (3.1- vs 17.1-fold growth of tumor). This study shows the potential clinical utility of a PSRCA armed with an apoptosis-inducing ligand.

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Bystander effect of Ad-IU2. A, marked apoptosis was induced in mRFP-labeled PC-3 cells co-cultured with CWR22rv cells 24 hours after infection with Ad-IU2. B, heat-inactivated, apoptotic body-enriched conditioned media from Ad-IU2 infected CWR22rv cells induced significant levels of apoptosis in PSA/PSMA-positive and -negative prostate cancer cells. C, heat treatment of conditioned media was sufficient to inactivate adenovirus, as indicated by a drastic reduction in GFP-positive CWR22rv cells following treatment with heat-inactivated Ad-E4PSESE1a conditioned media. D, TRAIL was not cleaved from the surface of Ad-IU2-infected CWR22rv cells and present in conditioned medium at physiologically relevant concentrations. CWR22rv cells were infected with 0.01 LDU/cell Ad-IU2 for 48 hours, and cell lysate and conditioned medium (CM) were collected. Cell lysate, CM and various concentrations of soluble rhTRAIL were separated by 12% SDS-PAGE and immunoblotted with anti-human TRAIL antibody. *** = p<0.001, **** = p<0.0001.
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Figure 4: Bystander effect of Ad-IU2. A, marked apoptosis was induced in mRFP-labeled PC-3 cells co-cultured with CWR22rv cells 24 hours after infection with Ad-IU2. B, heat-inactivated, apoptotic body-enriched conditioned media from Ad-IU2 infected CWR22rv cells induced significant levels of apoptosis in PSA/PSMA-positive and -negative prostate cancer cells. C, heat treatment of conditioned media was sufficient to inactivate adenovirus, as indicated by a drastic reduction in GFP-positive CWR22rv cells following treatment with heat-inactivated Ad-E4PSESE1a conditioned media. D, TRAIL was not cleaved from the surface of Ad-IU2-infected CWR22rv cells and present in conditioned medium at physiologically relevant concentrations. CWR22rv cells were infected with 0.01 LDU/cell Ad-IU2 for 48 hours, and cell lysate and conditioned medium (CM) were collected. Cell lysate, CM and various concentrations of soluble rhTRAIL were separated by 12% SDS-PAGE and immunoblotted with anti-human TRAIL antibody. *** = p<0.001, **** = p<0.0001.

Mentions: Due to limited viral transduction efficiency in vivo and the heterogeneity of human prostate tumors with regards to PSA/PSMA-expression, the ability to target and destroy prostate cancer cells in which a PSRCA cannot replicate and lyse the cell is critical to prevent the development of foci of untreated cells within a tumor. The killing power of Ad-IU2 could be enhanced through cell-to-cell contact of neighboring cells with infected prostate cancer cells or cell contact with the apoptotic bodies from dying cells. To determine whether Ad-IU2 imparted a bystander killing effect on neighboring PSA/PSMA-negative prostate cancer cells, we co-cultured Ad-IU1- or Ad-IU2-infected CWR22rv cells with mRFP-stably transfected PC-3 cells and detected the level of apoptosis induction in the mRFP-labeled PC-3 cells. As depicted in Figure 4A, PC-3 cells, which failed to undergo apoptosis induction following direct infection by Ad-IU2, due to a lack of PSA and PSMA expression (Figure 2A), exhibited a 4-fold induction of apoptosis above the level induced by Ad-IU1 co-culture when co-cultured with Ad-IU2-infected CWR22rv cells.


Antitumor activity of Ad-IU2, a prostate-specific replication-competent adenovirus encoding the apoptosis inducer, TRAIL.

Jiménez JA, Li X, Zhang YP, Bae KH, Mohammadi Y, Pandya P, Kao C, Gardner TA - Cancer Gene Ther. (2009)

Bystander effect of Ad-IU2. A, marked apoptosis was induced in mRFP-labeled PC-3 cells co-cultured with CWR22rv cells 24 hours after infection with Ad-IU2. B, heat-inactivated, apoptotic body-enriched conditioned media from Ad-IU2 infected CWR22rv cells induced significant levels of apoptosis in PSA/PSMA-positive and -negative prostate cancer cells. C, heat treatment of conditioned media was sufficient to inactivate adenovirus, as indicated by a drastic reduction in GFP-positive CWR22rv cells following treatment with heat-inactivated Ad-E4PSESE1a conditioned media. D, TRAIL was not cleaved from the surface of Ad-IU2-infected CWR22rv cells and present in conditioned medium at physiologically relevant concentrations. CWR22rv cells were infected with 0.01 LDU/cell Ad-IU2 for 48 hours, and cell lysate and conditioned medium (CM) were collected. Cell lysate, CM and various concentrations of soluble rhTRAIL were separated by 12% SDS-PAGE and immunoblotted with anti-human TRAIL antibody. *** = p<0.001, **** = p<0.0001.
© Copyright Policy
Related In: Results  -  Collection

License
Show All Figures
getmorefigures.php?uid=PMC2821463&req=5

Figure 4: Bystander effect of Ad-IU2. A, marked apoptosis was induced in mRFP-labeled PC-3 cells co-cultured with CWR22rv cells 24 hours after infection with Ad-IU2. B, heat-inactivated, apoptotic body-enriched conditioned media from Ad-IU2 infected CWR22rv cells induced significant levels of apoptosis in PSA/PSMA-positive and -negative prostate cancer cells. C, heat treatment of conditioned media was sufficient to inactivate adenovirus, as indicated by a drastic reduction in GFP-positive CWR22rv cells following treatment with heat-inactivated Ad-E4PSESE1a conditioned media. D, TRAIL was not cleaved from the surface of Ad-IU2-infected CWR22rv cells and present in conditioned medium at physiologically relevant concentrations. CWR22rv cells were infected with 0.01 LDU/cell Ad-IU2 for 48 hours, and cell lysate and conditioned medium (CM) were collected. Cell lysate, CM and various concentrations of soluble rhTRAIL were separated by 12% SDS-PAGE and immunoblotted with anti-human TRAIL antibody. *** = p<0.001, **** = p<0.0001.
Mentions: Due to limited viral transduction efficiency in vivo and the heterogeneity of human prostate tumors with regards to PSA/PSMA-expression, the ability to target and destroy prostate cancer cells in which a PSRCA cannot replicate and lyse the cell is critical to prevent the development of foci of untreated cells within a tumor. The killing power of Ad-IU2 could be enhanced through cell-to-cell contact of neighboring cells with infected prostate cancer cells or cell contact with the apoptotic bodies from dying cells. To determine whether Ad-IU2 imparted a bystander killing effect on neighboring PSA/PSMA-negative prostate cancer cells, we co-cultured Ad-IU1- or Ad-IU2-infected CWR22rv cells with mRFP-stably transfected PC-3 cells and detected the level of apoptosis induction in the mRFP-labeled PC-3 cells. As depicted in Figure 4A, PC-3 cells, which failed to undergo apoptosis induction following direct infection by Ad-IU2, due to a lack of PSA and PSMA expression (Figure 2A), exhibited a 4-fold induction of apoptosis above the level induced by Ad-IU1 co-culture when co-cultured with Ad-IU2-infected CWR22rv cells.

Bottom Line: Ad-IU2 induced fivefold greater apoptosis selectively in PSA/PSMA-positive CWR22rv and C4-2 cells than an oncolytic adenoviral control.Furthermore, prolonged infection with Ad-IU2 reversed TRAIL resistance in LNCaP cells.This study shows the potential clinical utility of a PSRCA armed with an apoptosis-inducing ligand.

View Article: PubMed Central - PubMed

Affiliation: Department of Urology, Indiana University School of Medicine, Indianapolis, IN, USA.

ABSTRACT
In this study, we analyzed the preclinical utility and antitumor efficacy of tumor necrosis factor-related apoptosis-inducing ligand (TRAIL) delivered by Ad-IU2, a prostate-specific replication-competent adenovirus (PSRCA), against androgen-independent prostate cancer. Through transcriptional control of adenoviral early genes E1a, E1b and E4, as well as TRAIL by two bidirectional prostate-specific enhancing sequences (PSES), the expression of TRAIL and adenoviral replication was limited to prostate-specific antigen (PSA) and prostate-specific membrane antigen (PSMA)-positive cells. Ad-IU2 induced fivefold greater apoptosis selectively in PSA/PSMA-positive CWR22rv and C4-2 cells than an oncolytic adenoviral control. Furthermore, prolonged infection with Ad-IU2 reversed TRAIL resistance in LNCaP cells. Ad-IU2 showed superior killing efficiency in PSA/PSMA-positive prostate cancer cells at doses five- to eight-fold lower than required by a PSRCA to produce a similar effect; however, this cytotoxic effect was not observed in non-prostatic cells. As an enhancement of its therapeutic efficacy, Ad-IU2 exerted a TRAIL-mediated bystander effect through direct cell-to-cell contact and soluble factors such as apoptotic bodies. In vivo, Ad-IU2 markedly suppressed the growth of subcutaneous androgen-independent CWR22rv xenografts compared with a PSRCA at 6 weeks after treatment (3.1- vs 17.1-fold growth of tumor). This study shows the potential clinical utility of a PSRCA armed with an apoptosis-inducing ligand.

Show MeSH
Related in: MedlinePlus