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. 2010 Mar;17(3):180-91.
doi: 10.1038/cgt.2009.62. Epub 2009 Oct 2.

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

J A Jiménez  1 , X Li, Y-P Zhang, K H Bae, Y Mohammadi, P Pandya, C Kao, T A Gardner
Affiliations

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

J A Jiménez et al. Cancer Gene Ther. 2010 Mar.

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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Figures

Figure 1
Figure 1
Characterization of Ad-IU2. A, genomic structure of Ad-IU2. TRAIL cDNA was cloned into the left ITR under control of the bidirectional PSES enhancer. To avoid interference with the adenoviral packaging sequence (ψ), E1a was placed at the right ITR under the transcriptional control of PSES along with E4. Replication competent adenoviral control vector, Ad-IU1 was constructed by replacing the PSES-TRAIL cassette with a PSES-HSV-TK expression cassette (*). B, immunoblot confirming TRAIL expression in PSA/PSMA-positive CWR22rv cells following infection with 0.01 LDU/cell Ad-IU2 or transfection with pORF-hTRAIL. No endogenous TRAIL expression was detected following infection with Ad-ΔTATA-E1a. C, cell surface expression of TRAIL was confirmed in CWR22rv prostate cancer cells following infection with Ad-IU2. Overlapping histograms for Ad-IU1 and Ad-IU2 are depicted. *** = p<0.001.
Figure 2
Figure 2
Apoptosis induction by Ad-IU2. A, 0.01 LDU/cell Ad-IU2 induced 5-fold greater apoptosis at 24 hours than the PSRCA control, Ad-IU1, specifically in PSA/PSMA-positive prostate cancer cells. B, infection of TRAIL-resistant, PSA/PSMA-positive LNCaP cells with Ad-IU2 for 48 hours reversed resistance to TRAIL-mediated apoptosis, inducing apoptosis to similar levels as in TRAIL-sensitive PSA/PSMA-positive prostate cancer cells. *** = p<0.001.
Figure 3
Figure 3
Cytotoxicity of Ad-IU2 was specific to PSA/PSMA-positive prostate cancer cells. Crystal violet killing curves for CWR22rv (A), C4-2 (B), LNCaP (C) and human dermal fibroblasts (D). Cell killing was assayed once cytopathic effect was detected at 4 days (CWR22rv), 3 days (C4-2 and LNCaP) and 7 days post-infection (HDFa, cytopathic effect was not detected). * = p<0.05, *** = p<0.001 difference between Ad-IU2 and Ad-E4PSESE1a.
Figure 4
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.
Figure 5
Figure 5
Ad-IU2 suppressed the growth of androgen-independent human prostate tumors in athymic mice. A, subcutaneous androgen-independent CWR22rv xenografts were established in castrated male athymic mice and treated with intratumoral injections of PBS (vehicle control, n = 5), Ad-IU1 (PSRCA control, n = 6) or Ad-IU2 (n = 9). Mean tumor volumes at day 0 and study endpoints are listed. *** = p<0.001 (Ad-IU2 vs. Ad-IU1). B, fold tumor growths for individual mice at the 6-week end-point. Histological appearance of harvested tumors 6 weeks after treatment with PBS (C), Ad-IU1 (D) (large yellow arrows, necrotic centers of oncolysis; small yellow arrows, patches of healthy tumor cells) or Ad-IU2 (E) (large yellow arrows, necrotic centers of oncolysis; small yellow arrows, condensed nuclei) (200× magnification). In situ TUNEL assay detected no apoptosis in tumors treated with PBS (F) or Ad-IU1 (G) and marked apoptosis in tumors treated with Ad-IU2 (H).
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