Oral Herpes

Herpesvirus saimiri-based endothelin-converting enzyme-1 shRNA expression decreases prostate cancer cell invasion and migration – Hong – 2010 – International Journal of Cancer

Sexual history has consistently been found to be a risk factor for the development of prostate cancer. Pantuck, Andreas Hinkel, Jean deKernion, and Robert Figlin. “Suicide” gene therapy delivered alone or in combination with other forms of treatment could potentially provide simultaneous efficacy against localized and systemic disease via the generation of cytotoxic activity and/or systemic immunity to the cancer. We therefore hypothesized that FDG-PET can predict tumor response to oncolytic HSV therapy. Aggressive tumours were defined as Gleason score ≥7, poorly-differentiated, including tumours staged at T4, N + (lymph node positive), or M + (distant metastases) regardless of their Gleason score or grade of differentiation. These viral RNAs were expressed to relatively low levels in this subset; because one of these RNAs encodes a major viral capsid protein, these cells may be producing KSHV. Howard, Howard J.

Hernaiz Driever P. Approximately 50% of human miRNAs are encoded in genomic regions that are frequently altered in various types of cancers including prostate cancer (PCa) [3-5]. Nat Med 3: 639-645. In particular, the upregulation of protein components involved in the endothelin-1 (ET-1) signaling axis plays a key role in progression of many types of cancer, including ovarian, colorectal, breast and PC.3–6 Specifically, plasma levels of ET-1 are abnormally elevated in patients with advanced, androgen-independent PC7, and the potent ET receptor subtype A (ETAR) antagonist atrasentan (ABT-627) has been reported to suppress partially prostate tumor growth.8 Hence, inhibition of ET formation or downstream signaling could provide a novel strategy to treat advanced PC. A history of multiple sexual partners also appears to have no effect on the development of prostate cancer and interestingly smoking does not appear to have any effect unlike other forms of cancer. DNA from prostate tissue was purified and checked for integrity as described (Alexeyev et al, 2006). Slides were rinsed with TBST (0.1% (v/v) Tween 20) and overlaid with anti-HSVtK antibody (MTM Laboratories, 1 in 100 dilution) diluted in 0.05 M Tris-HCl buffer, pH 7.2, containing 1% bovine serum albumin (Sigma) for 30 minutes.


In this context, we have previously reported that using siRNA, which targeted a common sequence of all ECE-1 isoforms, could effectively block proliferation of oral squamous carcinoma cells.21 However, the siRNA oligonucleotides only provide a transient effect in transfected cells, and cellular uptake is generally limited in ex vivo and in vivo environments. Specimens were obtained from the Department of Pathology at Cedars Sinai Medical Center, Los Angeles, CA, and the Department of Medicine, Universita Cattolica, Rome, Italy. Although adenoviral, retroviral and adeno-associated viral short-hairpin RNA (shRNA) delivery systems have been successfully exploited to silence genes in vitro and in vivo,22–25 they possess a major drawback either in strong immunogenicity or in unexpected endogenous oncogene expression because of the potential integration of viral DNA into the host genome.26–28 As a consequence, we have used Herpesvirus saimiri (HVS), a dsDNA γ-2 herpesvirus, as the gene therapy vector, which is maintained in infected cells as a nonintegrated circular episome. HepG2 and MRC-5 cells were maintained in Dulbecco’s modified essential medium (Invitrogen, Life Technologies) containing 2 mM glutamine, 0.45% glucose, and 10% FBS. These attributes therefore support the use of an HVS-based vector as a potent, safe and efficient delivery vehicle in gene therapy research. About 2500-bp inserted fragment was detected in AdTCPtk as compared to pJM17. The cDNA coding bp 1–1149 of human Cx43 was amplified by PCR using the synthesized cDNA from KB cells as a template and the following Cx43-specific primers: Cx43 forward primer (5′ ATCAATGGaccATGGGTGACTGGAGCGCCT) and Cx43 reverse primer (5′ CATCTAGACTAGATCTCCAGGTCATCAG.).

In our study, by targeting the ET axis through ECE-1 depletion, we have examined this potential approach as a novel treatment of advanced stages of PC. We have previously used siRNA duplexes (nonisoform specific) transiently to successfully target ECE-1 and modulate proliferative behavior of oral squamous carcinoma cells.21 Independently, using ECE-1 siRNA in ovarian carcinoma cells, the suppressive effect of ECE-1 knockdown on cell invasiveness, MMP-2 activity and epithelial-to-mesenchymal transition has been demonstrated.45 Here, we have extended our studies to achieve sustained suppression by exploiting a novel HVS-based viral delivery system to facilitate ECE-1-shRNA transfer into PC cells. HVS is a prototype gamma-2 herpesvirus, 112 kb in length, with the “gutless” potential to package ≥120 kb of heterologous DNA.46 Deletion of the transforming genes, STP and Tip, has eliminated its oncogenic potential and prevented it from T cell transformation, which increases the biosafety of an HVS-based vector.47, 48 The use of F-factor-based BAC allows the easy and efficient cloning and modification of viral genome in E. coli cells.49 HVS vectors are attractive for their ability to establish episomal latent persistence and sustained transgene expression in human carcinoma cells.30, 50 HVS can efficiently infect solid tumor xenografts and three-dimensional spheroid cultures.51 These results suggest that HVS-based vectors may be ideal for gene therapy applications. We have successfully cloned, characterized and purified HVS-BAC-shRNA virus and, using an MOI of 2, achieved infection efficiencies of ≥80% for G7 stromal cells ex vivo and PC-3 cells in vitro. Invasion studies using PC-3 cells in coculture with G7 stroma infected with HVS-BAC-shRNA revealed a reduction in PC-3 invasive ability of 33%. The reciprocal study using HVS-BAC-shRNA-infected PC-3 cells in coculture with untreated G7 stromal cells revealed a similar reduced PC-3 invasive ability.

A >50% reduction in serum PSA was achieved in five out of 20 patients in this dose-escalation study: all responders were in the two highest dosage groups.35 Only mild adverse events were observed, indicating that the PSA promoter/enhancer restriction is stringent enough to limit replication of the virus in other cell types. The cultured wells were washed with phosphate-buffered saline and pelleted briefly for 2 minutes to harvest the cells. Migration studies using a scratch assay showed that PC-3 cells infected with HVS-BAC-shRNA had reduced ability to migrate across and close an artificial scratch created in the growing cell monolayer. A. The luciferase activity was not significantly observed in nonprostate cancer cells in the presence of ATRA (Fig. Reports from physicians and relatives were also collected when available. In addition, to ensure a more efficient literature search and maximize the use of the subject lists in the SRP and EXP, the snowballing search strategy was applied to manually search for the referred literature in each paper [37].

found that exogenous addition of ET-1 failed to counterbalance the inhibition of growth and proliferation elicited by ECE-1 inhibitors.52 Altogether, these and other53 findings suggest that mechanisms additional to big ET-1 activation may be involved in ECE-1 inhibition-mediated suppression of cell proliferation and migration. Lentiviruses with antibodies recognizing a prostate cell-specific surface antigen bound to their envelope can specifically infect prostate cells (). Several potential mechanisms for the development of androgen-independency have been described, including activation of epidermal (EGF), fibroblast (FGF), or other growth-factor pathways; alteration of genes such as p21waf1, fibronectin, IGFBP2, and insulin receptor, and overexpression of bcl-2 (suppressor of apoptosis) in prostate cancer cell lines (reviewed in [5]). Our rationale for using ECE shRNA gene therapy for advanced PC is strengthened by a recent study using a lentiviral system to deliver an ECE-related homolog, NEP, to suppress PC growth.54 As NEP, an ET-1-degrading enzyme, has a tumor-suppressing effect and is frequently lost in various cancers,55, 56 the effects of NEP overexpression reinforce the concept of exploitation of ECE-1 knockdown in treatment of PC although, again, the NEP effects may be mediated both by its catalytic and noncatalytic (signaling) actions.54 Our results confirm the potential of HVS as a candidate vector for ECE siRNA gene therapy applications using PC cells and suggest that it may be have applicability in impeding the cancer cell ability to invade and migrate. To investigate the potential for detection bias by infection-mediated PSA elevation or –induction of prostate abnormalities, we performed additional separate analyses for cases diagnosed by “for-cause” and “end-of-study” biopsy, and we stratified analyses by follow-up time (within one year of blood draw at visit 2 versus later in follow-up, and ≤2.5 versus >2.5 years of follow-up from visit 2) for men diagnosed by “for-cause” biopsy. In addition, its episomal existence and species incompatibility may render it a safer option as a prospective application for therapy in humans.