ABSTRACT
BACKGROUND: Hec1 (Highly Expressed in Cancer gene 1) has recently been shown to play an important role in the proper segregation of chromosomes during mitosis. Recently, an adenovirus delivery system carrying RNA interference (RNAi) of Hec1 has been reported in a cervical adenocarcinoma model. Adenoviral delivery systems, however, have the main limitation of poor viral infectivity due to lack of the native receptor, Coxsackie-Adenovirus Receptor (CAR), on the surface of tumor cells. We hypothesize that the viral infectivity of the adenovirus vector would be enhanced via a CAR-independent pathway by altering the targeting tropism, thus increasing the knockdown effect of Hec1 expression in ovarian carcinoma cells. METHODS: Two adenoviruses (Ad-siRNA-Hec1 and Ad-siRNA-Hec1.F5/3), along with a negative control (Ad-siRNA-GAPDH.F5/3), were created using homologous recombination. HEY and SKOV3.ip1 cell lines were used to perform experiments. The following assays were then used to determine RNAi knockdown efficiency: (1) quantitative PCR (QPCR), (2) Western blot, (3) MTS assay, (4) Annexin V-FITC FACS, (5) crystal violet staining. In all experiments, a negative control served as a baseline measure. RESULTS: QPCR demonstrated a 2-log viral infectivity enhancement with Ad-siRNA-Hec1.F5/3 over Ad-siRNA-Hec1. QPCR at 72 h revealed mRNA knockdown induced by Ad-siRNA-Hec1 and Ad-siRNA-Hec1.F5/3 in SKOV3.ip1 and HEY cells, respectively (71%/60%, and 32%/78% mRNA knockdown compared to negative control). Western blot revealed translational inhibition induced by both Hec1 Ads with the least knockdown seen with Ad-siRNA-GAPDH.F5/3. FACS analysis revealed increased annexin V positivity in RNAi-infected cells, suggesting a higher rate of apoptosis. MTS assay indicated increased cell death 8 days post-infection with Ad-siRNA-Hec1 and Ad-siRNA-Hec1.F5/3 in SKOV3.ip1 and HEY cell lines, respectively (75% vs. 35% and 43% vs. 12% viable cells). Crystal violet staining revealed increased cell death with Ad-siRNA-Hec1.F5/3 in all tested cell lines. CONCLUSIONS: RNAi against Hec1 results in gene expression knockdown and apoptosis in vitro. The infectivity-enhanced adenovirus as delivery mechanism shows potential application in future gene therapy models of RNAi in ovarian cancer.
Subject(s)
Adenoviridae/genetics , Genetic Therapy/methods , Nuclear Proteins/genetics , Ovarian Neoplasms/therapy , RNA Interference , RNA, Small Interfering/genetics , Adenoviridae/pathogenicity , Apoptosis/genetics , Cell Line, Tumor , Cytoskeletal Proteins , Female , Genetic Vectors/genetics , Humans , Nuclear Proteins/antagonists & inhibitors , Nuclear Proteins/biosynthesis , Ovarian Neoplasms/genetics , Ovarian Neoplasms/virology , RNA, Messenger/biosynthesis , RNA, Messenger/genetics , RNA, Small Interfering/biosynthesisABSTRACT
Cholangiocarcinoma is a highly malignant neoplasm with no effective treatment. Conditionally replicative adenoviruses (CRAds) represent a promising new modality for the treatment of cancer in general. A key contribution in this regard was the introduction of tumor-selective viral replication for amplification of the initial inoculum in the neoplastic cell population. Under ideal conditions following cellular infection, the viruses replicate selectively in the infected tumor cells and kill the cells by cytolysis, leaving normal cells unaffected. However, to date there have been two limitations to the clinical application of these CRAd agents, i.e. poor viral infectivity and tumor specificity. Here we report the construction of three new CRAd agents, CRAd-S.RGD, CRAd-S.F5/3 and CRAd-S.pk7, in which the tumor specificity is regulated by a tumor-specific promoter, the survivin promoter, and the viral infectivity is enhanced by incorporating a capsid modification (RGD, F5/3 or pk7) in the adenovirus fiber region. These CRAd agents effectively target cholangiocarcinoma cells, induce strong cytoxicity in these cells in vitro and inhibit tumor growth in a murine xenograft model in vivo. In addition, the survivin promoter has extremely low activity both in the non-transformed cell line, HMEC, and in human liver tissue. Our results suggest that the survivin-based CRAds are promising agents for targeting cholangiocarcinoma with low host toxicity. Such results should provide important insights into the identification of novel therapeutic strategies for cholangiocarcinoma.
