In the present study, we successfully constructed a novel replication-selective adenovirus, Ad315-E1A, and a replication-deficient adenovirus, Ad315-EGFP. Our data show a significant effect of Ad315-E1A against tumor growth both in vitro and in vivo. This observation suggests that replication-selective adenoviruses driven by the IGF2 imprinting system may be used as a novel anti-cancer agent with a high therapeutic potential.
Using viruses as a genetic carrier offers an attractive alternative or promising integration with traditional therapeutic regimens for cancer treatment
]. Among viral-based therapies and oncolytic agents, adenoviruses have emerged as a promising vector that is already being used for the treatment of solid tumors in humans
]. Adenoviruses are non-enveloped viruses with linear double-stranded DNA, which infect cells by binding to the coxsackie and adenovirus receptor expressed on the surface of target cells
]. As vectors for oncolytic therapies, these viruses have many advantages over other vectors, including the capability of transducing and replicating in dividing and non-dividing cells, their ease of manipulation, and a naturally lytic replication cycle
]. All of these features highlight the utility of adenoviruses for in vitro production and in vivo curative effects
]. Since 1993, more than 300 clinical trials based on adenoviral vectors have been performed
] with promising outcomes. The first clinical results of trials based on adenoviruses as an oncolytic therapeutic agent have been promising, and show the clinical safety and feasibility of this approach
]. In our study, we constructed an oncolytic adenoviral vector and demonstrated its effective killing of cancer cells both in vitro and in vivo.
IGF2 encodes the mitogenic anti-apoptotic peptide IGF-II that is overexpressed in several human cancers cells. In colorectal cancer, it has been reported that LOI of IGF2 occurs in 44% of informative colorectal cancers that are linked to microsatellite instability
]. Additional studies have reported a range from 33% to 87% of sporadic colon cancers that were related to LOI of IGF2
Based on these findings, we theorized that we could build a kind of oncolytic adenovirus specific for replication in LOI tumor cells for colorectal cancer therapy. In this proof-of-concept study, we constructed adenoviral vectors, Ad315-E1A and Ad315-EGFP, which carry the three enhancer elements, DMD and promoter based on IGF2 LOI. Initially the utility of our expression system was tested using EGFP reporter assays. The results showed that green fluorescence was detected only in LOI cells. By increasing the multiplicity of infection and extending the time of infection, we found that the results were unchanged. To further demonstrate the selective efficacy of the LOI system, we used the oncolytic adenovirus H101, which has been clinically approved in China to treat malignancies, as a positive control for specific lysis of tumor cells by targeting the inactivated p53 in tumors. We used RT-PCR and western blotting to detect E1A expression at mRNA and protein levels, respectively, in Ad315-E1A-infected cells, and confirmed E1A expression in LOI colorectal cell lines. Furthermore, in H101-infected cells, E1A mRNA and protein was only detected in the p53 mutant cell line HT-29. All of the above results indicate that the replication-selective system that we used is practicable and effective.
In this study, the therapeutic potential of Ad315-E1A was tested by an MTT assay and flow cytometry, and the results clearly support the rationale of our hypothesis, in which Ad315-E1A effectively suppressed the growth of tumor cells and induced obvious cytotoxicity in LOI tumor cells. Therefore, we compared the therapeutic effect between IGF2 regulatory sequences and H101 for colorectal cancer treatment. The data showed that there was no detectable cytotoxicity in HCT-116 and GES-1 cells infected with Ad315-E1A or H101 (10 PFU/cell), whereas Ad315-E1A infection achieved significant growth inhibition of the IGF2 LOI and p53-active cell line (HCT-8) relative to that by H101 infection. In addition, both oncolytic viruses exhibited superior efficiency to inhibit growth of the HT-29 (p53 mutant and LOI) cell line. These results suggest that the IGF2 LOI system in our viral vector enabled efficient adenovirus replication in host cells using the E1A endogenous promoter.
Using the human colorectal cancer cell line HCT-8 as a model, we showed that this therapeutic strategy significantly increased cancer cell death and apoptosis caused by virus replication in vivo. Despite Ad315-E1A treatment significantly inhibiting tumor growth and prolonging the survival of tumor-bearing mice, the tumors were not eliminated completely. Previous research also found that oncolytic viruses have a limited potential to eradicate tumors when used as a monotherapy
]. Thus, oncolytic viruses are often used in combination with other modalities, such as chemotherapy
], or arming oncolytic adenoviruses with therapeutic genes
], to improve the anti-tumor efficacy. In our next study, we will investigate appropriate methods to enhance the potency of the novel recombinant adenovirus for cancer gene therapy.