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Abstract
This study was aimed to investigate the effect of combined cancer gene therapy with exogenous tumor necrosis factor-alpha (TNF-α) and cytosine deaminase (CD) suicide gene on laryngeal carcinoma cell line Hep-2 in vitro and in vivo. Transfection of the recombinant eukaryotic vectors of pcDNA3.1 (+) containing TNF-α and/or CD into Hep-2 cells resulted in expression of TNF-α and/or CD gene in vitro. The significant increase in apoptotic Hep-2 cells and decrease of Hep-2 cell proliferation were observed using 5-FC treatment combined with TNF-a expression by CD/5-FC suicide system. Moreover, bystander effect was also observed in the TNF-α and CD gene co-expression group. Laryngeal squamous cell carcinoma (LSCC) mice model was established by using BALB/c mice which different transfected Hep-2 cells with pcDNA3.1 (+) containing TNF-α and/or CD were applied subcutaneously. So these mice are divided into four groups, namely, Hep-2/TIC group; Hep-2/CD group; Hep-2/TNF-α group; Hep-2/0 group. At day 29 after cell inoculation, volume of grafted tumor had significant difference between each two of them (P<0.05). These results showed that the products of combined CD and TNF-α genes inhibited the growth of transplanted LSCC in mice model. So by our observed parameters and many others results, we hypothesized that 5-FC combined gene therapy with TNF-αand CD suicide gene should be an effective treatment on Laryngeal carcinoma.
Highlights
Laryngeal carcinoma (LC) is one of the most prevalent malignant tumors in the head and neck area
Cloning of target genes and construction of vector According to the nucleotide sequence of Tumor Necrosis Factor-alpha (TNF-a) gene and cytosine deaminase (CD) gene provided in GenBank, EcoR I and Not I restriction enzyme sites were inserted into up-stream and down-stream of TNF-a gene; Xho I and Apa I restriction enzyme sites were inserted into the 59 terminal of the up-stream and down-stream primers of CD gene
Amplification of TNF-a gene and CD gene Electrophoresis analysis was performed on the RT-PCR
Summary
Laryngeal carcinoma (LC) is one of the most prevalent malignant tumors in the head and neck area. A promising treatment strategy should ensure treatment efficacy, reduce treatment-related toxicity reaction and improve quality of life. These aspects have been climbed into the top priority consideration. There is no research which has been reported using CD gene combined with TNF-a gene in treating cancer Based on these above background, we hypothesis that, by transfectting eukaryotic vector pcDNA3.1 (+) carrying suicide CD and TNF-a gene into laryngeal cancer cellHep-2, this method can combine the complementary advantage of each gene, thereby improve the efficacy of gene therapy, increase anti-tumor immune responses, reduce their toxicity, exert the synergic cytocidal effect of suicidal gene as well as cytokine gene on tumor cells
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