Dual-targeted Gene Therapy For Laryngeal Carcinoma

Background and Objective:Laryngeal carcinoma is a common cancer in upper respiratory tract. The survival rate is still not obviously improved in spite of the great advances in medical technology in recent30years. Advanced cancers are often unresectable, thus the treatment mainly rely on chemotherapy and radiotherapy, instead of radical surgery. The survival rate is low for these patients. It would be meaningful if we introduce modern biotechnology into laryngeal carcinoma treatment to explore new anti-cancer biotherapy with high efficiency and low toxicity. Gene therapy is an important part of biotherapy. It has been widely applied in cancer reseach. Gene therapy is a promising direction to probing into, which brings new hope for laryngeal cancer treatment.Adenovirus is a widely-applied gene vector in gene therapy. Using tumor specific promoter to regulate gene expression can help the recombinant adenovirus targeting to the tumor tissues. In a previous study, we demonstrated that the secretory leukoprotease inhibitor (SLPI) promotor controlled recombinant adenovirus could effectively target to laryngeal carcinoma.Uncontrolled cell proliferation and apoptosis malfunction are responsible for the formation of cancer. Hence, the strategy of anti-cancer gene therapy can be designed from the following two aspects:the inhibition of cell proliferation and induction of apoptosis. The over-expression of epidermal growth factor receptor (EGFR) in head and neck squamous cell carcinoma (HNSCC) is one of the important molecular mechanisms of malignancy, and is closely associated with tumor growth, metastasis and prognosis. Molecular targeted drugs often induce drug resistence result from increased expression, impaired degradation and mutation of EGFR. RNA interference technology can downregulate EGFR expression to block its growth-promoting functions, while avoid the above-mentioned drug resistence mechanism.Caspase-3plays a key role in the induction of apoptosis. A recombinant Caspase-3has been designed to spontaneously fold into an active three-dimensional structure, inducing cell apoptosis directly without the activation of the upstream elements. Our previous study found that the recombinant Caspase-3could effectively and safely inhibit laryngeal cancer, under the control of a tumor specific promotor SLPI.We plan to design an adenovirus vector, armed with EGFR targeted artificial microRNA and recombinant Caspase-3under the control of laryngeal carcinoma specific promoter SLPI, and then evaluate the in vitro and in vivo cancer inhibition effect to human laryngeal squamous cancer cell lineHep-2cells, compared with DDP and Cetuximab, the first-line agents for advanced laryngeal carcinoma.Methods:1. As for adenovirus packaging, shuttle plasmids pDC312-SLPI-EGFRamiR-pA- SLPI-revCasp3-TAG-pA and pDC312-SLPI-GFP-pA were constructed and then cotransfected into HEK293separately with the backbone plasmid of pBGHlox(delta)E1,3Cre. PCR was used to identify recombinant adenovirus of Ad-EC and Ad-GFP. Recombinant adenovirus were amplified and then purified by CsCl density gradient centrifugation. Adenoviruses’titers were assessed by TCID50. Hep-2and NF cells were infected separately, and then gene expression was detected by fluorescent microscope, flow cytometry and western blot.2. Cell growth inhibition of Hep-2by recombinant adenoviruses in vitro was evaluated by MTT, flow cytometry analysis and RTCA, comparing with DDP, Cetuximab, combination group (Ad-EC+DDP) and no treatment group. Relative proteins were detected by Western blot.3. The antitumor activity and side effects by recombinant adenoviruses in vivo were measured in human laryngeal carcinoma xenograft in nude mice, comparing with DDP, Cetuximab, combination group (Ad-EC+DDP) and PBS. Tumor metabolism was tested by microPET.Results:1Construction of recombinant adenovirusesRecombinant adenoviruses were harvested after about13days from cotransfection when an obvious cytopathic effect was observed in HEK293. In the identification of adenoviruses using PCR, the expected fragments of879bp could be found in the amplified products of the virus supernatants from Ad-EC and1482bp from Ad-GFP, proving that the recombinant adenoviruses were successfully constructed. After amplification, purification and concentration, the titers for Ad-EC and Ad-GFP reached 6.3×109pfu/ml, and3.89×1O10pfu/ml respectively.72hours after the infection of Ad-GFP, strong signals of green fluorescence were observed in most of Hep-2cells, using fluorescence microscopy. However, it showed absent expression in NF. In flow cytometry, green fluorescent positive rate in Hep-2cells was45%, and NF only12%. The expression of EGFR was downregulated and active Caspase-3was upregulated in Hep-2cells after a72hours infection of Ad-EC, but not in Ad-GFP group. These results indicated that the expression of target genes was specifically restricted in Hep-2cells and absent in normal fibroblast, under the control of SLPI promoter.72hours was the appropriate time for infection.2Cytotoxicity of Ad-EC to Hep-2cells in vitro study2.1MTT AssayHep-2cells proliferation was well inhibited by Ad-EC after72hours post-infection. The inhibition rate, proportional to MOI level, was44%at a MOI of50. The inhibition rate was strongly increased in the combined use of Ad-EC and DDP. Cetuximab and Ad-GFP could rarely inhibit the proliferation of Hep-2cells.2.2Analysis of apoptosis by flow cytometryThe apoptosis rate of Hep-2cells infected by Ad-EC for72hours at a MOI of50was36.1%, significantly higher than0.25μg/ml of DDP (13.5%, P<0.001),500μg/ml of Cetuximab (3.5%, P<0.001) and Ad-GFP at a MOI of50(6.5%, P<0.001). When combined with DDP, the apoptosis rate rised to61.2%, sighnificantly higher than the single use of DDP and Ad-EC (P<0.001for both).2.3Real Time Cellular Analysis using iCELLigence systemA unit-less parameter termed cell index (CI) is derived to represent cell status based on the measured relative change in electrical impedance that occurs in the presence and absence of cells in the wells. The plateau in Ad-EC group at a MOI of50had a CI level of3.3-3.5, and DDP group2.8-3.0, both higher than control group (3.5-3.7). This indicated that hep-2cell growth was inhibited by Ad-EC and DDP. In the combination of Ad-EC and DDP, CI kept a downward trend from36hours post-administration (CI=3.7) to the endpoint at96hours post-administration (CI=1.0), indicating a high-level cytotoxicity.2.4Western blotThe expression of EGFR was downregulated, active Caspase-3upregulated and the cleaved of PARP increased, after Hep-2cells were infected by Ad-EC at a MOI of50for72hours. These changes were enhanced by the combined use of Ad-EC and DDP.3The antitumor activity of Ad-EC to human laryngeal carcinoma xenograft in nude miceThe tumor volume and tumor weight in the Ad-EC group were significantly smaller than PBS group (P<0.05). The activity of glucose uptake was observed to be lower, too. There were no significant differences in body weight, activities and diets among them. DDP also significantly inhibited tumor volume and tumor weight (P<0.05), as well as metabolism. However, the growth of nude mice body weight was notably restrained. In the combined use of Ad-EC and DDP, the tumor inhibition was enhanced, but body weight was suppressed even worse when compared to DDP group (P<0.05).Conclusion:1. The Recombinant adenovirus of Ad-EC was successfully constructed.2. In vitro study, Ad-EC could well inhibit the proliferation of Hep-2cells and induce apoptosis, which is dose-related. The efficacy could be enhanced in the combined use with DDP.3. In xenograft nude mice, Ad-EC could effectively inhibit tumor growth. The combination with DDP would enhance the tumor inhibition.