Antitumor Efficacy Of Adenovirus-mediated Gene Therapy By Activated Caspase-3 Controlled By Tissue Specific Promoter In Laryngeal Carcinoma

Laryngeal carcinoma is one of the most common cancers in head and neck, with 12,250 new cases reported every year in the United States alone according to the data from Surveillance, Epidemiology and End Results (SEER) in 2009. The incidence is also gradually increasing in our country. Despite advances in conventional therapy including surgery, chemotherapy and radiation, the 5-year survival rate has not been significantly improved during the past three decades. The regional control for advanced laryngeal carcinoma is sometimes unsatisfactory, even under the combined therapy of radical surgery, adjuvant radiotherapy and chemotherapy. Big hurdles also lie in front of the therapeutic regimen for terminal relapsed cases:the tumor was unresectable due to invasion to the important blood vessels in head and neck or to the important cranial nerves, while the sensitivity to the administration of adjuvant radiotherapy and chemotherapy is rather low in squamous cell carcinoma. Therefore, local and regional disease control is paramount, underscoring an urgent need for more effective, minimally invasive and lower toxic biotherapy in laryngeal carcinoma.Currently adenovirus-based therapy is a useful approach for effective gene delivery and expression in laryngeal carcinoma, by targeting various kinds of genes, like tumor suppressor genes, antisense oncogenes, immune regulatory factors, suicide genes. Although these strategies could be effective in inhibiting the growth of laryngeal cancer to some extend, no ideal strategy has yet been developed to achieve specific and efficient anti-tumor effect and avoid any adverse reactions in normal cells. Therefore, effective targeting strategies are necessary to increase target transduction and reduce the risk of unspecific side effects to nontarget tissues. So far, modifying adenovirus by tissue specific promoters (TSPs) is one of the most effective ways for targeting gene therapy for adenovirus vectors. However, no effective tissue specific promoter has been reported in laryngeal carcinoma. As 95% of the laryngeal carcinoma is composed by squamous cell carcinoma, the TSPs for some squamous cell carcinomas could also be a suitable candidate for laryngeal carcinoma. In recent years, some researches have reported the successful appliance for secretory leukocyte protease inhibitor (SLPI) promoter and squamous cell carcinoma antigen 2 (SCCA2) promoter in the targeting gene therapy in ovarian cancer, lung cancer, and other squamous cell carcinomas. Meanwhile, SLPI, SCCA2 were confirmed as potential biomarkers for laryngeal carcinoma, with significant over-expression in tumor tissues and extremely low expression in normal hepatic tissues. These characteristics have provided strong evidences for the usage of the two above promoters for targeting gene therapy in laryngeal carcinoma.In addition, the selection of suitable effect genes to induce efficient apoptosis is an essential part in gene therapy. The latest research results indicate that Caspase-3, a member of aspartate-specific cysteine protease, has taken a central stage in the induction of apoptosis, which make it a potential candidate as an apoptosis-induced factor with wide range of applicability in almost all kinds of malignant tumors. Recently, a kind of activated Caspase-3 was addressed to spontaneously fold into three-dimensional structure, inducing cell apoptosis directly without activation of the upstream elements. It would be more convenient and efficient by using activated Caspase-3 to kill tumor cells. By taking all these evidence into consideration, activated Caspase-3 could also be a powerful effect gene in the practice for gene therapy in laryngeal carcinoma. ObjectiveTo specifically and efficiently induce apoptosis in laryngeal carcinomas by a novel strategy for adenovirus-based targeting gene therapyMethods1. The region of SCCA2, SLPI1-3 promoters (corresponding to the region of SLPI promoter -849-+22,-650-+22,-531-+22) was amplified by PCR. After sequence analysis, they were inserted separately into the multiple cloning sites (MCS) in luciferase reporter gene vector of pGL4.10.2. The recombinant plasmids of pGL4.10-SCCA2, pGL4.10-SLPI1-3, and pGL4.10-Survivin were separately cotransfected with pGL4.74 into Hep-2, HuVEC and MG-63. The efficiency of each promoter was detected by using Dual-Luciferase Reporter Gene Assay Kit (SLPI2 is the most efficient promoter in our study).3. The shuttle plasmids of adenovirus pDC312-SLPI-Caspase-3-TAG-pA and Ad-SLPI-GFP-pA were constructed and then separately cotransfected into HEK293 with the backbone plasmid of pBGHlox (delta) E1,3Cre for adenovirus packaging. Recombinant adenovirus of Ad-SLPI-Caspase-3 and Ad-SLPI-GFP were identified by PCR and western blot. Recombinant adenovirus were amplified and purified by CsCl density gradient centrifugation. The titers for recombinant adenovirus were assessed by TCID50.4. The inhibition of the growth for Hep-2 and HuVEC by recombinant adenoviruses in vitro was evaluated by the microscope, MTT, DNA fragmentation analysis and flow cytometry analysis. 5. The antitumor activity and side effects in vivo by recombinant adenoviruses were observed in human laryngeal carcinoma xenograft in Nude Mice.Results1. Construction of SCCA2, SLPI1-3 promoters and detection for the activity of different promoters in each cell lineRecombinant plasmids of pGL4.10-SCCA2, pGL4.10-SLPI1-3, and pGL4.10-Survivin were successfully constructed. Among all the promoters for Hep-2, promoter for SLPI2 showed the highest activity, the difference was statistically significant (P<0.05) by one-way ANOVA test. By comparing with the activities in the cell lines for HuVEC and MG-63, promoter for SLPI2 also showed the highest activity in Hep-2, with approximately 36.67 fold and 17.11 fold respectively.2. Construction for the recombinant adenoviruses for Ad-SLPI-Caspase-3, Ad-SLPI-GFPAn obvious cytopathic effect (CPE) was observed in HEK293 after about 13 days from cotransfection. Recombinant adenoviruses were then harvested and identified by PCR. The expected fragments of 330bp and 1482bp could be found in the amplified products of the virus supernatants from Ad-SLPI-Caspase-3 and Ad-SLPI-GFP respectively. The recombinant adenoviruses were successfully constructed. After amplification, purification and concentration, the titers for Ad-SLPI-Caspase-3 and Ad-SLPI-GFP reached 1×109pfu/ml,6.3×109pfu/ml respectively, which was sufficient for future studies both in vivo and in vitro.Western blot showed that the 17kd activated Caspase-3 was significantly increased after 72 hours by the infection of Ad-SLPI-Caspase-3 in Hep-2. Meanwhile, strong signals of green fluorescence could be seen in a large number of Hep-2 cells after 72 hours by the infection of Ad-SLPI-GFP. However, it showed absent expression in HuVEC. These results indicated SLPI promoter could effectively induce the expression of activated Caspase-3 in laryngeal carcinoma cells. The expression of green fluorescent protein was specifically restricted in Hep-2 cells and absent in normal human umbilical vein endothelial cells of HuVEC. And 72 hours is the right time for infection.3. In vitro Study on cytotoxicity of Ad-SLPI-Caspase-3 to human laryngeal carcinoma cells Hep-23.1 MTT AssayThe proliferation of Hep-2 cells was noticeable inhibited by Ad-SLPI-Caspase-3 after transfecting 72 hours, while no significant inhibition was observed in HuVEC cells. The cell survival rate in Hep-2(42.8%±0.007) was much lower than that in HuVEC (92.2%±0.146).3.2 Morphological changes by infecting with Ad-SLPI-Caspase-3After 72 hours, the infected Hep-2 cells showed an obvious change in morphology:cells became round and shrieked, some even floated in cluster. However, no significant morphological change was observed in the infected Hove cells.3.3 DNA fragmentation assayNoticeable DNA ladder was detected in the infected Hep-2 cells by Ad-SLPI-Caspase-3, which clearly indicated effective induction of apoptosis in laryngeal carcinoma cells, but none was detected in HuVEC cells, as well as the Hep-2 cells and HuVEC cells infected by Ad-SLPI-GFP. This revealed that Caspase-3 was an effective gene to induce apoptosis, while the adenovirus vector itself did not produce any effect on cell apoptosis. 3.4 Flow cytometry for quantitative analysis of apoptosisThe rate of late apoptosis in the infected Hep-2 cells by Ad-SLPI-Caspase-3 was 80.6%, which was significantly higher than that by Ad-SLPI-GFP and PBS. No significant difference was observed in the infected HuVEC cells by Ad-SLPI-Caspase-3, Ad-SLPI-GFP and the control PBS group. All these evidence confirmed that Caspase-3 could efficiently induce apoptosis in laryngeal cancer cells. And the apoptosis-inducing effect was specific in Hep-2 cells under the control of SLPI promoter.4. The antitumor activity by Ad-SLPI-Caspase-3 in human laryngeal carcinoma xenograft in Nude Mice4.1 The antitumor activity by Ad-SLPI-Caspase-3 in human laryngeal carcinoma in vivoStatistical analysis showed that Ad-SLPI-Caspase-3 achieved significant antitumor efficacy at the thirteenth and sixteenth day after first injection (P< 0.05). However, by the end of the whole observation period (twenty-five days after first injection), the difference of the average tumor sizes and tumor weights among the three groups was not statistically significant(P> 0.05), which might probably be related with the incomplete suppression of tumor cells, the residual tumor continued to proliferate and lead to tumor progression. Large area of typical necrotic appearance was observed in the paraffin sections from tumor tissues of the Ad-SLPI-Caspase-3 group by HE staining. However, there was no necrosis in large area in the other two control group.4.2 Observation on toxicity in vivo by recombinant adenovirusDuring the whole period of study, all of the three groups of nude mice survived in good condition. There was no significant difference in the activities and diets among them. No significant difference in body weight and liver weight was seen in the adenovirus treated groups, compared with the control group. And no obvious degeneration and necrosis of liver cells was found by HE-staining for the hepatic tissues in the three groups.Conclusion1. SLPI2 promoter is an efficient and tissue-specific promoter for human laryngeal squamous cell carcinoma cell line Hep-2.2. Replication-defective adenovirus of Ad-SLPI-Caspase-3 and Ad-SLPI-GFP could infect Hep-2 cells specifically and effectively, inducing the expression of target genes. The activated Caspase-3 could be successfully expressed, meanwhile the endogenous Caspase-3 could also be activated by Ad-SLPI-Caspase-3 in Hep-2 cells.3. Replication-defective adenovirus of Ad-SLPI-Caspase-3 could inhibit growth of laryngeal carcinoma xenografts and induce necrosis efficiently in tumor tissues, without any obvious toxicity to the liver.