| Esophageal cancer(EC) is one of the most common digestive system neoplasms and has a quite poor prognosis, its mortality rate ranks the sixth place in the total death related to tumor, and the disease incidence has kept increasing year by year. According to the epidemiological statistics, about 150,000 people die in each year from esophageal cancer in China, which account for 46.6% of mortality in the world and take the fourth place in morbidity and mortality in China(WHO,1997). At present, major therapeutic modality for esophageal cancer refers to surgical operation combined with chemo- and radio-therapy. But follow up data show long-term curative effect is not ideal, and the overall 5-year survival rate is less than 10%.In recent years, gene-targeting therapy has become the focus in the research of esophageal carcinoma, and tumor necrosis factor related apoptosis inducing ligand(TRAIL) is a promising agent. TRAIL can recognize and bind to death receptor DR4 and DR5 within cell surface, which can activate downstream apoptosis pathway containing caspase cascade reactions and eventually induce cell apoptosis. A large number of studies show that TRAIL can induce apoptosis in a wide range of cancer cells, including esophageal cancers. However, TRAIL also activates apoptotic pathway in normal cells with a lack of specificity.mi RNAs are a class of small non-coding RNA. They recognize and bind mi RNA response element(MRE) that is located within the 3’ untranslated region of target m RNA, and degrade the targeted m RNA. Numerous evidences have demonstrated that deregulation of mi RNAs is closely associated with the formation and progression of cancers. mi R-143 has been identified to be downregulated in esophageal cancers, and maybe it participates in the occurrence of EC. Researches indicate that it can regulate specific expression of therapeutic gene by emplying MREs of mi RNA, which have low expression in tumors. Recent studies have pointed out that some MREs of mi RNA can mediate selective expression of TRAIL in melanoma, glioma, and bladder cancer cells.Firstly, we study the expression of mi R-143 and mi R-122 in EC. We obtained the results below. The abundances of mi R-143 and mi R-122 were reduced in esophageal cancer cells in comparison with normal esophageal cells. Then we infected target cells with psi Check2-143-122, which is a luciferase reporter vector carried mi R-143 and mi R-122. Luciferase assays indicated that luciferase expression level was increased in esophageal cancer cells, whereas cannot be detected in normal cells, indicating MRE of mi R-143 and mi R-122 may effectively target exogenous gene to esophageal cancer cells. We infected esophageal cancer cells and normal cells with adenovirus vectors, Ad-TRAIL-143-122 and Ad-TRAIL, and investigated the expression profiles of m RNA and protein of TRAIL in each group cells with real-time quantitative PCR and immunoblot assays. The results showed m RNA and protein expression levels of TRAIL in esophageal cancer cells were higher in EC cells than in normal cells, when they were transfected with Ad-TRAIL-143-122. And the two vectors had the same expression efficiency for TRAIL.We used the mixed inhibitors and mimics of m RNA for esophageal cancer cells and normal cells treated with Ad-TRAIL-143-122 respectively, and then detected the abundance of mi RNA and TRAIL in each group cells with real-time q PCR assays. The expression of TRAIL was partially restored in normal cells infected with Ad-TRAIL-143-122 by the suppression of mi R-143 and mi R-122, while was suppressed in esophageal cancer cells under the co-treatment of Ad-TRAIL-143-122 and mimics of mi R-143 and mi R-122. This finding suggested that TRAIL expression was controlled by MRE of mi R-143 and mi R-122.In order to evaluate the cytotoxicity of constructed vectors for gene therapy and cytotoxic effect on normal cells, we detected the expression of caspase-3 and cleaved PARP in each group cells by Western blot and assessed cell viability by MTT assays. Western blot results showed that it had higher expression levels of leaved caspase-3 and PARP in esophageal cancer cells infected with Ad-TRAIL-143-122 and Ad-TRAIL, while they cannot be detected in normal cells L-02 infected with Ad-TRAIL-143-122. MTT assays showed that the extent of decrease in the viability of esophageal cancer cells is higher than normal cells when they were infected with Ad-TRAIL-143-122, while there is no difference between them when infected with Ad-TRAIL. That showed Ad-TRAIL-143-122 can effectively kill esophageal cancer cells and has no cytotoxicity to normal cells.In vivo animal experiments also confirmed that the effectiveness of our vector. PBS, Ad-EGFP, Ad-TRAIL or Ad-TRAIL-143-122 was intratumorally injected into tumor xenografts in mice, followed by periodic measurement of tumor diameters. PBS, Ad-EGFP, Ad-TRAIL or Ad-TRAIL-143-122 was also injected into tail vein of mice, followed by blood harvesting for the detection of serum ALT levels. And we evaluated TRAIL expression level in the hepatic tissues from the mice by immunohistological staining. The results showed that Ad-TRAIL-143-122 suppressed the growth of Eca109 xenograft in mice. Ad-TRAIL-143-122 suppressed the growth of xenograft by a similar extent to Ad-TRAIL. Ad-TRAIL-143-122 and Ad-TRAIL expression was detected in tumor section from the mice injected with Ad-TRAIL and Ad-TRAIL-143-122, suggesting the viral vector constructed by us can make TRAIL be expressed in tumor selectively, and can be used for tumor therapy. More importantly, serum ALT levels were found to be increased obviously in the mice injected with Ad-TRAIL, suggesting that Ad-TRAIL has strong liver toxicity, while serum ALT levels were found to be suppressed in the mice injected with Ad-TRAIL-143-122. Immunohistological staining showed that TRAIL expression was suppressed in the liver of mice injected with Ad-TRAIL-143-122, while was widely expressed in the liver of mice injected with Ad-TRAIL.In this study, we constructed a MRE-regulated viral vector that carries killer genes and had a tumor-specific cytotoxicity. By in vivo and in vitro experiments, we showed that this strategy can treat cancer in a tumor-specific manner, and more importantly, it can prevent hepatotoxicity during the treatment of tumor. Therefore, the gene therapy strategy may be an effective modality for esophageal cancer treatment. |
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