| Carcinoma is one of a diseases of serious damage to human health and the leading causes of mortality except cardiovascular and cerebrovascular diseases. The survival rate of patients suffering postoperative recurrence has little progression despite the development of conventional therapeutic strategies including chemotherapy, radiation and surgical oncology over the past two decades. This failure of conventional therapy is due mainly to resistance to chemotherapy, radiation and the relapse and spreading after surgical treatment. Tumor stem cells are those that have the characters of adult stem cells and few tumor stem cells can cause the event of tumor. At the same time, tumor stem cells are the rootstock of development, metastasis and relapse in tumor occurring. Human teratocarcinomas is a disease that have higher mortality and lower incidence. The survival rate of patients suffering postoperative recurrence is only 3% in 5 years. Human embryonic carcinoma (EC) cell line (NTERA-2) cells derived from a metastasis of a human testicular teratocarcinoma have the ability to self-renew in vitro and maintain their ability to differentiate into all three lineages:ectoderm, mesoderm and endoderm. At the same time, EC cells express a set of important pluripotent markers that are exclusive to human ES cells but are not expressed in their differentiated derivatives. Hence, EC cells are considered to be the malignant counterparts of human embryonic stem cells and regarded as a mode for study of cancer stem cells and human embryonic stem cells.Tumor necrosis factor-related apoptosis-inducing ligand (TRAIL/Apo2L) is a type II membrane protein belonging to tumor necrosis factor (TNF) family. TRAIL induces apoptosis in most neoplastic cell lines by binding to its specific functional receptor molecules on the surface of the target cells, while leaving intact most normal cells that can express non-functional decoy receptors. Hence, TRAIL is regarded as a promising molecular therapeutic weapon against malignancies. Membranous TRAIL is so called soluble TRAIL (sTRAIL114-281) forms a homotrimeric complex in serum with potent apoptosis-inducing activity.The promoter of rat progression-elevated gene-3 (PEG-3) is an available cancer-selective gene promoter. Many studies have shown that PEG-3 promoter can drive synthesis of apoptosis-inducing genes, which inhibits the growth of tumor, kills only cancer cells and spares normal cells. Our studies showed that the activity of PEG-3 promoter was very low in human embryonic stem cells and normal tissue cells. So, PEG-3 promoter is the perfect promoter in tumor gene therapy, especially in gene therapy of tumor stem cell.In our research, the CMV promoter of AAV vector was replaced by PEG-3 promoter, a therapeutic vector named AAV-PEG-sTRAIL was constructed. Transfected with AAV-PEG-sTRAIL in NTERA-2 cell line, sTRAIL was able to induce obvious apoptosis of NTERA-2 cells and apoptotic cell accounted for 23.1±2.3% of the population. In the absence of sTRAIL, the apoptotic rate was only 1.9±1.2%. Under microscopical examination, the condensed nuclei, a characteristic of apoptosis, could be found in NTERA-2 cells transfected with AAV-PEG-sTRAIL but not in the AAV-PEG transfected cells. NTERA-2 cells transfected with AAV-PEG-sTRAIL were subjected to quantitative reverse transcription-PCR (qPCR) and western blot analysis. DcRl, DcR2, DR4 and DR5 mRNA and cleaved caspase-3 protein were assessed in EC cells following treatment. Results of qPCR revealed that treatment with AAV-PEG-sTRAIL induced an (>3-fold) increase in DR5 and DR4 expression in NTERA-2 cell line. Interestingly, no significant change was found in the expression of either DcR1 or DcR2 following treatment. Western blot analysis indicated that activated caspase-3 (19kd) was also detected in EC cells treated with AAV-PEG-sTRAIL. These results indicate that the apoptosis of EC cells was caused by transfection with AAV-PEG-sTRAIL. Tumorigenicity experiment in vivo showed that 25%(one of four) was observed in 60 days when 1×106EC cell treated with AAV-PEG-sTRAIL group. In contrast,100%(four of four) tumor incidence was found in the untreated group at 40 day. Treatment with sTRAIL gene in models in nonobese diabetic/severe combined immunnodeficient (NOD/SCID) mice resulted in a suppression in tumor growth and statistically significant prolonged survival of the tumor-bearing mice (p=0.044). histopathologic studies showed no obvious lesions in liver, spleen, lung, or kidney. In addition, body weight, gross appearance, and behavior provided no signs of systemic toxicity. The successful inhibition of teratocarcinoma growth and the absence of detectable toxicty suggest a putative role for sTRAIL gene from PEG-3 promoter in treatment of tumor stem cells.
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