Construction, Identification And Experimental Immunization Of The Dual Specific Anti-tumor Recombinant Adenovirus

Malignant tumour affects everyone - the young and old, the rich and poor, men. women and children - and represents a tremendous burden on patients, families and societies. Malignant tumour is a generic term for a large group of diseases that can affect any part of the body. Other terms used are cancers and neoplasms. One defining feature of malignant tumour is the rapid creation of abnormal cells that grow beyond their usual boundaries, and which can then invade adjoining parts of the body and spread to other organs. Malignant tumour is one of the leading causes of death in the world, particularly in developing countries. The disease accounted for 7.9 million deaths (or around 13% of all deaths worldwide) in 2007. The main types of malignant tumour leading to overall malignant tumour mortality each year are: lung (1.4 million deaths/year); stomach (866,000 deaths); liver (653,000 deaths); colon (677.000 deaths); breast (548,000 deaths). About 72% of all malignant tumour deaths in 2007 occurred in low- and middle-income countries. Deaths from cancer worldwide are projected to continue rising, with an estimated 12 million deaths in 2030. The most frequent types of malignant tumour worldwide (in order of the number of global deaths) are: Among men - lung, stomach, liver, colorectal, oesophagus and prostate; among women - breast, lung, stomach, colorectal and cervical.Yet, many of these deaths can be avoided. Over 40% of all malignant tumours can be prevented. Others can be detected early, treated and cured. Even with late stage cancer, the suffering of patients can be relieved with good palliative care. Operation, chemtherapy and radiotherapy are the main treatments in the control of malignant tumours. Although high efficiency and prolonged life span do treatments decribed above, nowadays, they still have some problem need to solve, including the virulence reversion, drug resistance, organism wounded, et al. Gene therapy, widely used all over the world, has the potential to be developed as a cure of malignant tumours. However, recently increasing reports showed that safty, high performance, specificity hampered the development and application of gene therapy. Therefore, it is necessary to develop a safe, specific and effective succedaneous praeparatum in cancer gene therapy. In this study, we synthesized artificially PEG3p and hTERTp. The tumor specificity of PEG3p and hTERTp was identified in human hepatoma carcinoma (HepG-2) cells, human lung carcinoma (A549) cells, human cervical carcinoma (Hela) cells, human embryonic lung (WI-38) cells and african green monkey kidney (Vero) cells. The results showed that hTERTp and PEG3p states the expression of foreign gene in HepG-2,A549 and Hela cells, whereas not in WI-38 and Vero.E1a gene of hAd5 was amplified by PCR and placed downstream of hTERTp and PEG3p. Apoptin and HN gene were placed downstream of CMV promoter of shuttle plasmid of RAPAd. I system. Then the expression cassettes containing PEG3p/E1a and hTERTp/E1a were inserted into the shuttle plasmids containing Apoptin and HN respectively, and resulted in shuttle plasmids of dual specific recombinant adenovirus pAd-VT, pAd-VP, pAd-HT and pAd-HP.The above shuttle plasmids of dual specific recombinant adenovirus and genome skeleton of RAPAd. I system were co-transfected into HEG-293 cells. The recombinant adenoviruses Ad-VT, Ad-VP, Ad-HT and Ad-HP were obtained by homologous recombination. The expression of Apoptin, HN and E1a of the recombinant adenovirus was identified by RT-PCR, western blot and indirect immunofluorescence. The results showed that the foreign genes expressed effectively in A549 cells.The anti-tumor effects of Ad-VT, Ad-VP, Ad-HT and Ad-HP on A549 cells were detected by MTT. The results showed that, except for Ad-EGFP, Ad-VT, Ad-VP, Ad-HT, Ad-HP, Ad-GT, Ad-GP, Ad-Apoptin and Ad-HN killed A549 cells in varying degrees. The anti-tumor effects of replicating adenoviruses (Ad-VT, Ad-VP, Ad-HT, Ad-HP, Ad-GT and Ad-GP) were powerful than replication defective adenoviruses (Ad-Apoptin and Ad-HN). Among replicating adenoviruses, anti-tumor effects of Ad-VT, Ad-VP, Ad-HT and Ad-HP were the stronger than Ad-GT and Ad-GP, and Ad-VT and Ad-VP were stranger than Ad-HT and Ad-HP. In the certain time frame, the suppression rates of Ad-VT, Ad-VP, Ad-HT, Ad-HP, Ad-GT and Ad-GP on A549 cells were heightened with the stepping up of infective dose, and resulted in some dose-effect relationship. In the definition of infection doses, the suppression rates of Ad-VT and Ad-VP on A549 cells were improved with the extending of infection time, and resulted in some time-effect relationship.By using observation of light microscope. AO/EB staining, DAPI staining, Annexin V staining and caspase activity detecting, we approached the decease pathway of A549 cells reduced by the dual specific recombinant adenoviruses. The results showed that the recombinant adenovirus induced apoptosis of A549 cells. The apoptosis induce function of replicating adenoviruses (Ad-VT, Ad-VP, Ad-HT, Ad-HP, Ad-GT and Ad-GP) were powerful than replication defective adenoviruses (Ad-Apoptin and Ad-HN). Among replicating adenoviruses, apoptosis induce function of Ad-VT, Ad-VP, Ad-HT and Ad-HP were the stronger than Ad-GT and Ad-GP, and Ad-VT and Ad-VP were stranger than Ad-HT and Ad-HP.C57BL/6 mice model bearing LLC was constructed by transplanting LLC cells into the right hind limb of the mice and the anti-tumor effects of the recombinant adenoviruses described above were observed through this model. The results showed that, compared with saline and control groups, experiment groups displayed varying degree anti-tumor effects. The suppression rates of Ad-VT (76.99%), Ad-VP (74.02%), Ad-HT (81.46%) and Ad-HP (68.08%) obviously higher than Ad-GT (39.89%), Ad-GP (15.76%), Ad-Apoptin (45.57%) and Ad-HN (45.19%). The survival rates of Ad-VT-, Ad-VP- and Ad-HP-treated groups, all of six mice survived (survival rate was 100%), were significant higher than other groups. The survival rates of Ad-HN, five of six mice survived (survival rate was 83.33%), was significant higher than saline-, Ad-EGFP-, Ad-Apoptin-, Ad-GP- and Ad-GT-treated groups. The survival rates of Ad-HT, four of six mice survived (survival rate was 66.67%), was significant higher than saline-. Ad-EGFP-, Ad-Apoptin-. Ad-GP-and Ad-GT-treated groups. The survival rates of Ad-Apoptin-, Ad-GP- and Ad-GT-treated groups were 50%, and the survival rates of Ad-EGFP- treated groups was 33.33%. The pathological section showed that the tumor cells of experiment groups display vacuolization and necrosis.The results of the detection of cytokine of Th1/Th2 showed that the recombinant adenovirus up-regulated the level of some cytokines. The level of IL-2 and IFN-γof mice of Ad-HT-, Ad-HP- and Ad-HN-treated groups was much higher than other groups. This result illustrated that Ad-HT, Ad-HP and Ad-HN lead the immune response to Th1 dominant, just as the previous research of our team. On the other hand, perhaps, because of the immunostimulation of adenovirus itself, the levels of IL-4 and IL-10 of the mice of Ad-HT-, Ad-HP- and Ad-HN-treated groups were up-regulated. In the Ad-VT-, Ad-VP-. Ad-GT-, Ad-GP- and Ad-Apoptin-treated groups, the same phenomenon occurred.In summary, the dual specific anti-tumor recombinant adenovirus constructed by using RAPAd. I system basing on tumor specific promoter PEG3p/hTERTp and specific anti-tumor gene Apoptin/HN in the study, which has both tumor specific restrain and tumor specific replication abilities, can suppress A549 cells and prolong life span of animal model effectively. The researches can provide a useful platform for the exploration of more safer, specific, effective anti-tumor medicine and basic data for the studies on the anti-tumor, immunoregulation mechamsm of Apoptin, HN gene.