Study On The Effect And Feature Of Selective Oncolytic Bluetongue Viruses Targeting Anticancer In Tumor Bearing Models

Occurrence and prevention of tumor have become research hot field of modern medicine since cancer causes severe human diseases. The traditional anti-turmor artifices comprise of surgery, chemotherapy, radiation therapy, etc. These clinical therapies for anticancer always fail in the methods because they are not tumor-targeting and kill cancer and normal cells as well. Targeting anticancer therapies are the real means that kill cancer cells only without killing normal cells. These ideal treatments are the goal that scientists and clinicians who are aspiring to conquer cancer would struggle to pursue and patients and their families want to get.Oncolytic virus is a kind of viruses that propagate selectively in tumor cells and destroy them without causing excessive damage to normal non-cancerous tissue. This new anticancer strategy of oncolytic viruses is based on the advances in viral molecular biology, molecular oncology, molecular biology and molecular genetics. Scientists modify virus gene by biotechnology to improve the effect of targeting oncolytic viruses, meanwhile looking for wild type viruses, including the human viruses and animal viruses, with no pathogenicity to human.Bluetongue virus (BTV) belongs to the orbivirus genus of the Reoviridae Family and is associated with bluetongue disease (BD) in sheep and some wild ruminants. Bluetongue viruse does not infect normal human cells and its dsRNA genome induces IFNs, therefore BTV has valuable anti-tumor potential.The content of thesis includes:(ⅰ) the study on the different serotype BTVs selectively killing the human hepatocarcinoma cells in vitro;(ⅱ) the acute toxicity tests of BTV-16in normal mice;(ⅲ) the effect of BTV-16degrading the murine hepatic carcinoma H22bearing BALB/C mice;(ⅳ) the effect of BTV-16targeting degrade the HCC(human hepatic cellular cancer) Hep-3B xenograft in nude mice;(ⅴ) the investigations for the patterns and mechanisms of cell death on BTV-16selectively infect and destroy Hep-3B cells(human hepatocarcinoma cells), Siha cells (human cervical carcinoma cells) and MA782cells (mouse breast carcinoma cells).Firstly, we studied the effect of BTV-4, BTV-9, BTV-16and BTV-21infecting Hep-3B cancer cells. The results demonstrated that the four serotypes of BTV all caused Hep-3B cells lesion and induced cells into apoptosis. The results confirmed that BTV selective destroy the tumor cells in vitro. In the future, the alternative injections of different serotypes of BTVs will be applied to avoid immune reaction and resistance caused by injecting single serotype bluetongue virus if patients need repeated application in clinical trials.Secondly, the acute toxicity tests of BTV-16were done with Kunming mice and the titer of anti-BTV antibodies in mice was detected by ELISA (enzyme-linked immunosorbent assay). The results showed the BTV-16is safe in mice and the level of anti-BTV antibodies has a variety curve with time. This immunological curve may be used as a parameter for the potential clinical trials and clinical application.Next, we constructed the hybrid mice model with immunocompetent bearing the murie hepatic carcinoma H22and explored effection of BTV-16targeting cancer cells and its safety in this model. The results demonstrated that BTV-16suppressed the tumor growth and showed the efficacy and safety of BTV-16anticancer in vivo.Based on the above results, we established the nude mice model bearing the human hepatoma Hep-3B cells to explore BTV-16targeting anticancer systematically. We applied intratumoral injection of BTV-16in this nude model while using adriamycin (ADM) as positive control. The results indicated that BTV-16targeting kill Hep-3B cells by methods of pathologic anatomy, histopathology, ultrastructural study, blood routine test, and blood biochemistry. The results revealed the efficacy and safety of BTV-16anticancer and the potential oncolytic application in the new level. The data showed that the efficiency of BTV-16treated groups is one hundred percent (100%), and the nude mice are in good state with none dead. The ADM administration group was observed with obvious side effects although the growth of tumor was inhibited. The blank control group was observed with increased size of tumor. The blood routine test and blood biochemistry of the nude mice in different concentrations of BTV-16used showed no significant difference (P>0.05). In order to investigate the pattern and mechanisms of the cell death BTV-16mediated, we used different tumor cells, including human hepatocarcinoma Hep-3B cells, human cervical carcinoma Siha cells and mouse breast cancer MA782cells infected by BTV-16in vitro. The results from flow cytometry analysis and electron microscopy observation are:(ⅰ) BTV-16targeting infected these tumor cells and the limited proliferation in these cancer cells is the important molecular events of targeting anticancer;(ⅱ) The fact that BTV-16infected tumor cells and induced the apoptosis of these cancer cell is one of mechanisms for targeting antitumor;(ⅲ) The endoplasmic reticulum stress reaction may be the important pathway that the BTV-16induces the tumor cell apoptosis;(ⅳ) BTV-16targeting infect some types of cancer cells and limited propagate result in these tumor cells destroyed.We believe the potential application of BTV targeting anticancer since it needs no genetic modifications and attenuation and is not carrying the harmful material to human body. BTV grows fast and is infective to the many human cancer cells. Since there is no preexisting neutralizing antibody against BTV in humans, the immune compromise will not occur in human body when the initial injection alone or with anti-cancer agents. These findings will be the base for future BTV potential clinical application. We hope that BTV will be the winner member of the next generation oncolytic viruses.