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To evaluate the anticancer effect of measles vaccine virus in combination with Nimotuzumab in vitro. To evaluate the anticancer effect of measles vaccine virus in combination with Nimotuzumab on nude mouse model with head and neck cancer (in vivo).
MINISTRY OF EDUCATION AND TRAINING MINISTRY OF DEFENSE VIETNAM MILITARY MEDICAL UNIVERSITY NGÔ THU HẰNG ANTICANCER EFFECTS OF VACCINE STRAIN MEASLES VIRUS IN COMBINATION WITH NIMOTUZUMAB IN TREATMENT OF LARYNGEAL CANCER IN VITRO AND IN VIVO Major: Biomedical Science Code: 9720101 SUMMARY OF DOCTORAL THESIS IN MEDICINE HA NOI 2020 THE THESIS IS COMPLETED AT THE VIETNAM MILITARY MEDICAL UNIVESITY Supervisors: Prof. Dr. NGUYỄN LĨNH TỒN Assoc. Prof. Dr. HỒ ANH SƠN Reviewer 1: Prof. Dr. VĂN ĐÌNH HOA Reviewer 2: Assoc. Prof. Dr. TRỊNH TUẤN DŨNG Reviewer 3: Assoc. Prof. Dr. PHẠM TUẤN CẢNH The thesis is defended in front of the scientific committee at the Vietnam Military Medical University at on 2020 The thesis can be found at: National Library Library of the Vietnam Military Medical University INTRODUCTION Cancer is a major health problem and is increasingly concerned in all countries of the world. Head and neck cancer (HNS) is a group of malignant tumors that develop in this part of the body, and 90% of HNS has squamous cell carcinoma (HNSCC). Globally, HNS ranks seventh with 4.8% of all newly diagnosed cancers. Oncolytic virus (OLV) therapy is based on the main mechanism that OLVs have the ability to specifically enter and replicate in cancer cells of the tumor and cause cell lysis, promote cell apoptosis and stimulate immune response against cancers Nimotuzumab is a monoclonal antibody targeting the epidermal growth receptor (EGFR), which is effective against angiogenesis, inhibits cell proliferation, induces apoptosis, and promotes cell sensitivity to radiation and chemotherapy We conducted the project “Anticancer effects of vaccine strain measles virus in combination with nimotuzumab in the treatment of laryngeal cancer in vitro and in vivo” with two objectives To evaluate the anticancer effect of measles vaccine virus in combination with Nimotuzumab in vitro To evaluate the anticancer effect of measles vaccine virus in combination with Nimotuzumab on nude mouse model with head and neck cancer (in vivo) Necessity of the project: Investigation of the anticancer effect of measles vaccine virus (MeV) in combination with Nimotuzumab both in vitro and on a nude mouse model will serve as a basis for further studies on the mechanism of combined antitumor effect of MeV and Nimotuzumab, as well as for clinical trials using a MeV and Nimotuzumab combination to treat cancer patients in general and HNS in particular. New contribution of the thesis: This thesis is the first study to evaluate the anticancer effect of the combination of MeV and Nimotuzumab against HNS on Hep2 cells as well as on a nude mouse model with head and neck cancer. This is the basis for further experimental studies and clinical trials for cancer treatment Layout of the thesis: The thesis has 150 pages, including: Introduction (2 pages), Chapter 1: Literature review (36 pages), Chapter 2: Materials and Methods (27 pages), Chapter 3: Results (47 pages), Chapter 4: Discussion (35 pages), Conclusion (2 pages), Recommendations (1 page). The thesis has 175 references (171 references in English) CHAPTER 1: LITERATURE REVIEW 1.1. Head and neck cancer Head and neck cancer (HNS) is a group of malignant tumors that develop in this part of the body including mouth, nose, throat, larynx, sinuses, or salivary glands, cancer of the oral cavity, sinuses next to nose and tongue. The incidence of HNS is increasing both in Vietnam and in some parts of the world HNS has a bad prognosis, is dangerous and has many major complications The main risk factors for HNS include tobacco, alcohol consumption, HPV infection (for oral cancer), EBV infection (for throat cancer). The oncogenes in HNSCC are associated with four main functional pathways: cell proliferation, squamous epithelialization, cell survival and invasion/metastasis 1.2. Therapy targeting Epidermal Growth Factor Receptor 1.2.1. Role of EGFR in head and neck squamous cell carcinoma Epidermal growth factor receptor (EGFR) has a molecular weight of 170 kiloDaltons (kDa). When the epithelial growth factor (EGF) binds to its receptor (EGFR), two EGFR molecules bind to each other (dimerization), the tyrosine kinase region is then phosphorylated This phosphorylation leads to the activation of specific tyrosins and EGFR receptordependent intracellular signaling proteins subsequently leads to the transcription of target genes that promote cell proliferation, survival (apoptosis), invasion and metastases. EGFR is highly important in the pathogenesis of HNSCC and its expression was found in 92% of the HNSCC tumors. Moreover, the expression of EGFR is high in tumors in the advanced stage or in less differentiated tumors 1.2.2. Nimotuzumab in treatment of Head and neck cancer Nimotuzumab is a monoclonal antibody that specifically binds to EGFR and blocks the activation of this receptor Nimotuzumab recognizes the EGFR extracellular domain and competes for the binding site of EGF, prevents EGF to bind to its receptor, therefore, prevents the activation of EGFR, inhibits tyrosine kinase activity, consequently inhibiting the growth of tumor cells. In order to respond to EGFR blockaded by Nimotuzumab, tumor cells reduce the secretion of vascular proliferation factors, such as vascular endothelial growth factor (VEGF), which leads to reduced formation of vascular and increase the number of apoptotic cells. Nimotuzumab (Cimaher) has been shown to be effective in the treatment of advanced HNSCC and Nimotuzumab has been shown to be safe and has less serious complications 1.3. Measles vaccine virus (MeV) in virusbased cancer therapy 1.3.1. Measles virus Measles virus is a singlestranded RNA () virus with a diameter of 100300 nm, and belongs to the genus Morbillivirus, family Paramyxoviruses, surrounded by a helix capsid. The MeV envelope glycoproteins are the hemagglutinin (H) and fusion (F) proteins that mediate viral binding and integration with the host cells. In current OLV therapy, the use of Edmonston vaccine strains includes a lab strains, which is closely related to a clinical strain isolated from the throat of a baby named David Edmonston (1954) and was transplanted into different cells to create a less virulent and non pathogenic MeV strain 1.3.2. Receptors of MeV MeV uses three receptors, CD46, CD150 and nectin4, to enter the target cells, the most important receptor is the CD46, which is a type 1 transmembrane glycoprotein that is common in all cells. The CD46 receptor is found to be highly expressed in cancer cells. In normal cells with low CD46 expression, MeV is likely to be infectious but the syncytial formation is negligible. In cancer cells with high CD46 expression, MeV infection leads to a strong synaptic formation: MeV binds to the receptor to enter the cells, replicate and cause the cells to form the symplasm and consequently kill the cells through a CD46 mediated mechanism 1.3.3. Safety of attenuated Measles vaccine MeV meets the standards of an ideal OLV, which must have a high selection of tumors, nonpathogenicity, genetic stability and no disease transmission to the community 1.3.4. The mechanism of cancer cell lysis 1.3.4.1. MeV directly kills tumor cells through syncytial formation The fusion between the infected cells and the adjacent normal cells forms syncytia A virusinfected cell can merge 50100 neighbouring cells to form a syncytium. This is a mechanism for spreading viruses without releasing viral particles from the host cells. The process of cell consolidation reduces the exposure of viruses to neutralizing antibodies of the host, that avoids the control and neutralization of the immune system. 1.3.4.2. Lysis of tumor cells mediated by stimulating specific anti tumor immunity MeV produces two types of danger signals including damage associated molecular pattern molecules (DAMP) and pathogen associated molecular patterns (PAMP), which trigger specific immune responses that contribute to tumor cell lysis such as IFN, cytokines, activation of NK cells, macrophages, DCs, and T lymphocytes. 1.4 Combination of measles vaccine virus and Nimotuzumab monoclonal antibody in the treatment of cancer There were 2 clinical trials using MeV targeting EGFR to invade and lyze neuroblastoma cells and one trial with HNSCC cells. All three trials showed that MeV has a strong ability to inhibit and kill cancer cells by targeting EGFR both in vitro and in vivo. Nimotuzumab is a monoclonal antibody targeting EGFR and has been shown to be effective in the treatment of HNSCC. From the above evidence, we conducted a study using MeV in combination with monoclonal antibody Nimotuzumab in order to treat HNSCC in vitro and in vivo in order to improve the anticancer effects CHAPTER 2: MATERIALS AND METHODS 2.1. Subjects and Materials 2.1.1. Animals: Nude mice BALB/c strain, 68 weeks old, weighing 1822g, eligible for the experiment 2.1.2. Measles virus Vaccine (MeV): Measles vaccine virus strains Edmonston 2.1.3. Cell lines: head and head squamous cell carcinoma cells Hep2, monkey kidney cell (Vero cells) 2.1.4. Monoclonal antibody Nimotuzumab: Product CIMAher 2.1.5. Equipment used for the study 2.1.5.1 Equipment: NSK 150mm callipers, electronic scales TE3102S Sartorius, clean room, cell culture room, centrifuges, optical density reader, realtime PCR machine, flow cytometry, pipettes and etc 2.1.5.2 Consumables: 6 and 96 well plates, tips, culture plates, bacterial filter, falcon tube, Eppendorf and etc 2.1.5.3. Chemicals and Reagents: M199, EMEM cell culture media, kits for MTT, Annexin V/PI Fluorescein isothiocyanate; RNA, cDNA synthesis kit, primers, master Mix, alcohol 70, 90 and etc 2.2. Methods: The study was conducted according to the standard experimental, prospective methods, compared and evaluated before, during and after treatment 2.2.1. Evaluated the ability to inhibit cancer cells and apoptosis of MeV in combination with Nimotuzumab on Hep2 head and neck cancer cell line 2.2.1.1. Evaluation criteria Determined viral concentration by CCID50 method Evaluated inhibition of Hep2 cells by MTT Evaluated apoptosis and necrosis by flow cytometry method Evaluated apoptosis through the expression of STAT3 and ISG15 genes by realtime PCR technique 2.2.1.2. Techniques a, Cell Culture Hep2 and Vero cells were taken from freezers (80 0C), thawed quickly (
