| Nasopharyngeal Carcinoma (NPC) is a highly malignant tumor derived from nasopharyngeal epithelium. As the tumor progression, other important structures like skull base are vulnerable to involve, and early cervical lymph node metastasis and distant metastasis may occur. Nasopharyngeal carcinoma manifests as ethnic and regional distribution. It is characterized by a high incidence of malignant tumors and its incidence rate in China is about 20/100,000 which already caused serious problems. At the present time, the main treatment of NPC is still radiotherapy and adjuvant chemotherapy, notwithstanding the total five year survival rate of traditional radiotherapy and chemotherapy of NPC is less than 60%.To the advanced stage of NPC, this number is below 40%, let alone a series of side-effects caused by radiotherapy and chemotherapy. So it is an urgent need of new treatments of NPC as a supplementary. Nasopharyngeal carcinoma is a complex disease caused by Epstein-Barr virus (EBV) latent infection, environmental factors and genetic factors, containing a multi-step process of carcinogenesis. All nasopharyngeal carcinoma cells have the appearance of EBV genomes and express EBNA1, EBER1, 2, and LMP1, LMP2A, LMP2B, which makes these viral proteins as an ideal tumor marker, in which LMP1 (latent membrane protein1,LMP1) plays a significant part in nasopharyngeal epithelial cells transformation and oncogenesis and is now recognized as the vital oncoprotein. Its unique molecular structure of protein produces a marked effect in the occurrence and development of NPC. Theoretically, LMP1 could be an ideal target in treating NPC because of its unique character. Compared with traditional macromolecular antibody, a novel human antibody Fab against LMP1, could offer a new approach for diagnosis and targeted therapy in NPC.The main reason of NPC death is resistance of chemotherapy and radiotherapy for those who sufferd metastatic and recurrent NPC. Therefore,it is substantial to search an ideal drug which is sensitive in chemotherapy against NPC. Mitomycin C (MMC) is an anti-tumor antibiotic, which is acknowledged effective against a variety of solid tumors and has been used for clinical treatment in numerous kinds of cancers. Its mechanism is thought to undermine the structure of DNA and RNA by forming cross linkage through alkylating group and double stranded helix, and finally causing cell apoptosis. It is reported that MMC is capable of maintaining high activity even oxygen-poor, manifesting more sensitivity in G0 stage and reducing drug resistance. Structurally, MMC consists of benzoquinone loop, indole loop, ethylenimine loop and formylamine side chain, it is convenient to form certain conjugate in target therapy field for NPC treatment. Our team has completed the inhibitory role of MMC against NPC cell in vitro previously.In this study, expression, purification and functional identification of this Fab against LMP1 extracellular region, which screened by human phage antibody display technology, were conducted. Furthermore, we investigate the inhibitory activity by applying Fab combined with MMC on NPC LMP1 xenografts in nude mice, observe the effect and analysis the potential mechanism.Methods1. Fab was expressed in prokaryote; human sources of anti-LMP1 extracellular domain antibody Fab were obtained; Fab was purified; functions were identified. E. coli Top 10F'were infected with Fab phage. When the culture of bacteria came to logarithmic phase, they were induced to express; the expressed protein product were purified by use of Protein L affinity chromatography and characterized by SDS-PAGE,2. Establishment of nasopharyngeal carcinoma LMP1 xenografts in nude mice. Nasopharyngeal carcinoma LMP1 cell suspension 0.2ml with cell number 5×106/ml were subcutaneously transplanted in to 20 nude mice. After xenograft models were accomplished, 20 bearing-cancer mice were randomly divided into Fab group, MMC group, Fab+MMC group and control group. These mice were injected different drugs for 5 times, once every 3 days, 0.3ml each time. Treated drugs and dosages are as followed: Fab group3:mg/kg, MMC group:2mg/kg, Fab+MMC group: Fab 3mg/kg+MMC 2mg/kg and normal saline for negative control ,3. The mice were followed for observation of tumor volume and weight, tumor growth inhibitory rate and tumor growth curve,4. The expression of vascular endothelial growth factor (VEGF) in disparate groups which measured with immunohistochemistry,5. The apoptotic rate of tumor cells in various groups which analysed by flow cytometry,6. Statistical methods: all the data were analyzed statistically by using SPSS10.0 software for t-test, homogeneity test of variance and analysis of variance. Each value represents mean±SD, ANOVA was used to analyze the significance of the difference between the different apoptotic rate, Mann-Whitney rank test was used to analyze semiquantitative datas. Differences were considered significant when P<0.05. Results1. Fab was expressed and correctly assembled in prokaryotic expression system; high purity Fab was obtained by use of Protein L affinity chromatography purification of antibodies,2. To establish nasopharyngeal carcinoma LMP1 xenografts in nude mice successfully, achievement ratio 100%,3. Different Tumor volume (mm3): Fab group: 462.71±42.7916, MMC group: 407.846±51.1506, Fab+MMC group: 266.851±46.3747, control group: 61.975±47.7266, tumor volume of Fab+MMC group is smaller than any other groups(P<0.01); different tumor weight (g): Fab group: 0.37±0.0332, MMC group: 0.324±0.0385, Fab+MMC group: 0.232±0.0259, control group: 0.456±0.0488, the tumor weight of Fab+MMC group is much more depressed compared with other groups(P<0.01); inhibitory rate: Fab group 18.90%,MMC group 28.95%,Fab+MMC group 49.12%,4. The expression of vascular endothelial growth factor (VEGF) in disparate groups: VEGF in Fab group and Fab+MMC group showed low expression and significantly reduced compared with control group (P<0.05),while VEGF in MMC group manifested high expression and displayed no statistics differences compared with control group (P>0.05). Majority intra-cellular positive staining of VEGF are always located in cytoplasm, macrophage, vascular endothelial cell and fibroblast,5. The apoptotic rate of tumor cells in various groups: Fab group: 7.337%±2.6755%, MMC group: 11.843%±1.5022%, Fab+MMC group: 17.55%±3.2058%, control group: 3.9%±0.5456%. The apoptotic rates in MMC group and Fab+MMC group were dramatically decreased compared with control group (P<0.01). Conclusions:1. Fab against LMP1 was expressed, obtained, purified and identified,2. Fab and MMC can both inhibit the subcutaneous xenograft tumors of NPC LMP1 cells in nude mice,3. Fab may inhibits the angiogenesis of tumors while MMC presumably induces apoptosis of cancer cells.
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