Expression Of RON In Burkitt's Iymphoma And Molecular Mechanism Research

BackgroundBurkitt's lymphoma (BL) is a highly invasive B lymphocyte tumor, the pathogenesis is still not clear. Abnormal cell cycle proteins, apoptosis associated proteins, oncogene expression and cell signaling pathways were found in the BL cells, and the exact pathogenesis remains to be further studied. RON (Recepteur d'Origine Nantais) belong to the MET oncogene family, which is a subfamily of receptor tyrosine kinases with unique biological functions. RON belongs to the category of conserved protein in the species evolution, and also plays important roles in the malignant transformation of mammalian cells and carcinogenosis. RON is expressed mainly in human epithelial cells, such as the gut, lung and mammary epithelial cells, and partially expressed in immune cells. The ligand of RON is human macrophage stimulating protein (MSP). RON is highly heterogeneous expressed in lymphoma as well as in colorectal cancer, breast cancer and bronchioloalveolar carcinoma. RON can regulate cell migration, invasion and proliferation, and promote tumor malignant transformation. But it is unclear whether RON plays an important role in the pathogenesis of BL, and whether it can be used as the target of BL treatment.In view of the above problems, we performed researches from three part to exploit the role of RON in BL pathogenesis and its potential value for treatment target from three aspects.Part I The expression of the oncogene RON in lymphomaObjective:To explore the expression of RON in different lymphoid tumor tissues and cell strains.Methods:Expression of RON was detected by immunohistochemistry in high density of tissue chip from patients of plasma cell lymphoma, Hodgkin's lymphoma, diffuse large B cell lymphoma, Burkitt's lymphoma, follicular lymphoma, mantle cell lymphoma, MALT lymphoma and T cell lymphoma, normal and inflammatory tissue. The RON positive tissue proportion and expression intensity were calculated. Expression of RON was also detected by Western Blotting in the biopsy of normal tissue, inflammatory tissue, positive control tissue, negative control tissue, diffused large B cell lymphoma tissue, T cell lymphoma tissue, Burkitt's lymphoma tissue, Hodgkin's lymphoma tissue, chronic lymphocytic leukemia tissue and bone marrow tumor tissue, as well as in the lymphoid cell lines, including the negative control cells (3T3cells), positive control cell lines (RON gene transfection cell lines of3T3), Hodgkin's lymphoma cell lines (L428cells), T cell lymphoma cell lines (Jurkat cells), T cell lymphoma cell lines (Molt-4cells), Burkitt's lymphoma cell lines (Raji cells), diffused large B cell lymphoma cell lines (Pfeffier cells and Farage cells) and multiple myeloma cell line (RPMI8226cells). Test the positive of EBV with EBER in the Hodgkin and Burkitt lymphoma.Results:(1) The highest expression of RON in the tissue chip was found in Hodgkin's lymphoma and Burkitt's lymphoma tissues, rather than the low or no expression in other lymphoma tissues and normal lymph tissues(p<0.05). Furthermore, analysis of expression intensity revealed that the rate of over expression of RON was more than30%in the RON positive Hodgkin's lymphoma and Burkitt's lymphoma tissues(p<0.05).(2) The highest expression of RON in the cell lines was found in Hodgkin's lymphoma cell lines (L428cells) and Burkitt's lymphoma cell lines (Raji cells), rather than the low or no expression in other cell lines(p<0.05).(3) There have high positive rate of EBV in Hodgkin and Burkitt lymphoma and positive related with RON expression(p<0.05).Conclusion:(1) Different expression of RON exist in lymphoma tissues and cell lines, with the highest expression in Burkitt's lymphoma and Hodgkin lymphoma.(2) The highest expression of RON in Raji cells (Burkitt's lymphoma cell lines) and L428cells (Hodgkin lymphoma cell lines) which is similar to the test of tissues, demonstrates that Raji cell line is a good model in vitro for the study of Burkitt's lymphoma.(3) The EBV infection of Burkitt's lymphoma and hodgkin's positive related with RON expression.Part II RON mediated Raji cell biological behavior and the effection of ZT/F2(2F2) antibody on Raji cellsObjective:Study of RON special ligand of MSP activated RON, regulating the malignant biological behavior (cell proliferation and colony formation) in Raji cell, as well as ZT/F2(2F2) mAb inhibition of Raji cell proliferation, induction of apoptosis mechanism.Methods:The effect of MSP (2.0nM) and ZT/F2(2.0nM) antibody treatment for24and72hours on proliferation of RON positive Raji cells and RON negative Jurkat cells was detected by MTT colorimetry. The effect of MSP and ZT/F2treatment on colony formation of Raji cells and Jurkat cells was detected by observing the colony count. Apoptosis of Raji cells was tested through the Annexin V/PI double staining with flow cytometry. Apoptosis related proteins were tested by Western Blotting. Raji cell cycles were tested by flow cytometry and cycle proteins were tested using Western Blotting.Results:(1) Proliferation of RON positive Raji cells treated with MSP or MSP combined with negative antibody for72hours were fully activated(p<0.05), while proliferation of ZT/F2antibody or MSP and ZT/F2treated Raji cells was inhibited significantly(p<0.05). No significant difference was found for proliferation of MSP or ZT/F2treated Jurkat cells(p>0.05). The colony formation assay of MSP or ZT/F2treated Raji cells and Jurkat cells were further investigated. MSP could stimulate the colony formation of Raji cells, and ZT/F2antibody could inhibit the colony formation(p<0.05). No effect on colony formation was found in MSP or ZT/F2treated in Jurkat cells(p>0.05).(2) Apoptosis of Raji cells treated with ZT/F2or MSP combined with ZT/F2for72hours were much higher than blank contro(p<0.05)1. And the apoptosis protein Caspase-3, Caspase-8, caspase-9and PARP expression were significantly increased(p<0.05), while the anti-apoptosis gene of MCL-1and apoptosis inhibitor of XIAP were decreased significantly(p<0.05).(3) Percentage of apoptotic cells with ZT/F2antibody treatment and MSP+ZT/F2treatment group were significantly increased than the blank control(p<0.05), which was corresponding to the apoptosis of Raji cells. The percentage of Raji cells arrested in G1phase was increased in ZT/F2antibody treatment group(p<0.05). Levels of cyclinD1, CDK4and CDK6were decreased(p<0.05), while that of P27was increased with ZT/F2treatment group and MSP+ZT/F2treatment group(p<0.05).Conclusion:(1) MSP can stimulate the proliferation of Raji cells, and increase the colony formation, exhibiting high malignant biological behavior. ZT/F2can inhibit the proliferation and colony formation of Raji cell by MSP. While no such effect was found in RON negative Jurkat cell line.(2) ZT/F2can induce apoptosis of Raji cells and change the expression of apoptosis related proteins. (3) ZT/F2can inhibit proliferation of Raji cells by changing the expression of cyclin and arresting cell cycle of G1phage.Part â…¢ RON mediated cancer molecular of signal pathway activation and the inhibition of ZT/F2(2F2) mAbObjective:Study RON induced signaling pathways activation stimulated by MSP, and the mechanism of inhibition of Burkitt's lymphoma in vitro and in vivo by ZT/F2. We also aim to explore the drug target effect of RON in Burkitt's lymphoma.Methods:Western Blotting was used to test phosphorylation of RON and activation of AKT, ERK1/2and P38-MAPK and NK kappa B signaling pathways and beta catenin induced by MSP for0',15min,30min,60min and120min. Inhibition of phosphorylation of RON and activation of the signaling pathways and beta catenin by ZT/F2treatment at60min was detected. Specific inhibitors of the signaling pathways were used combined with ZT/F2. Tumor-bearing mice of Raji cells was used to observe inhibition of ZT/F2on tumor growth. Expression of TUNEL and RON, and AKT and ERK1/2of tumor tissue were detected by immunohistochemical detection.Results:(1) MSP could induce phosphorylation of RON, and activation of the PI3K/Akt, p38MAPK, ERK1/2and NF kappa B signal pathways and expression of beta-catenin. The ZT/F2significantly inhibited MSP induced phosphorylation of RON and activation of ERK1/2, AKT, P38and NF-kB cell signaling pathways, and decreased expression of beta-catenin.(2) Combination of specific inhibitors of signalling pathways and ZT/F2can enhance the inhibition. When combined with ZT/F2, AKT inhibitors or ERK1/2inhibitor can not only inhibit p-AKT or pERKl/2expression, but also inhibit other signaling pathways, which indicated that AKT and ERK1/2pathways played a key role in the inhibition of RON activation by ZT/F2. (3) ZT/F2treated tumor-bearing mice had the smallest tumor size and weight, and the longest tumor latency compared with the control group. The immunohistochemical staining showed that ZT/F2treatment could significantly enhance TUNEL expression in tumor cells, and reduce the RON and AKT and ERK1/2within tumor cells.Conclusion:(1) MSP can induce phosphorylation of RON and activate related signaling pathways and beta-catenin in Raji cells. ZT/F2can inhibit the RON phosphorylation and activation of signaling pathways and beta-catenin.(2) When combined with ZT/F2, inhibitors of AKT and ERK can inhibit the activation of other signaling pathways activated by MSP, indicating that AKT and ERK1/2pathways play a key role in RON mediated signal pathways.(3) ZT/F2can inhibit the tumor growth of tumor-bearing mice of Raji cells, extend the tumor latency, inhibit RON, AKT and ERK, and induce apoptosis of Raji cells. Zt/f2is a potential therapeutic mAb capable of inhibiting RON-mediated oncogenesis by Burkitt's lymphoma in vivo and in vitro. The inhibitory effect of Zt/f2maybe represent a novel strategy for future burkitt's lymphoma therapy.