The Correlation Between The Expression Of Death Receptor5and The Cell Cycle In The Esophageal Cancer Cell

BackgroundDeath receptor5(DR5) is one of the primarily death receptors of tumor necrosis factor-relatedapoptosis-inducing ligand (TRAIL), which plays an important role in promoting TRAIL-induced apoptosis,and which is one of the important and promising potentially targets for cancer treatment. Recently, thereare three kinds of anticancer drugs targeting to death receptor5which are in the midst of Phase II clinicaltrials. However, it is an important factor that, the amount of the expression of DR5on cell surface, whichlimits and affects the curative effect of death receptor5–targeting antineoplastic. Additonally, DR5onlyplays a role on the cell surface, all factors that can promot DR5increasing expression on cell surface,which can improve the sensitivity of the cells to TRAIL and anti-DR5agonist antibody-induced apoptosis.Currently, numerous studies have shown that TRAIL and apoptosis-inducing agonistic anibodies to theTRAIL death receptors which associates with various chemotherapy drugs have significant synergistic anti-tumor effect, and at the same time can reduce the dose of chemotherapy drugs and their side effects.Further study showed that most of these chemotherapy drugs can inhibit cell proliferation and cell cycle,and can effectively enhance the expression of DR5. Homever, whether the cell cycle affectes the expressionof DR5by these drugs, and it is not clear that, the contact between the cell cycle and the expression ofDR5. In this study, we will investigate and clear the relevance between the expression of DR5and the cellcycle, which will provide guidance to the anticancer treatment and DR5as a target for anticancer drugs inthe clinical applications, as well as provide theoretical guidance for elucidating, the molecular mechanismthat the cell cycle affecting the expression of DR5, and which has important theoretical and practical value.In addition, we also purified soluble sDR5, and producted DR5monoclonal antibody named mGZA-1byhybridoma technique, by which we exploit specificity of DR5mAb.Objective: To explore the relevance of the cell cycle in tumor cells and DR5expression anddetection of DR5expression and distribution, and to explore the purified soluble DR5protein (sDR5) andmethods for preparing sDR5monoclonal antibodies. Meanwhile，we preliminary explored the specificityof the antibody to DR5. Methods: We used the column of affinity chromatography to purify sDR5and adoptedhybridoma technology to product anti-DR5agonist antibody-mGZA-1, molecular cloning, genetransfection, meanwhile we employed immunofluorescence and FCM to detect the expression of DR5.Then, we treated EC109cell line with an excess Thymidin, and double block synchronization method toget different phases of the cell cycle, and adopted FCM to detect DR5expression of each phase.Immunohistochemistry, Western bloting, immunofluorescence, RT-PCR technique also are all used todetect each cycle DR5expression and localization. The pegfp-n2recombinant vector carrying DR5wastransfected into EC109cell line, and to observe the distribution of DR5expression and localization insuspension or adherent state.Results: We haved successfully purified sDR5protein and its purity is95%. Then, we adoptedhybridoma technology to product anti-DR5agonist antibody-mGZA-1. Meanwhile, we get a stable celllines with pegfp-n2with DR5. We found that DR5mainly distributed in the cytoplasm and around thenucleus, when cells were in spreading state. While in suspended state, DR5mainly was around the cellmembrane. We employed thymidin double block synchronization release method to process EC109cells,respectively, obtaining G1phase (90.04%), S phase (97.86%), G2phase (87.17%) of the cell cycle at0h,3.5h,7.5h three different times. According to the above conditions, we detected DR5expression levels ofthe three differernt cell cycle phases by flow cytometry: normal group DR5expression rate to99.44%; G1phase93.80%; S phase98.66%; G2phase99.58%. The result of the cell immunohistochemistry detectingdifferent cell cycle phases of EC109cells (Figure13), which shows the amount of DR5expression indifferent cell cycle phases: G0/G1phase <S phase <G2/M phase. While the result of western blot ofdifferent cell cycle phases (Figure14), the DR5expression of G1phase less than the S phase, and less thanthe G2phase. Furthermore, we used Real time-PCR to detect gene level of DR5expression (Figure16) ofdifferent cell cycle phases, the DR5expression of G0/G1and S phase less than the G2/M phase. In addition,we used2.5mM Thymidine double block synchronization method and10uM LJK-11and20nM colchicineto deal with EC109cells, by which we respectively obtained cells in G0/G1phase and G2/M phase. Andwe treated the cells of G0/G1phase and G2/M phase with a certain concentration of pro-apoptotic anti-DR5antibody--mDRA-6(Figure17), the apoptosis rate of the cells of G0/G1phase less than the G2/Mphase, the difference was significant, P <0.05, having statistical significance. Conclusions:1.We’v obtained an anti-DR5monoclonal antibody named mGZA-1which thespecificity and activity is higher.2. The spreading status of EC109cells affected the distribution of DR5.3.The expression level of DR5haved differences at different phases of the cell cycle EC109, which indicatsthat there is a close link between the expression level of DR5and cell cycles.