1.Effect Of TNF Receptor Blocking Peptide Loaded PEG-PLGA Nanoparticles On TNF-induced Activation Of Neutrophils In Vitro 2.Vincristine-induced Translation Of TRAF1 By IRES-dependent Mechanism In Lymphoid Malignancies

Part 1: Effect of TNF receptor blocking peptide loaded on PEG-PLGA nanoparticles on TNF-induced activation of neutrophils in vitroTumor necrosis factor-alpha (TNF-α) as a pleiotropic cytokine, is involved in many physiological and pathological process like anticancer, anti-infection, imunreaction regulation and inflammation. A lot of infection or noinfection diseases are also related to the cytokine, e.g., it's overexpression can lead to severe inflammatory reactions, resulting thrombosis, shock, hypotension, tissue damage and even death.TNF-αexerts biological function through binding and activating the TNF receptors on the cell membrane. There are two types of TNF receptors, TNFRI and TNFRII. The biological effects are mainly induced by TNFRI, while TNFRII plays a supporting and some regulating role. In our previous study a special kind of peptide was obtained from a phage random peptide library, which might combine with TNFRI without signal transformation, called as TNFR blocking peptide (TRBP).However, the peptide has short half-life, and filtering out easily through renal glomerulus. TRBP was loaded on the PEG-PLGA-modified nanoparticles as a cooperative work with College of Life Sciences and Drug Research Institute, Huazhong University of Science and Technology. The study is designed to observe the biological activity of such loaded TRBP in vitro maintains its biological effects and prolonged half-life.The results are summarized as follows.1 Study on the biological activity of PEG-PLGA nanoparticle-loaded TRBP in vitro.1.1 Inhibition to mononuclear/macrophage phage function by PEG-modified PLGA nano particlesWith flow cytometry test it was observed that the PEG- modified PLGA nanoparticles, on which GFP loaded, might effectively avoid the phagocytosis of the mononuclear and macrophage system. It is suggested that it contributes a long-circulation to load agents with PEG-PLGA nanoparticles as carriers. 1.2 Prolongation of the inhibition to TNF cytotoxic effect by PEG-PLGA nanoparticlesAs showed by MTT test the supernatant of TRBP carried by PEG-PLGA could inhibit the sTNF-αcytotoxicity more effectively, e.g., the inhibition half time at 37℃was 2 days and even 5 days at 4℃. In converse TRBP alone induced inhibition at 37℃only for 3-6 hours and at 4℃for 24 hours. It is indicated that the inhibition function of TRBP is prolonged obviously when using PEG-PLGA nanoparticles as carriers.2 Inhibition to TNF-αstimulated neutrophil activation by the PEG-PLGA carried TRBP2.1 Effect of the PEG-PLGA carried TRBP on the neutrophil phagocytosisIn this study we improved some steps of neutrophil phagocytosis test by Flow Cytometry in which instead of labeling bacteria, a BL-21 bacteria transformed GFP was used. Cells pretreated with Cyto-D were set as control to show the nonspecific adherent effect. The result showed that the improved method can more accurately detect the neutrophil phagocytosis. During the experiment it was showed that the supernatant released by the PEG-PLGA carried TRBP for 6 h at 37℃could obviously inhibit the neutrophil phagocytosis stimulated by TNF-α, while TRBP had only a slight inhibition at the same conditions.2.2 Inhibitive effect of the PEG-PLGA carried TRBP on the TNF-αstimulated neutrophil oxidative burstAs NBT test showed that the supernatant of PEG-PLGA carried TRBP released for 6h at 37℃could obviously inhibit the oxidative burst induced by TNF-α. However, the TRBP showed again only a slight inhibition effect.2.3 Effect of PEG-PLGA carried TRBP on the TNF-αstimulated neutrophil IL-1βmRNA expressionThe up regulating effect of TNF-αon the neutrophil IL-1βmRNA expression could be inhibited by the supernatant as above obviously, while almost no effect by TRBP alone.2.4 Effect of PEG-PLGA carried TRBP on the TNF-αinduced neutrophil NF-κB activation Using western blot to test the IκB degradation after its pathway stimulated, it was showed again that the PEG-PLGA carried TRBP could inhibit the degradation of IκB, while TRBP had significant less such effect.It is suggested by the experiment that the PEG-PLGA nanoparticles loaded TRBP could effectively inhibit the TNF-αinduced stimulation of neutrophil, reducing inflammation and resulting TRBP life time prolonged. It provides some basis to develop TNF-related anti-inflammation drugs and to use TRBP in clinics in future.Part 2: Vincristine-induced translation of TRAFl by IRES-dependent mechanism in lymphoid malignanciesTRAF1 (Tumor necrosis factor receptor-associated factor) is a member of the TRAF family that was originally identified based on its ability to interact with the cytosolic domain of TNF receptor type 2 (TNFR2). TRAF1 plays important roles in signal transduction that mediate cell life and death in the immune response, inflammatory and malignant diseases. It was reported recently that TRAF1 could inhibit the TNF-αor TCR induced apoptosis. When the combination of TNF-αand TNFR1, TRADD recruits conjunction molecular RIP1,TRAF2,TRAF5 to activate IKK complex, thereby activating NF-κB. The constructive activation of NF-κB, plays important role in resistance of chemotherapy. In our study, VCR can significantly stimulate TRAF1 expression in primary leukemia cells which were obtained from eight patients with B-cell precursor (BCP) ALL. It is indicated that TRAF1 is a highly inducible protein. In this study, we will explore the mechanism in which VCR induce TRAF1 expression. After VCR stimulation, TRAF1 mRNA stays same. It means that the regulation is not in the level of transcription. Cap-dependent translation was depressed after VCR stimulation too. Therefore, this study is to investigate whether the VCR increased the expression of TRAF1 by IRES-dependatant mannar and the regulation mechanism. The results are summarized as follows. 1 Induction of protein expression in BCP-ALL cell by vincristine. VCR can induce TRAF1 expression in BCP-ALL cell the same as TNF-α. In addition, we found the VCR induced TRAF1 expression in a dose- and time-dependent manner.2 Vincristine-induced TRAF1 expression is not in transcriptional level. Weperformed RT-PCR demonstrated that VCR did not induce TRAF1 transcription, whereas TNF-αinduced TRAF1 mRNA expression. These results suggest that VCR and TNF-αinduce TRAF1 expression via different mechanisms.3 Identification of the long 5'-UTR within TRAF1 mRNA. TRAF1 has an unusually long 5'-UTR. We performed RT-PCR on cancer cell lines(Raji and HDLM) and primary leukemia cells(UPN 1 and UPN 2), demonstrating and confirming the presence of the 5'-UTR of TRAF1 mRNA in all cancer cells studied.4 Analysis of IRES activity within the 5'-UTR of TRAF1 mRNA. We divided the entire 2446 bp TRAF1 5'-UTR into three parts (-2446 to -1587, -1586 to -573 and -572 to -1) and cloned each of them into the pGL3 control plasmid. As control, -572 to -1 fragment in a reverse orientation was inserted into pGL3 control plasmid. This suggests that the forward -572 to -1 sequence of the TRAF1 5'-UTR has IRES activity. We cloned -572 to -1 fragment into the dicistronic pRL-FL vector or phpRL-FL vector. The result confirmed that -572 to -1 sequence of the TRAF1 5'-UTR has IRES activity. Transfection of the plasmids into Raji cells showed FL activity induced in the dicistronic plasmid was indeed induced by that-572 to-1 fragment.5 Mapping the TRAF1 IRES. To further determine the core IRES region within the TRAF1 5'-UTR, we generated a series of deleted constructs of the -572 to -1 fragment, to clone into the dicistronic vector pRL-FL. We discovered that the core IRES region resided between -392 and -322. We cloned this 71 bp fragment into the pGL3-basic vector and found that it did not stimulate FL activity in the pGL3-basic vector. It confirm that the sequence between -392 and -322 is critical for TRAF1 IRES activity,6 Induction of TRAF1 IRES activity and TRAF1 protein expression by vincristine. Last, we performed gene transfections and reporter assays to see if VCR induced TRAF1 IRES activity. We found that VCR increased the FL (TRAF1 IRES) activity, whereas Dox decreased the FL activity. 7 Subcellular redistribution of PTB induced by vincristine confers TRAF1 translation. Western blot to demonstrate that VCR could induce PTB protein to transfer from nuclear to cytoplasmic. After a protein-RNA binding assay with UV cross-linking, it showed that the PTB protein bound to the TRAF1 IRES RNA -392 to -322. Transfection of PTB siRNA (pSUPER/PTB) not only suppressed endogenous PTB expression, but also depressed the expression of TRAF1. We also found that co-transfection of the PTB siRNA plasmid with the pRL-392-FL reporter plasmid into cells remarkably decreased the activity of FL. The result comfirmed that PTB promotes the IRES-dependent translation.