The Preparation And Targeting Anti-colon Cancer Effects Of Recombinant INGR-GRIM-19 Fusion Protein

Colon cancer is a common and serious digestive tract malignant that threat to the health.The morbidity and mortality of colon cancer account for the forefront of malignant tumors.The commonly treatment for colon cancer is surgery combined with systemic or local chemotherapy and radiotherapy.However,the side effect of radiotherapy and chemotherapy is serious,because normal cells were simultaneously killed.Tumor targeting therapy is a novel method which selectively kills tumor cells by means of specific tumor targeting vehicles,without harming normal cells.In the research of tumor targeting therapy,how to select therapy target,tumor-homing vehicles and antitumor element is the most important.Gene associated with retinoid-interferon induced mortality-19(GRIM-19)is a tumor suppressor gene.GRIM-19 has important roles in cell cycle regulation,apoptosis,innate immune and energy metabolism.Normal expression of GRIM-19 can maintain a normal cell cycle so that cells enter apoptosis.On the contrary,low expression of certain cells should not enter apoptosis,and thus trigger the occurrence of tumor.Studies have shown that the abnormal expression of GRIM-19(low expression or null expression)exist in many malignant carcinomas.The results demonstrated that GRIM-19 is safe and exerted an efficient antitumor activity,while has no cytotoxicity on normal cells.Based on this evidence,it appears that overexpression of GRIM-19 might offer a novel therapeutic strategy for the management of cancer.CD13 is a glycosylated type Ⅱ Zn2~+-dependent membrane-bound exopeptidase.CD13 is highly expressed on endothelial cells of tumor blood vessels or tumor cells,which is little or no express on normal cells.CD13 plays and important role in tumor angiogenesis,tumor growth and metastasis,it is a new target for cancer therapy.The NGR(Asparagine–glycine–Arginine)sequence is known to serve as a recognition moiety between the ligands and integrins.Present data obtained from phagedisplay techniques found that NGR can bind selectively to CD13 integrin.So NGR is a promising homing peptide for tumor targeting therapy.Internalizing-NGR(i NGR)is a cell penetrating homing peptide,which possesses the targeting ability as NGR,supplemented with cell-internalizing and tumor-penetrating properties.Previous studies have confirmed that CD13 is highly expressed in colon cancer.Based on the above,we intend to construct a new i NGR-GRIM-19 fusion protein,expect that the i NGR peptide take along GRIM-19 homed to tumor vessels and penetrated into the tumor mass so as to inhibit tumor growth.We first analyzed GRIM-19 expression in colon cancer patient tissues vs.normal tissues,and analyzed the correlation between GRIM-19 expression level and clinicopathological characteristics.In this work,we fused GRIM-19 with the i NGR peptide,a ligand of CD13 integrin in colon cancer,by recombinant DNA technology.We constructed E.coli expression system of the i NGR-GRIM-19 fusion protein.In this research,anti-tumor effects and mechanism of i NGR-GRIM-19 fusion protein on human colon cancer cells in vitro and in vivo were investigated with colon cancer Colo205 cell and colon cancer-bearing nude mice models through Flow Cytometry(FCM),transmission electron microscopy(TEM),Western blot,reverse transcription-polymerase chain reaction(RT-PCR),and immunofluorescence methods.At last,we explored the safety and toxicity of i NGR-GRIM-19 fusion protein.The study included the following five parts: Part I Expression and significance of GRIM-19,p-STAT3,and p-Akt in colon cancer tissue and cellObjective: To detect the expression of GRIM-19,p-STAT3,and p-Akt in colon cancer tissue and Colo205 cell,and explore their effects and possible mechanism during the occurrence and development of colon cancer.Methods: Colon cancer tissues and adjacent non-tumor colon tissues were collected from 72 in-patients from the First Hospital of Jilin University.GRIM-19,p-STAT3,and p-Akt expression in the colon cancer tissues and adjacent non-tumor colon tissues were analyzed by immunohistochemistry(IHC),western blot,and RT-PCR.Collected clinical and pathological parameters of 72 cases of colon cancer and analyzed the correlation of GRIM-19,p-STAT3,and p-Akt expression with clinicopathological parameters.Applying RT-PCR to detect the m RNA expression of GRIM-19,p-STAT3,and p-Akt genes in colon cancer cell Colo205 and normal colon epithelial cell HCEpic.Results: The protein and m RNA levels of GRIM-19 in colon cancer tissues were significantly lower than that in adjacent non-tumor colon tissues.There was a negative correlation between GRIM-19 and tumor tissue differentiation,TNM staging.By comparison,the protein and m RNA levels of p-STAT3 and p-Akt in colon cancer was significantly higher than in adjacent non-tumor colon tissues,and with poorer differentiation of colon cancer,occurring of lymph node metastasis,and higher TNM stage.GRIM-19 expression in colon cancer had negative significant correlation with p-STAT3 and p-Akt.The level of GRIM-19 m RNA in colon cancer Colo205 cell was lower than that in human normal colon epithelium cell line(HCEpic),while the m RNA levels of p-STAT3 and p-Akt in Colo205 cell were higher than that in HCEpic.Conclusion: The low expression of GRIM-19 and the high expression of p-STAT3 and p-Akt co-exist in colon cancer tissues and Colo205 cell,which may play an important role in the carcinogenesis and progression of human colon cancer.Part Ⅱ The expression and purification of recombinant i NGR-GRIM-19 fusion proteinObjective: To construct a prokaryotic expression vector of i NGR-GRIM-19 fusion protein,induce its expression and purify the expression products.Methods: Using the PCR method,we amplified fusion gene i NGR-GRIM-19.The i NGR-GRIM-19 was then cloned into the expression vector p ET32a(+)and expressed in E.coli BL21.Expressed product was purified by Ni-NTA chelating agarose,refolded by dialysis and detected by sodium dodecyl sulfate-polyacrylamide gel electrophoresis(SDS-PAGE)and Western blot.Results: We correctly constructed the i NGR-GRIM-19 fusion gene.The fusion protein was successfully expressed in E.coli BL21 induced by isopropylthiogalactoside(IPTG)in the form of inclusion body.We identified the fusion protein by SDS-PAGE and Western blot.We obtained a purity of about 90% of the target protein by using Ni-NTA affinity chromatography.Conclusion: The recombinant expression plasmid p ET32a-i NGR-GRIM-19 was constructed and expressed in E.coli BL21 successfully.Part ⅡI Effects and mechanism of recombinant i NGR-GRIM-19 fusion protein on colon cancer in vitroObjective: To observe the trans-membrane activity of i NGR-GRIM-19 fusion protein towards Colo205 cell and detect the position of the fusion protein in this cell.Meanwhile,the effect and mechanism of i NGR-GRIM-19 fusion protein on proliferation,apoptosis,invasion,and metastasis of Colo205 cell were studied.Methods: Purified i NGR-GRIM-19 and GRIM-19 were added to cultured colon cancer cell lines Colo205 and normal colon epithelium cell HCEpic,the trans-membrane effect of i NGR-GRIM-19 and GRIM-19 were determined by fluorescence microscope and Western blot.Detected the effect of i NGR-GRIM-19 fusion protein on the proliferation,cell cycle,apoptosis levels and invasive ability of Colo205 cells by MTT assay,colony formation assay,TEM,FCM analysis with PI staining,FCM with an Annexin-V-FITC apoptosis kit,Terminalnucleotidyltransferase(Td T)-mediated nick end labeling(TUNEL)analysis,Hoechst 33258 staining,transwell test and wound-healing assay.CDK4,Cyclin E,Cyclin D1,Bcl-2,Cleaved Caspase-3,-8,-9,Survivin,VEGF,MMP-2,MMP-9,p-STAT3 and p-Akt were detected by Western blot and RT-PCR.Results: Fluorescent microscope and Western blot analysis showed that i NGR-GRIM-19 only translocate into Colo205 cells,but not HCEpic cells.GRIM-19 could not across cell membrane into Colo205 or HCEpic cells.MTT and colony formation assay showed that i NGR-GRIM-19 significantly inhibited the growth and proliferation of Colo205,has a dose-and time-dependent relationship.The i NGR-GRIM-19 fusion protein could make Colo205 cell morphological changes,showing the typical characteristics of apoptotic cells.FCM analysis showed that Colo205 cells were arrest in G1 phase after i NGR-GRIM-19 treatment,and the degree of arrest was concentration dependent.The results of Annexin V-FITC/PI assay indicated the dosedependent proapoptotic effect of i NGR-GRIM-19 on Colo205 cells.The results of Transwell test and wound-healing assays demonstrated that i NGR-GRIM-19 showed remarkable suppression of migration and invasion.In addition,i NGR-GRIM-19 can reduced the expression of CDK4,Cyclin E and Cyclin D1,which were associated with cell cycle;elevated the expression of Cleaved Caspase-3,-8-9,reduced Bcl-2 and Survivin,which were associated with cell apoptosis;reduced the expression of VEGF,MMP-2 and MMP-9,which were associated with cell invasion;reduced the expression of p-STAT3 and p-Akt.Conclusion : The i NGR-GRIM-19 fusion proteins have efficient targeting Colo205 cell and trans-membrane activity,also can specific induce apoptosis,inhibit proliferation and metastasis of Colo205 cell.The mechanism of i NGR-GRIM-19 suppressed colon cancer may involve the negatively regulation of JAK/STAT3 and PI3K/Akt signaling pathway.Part IV Effects of recombinant i NGR-GRIM-19 fusion protein on human colon cancer Colo205 nude mice in vivoObjective: Tumor-bearing mode was established with BALB/c nude mice by subcutaneous injection of Colo205 cell.The existence of i NGR-GRIM-19 in different tissues and its anti-tumor activity in vivo were studied.Methods: Mice colon cancer cells of the line Colo205 were inoculated subcutaneously into BALB/c nude mice to establish model of colon cancer.Four mice were randomly divided into two groups to be injected with the i NGR-GRIM-19 or GRIM-19 protein labeled with fluorescein isothiocyanate(FITC).The location of the protein in the colon cancer bearing mice tissue was analyzed by the optical in vivo imaging two hours after the injection and fluorescence microscopy twenty-four hours after the injection.Detected the effect of i NGR-GRIM-19 fusion protein on the proliferation,cell cycle and apoptosis levels of colon cancer of nude mice by HE staining,TEM,FCM analysis with PI staining,FCM with an Annexin-V-FITC apoptosis kit and TUNEL analysis.Protein and m RNA expression levels of CDK4,Cyclin E,Cyclin D1,Bcl-2,Cleaved Caspase-3,-8,-9,Survivin,VEGF,MMP-2,MMP-9,p-STAT3,p-Akt were analyzed by Western blot and RT-PCR.Results: The optical in vivo imaging and fluorescence microscopy analyses showed that FITC fluorescence labeling i NGR-GRIM-19 fusion protein was assembling in the tumor of the colon cancer bearing mice,but not in normal tissues.However,GRIM-19 showed no translocation activity in colon cancer or normal tissues.The in vivo results showed that i NGR-GRIM-19 significantly suppresses the growth of xenografted tumors,and induced tumor cell cycle arrest in G1 phase,increased tumor cell apoptosis.Tissue sample analysis shows that changes observed in cell cycle arrest related-proteins,cell apoptosis related-proteins,cell metastasis related-proteins,and key proteins in signaling pathways were consistent with the changes observed in vitro experiment.The i NGR-GRIM-19 treatment did not influence nude mice blood system,liver and kidney organs.Conclusion: The i NGR-GRIM-19 had strong binding affinity to Colo205 cells and proliferation-inhibiting activity to colon cancer tumor-burdened mice.Part V The study on the security and pharmacokinetics of recombinant i NGR-GRIM-19 fusion proteinObjective: To explore the safety and toxicity of i NGR-GRIM-19 fusion protein,as well as preliminary calculate its pharmacokinetics.Methods: The safety tests include the allergic test of i NGR-GRIM-19 fusion protein for injection in guinea pigs,rabbit pyrogen test and rabbit muscle stimulation test.To determince the toxicity of i NGR-GRIM-19 fusion protein,organ index,blood T lymphocytes and maximum tolerated dose of i NGR-GRIM-19 were measured.Pharmacokinetic parameters were also calculated.Results: There were no allergic reaction occurred;rabbit pyrogen test was qualified;there were no manifest abnormality occuuued in rabbit muscle stimulation test.i NGR-GRIM-19 fusion protein didn’t adversely affect body weight and food intake.No significant organ treatment-related changes and the proportion of T cells were observed.Pharmacokinetics of i NGR-GRIM-19 fusion protein following i.v.administration in mice showed: the distribution half-life is 0.16 h and the elimination half-life is 22.4 h,the maximal blood drug concentration is 76.1 mg/m L,the area under the curve of drug concentration-time is 86.6 mg/m L/h,and the apparent distribution volume is 38.2 L/kg.Conclusion: The tolerance and safety of i NGR-GRIM-19 fusion protein are excellent and the i NGR-GRIM-19 has a promising clinic application value.In summary,the low expression of GRIM-19 may play an important role in the tumorigenes of colon cancer.We constructed a new fusion protein i NGR-GRIM-19.The fusion protein exert anti-tumor effect by inducing apoptosis,inhibiting invasion and metastasis in vitro and in vivo efficiently,but has no significant effects on the growth of normal cells and tissues.The tolerance and safety of i NGR-GRIM-19 fusion protein are also excellent.Our studies will lay the foundation theoretical research and application development of targeting drugs of i NGR-GRIM-19 in colon cancer.