Experimental Study On Pathway Of Recombinant Macrophage Metalloelastase Suppressing Tumor Angiogenesis In Murine Colon Cancer

BackgroundMacrophage metalloelastase(MME) was secreted by activation macrophages or umor cell.MME is responsible for the inhibition of tumor growth and angiogenesis.It is different with other members of the matrix metalloproteinase(MMP) family.In previously study,we found that MME suppressing growth and metabasis was concerned with the mechanism of restraining tumor angiogenesis in murine colon carcinoma. Accordingly,more superiority method should been adopted for confirming the function and pathway of MME in colon carcinoma.ObjectiveImprove the previously methods in construction,transfection and identification of the recombinant MME plasmid.The GFP-tagged MME recombinant plasmid should be constructed and transfected into CT-26 colon cancer cells.These techniques should be established of preparation of radioiodine-labeled plasminogen and radioactivity racer. The current study was designed to determine whether tumor cells engineered with MME could generate activity angiostatin by decomposing plasminogen in vitro and in vivo,and to further investigate the correlation between MME and basic fibroblast growth factor(bFGF) expression involved in growth and microvessel density of colon cancer.Methods To generate a GFP-tagged MME,pcDNA3.1-MME and pEGFP-C1 were cut with XbaI and BamHI and MME inserted into the green fluorescent fusion expression vector pEGFP-C1 between the XbaI and BamHI sites.The recombinant plasmid pEGFP-C1-MME was transfected into CT-26 colon cancer cells.RT-PCR,Western blot, soluble typeâ… collagen and gelatin zymography were analyzed to validate the presence and its enzymatic activity of MME protein in pEGFP-C1-MME-transfected cells.Murine CT-26 colon cancer cells stably transfected with MME were inoculated subcutaneously.The cell suspensions were pipetted to produce a single-cell suspension; cell viability was determined by Propidium Iodide,and only single-cell suspensions of greater than 90%viability were used.Immunoblotting and immunohistochemistry were used to detect the expression of MME in tumor tissue.Immunohistochemical staining of CD34 was performed to determine the microvessel density(MVD).Fluorescence intensity of VEGF was measured by immunofluorescence analysis. Reverse-transcriptase polymerase chain reaction(RT-PCR),immunoblotting,and immunohistochemistry were used to explore the bFGF mRNA and protein expression.For in vivo biodistribution studies,tumor-bearing mice were injected via the tail vein with ¹²⁵I-plasminogen.Radioisotope tracer,immunoblotting were used to explore the pathway of angiostatin generation.To evaluate the effect of MME on vascular endothelial cell proliferation and migration ability,MTT and Chemotaxis Study were performed in vitro.Results1.construction,transfection and identification of the recombinant MME plasmidDirect DNA sequencing and enzyme digestion showed pEGFP-C1-MME were constructed.After transfection with the pEGFP-C1 vector and pEGFP-C1-MME and elected by G418,GFP expression could be observed under fluorescent microscopy.The pattern of intracellular GFP distribution appeared different:GFP in control vector-transfected cells spread throughout the cells,whereas GFP in pEGFP-C1-MME-transfected cells was mainly localized in the cytosol.To validate the presence of MME protein in pEGFP-C1-MME-transfected cells,conditioned medium from cells growing in serum-free medium was analyzed by Western blotting.A 57 kDa band,corresponding to the domainsâ… andâ…¡plus GFP,was detected in MME-transfected cells,but not in vector-transfected cells or nontransfected cells.The protein with 30 kDa molecular mass corresponded to the domainsâ… andâ…¡of MME.To determine if the recombinant MME expressed in CT-26 cells was capable of enzymatic activity in vitro,1000μg of insoluble typeâ… collagen was incubated with the supernatants of transfected or nontransfected cells lysed by sonication. MME-transfected cells exhibited soluble cleavage of typeâ… collagen by MME.In contrast,degraded soluble products of typeâ… collagen were not seen in vector-transfected and nontransfected cells.Gelatin zymography was also performed.To determine whether the three base mutations in the recombinant MME altered its proteolytic activity,gelatin zymography was performed.A 22 kDa lyric band was detected only in MME-transfected cells but not in the controls.The 22 kDa band corresponded to the final active form of MME after cleavage of domain I(8 kDa) and GFP(27 kDa).2.Effects of recombinant MME on tumor growth and correlation factor of tumor angiogenesisProbability of formed tumor was 94.8%(91/96).There was a statistically significant difference in the survival period of the CT-26-EGFP-MME transfected group (1 of 30 mice died within 4 weeks) and the control groups(8 of 30 and 6 of 30 mice died within 4 weeks in the CT-26-EGFP transfected group and nontransfected group, respectively;P＜0.05).The CT-26-EGFP transfected and nontransfected cells formed large subcutaneous tumors with volumes of 2071.80.50±608.96 and 2231.90±496.91 mm³ 4 weeks after implantation,respectively.In contrast,CT-26-EGFP-MME transfected cells formed smaller tumors,with volumes of 816.56±234.02 mm³. Therefore,the growth of MME-transfected tumors was significantly retarded in comparison with the control tumors(P＜0.001). Forty-six of 60 mice(76.6%) in the control groups developed thoracic cavity metastases;only 11 of 30 mice(36.6%) with thoracic cavity metastases were found in the MME-transfected group.No liver metastasis was found in any mouse in the three groups.Tumors derived from CT-26-EGFP-MME transfected cells demonstrated a lower microvessel density(9.35±2.79) compared with control tumors derived from CT-26-EGFP transfected(22.85±3.80) and nontransfected(23.45±4.49) cells(P＜0.001). There was no significant difference between the two control groups.A differential expression of bFGF mRNA in among tumors from control and MME-transfected cells was shown in our study.Quantitative real-time RT-PCR analysis revealed that the bFGF mRNA levels were 2.7-fold(0.56±0.063 versus 0.21±0.042) and 2.5-fold(0.53±0.066 versus 0.21±0.042) higher in tumors from CT-26-EGFP and nontransfected cells, respectively,in comparison with that in tumors from CT-26-EGFP-MME cells(P＜0.01). Similarly,immunohistochemical staining showed that bFGF protein expression was significantly reduced in tumors from CT-26-EGFP-MME cells compared with those in tumors from CT-26-EGFP and parental cells(P＜0.01).Western blot analysis of bFGF was also performed in tumor tissues to quantify difference in protein levels.A strong 19 kDa bFGF protein band was present in CT-26-EGFP and nontransfected tumor tissues, whereas the intensity of this bFGF band was significantly reduced in CT-26-EGFP-MME tumors.3.Study on pathway of recombinant MME for angiostatin generationSDS-PAGE electrophoresis analysis of tumor tissue shows that,in the PAGE gel contained the protein with molecular weights of 35 and 38 kDa,radioactivity of the CT-26-EGFP-MME group(6891.88±768.86 cpm) was significantly higher than control groups,P＜0.01.Radioactivity of the nontransfected and CT-26-EGFP group were 1503.9±304.42 cpm and 1376.21±186.36 cpm respectively.On Western blot analysis, we also detected the generation of angiostatin in the MME-transfected group.In contrast,the control groups showed two very faint bands for angiostatin.Quantification of the protein signals revealed that the angiostatin protein levels were obviously increased(9.32±1.52 and 5.61±2.24) in CT-26-EGFP-MME transfected tumor tissues, compared with CT-26-EGFP transfected(2.47±0.23 and 0.67±0.12) and nontransfected tumor tissues(1.21±0.69 and 0.86±0.44),P＜0.01.In vitro,we detected the generation of angiostatin 24h after adding MME lysate and plasminogen into PIEC.The 38-kDa fragment was found in the supematant of the PIEC cocultures incubated in medium plus MME and plasminogen.In contrast,the control groups showed no obviously bands for angiostatin.PIEC proliferation activity begin weaken for 24h after plus MME and plasminogen.After 72 h,Cell proliferation decreased obviously and appeared cell accumulation.Migration experiment was perform with Boyden cultivante cabin.Inhibition ratio of PIEC was 25.5%in MME group,the control groups were 4.2%and 3.5%respectively.It indicated expression of MME could degrade invasiveness of vascular endothelial cell in vitro.ConclusionUnder the foundation of previously study,we successfully constructed a vector, pEGFP-C1-MME,and transfected the MME gene into murine colon cancer cells.MME is responsible for the inhibition of tumor growth,angiogenesis and pleural metastasis in this tumor model.In addition,expression of MME inversely correlates with the expression of bFGF and tumor angiogenesis in the model of murine colon cancer. Moreover,preparation of radioiodine-labeled plasminogen and radioactivity racer were established.It was confirmed the biologic properties of MME,plasminogen,and angiostatin appear to be tightly linked in vivo,and MME was able to restrain proliferation and migratory in endothelial cell.