Transfection Of Mouse Macrophage Metalloelastase Gene Into Murine CT-26 Colon Cancer Cells Suppresses Orthotopic Tumor Growth, Angiogenesis And Vascular Endothelial Growth Factor Expression

Objective Mouse macrophage metalloelastase (MME), a member of the matrix metalloproteinase family, is believed to play an important role in the generation of angiostatin, an internal fragment of plasminogen, which shows inhibition of tumor angiogenesis. The current study was designed to determine the correlation between MME and vascular endothelial growth factor (VEGF) expression involved in growth and angiogenesis of colon cancer.Methods A cDNA fragment coding for domains I and II of MME was transfected into murine CT-26 colon cancer cells that are MME deficient. The enzymatic activity of recombinant MME was confirmed by cleavage of native substrate in vitro. An orthotopic implantation model was established by using MME-transfected cells and control cells. Tumor samples were subjected to in situ hybridization (ISH), immunohistochemical staining (IHC) and Western blot to detect expressions of MME and VEGF. The microvessel counting was used to assess angiogenesis of murine colon tumors.Results pUC9-MME cDNA was subjected to 35 cycles of PCR amplification and produced a 840bp MME cDNA fragment coding for domains I and II of MME. The pGEM-T-MME plasmid was digested into two fragments by 1% agarose gel electrophoresis with BamH I and Xba I . They were consistent with theoretic values 3.0kb and 832bp. indicating that PCR products have been cloned into pGEM-T vector. The sequence of the insert was 99.63% identical to that in GeneBank. The recombinant pcDNA3.1-MME plasmid was separated into two bands(about 5.4kb and 832bp,respectively) in a 1% agarose gel using BamW I and Xba I , suggesting that MME gene fragment has been cloned into pcDNA3.1 vector correctly. CT-26 transfectants were selected by G418 and MME mRNA expression was analyzed by RT-PCR. The 840bp amplification products expected for MME were identified in MME-transfected clones. In contrast. MME mRNA was not detected in pcDNA3.1-transfected clone and nontransfected cells. To detect the presence of MME protein, immunocytochemistry was performed. Positive immunostaining for MME protein was present in the cytoplasm of MME-transfected cells, but not in those of pcDNA3.1-transfected and nontransfected cells. MME protein was also confirmed by Western blot analysis in CT-26 cells. A 30 kDa band was detected in MME-transfected cells, but not in pcDNA3.1-transfected cells and nontransfected cells. The protein with 30 kDa molecular mass corresponded to the domains I and II of MME. To determine if the recombinant MME expressed in CT-26 cells was capable of enzymatic activity in vitro. 500ug of insoluble type I collagen was incubated with the supernatants of transfected or nontransfected cells lysed by sonication. MME-transfected cells exhibited soluble cleavage of type I collagen by MME. In contrast, degraded soluble products of type I collagen were not seen in pcDNA3.1-transfected and nontransfected cells. Gelatin zymography was also performed. A 22 kDa lytic band was identified in MME-transfected cells, whereas no zone of lysis was indicated in the controls (pcDNA3.1-transfected and nontransfected cells). The 22 kDa band corresponded to the final active form of MME after cleavage with loss of domain I (8kDa). Mice carrying pcDNA3.1-transfected and nontransfected cells formed large primary tumors with volumes of 1151.07±35.91mm3 and 1201.13± 42.15mm3, respectively. In contrast, mice implanted with MME-transfected cells formed smaller tumors with volumes of 384.83 ±4.76mm3.Growth of MME-transfected tumors was significantly inhibited compared with control tumors (MME-transfected group versus pcDNA3.1 -transfected group, PO.001; and versus nontransfected group, PO.001). There is on significant difference between control groups, />=0.374. In addition, 2 of 30 mice (6.7%)developed liver metastases in control groups, and no livermetastasis was found in MME-transfected group. 18 of 30 mice (60.0%) developed peritoneum metastases in control groups, and 4 of 15 mice (26.7%) with peritoneum metastases were found in MME-transfected group. P=0.035. Tumors derived from MME-transfected cells demonstrated a lower microvessel density (8.48 ± 0.53) compared with control tumors derived from pcDNA3.1-transfected (21.87±0.47) and nontransfected (22.56 ± 0.71) cells (MME-transfected group versus pcDNA3.1-transfected group, P<0.001; and versus nontransfected group, PO.001). There is no significant difference between control groups, P=0A23. VEGF mRNA expression by ISH was significantly lower in MME-transfected tumors than that in pcDNA3.1-transfected and nontransfected tumors (MME-transfected group versus pcDNA3.1-transfected group, /)=0.028; and versus nontransfected group. P=0.003). There is no significant difference between control groups (P=0A09). VEGF protein expression by IHC was significantly lower in MME-transfected tumors than that in pcDNA3.1-transfected and nontransfected tumors (MME-transfected group versus pcDNA3.1-transfected group, />=0.025; and versus nontransfected group. .P=0.008). There is no significant difference between control groups (P=0.624). Western blot analysis of VEGF was also performed in the tumor tissue samples to quantify difference in protein level. A strong VEGF protein band was present in pcDNA3.1-transfected and nontransfected tumor tissues, whereas in MME-transfected tumor tissues a weaker band was observed. Quantification of the protein signals by image analysis revealed that the VEGF protein levels were 2.6-fold (7.83 + 1.14 versus 3. O4±0. 52) and 2.2-fold (6.57 ±0.76 versus 3.04+0.52) increased in pcDNA3.1-transfected and nontransfected tumor tissues, respectively, compared with MME-transfected tumor tissues. PO.01. Conclusion Our data show that the recombinant mammalian cell expression vector pcDNA3.1-MME is successfully constructed. The MME gene transfected into murine colon cancer cells can effectively suppresses the growth of orthotopic tumors by inhibition of vascularity. Both MME and VEGF gene expression is highly associated with the vascularity of tumors, which may depend on a balance between MME and...