The investigation of the effect of endostatin on matrix metalloproteinase-2 in colon and rectum cancers

BackgroundColorectal cancer, one of the most prevalent cancers worldwide, is the second leading cause of cancer-related mortality in developed countries. Tumor metastasis and progression, which depend on angiogenic process, include the proteolytic degradation of the basement membrane (BM), and the extracellular matrix (ECM), altered cell adhesion and the physical movement of tumor cells. Among these steps, excessive degradation of the matrix is one of the hallmarks of this process. Many proteinases are capable of degrading ECM components, but the proteinase system primarily responsible for ECM degradation is matrix metalloproteinase (MMPs). MMP-2 and MMP-9 have been implicated to play a role in colorectal cancer progression, invasion and metastasis in animal models and patients. The enzyme activity is regulated extracellularly and its regulation is mainly based on the balance between pro-enzyme activation and inhibition by tissue inhibitors of MMPs (TIMPs). Furthermore previous studies have shown that MMP-2 and MMP-9 may directly regulate angiogenesis by generating pro-angiogenic factors, such as vascular endothelial growth factor (VEGF), as well as the anti-angiogenic factor endostatin. Hence the regulation of the activity of these proteinases appears to be of great importance in tumor progression at different stages.AimSince observations support the theory that development of colon and rectal cancers may involve different mechanisms, we classified colorectal cancer into colon and rectal cancer. We focused on the activity level of MMP-2, MMP-9, TIMP-2, MT-1 MMP, VEGF and endostatin protein level between tumor areas compared with normal tissue specimens and investigated potential relationships between these parameters and clinicopathological variables in colon and rectal cancers. With in vitro experiments, we also examined how these MMP-2 secretion and activity may be regulated by recombinant endostatin and we also determined the possible role of endostatin on cell viability and invasion of human colorectal adenocarcinoma cell line, HT-29.MethodsTumor and normal tissue specimens were obtained from colon (n=50) and rectal carcinoma (n=34). MMP-2 and MMP-9 activity levels were examined using gelatin zymography. MT-1 MMP activity level was analysed by activity assay. TIMP-2, VEGF and endostatin protein levels were examined using enzyme-linked immunosorbent assay (ELISA). Also the protein expression level of endostatin was assessed by Western Blotting. The in vitro invasion assay was carried out using Transwell chamber with 8 mm diameter polycarbonate membrane precoated with Matrigel.ResultsIn colon tumors activity levels of pro and active MMP-2 and also active MMP-9 were significantly higher than in the corresponding normal tissue (p<0.05). The protein levels of TIMP-2, MT-1 MMP, VEGF and endostatin did not reach a statistically significant difference between tumor and normal tissue. Significant relationships were found between colon tumor active MMP-9 and tumor invasion depth ?pT1-2 vs pT3-4? (p=0.011) and distant metastase. (p=0.047). However, MMP-2 activity in colon tumor tissue did not correlate with any of the clinicopathological parameters investigated. In rectal tumors, the activity levels of both pro and active MMP-2 and MMP-9 were significantly higher than those in normal tissues, the differences being significant for all (p<0.05). No significant differences between tumor and normal tissue were found for the protein level of TIMP-2, MT-1 MMP, VEGF and endostatin. Tumor active MMP-9 levels and the ratio between active and proMMP-2 (rMMP-2) showed statistically significant relationships with metastatic disease during the diagnosis (p=0.002, p=0.006, respectively) and also tumor active MMP-9 level showed a significant relationship with perineural invasion (p=0.042). Also we found statistically significant relationship between tumor proMMP-9, proMMP-2 activity levels and lymph node metastasis (p= 0.012, p=0.021, respectively). Tumor TIMP-2 levels showed a significant relationship with recurrence (p=0.011). The significant relation was found between tumor VEGF level and perineural invasion (p=0.044). Also there was a statistically significant correlation between tumor VEGF and tumor size (p=0.009 r=0.454). Cell culture experiments revealed that recombinant endostatin decreased the cell viability of HT-29 cells. It was found that both proMMP-2 secretion and activity were significantly lower in endostatin treated groups than the control group (p<0.05). There was a significant positive correlation between cell viability and proMMP-2 (p=0.007 r= .423). In the study group, MT-1 MMP level and cellular invasion were found lower compared with the control group, but these differences were not statistically significant. However, the ratio between TIMP-2 and MT-1 MMP in experiment groups were significantly higher than in the corresponding control group (p<0.05). There was a very strong negative and statistically significant correlation between invasion and TIMP-2/MT-1 MMP ratio (p=0.00, r= -.962). Also the very strong positive and statistically significant correlation was found between invasion and MT-1 MMP level (p=0.000, r= .991).ConclusionThe significant relationships between the increased activity of MMP-9 and tumor invasion depth ?pT1-2 vs pT3-4?and distant metastases reflect that MMP-9 may play a role in predicting the agressive behavior of colon cancer. In addition, a number of significant relationships demonstrated between MMP-2, MMP-9 activity level, TIMP-2, VEGF levels and tumor pathology including metastatic disease during the diagnosis, perineural invasion, lymph node metastasis, recurrence might contribute to further investigation of possible prognostic significance in rectal cancer. Also the results of the cell culture experiments on the effect of recombinant endostatin on cell viability and MMP secretion of the HT-29 are expected to contribute to further investigation for anticancer strategies in colon cancer.