Circulating gelatinases and tissue inhibitor of metalloproteinase-1 in colorectal cancer metastatic liver disease.

AIMS: The degradation of the extracellular matrix is intrinsic to the invasion and progression of cancer. Matrix metalloproteinase (MMP)-2 and -9 and their natural inhibitors are involved in this process. The study aims to investigate if plasma MMP-2, -9 and tissue inhibitor of metalloproteinase-1 (TIMP-1) can be useful markers in the diagnosis and prognosis of colorectal cancer (CRC) metastatic liver disease. METHODS: Fifty-seven patients undergoing liver metastasis operation were followed prospectively. ProMMP-2, -9 and TIMP-1 plasma levels were determined by zymography and ELISA, before and after the resection of liver metastases. Data were compared with those of healthy controls (n=51) and primary CRC patients (n=94). The diagnostic and prognostic potential was investigated with ROC-curves and Kaplan-Meier survival analysis. RESULTS: Plasma proMMP-2 levels were lower (P<0.001), and TIMP-1 levels higher (P<0.001) in CRC metastatic liver disease than in healthy controls. If compared to those in primary CRC patients, no differences were found. In ROC-curves, the area under the curve was 0.48 and 0.61 for proMMP-2 and -9, respectively. Plasma proMMP-2, -9 and TIMP-1 levels were unsuitable to predict survival. In both diagnostic and prognostic examinations, CEA proved to be a better marker. In the postoperative follow-up, protracted low levels of proMMP-2 seemed related to disease recurrence. CONCLUSION: The preoperative plasma proMMP-2, -9 and TIMP-1 levels have no potential value as diagnostic or prognostic markers in CRC liver metastatic disease.

Plasma gelatinase activity does not reflect disease activity after operation for colorectal cancer.

OBJECTIVE: To investigate if plasma matrix metalloproteinase (MMP)-2 or -9 are better markers for disease activity than carcinoembryonic antigen (CEA) in the postoperative follow-up of colorectal cancer patients. METHODS: A prospective study was performed including 61 patients operated for primary colorectal cancer. The follow-up was for at least 2 years and postoperative blood samples were obtained periodically with 3-month intervals. Plasma gelatinase activity was measured with quantitative gelatin zymography and serum CEA with a specific immunoassay. RESULTS: Zymographic analysis of plasma samples revealed the presence of the proforms, but not the active forms, of both MMP-2 and -9. Prior to the detection of recurrent disease or metastasis in potentially curatively operated colorectal cancer patients, the changes in proMMP-2, -9 and CEA blood levels were determined. ProMMP-2 and -9 plasma levels changed little in this period and changes between patients with and without disease relapse were not statistically significant. In contrast, patients with disease relapse showed a significant increase (p = 0.002) in CEA in the two consecutive serum samples prior to the detection of recurrent disease or metastasis. Similarly, prior to death due to colorectal cancer, proMMP-2 and -9 plasma levels showed no significant change, whereas CEA levels increased considerably and significantly (p < 0.001) when compared to changes found in survivors. CONCLUSION: Plasma proMMP-2 and -9 activities show no potential value as prognostic markers in the follow-up of colorectal cancer.

Circulating matrix metalloproteinase-9 is transiently elevated after colorectal surgery.

BACKGROUND AND AIMS: Plasma levels of matrix metalloproteinases (MMPs) may yield important information in patients suffering from colorectal cancer but the effect of surgery, a common treatment modality in these patients, on circulating MMP levels is currently unknown. The aim of this study was to assess whether plasma MMP-2 and MMP-9 levels are affected by operative procedures. MATERIALS AND METHODS: In total 128 patients undergoing elective surgery for colorectal cancer (n = 66), liver metastases from colorectal origin (n = 50) and arthrosis of the hip (n = 12) were included in the study. Gelatinase activity was measured, using quantitative gelatin zymography, in plasma obtained before operation and 1 week, 1 month and 3 months postoperatively. RESULTS: One week after operation a significant increase in proMMP-9 activity was measured after colorectal surgery (260%, p = 0.0038), liver surgery (285%, p < 0.0001) and hip surgery (217%, p = 0.012) as compared with preoperative levels. After 1 month proMMP-9 activity had returned to preoperative levels. No effect on proMMP-2 activity was measured. CONCLUSION: Operative procedures have a profound but transient effect on plasma MMP-9 activity. If used to assess disease status, postoperative plasma MMP levels should be interpreted with caution.

Matrix metalloproteinase 2 and 9 activity in patients with colorectal cancer liver metastasis.

BACKGROUND: Matrix metalloproteinases (MMPs) have been reported to play an important role in tumour cell invasion and metastasis. The bioactivity of MMPs in liver metastasis from colorectal cancer was investigated and correlated with clinicopathological variables. METHOD: Thirty-two patients underwent resection of colorectal cancer liver metastases. Latent and active forms of MMP were measured in tissue extracts, by means of quantitative gelatin zymography and a fluorometric activity assay. RESULTS: Broad-spectrum MMP activity, and levels of both active and latent forms of MMP-2 and MMP-9, were higher in tissues containing metastatic tumour than in normal liver tissue. Median metastatic to normal tissue ratios were 15.0 and 17.6 for active and proMMP-2 respectively, and those for active and proMMP-9 were 6.2 and 2.9. The ratios of active to latent enzyme were higher in metastatic tissue than in normal tissue. Lowered MMP-2 activity was associated with large metastatic lesions and increased proMMP-9 levels with preoperative chemotherapy. Both MMP-2 and MMP-9 activity were linked unfavourably to early recurrent disease. CONCLUSION: These data suggest a role for MMPs in colorectal cancer liver metastasis, but indicate different roles for individual MMPs.

Tissue levels of active matrix metalloproteinase-2 and -9 in colorectal cancer.

The bioactivity of matrix metalloproteinases was studied in tissues from colorectal cancer patients by means of both quantitative gelatin zymography and a fluorometric activity assay. Next to paired samples of tumour tissue and distant normal mucosa (n=73), transitional tissue was analysed from a limited (n=33) number of patients. Broad-spectrum matrix metalloproteinase activity and both the active and latent forms of the gelatinases matrix metalloproteinase-2 and -9 were higher in tumour than in normal mucosa. The ratio's between active and latent forms of matrix metalloproteinase-2 and -9 were highest in tumour tissue and normal mucosa, respectively. Matrix metalloproteinase-2 levels, both active and latent forms, correlated inversely with stage of disease, the tumours without synchronous distant metastases containing significantly (P=0.005) more active matrix metalloproteinase-2 than the others. At much lower levels of activity, the same trend was observed in distant normal mucosa. The level of latent form of matrix metalloproteinase-9 in tumour depended on tumour location. Neither the active form of matrix metalloproteinase-9 nor broad-spectrum matrix metalloproteinase activity in tumour tissue did correlate with any of the clinicopathological parameters investigated. The results demonstrate explicit differences between the activity of matrix metalloproteinase-2 and -9, indicating different roles for both gelatinases in tumour progression. Such data are necessary in order to develop rational anti-cancer therapies based on inhibition of specific matrix metalloproteinases.

Is mutated serine hydroxymethyltransferase (SHMT) involved in the etiology of neural tube defects?

Neural tube defects (NTD) arise in the first weeks of pregnancy due to a combination of environmental and genetic factors. In mothers of children with NTD elevated homocysteine (Hcy) levels and decreased plasma folate levels were observed, which suggests a defect in the folate-dependent Hcy metabolism. Therefore, mutations in genes coding for enzymes of this metabolism could be involved in NTD. Serine hydroxymethyltransferase (SHMT) catalyzes the reversible reaction of serine and tetrahydrofolate (THF) to glycine and 5,10-methylene THF. Two different isoforms of SHMT are known, one is present in the cytosol (cSHMT) and the other in the mitochondrion (mSHMT). Theoretically, mutated SHMT could lead to elevated Hcy levels and to an altered distribution of the different folate derivatives and might therefore become a risk factor for NTD. This study concerns the molecular genetic analysis of genes coding for both isoforms of the SHMT enzyme by single-stranded conformation polymorphism analysis. Several mutations as well as polymorphisms were found in both genes. The relevance of two variations, the 1420 C>T mutation of the cytosolic isoform and the 4-bp deletion of the mitochondrial isoform (delTCTT 1721-1724), to NTD risk was tested in a study group, which consisted of 109 NTD patients, 120 mothers of children with NTD, and 420 controls. Neither of the two polymorphisms led to an increased risk of NTD. In mothers with the 1420 CC genotype, significant increased Hcy levels are present. Also, significantly decreased red blood cell folate and plasma folate levels were present in individuals with the 1420 CC genotype. Probably, the 1420 C>T polymorphism causes a shift in distribution of the different folate derivatives. The 4-bp deletion of the mSHMT gene did not lead to altered Hcy or folate levels. So far, the results of this study provide no direct evidence for a role of defective SHMT functioning in NTD. Still, the influence of the 1420 C>T polymorphism of the cSHMT gene on the folate-related risk of NTD needs further investigation.

Coexpression of integrin alpha(v)beta3 and matrix metalloproteinase-2 (MMP-2) coincides with MMP-2 activation: correlation with melanoma progression.

Tumor cell invasion and metastasis formation depend on both adhesive and proteolytic mechanisms. Previous studies have shown that expression of matrix metalloproteinase-2 and integrin alphavbeta3 correlate with melanoma progression. Recently, direct binding of matrix metalloproteinase-2 to alpha(v)beta3 was implicated in presenting activated matrix metalloproteinase-2 on the cell surface of invasive cells. In this study we investigated this, using the highly metastatic, alpha(v)beta3-negative melanoma cell lines MV3 and BLM, their beta3-transfected alpha(v)beta3 expressing counterparts, xenografts derived from these cell lines, and fresh human cutaneous melanoma lesions comprising all stages of melanoma progression. Expression and activation status of matrix metalloproteinase-2 were studied by reverse transcription-polymerase chain reaction, immunohistochemistry, western blotting, and zymographic analysis, respectively. Matrix metalloproteinase-2 protein expression in vitro was similar in both alpha(v)beta3-negative and alpha(v)beta3-positive cell lines Remarkable differences, however, exist in the localization of inactive and active matrix metalloproteinase-2. Soluble active matrix metalloproteinase-2 was detectable only in the conditioned medium of alpha(v)beta3-negative cell lines and undetectable in the alpha(v)beta3-positive cell lines. Conversely, active matrix metalloproteinase-2 was present exclusively on the cell surface of the alpha(v)beta3 expressing transfectants. Western blot analysis of other components that are involved in matrix metalloproteinase-2 activation showed that processing of proMT1-matrix metalloproteinase to the activated form was enhanced in beta3 transfectants, whereas secretion of tissue inhibitor of metalloproteinase-2 was decreased. In vivo, the presence of functionally active matrix metalloproteinase-2 was significantly higher in xenografts derived from the alpha(v)beta3 expressing MV3 and BLM cell lines. In human cutaneous melanoma lesions, neither matrix metalloproteinase-2 nor integrin alpha(v)beta3 is detectable in melanoma in situ as determined by immunohistochemistry. In contrast, the number of matrix metalloproteinase-2-positive and alphavbeta3-positive tumor cells was clearly increased in primary melanomas, and melanoma metastases. Double staining experiments and confocal laser microscopy demonstrated that the percentage of cells coexpressing matrix metalloproteinase-2 and alpha(v)beta3 increased in advanced primary melanomas and melanoma metastases. In addition, zymography showed that functionally active matrix metalloproteinase-2 was frequently present in melanoma metastases. In these lesions a high proportion of matrix metalloproteinase-2- and alphavbeta3-double-positive melanoma cells were detectable. Our study demonstrates that the presence of activated matrix metalloproteinase-2 correlates with expression of alpha(v)beta3 in human melanoma cells both in vitro and in vivo, and also in fresh human melanoma lesions. These findings strongly suggest that co-ordinated expression of both factors may be required for melanoma cell invasion and metastasis formation.

Matrix metalloproteinases in human melanoma cell lines and xenografts: increased expression of activated matrix metalloproteinase-2 (MMP-2) correlates with melanoma progression.

Matrix metalloproteinases (MMPs) and their tissue inhibitors (TIMPs) are involved in tumour progression and metastasis. In this study, we investigated the in vitro and in vivo expression patterns of MMP-1, MMP-2, MMP-3, MMP-9, TIMP-1 and TIMP-2 mRNA and protein in a previously described human melanoma xenograft model. This model consists of eight human melanoma cell lines with different metastatic behaviour after subcutaneous (s.c.) injection into nude mice. MMP-1 mRNA was detectable in all cell lines by reverse transcription polymerase chain reaction (RT-PCR), but the expression was too low to be detected by Northern blot analysis. No MMP-1 protein could be found using Western blotting. MMP-2 mRNA and protein were present in all cell lines, with the highest expression of both latent and active MMP-2 in the highest metastatic cell lines MV3 and BLM. MMP-3 mRNA was expressed in MV3 and BLM, and in the non-metastatic cell line 530, whereas MMP-3 protein was detectable only in MV3 and BLM. None of the melanoma cell lines expressed MMP-9. TIMP-1 and TIMP-2 mRNA and protein, finally, were present in all cell lines. A correlation between TIMP expression level and metastatic capacity of cell lines, however, was lacking. MMP and TIMP mRNA and protein expression levels were also studied in s.c. xenograft lesions derived from a selection of these cell lines. RT-PCR analysis revealed that MMP-1 mRNA was present in MV3 and BLM xenografts, and to a lesser extent in 530. Positive staining for MMP-1 protein was found in xenograft lesions derived from both low and high metastatic cell lines, indicating an in vivo up-regulation of MMP-1. MMP-2 mRNA was detectable only in xenografts derived from the highly metastatic cell lines 1F6m, MV3 and BLM. In agreement with the in vitro results, the highest levels of both latent and activated MMP-2 protein were observed in MV3 and BLM xenografts. With the exception of MMP-9 mRNA expression in 530 xenografts, MMP-3, MMP-9, and TIMP-1 mRNA and protein were not detectable in any xenograft, indicating a down-regulated expression of MMP-3 and TIMP-1 in vivo. TIMP-2 mRNA and protein were present in all xenografts; interestingly, the strongest immunoreactivity of tumour cells was found at the border of necrotic areas. Our study demonstrates that of all tested components of the matrix metalloproteinase system, only expression of activated MMP-2 correlates with increased malignancy in our melanoma xenograft model, corroborating an important role of MMP-2 in human melanoma invasion and metastasis.