Analysis of MMP-2-735C/T (rs2285053) and MMP-9-1562C/T (rs3918242) Polymorphisms in the Risk Assessment of Developing Lung Cancer.

Matrix metalloproteinase (MMP)-2 and -9 are gelatinases which are capable of degrading type IV collagen and have been linked to cancer invasion and metastatic development. MMP-2 and MMP-9 gene polymorphisms may affect their biological function, and thus their role in cancer development and progression. We analyzed the association of the polymorphism frequencies of MMP-2-735C/T and MMP-9-1562C/T with MMP-2 and MMP-9 serum concentrations, as well as their potential effects in lung cancer patients. We conducted a retrospective, case-control study consisting of 112 lung cancer patients and 100 healthy individuals from a Caucasian population in Poland. Polymerase chain reaction with restriction fragment length polymorphism (PCR/RFLP) and electrophoresis was used to genotype genomic DNA from whole blood samples. MMP-2 and MMP-9 serum concentrations were then determined using ELISA. For statistical analysis, Statistica version 13 from TIBCO Software Inc. was utilized with a significance level <0.05. Logistic regression analysis revealed that MMP-2-735CC (OR = 5.39; 95% CI = 0.62-47.17; p = 0.238504) and -735CT genotype (OR = 7.22; 95% CI = 0.78-67.14; p = 0.072836), as well as MMP-9-1562CC (OR = 1.45; 95% CI = 0.31-6.70; p = 0.757914) and -1562CT genotype (OR = 1.60; 95% CI = 0.33-7.83; p = 0.548801) were associated with a higher risk of lung cancer. There were statistically significant differences observed in the MMP-2 concentration between individuals with the -735CC genotype and the -735CT genotype (non-smoking control: 204.04 ng/mL vs. 237.00 ng/mL, respectively, p = 0.041479; adenocarcinoma patients: 157.69 ng/mL vs. 126.37 ng/mL, respectively, p = 0.013222), as well as differences in the MMP-9 concentration between individuals with the -1562CC genotype and the -1562CT genotype (smoking control: 385.67 ng/mL vs. 562.80 ng/mL, respectively, p = 0.000936; patients with other lung neoplasms: 821.64 ng/mL vs. 928.88 ng/mL, respectively p = 0.023315). The role of MMP-2-735C/T and MMP-9 -1562C/T polymorphisms in an increased risk of lung cancer cannot be dismissed. Specific genotypes affect MMP-2 and MMP-9 concentrations in both lung cancer patients and healthy controls, which may thereby increase lung cancer risk, disease aggressiveness, and patient survival outcomes.

Melatonin has a stimulatory effect on osteoblasts by upregulating COL-I and OPN expression/secretion / Melatonina tem efeito estimulador em osteoblastos, pela regulação positiva da expressão/secreção de COL-I e OPN

ABSTRACT Melatonin (MLT) is a potential signaling molecule in the homeostasis of bone metabolism and may be an important mediator of bone formation and stimulation. The aim of this in vitro study was to evaluate the effect of MLT on the viability, mRNA/protein expression and mineralization of pre-osteoblastic cells. The concentrations 5, 2.5, 1, 0.1 and 0.01 mM MLT were tested on pre-osteoblastic cells (MC3T3) compared to control (no MLT), evaluating proliferation and cell viability (C50), gene expression (RT-PCR) and secretion (ELISA) of COL-I and OPN at 24h, 48h and 72h, and the formation of mineral nodules (alizarin red and fast red) after 10 days of treatment. MLT at 5 and 2.5 mM proved to be cytotoxic (C50), so only 0.01, 0.1 and 1 mM were used for the subsequent analyses. OPN mRNA expression increased with MLT at 0.1 mM - 1 mM, which was followed by increased secretion of OPN both at 24h and 72h compared to the remaining groups (p <0.05). COL-I mRNA and COL-1 secretion followed the same pattern as OPN at 0.1 mM MLT at 72h of treatment (p <0.05). Regarding mineralization, all MLT doses (except 1mM) caused an increase (p <0.05) in the formation of mineral nodules compared to the control. Melatonin at 0.01mM - 1mM had a stimulatory effect on osteoblasts by upregulating COL-I and OPN expression/ secretion and mineralization, thereby fostering osteogenesis.

RESUMO A melatonina (MLT) é uma molécula potencial de sinalização na homeostase do metabolismo ósseo e pode ser um importante mediador da formação e estimulação óssea. O objetivo deste estudo in vitro foi avaliar o efeito da MLT na viabilidade, na expressão do mRNA da proteína e mineralização de células préosteoblásticas. As concentrações de MLT 5, 2,5, 1, 0,1 e 0,01 mM foram testadas em células pré-osteoblásticas da linhagem MC3T3 em comparação ao controle (sem MLT), avaliando a proliferação e a viabilidade celular (C50), expressão gênica (rtPCR) e secreção (Elisa) de Colágeno tipo 1 (COL-I) e osteopontina (OPN) às 24, 48 e 72 horas, além da formação de nódulos minerais por meio do teste vermelho de Alizarina fast red após 10 dias de tratamento. MLT a 5 e 2,5 mM provou ser tóxico (C50). Portanto, as concentrações de 0,01, 0,1 e 1 mM foram utilizadas para as análises subsequentes. A expressão do mRNA da OPN aumentou com MLT a 0,1 mM-1mM, seguida pela secreção aumentada de OPN às 24 e 72 horas em comparação aos demais grupos (p<0,05). O mRNA de COL-I e a secreção de COL-I seguiram o mesmo padrão do OPN a 0,1 mM de MLT em 72 horas de tratamento (p<0,05). Em relação à mineralização, todas as doses de MLT (exceto 1mM) causaram aumento (p<0,05) na formação de nódulos minerais em comparação ao controle. A MLT na concentração entre 0,01mM a 1 mM teve um efeito estimulador sobre os osteoblastos, ao regular positivamente a expressão e secreção de COL-I e OPN, além da mineralização, favorecendo a osteogênese.

Immunohistochemical assessment of metalloproteinases MMP2 and MMP9 expression in canine various subtypes of lymphomas in relation with proliferative and apoptotic markers.

Matrix metalloproteinases 2 and 9 (MMP2 and MMP9) are proteolytic enzymes involved with extracellular matrix degradation. They play a role in tumor invasion and metastases. Because of their ability to degrade signaling molecules presented in extracellular matrix, MMPs contribute to tumor proliferation and apoptosis. The aim of this study was to evaluate expression of MMP2 (latent and both active and latent forms) and MMP9 (active, latent, active and latent forms) in different subtypes of canine lymphomas and their relationship with proliferative (mitotic index and percentage of Ki67-positive cells) and apoptotic (apoptotic index) markers. Expression of MMPs was assessed immunohistochemically using an immunoreactive score system. Expression of both MMPs was found in all 20 examined lymphomas belonging to six subtypes. Most cases showed a moderate level of all analyzed forms of MMP2 and MMP9. High expression of MMPs was found in single cases. Except for a positive correlation between the active form of MMP9 and the mitotic index for all lymphoma cases, no other correlations between any remaining forms of MMPs and neither proliferative nor apoptotic markers were found, irrespective of whether the analysis encompassed all cases or the most numerous lymphoma subtypes i.e. centroblastic and Burkitt-like. Our results were not able to clearly confirm the influence of MMPs on the proliferation and apoptotic activity of canine lymphoma cells. However, further studies examining MMPs activity by zymography, expression of their inhibitors and other factors involved in activation of cell proliferation and apoptosis inhibition are needed to clarify the role of MMPs, especially the active form of MMP9, in the behavior of canine lymphoma cells.

Matrix-assisted refolding, purification and activity assessment using a 'form invariant' assay for matrix metalloproteinase 2 (MMP2).

Matrix metalloproteinases expression is used as biomarker for various cancers and associated malignancies. Since these proteinases can cleave many intracellular proteins, overexpression tends to be toxic; hence, a challenge to purify them. To overcome these limitations, we designed a protocol where full length pro-MMP2 enzyme was overexpressed in E. coli as inclusion bodies and purified using 6xHis affinity chromatography under denaturing conditions. In one step, the enzyme was purified and refolded directly on the affinity matrix under redox conditions to obtain a bioactive protein. The pro-MMP2 protein was characterized by mass spectrometry, CD spectroscopy, zymography and activity analysis using a simple in-house developed 'form invariant' assay, which reports the total MMP2 activity independent of its various forms. The methodology yielded higher yields of bioactive protein compared to other strategies reported till date, and we anticipate that using the protocol, other toxic proteins can also be overexpressed and purified from E. coli and subsequently refolded into active form using a one step renaturation protocol.

Safety assessment of ovarian cryopreservation and transplantation in nude mice bearing human epithelial ovarian cancer.

OBJECTIVE: Nude mice with orthotopic transplantation of human ovarian epithelial cancer were used to investigate screening criteria for paraneoplastic normal ovarian tissue and the security of the freezing and thawing for ovarian tissue transplantation. METHODS: Expression of CK-7, CA125, P53, survivin, MMP-2/TIMP- 2 in paraneoplastic normal ovarian tissues were detected by RT-PCR as well as immunohistochemistry. The tissues of the groups with all negative indicators of RT-PCR, all negative indicators of immunohistochemistry, negative expression of CK-7, CA125 and survivin, positive expression of CK-7, CA125 and survivin, cancer tissues and normal ovarian tissues of nude mice were used for freezing and thawing transplantation, to analyze overt and occult carcinogenesis rates after transplantation. RESULTS: When all indicators or the main indicators, CK-7, CA125 and survivin, were negative, tumorigenesis did not occur after transplantation. In addition the occult carcinogenesis rate was lower than in the group with positive expression of CK-7, CA125 and survivin (P<0.01). After subcutaneous and orthotopic transplantation of ovarian tissues, rates did not change (P>0.05). There was no statistical significance among rates after transplantation of ovarian tissues which were obtained under different severity conditions (P>0.05). CONCLUSION: Negative expression of CK-7, CA125 and survivin can be treated as screening criteria for security of ovarian tissues for transplantation. Immunohistochemical methods can be used as the primary detection approach. Both subcutaneous and orthotopic transplantation are safe. The initial severity does not affect the carcinogenesis rate after tissue transplantation. Freezing and thawing ovarian tissue transplantation in nude mice with human epithelial ovarian carcinoma is feasible and safe.

Quantitative assessment of the effects of MMP-2 polymorphisms on lung carcinoma risk.

BACKGROUND: Previous studies assessing associations between matrix metalloproteinase 2 (MMP-2) polymorphisms and lung cancer risk reported conflicting results. A meta-analysis was therefore performed to derive a more precise estimation. METHOD: Case-control studies assessing associations between MMP-2 C735T and C1306T polymorphisms and lung cancer risk were included. Pooled odds ratios (ORs) with 95% confidence intervals (CIs) were estimated. RESULTS: 7 studies with a total of 3,189 lung cancer cases and 3,013 controls were finally included into this meta-analysis. Overall, the MMP-2 C735T polymorphism was associated with lung cancer risk under the homozygote model (CC versus TT: OR =1.44, 95% CI = 1.03-2.02, I2 = 0%), while the MMP- 2 C1306T polymorphism also associated demonstrated links with all four models (all P values less than 0.05). Subgroup analyses by race suggested obvious associations between MMP-2 C735T and C1306T polymorphisms and lung cancer risk in Asians but not in Caucasians. There was no evidence for publication bias. CONCLUSION: Currently available evidence supports teh conclusion that MMP-2 C735T and C1306T polymorphisms influence susceptibility to lung cancer in Asians.

Assessment of control tissue for gene and protein expression studies: a comparison of three alternative lung sources.

The use of an appropriate control group in human research is essential in investigating the level of a pathological disorder. This study aimed to compare three alternative sources of control lung tissue and to determine their suitability for gene and protein expression studies. Gene and protein expression levels of the vascular endothelial growth factor (VEGF) and gelatinase families and their receptors were measured using real-time reverse transcription polymerase chain reaction (RT-PCR) and immunohistochemistry. The gene expression levels of VEGFA, placental growth factor (PGF), and their receptors, fms-related tyrosine kinase 1 (FLT1), and kinase insert domain receptor (KDR) as well as matrix metalloproteinase-2 (MMP-2) and the inhibitors, tissue inhibitor of matrix metalloproteinase-1 (TIMP-1) and TIMP-2 were significantly higher in lung cancer resections. The gene expression level of MMP-9 was significantly lower in the corresponding samples. Altered protein expression was also detected, depending on the area assessed. The results of this study show that none of the three control groups studied are completely suitable for gene and protein studies associated with the VEGF and gelatinase families, highlighting the need for researchers to be selective in which controls they opt for.

Matrix metalloproteinases 2 and 3 gene polymorphisms and the risk of target vessel revascularization after percutaneous coronary intervention: Is there still room for determining genetic variation of MMPs for assessment of an increased risk of restenosis?

OBJECTIVE: Mixed results have been reported of matrix metalloproteinases (MMP) and their association with restenosis after percutaneous coronary intervention (PCI). The current study examines whether multiple single nucleotide polymorphisms (SNPs), covering the full genomic region of MMP2 and MMP3, were associated with restenosis in the GENDER study population. METHODS AND RESULTS: The GENetic DEterminants of Restenosis (GENDER) study enrolled 3104 consecutive patients after successful PCI. The primary endpoint was clinical restenosis, defined as target vessel revascularization (TVR), occurring in 9.8% of the patients. From the Hapmap database, 19 polymorphisms of MMP2 and 11 of MMP3 were selected. Furthermore, in a subpopulation, a genome-wide association analysis (GWA) was performed. No significant association was found with any of the investigated SNPs, including the previously reported 5A/6A polymorphism (rs3025058), with regard to TVR using single SNP analysis or haplotype analysis. CONCLUSION: We found no significant association of MMP2 or MMP3 with TVR with this SNP-broad gene approach. Although we did not test all the known polymorphisms of these genes, using tagging analyses we examined those SNPs covering all known haplotypes of MMP2 and MMP3 to conclude that these genes do not correlate with a genetic risk of coronary restenosis after successful PCI.

Targeted deletion of class A macrophage scavenger receptor increases the risk of cardiac rupture after experimental myocardial infarction.

BACKGROUND: Class A macrophage scavenger receptor (SR-A) is a macrophage-restricted multifunctional molecule that optimizes the inflammatory response by modulation of the activity of inflammatory cytokines. This study was conducted with SR-A-deficient (SR-A(-/-)) mice to evaluate the relationship between SR-A and cardiac remodeling after myocardial infarction. METHODS AND RESULTS: Experimental myocardial infarction (MI) was produced by ligation of the left coronary artery in SR-A(-/-) and wild-type (WT) male mice. The number of mice that died within 4 weeks after MI was significantly greater in SR-A(-/-) mice than in WT mice (P=0.03). Importantly, death caused by cardiac rupture within 1 week after MI was 31% (17 of 54 mice) in SR-A(-/-) mice and 12% (6 of 51 mice) in WT mice (P=0.01). In situ zymography demonstrated augmented gelatinolytic activity in the infarcted myocardium in SR-A(-/-) mice compared with WT mice. Real-time reverse transcription-polymerase chain reaction at day 3 after MI showed that the expression of matrix metalloproteinase-9 mRNA increased significantly in the infarcted myocardium in SR-A(-/-) mice compared with WT mice. Furthermore, SR-A(-/-) mice showed augmented expression of tumor necrosis factor-alpha and reduction of interleukin-10 in the infarcted myocardium at day 3 after MI. In vitro experiments also demonstrated increased tumor necrosis factor-alpha and decreased interleukin-10 expression in activated SR-A(-/-) macrophages. CONCLUSIONS: The present findings suggest that SR-A deficiency might cause impairment of infarct remodeling that results in cardiac rupture via insufficient production of interleukin-10 and enhanced expression of tumor necrosis factor-alpha and of matrix metalloproteinase-9. SR-A might contribute to the prevention of cardiac rupture after MI.

SNP subset selection for genetic association studies.

Association studies for disease susceptibility genes rely on the high density of SNPs within candidate genes. However, the linkage disequilibrium between SNPs imply that not all SNPs identified in the candidate region need be genotyped. Here we develop several approaches to SNP subset selection, which can substantially reduce the number of SNPs to be genotyped in an association study. We apply clustering algorithms to pairwise linkage disequilibrium measures, with SNP subsets determined for different cut-off values of Delta using nearest and furthest neighbour clusters. Alternatively, SNP subsets may be determined by the proportion of haplotypes they identify. We also show how power calculations, based on the average power to identify a SNP as the disease susceptibility mutation using haplotype-based or logistic regression based statistical analyses, can be used to choose SNP subsets. All these methods provide a ranking method for subsets of a specific size, but do not provide criteria for overall choice of SNP subset size. We develop such criteria by incorporating power calculations into a decision analysis, where the choice of SNP subset size depends on the genotyping costs and the perceived benefits of identifying association. These methods are illustrated using eleven SNPs in the MMP2 gene.