MMP-14 Exhibits Greater Expression, Content and Activity Compared to MMP-15 in Human Renal Carcinoma.

Membrane-type metalloproteinases (including MMP-14 and MMP-15) are enzymes involved in the degradation of extracellular matrix components. In cancer, they are involved in processes such as cellular invasion, angiogenesis and metastasis. Therefore, the aim of this study was to evaluate the expression, content and activity of MMP-14 and MMP-15 in human renal cell carcinoma. Samples of healthy kidney tissue (n = 20) and tissue from clear-cell kidney cancer (n = 20) were examined. The presence and contents of the MMPs were assessed using Western blot and ELISA techniques, respectively. Their activity-both actual and specific-was evaluated using fluorimetric analysis. Both control and cancer human kidney tissues contain MMP-14 and MMP-15 enzymes in the form of high-molecular-weight complexes. Moreover, these enzymes occur in both active and latent forms. Their content in cancer tissues is very similar, but with a noteworthy decrease in content with an increase in the kidney cancer grade for both membrane-type metalloproteinases. Even more notable is the highest content of the investigated enzymes represented by MMP-14 in the control tissues. Considering the actual and specific activity outcomes, MMP-14 dominates over MMP-15 in all of the investigated tissues. Nevertheless, we also noted a significant enhancement of the activity of both metalloproteinases with an increase in the grade of renal cancer. The expression and activity of both enzymes were detected in all examined renal cancer tissues. However, our findings suggest that transmembrane metalloproteinase 14 (MMP-14) plays a much more significant and essential role than MMP-15 in the studied renal carcinoma tissues. Therefore, it seems that MMP-14 could be a promising target in the diagnosis, prognosis and therapy of renal cell carcinoma.

A New Approach to the Quantification of Fibroblast Growth Factor 23-An Array Surface Plasmon Resonance Imaging Biosensor.

A new biosensor based on the "surface plasmon resonance imaging (SPRi)" detection technique for the quantification of "fibroblast growth factor 23 (FGF23)" has been developed. FGF23 is mainly produced in bone tissues as a phosphaturic hormone that forms a trimeric complex with "fibroblast growth factor receptor 1 (FGFR1)" and αKlotho upon secretion. FGF23 stimulates phosphate excretion and inhibits the formation of active vitamin D in the kidneys. FGF23 has been shown to play a role in bone carcinogenesis and metastasis. The newly developed method, based on the array SPRi biosensor, was validated-the precision, accuracy, and selectivity were acceptable, and yielded less than ±10% recovery. The rectilinear response of the biosensor ranges from 1 to 75 pg/mL. The limit of detection was 0.033 pg/mL, and the limit of quantification was 0.107 pg/mL. The biosensor was used to determine FGF23 concentrations in the blood plasma of healthy subjects and patients with "clear cell" renal cell carcinoma (ccRCC). The obtained results were compared with those measured through an "enzyme-linked immunosorbent assay (ELISA)". The determined Pearson correlation coefficients were 0.994 and 0.989, demonstrating that the newly developed biosensor can be used as a competitive method for the ELISA.

A New Analytical Method for Determination of Cathepsin L Based on the Surface Plasmon Resonance Imaging Biosensor.

The purpose of this study was to develop a new method for a determination of the cathepsin L-biosensor based on the Surface Plasmon Resonance Imaging technique. The cathepsin L is an endopeptidase, which degrades proteins and plays an important role in various processes occurring in the human body. The detection technique, Surface Plasmon Resonance Imaging, is an optical, label-free technique, which can be used for quantitative determination of the different proteins. In order to bind the enzyme, the cathepsin L inhibitor-RKLLW-NH2 was used. The validation process showed that parameters: precision, accuracy, and selectivity of the method were acceptable. The analytically useful range of the standard curve was 0.50 ng/mL-15.00 ng/mL. The detection and quantification limit of method was 1.67 pg/mL and 5.07 pg/mL, respectively. The usefulness of the developed method was confirmed by the determination of the cathepsin L concentration in the blood plasma of some healthy persons and in the blood plasma of patients. The obtained results were compared with the results obtained by the ELISA. It was found that the correlation between these two methods was very strong, what suggest that the developed method can be used as the competitive method to the ELISA.

Utility of cystatin C as a potential bladder tumour biomarker confirmed by surface plasmon resonance technique.

BACKGROUND & OBJECTIVES: The determination of cystatin C (cysC) may be helpful in diagnosis and monitoring of cancer because the pathogenesis of cancer is linked with an increased activity of cysteine peptidases (cathepsins) and a decrease of cysC concentration. This study was aimed to examine the utility of cysC as a marker of bladder cancer (BCa) to be used in the diagnosis. METHODS: This study was conducted with 90 patients with BCa and 27 healthy people. Patients with other cancers, inflammation process and impaired renal function were excluded from the study. The concentrations of cysC in the plasma and urine were measured by surface plasmon resonance imaging technique. RESULTS: The concentration of cysC in the serum taken from the patients with BCa [0.35±0.02 µg/ml (range: 0.20-0.78 µg/ml)] was significantly (P<0.001) lower than the serum cysC concentration of the healthy people [0.68±0.05 µg/ml (range: 0.52-0.89 µg/ml)]. The urinary cysC concentration of the BCa patients [0.19±0.01 µg/ml (range: 0.09-0.34 µg/ml)] was not significantly different from the urinary cysC concentration of the healthy people [0.24±0.02 µg/ml (range: 0.16-0.33 µg/ml)]. Receiver operating characteristic (ROC) curve showed that BCa patients with cysC concentration <0.54 µg/ml [sensitivity: 87%; specificity: 92%; area under the curve (AUC) of ROC: 0.927; P=0.02] could be optimally separated from healthy people. The ROC curve further showed that superficial low-grade patients with cysC concentration lower than 0.36 µg/ml (sensitivity: 0.63%; specificity: 0.58%; AUC of ROC: 0.635; P=0.08) could not be optimally separated from high-risk tumour patients. INTERPRETATION & CONCLUSIONS: BCa patients have lower serum cysC concentration than the control group. Serum cysC may be considered as a potential marker of BCa but not its aggressiveness.

Higher Content but No Specific Activity in Gelatinase B (MMP-9) Compared with Gelatinase A (MMP-2) in Human Renal Carcinoma.

Gelatinases belong to a group of enzymes known as matrix metalloproteinases (MMPs). Gelatinases A and B (MMP-2 and MMP-9, respectively) are the enzymes with the highest ability to destroy collagen, primarily type IV collagen, which is an essential component of the base membrane. Hence, it can be assumed that they are involved, among other things, with the metastasis process of cancer. As a result, the objective of this study was to assess the presence, activity, and expression of selected gelatinases in human renal cancer. Healthy (n = 20) and clear-cell kidney cancer tissue samples (G2 n = 10, G3 n = 10) were analyzed. The presence and content of MMPs were measured using the Western blot and ELISA methods, respectively. The activity (actual and specific) was analyzed with a fluorimetric method. The presence of both investigated enzymes was demonstrated in the representative zymogram. MMP-9 showed the most intensive saturation. It has been observed that both gelatinases occur primarily in high molecular complexes in the human kidney, regardless of whether it is a control or tumor tissue. Both gelatinases were present in comparable amounts in healthy tissues of the kidney. MMP-9 showed a higher content than MMP-2 in both renal cancer grades, but we observed the enhanced activity of both gelatinases with an increase in the grade of renal cancer. A higher MMP-9 content and, on the other hand, lower specific activity in the cancer tissue suggest that MMP-9 is predominantly present in an inactive form in renal cancer. The higher activity of MMP-9 demonstrated using the zymography method may be a cause of different values of activity that depend on the phase of the carcinogenic process. The present study revealed changes in the tested gelatinases in healthy and cancerous tissues of renal cell carcinoma. Therefore, it can be concluded that matrix metalloproteinases 2 and 9 are enzymes directly involved in carcinogenesis, and hence, it seems that MMPs may have potential in the diagnosis and treatment of renal carcinoma.

The Significance of Matrix Metalloproteinase 9 (MMP-9) and Metalloproteinase 2 (MMP-2) in Urinary Bladder Cancer.

INTRODUCTION: Urinary bladder cancer is a serious oncological problem that is the cause of many deaths worldwide. The processes of metastasis and origination of local tumor invasion depend on the extracellular matrix (ECM) degradation. The cancer microenvironment, particularly the ECM, may be considered a key factor in cancer progression. Matrix metalloproteinases (MMPs) are classified as the main factors responsible for the degradation of ECM components. Therefore, the aim of the study was to evaluate the expression and activity of matrix metalloproteinase 2 and 9 (MMP-2 and MMP-9) in urinary bladder cancer according to different stages. MATERIAL AND METHODS: Urinary bladder tissue samples were analyzed. Cancer patients were divided into two groups: low-grade tumors (LG; Group I) and high-grade tumors (HG; Group II). Control tissue was obtained from the opposite site to the tumor. MMPs content and activity (actual and specific) were evaluated using ELISA and Western blot methods, respectively. RESULTS: Both MMPs are present in high and low molecular complexes in healthy or bladder cancer tissues. The content of MMP-9 is enhanced in comparison with MMP-2, particularly in HG cancer tissue. The actual activity of MMP-2 was highest in LG cancer tissue whereas the actual activity of MMP-9 was highest in HG cancer. Specific activity of both MMPs was highest in LG cancer, but the activity of MMP-9 was higher in comparison with MMP-2. CONCLUSIONS: In conclusion, the content and specific activity of MMP-9 were increased in comparison with MMP-2. The revealed differences in content and activity of both MMPs demonstrate their different participation in ECM remodeling at different stages of cancer development. Moreover, it seems that MMP-9 has higher clinical utility than MMP-2 as a potential therapeutic option and a diagnostic biomarker of urinary bladder cancer.

Concentration of UHCL1 in the Serum of Children with Acute Appendicitis, Before and After Surgery, and Its Correlation with CRP and Prealbumin.

Ubiquitin-mediated protein degradation plays a crucial role in various cellular processes, including signal transduction, cell differentiation, and stress response. Ubiquitin C-terminal hydrolase 1 (UCHL1) is a unique deubiquitinating enzyme that has both hydrolase and ligase activities. The aim of this study was the determination of UCHL1 concentration in serum of children with appendicitis, before and after the surgery. MATERIAL AND METHODS: 42 children with acute appendicitis, who were managed at the Pediatric Surgery Department, between 2013 and 2014, were randomly included into the study (age 9 months up to 14 years, mean age 2.5 + 1 years). There were 15 girls and 27 boys. 18 healthy, age-matched subjects, admitted for planned surgeries served as controls. Exclusion criteria were: severe preexisting infections, immunological or cardiovascular diseases that required long-term medication, and complicated cases of appendicitis with perforation of appendix and/or peritonitis. RESULTS: The UCHL1 concentrations in the blood plasma of patients with acute appendicitis, were highest before the surgery, and were above the range of concentrations measured in controls, the difference was statistically significant. The UCHL1 concentration measured 24 and 72 h after the operation, slowly decreased over time, and still did not reach the normal range, when compared with the concentration measured in controls (p < 0.05). CONCLUSIONS: UCHL1 concentration may reflect the metabolic response to acute state inflammation, and the process of gradual ebbing of the inflammation. The method of operation-classic open appendectomy, or laparoscopic appendectomy, does not influence the general trend in UCHL1 concentration in children with appendicitis. There is strong negative correlation between prealbumin and UCHL1 concentrations.

Determination of the concentration of cathepsin B by SPRI biosensor in children with appendicitis, and its correlation with proteasomes.

BACKGROUND: Cathepsin B (CatB) belongs to a family of lysosomal cysteine proteases and plays an important role in intracellular proteolysis. OBJECTIVES: The concentration of CatB and 20S proteasome was evaluated in the serum of children with appendicitis, before and after surgery, on a basis of an innovative method for determining biomolecules concentration - surface plasmon resonance imaging (SPRI) biosensor. MATERIAL AND METHODS: Forty-two children with acute appendicitis, who were treated at the Department of Pediatric Surgery (Medical University of Bialystok, Poland), were randomly included into the study (age: 5-17 years, mean age: 11.5 ±1 year). There were 15 girls and 27 boys in the study group. Eighteen healthy, age-matched subjects, admitted for planned surgeries, served as controls. Exclusion criteria were the following: severe preexisting infections, immunological or cardiovascular diseases that required longterm medication, and complicated cases of appendicitis with perforation of the appendix and/or peritonitis. RESULTS: The CatB concentrations in the blood plasma of patients with acute appendicitis were elevated before surgery, they were the highest 24 h after surgery, and were above the range of concentrations measured in controls; the difference was statistically significant. The CatB concentration measured 72 h after the operation was decreased, but still did not reach the normal range when compared with the concentration measured in controls (p < 0.05). CONCLUSIONS: Cathepsin B concentration may reflect the metabolic response to acute state of inflammation, surgical intervention in the abdominal cavity and the process of gradual ebbing of the inflammation. The method of operation - classic open appendectomy or laparoscopic appendectomy - does not influence the general trend in the CatB concentration in children with appendicitis. There is a strong positive correlation between the CatB and 20S proteasome concentrations 24 h after surgery. The SPRI method can be successfully used for determining the concentration of active forms of enzymes presented in lysosomes in the diagnosis of inflammatory conditions in the abdominal cavity.

Matrix metalloproteinase-2 and its correlation with basal membrane components laminin-5 and collagen type IV in paediatric burn patients measured with Surface Plasmon Resonance Imaging (SPRI) biosensors.

The purpose of this study was the determination of matrix metalloproteinase-2 and its correlation with basal membrane components laminin-5 and collagen type IV in the blood plasma of burn patients measured with Surface Plasmon Resonance Imaging (SPRI) biosensors. MATERIAL AND METHODS: 31 children scalded by hot water who were managed at the Department of Paediatric Surgery between 2014-2015, after primarily presenting with burns in 4-20% TBSA were included into the study (age 9 months up to 14 years, mean age 2,5+1 years). There were 10 girls and 21 boys. Venous blood samples were drawn 2-6h, and 12-16h after the thermal injury, and on the subsequent days 3, 5 and 7. The matrix metalloproteinase-2, collagen type IV and laminin-5 concentrations were assessed using Surface Plasmon Resonance Imaging by the investigators blinded to the other data. RESULTS: The MMP-2, laminin-5 and collagen type IV concentrations in the blood plasma of patients with burns, were highest 12-16h after thermal injury, the difference was statistically significant. The MMP-2, laminin-5 and collagen type IV concentrations measured 3 days, 5 days and 7 days after the thermal injury, slowly decreased over time, and on the 7th day reached the normal range, when compared with the concentration measured in controls. CONCLUSION: Current work is the first follow-up study regarding MMP-2 in burns. MMP-2, laminin-5 and collagen type IV levels were elevated early after burn injury in the plasma of studied patients, and were highest 12-16h after the injury. MMP-2, laminin-5 and collagen type IV levels were not proportional to the severity of the burn. We believe in the possibility that the gradual decrease of MMP-2, collagen type IV and laminin-5 concentrations could be connected with the process of healing, but to prove it, more investigation is needed in this area. The SPR imaging biosensor is a good diagnostic tool for determination of MMP-2, laminin-5 and collagen type IV in blood plasma of patients with burns.

Cystatin C: a kidney function biomarker.

Renal function is essential for homeostasis. The kidneys play important pleiotropic roles including removal of metabolic waste products and maintenance of water-electrolyte balance and blood pressure. Early diagnosis of renal dysfunction and institution of appropriate therapy are vital to survival. Unfortunately, common indicators of renal function lack necessary sensitivity and specificity. Recent evidence has, however, suggested that cystatin C (cysC) may be useful as a marker for glomerular filtration. CysC is a protein belonging to a group of cysteine proteases inhibitors produced primarily by nucleated cells. Due to low molecular weight and positive pI, it is easily filtered. Moreover, its serum concentration is independent of gender, age, or muscle mass, i.e., typical confounders in assessing glomerular filtration rate (GFR). This chapter discusses the structure and biologic function of cysC, its role as an indicator of GFR, and the most frequently used methods for its determination.