Relationship between Trace Elements and Matrix Metalloproteinases 2 and 9 and their Tissue Inhibitors in Medullary Thyroid Carcinoma.

Medullary Thyroid Carcinoma (MTC) constitutes around 5% of all thyroid cancers. Trace elements assessment has emerged as a useful strategy in the diagnostics of MTC combined with Matrix Metalloproteinases (MMPs) and Tissue Inhibitors of Matrix Metalloproteinases (TIMPs) analysis. The aim of this study was to compare the presence and content of trace elements (i.e., Copper (Cu), Zinc (Zn), Iron (Fe), and Manganese (Mn)) in MTC with respect to control samples and their potential relationship with markers of MTC in tissues. The study included 26 patients who had undergone thyroidectomy, due to the diagnosis of MTC and 17 patients as control. We combined tumour pathology and staging, immunohistochemical analysis of calcitonin, MMPs, and TIMPs, with analytical biochemistry using Inductively Coupled Plasma - Mass Spectrometry (ICP-MS) to determine the levels of trace elements. No differences by MTC type for MMPs and their TIPMs, although strong TIMP-1 and TIMP-2 immunohistochemical expression of MTC were unveiled. Additionally, Zn, Fe, and Mn tended to be decreased, and Cu to be increased in samples presenting MTC with respect to controls. Moreover, Zn was the unique trace element which seemed to be correlated with MMPs and TIMPs. Trace elements such as Zn, Fe, and Mn are decreased in tissues affected by MTC. In addition, Zn may be the trace element which saves more relationship with the proportion and intensity of MMPs, being considered altogether useful biomarkers of MTC. We therefore suggest the analysis of novel and traditional markers of MTC as a novel approach in this pathology.

Matrix Metalloproteinases 2 and 9 and Their Tissue Inhibitors in the Diagnostics of Medullary Thyroid Carcinoma.

Medullary Thyroid Carcinoma (MTC) is a tumor of the neuroendocrine system. In recent years, the need to assess the MTC diagnostic-related parameters has emerged with the aim to elucidate the mechanisms involved in this pathology. The objective of this study was to evaluate the role of Matrix Metalloproteinases (MMPs) 2 and 9, their tissue inhibitors of matrix metalloproteinases (TIMPs), S100 protein, and amyloid in the diagnostic of MTC. Thirty-two samples with MTC (72% women) were included in this cross-sectional study and divided by groups: T category 1 (T1)≤20 mm and T category 2 (T2) 20 to 40 mm of tumor size. MMPs 2 and 9, TIMPs 2 and 1, S100 protein, and calcitonin in tissues were obtained by immunohistochemical techniques. The presence of amyloid in tissue sections was detected on Thioflavin T-stained slides under fluorescent microscope. Percentage of positive cells (P) observed for MMP-2 was higher in those samples presenting T2 MTC with respect to those with T1 MTC ( P <0.05). Moreover, P-MMP-2 showed a direct correlation with higher T category of MTC (Rho=0.439, P < 0.001), whereas P-MPP-9 was directly correlated with S100 protein and the intensity of calcitonin in tissues (Rho=0.419, P =0.017; Rho=0.422, P =0.016, respectively. Therefore, MMPs were directly correlated with some traditional biomarkers of MTC. In this regard, P-MMP-2 was more expressed in type 2 MTC. Combining the analysis of traditional and other useful biomarkers of MTC as MMPs 2 and 9 could be a useful strategy in the diagnostic of MTC.

Effects of subchronic methionine stimulation on oxidative status and morphological changes in the rat ileum.

This study was conducted to explore the effects of sulfur containing amino acids on redox status and morphological parameters in the rat ileum tissue. Male Wistar albino rats were randomly divided into the following groups: Group K (saline (1 ml/day, i.p.)), Group M (methionine (0.8 mmol/kg/day, i.p.)), Group C (methionine (0.8 mmol/kg/day) + L-cysteine (7 mg/kg/day), i.p.) and Group N (methionine (0.8 mmol/kg/day) + N-acetyl-L-cysteine (50 mg/kg/day), i.p.). Activities of antioxidant enzymes in the ileum were analyzed to profile oxidative status. Morphometric analysis included measurement of villus height (µm), tunica mucosa thickness (µm), tunica muscularis thickness (µm), the total thickness of the ileal wall (µm) and the number of cells in the lamina propria (per 0.1 mm2 of tissue). Results showed that methionine treatment reduced the activity of antioxidant enzymes (SOD, GPx, CAT) and the GSH content compared to the control group (p > 0.05). The application of methionine reduced the following parameters statistically significant compared to the control group: length of the ileal villi (p < 0.01), tunica mucosa thickness (p < 0.01), and ileal wall thickness (p < 0.01). We concluded that methionine induced the changes in the gut redox status, which implied oxidative stress occurrence. L-cysteine and N-acetyl-L-cysteine both exhibited antioxidant properties.

Betaine modulates oxidative stress, inflammation, apoptosis, autophagy, and Akt/mTOR signaling in methionine-choline deficiency-induced fatty liver disease.

We examined the effects of betaine, an endogenous and dietary methyl donor essential for the methionine-homocysteine cycle, on oxidative stress, inflammation, apoptosis, and autophagy in methionine-choline deficient diet (MCD)-induced non-alcoholic fatty liver disease (NAFLD). Male C57BL/6 mice received standard chow (control), standard chow and betaine (1.5% w/v in drinking water), MCD, or MCD and betaine. After six weeks, serum and liver samples were collected for analysis. Betaine reduced MCD-induced increase in liver transaminases and inflammatory infiltration, as well as hepatosteatosis and serum levels of low-density lipoprotein, while it increased that of high-density lipoprotein. MCD-induced hepatic production of reactive oxygen and nitrogen species was significantly reduced by betaine, which also improved liver antioxidative defense by increasing glutathione content and superoxide-dismutase, catalase, glutathione peroxidase, and paraoxonase activity. Betaine reduced the liver expression of proinflammatory cytokines tumor necrosis factor and interleukin-6, as well as that of proapoptotic mediator Bax, while increasing the levels of anti-inflammatory cytokine interleukin-10 and antiapoptotic Bcl-2 in MCD-fed mice. In addition, betaine increased the expression of autophagy activators beclin 1, autophagy-related (Atg)4 and Atg5, as well as the presence of autophagic vesicles and degradation of autophagic target sequestosome 1/p62 in the liver of NAFLD mice. The observed effects of betaine coincided with the increase in the hepatic phosphorylation of mammalian target of rapamycin (mTOR) and its activator Akt. In conclusion, the beneficial effect of betaine in MCD-induced NAFLD is associated with the reduction of liver oxidative stress, inflammation, and apoptosis, and the increase in cytoprotective Akt/mTOR signaling and autophagy.

Effects of Vitamin D3 on the NADPH Oxidase and Matrix Metalloproteinase 9 in an Animal Model of Global Cerebral Ischemia.

Decreased blood flow in the brain leads to a rapid increase in reactive oxygen species (ROS). NADPH oxidase (NOX) is an enzyme family that has the physiological function to produce ROS. NOX2 and NOX4 overexpression is associated with aggravated ischemic injury, while NOX2/4-deficient mice had reduced stroke size. Dysregulation of matrix metalloproteinases (MMPs) contributes to tissue damage. The active form of vitamin D3 expresses neuroprotective, immunomodulatory, and anti-inflammatory effects in the CNS. The present study examines the effects of the vitamin D3 pretreatment on the oxidative stress parameters and the expression of NOX subunits, MMP9, microglial marker Iba1, and vitamin D receptor (VDR), in the cortex and hippocampus of Mongolian gerbils subjected to ten minutes of global cerebral ischemia, followed by 24 hours of reperfusion. The ischemia/reperfusion procedure has induced oxidative stress, changes in the expression of NOX2 subunits and MMP9 in the brain, and increased MMP9 activity in the serum of experimental animals. Pretreatment with vitamin D3 was especially effective on NOX2 subunits, MMP9, and the level of malondialdehyde and superoxide anion. These results outline the significance of the NOX and MMP9 investigation in brain ischemia and the importance of adequate vitamin D supplementation in ameliorating the injury caused by I/R.

Suppression of methionine-induced colon injury of young rats by cysteine and N-acetyl-L-cysteine.

Changes in the methionine metabolism can cause a state called hyperhomocysteinemia, inducing oxidative stress in the gut. The production of free radicals is important in the colon damage caused by methionine. This study aimed at evaluating the effect of the use of L-cysteine and N-acetyl-L-cysteine on the colon morphometry of young rats treated with methionine. A total number of 32 male rats were distributed in a randomized experimental design in 4 groups: control group treated with saline; methionine group; cysteine + methionine group, and N-acetyl-L-cysteine + methionine group. After 21 days of treatment, rats were sacrificed and the colon samples were taken for histological and biochemical analysis. Methionine load increased depth of crypts, the lamina muscularis mucosae thickness, the mucosal height, and the number of cells in lamina propria (p < 0.01). Combination of methionine with L-cysteine (C group) and with N-acetyl-L-cysteine (N group) reversed methionine effects. Methionine treatment increased the GPx activity and MDA concentration, while L-cysteine and N-acetyl-L-cysteine increased the catalase activity compared to methionine group. It was concluded that the use of L-cysteine and N-acetyl-L-cysteine was beneficial to decrease intestinal mucosal height and oxidative damage when methionine was used in combination with them.

Biochemical Markers of Renal Function.

Kidney damage can be induced by ischemia, autoimmune diseases, hypertension, allograft rejection, metabolic or genetic disorders, infections or toxins. The influence of these factors could result in acute kidney injury (AKI) defined as an unexpected decrease in urine output or renal function, or encourage the development of chronic kidney disease (CKD). Biomarkers of renal function, measured in urine and serum, are in increasing use in order to estimate the severity and nature of kidney injury, and consequently apply appropriate therapy and improve patient management. The determined values of biomarkers can suggest the potential risk of kidney disease and the type of renal injury, predict the disease progression, as well as be helpful for assessing the response to an applied therapy. Although novel biomarkers are in practical use, serum creatinine, the indicator of glomerular filtration rate is still the most frequently used biomarker of renal function despite its known limitations. In recent decades, numerous studies resulted in discovering urinary and serum proteins that can serve as biomarkers for early and accurate detection of AKI and its development, as well as the identification of CKD. This review gives an overview of the most important renal biomarkers investigated in kidney diseases, classified in following types: functional biomarkers, up-regulated proteins, enzymes, and cycle arrest biomarkers. It describes their properties, physiological roles, and discusses the utility of these molecules in different clinical settings.

Rimonabant Improves Oxidative/Nitrosative Stress in Mice with Nonalcoholic Fatty Liver Disease.

The present study deals with the effects of rimonabant on oxidative/nitrosative stress in high diet- (HFD-) induced experimental nonalcoholic fatty liver disease (NAFLD). Male mice C57BL/6 were divided into the following groups: control group fed with control diet for 20 weeks (C; n = 6); group fed with HFD for 20 weeks (HF; n = 6); group fed with standard diet and treated with rimonabant after 18 weeks (R; n = 9); group fed with HFD and treated with rimonabant after 18 weeks (HFR; n = 10). Daily dose of rimonabant (10 mg/kg) was administered to HFR and R group by oral gavage for two weeks. Treatment induced a decrease in hepatic malondialdehyde concentration in HFR group compared to HF group (P < 0.01). The concentration of nitrites + nitrates in liver was decreased in HFR group compared to HF group (P < 0.01). Liver content of reduced glutathione was higher in HFR group compared to HF group (P < 0.01). Total liver superoxide dismutase activity in HFR group was decreased in comparison with HF group (P < 0.01). It was found that rimonabant may influence hepatic iron, zinc, copper, and manganese status. Our study indicates potential usefulness of cannabinoid receptor type 1 blockade in the treatment of HFD-induced NAFLD.

Copper as ancillary diagnostic tool in preoperative evaluation of possible papillary thyroid carcinoma in patients with benign thyroid disease.

Preoperative diagnosis of papillary thyroid carcinoma (PTC) comprises numerous diagnostic procedures which are mostly applicable in tertiary institutions. Normal thyroid function depends on the presence of many trace elements and copper (Cu) and zinc (Zn) are some of those. The study is based on retrospective review of 118 patients with preoperatively diagnosed benign thyroid disease (BTD) and 12 with PTC, who underwent thyroid surgery at the Center for Endocrine Surgery Clinical Center of Serbia, Belgrade, between 2010 and 2012. The objective was to evaluate concentrations of Cu and Zn in serum as possible prediction markers for PTC in patients who underwent surgery for preoperatively diagnosed BTD. Concentrations of Cu and Zn ions in serum were measured using atomic absorption spectrophotometer. Data were analyzed using methods of descriptive statistics, Anova and t-test (p < 0.05 was considered statistically significant). Definitive pathohistological findings revealed PTC in 23 (19.5%) and papillary microcarcinoma-mPTC in 13 (11.0%) of BTD patients. The concentrations of Cu ions in serum of PTC patients as well as in serum of patients with mPTC were significantly higher than in serum of BTD patients (p < 0.05). The concentrations of Zn ions and Cu/Zn ratio in serum of PTC and mPTC patients were not significantly higher than in serum of BTD patients. The concentration of Cu ions in serum of patients before thyroid surgery can be useful, easy available, and a low-cost tool in prediction of preoperatively undiagnosed PTC in patients with BTD.

Activity of superoxide dismutase, catalase, glutathione peroxidase, and glutathione reductase in different stages of colorectal carcinoma.

BACKGROUND: Reactive oxygen species are involved in the pathogenesis of colorectal carcinoma. Clarification of oxidative/antioxidant specificities of different stages of colorectal carcinoma is of special importance. AIM: To determine oxidative/antioxidant status in plasma of patients with different stages of colorectal carcinoma using malondialdehyde concentration, superoxide dismutase, catalase, glutathione peroxidase, and glutathione reductase activities and distribution of superoxide dismutase isoforms. METHODS: Lipid peroxidation and antioxidant enzymes activity were estimated using spectrophotometric methods. Reverse zymography was applied for characterization of superoxide dismutase isoforms. RESULTS: Lipid peroxidation is increased in all groups compared to the control, but without differences between different stages of colorectal carcinoma. Total superoxide dismutase activity is lower in all colorectal carcinoma groups than in control, and there is a significant increase in tumor stage IV when compared with tumor stage II. Manganese superoxide dismutase isoform is dominant in all groups and its relative activities are significantly higher than activities of a copper/zinc isoform. Total peroxidase potential reflected in catalase and glutathione peroxidase activity is increased when compared to the control, but without any significant differences between colorectal carcinoma groups. Glutathione reductase activity is lower in all colorectal carcinoma groups than in control, and a significant decrease in glutathione reductase activity was obtained between patients in tumor stage II and III compared to tumor stage IV. CONCLUSIONS: Colorectal carcinoma is characterized by increased oxidative stress and antioxidant disbalance. Progression of disease is followed by an increase in redox disbalance.

Matrix metalloproteinase-9 and the Cu/Zn ratio as ancillary diagnostic tools in distinguishing between the classical and follicular variants of papillary thyroid carcinoma.

The most common histological variants of papillary thyroid carcinoma (PTC), classical (CPTC) and follicular (FPTC), have different diagnostic features, molecular biology, and prognosis. Matrix metalloproteinase-9 (MMP-9) endopeptidase which degrades the components of the extracellular matrix is essential in the invasive growth and metastasizing of malignant tumors. The serum copper (Cu)/zinc (Zn) ratios are sensitive diagnostic and prognostic indicators in oncology since Cu- and Zn-dependent enzymes play important roles in the genesis and the progression of tumors. The aim of this study was to examine the expressions of MMP-9 in tissues of CPTC and FPTC, as well as to determine the Cu/Zn ratios in the same samples. MMP-9 was determined immunohistochemically, and the concentrations of copper and zinc in thyroid tissue were determined by means of flame atomic absorption spectrometry. The results obtained revealed significantly higher expressions of MMP-9 in CPTC in comparison with FPTC, as well as higher Cu/Zn ratios in CPTC than in FPTC. Thus, determining MMP-9 activities and the Cu/Zn ratios could improve the accuracy of the standard histopathological diagnosis of these two types of PTC.

Matrix metalloproteinase-2 (MMP-2) and -9 (MMP-9) in preoperative serum as independent prognostic markers in patients with colorectal cancer.

Colorectal cancer is one of the leading causes of cancer related death in developed countries. One of the reasons is the absence of tumor specific diagnostic and prognostic markers. The aim of this study was to examine the correlation of matrix metalloproteinase-2 (MMP-2) and -9 (MMP-9) expressions in serum and clinicopathological features of the colorectal adenocarcinoma. Another aim was to examine expression of MMP-9 in the tissue of the colorectal carcinoma in MMP-9 serum positive patients. In addition, we tried to establish the correlation between preoperative levels of serum markers (CEA and CA 19-9) and presence of MMP-2 or MMP-9. The study was performed on 32 patients with colorectal adenocarcinoma who underwent surgery and 11 patients in a control group who were operated for benign diseases. The samples were analyzed by SDS-PAGE to determine the molecular mass and SDS-PAGE zymography to determine levels of MMP-2 and MMP-9. Expression of MMP-9 was determined immunohistochemically in the tissue of the colorectal carcinoma of MMP-9 serum positive patients. MMP-2 and MMP-9 levels were increased in the serum of the patients with colorectal cancer compared to the control group. There was significant correlation in MMPs levels among the patients with tumor stage I and II and the patients with tumor stage III and IV. Obtained results did not demonstrate correlation between levels of CEA, CA 19-9 and presence of MMP-2 or MMP-9. MMP-9 expression was positive in 85% of MMP-9 serum positive patients with colorectal carcinoma. The overexpression of MMP-2 and MMP-9 strongly suggests its association with colorectal adenocarcinoma. Detection of MMP-2 and MMP-9 in serum might be useful for identification of patients with higher risk for colorectal cancer recurrence.

Expression of matrix metalloproteinase-2 and its tissue inhibitor-2 in fetal and neoplastic thyroid tissue and their significance as diagnostic and prognostic markers in papillary carcinoma.

Matrix metalloproteinases (MMPs) and their tissue inhibitors (TIMPs) have roles in physiological and pathological processes. We evaluated immunohistochemical expression of MMP-2 and TIMP-2 in paraffin sections of 12 human fetal thyroids at mid-term gestation and 79 thyroid tumors of follicular origin. Besides evaluating expression of these proteins during fetal development and neoplastic transformation, we determined whether expression of MMP-2 and TIMP-2 may help to differentiate papillary thyroid carcinoma (PTC) from follicular thyroid adenoma (FTA) and/or peritumoral tissue (PT). We also investigated their relationship with prognostically important clinicopathological parameters of PTC. Immunoreactive MMP-2 and TIMP-2 were found in all fetal thyroid tissues examined. Tumor tissues contained variable amounts of MMP-2 and TIMP-2, with overexpression of these proteins in PTC compared to FTA and PT tissue. According to the statistical analysis, MMP-2 distinguished follicular variant of PTC from FTA and overall PTC from total nonmalignant lesions. In PTC, high MMP-2 expression correlated with lymph node metastasis (P=0.022), while high TIMP-2 expression was positively correlated with tumor size (P=0.049) and extrathyroid invasion (P=0.017). Overall, these results indicate a role for MMP-2 and TIMP-2 in both thyroid development and malignant transformation and suggest that positive immunohistochemistry for MMP-2 and TIMP-2 might support diagnosis of PTC and predict unfavorable biological behavior.

Detection of gelatinase B activity in serum of gastric cancer patients.

AIM: To determine the proteolytic activity and expression of gelatinase B in serum of gastric cancer patients and their correlation with the stage of the tumor. METHODS: Sera from 23 patients who underwent surgery for primary gastric cancer as the experimental group and from 11 as the control group were used to determine the proteolytic activity and its inhibition by EDTA and 1,10-phenanthroline. Gelatinase B activity was detected by SDS polyacrylamide gel electrophoresis (SDS-PAGE) and SDS-PAGE zymography. RESULTS: Proteolytic enzyme activity was increased in gastric cancer patients when compared to the control group (P < 0.05). The proteinases were determined to be metalloproteinases upon inhibition test with specific metalloproteinase inhibitors 1,10-phenanthroline (P < 0.05) and EDTA (P < 0.01). SDS-PAGE and SDS-PAGE zymography revealed gelatinase B (proMMP-9) activity and its molecular mass of 92 ku. CONCLUSION: Proteinase activity is overexpressed in serum of gastric cancer patients. Gelatinase B in serum plays an important role in the progression of gastric cancer. ProMMP-9 can be used as a marker for invasiveness of gastric cancer.