Tetracaine downregulates matrix metalloproteinase activity and inhibits invasiveness of strongly metastatic MDA-MB-231 human breast cancer cells.

Tetracaine, a long-acting amino ester-type local anesthetic, prevents the initiation and propagation of action potentials by reversibly blocking voltage-gated sodium channels (VGSCs). These channels, which are highly expressed in several carcinomas (e.g. breast, prostate, colon and lung cancers) have been implicated in promoting metastatic behaviours. Recent evidence suggests that local anesthetics can suppress cancer progression. In this paper, we aimed to explore whether tetracaine would reduce the invasive characteristics of breast cancer cells. In a comparative approach, we used two cell lines of contracting metastatic potential: MDA-MB-231 (strongly metastatic) and MCF-7 (weakly metastatic). Tetracaine (50 µM and 75 µM) did not affect the proliferation of both MDA-MB-231 and MCF-7 cells. Importantly, tetracaine suppressed the migratory, invasive, and adhesive capacities of MDA-MB-231 cells; there was no effect on the motility of MCF-7 cells. Tetracaine treatment also significantly decreased the expression and activity levels of MMP-2 and MMP-9, whilst increasing TIMP-2 expression in MDA-MB-231 cells. On the other hand, VGSC α/Nav1.5 and VGSC-ß1 mRNA and protein expression levels were not affected. We conclude that tetracaine has anti-invasive effects on breast cancer cells and may be exploited clinically, for example, in surgery and/or in combination therapies.

The expression of HDAC9 and P300 in papillary thyroid carcinoma cell line.

PURPOSE: Papillary thyroid carcinoma (PTC) is the most common type of thyroid cancer and accounts for 85-90% of all thyroid cancers. Metastatic differentiated thyroid cancer, radioiodine-refractory thyroid cancer, and anaplastic thyroid cancer still lack effective therapeutic options. Here, we aimed to assess HDAC9 and P300 expression in the papillary thyroid carcinoma cell line and compare them with normal thyroid cells. METHODS: Nthy-ori-3-1, a normal thyroid cell line, and BCPAP, a PTC cell line, were cultured for 24 and 48 h and immunofluorescence staining was used to determine the levels of HDAC9 and P300 protein expression. HDAC9 paracrine release was assessed using an ELISA assay. RESULTS: HDAC9 protein expression was higher in both cell groups at the 48th hour than at the 24th hour; however, P300 protein expression was lower in BCPAP cells at the 48th hour than at the 24th hour. In comparison to Nthy-ori-3-1, BCPAP expressed more HDAC9 and P300 proteins. HDAC9 secretion slightly increased in Nthy-ori-3-1 cells from 24 to 48 h. Furthermore, HDAC9 secretion in BCPAP cells dramatically decreased from 24 to 48 h. CONCLUSION: Our findings revealed that the expression of HDAC9 and P300 was higher in the PTC cell line than in normal thyroid cells. This indicates that the acetylation mechanism in thyroid cancer cells is not the same as it is in healthy cells. Epigenetic studies may reveal the mechanisms underlying PTC with further analysis.

Stemness potency and structural characteristics of thyroid cancer cell lines.

BACKGROUND: Thyroid cancer is the most frequent type of endocrine malignancy. Thyroid carcinomas are derived from the follicular epithelium and classified as papillary (PTC) (85%), follicular (FTC) (12%), and anaplastic (ATC) (<3%). Thyroid cancer could arise from thyroid cancer stem-like cells (CSCs). CSCs are cancer cells that feature stem-like properties. Kruppel-like factor (KLF4) and Stage-spesific embryonic antigen 1 (SSEA-1) are types of stem cell markers. Filamentous actin (F-actin) is an essential part of the cellular cytoskeleton. The purpose of this study was to evaluate the stem cell potency and the spatial distribution of the cytoskeletal element F-actin in PTC, FTC, and ATC cell lines. MATERIALS AND METHODS: Normal thyroid cell line (NTC) Nthy-ori-3-1, PTC cell line BCPAP, FTC cell line FTC-133 and ATC cell line 8505c were stained with SSEA-1 and KLF4 for stem cell potency and F-actin for cytoskeleton. The morphological properties of cells were assessed by a scanning electron microscope (SEM) and elemental ratios were compared with EDS. RESULTS: PTCs had greater percentages of SSEA-1 and KLF4 protein intensity (0.32% and 0.49%, respectively) than NTCs. ATCs had a greater proportion of KLF4 expression (0.8%) than NTCs. NTCs and FTCs had increased F-actin intensity across the cell, but PTCs had the lowest among these four cell lines. NTCs and PTCs, as well as NTCs and FTCs, have statistically identical aspect ratios and round values. These values, however, were statistically different in ATCs. CONCLUSION: The study of stem cell markers and the cytoskeletal element F-actin in cancer and normal thyroid cell lines may assist in the identification of new therapeutic targets and contribute in the understanding of treatment resistance mechanisms.

Melatonin Prevents UVB-Induced Skin Photoaging by Inhibiting Oxidative Damage and MMP Expression through JNK/AP-1 Signaling Pathway in Human Dermal Fibroblasts.

Exposure to ultraviolet (UV) irradiation causes damage to the skin and induces photoaging. UV irradiation stimulates production of reactive oxygen/nitrogen species, which results in activation of epidermal growth factor receptor (EGFR) and mitogen-activated protein kinases (MAPK) in fibroblasts. MAPKs are responsible for activation of activator protein-1 (AP-1), which subsequently upregulates expression of matrix metalloproteinases (MMPs). Melatonin is a potent free radical scavenger which is known to have photoprotective effects. The aim of this study is to investigate the underlying molecular mechanisms for the photoprotective effects of melatonin in UVB-irradiated primary human dermal fibroblasts (HDFs) in terms of EGFR activation, oxidative/nitrosative damage, JNK/AP-1 activation, MMP activities, and the levels of tissue inhibitors of metalloproteinase-1 (TIMP-1) and type I procollagen (PIP-C). In this study, HDFs were pretreated with 1 µM of melatonin and then irradiated with 0.1 J/cm2 of UVB. Changes in the molecules were analyzed at different time points. Melatonin inhibited UVB-induced oxidative/nitrosative stress damage by reducing malondialdehyde, the ratio of oxidized/reduced glutathione, and nitrotyrosine. Melatonin downregulated UV-induced activation of EGFR and the JNK/AP-1 signaling pathway. UVB-induced activities of MMP-1 and MMP-3 were decreased and levels of TIMP-1 and PIP-C were increased by melatonin. These findings suggest that melatonin can protect against the adverse effects of UVB radiation by inhibiting MMP-1 and MMP-3 activity and increasing TIMP-1 and PIP-C levels, probably through the suppression of oxidative/nitrosative damage, EGFR, and JNK/AP-1 activation in HDFs.

Effects of vertebral fusion on levels of pro-inflammatory and catabolic mediators in a rabbit model of intervertebral disc degeneration.

OBJECTIVE: The aim of this study was to explore the alterations in levels of pro-inflammatory and catabolic mediators following vertebral fusion in a rabbit model of intervertebral disc degeneration. METHODS: In this study, 24 female New Zealand albino rabbits (aged 4 to 5 months and weighing 3 to 3.5 kg) were used. All the animals were randomly categorized into four groups, and dorsal spinal exposure of all lumbar vertebrae was routinely performed in each group. While disc degeneration was created in groups B, C, and D, spinal fusion was added to disc degeneration in groups C and D. Disc degeneration was typically created by puncturing the discs with an 18-gauge needle under the guidance of C-arm imaging. Fusion was achieved with posterior/posterolateral decortication and iliac bone grafts. The rabbits in groups A, B, and C were euthanized, and the discs were removed in the first week after the surgery. The rabbits in Group D were sacrificed, and the discs were harvested at 5 weeks after the surgery. The levels of Interleukin (IL)-1ß, IL-6, Nitric Oxide (NO), Matrix Metalloproteinase (MMP)-3, MMP-13, and Tissue Inhibitor of Metalloproteinases-1 (TIMP-1) in the discs were analyzed using enzyme-linked immunosorbent assay kits. RESULTS: Significant increase was observed in the protein levels of both pro-inflammatory and catabolic mediators in disc degeneration groups (Group B, C, and D) compared to Group A. In the fusion groups (Group C and D), these increased mediators decreased, compared to non-fusion group (Group B), (IL1-ß P = 0.017, TIMP-1 P = 0.03, NO P = 0.03). However, there was no statistically significant difference in mediator levels between the short- and long-term fusion (Group C versus D). CONCLUSION: The results of this study have shown that a significant decrease in pro-inflammatory and catabolic mediators may be expected after vertebral fusion whereas there may be no significant difference between the first and fourth week of fusion surgery. These findings may contribute to clarifying the mechanism of action of vertebral fusion in the treatment of low back pain.

Effects of simvastatin on matrix metalloproteinase regulation in IL-1ß-induced SW1353 cells.

The present study shows the basis for the anti-inflammatory effects of statins in interleukin 1ß (IL-1ß) induced SW1353 chondrosarcoma cell-line. The cells were pre-treated with simvastatin (5 µM, 10 µM, and 50 µM), followed by IL-1ß (5 ng/mL) stimulation. Effects of simvastatin on cell viability and cytotoxicity of chondrocytes were measured with WST-1 and lactate dehydrogenase (LDH) assays, respectively. Under inflammatory conditions, in the absence/presence of simvastatin, the changes in matrix metalloproteinases (MMPs) and tissue inhibitor of metalloproteinase-1 (TIMP-1) expression levels were examined. Expression levels of MMP-1, -2, -3, -9, -13, and TIMP-1 and -2 were examined by qPCR. MMP-1, -9, -13, TIMP-1, and -2 levels were also determined by Western blotting. Gelatin zymography was performed to analyze the released and intracellular MMP-2 and MMP-9 activity levels. The results showed that simvastatin downregulated the degradation related genes MMP-3, MMP-13, MMP-2, MMP-9 and TIMP-2 in a dose-dependent manner.

Melatonin attenuates the detrimental effects of UVA irradiation in human dermal fibroblasts by suppressing oxidative damage and MAPK/AP-1 signal pathway in vitro.

BACKGROUND: People living in Mediterranean countries are mostly exposed to solar ultraviolet (UV) radiation that damages skin and results in photoaging which involves activation of epidermal growth factor receptor (EGFR) and downstream signal transduction through mitogen-activated protein kinases (MAPKs) in fibroblasts. Generation of reactive oxygen/nitrogen species by UV radiation is also critical for EGFR and MAPKs activation. MAPKs are responsible for activation of AP-1 subunits in the nucleus which induce matrix metalloproteinases. Melatonin, along with its metabolites, are known to be the most effective free radical scavenger and protective agent due to its ability to react with various radicals, lipophilic/hydrophilic structures. OBJECTIVES: In this study, we investigated the effects of melatonin on UVA-irradiated primary human dermal fibroblasts (HDFs) by following the alteration of molecules from cell membrane to the nucleus and oxidative/nitrosative damage status of the cells in a time-dependent manner which have not been clearly elucidated yet. METHODS: To mimic UVA dosage in Mediterranean countries, HDFs were exposed to UVA with sub-cytotoxic dosage (20 J/cm2 ) after pretreatment with melatonin (1 µmol/L) for 1 hour. Changes in the activation of the molecules and oxidative/nitrosative stress damage were analyzed at different time points. RESULTS: Our results clearly show that melatonin decreases UVA-induced oxidative/nitrosative stress damage in HDFs. It also suppresses phosphorylation of EGFR, activation of MAPK/AP-1 signal transduction pathway and production of matrix metalloproteinases in a time-dependent manner. CONCLUSION: Melatonin can be used as a protective agent for skin damage against intracellular detrimental effects of relatively high dosage of UVA irradiation.

Evaluation of Matrix Metalloproteinase-2 (MMP-2) and -9 (MMP-9) and Their Tissue Inhibitors (TIMP-1 and TIMP-2) in Plasma from Patients with Neurodegenerative Dementia.

Matrix metalloproteinases (MMPs) are substantial regulators of learning and memory and might be involved in neurodegeneration. It is known that MMPs are involved in pathogenesis of Alzheimer's disease (AD) and are particularly involved in the amyloid-ß processing pathway. However, information on circulating levels of these proteins and their tissue inhibitors (TIMPs) in AD and other neurodegenerative dementia (ND) diseases such as dementia with Lewy bodies (DLB) and frontotemporal dementia (FTD) is not clear. Therefore, this study was directed toward finding out how plasma levels of MMP-2, MMP-9, TIMP-1, and TIMP-2 vary in AD, DLB, and FTD; and investigating the correlation of the levels of MMPs and their inhibitors with clinical parameters of the patients. MMP-2, MMP-9, TIMP-1, and TIMP-2 levels were measured by enzyme linked immunosorbent assay (ELISA). Plasma MMP-2 levels were significantly lower in all the patient groups than in the age-matched healthy controls (HCs) (p < 0.05). MMP-9 levels were significantly lower in the FTD patients than in the HCs (p < 0.05). Also, TIMP-1 levels were lower in the AD and FTD patients than in the HCs (p < 0.05). TIMP-2 levels were similar in all the groups. These findings highlight the importance of circulating MMPs in ND and suggest that MMPs and their inhibitors might play a role in impaired amyloid-ß peptide metabolism which is responsible for the genesis and progression of ND. Furthermore, measurement of MMP-2 and MMP-9 and their inhibitors may be of great importance for large scale basic research and clinical studies of ND.

Matrix metalloproteinase-2 and -9 activity levels increase in cutaneous lupus erythematosus lesions and correlate with disease severity.

Lupus erythematosus is a chronic autoimmune disease characterized by remissions and exacerbations. Accumulated evidence indicated that matrix metalloproteinases (MMPs) are upregulated in inflammatory cells of cutaneous lupus erythematosus (CLE); however, the activity levels of these proteases have remained uncharacterized. To elucidate the significance of MMP-2, MMP-9, and TIMP-1 in CLE pathogenesis, gelatin zymography was used to investigate pro and active levels of MMP-2 and MMP-9 in lesional and perilesional skin biopsies obtained from twenty-two CLE patients. TIMP-1 protein levels were detected by ELISA in the biopsy specimens. The correlation between biochemical parameters and clinical characteristics of the disease was also evaluated. Significantly higher levels of active MMP-2, active MMP-9, proMMP-9, active/proMMP-2, and TIMP-1 were detected in lesional skin samples. Besides, the active/proMMP-9 was elevated in female and smoking patients. Active MMP-9 levels and active/proMMP-9 were also increased in elderly patients. Active MMP-9 levels were lower in patients who had smaller total damage score. Consistently, active/proMMP-9 and active/proMMP-2 were positively correlated with CLASI. Interestingly, in hydroxychloroquine or topical corticosteroid-treated patients, MMP-2/-9 activity levels were found to be higher compared to untreated patients. These findings suggest that increased MMP-2 and MMP-9 activities may contribute to the pathogenesis of CLE and cutaneous disease severity.

An improved collagen zymography approach for evaluating the collagenases MMP-1, MMP-8, and MMP-13.

Collagen zymography is an SDS-PAGE-based method for detecting both the proenzyme and active forms of collagenases. Although collagen zymography is used for assessment of the matrix metalloproteinases MMP-1 and MMP-13, it can be difficult to detect these collagenases due to technical issues. Moreover, it remains unclear whether the collagenase activity of MMP-8 can be detected by this method. Here, we present an improved collagen zymography method that allows quantification of the activities of MMP-1, MMP-8, and MMP-13. Activities of recombinant collagenases could be detected in collagen zymogram gels copolymerized with 0.3 mg/mL type I collagen extracted from rat tail tendon. This improved method is sensitive enough to detect the activity of as little as 1 ng of collagenase. We generated standard curves for the three collagenases to quantify the collagenolytic activity levels of unknown samples. To validate our improved method, we investigated MMP-1 activity levels in human thyroid cancer (8505C) and normal thyroid (Nthy-ori-3-1) cell lines, finding that the proenzyme and active MMP-1 levels were greater in 8505C cells than in Nthy-ori-3-1 cells. Taken together, our data show that collagen zymography can be used in both molecular and clinical investigations to evaluate collagenase activities in various pathological conditions.

18F-FDG-PET/CT in Initiation and Progression of Inflammation and Infection.

OBJECTIVE: Detection/localization of infection and inflammation is important for the initiation of correct treatment as well as its maintenance. Nuclear medicine imaging methods play an important role in determining infection and inflammation. 18F-2'-deoxy-2-fluoro-d-glucose (18F-FDG) positron emission tomography/computed tomography (PET/CT) is highly sensitive in such cases when used with tomographic cross-sections. In this study, the development and progression of infection and inflammation were monitored on rats by using 18F-FDG via PET/CT. METHODS: Sterile and infected abscesses were formed on rats using turpentine and S. aureus, respectively. For evaluation of the formation and progression of the abscess, 18F-FDG was injected into the rats and they were imaged by PET/CT at intervals of twenty-four hours for five days. Maximum standard uptake value (SUVmax) of 18F-FDG was calculated. RESULTS: The highest activity involvement was seen on the first day of abscess formation. On the first day, SUVmax of the S. aureus abscess was 3.9±0.9 while in the sterile abscess SUVmax in the first day was 2.2±0.8. 18F-FDG uptake decreased day by day and it reached the background level on the fourth and fifth days. There were statistically significant differences between S. aureus and sterile abscess, and between sterile abscess and background activity in terms of SUVmax values during the first three days (p<0.05). On the fourth and fifth days, there was no statistically significant difference between S. aureus and sterile abscess, and between sterile abscess and background activity (p>0.05). CONCLUSION: The results demonstrated that the SUVmax value for 18F-FDG can be useful in the early differentiation of sterile and infected abscess. In addition, 18F-FDG-PET imaging has the advantage of local availability of equipment and labeled agents leading rapid diagnosis of differentiation of infection and inflammation.

Anti-inflammatory and Anti-apoptotic Effect of Valproic Acid and Doxycycline Independent from MMP Inhibition in Early Radiation Damage.

BACKGROUND: Matrix metalloproteinase (MMP) inhibitors decrease inflammation in normal tissues and suppress cancer progress in normal tissues. Valproic acid (VA) and doxycycline (DX) are MMP inhibitors that have radio-protective effects. Their ability to inhibit MMPs in irradiated tissue is unknown and the role of MMPs in radio-protective effects has not been tested to date. AIMS: The purpose of this study was to examine whether administration of VA and DX to rats before irradiation affects tissue inflammation and apoptosis in the early phase of radiation, and whether the effect of these drugs is mediated by MMP inhibition. STUDY DESIGN: Animal experimentation. METHODS: Twenty-six Wistar rats were randomized into four groups: control (CTRL), radiation (RT), VA plus radiation (VA+RT), and DX plus radiation (DX+RT). Three study groups were exposed to a single dose of abdominal 10 Gy gamma radiation; the CTRL group received no radiation. Single doses of VA 300 mg/kg and DX 100 mg/kg were administered to each rat before radiation and all rats were sacrificed 8 hours after irradiation, at which point small intestine tissue samples were taken for analyses. Levels of inflammatory cytokines (TNF-α, IL-1ß, and IL-6) and matrix metal-loproteinases (MMP-2 and MMP 9) were measured by ELISA, MMP activities were measured by gelatin and casein zymography and apoptosis was assessed by terminal deoxynucleotidyl transferase dUTP nick end labeling assay. RESULTS: VA decreased the levels of TNF-α and IL-1ß proteins insignificantly and decreased apoptosis significantly in the irradiated tissue, but did not inhibit MMPs. In contrast, VA protected the basal MMP activities, which decreased in response to irradiation. No effect of DX was observed on the levels of inflammatory cytokines or activities of MMPs in the early phases of radiation apoptosis. CONCLUSION: Our findings indicated that VA protects against inflammation and apoptosis, and DX exhibits anti-apoptotic effects in early radiation and these effects are independent from MMP inhibition.

Expressions of TIMP-1, COX-2 and MMP-7 in Colon Polyp and Colon Cancer.

OBJECTIVE: We aimed to investigate the relationship of expression of matrix metalloproteinase-7 (MMP-7), tissue inhibitor of metalloproteinase-1 (TIMP-1) and cyclooxygenase-2 (COX-2) in colon cancer and its predecessor colon polyp. MATERIALS AND METHODS: This study included 29 patients with colon polyp, 19 patients with colon cancer and 65 healthy control subjects. The expressions of MMP-7, TIMP-1 and COX-2 were investigated by real time-polymerase chain reaction (RT-PCR). RESULTS: The expressions of TIMP-1, COX-2 and MMP-7 levels were significantly higher in polyp tissue compared to normal tissue (p = 0.024, p < 0.001, p = 0.009, respectively). Expression of TIMP-1, COX-2 and MMP-7 in cancer tissues were higher than both normal tissue and polyp tissue (p = 0.009 and p = 0.001; p < 0.001 and p < 0.001; p = 0.029 and p = 0.008, respectively). In the cancer group, no significant relationship was detected between metastasis and MMP-7, TIMP-1 and COX-2 expressions (p > 0.05). In the polyp tissues, no significant relationship was detected between the histologic type and size of polyps and MMP-7, TIMP-1 and COX-2 levels (p > 0.05). The areas under the receiver operating characteristic (ROC) curve for the cancer group were 0.821 for TIMP-1, 0.888 for COX-2, and 0.880 for MMP-7 (p = 0 < 0.001). CONCLUSION: A role and implication of expressions of MMP-7, COX-2 and TIMP-1 in colon cancer is predicted. HOW TO CITE THIS ARTICLE: Bengi G, Keles D, Topalak Ö, Yalçin M, Kiyak R, Oktay G. Expressions of TIMP-1, COX-2 and MMP-7 in Colon Polyp and Colon Cancer. Euroasian J Hepato-Gastroenterol 2015;5(2):74-79.

Expression and activity levels of matrix metalloproteinase-7 and in situ localization of caseinolytic activity in colorectal cancer.

OBJECTIVES: Matrix metalloproteinase-7 is capable of degrading several ECM and non-ECM molecules and contributes to colorectal cancer progression and metastasis. Here, we examined the significance of MMP-7 in colorectal tumors by detecting active and latent MMP-7 levels and localization of its caseinolytic activity. DESIGN AND METHODS: We investigated expression levels, localization, and proteolytic activity of MMP-7 and local caseinolytic activity in colorectal tumor and paired normal tissues by using real time PCR, casein zymography, immunohistochemistry and in situ casein zymography, respectively. In addition the results were compared with clinicopathological variables. RESULTS: Real time PCR and immunohistochemistry showed that MMP-7 expressions were higher in colorectal tumor tissues than in normal tissues. Also, mRNA expressions of MMP-7 were positively correlated with tumor and pathological stages and negatively correlated with age. Furthermore, MMP-7 mRNA expression had a sensitivity of 81.3% and a specificity of 81.2% at a cut-off value of 0.0006, making it a potential marker for diagnosis of colorectal cancer. According to casein zymography, pro- and active MMP-7 levels were also elevated in tumor tissues. In addition, we assessed local caseinolytic activity using in situ casein zymography. Increased immunoreactivity of MMP-7 and local caseinolytic activity were found in neoplastic cells but not in stromal cells. CONCLUSION: We emphasized the significant role of MMP-7 in diagnosis and progression and/or development of colorectal cancer.

Improvement of colonic healing by preoperative oral partially hydrolyzed guar gum (Benefiber) in rats which underwent preoperative radiotherapy.

BACKGROUND: Neoadjuvant radiotherapy in rectal cancer could interfere with anastomotic healing. We investigated the effects of preoperative oral administration of Benefiber on the healing irradiated colonic anastomosis. METHODS: Forty male Wistar rats were divided into four groups. Group I (control group), Group II (Benefiber® pretreatment group), Group III (preoperative radiotherapy group) and Group IV (preoperative radiotherapy and Benefiber® pretreatment group). All animals underwent 1 cm left colon resection and primary anastomosis. On the 3rd and 7th postoperative days, all the rats were anesthetized to assess the anastomotic healing clinically, mechanically, histologically and biochemically. RESULTS: The mean bursting pressure was significantly lower in-group III and significantly higher in-group II on day 7. The histologic parameters of anastomotic healing, such as epithelial regeneration and formation of granulation tissue, were significantly improved by use of preoperative Benefiber® on day 7. The amount of acid-soluble collagen concentrations significantly increased in-group IV compared to group III on day 3. The amount of salt-soluble collagen concentrations significantly increased in group II compared to group III on day 3. CONCLUSIONS: Colonic anastomotic healing can be adversely affected by preoperative radiotherapy, but orogastric feeding with Benefiber may improve the healing process.

Protective Effects of Resveratrol against Chronic Immobilization Stress on Testis.

Objective. The aim of this study was to investigate protective effects of resveratrol, a strong antioxidant, against possible negative effects of chronic immobilization stress on testes of male rats histochemically, immunohistochemically, ultrastructurally, and biochemically. Material and Methods. Male Wistar rats were divided into 4 groups (n = 7). Group I, control group (C), was not exposed to stress. Group II, stress group (S), was exposed to chronic immobilization stress. In Group III, low dose resveratrol + stress group (LRS), rats were given 10 mg/kg/day resveratrol just before the stress application. In Group IV, high dose resveratrol + stress group (HRS), rats were given 20 mg/kg/day resveratrol just before the stress application. For chronic immobilization stress application animals were put in the plastic tubes (6 cm in diameter, 15 cm in length) during 32 days for 6 hours. All animals were sacrificed 18 hours after the last stress application. Results. Histochemical and ultrastructural investigations showed that in stress group there was germ cell deprivation in seminiferous tubules and increase of connective tissue on interstitial area. No significant changes were seen in low and high dose resveratrol groups. After immunohistochemical investigations, TUNEL (+) and Active Caspase-3 (+) cells were increased in seminiferous tubules of stress group compared with those control group, but they were decreased in low and high dose resveratrol groups. According to biochemically results, MDA, GSH, and testosterone levels in stress group showed no significant difference when compared with those of the other groups. Conclusion. The chronic immobilization stress increases oxidative stress and apoptosis and causes histological tissue damages; resveratrol can minimize the histological damage in testes significantly.

Taurine inhibits increased MMP-2 expression in a model of oxidative stress induced by glutathione depletion in rabbit heart.

Matrix metalloproteinase enzymes (MMPs) activated by oxidative stress are involved in the pathogenesis of cardiovascular diseases. Glutathione (GSH) plays an important protective role against oxidatively induced damage in mammalian tissues. We investigated the possible role of gelatinases and the effect of the semiessential amino acid 2-aminoethanesulfonic acid (taurine) in oxidatively induced damage by GSH depletion in rabbit cardiac tissues. Rabbits were treated with buthionine sulfoximine (BSO), an effective GSH-depleting compound. BSO treatment significantly reduced GSH and increased MDA (malondialdehyde) levels. BSO treatment caused significant increase in proMMP-2 levels. MMP-9 (pro and active) expressions were not found in either treated- or untreated heart tissues. TIMP-1(endogenous inhibitor of MMP-9) and MT-MMP1 (endogenous activator of MMP-2) were not affected by BSO. Immunoscoring showed that MMP-2 expression significantly increased in hearts from BSO treated group but MMP-9 antibody did not show any significant positive immunostaining from all groups. Type I procollagen and total collagen did not significantly alter in heart tissues from all treatment groups. Taurine restored the increased MDA and the diminished GSH levels by BSO treatment. Pro MMP-2 expression was prevented by taurine. These results suggest that MMP-2 is a major gelanitase in rabbit hearts under oxidative stress and pharmacological inhibition of MMP-2 activation by taurine could represent a useful strategy for the prevention and/or treatment of different cardiovascular disorders.

Resveratrol reduces matrix metalloproteinase-2 activity induced by oxygen-glucose deprivation and reoxygenation in human cerebral microvascular endothelial cells.

The main pathophysiology in cerebral ischemia is the structural alteration in the neurovascular unit, coinciding with neurovascular matrix degradation. Among the human matrix metalloproteinases (MMPs), MMP-2 and -9, known as gelatinases, are the key enzymes for degrading type IV collagen, which is the major component of the basal membrane that surrounds the cerebral blood vessel. In the present study, we investigated the effects of resveratrol on cytotoxicity, reactive oxygen species (ROS), and gelatinases (MMP-2 and -9) in human cerebral microvascular endothelial cells exposed to 6 hours of oxygen-glucose deprivation and a subsequent 24 hours of reoxygenation with glucose (OGD/R), to mimic ischemia/reperfusion in vivo. Lactate dehydrogenase increased significantly, in comparison to that in the normoxia group. ROS was markedly increased in the OGD/R group, compared to normoxia. Correspondingly, ROS was significantly reduced with 50 µM of resveratrol. The proMMP-2 activity in the OGD/R group showed a statistically significant increase from the control cells. Resveratrol preconditioning decreased significantly the proMMP-2 in the cells exposed to OGD/R in comparison to that in the OGD/R group. Our results indicate that resveratrol regulates MMP-2 activity induced by OGD/R via its antioxidant effect, implying a possible mechanism related to the neuroprotective effect of resveratrol.

Pioglitazone reduces peritoneal fibrosis via inhibition of TGF-ß, MMP-2, and MMP-9 in a model of encapsulating peritoneal sclerosis.

OBJECTIVE: Matrix metalloproteinases (MMPs) and transforming growth factor beta (TGF-ß) were increased in peritoneal dialysis patients with encapsulating peritoneal sclerosis (EPS) and in chlorhexidine gluconate (CG)-induced peritoneal sclerosing animal models. Peroxisome proliferator-activated receptors (PPARs) are the major regulators of key metabolic pathways of various inflammatory responses in fibrosing processes in most tissues. The objective of this study was to investigate the effect of pioglitazone (Pio), a synthetic PPAR-γ ligand, on the development of peritoneal fibrosis in CG-induced EPS rats. METHODS: Thirty-two Wistar albino rats were intraperitoneally injected with saline (C group n = 8) or with CG (1.5 mL/100 g; CG group, n = 8). Pio (30 mg/kg/day) was administered orally to another group of CG injected rats (the CG + Pio group, n = 8) and to another control group (Pio group, n = 8) from initiation to the end of this study. After 14 days of Pio administration, the rats were killed and the parietal and visceral peritoneum were harvested. TGF-ß, MMP-2, MMP-9, tissue inhibitor of metalloproteinases (TIMP)-1, and TIMP-2 activity assays and a morphological examination of the peritoneal tissues were performed. RESULTS: Pio significantly inhibited thickening of the submesothelial layer, fibrosis, and inflammation in the peritoneum. It also prevented increases in pro-MMP-2, pro-MMP-9, TIMP-1, and TGF-ß activities. CONCLUSION: Pio, via MMP and TGF-ß inhibition, may lessen accumulation of peritoneal extracellular matrix and fibrosis to some extent in an EPS model and might be a new approach to the amelioration of EPS.

Evidence for upregulated repair of oxidatively induced DNA damage in human colorectal cancer.

Carcinogenesis may involve overproduction of oxygen-derived species including free radicals, which are capable of damaging DNA and other biomolecules in vivo. Increased DNA damage contributes to genetic instability and promote the development of malignancy. We hypothesized that the repair of oxidatively induced DNA base damage may be modulated in colorectal malignant tumors, resulting in lower levels of DNA base lesions than in surrounding pathologically normal tissues. To test this hypothesis, we investigated oxidatively induced DNA damage in cancerous tissues and their surrounding normal tissues of patients with colorectal cancer. The levels of oxidatively induced DNA lesions such as 4,6-diamino-5-formamidopyrimidine, 2,6-diamino-4-hydroxy-5-formamidopyrimidine, 8-hydroxyguanine and (5'S)-8,5'-cyclo-2'-deoxyadenosine were measured by gas chromatography/isotope-dilution mass spectrometry and liquid chromatography/isotope-dilution tandem mass spectrometry. We found that the levels of these DNA lesions were significantly lower in cancerous colorectal tissues than those in surrounding non-cancerous tissues. In addition, the level of DNA lesions varied between colon and rectum tissues, being lower in the former than in the latter. The results strongly suggest upregulation of DNA repair in malignant colorectal tumors that may contribute to the resistance to therapeutic agents affecting the disease outcome and patient survival. The type of DNA base lesions identified in this work suggests the upregulation of both base excision and nucleotide excision pathways. Development of DNA repair inhibitors targeting both repair pathways may be considered for selective killing of malignant tumors in colorectal cancer.