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.

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.

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.

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.

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.

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.