Tissue Inhibitor Of Metalloproteinase-1 mRNA Research Articles

Tissue inhibitor of metalloproteinases 3 promoter methylation and mRNA expression in cardia carcinoma and its relationship with prognosis

Objective To explore the relationship of the methylation levels of tissue inhibitor of metalloproteinases 3(TIMP-3) gene promoter CpG islands with the invasiveness and prognosis in cardia carcinoma. Methods The tumors tissues were collected from 65 patients with cardia carcinoma. The methylation levels of the promoter of TIMP-3 gene were detected by methylation-specific PCR(MSP), the mRNA expression levels of TIMP-3 gene were detected by RT-PCR. The relationship of TIMP-3 gene ectopic methylation with invasiveness and prognosis of the cardia carcinoma patients was analyzed. Results The positive expression rates of TIMP-3 mRNA in gastric cancer and normal gastric tissues were 53.8 % (35/65) and 96.9 % (63/65), respectively. The difference was not statistically significant(Fisher exact test, P=0.912). The positive rate of TIMP-3 mRNA was negatively correlated with the depth of tumor invasion and lymph node metastasis [mucosa and muscular vs. serosa and mucosa outside of the serosa and muscula: 83.3 % (10/12) vs. 45.3 % (24/53); with lymph node metastasis vs. without lymph node metastasis: 73.9 % (17/23) vs. 40.5 % (17/42)] (both P<0.05). There was a negative correlation between TIMP-3 gene promoter methylation and TIMP-3 mRNA expression (r=-0.276, P=0.026). The size of tumor and TIMP-3 gene promoter methylation were both the independent influencial factors of prognosis in cardia carcinoma(both P<0.05). Conclusion The methylation of promoter region in CpG islands plays an important role in the invasion and metastasis of cardia carcinoma, and it can be used as an independent predictor of biological behavior and prognosis. Key words: Stomach neoplasms; Tissue inhibitor of metalloproteinase 3; Methylation; RNA, messenger; Prognosis

Changes in miR-221/222 Levels in Invasive and In Situ Carcinomas of the Breast: Differences in Association with Estrogen Receptor and TIMP3 Expression Levels

Breast cancer (BC) is a heterogeneous group of diseases that still represents a major cause of death in the female population. MicroRNAs (miRNAs, miRs), such as miR-221 and miR-222, have been shown to be involved in BC pathology by acting via its target genes such as tissue inhibitor of metalloproteinase 3 (TIMP3). The main goals of this study were to find differences in miR-221/222 levels of expression in BC groups based on invasiveness, and to investigate the association with estrogen receptor (ER), TIMP3 messenger RNA (mRNA) levels, and clinicopathological characteristics of patients and tumors. In this study, we measured levels of miR-221/222 in 63 breast tissue samples by quantitative reverse transcription-polymerase chain reaction (qRT-PCR) using TaqMan® technology and immunohistochemistry. miR-221/222 levels varied significantly across groups based on invasiveness (P<0.001). In in situ tumors, miR-221 and miR-222 were negatively associated with ER (P=0.001, r=-0.714, and P=0.013, r=-0.585, respectively). In invasive breast carcinomas associated with non-invasive tumors, miR-222 was inversely associated with ER (P=0.039, r=-0.620). Pure invasive BCs showed a positive correlation of miR-221 and miR-222 with TIMP3 mRNA levels (P=0.008, r=0.508, and P=0.010, r=0.497, respectively). An increase in miR-221/222 might be an important event for in situ carcinoma formation, and miR-221/222 may be important molecules that highlight potential differences between invasive breast carcinomas associated with non-invasive and pure invasive BCs.

Trichostatin A increases the TIMP-1/MMP ratio to protect against osteoarthritis in an animal model of the disease.

The histone deacetylase inhibitor trichostatin A (TSA) has been demonstrated to alleviate certain symptoms associated with osteoarthritis (OA). However, the exact mechanisms underlying this protective effect remain to be elucidated. The present study therefore examined the effects of TSA on the expression levels of interleukin-1β (IL-1β)-induced matrix metalloproteinases (MMPs) and tissue inhibitor of metalloproteinases-1 (TIMP-1) in vitro and in vivo. In vitro, reverse transcription-quantitative polymerase chain reaction was performed to investigate alterations in mRNA expression levels in TSA-treated chondrocytes in the presence or absence of IL-1β; in addition, protein expression and acetylation levels were assessed by western blotting. In vivo, TSA was administered to rats by intra-articular injection, following which the mRNA and protein expression levels were analyzed. In addition, macroscopic and histological observations were conducted. Chondrocytes treated with IL-1β demonstrated increased mRNA and protein expression levels of MMP-1, MMP-3 and MMP-13, and decreased expression levels of TIMP-1 mRNA and protein; these alterations were significantly attenuated by TSA treatment. In addition, increased MMPs and decreased TIMP-1 expression levels were observed in vivo in the OA rat model. TSA treatment demonstrated in vivo efficacy through the attenuation of various OA-associated molecular and physiological changes. Taken together, the results of the present study suggest that TSA has potential therapeutic value for the treatment of OA.

Abstract 676: Tissue inhibitor of metalloproteinase-2 (TIMP2) deficiency enhances tumor burden via increasing HIF-2á expression

Abstract The tissue inhibitor of metalloproteinase family of proteins (TIMPs 1-4) function as natural MMP inhibitors, and have been shown to play a role in maintenance and remodeling of the ECM as well as other cellular processes including proliferation, apoptosis and angiogenesis. A number of studies have shown that the down-regulation or silencing of TIMP2 accelerates tumor development, however, the mechanism is not well understood. High HIF-2á levels in non-small cell lung cancer (NSCLC) correlate with decreased overall survival, while inhibition of HIFs targeted genes VEGF or VEGFR2 are associated with improved clinical outcome. Similarly, TIMP2 mRNA levels were found to be low in NSCLC compared to the corresponding non-neoplastic surrounding lung (p&amp;lt;0.05). The current study was undertaken to examine the molecular mechanisms associated with tumor hypoxia and TIMP2 expression in TIMP2 deficient mice (T2D-/-) and wild type (WT) littermates. Mice were given (1×106)/50ìl Lewis lung carcinoma cells transfected with luciferase (LL/2-Luc-M38, Caliper) via intratracheal installation. Tumor progression was monitored by IVIS imaging 2 weeks after administration and continued until week 4 when mice were sacrificed and lungs harvested for further analysis. The IVIS analysis shows higher tumor burden in T2D-/- compared to WT littermates, suggesting that TIMP2 deficiency augments tumor development in these mice (p&amp;lt;0.05). H&amp;E analysis of lung tissue sections revealed a significant increase in the number of tumor nodules in lungs of T2D mice compared to controls (p = 0.003). In an effort to investigate the relationship between TIMP2 and HIF-2á expression; we determined the expression levels of HIF-2á in healthy non-tumor bearing T2D mice compared to WT littermates. Using semi-quantitative real-time PCR, we confirmed a strong basal up regulation of HIF-2á (but not HIF-1á) mRNA in T2D mice lungs compared to WT (p&amp;lt;0.002). In tumor bearing mice, this increase was even higher in the lungs of T2D mice compared to WT (p&amp;lt;0.007). Since VEGF is the direct downstream target of HIF-2á, we determined the expression of VEGF in these mice. As expected, we found a similar trend of VEGF expression in T2D mice. The basal level of VEGF expression was increased in the lungs of healthy T2D mice compared to WT (p&amp;lt;0.006) and even higher in tumors of T2D mice compared WT (p&amp;lt;0.002). Given that the HIF-2á−VEGF signaling pathway plays a major role in neovascularization and cancer progression, microvessel density was assessed in tumor tissue by calculating the mean number of CD31+ vessels. Accordingly, we found a higher number of CD31+ vessels in T2D mice compared to WT (p&amp;lt;0.0001). Collectively, these findings support our previous work on demonstrating TIMP2 as an inhibitor of tumor angiogenesis and also suggest that TIMP2 regulates hypoxic mediators within the tumor microenvironment, therefore offering TIMP2 as a novel bio-therapeutic molecule for lung cancer therapy. Citation Format: Sarvesh Kumar, Sandra M. Jensen, Ananda Chowdhury, Beiyang Wei, William G. Stetler-Stevenson. Tissue inhibitor of metalloproteinase-2 (TIMP2) deficiency enhances tumor burden via increasing HIF-2á expression. [abstract]. In: Proceedings of the 107th Annual Meeting of the American Association for Cancer Research; 2016 Apr 16-20; New Orleans, LA. Philadelphia (PA): AACR; Cancer Res 2016;76(14 Suppl):Abstract nr 676.

Tissue Inhibitor of Metalloproteinase-1 Is Confined to Tumor-Associated Myofibroblasts and Is Increased With Progression in Gastric Adenocarcinoma.

The tissue inhibitor of metalloproteinase-1 (TIMP-1) inhibits the extracellular matrix-degrading activity of several matrix metalloproteinases, thereby regulating cancer cell invasion and metastasis. Studies describing the expression pattern and cellular localization of TIMP-1 in gastric cancer are, however, highly discordant. We addressed these inconsistencies by performing immunohistochemistry and in situ hybridization analyses in a set of 49 gastric cancer lesions to reexamine the TIMP-1 localization. In addition, we correlated these findings to clinicopathological parameters. We show that strong expression of TIMP-1 protein and mRNA was observed in a subpopulation of stromal fibroblast-like cells at the periphery of the cancer lesions. In a few cases, a small fraction of cancer cells showed weak expression of TIMP-1 protein and mRNA. The stromal TIMP-1-expressing cells were mainly tumor-associated myofibroblasts. In the normal-appearing mucosa, scattered TIMP-1 protein was only found in chromogranin A positive cells. TIMP-1-positive myofibroblasts at the invasive front of the tumors were more frequently seen in intestinal than in diffuse histological subtype cases (p=0.009). A significant trend to a higher number of cases showing TIMP-1 staining in myofibroblasts with increasing tumor, node, metastasis (TNM) stage was also revealed (p=0.041). In conclusion, tumor-associated myofibroblasts are the main source of increased TIMP-1 expression in gastric cancer.

Tissue inhibitor of metalloproteinases 3-dependent microvascular endothelial cell barrier function is disrupted under septic conditions.

Sepsis is associated with dysfunction of microvascular endothelial cells (MVEC) leading to tissue edema and multiple organ dysfunction. Metalloproteinases can regulate MVEC function through processing of cell surface proteins, and tissue inhibitor of metalloproteinases 3 (TIMP3) regulates metalloproteinase activity in the lung following injury. We hypothesize that TIMP3 promotes normal pulmonary MVEC barrier function through inhibition of metalloproteinase activity. Naive Timp3(-/-) mice had significantly higher basal pulmonary microvascular Evans blue (EB) dye-labeled albumin leak vs. wild-type (WT) mice. Additionally, cecal-ligation/perforation (CLP)-induced sepsis significantly increased pulmonary microvascular EB-labeled albumin leak in WT but not Timp3(-/-) mice. Similarly, PBS-treated isolated MVEC monolayers from Timp3(-/-) mice displayed permeability barrier dysfunction vs. WT MVEC, evidenced by lower transendothelial electrical resistance and greater trans-MVEC flux of fluorescein-dextran and EB-albumin. Cytomix (equimolar interferon γ, tumor necrosis factor α, and interleukin 1β) treatment of WT MVEC induced significant barrier dysfunction (by all three methods), and was associated with a time-dependent decrease in TIMP3 mRNA and protein levels. Additionally, basal Timp3(-/-) MVEC barrier dysfunction was associated with disrupted MVEC surface VE-cadherin localization, and both barrier dysfunction and VE-cadherin localization were rescued by treatment with GM6001, a synthetic metalloproteinase inhibitor. TIMP3 promotes normal MVEC barrier function, at least partially, through inhibition of metalloproteinase-dependent disruption of adherens junctions, and septic downregulation of TIMP3 may contribute to septic MVEC barrier dysfunction.

Pathophysiology of chronic pancreatitis induced by dibutyltin dichloride joint ethanol in mice.

To search for a new chronic pancreatitis model in mice suitable for investigating the pathophysiological processes leading to pancreatic fibrosis. The mice were randomly divided into 2 groups (n = 50), control group and model group. The mice in model group were given ethanol (10%) in drinking water after injection of dibutyltin dichloride (DBTC) (8 mg/kg BW) in tail vein. The mice in control group were injected with only solvent into tail vein (60% ethanol, 20% glycerine and 20% normal saline) and drank common water. At days 1, 7, 14, 28, and 56 after application of DBTC or solvent, 10 mice in one group were killed at each time point respectively. Blood was obtained by inferior vena cava puncture. The activity of amylase, concentration of bilirubin and hyaluronic acid in serum were assayed. The pancreas was taken to observe the pancreatic morphology by HE staining, and to characterize the pancreatic fibrosis by Masson staining. The expression of F4/80, CD3 and fibronectin (FN) were assayed by immuno-histochemistry or Immunofluorescence technique. Collagen type I (COL1A1) in pancreas were detected by Western blot. The expression of matrix metalloproteinase-1 (MMP-1) and tissue inhibitor of metalloproteinases-1 (TIMP-1) mRNA in the pancreas was assessed by real time PCR. DBTC induced an acute edematous pancreatitis within 1 d. The dilated acini, scattered acinar cell necrosis, and inflammatory cells were found at day 7. Extensive infiltration with inflammatory cells following deposition of connective tissue was observed at day 14. At day 28, level of pancreatic fibrosis was aggravated. The pancreatic tissue was replaced by an extended interstitial fibrosis at the end of 2 mo. There was significant difference in the level of amylase, bilirubin and hyaluronic acid in serum between control group and model group (P < 0.05). The level of COL1A1 and FN in pancreas increased. The expression of MMP-1 mRNA in pancreas decreased, but TIMP-1 mRNA increased at model group. DBTC joint Ethanol drinking can induce chronic pancreatitis in accordance with the pathophysiological modification of human. DBTC joint Ethanol-induced pancreatitis in mice is an effective and handy experimental method. The model is suitable to study the mechanism of pancreatic fibrosis in chronic pancreatitis.

The Clinical Utility of TIMP3 Expression in ACTH-Secreting Pituitary Tumor.

In recent years, the tissue inhibitor of metalloproteinase-3 (TIMP3) plays a pivotal role in tumorigenesis, while the role of TIMP3 in adrenocorticotrophic hormone (ACTH)-secreting pituitary adenomas remains unclear. In this study, 86 sporadic pituitary tumor specimens, including ACTH (40), GH (18), PRL-secreting (8), and non-functioining (20) and non-tumorous pituitary samples (n = 10) were available, and then, the mRNA and protein expression of TIMP3 was quantified by quantitative reverse transcriptase polymerase chain reaction (RT-PCR), western blotting, and immunohistochemistry, respectively. Our findings showed that TIMP3 expression was significantly correlated with Ki-67 expression and the invasiveness of pituitary adenomas. TIMP3 mRNA and protein expression were reduced in ACTH-secreting pituitary adenomas and the other three types of pituitary adenomas compared to adjacent non-tumorous pituitary tissues (all p < .01). On the other hand, the expression of TIMP3 was negatively correlated with tumor size and Ki-67 in ACTH-secreting pituitary adenomas. TIMP3 mRNA expression was significantly lower in invasive pituitary adenomas than that in noninvasive ones (1.92-fold, p < .05). TIMP3 protein levels were also significantly lower in the majority of invasive adenomas (1.41-fold, p < .05) Furthermore, TIMP3 mRNA and protein expression were significantly lower in pituitary giant adenomas than those in microadenomas (2.58-fold, p < .05). In conclusion, the expression of TIMP3 is low in pituitary adenomas including ACTH-secreting pituitary adenomas and negatively associated with tumor aggressiveness. TIMPs may play a potential role in the progression of ACTH-secreting pituitary adenomas and be useful as a biomarker of invasiveness.

Antiadhesive and anti-inflammatory effects of pirfenidone in postoperative intra-abdominal adhesion in an experimental rat model

BackgroundPirfenidone (PF) is a potent antifibrotic and anti-inflammatory agent. We investigated the protective effect of PF against postoperative intra-abdominal adhesions. Material and methodsThirty male Sprague–Dawley rats were divided into three groups (n = 10 in each group). In group 1 (control), adhesion induction was performed by cecal abrasion, and no treatment was administered. In group 2 (vehicle), for 2 wk after adhesion induction, 0.4%-carboxymethylcellulose was administered by gavage. In group 3 (PF treatment), for 2 wk after adhesion induction, 500-mg/kg/d PF was administered by gavage. On the 15th postoperative day, the animals were killed, and cecal and peritoneal tissues were excised. The adhesions were graded macroscopically. The protein concentrations and mRNA expression levels of the following genes were measured in the tissues: matrix metallopeptidase-9 (MMP-9); tissue inhibitor of metalloproteinase-1 (TIMP-1); tumor necrosis factor-alpha (TNF-α); and transforming growth factor-beta 1 (TGF-β1). The tissue samples were also evaluated histopathologically. ResultsMacroscopic and histopathologic evaluation showed that PF-reduced adhesion and inflammation (P < 0.001, P = 0.004, respectively). Pretreatment with PF-reduced TIMP-1, TNF-α, and TGF-β1 protein concentrations (P < 0.001, P < 0.001, and P < 0.001, respectively) and mRNA expression levels (P = 0.030, P = 0.005, and P = 0.016, respectively) and increased MMP-9 protein concentrations (P < 0.001) and mRNA expression (P = 0.021). ConclusionsThe findings of this study suggest that PF can be used as a protective agent to prevent the development of peritoneal adhesions and inflammation during the postoperative period.

In vitro effects of phenytoin and DAPT on MDA-MB-231 breast cancer cells.

Voltage-gated sodium channel (VGSC) activity enhances cell behaviors related to metastasis, such as motility, invasion, and oncogene expression. Neonatal alternative splice form of Nav1.5 isoform is expressed in metastatic breast cancers. Furthermore, aberrant Notch signaling pathway can induce oncogenesis and may promote the progression of breast cancers. In this study, we aimed to analyze the effect of the nNav1.5 inhibitor phenytoin and Notch signal inhibitor N-[N-(3,5-difluorophenacetyl)-L-alanyl]-S-phenylglycine-t-butyl ester (DAPT) on triple negative breast cancer cell line (MDA-MB-231) via inhibition of nNav1.5 VGSC activity and Notch signaling, respectively. In order to determine the individual and combined effects of these inhibitors, the 4-[3-(4-iyodophenyl)-2-(4-nitrophenyl)-2H-5-tetrazolio]-1,3-benzene disulfonate (WST-1) test, wound healing assay, and zymography were performed to detect the proliferation, lateral motility, and matrix metalloproteinase-9 (MMP9) activity, respectively. The expressions of nNav1.5, Notch4, MMP9, and tissue inhibitor of metalloproteinases-1 (TIMP1) were also detected by quantitative real-time reverse transcriptase-polymerase chain reaction. DAPT caused an antiproliferative effect when the doses were higher than 10 µM, whereas phenytoin showed no inhibitory action either alone or in combination with DAPT on the MDA-MB-231 cells. Furthermore, it was found that the lateral motility was inhibited by both inhibitors; however, this inhibitory effect was partially rescued when they were used in combination. Meanwhile, the results showed that the MMP9 activity and the ratio of MMP9 mRNA to TIMP1 mRNA were only decreased by DAPT. Thus, we conclude that the combined effect of DAPT and phenytoin is not as beneficial as using DAPT alone on MDA-MB-231 breast cancer cells.

Expression and regulative function of tissue inhibitor of metalloproteinase 3 in the goat ovary and its role in cultured granulosa cells

Tissue inhibitor of metalloproteinase 3 (TIMP3) played a key role in female reproduction. However, its expression and function in goat are still unclear. In the present study, the full-length cDNA of goat TIMP3 was cloned from adult goat ovary; meanwhile, we demonstrated that putative TIMP3 protein shared a highly conserved amino acid sequence with known mammalian homologs. Real-time PCR results showed that TIMP3 was widely expressed in the tissues of adult goat. In the ovary, increasing expression of TIMP3 mRNA was discovered during the growth process of follicle and corpus luteum. Immunohistochemistry results suggested that TIMP3 protein existed in oocytes of all types of follicles, corpus luteum and granulosa and theca cells of primary, secondary, and antral but not primordial follicles. In vitro, human chorionic gonadotropin (hCG) stimulated the expression of TIMP3 in goat granulosa cells. hCG-induced TIMP3 mRNA expression was reduced by the inhibitors of protein kinase A, protein kinase C, MAPK kinase, or p38 kinase. Functionally, over-expression of TIMP3 significantly increased apoptosis and decreased the viability of cultured granulosa cells. Knockdown of TIMP3 could decrease hCG-induced progesterone secretion and the mRNA abundance of key steroidogenic enzymes (StAR, p450scc and HSD3B) as well as ECM proteins (DCN and FN). These findings provided evidence that the hCG induced expression of TIMP3 may play an important role in regulating goat granulosa cell survival and steroidogenesis.

Effect of tissue inhibitor of metalloproteinases 3 on DLK1 shedding in cultured human pre-adipocytes and implications for adipose tissue remodelling

BackgroundMetabolically unhealthy obesity is associated with adipose tissue inflammation and increased risk of type 2 diabetes. Dysfunctional adipose tissue remodelling has been implicated in development of metabolically unhealthy obesity, but the pathogenesis remains poorly characterised. We hypothesised that in health, tissue inhibitor of metalloproteinases 3 (TIMP3) modulates adipose tissue remodelling by regulating extracellular matrix turnover and shedding of the adipogenic regulator DLK1, but that in adipose tissue inflammation it might drive development of metabolically unhealthy obesity. MethodsPrimary pre-adipocyte and in-vitro-differentiated adipocyte cultures were established from abdominal subcutaneous adipose tissue donated by healthy women undergoing breast reconstruction. Cells were seeded onto collagen I, and subsequently treated with differentiation medium or tumour necrosis factor (TNF) alpha (50 ng/mL). Adenoviral transduction allowed TIMP3 overexpression. Media and lysates were collected for quantitative RT-PCR, and immunoblot and hydroxyproline release assays. Statistical analysis was performed with t testing or ANOVA. FindingsInduction of differentiation in human pre-adipocytes reduced TIMP3 mRNA levels by 75% (n=3, p<0·0001). Hydroxyproline release by differentiating pre-adipocytes was 2·3 times greater than that by control-treated cells (mean 5·66 μg/mL [SD 0·77] vs 2·45 [0·36], p<0·0001) indicating greater collagen I degradation. TNF alpha reduced TIMP3 mRNA levels by 66% in in-vitro-differentiated adipocytes (n=3, p<0·0001); reduced TIMP3 expression was confirmed by western blot. Shedding of soluble DLK1 (sDLK1) by pre-adipocytes was increased by TNF alpha and by overexpression of adenovirally delivered TIMP3 compared with control conditions, as confirmed by immunoblot (n=3). Addition of recombinant human sDLK1 (500 pM) to pre-adipocyte cultures reduced adipogenesis, as assessed by oil red O staining (n=2). InterpretationWe have shown that TIMP3 is downregulated in adipogenesis, and by inflammatory signals in adipocytes. Furthermore, TIMP3 modulates sDLK1 shedding and collagen I degradation. TIMP3 is known to inhibit ADAM17 (DLK1 sheddase) and MMP14 (implicated in extracellular matrix turnover). TIMP3 might therefore integrate inflammatory signals with adipose remodelling. Subversion of remodelling pathways by chronic adipose inflammation might lead to maladaptive adipose expansion and metabolically unhealthy obesity. FundingBritish Heart Foundation, Diabetes Research and Wellness Foundation Open Funding 2011.

Alterations in enhancer of zeste homolog 2, matrix metalloproteinase-2 and tissue inhibitor of metalloproteinase-2 expression are associated with ex vivo and in vitro bone metastasis in renal cell carcinoma.

Renal cell carcinoma (RCC) has a high potential for bone metastasis; however, the molecular mechanisms underlying this metastasis have remained to be elucidated. The present study aimed to explore the expression levels of enhancer of zeste homolog 2 (EZH2), matrix metalloproteinase-2 (MMP2) and tissue inhibitor of metalloproteinase-2 (TIMP2) as determinants of RCC-associated bone metastasis. Their expression was evaluated in a newly generated RCC cell subline that has a high potential for bone metastasis, in tissue specimens from metastasized bone tissues from patients with RCC and in RCC tissues without metastasis. A total of 25 RCC tissue specimens without metastasis and 13 RCC tissue specimens with bone metastasis were acquired for immunohistochemical analysis of EZH2, MMP2 and TIMP2 protein expression. The expression levels of EZH2, MMP2 and TIMP2 mRNA and protein were analyzed in the ACHN and ACHN-BO5 cell lines using western blot and reverse transcription polymerase chain reaction (PCR) analyses. Methylation-specific PCR was also used to analyze TIMP2 promoter methylation. EZH2 and MMP2 proteins were found to be expressed at higher levels in tissues from patients where RCC had metastasized to the bone as compared with those in RCC patients without metastasis, whereas there was no significant difference in the expression of TIMP2 protein between the two tissues. Furthermore, the expression of EZH2 protein was correlated with MMP2 expression, but there was no significant correlation between the expression of EZH2 and TIMP2 proteins. The in vitro results using cell lines confirmed the ex vivo findings, indicating that the expression levels of EZH2 and MMP2 protein and mRNA were higher in ACHN-BO5 cells than those in ACHN cells. By contrast, TIMP2 protein and mRNA expression levels were lower in ACHN-BO5 cells than those in the parental ACHN cells. The TIMP2 promoter was highly methylated in ACHN-BO5 cells compared with that in ACHN cells. Upregulation of EZH2, MMP2 and TIMP2 expression was correlated with metastasis of RCC to bone tissues ex vivo and in vitro. Further studies are required in order to elucidate the mechanism underlying the altered expression of these genes.

Elevated circulating tissue inhibitor of metalloproteinase 1 (TIMP-1) levels are associated with neuroendocrine differentiation in castration resistant prostate cancer.

Tissue inhibitor of metalloproteinase-1 (TIMP-1) is a 28.5 kDa secreted glycoprotein that inhibits matrix metalloproteinase (MMP) activity. Our group has previously shown that elevated plasma TIMP-1 levels predict poor survival in metastatic castration-resistant prostate cancer (CRPC) patients; however, the underlying source and impact of elevated circulating TIMP-1 protein is unknown. In this study, we used qRT-PCR, ELISA and immunohistochemistry to evaluate TIMP-1 expression in androgen-sensitive and resistant prostate cancer (PC) cell lines, tumor tissues and patient sera, and to correlate TIMP-1 levels to expression of chromogranin A (CGA), an established marker of neuroendocrine differentiation (NED). We also explored the relationship between TIMP-1 overexpression and induction of NED by overexpressing TIMP-1 in androgen-sensitive LNCaP cells, as well as by inducing NED of LNCaP cells with IL-6. Patients with CRPC have significantly higher serum TIMP-1 levels compared to patients with hormone-sensitive disease. Although circulating TIMP-1 levels were increased, peripheral blood cells were not the source of elevation. Instead, elevated TIMP-1 expression was associated with higher expression of CGA in both blood and metastatic tumor tissue. We further show that androgen receptor (AR) and PSA non-expressing prostate cancer cell lines known to display NED phenotypes such as PC-3, PC-3M, and DU145 cells, expressed high levels of TIMP-1, in contrast to AR (+) and PSA (+) adenocarcinoma cell lines such as LNCaP, VCaP, and LAPC-4, which had barely detectable levels of TIMP-1. In addition, ectopic overexpression of TIMP-1 in LNCaP cells did not induce NED. However, TIMP-1 mRNA expression was elevated >10-fold during IL-6-induced NED of LNCaP cells, suggesting that TIMP-1 overexpression accompanies, but is not the driving force for NED. Finally, we show that conditioned media from androgen-resistant PC-3, PC-3M, and DU145 cells induced TIMP-1 mRNA expression in primary prostate stromal fibroblasts in an ERK and NF-κB dependent manner. We provide in vitro and clinical evidence to support the association between NED and elevated circulating TIMP-1 expression in CRPC. Our observation supports further evaluation of TIMP-1 as a tissue and serum biomarker for NED in CRPC.

Thalidomide Accelerates the Degradation of Extracellular Matrix in Rat Hepatic Cirrhosis via Down-Regulation of Transforming Growth Factor-β1.

PurposeThe degradation of the extracellular matrix has been shown to play an important role in the treatment of hepatic cirrhosis. In this study, the effect of thalidomide on the degradation of extracellular matrix was evaluated in a rat model of hepatic cirrhosis.Materials and MethodsCirrhosis was induced in Wistar rats by intraperitoneal injection of carbon tetrachloride (CCl4) three times weekly for 8 weeks. Then CCl4 was discontinued and thalidomide (100 mg/kg) or its vehicle was administered daily by gavage for 6 weeks. Serum hyaluronic acid, laminin, procollagen type III, and collagen type IV were examined by using a radioimmunoassay. Matrix metalloproteinase-13 (MMP-13), tissue inhibitor of metalloproteinase-1 (TIMP-1), and α-smooth muscle actin (α-SMA) protein in the liver, transforming growth factor β1 (TGF-β1) protein in cytoplasm by using immunohistochemistry and Western blot analysis, and MMP-13, TIMP-1, and TGF-β1 mRNA levels in the liver were studied using reverse transcriptase polymerase chain reaction.ResultsLiver histopathology was significantly better in rats given thalidomide than in the untreated model group. The levels of TIMP-1 and TGF-β1 mRNA and protein expressions were decreased significantly and MMP-13 mRNA and protein in the liver were significantly elevated in the thalidomide-treated group.ConclusionThalidomide may exert its effects on the regulation of MMP-13 and TIMP-1 via inhibition of the TGF-β1 signaling pathway, which enhances the degradation of extracellular matrix and accelerates the regression of hepatic cirrhosis in rats.

Abstract 1098: TIMP-1: A potential biomarker of neuroendocrine differentiation in metastatic castration-resistant prostate cancer (mCRPC)

Abstract BACKGROUND: Tissue inhibitor of metalloproteinases-1 (TIMP-1) is a 28.5 kDa secreted glycoprotein that inhibits matrix metalloproteinase (MMP) activity. Recent studies have demonstrated that TIMP-1 promotes tumor growth in an MMP-independent manner. We have previously shown that elevated plasma TIMP-1 predicts worse survival in mCRPC patients; however, the mechanism underpinning this association is unknown. Normal and malignant neuroendocrine (NE) cells, from various organs, express TIMP-1 as measured by immunohistochemistry (IHC). We hypothesized that elevated blood levels of TIMP-1 in mCRPC patients results from androgen deprivation therapy-induced NE differentiation. METHODS: In this study, we used qPCR, ELISA and IHC to correlate the expression of TIMP-1 and NE markers such as chromogranin A (CGA) in 7 prostate cancer cell lines, 37 patient serum samples and prostate cancer (adenocarcinoma and NE cancer) tissue microarrays. RESULTS: We first examined TIMP-1 and NE marker (NSE and PTHLH) mRNA expression in 7 prostate cancer cell lines. Our results show that PC-3, PC-3M, DU145 cells express high levels of TIMP-1 as well as NE markers. On the other hand, LNCAP, VCAP and LAPC-4 cells had barely detectable levels of TIMP-1 and much lower expression of NE markers. We then measured serum TIMP-1 and CGA levels from CRPC patient samples by ELISA. Patients with castration resistant disease had significantly higher serum TIMP-1 levels (mean value of 373.5 ng/ml) than patients with hormone sensitive disease (mean value of 304.8 ng/ml) (p=0.01), suggesting that castration resistance in prostate cancer patients is associated with increased TIMP-1 production. Interestingly, when we divided patients using a cutoff of 100 ng/ml (based on cutoffs reported in published literature), serum TIMP-1 was significantly higher in the high CGA group (p=0.018), indicating an association between TIMP-1 production and existing NE markers. We also compared PSA values between low and high CGA groups and found that the high CGA group was associated with lower PSA levels (p=0.029). Next, we explored TIMP-1 expression in NE tumor tissues. We performed TIMP-1 and CGA IHC staining in archival liver neuroendocrine and thyroid cancers and found that TIMP-1 expression was associated with CGA expression. In prostate cancer specimens, normal prostate epithelial cells expressed high levels of TIMP-1, but the expression was lost in adenocarcinomas. In contrast, prostatic neuroendocrine cancers expressed high levels of TIMP-1 as well as the NE marker CGA. CONCLUSIONS: These observations support the further evaluation of TIMP-1 as a tissue and serum biomarker for NE differentiation in CRPC. Note: This abstract was not presented at the meeting. Citation Format: Yixuan Gong, Uma Chippada-Venkata, Xudong Dai, Matthew D. Galsky, Jiaoti Huang. TIMP-1: A potential biomarker of neuroendocrine differentiation in metastatic castration-resistant prostate cancer (mCRPC). [abstract]. In: Proceedings of the 105th Annual Meeting of the American Association for Cancer Research; 2014 Apr 5-9; San Diego, CA. Philadelphia (PA): AACR; Cancer Res 2014;74(19 Suppl):Abstract nr 1098. doi:10.1158/1538-7445.AM2014-1098

Losartan attenuates paraquat-induced pulmonary fibrosis in rats.

Paraquat (PQ) is one of the most widely used herbicides in the world and can cause pulmonary fibrosis in the cases with intoxication. Losartan, an angiotensin II type 1 receptor antagonist, has beneficial effects on the treatment of fibrosis. The aim of this study was to examine the effect of losartan on pulmonary fibrosis in PQ-intoxicated rats. Adult male Sprague Dawley rats (n = 32, 180-220 g) were randomly assigned to four groups: (i) control group; (ii) PQ group; (iii) PQ + losartan 7d group; and (iv) PQ + losartan 14d group. Losartan treatment (intragastrically (i.g.), 10 mg/kg) was performed for 7 and 14 days after a single i.g. dose of 40 mg/kg PQ. All rats were killed on the 16th day, and hematoxylin-eosin and Masson's trichrome staining were used to examine lung injury and fibrosis. The levels of hydroxyproline and transforming growth factor β1 (TGF-β1), matrix metallopeptidase 9 (Mmp9), and tissue inhibitor of metalloproteinase 1 (TIMP-1) messenger RNA (mRNA) expression and relative expression levels of collagen type I and III were also detected. PQ caused a significant increase in hydroxyproline content, mRNA expression of TGF-β1, Mmp9, and TIMP-1, and relative expression levels of collagen type I and III ( p < 0.05), while losartan significantly decreased the amount of hydroxyproline and downregulated TGF-β1, Mmp9, and TIMP-1 mRNA and collagen type I and III expressions ( p < 0.05). Histological examination of PQ-treated rats showed lung injury and widespread inflammatory cell infiltration in the alveolar space and pulmonary fibrosis, while losartan could markedly reduce such damage and prevent pulmonary fibrosis. The results of this study indicated that losartan could reduce lung damage and prevent pulmonary fibrosis induced by PQ.

Effects of captlpril on tissue inhibitorof metalloproteinase-1 in rat hepatic stellate cells

Objective To investigate the effects of angiotensin Ⅱ (Ang Ⅱ) and captlpril on tissue inhibitorof metalloproteinase-1 (TIMP-1) mRNA expression in culured hepatic stellate cells (HSCs) in vivo.Methods HSC-T6 rat hepatic stellate cell line was chosen as the study model of the activated HSCs.The cultured HSCs were randomized into control group,Ang Ⅱ group (1 × 10-5,1 × 10-7 mol/L) and AngⅡ + captlpril group (1 × 10-5,1 × 10-7 mol/L).HSCs were harvested to measure the TIMP-1 mRNA expression by reverse transcription-polymerase chain reaction (RT-PCR).Results TIMP-1 gene expression levels in different groups of Ang Ⅱ (1 × 10-5 mol/L,1 × 10-7 mol/L) were 1.145 ±0.219 and 0.860±0.115 respectively,and those in control group was 0.523 ± 0.056.TIMP-1 expression levels in different groups of Ang Ⅱ ± captlpril (1 × 10-5 mol/L,1 × 10-7 mol/L) were 0.740 ± 0.089 and 0.590 ± 0.073 respectively,and those in Ang Ⅱ group were 0.850 ± 0.107 and 0.620 ± 0.103 respectively.Conclusion Angiotensin Ⅱ can increase the type Ⅰ collage mRNA expression of HSCs,which can be inhibited by captlpril. Key words: Angiotensin Ⅱ ; Captlpril; Hepatic stellate cells; Tissue inhibitorof metalloproteinase-1

Preventive effects of punicalagin against ultraviolet B-induced damage to human HaCaT keratinocytes

Objective To investigate the mechanisms underlying the protection by punicalagin against ultraviolet B (UVB)-induced damage to keratinocytes.Methods Cultured human HaCaT keratinocytes were divided into several groups:blank control group receiving no treatment,punicalagin groups treated with various concentrations of punicalagin,UVB group irradiated with UVB at 30 mJ/cm2,combination groups pretreated with different concentrations of punicalagin followed by UVB radiation at 30 mJ/cm2.The concentrations of punicalagin were 5,10,20,40 and 80 μmol/L in the cell proliferation assay,10,20 and 40 μmol/L in the other assays.After additional culture for different durations,methyl thiazolyl tetrazolium (MTT) assay was performed to evaluate the proliferation of HaCaT cells,Hoechst and propidium iodide (PI) staining as well as flow cytometry to detect the apoptosis in cells,reverse transcription-PCR to quantify the mRNA expressions of matrix metalloproteinase-1 (MMP1) and tissue inhibitor of metalloproteinase-1 (TIMP1) in HaCaT cells,Western blot to determine the phosphorylation levels of the mitogen-activated protein kinase (MAPK) pathway-related proteins including P38,JNK and ERK.Statistical analysis was carried out by t test,one-way analysis of variance,and Dunnett's t-test.Results As the MTT assay showed,punicalagin at 10-40 μmol/L showed stronger pre-protective effects against UVB-induced damage to HaCaT cells compared with punicalagin at the other concentrations.The number of cells highly positive for both Hoechst and PI staining was larger in the UVB group than that in the blank control group,but smaller in the combination groups than in the UVB group.The percentage of apoptotic cells increased significantly in the UVB group compared with the blank control group (9.82％ ± 0.11％ vs.1.24％ ± 0.91％,P ＜ 0.01),but decreased significantly in the three combination groups (punicalagin (10,20 and 40 μmol/L) + UVB) compared with the UVB group (6.38％ ± 0.14％,5.24％ ± 0.17％ and 3.77％ ± 0.11％ vs.9.82％ ± 0.11％,all P＜ 0.01).Theexpression of MMP1 mRNA was significantly higher,but that of TIMP1 mRNA was significantly lower in the UVB group than in the blank control group (both P ＜ 0.01),whereas no statistically significant difference was observed in the expression of MMP1 or TIMP1 mRNA between the punicalagin groups and blank control group (all P ＞ 0.05).The pretreatment with punicalagin significantly reduced the expression level of MMP1 mRNA (P ＜ 0.01),but elevated that of TIMP1 mRNA (P ＜ 0.01) in the combination groups compared with the UVB group.As Western blot showed,the phosphorylation levels of P38,JNK and ERK were markedly increased in the UVB group (all P ＜0.01),but experienced no significant changes in the punicalagin groups (all P ＞ 0.05) compared with the blank control group,and decreased to different degrees in the combination groups compared with the UVB group (all P ＜0.01).Conclusion Punicalagin has a pre-protective effect on UVB-induced damage to HaCaT cells. Key words: Keratinocytes; Ultraviolet rays; Matrix metalloproteinase 1; Extracellular signal-regulated MAP kinases; Punicalagin; Matrix metalloproteinase inhibitory factor

Low sputum MMP-9/TIMP ratio is associated with airway narrowing in smokers with asthma.

Asthmatic smokers have poor symptom control and accelerated decline in lung function. A reduced ratio of matrix metalloproteinase (MMP)-9/tissue inhibitors of metalloproteinases (TIMPs) in nonsmokers with asthma has been implicated in airway remodelling. We tested the hypothesis that sputum MMP-9 activity/TIMPs ratios are reduced in smokers compared with never-smokers with asthma and are associated with reduced lung function and altered computed tomography (CT) measures of airway wall dimensions. Lung function, airway dimensions by CT, and induced sputum concentrations (and activity) of MMP-9 and TIMP-1 and -2 were measured in 81 asthmatics and 43 healthy subjects (smokers and never-smokers). Respiratory epithelial MMP9 and TIMP mRNA was quantified in 31 severe asthmatics and 32 healthy controls. Sputum MMP-9 activity/TIMP-1 and TIMP-2 ratios, and nasal epithelial MMP9/TIMP1 and MMP9/TIMP2 expression ratios were reduced in smokers with asthma compared with never-smokers with asthma. Low sputum ratios in asthmatic smokers were associated with reduced post-bronchodilator forced expiratory volume in 1 s (FEV1), FEV1/forced vital capacity ratio and segmental airway lumen area. The association of a low sputum MMP-9 activity/TIMP-1 ratio with persistent airflow obstruction and reduced CT airway lumen area in smokers with asthma may indicate that an imbalance of MMP-9 and TIMPs contributes to structural changes to the airways in this group.