MMP-9 Research Articles

Flavocoxid mitigates cadmium-induced toxicity: structural, immunohistochemical and molecular analysis in mice kidney

Background: Cadmium (Cd), a diffused environmental pollutant, has adverse effects on urinary apparatus [1]. The role of flavocoxid, a flavonoid with antioxidant activity [2], on the morphological and biochemical changes induced in vivo by Cd in mice kidneys was evaluated. Methods: C57 BL/6J mice received 0.9% NaCl alone, flavocoxid (20 mg/kg/day i.p.) alone, Cd chloride (CdCl2) (2 mg/kg i.p.) alone, or CdCl2 plus Flavocoxid (2 mg/kg i.p. plus 20 mg/kg/day i.p.) for 14 days. At the end of experiment, the mice were killed with an overdose of ketamine and xylazine and the kidneys were collected and processed for structural, immunohistochemical and biochemical analysis. Results: Cd treatment alone significantly increased iNOS, TNF-α and MMP-9 expression, induced structural damages in the glomeruli and in the proximal tubule epithelium, and reduced claudin-11, occludin and N-cadherin immunoreactivity. Flavocoxid administration reduced iNOS, TNF-α and MMP-9 expression, ameliorated glomerular and tubular changes and enhanced claudin-11, occludin and N-cadherin immunoreactivity. Conclusions: We demonstrated for the first time that flavocoxid has a protective role against Cd-induced damages in mice kidney. Therefore, flavocoxid may have a promising role against environmental Cd, in particular against its harmful effects on glomerular and tubular lesions.

Low frequency ultrasound combined with baicalein can reduced the invasive capacity of breast cancer cells by down regulating the expression of MMP-2, MMP-9, and u-PA

Background: Over the past three decades, a large number of patients have died owing to breast cancer metastasis and recurrence. Therefore, there is an urgent need to develop effective and comprehensive strategies for the treatment of breast cancer. Earlier studies have demonstrated that low frequency ultrasound and baicalein can arrest the invasive activity of breast cancer cells and inhibit the migration and metastasis of breast cancer. However, the exact mechanism of action of this combination remains elusive. Furthermore, the inhibitory effect of the combination treatment with low frequency ultrasound and baicalein on breast cancer has not been reported. Methods: In this study, human breast cancer cell line MDA-MB-231 was cultured and subjected to one of the following four treatment: low frequency ultrasound, baicalein, combination of the two, or no treatment. Cells in the low frequency group were irradiated with ultrasound for 5 min at a frequency of 840 kHz, 0.75 W/cm2. Cells in the baicalein group were treated with baicalein for 24 h at a concentration of 150 μmol/L. In the combination group, cells were treated with both low frequency ultrasound and baicalein. Cells receiving no treatment were designated as the control group. Invasive capacity of cells in the four groups was examined by transwell assay. matrix metalloproteinase 2 (MMP-2), MMP-9, and urokinase type plasminogen activator (u-PA) mRNA and protein levels were examined using Real-time PCR and Western blotting, respectively. Results: The invasive capacity of cells in the low frequency group, baicalein group and the combination group was significantly reduced by 21.6%±1.68%, 31.9%±2.19% and 51.6%±2.01%, respectively. The expression levels of both mRNA and protein of MMP-2, MMP-9, and u-PA were significantly decreased in all three treatment groups, compared to that in the control group. The decline in the expression of MMP-2, MMP-9 and u-PA was highest in the combination group at both mRNA and protein levels. Conclusions: Low frequency ultrasound combined with baicalein can effectively reduce the invasive capacity of breast cancer, probably by downregulating the expression of MMP-2, MMP-9, and u-PA.

Gemcitabine induces epithelial-mesenchymal transition and promotes metastasis in cholangiocarcinoma through CXC chemokine ligand 12/CXC chemokine receptor 4 axis

Objective To explore the effect of gemcitabine (GEM) in cholangicocarcinoma cells on metastatic potential and epithelial-mesenchymal transition (EMT) through CXC chemokine ligand 12 (CXCL12)/CXC chemokine receptor 4 (CXCR4) axis. Methods The in vitro model was established using stromal cell derived factor 1 (SDF-1, 100 mg/ml) to activate the expression of CXCL12/CXCR4 biological axis in cholangicocarcinoma cells with a high CXCR4 expression. The proliferation rate was measured by methyl thiazol tetrazolium (MTT) method. Cholangicocarcinoma RBE cells were divided into control group and GEM group. Transwell chamber was used to observe the invasion and metastasis of the cells. Real-time quantitative polymerase chain reaction (Real-time PCR) was applied to detect the CXCR4, and Western blotting was used to detect the expression of CXCR4 and its downstream invasion and EMT related factors vascular endothelial growth factor C (VEGF-C), matrix metalloproteinase (MMP)-9, N-cadherin and E-cadherin. Results The cell survival rate in GEM group was significantly decreased (76.65%, 71.40%, and 52.25%; P<0.05). GEM promoted metastasis potential ability of cells (282.00±3.45 vs. 168.00±2.49), and promoted the overexpression of CXCR4 protein (0.965±0.163, 1.895±0.191, 3.975±0.120, 3.355±0.163, 3.572±0.198 and 6.154±0.113) in and mRNA (1.021±0.210, 2.442±0.556, 2.774±0.146, 2.961±0.275, 3.279±0.228 and 5.010±0.826), and also promoted the ability of metastasis [VEGF-C (3.250±0.212 vs. 1.025±0.318), MMP-9 (1.980±0.212 vs. 1.025±0.318)] and EMT [N-cadherin (2.162±0.254 vs. 1.025±0.318), E-cadherin (0.430±0.154 vs. 1.025±0.318), P<0.05]. Conclusion GEM may induce metastasis and EMT in cholangiocarcinoma through CXCL12/CXCR4 axis. Key words: Gemcitabine; Cholangiocarcinoma; CXC chemokine receptor 4; CXC chemokine ligand 12; Epithelial-mesenchymal transtion; Metastasis

Effects of cyclic tensile stress on metabolism and apoptosis of chondrocytes inrat

Objective To investigate the effect of cyclic tensile stress (CTS) on the metabolism inand apoptosis of rat chondrocytes. Methods Primary rat chondrocytes were cultured on a Bioflex plate for one day and then stretched cyclically for 24 hours at a frequency of 0.5 Hz using a Flexcell-5000T apparatus. The cells were divided into 5 groups according to their stretching ratio: 0% (the control group), 2%, 6%, 10% and 14%. After the stretching, Col II, Aggrecan, MMP-13 and ADAMTS-5 mRNA were measured using qPCRs, and the NO and PGE2 levels were measured using ELISA kits. Moreover, TUNEL staining and Annexin V-FITC/PI were used to analyze the apoptosis of chondrocytes. Results Compared with the control group, the average levels of Col II and Aggrecan mRNA decreasedin 10% and 14% groups [(0.738±0.11) and (0.58±0.13), (0.75±0.11) and (0.55±0.09)]. In those groups, the MMP-13 [(2.47±0.47) and (2.88±0.36)] and ADAMTS-5 mRNA level [(2.39±0.33) and (2.75±0.49)], the NO [(6.96±0.96) and (8.28±0.82)] and PGE2 level [(6.83±0.66) and (7.15±0.71)] had increased significantly. In the 6% group the average levels of Col II(1.76±0.30) and Aggrecan mRNA (1.93±0.14) of 6% group were significantly higher than the control group, but the NO level of the former (3.07±0.20) was significantly lower than the control group (3.89±0.33). The apoptosis rate of chondrocytesin 2% and 6% groups were (0.065±0.013) and (0.063±0.147), without significant differences to that of the control group (0.045±0.008). However, compared with the control group, apoptosis in the 10% and 14% groups [(0.135±0.026) and (0.184±0.020)] increased significantly. Conclusion The effect of cyclic tensile stress on chondrocyte metabolism and apoptosis was magnitude-dependent. Ten percent and 14% CTS can increase the catabolism and apoptosis of chondrocytes.Ten percent and 14% strain can increase the catabolism and apoptosis of chondrocytes.Cyclic 6% strain can increase the anabolism of chondrocytes, but 2% strain has no impact on metabolism or apoptosis. Key words: Cyclic tensile stress; Chondrocyte; Apoptosis; Metabolism; Rat

The expression changes and clinical significance of PEDF in bladder mucosa and bladder cancer

Objectives To investigate the expression changes and clinical significance of pigment epithelium derived factor(PEDF) in bladder mucosa and bladder cancer. Methods Bladder cancer tissue and tissue adjacent to carcinoma from patients in our hospital diagnosed with bladder cancer were collected, mRNA expression level of PEDF and protein levels in different clinical stages of bladder cancer tissue were tested by using RT-PCR, Western Blot and immunohistochemistry. The relevant expression level of VEGF 、MMP-2、MMP-9 and MMP-12 were also analyzed by IHC. The relevance between PEDF and different physiological parameters was analyzed by collecting clinical data. Results mRNA level, protein expression level of PEDF in bladder cancer tissues were significantly lower than normal tissue. Correlation analysis showed that the positive rate of PEDF significantly associated with clinical stages, lymph node metastasis of bladder cancer (P 0.05). Conclusions There may be some relativity between PEDF expression differences and development of bladder cancer. Key words: Urinary Bladder Neoplasms; Vascular Endothelial Growth Factor A

Suppression of cartilage degeneration by intra-articular injection of heparan sulfate 6-O endosulfatase in a mouse osteoarthritis model.

We previously reported that heparan sulfate 6-O endosulfatases (Sulfs) were expressed in articular cartilage, and that the Sulf-1 knockout mouse developed severe knee osteoarthritis. In this study, we hypothesised that intra-articular injection of Sulf-1 would prevent cartilage degeneration. After confirming that 1 mg/ml Sulf-1 did not induce ATDC5 cell death in vitro, gene expression of type II collagen and matrix metalloproteinase (MMP)-13 in the presence of Sulf-1 (1 100 ng/ml) were determined by quantitative real-time polymerase chain reaction. Sulf-1 was also injected intra-articularly into mice following surgical destabilisation of the medial meniscus to produce a model of osteoarthritis, and cartilage degeneration was evaluated by safranin O and MMP-13 staining. We also investigated fibroblast growth factor 2 (FGF2)/extracellular signal-regulated kinase (Erk) cell signalling by western blotting. Exposure to Sulf-1 in vitro increased type II collagen expression and decreased MMP-13 expression in a concentration-dependent manner. Sulf-1 injection into the mouse osteoarthritic knee significantly suppressed glycosaminoglycan loss and MMP-13 expression. Erk1/2 signalling pathway activation was significantly reduced by Sulf-1 and FGF2. These findings indicate that Sulf-1 prevents cartilage degeneration by suppressing MMP-13 via an effect on FGF2/Erk1/2 signalling.

LINC00312 inhibits the migration and invasion of bladder cancer cells by targeting microRNA-197-3p

To investigate the influence of the long non-coding RNA LINC00312 on bladder cancer (BC) cell invasion and metastasis by targeting miR-197-3p. BC and corresponding adjacent tissues were collected. LINC00312 and miR-197-3p were measured, and their correlation was detected through quantitative real-time PCR (qRT-PCR). BC cell line T24 was transfected and grouped (five groups) according to different transfection conditions. A scratch test was applied to analyze cell migration, and a Transwell assay was used to test cell invasion ability. Western blotting was to measure matrix metalloproteinase (MMP)-2, MMP-9, and the tissue inhibitor of metalloproteinase 2 (TIMP2) protein levels. qRT-PCR indicated that LINC00312 expression was lower but miR-197-3p expression was higher in BC tissues compared with adjacent tissues; LINC00312 was negatively correlated with miR-197-3p. The migration test revealed that the downregulation of miR-197-3p and overexpression of LINC00312 inhibited cell migration and invasion abilities, while the overexpression of miR-197-3p and the upregulation of LINC00312 promoted cell migration and invasion. BC cells with downregulated miR-197-3p or upregulated LINC00312 had low MMP-2 and MMP-9 levels but high TIMP2. LINC00312 inhibited BC cell invasion and metastasis through mediating miR-197-3p.

Effect of quercetin on the biological characteristics of human glioma U251 cells

Objectives To Study the effect of quercetin on the biological characteristics of human glioma U251 cells and to investigate its possible mechanisms. Methods The U251 cells were cultured in vitro. The effect of quercetin on the proliferation of U251 cells was detected by CCK8 assay. Flow cytometry was used to detect the effect of quercetin on the U251 cell cycle and apoptosis. Scratch test and Transwell test were used to observe the effect of quercetin on metastasis and invasion of U251 cells. Western blot was used to detect the effects of quercetin on protein expression levels of U251 cell Bax, Bcl-2, MMP-9, and MMP-2. Results Quercetin could inhibit the proliferation ability in vitro of U251 cells. IC50 at 24 h and 48 h were 113.65 μg/ml and 48.61 μg/ml respectively. Flow cytometry showed that quercetin acting on U251 cells might have some changes, such as the upregulation of peak ratio, the downregulation of G0/G1 phase rate, and upregulation of G2/M phase rate. The migration and invasion abilities of the U251 cells pretreated by quercetin for 24 h were decreased significantly compared with the control group, and the 20 μg/ml group was lower than the10 μg/ml group (P<0.05). After the U251 cells being treated with quercetin, the Bax expression level was upregulated, the Bcl-2, MMP-9, and MMP-2 protein expression levels were decreased, and there was significant difference between the 10 μg/ml group and the 20 μg/ml group (P<0.05). Conclusions Quercetin has significant cytotoxic effect in vitro on U251 cells. It can induce apoptosis of U251 cells, causing G2/M phase arrest, decreasing the cell migration and invasion abilities. These effects may be associated with the decrease of Bcl-2/Bax ratio and the downregulation of MMP-9 and MMP-2 protein expression levels. Key words: Glioma; Quercetin; Apoptosis; Cell migration assays; Neoplasm invasiveness

Experimental study on evaluating testicular ischemia/reperfusion injury with novel lipid microbubbles with matrix metalloproteinase-9 targetin in rabbit

Objective To investigate the change of MMP-9 on moderate ischemia / reperfusion injury in rabbit testicles, and to explor the the feasibility of evaluating complete ischemical reperfusion injury(IRI) of the testis by contrast-enhanced ultrasonography with microbubbles(MB) targeted to TMB9 in rabbits. Methods MB and TMB9 were prepared. Twenty-four white rabbits were randomly divided into control group (group SH-1 and group SH-2, threaded but not ligated), ischemia / reperfusion group(group IRI-1 and group IRI-2, selected one side randomly to have testicular complete torsion surgery 4 h after ligation, released to reperfusion 2 h respectively), every group include 6 rabbits. The operated testes of group SH-1 and gruoup IRI-1 were resected after operating for immunohistochemical staining and the mean optical density of MMP-9. Contrast-enhanced ultrasonography of the bilateral testes were performed with MB or TMB9 in group SH-2 and group IRI-2 before IRI, and after IRI. All images were stored in the machine and using professional CEU software to analyse the contrast images. The contrast curve of rabbits′ testis were evaluated [include slope, time to peak(TP), peak intersity(PI), half time(DT/2), the mean transit time(MTT) and area under time-intensity curve]. Then the operated testes of group IRI-2 were resected after operating for the mean optical density of MMP-9. Results Preoperative, the ultrasonic imaging manifestations and the imaging parameters with MB and TMB9 did not see obvious difference between group SH-2 and group IRI-2. After IRI, the testicle of group IRI-2 with TMB9 showed TP forward, PI rise and DT/2 delay compared with group IRI-2 with MB. Immunohistochemistry showed a significantly increase in the expression of MMP-9 in the vascular basement membrane and interstitial of the IRI testes, but did not in those of the control. The mean optical density of MMP-9 of the testicle in group IRI-2 showed lower than that in group IRI-1 after IRI. Conclusions In a testicular IRI model, MMP-9 can be found in vascular basement membrane and interstitial. Contrast-enhanced ultrasonography with TMB9 can be used to evaluate tesicular ischemia-reperfusion injury. Key words: Testis; Reperfusion injury; Microbubbles; Matrix metalloproteinase 9

CLC-3 Cl- channel-mediated invasion and migration of human ovarian cancer cells.

To investigate the potential role of CLC-3, a member of the voltage-gated chloride channel (CLC) superfamily, in invasion and migration of ovarian cancer cell line SKOV3. CLC-3 antisense oligonucleotides were transfected into ovarian cancer cell line SKOV3, and its effects on cell invasion and migration were analyzed by using Transwell chamber assay and wound healing assay in vitro. The efficiency of CLC-3 antisense was determined with RT-PCR and Western blotting. The protein concentrations of matrix metalloproteinase (MMP)-2, MMP-9, and vascular endothelial growth factor (VEGF) were determined using ELISA kits. Cell volume measurements were performed. Studies in vitro revealed that the CLC-3 antisense inhibited invasion and migration of ovarian cancer cell line SKOV3. CLC-3 antisense treatment decreased protein levels of MMP-2, MMP-9, and VEGF in culture medium of SKOV3 cells. In addition, the authors found that the capability for regulatory volume decrease (RVD) was much attenuated in SKOV3 cells transfected with CLC-3 antisense. These results strongly suggest that CLC-3 may get involved in proliferation, invasion, and migration of ovarian cancer cells and thus may be a useful therapeutic target.

Effects of recombinant adenovirus with high expression of osteopontin gene on malignant biological behaviors of hepatocellular carcinoma cells

Objective To study the effect of recombinant adenovirus with high expression of osteopontin (OPN) gene on malignant biological behaviors of hepatocellular carcinoma (HCC) cells. Methods HCC (HepG2) cells were cultured and divided into rAd-OPN group and rad NC group. HCC cells in rAd-OPN group were transfected with recombinant adenovirus with high expression of OPN gene, and thsose in NC group were transfected with recombinant adenovirus containing invalid fragment. The expression of OPN, and cell migration, invasion and metastasis associated genes was detected. Results At 12, 24, 36, and 48 h after adenovirus transfection, mRNA expression of OPN in rAd-OPN group was 1.93±0.25, 2.58±0.30, 3.61±0.57 and 3.88±0.63, cell migration rate was (18.20±2.76)%, (43.76±7.17)%, (62.48±9.25)% and (77.33±10.24)%, and transmembrane cell number was (25.24±4.26), (32.13±4.97), (40.27±6.24) and (47.95±7.25) cells respectively, significantly higher than in rAd-NC group (P<0.05). At 48 h after transfection of adenovirus, mRNA expression of matrix metalloproteinase (MMP)-2, MMP-9, urokinase-type plasminogen activator (uPA) in rAd-OPN group was respectively 2.32±0.49, 1.78±0.28, 3.14±0.51, significantly higher than that in rAd NC group (P<0.05). Conclusion Recombinant adenovirus with high expression of OPN gene can promote the migration, invasion and up-regulate the metastasis-related gene expression. Key words: Carcinoma, hepatocellular; Osteopontin; Migration; Invasion

Mechansim of resveratrol inhibiting interleukin-6-induced invasion of human gastric cancer SGC7901 cells

Objective To elucidate the inhibitory effect of resveratrol on interleukin (IL)-6-induced invasive ability of SGC7901 cells and the molecular mechanism. Methods The cell viability of cells treated with resveratrol was evaluated by methyl thiazolyltetrazolium (MTT) assay. The SGC7901 cells were treated with IL-6 and non-cytotoxic concentration of resveratrol. The invasion of SGC7901 cells was detected by Boyden chamber assay. Matrix metalloproteinase (MMP)-2 activity was determined by gelatine zymography assay. The changes of the related proteins were detected by Western blotting. Results Resveratrol was not toxic to SGC7901 cells at the concentration under 30 μmol/L. The invasion ability of IL-6-induced SGC7901 cells was decreased about 20% after treatment with 20 μmol/L resveratrol for 24 h, accompanied by the inhibition of MMP-2 secretion for 50%, as well as the levels of phosphorylated extracellular regulated protein kinases 1/2 (p-ERK1/2). Conclusion Resveratrol of 20 μmol/L inhibits invasion of SGC7901 cells possibly through the suppression of the activation of ERK signaling pathway, subsequently exerting inhibitory effect on MMP-2. Key words: Resveratrol; Extracellular signal regulated kinase 1/2; Gastric carcinoma

7 Association between low-dose aspirin use, monocyte biomarkers, matrix metalloproteinases and outcome in HFpEF

Introduction Most community heart failure patients present with preserved ejection fraction (HFpEF) associated with inflammation, monocytosis, endothelial dysfunction and platelet activation. We investigated the clinical relationship between low-dose aspirin and outcome in HFpEF and the effects of aspirin on monocyte function and cell-cell interactions in-vitro. Methods In a retrospective analysis of HFPEF patients, we identified 150 patients taking low-dose (75 mg/day) aspirin and age/sex-matched HFPEF controls not taking aspirin over a 3 year follow-up period. From this cohort, we also studied HFPEF age/sex-matched patients (14 aspirin, 14 non-aspirin) using primary monocyte isolation, monocyte qPCR, serum matrix metalloproteinase (MMP) and inflammatory marker assays. Finally, primary monocytes were isolated from 6 healthy volunteers, analysed using flow cytometry for monocyte-platelet complexes, monocyte chemotaxis assays, and co-cultured with platelet releasate (PR, 16h) with and without aspirin. Results Aspirin was associated with higher overall survival and lower HF hospitalisations over the 3-year follow up period (HR = 0.605, 95% confidence interval, 0.389–0.961). Serum MMP2 and sCD163 were significantly reduced in aspirin HFPEF versus matched HFPEF controls. The majority (88%) of donor monocytes were complexed with platelets. Monocyte incubation with PR caused cell activation, increased release of matrix metalloproteinas and MCP1 as well as increased monocyte invasiveness in an in-vitro transwell assay. Inflammatory cytokines (IL1alpha, IL1beta, CCL17) and gene expression of PSGL was reduced by aspirin as was monocyte invasion was reduced by 50% with aspirin treatment (p Conclusion This retrospective study shows an association between the use of low-dose aspirin and better clinical outcome in HFpEF, associated with reduced sCD163 and circulating matrix metalloproteinase. These effects may be related to antiplatelet mediated modulation of monocyte function. Further work is required to evaluate antiplatelet strategies in HFpEF.

Research progress of genetic susceptibility genes associated with intervertebral disc degeneration

The process of intervertebral disc degeneration, which could result in intervertebral disc structural and functional change, is a chronic one with multiple factors. The pathophysiologic process is still not completely find out. More and more research reports manifest that certain gene polymorphism also lead to increased risk of intervertebral disc degeneration except environmental factors. Discussions about related genetic factors and their pathophysiological role in the process of degeneration could have a further understanding to disease development. Elucidating genetic components which are associated with degeneration could not only provide insights into the mechanism of the process, but also have clinical significance for early diagnosis and prevention. In order to have a thorough understanding of functional role played by different genes, this paper summarize polymorphism and disease correlation by selecting 15 genes after reviewed the related literature published in recent years. Genetic polymorphisms in 15 genes have been analyzed in association with intervertebral disc degeneration, including aggrecan, collagen Types Ⅰ, Ⅸ and Ⅺ, fibronectin, HAPLN1, CILP, MMP-1, 2 and 3, PARK2, IL-1, 6 and VDR. Each genetic polymorphism codes for a protein which has a functional role in the pathogenesis of disease. Among the 15 genes analyzed, polymorphisms in aggrecan, Type Ⅸ collagen, MMP3, IL1, IL6 and VDR show the most promise as functional variants. Genetic studies are necessary for understanding the mechanism of the degeneration. Relevant genetic information could be used as a predictive model for determining individuals’ risk for intervertebral disc degeneration eventually.

Effect of triggering receptor expressed on myeloid cells 2 overexpression on airway inflammation and remodeling in mice with allergic asthma

To investigate the effect of triggering receptor expressed on myeloid cells 2 (TREM-2) overexpression on airway inflammation and remodeling in mice with asthma. A total of 40 BALB/c mice were randomly divided into normal control, asthma, empty vector, and TREM-2 overexpression groups (n=10 each). Ovalbumin (OVA) sensitization and challenge were performed to establish the model of asthma. The mice in the control group were given normal saline, and those in the empty vector and TREM-2 overexpression groups were transfected with adenovirus vector and TREM-2 adenovirus, respectively. RT-PCR and Western blot were used to measure the expression of TREM-2, MMP-2, MMP-9, ADAM33, and ADAM8. Bronchoalveolar lavage fluid (BALF) was collected to perform cell counting and classification. ELISA was used to measure the total serum level of IgE and the levels of cytokines in BALF. Compared with the control group, the asthma group showed significant reductions in the mRNA and protein expression of TREM-2 (P<0.05), a significantly increased level of Th2 cytokine (P<0.05), and significantly increased numbers of total cells and classified cells. Compared with the asthma group, the TREM-2 overexpression group showed a significantly reduced level of Th2 cytokine (P<0.05), a significantly reduced level of IgE (P<0.05), and significantly reduced numbers of total cells and classified cells (P<0.05), as well as significantly downregulated expression of the inflammatory factors and growth factors MMP-2, MMP-9, TGF-β1, ADAM8, and ADAM33 (P<0.05). TREM-2 overexpression significantly alleviates airway inflammation and airway remodeling in mice with asthma and may become a potential target for the prevention and treatment of childhood asthma.

Expression of Sp1 in NK/T cell lymphoma cell lines and its influence on cell invasion

Objective To identify the expression of transcription factor Sp1 in NK/T-cell lymphoma (NK/TCL) cell lines and to investigate the role of Sp1 in regulation of cell invasion. Methods Real-time PCR, immunofluorescence and Western blot were performed to detect the expression of Sp1 in NK/TCL cell lines SNK-1 and SNK-6 and normal NK cells. Expression levels of IGF-1R and MMP-2 were measured by real-time PCR and Western blot, respectively. Transwell assay was applied to observe the effects of mythramycin A(MIT) on cell invasion. Results Sp1 expression in mRNA and protein were over-expression in NK/TCL cell lines SNK-1 and SNK-6 when compared with normal NK cells. Inhibition of Sp1 by MIT remarkably reduced expression of IGF-1R and MMP-2 in SNK-1, SNK-6 and as a result, or significantly suppressed cell invasion. Expression levels of Sp1 mRNA in SNK-1 and SNK-6 were (9.4±0.3) and (10.6±0.3) foldsincrease as compared with that of control group, respectively(P=0.005 2, P=0.003 7). Levels of Sp1 protein were (5.4±0.3) and (8.6±0.5) foldsincrease times than control groups, respectively(P=0.008 3, P=0.006 9). Inhibition of Sp1 by MIT (100 nmol/L) remarkably reduced expression levels of IGF-1R mRNA by (83.9±3.7)% and (65.8±4.2)% (P= 0.008 2, P= 0.009 7) as compared with controls. Meanwhile, levels of IGF-1R protein were reduced by (51.5±7.1)% and (49.6±9.1)% (P= 0.017 8, P= 0.015 5) as compared with control group. Inhibition of Sp1 by MIT (100 nmol/L) significantly reduced cell invasion and MMP-2 expression in the two cell lines,the cell invasion rates were reduced by (29.6±6.4)% and (37.2±7.6)% (P=0.041 8, P= 0.037 2) in SNK-1 and SNK-6 as compared with control group. The MMP-2 protein levels were found to be (52.7±4.7)% and (29.7±5.6)% (P= 0.028 6, P= 0.020 2) of control group. Conclusion Sp1 is over-expressed in NK/TCL cell lines, and it promotes NK/TCL cell invasion by up-regulating IGF-1R and further increasing MMP-2 expression. Key words: Natural killer/T-cell lymphoma; Transcription factor Sp1; Insulin-like growth factor 1 receptor; Cell invasion

Matrix metalloproteinase and colorectal cancer

Matrix metalloproteinase (MMP) plays an important role in the degradation of extracellular matrix components, which is crucial for tumor growth, invasion and metastasis. As one with the largest relative molecular mass in MMP family, MMP-9 takes part in the generation and progression of colorectal cancer. Research shows that MMP-9 can be used for the early diagnosis of colorectal cancer. The expression levels of MMP-9 in tissue and plasma are negatively correlated with prognosis of the postoperative patients with colorectal cancer. In addition, targeted therapy drugs will be designed on the basis of MMP-9 expression increased in colorectal cancer. Key words: Matrix metalloproteinase 9; Colorectal neoplasms; Diagnosis; Prognosis; Therapy

Effect of tumor-associated macrophages on invasion and metastasis of gastric cancer cells

To explore the influence of tumor-associated macrophages(TAMs) on the ability of invasion and metastasis of gastric cancer cells, and its associated mechanism. Immunohistochemistry was used to examine the expression of TAM in 10 samples of normal gastric mucosa and 15 samples of gastric cancer tissues from sample bank of Department of Pathology, Union Hospital. Phorbol 12-myristate 13-acetate(PMA) and macrophage colony stimulating factor (M-CSF) were used to make THP-1 monocytes differentiate into TAMs. AGS gastric cancer cells were divided into two groups: experiment group was cultured with RPMI/1640 condition medium containing 50% TAM and control group was cultured with RPMI/1640 complete medium. The ability of invasion and metastasis of gastric cancer cells was measured by Transwell assays. Real-time PCR and Western blot were applied to detect the expression of MMPs and its inhibitor TIMPs before and after stimulation of TAMs. Immunohistochemistry results showed that CD68(+) cell number in normal gastric mucosa tissue was significantly less than that in gastric cancer tissue [(11.3±0.8)/HP vs. (31.6±1.4)/HP, P<0.000 1]. When treated with PMA and M-CSF, THP-1 cells were differentiated into type M2 TAMs with high expression of specific markers CD68, CD163, CD204 and CD206. Transwell test revealed that the number of piercing cells in the experimental group was significantly more than that in control group [(36.8±1.1)/HP vs. (12.8±0.9)/HP, t=17.5, P=0.000). Compared to control group, the expression of MMP-2, MMP-9 mRNA in experimental group respectively increased by 1.61 and 1.87 folds(P=0.017 and P=0.009). Protein level of MMP-2, MMP-9 was up-regulated accordingly. The expression of TIMP-1 and TIMP-3 mRNA was not significantly different between two groups(P=0.120 and P=0.096). TAMs may promote the invasion and metastasis of gastric cancer cells through increasing expression of MMP-9 and MMP-2, which may be one of the mechanisms of gastric cancer development.

Effect of interleukin 17 on invasion of human colon cancer cells

To investigate the effect and its possible mechanism of interleukin-17 (IL-17) on invasion and metastasis of human colon cancer cells. IL-17 was added into the culture media of human colon cancer cells SW480 and LOVO. Cells were divided into 4 groups: SW480 control group (SW480 cells), LOVO control group (LOVO cells), SW480 experiment group (50 μg/L IL-17+SW480 cells), and LOVO experiment group (50 μg/L IL-17+LOVO cells). Cell growth was measured by CCK-8 assay. The proliferation rate(%)=[(Aexperiment group-Ablank)/(Acontrol group-Ablank)]×100%). The ability of cell invasion and migration was measured by transwell assay. Real time-PCR was used to detect mRNA expression of VEGF and MMP-9. Western blot was performed to detect protein expression of STAT3, p-STAT3, VEGF and MMP-9. Enzyme-linked immunosorbent assay (ELISA) was applied to measure the protein content of VEGF and MMP-9 in the supernatant. After cultivation for 24, 48 and 72 hours, CCK-8 assay revealed that the proliferation rate of SW480 was 1.18%±0.07%, 1.42%±0.09%, and 1.62%±0.08%; the proliferation rate of LOVO was 1.13%±0.02%, 1.32%±0.05% and 1.73%±0.02% in experiment group. Transwell experiments showed that after cultivation with IL-17 for 24 hours, number of invasion cell in experimental groups (SW480: 34.00±0.45, LOVO: 41.60±0.51) was higher as compared to corresponding control groups (SW480: 4.53±0.14; LOVO: 3.67±0.33) with significant differences (SW480: t=-76.026, P=0.001; LOVO: t=-81.580, P=0.005). The number of migration cell in experimental groups (SW480: 36.40±0.51, LOVO: 46.40±0.68) was higher as compared to corresponding control groups (SW480: 7.83±0.69; LOVO: 6.67±0.48) with significant differences (SW480: t=-51.542, P=0.003; LOVO: t=-49.265, P=0.005). Real-time PCR results revealed that after cultivation with IL-17 for 24 hours, VEGF and MMP-9 mRNA relative expression levels in experimental groups (SW480: VEGF:1.53±0.12, MMP-9: 2.44±0.23; LOVO: VEGF: 2.96±0.35, MMP-9: 3.38±0.55) were higher than those in control groups (both 1) with significant differences (VEGF: t=3.799, P=0.043; MMP-9: t=5.254, P=0.039). Western blot illustrated that after cultivation with IL-17 for 24 hours, STAT3, p-STAT3, VEGF and MMP-9 proteins relative expression levels in experimental groups were significantly higher that those in control groups (SW480:STAT3: t=3.233, P=0.023; p-STAT3: t=3.954, P=0.032; VEGF: t=3.201, P=0.025; MMP-9: t=3.154, P=0.029; LOVO: STAT3: t=3.788, P=0.012; p-STAT3: t=2.662, P=0.040; VEGF: t=4.118, P=0.035; MMP-9: t=4.268, P=0.030). ELISA indicated that content of VEGF and MMP-9 in the supernatant of experimental groups (SW480: VEGF 5 491.41±63.22, MMP-9: 21.43±1.35. LOVO: VEGF: 8 631.46±129.59, MMP-9: 178.32±3.20) were higher than those in control groups (SW480: VEGF:4 456.32±87.56, MMP-9:18.57±2.44. LOVO: VEGF: 8 122.38±108.66, MMP-9: 163.22±6.89) with significant differences (SW480: VEGF: t=6.993, P=0.037; MMP-9: t=5.587, P=0.040. LOVO: VEGF: t=7.013, P=0.044; MMP-9: t=6.762, P=0.043). IL-17 may be able to activate STAT3 signal transduction pathway in vitro through up-regulation of VEGF and MMP-9 expression, thereby enhancing the invasion and migration of colon cancer SW480 and LOVO cells.

Shear stresses modulate chondrogenic and osteogenic genes expression in human bone marrow derived stem cells

Background: Mechanical stimuli in the microenvironment may provide cues for differentiation, maintenance of phenotypes, and extracellular matrix homeostasis. This study investigated bone marrow-derived stem cells (BMSCs) response to mechanical stresses in terms of differentiation and the extracellular matrix/matrix metalloproteinase genes expression. Materials & Methods: Human BMSCs were exposed to shear stresses (20 rpm or 50 rpm) for 2, 5, or 10 days with or without osteogenic induction medium. The expression of chondrogenic (type II collagen and aggrecan) and osteogenic (type I collagen, alkaline phosphatase, osteopontin, osteocalcin) phenotypic genes and matrix metalloproteinase genes (aggrecanse, MMP-2, and MMP-9) were analyzed by reverse transcription polymerase chain reaction. In vitro differentiation of BMSCs after shear stress treatment were analyzed by von Kossa staining on monolayer cultures and safarin-O staining on pelleted cells. Results: Shear stress at 20 rpm significantly upregulatedexpression of type II collagen on the 5th day, type I collagen on the 2nd, 5th, and 10th day, osteopontin on the 2nd and 5th day, and osteocalcin on the 5th day as compared to shear stress at 50 rpm. Combination of osteogenic induction medium and shear stress at 20 rpm decreased the mRNA expression levels of chondrogenic genes (type II collagen on all time points and aggrecan on the 5th and 10th day) as compared with shear stress alone. It also decreased the expression of osteogenic genes (type I collagen, alkaline phosphatase, and osteopontin) on the 10th day as compared to osteogenic induction medium. Concomitantly, shear stress upregulated aggrecanase on the 10th day, MMP-2 on the 10th day, and MMP-9 on the 5th and 10th day as compared with osteogenic induction medium. Shear stress increased mineralization of the extracellular matrix as shown by von Kossa staining on the 2nd, 5th, and 10th day. Chondrogenic differentiation by cell pellet analysis found similar amount of proteoglycans but less organized extracellular matrix in shear stress-treated BMSCs as compared to control cells. Discussion: Although shear stress could modulate chondrogenic gene expression as well as osteogenic genes expression in BMSCs, the combination of shear stress inhibited the expression of osteogenic phenotypic genes induced by osteogenic induction medium. Parallel studies revealed that shear stress concomitantly modulate the expression of extracellular matrix genes and the matrix metalloproteinase genes. In vitro differentiation analysis found that shear stress may enhance osteogenesis but interfere with chondrogenesis. Our results may provide data for the design or development of in vitro bioreactor systems to control lineage differentiation of stem cells for tissue engineering. More studies on the mechanotransduction pathways are needed in the future.