ST14/MT-SP1 influences expression of MT2-MMP and TIMP-2 to promote invasiveness of colorectal cancer cells

AIM: To evaluate how ST14/MT-SP1 overexpression alters invasiveness of colorectal cancer cells.METHODS: Human full length ST14/MT-SP1 gene was transiently transfected into colorectal cancer (RKO) cell lines.The expression products were purified by chromatography on Ni2+-affinity resin column and analyzed for gelatinase activity by gelatin zymography.Cell invasion and migration were determined by ECM invasion assay in vitro.RNA was isolated from stable ST14-transfected RKO cells and a cDNA microarray was utilized to detect the change in expression of MMPs and TIMPs.Real-time quantitative PCR was used to validate the change of expression.RESULTS: The full length ST14/MT-SP1 was transfected and expressed in RKO cells.The expressed protein showed a gelatinase activity.In addition,invasiveness of RKO was significantly enhanced by ST14/MT-SP1 overexpression (P<0.01).Blockage of ST14/MT-SP1 resulted in a decrease in the invasiveness of SW480 and SW620 (P<0.01).Furthermore,MT2-MMP (MMP-15) expression was significantly up-regulated (2.5-fold change) and TIMP2 down-regulated (0.35-fold change) by ST14/MT-SP1 overexpression in RKO.CONCLUSION: ST14/MT-SP1 overexpression results in an increase in invasiveness of colorectal cancer RKO cells.Increased invasiveness is due to an increase in the gelatinase activity of ST14/MT-SP1 and a change in up-regulated MT2-MMP and down-regulated TIMP-2 expression.Therefore,ST14/MT-SP1 overexpression enhances invasiveness of colorectal cancer cells.     

Regulation of MMP-2 and TIMP-3 expression by uPA signal transduction system in human bone giant cell tumor

AIM: To study effects of urokinase-type plasminogen activator (uPA) signal transduction on expression of matrix metalloproteinase-2 (MMP-2) and tissue inhibitor of matrix metalloproteinase-3 (TIMP-3) in giant cell tumor of bone (GCT). METHODS: Expression of uPAR, MMP-2 and TIMP-3 in GCT tissue was detected by immunohistochemistry. Phosphorylation level of mitogen-activated protein kinase (p44) in uPA/uPAR signal pathway in cultured GCT cells was detected by immunoprecipitation. The expression of MMP-2 and TIMP-3 in cultured cells after treatment with uPA-ATF or anti-uPAR antibody was also detected by Western blotting. RESULTS: 1) Urokinase-type plasminogen activator receptor (uPAR) was positive on the cell membrane and in cytoplasm of some mononuclear stromal cells (MSCs) and multinucleated giant cells (MGCs); 2) MMP-2 was positive in the cytoplasm and on the cell membrane of almost all of MSCs and some of MGCs. The polar distribution of MMP-2 in the cytoplasm of MGCs was especially obvious; 3) The expression of TIMP-3 of some MSCs and MGCs in GCT was much lower than MMP-2. The positive signal also showed a prominent polarity; 4) After treatment with uPA-ATF, the phosphorylation level of p44 in GCT cultured cells was much higher than the control. Addition of anti-uPAR antibody in the cells remarkably down-regulated the phosphorylation level of p44 as compared with the control group, suggesting that uPA-ATF participates cell signal transduction and this reaction can be inhibited by anti-uPAR antibody; 5) uPA-ATF cell signal pathway up-regulated expression of MMP-2 and TIMP-3, while anti-uPAR antibody down-regulated the expression of MMP-2 and TIMP-3. CONCLUSION: These results demonstrate for the first time that uPA-ATF directly regulates the expression of MMP-2 and TIMP-3 by signal transduction pathway, and the over-expression of MMP-2 and TIMP-3 may play an important role in local osteolysis of GCT.     

Effects of chloroquine on autophagy and collagen metabolism in activated hepatic stellate cells in vitro

AIM: To explore the effects of chloroquine (CQ) on collagen Ⅰand collagen Ⅲ expression in activated rat hepatic stellate cell line HSC-T6 and the possible mechanism.METHODS: Transforming growth factor-β1 (TGF-β1) was used to activate HSC-T6 cells and 3 doses of CQ was administered for 24 h. The cells were divided into 5 groups as follows:control group, TGF-β1 group, TGF-β1+CQ (15 μmol/L) group, TGF-β1+CQ (30 μmol/L) group and TGF-β1 + CQ (60 μmol/L) group. Western blot was used to determine the expression of LC3-Ⅱ/LC3-I, P62 and α-SMA in activated HSC-T6 cells. The expression of collagen I and collagen Ⅲ was detected by immunocytochemical staining, Western blot and RT-qPCR. Western blot and RT-qPCR were also used to detect the expression of matrix metalloproteinase-13 (MMP-13), tissue inhibitor of metalloproteinase-1 (TIMP-1) and TIMP-2 at mRNA and protein levels.RESULTS: The ratio of LC3-Ⅱ/LC3-Ⅰ and P62 expression were increased after CQ intervention. Moreover, they were significantly higher in the TGF-β1+CQ groups than those in TGF-β1 group (P<0.01). The expression of collagen I and collagen Ⅲ at mRNA and protein levels was significantly increased in all TGF-β1+CQ groups as compared with TGF-β1 group (P<0.01), and it was markedly increased among TGF-β1+CQ groups in a dose-dependent manner. The expression of MMP-13 at mRNA and protein levels was significantly lowered and that of TIMP-1 and TIMP-2 was significantly increased in TGF-β1+CQ groups as compared with TGF-β1 group (P<0.05).CONCLUSION: Inhibition of autophagy by CQ in activated HSC-T6 cells up-regulates the expression of collagen I and collagen Ⅲ in a dose-dependent way, probably due to reduction of MMP-13 and enhancement of TIMP-1 and TIMP-2 expression.     

Regulation by reactive oxygen species of matrix metalloproteinase-1, 3 and tissue inhibitor of matrix metalloproteinase-1 in smooth muscle cells

AIM: To understand whether reactive oxygen species promote the rupture of atherosclerotic plaques by regulating the balance of matrix metalloproteinase-1, 3 (MMP-1, 3) and tissue inhibitor of matrix metalloproteinase-1 (TIMP-1) in smooth muscle cells. METHODS: Aortic smooth muscle cells from 4-6months-healthy abortive fetuses were incubated for 24 hours with xanthine (100 μmol/L) and xanthine oxidase (5 U/L) in vitro . MMP-1, 3 and TIMP-1 in the concentrated culture media were measured by Western blotting ( n =3 independent experiments). RESULTS: Incubation with xanthine/xanthine oxdiase decreased the amount of MMP-1 in the aortic smooth muscle cells (21.2%±5.5% of the control group), and pro-MMP-1 was activated completely. Reactive oxygen species (ROS) also activated pro-MMP-3, and increased the production of MMP-3 in the aortic smooth muscle cells. On the other hand, ROS inhibited the production of TIMP-1 in the aortic smooth muscle cells. CONCLUSION: It is complicated that ROS regulates the balance of MMPs and TIMPs. ROS may contribute to matrix degradation and the rupture in the atherosclerotic plaques.     

Effects of genistein on proliferation,apoptosis and invasiveness in methotrexate-resistant human choriocarcinoma JAR/MTX cells and it s mechanism

AIM:To study the effects of genistein on JAR/MTX cell proliferation, apoptosis and invasion and it's mechanism in vitro. METHODS:MTT assay, Annexin-Ⅴ and propidium iodide label analysis and invasion assay were used to determine the effects of genistein on proliferation, apoptosis and invasiveness in JAR/MTX methotrexate- resistant human choriocarcinoma cells. RT-PCR was used to estimate the relative mRNA amounts of estrogen receptor(ER), MTA3 and snail in the cells. Western blotting and gelatin zymography assay were used to estimate the relative protein amounts of MMP-2, MMP-9 and E-cadherin in the cells. RESULTS:After treatment of genistein, the proliferation and invasiveness of JAR/MTX cells were decreased significantly in a dose-dependent manner. 10 μmol/L genistein induced apoptosis, whereas 25, 50, 100 μmol/L genistein induced apoptosis and necrosis significantly. Genistein led to an increase in ERβ, MTA3 mRNA and E-cadherin protein expression, and decreases in the amounts for snail mRNA and MMP-2 and MMP-9 protein expression of JAR/MTX cells. CONCLUSIONS:Genistein inhibits the cell proliferation by inducing cell apoptosis and necrosis. Genistein also may inhibit JAR/MTX cell invasion in part through the upregulation of E-cadherin and downregulation of MMP-2 and MMP-9. The signal transduction pathway of invasion suppression induced by genistein in JAR/MTX cells may be as follows: MTA3→snail→ E-cadherin.     

Effects of pirenzepine on expression of MMP-2 and TIMP-2 in stroma of sclera in chick myopic eyes

AIM:To observe the effect of intravitreal injection of pirenzepine on form-deprivation myopia in chicks. METHODS:Forty-one day old chicks were randomly divided into 4 groups:normal control group, form deprivation group, vehicle control group and pirenzepine injection group. The right eyes of all chicks were used as experimental eyes. The deprived eyes in vehicle control group and pirenzepine injection group received daily intravitreal injection of vehicle control solution(0.01 mol/L PBS) and pirenzepine(1%in PBS), respectively. Optical examinations such as refraction, axial length and equatorial diameter were made at the end of the 5th day. The mRNA and protein levels of matrix metalloproteinase 2(MMP-2) and tissue inhibitor of metalloproteinase-2(TIMP-2), and the activity of MMP-2 were detected by RT-PCR, Western blotting and zymography analysis,respectively. RESULTS:Refraction, axial length and equatorial diameter of the eyes in pirenzepine injection group were significantly lower than those in form deprivation group and vehicle control group(P<0.05), but those were higher(P<0.05) and the eyes were relatively myopic as compared with normal control group. The mRNA expression, protein le vels and activity of MMP-2 in pirenzepine group were significantly higher than those in normal control group(P<0.01), and were significantly lower than those in form deprivation group and vehicle control group(P<0.01, P<0.01). The mRNA and protein levels of TIMP-2 in pirenzepine group were significantly lower than those in normal control group(P<0.01), and was significantly higher than those in form deprivation group and vehicle control group(P<0.01, P<0.01). CONCLUSION:Intravitreal injection of pirenzepine may partly prevent form-deprivation myopia by modulating the expression of MMP-2 and TIMP-2 in the fibrous layer of sclera.     

Oxysterols downregulate tissue inhibitor of metalloproteinase -1 expression in vascular smooth muscle cells

AIM:To investigate the effects of oxysterols on matrix metalloproteinase (MMP) and tissue inhibitor of metalloproteinase-1 (TIMP-1) expression in vascular smooth muscle cells. METHODS:Rabbit aortic vascular smooth muscle cells were culturedin vitroand incubated with cholesterol, Triol and 25-hydroxycholesterol (25-OH), respectively. Slot blot was used to detect the mRNA expression level of TIMP-1 in vascular smooth muscle cells (VSMCs); meanwhile the protein expression level of MMP-9 and TIMP-1 was detected by immunohistology. RESULTS:Triol and 25-OH inhibited the expression of TIMP-1 compared with control and cholesterol, but have no effect on expression of MMP-9. CONCLUSION:Both Triol and 25-OH downregulated TIMP-1 expression in VSMCs.     

Relationship between the expression of MMP-2, TIMP-2 and VEGF-C in hepatocellular carcinoma with or without lymph node metastasis

AIM: To investigate the relationship between the expression of MMP-2, TIMP-2 and VEGF-C in hepatocellular carcinoma (HCC) with or without lymph node metastasis. METHODS: The expression of MMP-2, TIMP-2 and VEGF-C in 44 cases of HCC were examined using immunohistochemistry methods (SP).RESULTS: The positive expression of MMP-2, TIMP-2 and VEGF-C was associated with lymph node metastasis of HCC (P<0.05). CONCLUSIONS: There was a significant correlation between the high-expression of MMP-2 and VEGF-C, and the low-expression of TIMP-2 in lymph node metastasis of HCC. VEGF-C is an important factor promoting lymph node metastasis of HCC. The expression of MMP-2, TIMP-2 and VEGF-C may have important value in determining lymph node metastasis and prognosis of HCC.     

Effects of arsenic trioxide on activities of MMPs and expression of TIMP-1 and TGF-β1 in skin fibroblasts and polymorphonuclear neutrophils

AIM: To observe the effects of arsenic trioxide (As₂O₃) on activities of matrix metalloproteinases (MMPs), expression of tissue inhibitor of metalloproteinase-1 (TIMP-1) and transforming growth factor beta1 (TGF-β₁) in human fibroblast (hFb), and to discuss weather As₂O₃ promotes the healing of chronic skin ulcer through regulating collagen metabolism. METHODS: Zymography was used for testing activity of MMP-9 deriving from rat polymorphonuclear neutrophils (PMNs) and activities of MMP-1, MMP-2 secreted by hFb. Immunocytochemical method was used to determine the expressions of TIMP-1 and TGF-β₁. RESULTS: At the concentration of 50 mg/L, As₂O₃ elevated the activity of MMP-9 (P<0.01). At the concentration of 0.8 mg/L, As₂O₃ increased the activities of MMP-1 and MMP-2 (P<0.01, respectively). After hFb was cultured with As₂O₃ for 6 h, 12 h and 18 h, the expressions of TIMP-1 and TGF-β₁ decreased continuously (P<0.01). CONCLUSION: As₂O₃ elevates the activities of MMP-1, MMP -2 and MMP-9, also inhibits the expressions of TIMP-1 and TGF-β₁, suggesting that arsenic preparation may exert positive effect on healing chronic skin ulcer through regulating collagen metabolism.     

Effect of idazoxan on permeability of inflammatory blood-brain barrier model in vitro

AIM: To study the effect of idazoxan on the permeability of inflammatory blood-brain barrier (BBB) model in vitro and the expression of tight junction protein ZO-1.METHODS: In vitro BBB model was established by murine brain endothelial cell line bEnd.3 incubated for 7 d. The cells were treated with TNF-α (10 nmol/L) for additional 24 h to establish the inflammatory BBB model, which was pretreated with IDA at doses of 50, 100 and 200 μmol/L, respectively. The permeability was measured using fluorescein isothiocyanate-conjugated dextran (FD-40, MW 40,000), the expression of ZO-1 was detected by Western blot analysis, the distribution of ZO-1 was observed by immunofluorescence, and the mRNA expression of MMP-9/TIMP-1 was measured by RT-PCR.RESULTS: After incubated for 7 d, b.End3 cells converged to be confluent monolayer with low permeability. The inflammatory BBB model induced by TNF-α treatment displayed much higher permeability with decreased expression of tight junction protein ZO-1, destroyed distribution of ZO-1 and increased mRNA expression of MMP-9. When pretreated with IDA, the permeability was greatly decreased, the expression of ZO-1 was greatly increased, the abnormal distribution of ZO-1 was greatly ameliorated and the mRNA expression of MMP-9 was obviously reduced. The effect was most significant in IDA (200 μmol/L)-pretreated group (P<0.01).CONCLUSION: IDA directly acts on brain endothelial cells to reduce the expression of MMP-9, increase the expression of tight junction protein ZO-1 and ameliorate the destroyed distribution of ZO-1 in the inflammatory BBB, thus reversing the abnormally elevated permeability in a inflammatory BBB model in vitro induced by TNF-α.     

Effects of Maxing-Shigan decoction on airway remodeling and expression of MMP-9 and TIMP-1 in lung tissues of asthmatic mice

AIM:To investigate the effects of Maxing-Shigan decoction on airway remodeling and expression of matrix metalloproteinase-9 (MMP-9) and tissue inhibitor of metalloproteinase-1 (TIMP-1) in the lung tissues of asthmatic mice, and to explore its possible mechanism in treatment of asthma. METHODS:The BALB/c mice were divided into blank control group, model group, low-dose Maxing-Shigan decoction group, middle-dose Maxing-Shigan decoction group, high-dose Maxing-Shigan decoction group and positive control group. The mice were sensitized and challenged with ovalbumin to establish asthma model. The mice in blank control group and model group were given saline by oral administration before 30 min of suscitation. The mice in low-dose, middle-dose and high-dose Maxing-Shigan decoction groups were given Maxing-Shigan decoction at 5.0 g/kg, 10.0 g/kg and 20.0 g/kg, respectively, by oral administration before 30 min of suscitation. The mice in positive control group was given dexamethasone at 0.005 g/kg by oral administration before 30 min of suscitation. After consecutive administration for 7 d, the variations of airway responsiveness, the percentage of the goblet cells, the collagen deposition, and the eosinophil (EOS) counts in bronchoalveolar lavage fluid (BALF) of each group were observed. The protein levels of MMP-9 and TIMP-1 in the lung tissues were determined by ELISA and Western blot. The mRNA expression of MMP-9 and TIMP-1 was detected by RT-qPCR. RESULTS:Compared with blank control group, the airway responsiveness, the goblet cell percentage, the collagen deposition, the EOS counts in BALF, the protein levels of MMP-9 and TIMP-1, and the mRNA expression of MMP-9 and TIMP-1 were significantly increased in model group (P<0.01). Compared with model group, all of the indexes were reversed in low-dose, middle-dose and high-dose Maxing-Shigan decoction groups and positive control group (P<0.05 or P<0.01). CONCLUSION:Maxing-Shigan decoction improves airway remodeling in asthma model mice by down-regulating the expression of MMP-9 and TIMP-1.     

Effect of Kechuanning on airway remodeling and protein level of p-ERK1/2 in lung tissues of asthmatic rats induced by virus

AIM:To investigate the effect of Kechuanning on airway remodeling and the protein level of p-ERK1/2 in lung tissues of asthmatic rats induced by virus. METHODS:The asthmatic rat model induced by respiratory syncytial virus was established. The experimental rats were divided into normal group, asthma model group, low dose (0.33 mL/kg), middle dose (3.0 mL/kg) and high dose (10 mL/kg) of Kechuanning groups, and PD98059 (3 mg/kg) group. The airway responsiveness of the rats was measured by animal ventilator. The pathological changes of the lung tissues were observed by HE staining. PAS staining and Masson staining were used to observe goblet epithelial cells metaplasia and airway collagen deposition. The expression of matrix metalloproteinases-9 (MMP-9) and tissue inhibitor of matrix metalloproteinase-1 (TIMP-1) in the lung tissues of the rats was detected by immunohistochemical staining. The protein levels of ERK1/2 and p-ERK1/2 were determined by Western blot. RESULTS:Compared with model group, the airway responsiveness of the rats in middle dose and high dose of Kechuanning groups was significantly decreased (P<0.01), the injury of lung tissues was significantly decreased, the goblet epithelial cells metaplasia and airway collagen deposition were significantly reduced (P<0.01), and the expression of MMP-9 and TIMP-1 in the lung tissues was also significantly decreased (P<0.01). In addition, the protein level of p-ERK1/2 in high dose of Kechuanning group was significantly decreased compared with model group (P<0.01). CONCLUSION:Kechuanning may treat asthma by regulating the expression of p-ERK1/2 in the lung tissues and improving the airway remodeling symptoms of asthmatic rats induced by virus.