Long noncoding RNAs HAND2-AS1 ultrasound microbubbles suppress hepatocellular carcinoma progression by regulating the miR-873-5p/tissue inhibitor of matrix metalloproteinase-2 axis

BACKGROUND Increasing data indicated that long noncoding RNAs(lncRNAs)were directly or indirectly involved in the occurrence and development of tumors,including hepatocellular carcinoma(HCC).Recent studies had found that the expression of lncRNA HAND2-AS1 was downregulated in HCC tissues,but its role in HCC progression is unclear.Ultrasound targeted microbubble destruction mediated gene transfection is a new method to overexpress genes.AIM To study the role of ultrasound microbubbles(UTMBs)mediated HAND2-AS1 in the progression of HCC,in order to provide a new reference for the treatment of HCC.METHODS In vitro,we transfected HAND2-AS1 siRNA into HepG2 cells by UTMBs,and detected cell proliferation,apoptosis,invasion and epithelial-mesenchymal transition(EMT)by cell counting kit-8 assay,flow cytometry,Transwell invasion assay and Western blotting,respectively.In addition,we transfected miR-837-5p mimic into UTMBs treated cells and observed the changes of cell behavior.Next,the UTMBs treated HepG2 cells were transfected together with miR-837-5p mimic and tissue inhibitor of matrix metalloproteinase-2(TIMP2)overexpression vector,and we detected cell proliferation,apoptosis,invasion and EMT.In vivo,we established a mouse model of subcutaneous transplantation of HepG2 cells and observed the effect of HAND2-AS1 silencing on tumor formation ability.RESULTS We found that UTMBs carrying HAND2-AS1 restricted cell proliferation,invasion,and EMT,encouraged apoptosis,and HAND2-AS1 silencing eliminated the effect of UTMBs.Additionally,miR-873-5p targets the gene HAND2-AS1,which also targets the 3’UTR of TIMP2.And miR-873-5p mimic counteracted the impact of HAND2-AS1.Further,miR-873-5p mimic solely or in combination with pcDNA-TIMP2 had been transformed into HepG2 cells exposed to UTMBs.We discovered that TIMP2 reversed the effect of miR-873-5p mimic caused by the blocked signalling cascade for matrix metalloproteinase(MMP)2/MMP9.In vivo results showed that HAND2-AS1 silencing significantly inhibited tumor formation in mice.CONCLUSION LncRNA HAND2-AS1 promotes TIMP2 expression by targeting miR-873-5p to inhibit HepG2 cell growth and delay HCC progression.

Matrix Metalloproteinase-2,Matrix Metalloproteinase-9,and Transforming Growth Factor Beta 1 Levels in Escherichia coli-Infected Rats with Acute Pyelonephritis

Objective:To investigate the expression of matrix metalloproteinase-2(MMP-2),matrix metalloproteinase-9(MMP-9),and transforming growth factor beta 1(TGF beta 1)in the kidney tissue of rats with pyelonephritis and their relationship with pyelonephritis by establishing a rat model of acute pyelonephritis.Methods:80 male Wistar rats were randomly divided into a control group and an experimental group,with 40 rats each.The rats of the control group were injected with and saline and those of the experimental group were injected with 10μg/mL Escherichia coli(E.coli)and saline(1:100);the solutions for both groups were administered every 3 days for 7 days.The expressions of MMP-2,MMP-9 and TGF beta 1 in the kidney tissues of rats in each group were observed.Results:The expression of MMP-9 and TGF beta 1in the kidney tissue of rat acute pyelonephritis model rats was significantly higher than those of the control group(P<0.01);the MMP-9 mRNA content in the kidney tissue of the experimental group was significantly higher than that of the control group(P<0.05);the TGF beta 1 mRNA content in the renal tissue of the experimental group increased significantly compared to the(P<0.05);MMP-2,MMP-9 and TGF beta 1 began to express in the early stage of pyelonephritis until the complete formation of renal pelvic edema.The difference between groups was statistically significant(P<0.01).Conclusion:MMP-9 and TGF beta 1 are important factors regulating renal tubular epithelial cell injury and inflammatory response.

Suppression of matrix metalloproteinase-2 via RNA interference inhibits pancreatic carcinoma cell invasiveness and adhesion

AIM:To investigate the inhibitory effects of RNA interference (RNAi) on expression of matrix metalloproteinase-2 (MMP-2) gene and invasiveness and adhesion of human pancreatic cancer cell line,BxPC-3.METHODS:RNAi was performed using the vector (pGPU6)-based small interference RNA (siRNA) plasmid gene silence system to specifically knock down MMP-2 expression in pancreatic cancer cell line,BxPC-3. Four groups of different specific target sequence in coding region of MMP-2 and one non-specific sequence were chosen to construct four experimental siRNA plasmids of pGPU6-1,pGPU6-2,pGPU6-3 and pGPU6-4,and one negative control siRNA plasmid of pGPU6 (-). MMP-2 expression was measured by reverse transcription polymerase chain reaction (RT-PCR) and Western blot. Cell proliferation and apoptosis were examined by methyl thiazolyl tetrazolium (MTT) and flow cytometry,respectively. The abilities of adhesion and invasion were detected by cell adhesion assay and cell invasion assay using Transwell chambers.RESULTS:The expression of MMP-2 was inhibited and the inhibitory effects of different sequence varied. pGPU6-1 group had the most efficient inhibitory effect,followed by pGPU6-2 and pGPU6-3 groups.Invasiveness and adhesion were more significantly reduced in pGPU6-1,pGPU6-2 and pGPU6-3 groups as compared with pGPU6 (-) and blank control groups. However,no difference concerning cell proliferation and apoptosis was observed after transfection between experiment groups and control groups.CONCLUSION:RNAi against MMP-2 successfully inhibited the mRNA and protein expression of MMP-2 in the pancreatic cancer cell line,BxPC-3,leading to a potent suppression of tumor cell adhesion and invasion without affecting cell proliferation and apoptosis. These findings suggest that the RNAi approach towards MMP-2 may be an effective therapeutic strategy for the clinical management of pancreatic tumor.

Effects of hypoxia,hyperoxia on the regulation of expression and activity of matrix metalloproteinase-2 in hepatic stellate cells

AIM: To study the effects of hypoxia, hyperoxia on the regulation of expression and activity of matrix metalloproteinase-2 (MMP-2) in hepatic stellate cells (HSC). METHODS: The expressions of MMP-2, tissue inhibitor of matrix metalloproteinase-2 (TIMP-2) and membrane type matrix metalloproteinase-1 (MT1-MMP) in cultured rat HSC were detected by immunocytochemistry (ICC) and in situ hybridization (ISH). The contents of MMP-2 and TIMP-2 in culture supernatant were detected with ELISA and the activity of MMP-2 in supernatant was revealed by zymography. RESULTS: In the situation of hypoxia for 12h, the expression of MMP-2 protein was enhanced (hypoxia group positive indexes: 5.7 +/- 2.0, n=10; control: 3.2 +/- 1.0, n = 7; P【0.05), while TIMP-2 protein was decreased in HSC (hypoxia group positive indexes: 2.5 +/- 0.7, n = 10; control: 3.6 +/- 1.0, n = 7; P 【 0.05), and the activity (total A) of MMP-2 in supernatant declined obviously (hypoxia group: 7.334 +/- 1.922, n = 9; control: 17.277 +/- 7.424, n = 11; P 【 0.01). Compared the varied duration of hypoxia, the changes of expressions including mRNA and protein level as well as activity of MMP-2 were most notable in 6h group. The highest value(A(hypoxia)-A(control)) of the protein and the most intense signal of mRNA were in the period of hypoxia for 6h, along with the lowest activity of MMP-2. In the situation of hyperoxia for 12h, the contents (A(450)) of MMP-2 and TIMP-2 in supernatant were both higher than those in the control, especially the TIMP-2 (hyperoxia group: 0.0499 +/- 0.0144, n = 16; control: 0.0219 +/- 0.0098, n = 14; P 【 0.01), and so was the activity of MMP-2 (hyperoxia group: 5.252 +/- 0.771, n = 14; control: 4.304 +/- 1.083, n = 12; P 【 0.05), and the expression of MT1-MMP was increased. CONCLUSION: HSC is sensitive to the oxygen, hypoxia enhances the expression of MMP-2 and the effect is more marked at the early stage; hyperoxia mainly raises the activity of MMP-2.

Relationship between matrix metalloproteinase-2 mRNA expression and clinicopathological and urokinase-type plasminogen activator system parameters and prognosis in human gastric cancer

AIM: To investigate the relationship between matrix metalloproteinase-2 (MMP-2) mRNA expression and clinicopathologic and urokinase-type plasminogen activator (uPA) system parameter and prognosis in human gastric cancer. METHODS: Expression of MMP-2 mRNA, uPA, and uPA-R mRNA in tumor tissues and ≥5 cm adjacent normal tissues from 67 cases of gastric cancer was studied using RT-PCR and Northern blot respectively.Survival analyses were done using the Kaplan-Meier method. RESULTS: The expression rates of MMP-2 mRNA,uPA and uPA-R mRNA in tumor tissues (31%,41%,and 51%, respectively) were significantly higher than those in ≥5 cm adjacent tissues (19%, 11%, and 9%; X2=4.59,43.58, and 53.24 respectively, P<0.05,0.0001,and 0.0001, respectively). Expression of MMP-2 mRNA was significantly correlated with lymph node metastasis (metastasis: 61.9%, no metastasis: 39.1%, X2= 7.61, P<0.05),Lauren's classification of diffuse/mixed types:54.2%,intestinal type: 26.3%,X2 = 4.25, P<0.05, expression of uPA and uPA-R mRNA (uPA+: 55.1%, uPA-: 22.2% and uPA-R+: 54.9%, uPA-R-: 18.8%, X2=5.72 and 6.40 respectively, P<0.05).Kaplan-Meier survival analysis of MMP-2 mRNA expression did not show significant difference in all 67 cases, but revealed an association of the expression of MMP-2 mRNA, uPA, and uPA-R mRNA with worse prognosis (P= 0.0083, 0.0160, and 0.0094, respectively). CONCLUSION: MMP-2 may play an important role in the development of invasion and metastasis of gastric cancer.

Correlation of RECK with Matrix Metalloproteinase-2 in Regulation of Trophoblast Invasion of Early Pregnancy

To study the role of the reversion-inducing-cysteine-rich protein with Kazal motifs （RECK） gene and matrix metalloproteinase-2 （MMP-2） in the regulation of trophoblast invasion of early pregnancy. Immunohistochemistry, Western blot and gelatin zymography were used to detect the RECK protein expression localization, expression level and MMP-2 activation level in the placental tissues harvested from 52 normal pregnant women （27 in the early pregnancy, 25 in the term pregnancy）. Immunohistochemistry showed that RECK expression was found both in villous tissues of early pregnancy group and term pregnancy group and was mainly observed in cell membrane and cytoplasm of cytotrophoblasts and syneytiotrophoblasts. RECK expression increased with gestational time. RECK expression of early pregnancy group was significantly lower than that of term pregnancy group （P〈0.05）. RECK expression was significantly lower in cellular column （CC） with invasion ability. Western blot showed that the RECK protein expression in early pregnancy group was significantly lower than that in term pregnancy （P〈0.05）. The optical density values of RECK protein expression in early pregnancy group and term pregnancy group were 1.35±0.14 and 2.68±0.26, respectively, while MMP-2 activation ratio was contrary to RECK protein expression and decreased with the gestation time （P〈0.01）. The MMP-2 activation ratios of early pregnancy group and term pregnancy group were 0.46±0.05 and 0.10±0.02, respectively. The expression of the tumor inhibitory gene RECK was positively related with the invasion ability of trophoblasts, while the invasion gene MMP-2 was negatively related with the ability. The interaction between RECK and MMP-2 may play an important role in the regulation of the trophoblast invasion in early pregnancy.

Distribution and relative activity of matrix metalloproteinase-2 in human coronal dentin

The presence of matrix metalloproteinase-2 （MMP-2） in dentin has been reported, but its distribution and activity level in mature human coronal dentin are not well understood. The purpose of this study was to determine the MMP-2 distribution and relative activity in demineralized dentin. Crowns of twenty eight human molars were sectioned into inner （ID）, middle （MD）, and outer dentin （OD） regions and demineralized. MMP-2 was extracted with 0.33 mol·L-1 EDTA/2 mol·L-1 guanidine-HCl, pH 7.4, and MMP-2 concentration was estimated with enzyme-linked immunoabsorbant assay （ELISA）. Further characterization was accomplished by Western blotting analysis and gelatin zymography. The mean concentrations of MMP-2 per mg dentin protein in the dentin regions were significantly different （P=0.043）： 0.9 ng （ID）, 0.4 ng （MD）, and 2.2 ng （OD）, respectively. The pattern of MMP-2 concentration was OD〉ID〉MD. Western blotting analysis detected -66 and -72 kDa immunopositive proteins corresponding to pro- and mature MMP-2, respectively, in the ID and MD, and a -66 kDa protein in the OD. Gelatinolytic activity consistent with MMP-2 was detected in all regions. Interestingly, the pattern of levels of Western blot immunodetection and gelatinolytic activity was MD〉ID〉OD. The eoneentration of MMP-2 in human coronal dentin was highest in the region of dentin that contains the dentinoenamel junction and least in the middle region of dentin. However, levels of Western blot immunodetection and gelatinolytic activity did not correlate with the estimated regional concentrations of MMP-2, potentially indicating region specific protein interactions.

Preparation and in vitro studies of microencapsulated cells releasing human tissue inhibitor of metalloproteinase-2

Objective： To prepare microencapsulated cells releasing human tissue inhibitor ofmetalloproteinase-2 （TIMP-2）, and investigate their biological characteristics in vitro. Methods： Chinese hamster ovary （CHO） cells were stably transfected with a human TIMP-2 expression vector, encapsulated in barium alginate microcapsules and cultured in vitro. Morphological appearance of the microcapsules was observed under a light microscope. Cell viability was assessed using MTT （3-（4,5-dimethylthiazol-2-yl）-2,5-diphenyltetrazolium bromide） assay. Enzyme linked immunosorbent assay （ELISA） and reverse zymography were used to confirm the release of biologically active TIMP-2 from the microcapsules. Cryopreservation study of the microencapsulated cells was carried out using dimethyl sulfoxide （DMSO） as preservative agent. Results： The microcapsules appeared like a sphere with diameter of 300-600 ~tm. The surface of the capsule wall was clearly smooth. The microencapsulated cells survived well and kept proliferating over the 6 weeks observed. No significant difference in TIMP-2 secretion was found between encapsulated and unencapsulated cells. Reverse zymography confirmed the bioactivity of MMP （matrix metalloproteinase） inhibition of TIMP-2. The cryopreservation process did not damage the microcapsule morphology nor the viability of the cells inside. Conclusion： Microencapsulated engineered CHO cells survive at least 6 weeks after preparation in vitro, and secrete bioactive TIMP-2 freely from the microcapsules.

Vascular Endothelial Growth Factorl65-regulated Nasopharyngeal Carcinoma Cell Lines Invasion and Migration Involve Expression and Activation of Matrix Metalloproteinase-2

The effect of vascular endothelial growth factor （VEGF） overexpression on matrix metalloproteinase-2 （MMP-2） in nasopharyngeal carcinoma （NPC） cells in vitro and the possible mechanism involved were investigated, and the correlation between the expression of VEGF and MMP-2 in NPC evaluated. The NPC cells were transfected with PAd-trackVEGF165 plasmid. The expression levels of VEGF and MMP-2 mRNA and protein in NPC cells were detected by semi-quantitative RT-PCR and Western blot respectively. It was found that the expression of VEGF and MMP-2 mRNA and protein was significantly increased in NPC cells after transfection of VEGF 165. It was concluded that the expression of VEGF was correlated to the in vitro invasion of NPC cells, and the induction of MMP-2 by VEGF was a key process of NPC cell invasion.

Influence of hydrogen sulfide on zymogen activation of homocysteineinduced matix metalloproteinase-2 in H9C2 cardiocytes

Objective: To observe the influence of different concentrations of homocysteine(Hcy) and hydrogen sulfide(H2S) on the secretion and activation of matix metalloproteinase-2(MMP-2) in cardiocytes so as to search for new ways to fight against myocardial tissue fibrosis. Methods: Cardiocytes H9C2 was cultured in vitro and different concentrations of Hcy and H2 S were added for 6-h and 24-h cultivation. MTT cell proliferation assay was applied to test the activation change of cardiocytes H9C2 after affecting by different concentrations of Hcy and H2 S. ELISA and MTT were employed to detect the expression and enzymatic activity of MMP-2. Results: The H9C2 cell inhibition of activity was more significant with 1 000 μmol/L of Hcy as compared with other concentrations(P<0.001). With 2.5-100.0 μmol/L Hcy and 0.1, 1.0 and 10.0 mmol/L H2 S, the activity of H9C2 did not change significantly(P>0.05). Hcy with concentrations of 10, 50 and 100 μmol/L could increase the quantity of MMP-2 secreted by cardiocytes H9C2, and the interaction strength was concentration-dependent(P<0.05). After interacting with 100 μmol/L of Hcy for 6 h, the zymogen activation effect of MMP-2 was stronger than that of the 2.5-25 μmol/L group(P<0.05). After interacting with Hcy and H2S(1.0 mmol/L) for 6 h and 24 h, the activation effect of MMP-2 was stronger than those interacted with 10, 25, 50 and 100 μmol/L of Hcy(P<0.05). Conclusions: Hcy can increase the production of MMP-2 secreted by H9C2 cell and improve its zymogen activation. Besides, the interaction strength is concentration-dependent; while H2 S can up-regulate the activation of MMP-2 and co-promote the activation of MMP-2 with Hcy as well.

Rosiglitazone uppresses lipopolysaccharide-induced matrix metalloproteinase-2 activity in rat aortic endothelial cells via rasMEK1/2 signaling

ix metalloproteinase(MMPs) plays a key role in the pathogenesis of chronic inflammatory disease,such as atherosclerosis.Among MMPs,MMP-2 is regarded as a major proteinase in atherosclerotic plaque lesions.Peroxisome proliferator activated receptor-gamma(PPARg) ameliorates oxidative stress and the inflammatory response.The aim of the present study was to evaluate the effect of Rosiglitazone on Lipopolysaccharide(LPS)-induced MMP-2 activation as well as its possible mechanism.LPS-induced MMP-2 activity was inhibited by Rosiglitazone(PPARg agonist) in the rat aortic endothelial cells(RAEC).LPS-induced MMP-2 activation was diminished no matter exposure to NF-kB Activation Inhibitor II(JSH-23)or Ras inhibitor,farnesylthiosalicylic acid(FTS). Further study shows that LPS-induced activation of Phospho-Rho A and Phospho-MEKl/2 were significantly inhibited by Rosiglitazone.The activation of NF-kB p65 in the nuclear extract of cells was also significantly suppressed by Rosiglitazone, moreover,the expression of NF-κB p65 was partly activated by GW9662(PPARg antagonist).NF-kB DNA binding activity was also demolished by Rosiglitazone.In summary,our data showed that PPARg agonist,Rosiglitazone suppresses LPS-activated MMP-2 secretion via Ras-MEK1/2 signaling pathways and NF-kB activation.PPARg agonist and Ras-MEK1/2 pathway may be another potential therapeutic target for the disease induced by chronic inflammation.

Cryptotanshinone attenuates isoprenaline-induced cardiac fibrosis in the mouse associated with upregulation and activation of matrix metalloproteinase-2

Objectives The impairment of matrix metallopro- teinase-2(MMP-2)has been associated with the development of cardiac fibrosis.Although the Chinese herb Salvia miltior-rhiza has been widely used in patients with cardiovascular disorders,the mechanisms involved have not been elucidated. The purpose of the present study was to determine whether the administration of cryptotanshinone,an active ingredient of Salvia miltiorrhiza,could prevent the cardiac fibrosis induced by isoprenaline and to investigate the underlying mechanisms. Methods and Results Male C57BL/6 mice were submitted to receive daily injection of 0.9%saline,3 mg/kg isoprenaline, or isoprenaline plus 20 mg/kg cryptotanshinone by gastric gavage for 2 weeks.Herein,we demonstrate that cryptotanshinone can significantly ameliorate the isoprenaline-induced cardiac fibrosis,which was associated with marked up-regulation and activation of MMP-2 in ventricular myocardium. Additionally,we demonstrate that cryptotanshinone can dose-dependently upregulate and activate MMP-2 in cultured cardiac fibroblast.Moreover,incubation with cryptotanshinone also can prevent isoprenaline-induced downregulation and inactivation of MMP-2 in cultured cardiac fibroblast. Conclusions Taken together,our data suggest that cryptotanshinone may become a novel and potent antifibrotic agent. The present findings might further our understanding of the role of MMP-2 in cardiac fibrosis and antifibrotic mechanisms of cryptotanshinone.

Study on matrix metalloproteinase-2, 9 in peri-implant sulcular fluid

Objective: To study the expression of matrix metalloproteinases-2, 9 (MMP-2, MMP-9) of healthy implant and peri-implant sulcular fluid (PISF) by enzyme-linked immunosorbent assay (ELISA) method, and evaluate the level of MMP-2 and MMP-9 in sulcular fluid as an objective indicator of tissue inflammation around implants. Methods: A total of 40 implants were selected from 30 patients who were treated with dental implants and were divided into two groups: the inflammatory group and the healthy control group with 20 pieces respectively. ELISA double antibody sandwich method was used to detect the levels of MMP-2 and MMP-9 in PISF. Results: The MMP-2 and MMP-9 expressions were significantly different between the healthy implant group and the peri-implant group (p < .05). The concentration of MMP-2, MMP-9, and the amount of sulcular fluid in the inflammatory implant group were positively correlated with the clinical parameters (probing depth [PD], modified sulcus bleeding index [mSBI]). Conclusions: Under physiological conditions, the levels of MMP-2 and MMP-9 were low. When the periodontal tissue was stimulated by inflammation, the expression levels of MMP-2 and MMP-9 were increased, which could reflect the severity of inflammation. The increase levels of MMP-2 and MMP-9 in PISF could better reflect the health status of peri-implant tissues, which could be used as an objective indicator to assist in the diagnosis of peri-implant inflammation.

Expression of matrix metalloproteinase-2 and tissue inhibitor of metalloproteinase-1 in hepatic stellate cells during rat hepatic fibrosis and its intervention by IL-10

AIM: To investigate the expression of matrix metallopr-oteinase-2 and tissue inhibitor of metalloproteinase-1 in hepatic fibrosis and the antifibrogenic role of exogenous interleukin-10 (IL-10). METHODS: Hepatic fibrosis was induced by CCI4 administration and 60 male Sprague-Dawley rats were randomly divided into normal control group (group N, 8 rats), CCI4-induced group (group C, 28 rats) and IL-10-treated group (group I, 24 rats). At the beginning of the 7th and 11th wk, rats in each group were routinely perfused with pronase E and type IV collagenase through portal vein catheter and the suspension was centrifuged by 11% Nycodenz density gradient to isolate hepatic stellate cells (HSCs). RT-PCR was used to analyze mRNA of MMP-2 and TIMP-1 from freshly isolated cells. Densitometric data were standardized with β-actin signals. Immunocytochemistry was performed to detect MMP-2 and TIMP-1 expression in HSC cultured for 72 h. RESULTS: Compared to group N in the 7th wk, MMP-2 and TIMP-1 mRNA increased in group C (P= 0.001/0.001) and group I (P= 0.001/0.009). The level of MMP-2 and TIMP-1 mRNA in group I was significantly lower than that in group C (P= 0.001/0.001). In the 11th wk, MMP-2 mRNA in group I was still lower than that in group C (P = 0.005), but both dropped compared with that in the 7th week (P = 0.001/0.004). TIMP-1 mRNA in group I was still lower than that in group C (P= 0.001), and increased in group C (P= 0.001) while decreased in group I (P = 0.042) compared with that in the 7th wk. Same results were found by immunocytochemistry. CONCLUSION: Expression of MMP-2 and TIMP-1 is increased in hepatic fibrosis. IL-10 exhibits an antifibrogenic effect by suppressing MMP-2 and TIMP-1 expression.

Antiangiogenic effect of somatostatin receptor subtype 2 on pancreatic cancer cell line:Inhibition of vascular endothelial growth factor and matrix metalloproteinase-2 expression in vitro

AIM:To investigate the anti-angiogenic effect of somatostatin receptor subtype 2 (SSTR2) gene transfer into pancreatic cancer cell line PC-3, and the mechanisms involved in this effect.METHODS: The full length human SSTR2 cDNA was introduced into pancreatic cancer cell line PC-3 by lipofectamine-mediated transfection. Positive clones were screened by G418 and stable expression of SSTR2 was detected by immunohistochemistry SABC methods and RT-PCR. Enzyme-linked immunosorbent assay (ELISA) was used to detect vascular endothelial growth factor (VEGF) levels in the cell culture supernatants of SSTR2-expressing cells, vector control and mock control cells. Furthermore, the expressions of VEGF and matrix metalloproteinase-2 (MMP-2) were detected by immunohistochemistry SABC methods and RT-PCR in these cells.RESULTS: VEGF levels in the cell culture supernatants were significantly reduced in the SSTR2-expressing cells (first week,172.63±21.2ng/L and after two months, 198.85±26.44ng/L)compared with the vector control (first week, 790.39±86.52ng/L and after two months, 795.69±72.35ng/L) and mock control (first week, 786.42±90.62ng/L and after two months,805.32±84.36ng/L) (P<0.05).The immunohistochemical assay showed a significant reduction of the integral optical density of VEGF and MMP-2 in the SSTR2-expressing cells (42.25±8.6 and 70.5±6.25, respectively) compared with the vector control (85.75±12.9 and 110.52±13.5, respectively) and mock control (82.6±9.28 and 113.56±9.62,respectively) (P<0.05).Conversely, the average gray value of VEGF and MMP-2 was significantly increased in the SSTR2-expressing cells (121.56±8.43 and 134.46±19.95, respectively) compared with the vector control (55.72±5.6 and 62.26±12.68,respectively) and mock control cells (58.48±6.2 and 65.49±9.16, respectively) (P<0.05). Moreover, the expressions of VEGF mRNA and MMP-2 mRNA were significantly reduced in the SSTR2-expressing cells (0.1384±0.017 and 0.2343±0.070, respectively) compared with the vector control (1.024±0.117 and 0.806±0.119,respectively) and mock control (1.085±0.105 and 0.714±0.079,respectively) (P<0.05).CONCLUSION: The expression of reintroduced human SSTR2 gene exerts its antiangiogenic effects by downregulating the expressions of the factors involved in tumor angiogenesis and metastasis, suggesting SSTR2 gene transfer as a new strategy of gene therapy for pancreatic cancer.

ADENOVIRUS-MEDIATED EXPRESSION OF PEX, A NONCATALYTIC FRAGMENT OF MATRIX METALLOPROTEINASE-2, AND IT’S INHIBITION ON ANGIOGENESIS AND TUMOR GROWTH

Objective： To develop an adenovirus system to deliver biologically active peptides or proteins such as angiogenesis inhibitors in vivo for the treatment of cancer. Methods： DNA recombination techniques were employed to construct adenovirus shuttle vector, in which angiogenesis inhibitor was put downstream of rat growth hormone signal peptide, and the C-terminal was the myc-epitope 10-amino-acid peptide for the following up of the protein. Adenovirus was made using the bacteria recombination method. We tested this system using an angiogenesis inhibitor chick MMP-2 C-terminal hemopexin-like fragment （PEX） in Sarcoma 180 （S-180） bearing Kunming mice. The anti-angiogenie effect was performed by chick chorioallantoic membrane assay. Results： PEX was readily secreted outside human stomach carcinoma BGC823 cells as demonstrated by immunofluorcscent staining and western blot infected by adenovirus with rat growth hormone signal peptide （E-T-rGH-PEX）. However, without signal pcptide （E-T-PEX）, PEX was expressed and localized in the cytoplasm of the infected cells, and formed large aggregates, which suggested that PEX was insoluble. The adenovirus E-T-rGH-PEX could inhibit angiogenesis, while E-T-rGH-PEX not. The adenoviruses of E-T-rGH-PEX inhibited the growth of S-180 tumor significantly compared with the empty virus control group E-T （P=0.026） and without signal peptide group E-T-PEX （P=0.006） respectively, while E-T-PEX had little effect. Conelusion： These results suggest that this adenoviral system is likely to be used in the gene therapy of cancer to deliver angiogenesis inhibitors.

Integrin Alpha-V Beta-3-Matrix Metalloproteinase-2 (MMP-2), Cross-Talk

The present study aimed to detect comparative expression of integrin αVβ3 and its involvement in expression and activation of matrix metalloproteinase-2 (MMP-2) in 25 malignant human breast tumor and adjacent normal breast tissues from different clinical TNM stages (DCIS to T4) of the disease and possible involvement of known regulating parameters of MMP-2 like TIMP-2, MT1-MMP and EMPRIN. Integrin αVβ3 was highly expressed in tumors than adjacent normal breast tissues. Pro-MMP-2(72-KD) was mainly expressed in adjacent normal tissues compared to tumors. The mature forms of MMP-2 (68 KD and 64 KD) were found only in tumors. Appreciable expression of TIMP-2 and induction of MT1-MMP and EMPRIN in T2-T4 stages suggested their possible role in MMP-2 activation. Over expression Integrin αVβ3 in tumors than adjacent normal breast tissues was an indication of cancer progression with involvement of integrin signaling. We conclude that, the co-precipitation of MMP-2 with αvβ3 by anti-αv antibody is a strong indication that integrin αvβ3 is a surface receptor for MMP-2 and αvβ3-MMP-2 complex on the surface of tumor cells may play a very important role in determining the invasive property and malignant behavior of tumor tissues. The positive expression of endogenous inhibitor of MMP-2, TIMP-2 may have an appreciable role in activation of this protease and risk of malignancy in advanced stage of the disease. The enhanced expression of MT1-MMP and EMPRIN suggested a role for these factors in gelatinase regulation. However the exact mechanism(s) remains to be investigated. Finally, evaluation of integrin αVβ3 associated MMP-2 expression and activity may add valuable information and can possibly be therapeutic target. The clinical exploitation of integrins will provide oncologists with novel therapeutic strategies for the treatment of malignancy in breast cancer.

EXPRESSION OF MATRIX METALLOPROTEINASE-2 AND VASCULAR ENDOTHELIAL GROWTH FACTOR IN HUMAN GLIOMA AND THEIR RELATION TO THE INVASION OF THE TUMOR

Objective To study the expression of vascular endothelial growth factor (VEGF) and matrix metalloproteinase-2 (MMP-2) in different grade human glioma. To investigate their relation to the pathological grade and invasion of the tumor. Methods The expression of MMP-2 and VEGF were determined by immunohistochemical technique in 48 cases of human glioma and 10 specimens of normal brain tissue. Results The expression levels of MMP-2 and VEGF in human glioma were positively related to tumor grades (P<0.01), and their expressions in the glioma of grade Ⅲ and Ⅳ were significantly different from those in the glioma of grade Ⅰ-Ⅱand normal brain tissue (P<0.01). The expression of MMP-2 was positively correlated to that of VEGF (P<0.01). Conclusion MMP-2 and VEGF were highly expression in human glioma and were positively related to the tumor grades. The synergic interaction of MMP-2 and VEGF promoted the angiogenesis and invasion of human glioma.