Induction of proline-rich tyrosine kinase 2 activation-mediated C6 glioma cell invasion after anti-vascular endothelial growth factor therapy.

BACKGROUND: Anti-angiogenic therapy inhibits tumor growth and is considered as a potential clinical therapy for malignant glioma. However, inevitable recurrences and unexpected tumor resistance, particularly increased invasion ability of glioma cell, were observed after anti-angiogenic treatment. The underlying mechanism remains undetermined. Focal adhesion kinase (FAK) and proline-rich tyrosine kinase 2 (Pyk2) are closely associated with cell migration; therefore, we investigated the possible role of these kinases in rat C6 glioma cell invasion induced by bevacizumab, a recombinant monoclonal antibody against vascular endothelial growth factor (VEGF). METHODS: The effects of bevacizumab on migration and invasion of C6 glioma cells were investigated in vitro and in vivo. The cells proliferation, migration, and invasion were determined by MTT assay, wound healing, and transwell assay, respectively. Invasive potential of glioma cells in vivo was assessed by counting vimentin-positive cells crossing the solid tumor rim by immunohistochemical staining. The total and phosphorylated protein levels of FAK and Pyk2 were detected by Western blotting. RESULTS: Bevacizumab exposure increased migration and invasion of cultured C6 cells in a concentration-dependent manner. In addition, the continuous bevacizumab treatment also promoted tumor invasion in rat C6 intracranial glioma models. Bevacizumab treatment enhanced Pyk2 phosphorylation at Tyr402, but no effect on FAK phosphorylation at Tyr397 both in vitro and in vivo. Knockdown of Pyk2 by siRNA or inhibition of Pyk2 phosphorylation by Src kinase specific inhibitor PP1 partially inhibited bevacizumab-induced cell invasion in cultured C6 glioma cells. Furthermore, the combined administration of bevacizumab and PP1 significantly suppressed glioma cell invasion into surrounding brain tissues compared to bevacizumab treatment alone in experimental rats. CONCLUSIONS: These results suggest that anti-VEGF treatment promotes glioma cell invasion via activation of Pyk2. Inhibition of Pyk2 phosphorylation might be a potential target to ameliorate the therapeutic efficiency of anti-VEGF treatment.

Delta-like ligand 4 correlates with endothelial proliferation and vessel maturation in human malignant glioma.

AIM: To investigate the role of delta-like ligand 4 (DLL4) in the angiogenesis of high-grade malignant glioma. MATERIALS AND METHODS: DLL4 expression and microvessel density (MVD) were detected by immunohistochemistry in 51 human high-grade malignant glioma tissue samples. The vessel maturation index (VMI) was calculated as the percentage of a-smooth muscle actin (a-SMA)-positive vessels in relation to the amount of CD31-positive vessels. Double fluorescent immunostaining for CD31 and EphrinB2 or EphB4 was performed to identify the arterial (EphrinB2) or venous (EphB4) origins of glioma microvessels. RESULTS: Strong immunostaining of DLL4 and a positive correlation of DLL4 with the MVD were observed in high-grade malignant gliomas. The VMI of the DLL4-positive group was significantly higher than that of the DLL4-negative group. However, no significant association was found between DLL4 and EphrinB2 or EphB4 in high-grade gliomas. CONCLUSION: DLL4 may be an important regulator for vessel proliferation and maturation in human high-grade malignant gliomas.

Chitosan nanoparticle as gene therapy vector via gastrointestinal mucosa administration: results of an in vitro and in vivo study.

This study was designed to investigate the in vitro and in vivo transfection efficiency of chitosan nanoparticles used as vectors for gene therapy. Three types of chitosan nanoparticles [quaternized chitosan -60% trimethylated chitosan oligomer (TMCO-60%), C(43-45 KDa, 87%), and C(230 KDa, 90%)] were used to encapsulate plasmid DNA (pDNA) encoding green fluorescent protein (GFP) using the complex coacervation technique. The morphology, optimal chitosan-pDNA binding ratio and conditions for maximal in vitro transfection were studied. The in vivo transfection was conducted by feeding the chitosan/pDNA nanoparticles to 12 BALB/C-nu/nu nude mice. Both conventional and TMCO-60% could form stable nanoparticles with pDNA. The in vitro study showed the transfection efficiency to be in the following descending order: TMCO-60%>C(43-45 KDa, 87%)>C(230 KDa, 90%). TMCO-60% proved to be the most efficient and the optimal chitosan/pDNA ratio being 3.2:1. In vivo study showed most prominent GPF expression in the gastric and upper intestinal mucosa. GFP expression in the mucosa of the stomach and duodenum, jejunum, ileum, and large intestine were found, respectively, in 100%, 88.9%, 77.8% and 66.7% of the nude mice examined. TMCO-60%/pDNA nanoparticles had better in vitro and in vivo transfection activity than the other two, and with minimal toxicity, which made it a desirable non-viral vector for gene therapy via oral administration.

[Characteristics of rainfall and runoff in urban drainage based on the SWMM model.] / 基于SWMM模型的城市排水区域降雨及地表产流特征.

The characteristics of 235 rainfall and surface runoff events, from 2009 to 2011 in a typical urban drainage area in Shanghai were analyzed by using SWMM model. The results showed that the rainfall events in the region with high occurrence frequency were characterized by small rainfall amount and low intensity. The most probably occurred rainfall had total amount less than 10 mm, or mean intensity less than 5 mm·h-1,or peak intensity less than 10 mm·h-1, accounting for 66.4%, 88.8% and 79.6% of the total rainfall events, respectively. The study was of great significance to apply low-impact development to reduce runoff and non-point source pollution under condition of less rainfall amount or low mean rainfall intensity in the area. The runoff generally increased with the increase of rainfall. The threshold of regional occurring runoff was controlled by not only rainfall amount, but also mean rainfall intensity and rainfall duration. In general, there was no surface runoff when the rainfall amount was less than 2 mm. When the rainfall amount was between 2 to 4 mm and the mean rainfall intensity was below 1.6 mm·h-1, the runoff was less than 1 mm. When the rainfall exceeded 4 mm and the mean rainfall intensity was larger than 1.6 mm·h-1, the runoff would occur generally. Based on the results of the SWMM simulation, three regression equations that were applicable to regional runoff amount and rainfall factors were established. The adjustment R2 of the three equations were greater than 0.97. This indicated that the equations could reflect well the relationship between runoff and rainfall variables. The results provided the basis of calculations to plan low impact development and better reduce overflow pollution in local drainage area. It also could serve as a useful reference for runoff study in similar drainage areas.

TGF beta1 expression and angiogenesis in colorectal cancer tissue.

AIM: Transforming growth factor TGF beta1 is involved in a variety of important cellular functions,including cell growth and differentiation, angiogenesis, immune function and extracellular matrix formation. However, the role of TGF beta(1) as an angiogenic factor in colorectal cancer is still unclear. We investigate the relationship between transforming growth factor beta(1) and angiogenesis by analyzing the expression of transforming growth factor TGF beta(1) in colorectal cancer, as well as its association with VEGF and MVD. METHODS: The expression of TGF beta(1),VEGF, as well as MVD were detected in 98 colorectal cancer by immunohistochemical staining. The relationship between the TGF beta(1) expression and VEGF expression,MVD was evaluated. To evaluate the effect of TGF beta(1) on the angiogenesis of colorectal cancers. RESULTS: Among 98 cases of colorectal cancer,37 were positive for TGF beta(1) 37.8% 36 for VEGF 36.7% respectively. The microvessel counts ranged from 19 to 139.8, with a mean of 48.7(standard deviation,21.8). The expression of TGF beta(1) was correlated significantly with the depth of invasion, stage of disease, lymph node metastasis, VEGF expression and MVD. Patients in T3-T4, stage III-IV and with lymph node metastasis had much higher expression of TGF beta(1) than patients in T1-T2, stage I-II and without lymph node metastasis (P<0.05). The positive expression rate of VEGF(58.3%) in the TGF-beta(1) positive group is higher than that in the TGF-beta(1) negative group(41.7%, P<0.05). Also, the microvessel count (54+/-18) in TGF-beta(1) positive group is significantly higher than that in TGF-beta(1) negative group(46+/-15, P<0.05). The microvessel count in tumors with both TGF-beta(1) and VEGF positive were the highest (58+/-20 36-140, P<0.05). Whereas that in tumors with both TGF-beta(1) and VEGF negative were the lowest (38+/-16, 19-60, P<0.05). CONCLUSION: TGF beta(1) might be associated with tumor progression by modulating the angiogenesis in colorectal cancer and TGF beta(1) may be used as a possible biomarker.

Effects of low molecular weight heparin on platelet surface P-selectin expression and serum interleukin-8 production in rats with trinitrobenzene sulphonic acid-induced colitis.

AIM: To observe the effects of low molecular weight heparin (LMWH) on platelet surface P-selectin expression and serum interleukin-8 production in rats with trinitrobenzene sulphonic acid (TNBS) induced colitis. METHODS: Colitis was induced in female Sprague-Dawley rats by colonic administration of 2, 4, 6-TNBS. LMWH, a dalteparin (150 U/kg, 300 U/kg), was subcutaneously administrated one hour before induction of colitis and went on once a day for 6 days. Then a half dose was given for the next 7 days. Control animals received the same volume of normal saline once a day for 14 days after treated by TNBS. Animals were sacrificed at 24 h, days 7 and 14 after induction of colitis. The colon was excised for the evaluation of macroscopic and histological findings and TNF-alpha immunohistochemical assay. Platelet surface P-selectin expression was determined by radioimmunoassay and serum IL-8 production was assayed by ELISA method. RESULTS: LMWH treatment in a dose of 300 U/kg for 14 days significantly improved colonic inflammation by histological examination. Serum IL-8 production in the 300 U/kg treatment group was more significantly decreased at day 14 than that at 24 h (P<0.05). However, platelet surface P-selectin expression and TNF-alpha staining in colonic tissue were not significantly different among the three groups. CONCLUSION: LMWH has an anti-inflammatory effect on TNBS induced colitis in rats. The effect is possibly related to inhibition of proinflammatory cytokine IL-8, but not involved platelet surface P-selectin expression.

Expression of nuclear factor-kappa B and target genes in gastric precancerous lesions and adenocarcinoma: association with Helicobactor pylori cagA (+) infection.

AIM: To examine the expression of nuclear factor kappaB (NF-kappaB) and its target genes in intestinal metaplasia (IM), dysplasia (DYS) and gastric carcinoma (GC) infected with Helicobacter pylori (H pylori) and to investigate the mechanism underlying H pylori cytotoxin associated gene A (cag A) infection leading to gastric adenocarcinoma. METHODS: Expressions of NF-kappaB/p65 and its target genes: c-myc, cyclinD1 and bcl-xl were immunohistochemically examined in 289 cases of gastric biopsy and resection specimens from patients with IM, DYS and GC infected with H pylori. H pylori in the above mentioned tissues was detected by Warthin-Starry stain and rapid urease tests. IgG antibody to cagA in sera of the patients was measured by ELISA. RESULTS: The positive rates of NF-kappaB/p65 were significantly higher in groups with cagA of IMI-II(28/33), IM III(48/52), DYSI(27/31), DYS II-III(28/32), GC(35/40) than in groups without cagA of IMI-II(4/17), IMIII(3/20), DYSI(3/20), DYSII-III(6/21), GC(10/23). The expressions of c-myc, cyclinD1, and bcl-xl were significantly higher in groups with cagA of IM III(47/52, 49/52, 46/52), DYSII-III(29/32, 26/32, 25/32) than in groups without cagA of IM III(8/20, 7/20, 5/20), DYSII-III(10/21, 8/21, 3/21), which were in conformity with the expression of NF-kappaB in IM III, and DYSII-III. A significantly higher expression level of NF-kappaB/p65, c-myc, cyclinD1 and bcl-xl was detected in intestinal type GC(27/28, 18/28, 22/28, 24/28) than in diffuse type GC(8/12, 3/12, 3/12, 6/12), respectively. CONCLUSION: There may be two different molecular mechanisms in the occurrence of intestinal and diffuse type gastric carcinomas. Intestinal type gastric carcinoma is strongly associated with high expression of c-myc, cyclinD1 and bcl-xl through NF-kappaB/p65 activated by H pylori cagA. Inhibiting the activity of NF-kappaB is an effective and promising way to prevent intestinal type gastric carcinoma.

Helicobacter pylori infection generated gastric cancer through p53-Rb tumor-suppressor system mutation and telomerase reactivation.

AIM: To investigate the relationship between Helicobacter pylori (H.pylori) infection and the expressions of the p53, Rb, c-myc, bcl-2 and hTERT mRNA in a series of diseases from chronic gastritis (CG), intestinal metaplasia type I or II(IMI-II), intestinal metaplasia type III (IMIII), mild or modest dysplasia (DysI-II), severe dysplasia (DysIII) to gastric cancer(GC) and to elucidate the mechanism of gastric carcinogenesis relating to H.pylori infection. METHODS: 272 cases between 1998 and 2001 were available for the study including 42 cases of CG, 46 cases of IMI-II, 25 cases of IMIII, 48 cases of DysI-II, 27 cases of DysIII, 84 cases of GC. H.pylori infection and the expressions of p53, Rb, c-myc, bcl-2 were detected by means of streptavidin-peroxidase (SP) immunohistochemical method. HTERT mRNA was detected by in situ hybridization (ISH). RESULTS: The expressions of p53, Rb, c-myc, hTERT mRNA and bcl-2 were higher in the GC than in CG, IM, Dys. The expression of c-myc was higher in IMIII with H.pylori infection (10/16) than that without infection (1/9) and the positive rate in DysI-II and DysIII with H.pylori infection was 18/30 and 13/17, respectively, higher than that without infection (4/18 and 3/10, respectively). In our experiment mutated p53 had no association with H.pylori infection, the expression of Rb was associated with H.pylori infection in GC, but the p53-Rb tumor-suppressor system abnormal in DysI-II cases, DysIII and GC cases with H.pylori infection was 21/30, 15/17 and 48/48 respectively, higher than non-infection groups (4/18, 3/10, 28/36). Furthermore the level of hTERT mRNA in GC with H.pylori infection (47/48) was higher than that without infection (30/36), however the relationship between bcl-2 and H.pylori was only in IMIII. C-myc had a close association with hTERT mRNA in DysIII and GC (P=0.0 253,0.0 305 respectively). CONCLUSION: In the gastric carcinogenesis, H.pylori might cause the severe imbalance of proliferation and apoptosis in the precancerous lesions (IMIII and GysIII) first, leading to p53-Rb tumor-suppressor system mutation and telomerase reactivation, and finally causes gastric cancer.

Transforming growth factor-beta1 in invasion and metastasis in colorectal cancer.

AIM: To investigate the role of TGFbeta1 in invasion and metastasis in colorectal cancer by analysing TGFbeta1 correlated with depth of tumor invasion, stage and metastasis. METHODS: Serum TGFbeta1 levels were determined in 50 patients with colorectal cancer and 30 healthy volunteers using a TGFbeta1 enzyme-linked immunosorbent assay. TGFbeta1 expression in primary and lymph node metastatic lesions were detected in 98 cases of colorectal cancer by immunohistochemical staining and in situ hybridization. RESULTS: Serum levels of TGFbeta1 in patients with colorectal cancer(40+/-18 microg.L(-1)) were significantly higher than those in the healthy control group(19+/-8 microg.L(-1)), P<0.05. Elevated levels of serum TGFbeta1 were found in 60 % of patients with colorectal cancer when the mean +2 s was used as the upper limit of the normal range (35.1 microg.L(-1)). Increases in serum TGFbeta1 levels were significantly associated with Duke's stage (P<0.05), but there was no significant difference between Duke's stage B patients and Duke's stage C patients. In the cytoplasm of cancer cells, TGFbeta1 was immunostained in 37.8 % (37/98) of colorectal cancer, and this expression was confirmed by in situ hybridization. Among 35 cases of colorectal cancer with lymph node metastatic lesions, TGFbeta1 positive staining was found in 18 (51.4 %) cases of primary tumor, and 25 (71.4 %) cases with lymph node metastatic lesions, respectively. Of 17 cases w ith no staining in the primary lesion, 7 (41.2%) casesshowed TGFbeta1 staining in the metastatic lesion. Serum TGFbeta1 levels and TGFbeta1 expression in colorectal cancer tissues were correlated significantly with depth of tumor invasion, stage and metastasis. Patients in stage C-D,T3-T4 and with metastasis had significantly higher TGFbeta1 levels than patients in stage A-B,T1-T2 and without metastasis (P<0.05). CONCLUSION: These results suggest that transforming growth factor-beta1 is closely related to the invasion and metastasis of colorectal cancer. It increased the invasive and metastatic potential of tumor by altering a tumor microenvironment. TGFbeta1 may be used as a possible biomarker.