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Cancer stem cells (CSCs), a special subpopulation of tumor cells, are considered as tumor initiating cells. More recently, these cells have also been identified as initiators of tumor neovascularization. A better understanding of the contribution of CSCs to neovascularization should elucidate the mechanisms of cancer initiation and progression as well as establish new concepts for cancer diagnosis and treatment. In this review, we discuss the evidence for the roles of CSCs in tumor vascularization, including production of proangiogenic factors, transdifferentiation into vascular mural cells such as endothelial and smooth muscle-like cells, and formation of nonendothelium-lined vasculogenic mimicry. In addition, the potential therapeutic significance of targeting CSCs is envisaged., (Copyright © 2011 AlphaMed Press.)

Interleukin-2 (IL-2) has been shown to possess antitumor activity in numerous preclinical and clinical studies. However, the short half-life of recombinant IL-2 protein in serum requires repeated high-dose injections, resulting in severe side effects. Although adenovirus-mediated IL-2 gene therapy has shown antitumor efficacy, the host antibody response to adenoviral particles and potential biosafety concerns still obstruct its clinical applications. Here we report a novel nanopolymer for IL-2 delivery, consisting of low molecular weight polyethylenimine (600 Da) linked by β-cyclodextrin and conjugated with folate (named H1). H1 was mixed with IL-2 plasmid to form H1/pIL-2 polyplexes of around 100 nm in diameter. Peritumoral injection of these polyplexes suppressed the tumor growth and prolonged the survival of C57/BL6 mice bearing B16-F1 melanoma grafts. Importantly, the antitumor effects of H1/pIL-2 (50 μg DNA) were similar to those of recombinant adenoviruses expressing IL-2 (rAdv-IL-2; 2 × 10(8) pfu). Furthermore, we showed that H1/pIL-2 stimulated the activation and proliferation of CD8+, CD4+ T cell, and natural killer cells in peripheral blood and increased the infiltration of CD8+, CD4+ Tcells, and natural killer cells into the tumor environment. In conclusion, these results show that H1/pIL-2 is an effective and safe melanoma therapeutic with an efficacy comparable to that of rAdv-IL-2. This treatment represents an alternative gene therapy strategy for melanoma.

Purpose: This study was to investigate the clinicopathologic significance and potential role of HOXB7 in the development and progression of colorectal cancer (CRC)., Experimental Design: The relationship between HOXB7 expression and clinical characteristics of CRC was analyzed in 224 paraffin-embedded archived CRC specimens by immunohistochemistry (IHC). The effects of HOXB7 on cell growth and proliferation, as well as on tumorigenesis, were examined both in vitro and in vivo, using MTT assay, colony formation assay, cell cycle analysis, soft agar assay, and tumorigenesis in nude mice. Western blotting and real-time reverse transcriptase-PCR were performed to examine the impact of HOXB7 on the PI3K/Akt and MAPK signaling pathways., Results: HOXB7 protein level was significantly correlated with advanced Dukes stage (P < 0.001), T stage (P = 0.012), distant metastasis (P = 0.042), higher proliferation index (P = 0.007) and poor survival of patients (P = 0.005). Enforced expression of HOXB7 in CRC cell lines significantly enhanced cell growth, proliferation and tumorigenesis. Conversely, knockdown of HOXB7 caused an inhibition of cell growth, proliferation, and tumorigenesis. We also showed that HOXB7 accelerated G(0)-G(1) to S-phase transition concomitantly with upregulation of cyclin D1 and downregulation of p27Kip1. On the contrary, knockdown of HOXB7 caused G(1)-S-phase arrest, downregulation of cyclin D1 and upregulation of p27Kip1. Enforced expression of HOXB7 could enhance PI3K/AKT and MAPK pathway activity., Conclusion: Our findings suggest that HOXB7 protein, as a valuable marker of CRC prognosis, plays an important role in the development and progression of human CRC., (©2011 AACR.)

Purpose: To elucidate the role of Semaphorin-3F (SEMA3F), originally described as an axon guiding chemorepulsant implicated in nerve development, in the progression of colorectal carcinoma., Experimental Design: SEMA3F and its receptor NRP2 were examined in 72 cases of human colorectal carcinoma specimens and cell lines LoVo, SW480, and SW620 with immunohistochemistry and Western blotting. SEMA3F mRNA expression in the frozen tissue specimens and cell lines was examined with quantitative reverse transcriptase-PCR. Confocal laser scanning microscopy was used for detection of cellular localization of the proteins by immunofluorescent staining. MTT assay, flow cytometry, cell adhesion and migration, and xenografts were used to evaluate biological significance of SEMA3F., Results: SEMA3F was significantly reduced in colorectal carcinoma tissues and cell lines. Overexpression of SEMA3F resulted in reduced proliferation, adhesion to fibronectin, and migratory capability as well as reduced S-phase population and integrin αvβ3 expression of SW480 colon cancer cells. In addition, SEMA3F-overexpressing cells exhibited diminished tumorigenesis when transplanted orthotopically in nude mice and reduced liver metastases. Moreover, transfection of siRNA targeting SEMA3F in colon cancer cells increased their tumorigenicity in vivo., Conclusions: Endogenous SEMA3F acts as a suppressor of the growth and metastasis of human colorectal cancer cells., (©2011 AACR.)

Vasculogenic mimicry (VM), a newly-defined pattern of tumor blood supply, provides a special passage without endothelial cells and is conspicuously different from angiogenesis and vasculogenesis. The biological features of the tumor cells that form VM remain unknown. Cancer stem cells (CSCs) are believed to be tumor-initiating cells, capable of self-renewal and multipotent differentiation, which resemble normal stem cells in phenotype and function. Recently CSCs have been shown to contribute to VM formation as well as angiogenesis. These findings challenge the previous understanding of the cellular basis of VM formation. In this review, we present evidence for participation of CSCs in VM formation. We also discuss the potential mechanisms and possible interaction of CSCs with various elements in tumor microenvironment niche. Based on the importance of VM in tumor progression, it constitutes a novel therapeutic target for cancer.

It is believed that cancer stem cells (CSCs) are precursors for the formation, development, and recurrence of malignant tumors. However, it has proven difficult to isolate and enrich these rare, undifferentiated cells from heterogeneous tumor masses. With some existing reports and preliminary results in mind, we hypothesized that the mitochondrial membrane potential within a tumor mass was heterogeneous and could be used as a tool to isolate and enrich CSCs.

Endothelial progenitor cells (EPCs) are involved in tumor neovascularization with undefined mechanisms. In this study, we explored the role of formylpeptide receptor, a G protein-coupled receptor, expressed by human malignant glioma cells in neovascularization of malignant glioma. EPCs were isolated from human umbilical cord blood and their migratory capability and tubulogenesis induced by the supernatant of U87 glioblastoma (GBM) cell line were examined. We also assessed the recruitment and incorporation of EPCs into orthotopic intracranial tumors formed by implanted U87 GBM cells. The supernatant of control U87 cells induced high levels of migration and tubule-formation in vitro by EPCs. In contrast, the chemotactic and tubule-stimulating activities on EPCs in the supernatant of U87 cells with FPR knocking down by small interference (si) RNA were significantly attenuated. In addition, the number of EPCs recruited and incorporated into intracranial glioma xenografts was significantly higher in tumors formed by control U87 cells than tumors formed by U87 cells containing FPR-siRNA. Our results suggest that expression of functional FPR in glioma cells plays an important role in regulating vasculogenesis by EPCs, which constitute a novel target for anti-angiogenic therapy in gliomas., (Copyright © 2010. Published by Elsevier B.V.)

The emerging concept of generating cancer stem cells from epithelial-mesenchymal transition has attracted great interest; however, the factors and molecular mechanisms that govern this putative tumor-initiating process remain largely elusive. We report here that miR-200a not only regulates epithelial-mesenchymal transition but also stem-like transition in nasopharyngeal carcinoma cells. We first showed that stable knockdown of miR-200a promotes the transition of epithelium-like CNE-1 cells to the mesenchymal phenotype. More importantly, it also induced several stem cell-like traits, including CD133(+) side population, sphere formation capacity, in vivo tumorigenicity in nude mice, and stem cell marker expression. Consistently, stable overexpression of miR-200a switched mesenchyme-like C666-1 cells to the epithelial state, accompanied by a significant reduction of stem-like cell features. Furthermore, in vitro differentiation of the C666-1 tumor sphere resulted in diminished stem-like cell population and miR-200a induction. To investigate the molecular mechanism, we demonstrated that miR-200a controls epithelial-mesenchymal transition by targeting ZEB2, although it regulates the stem-like transition differentially and specifically by β-catenin signaling. Our findings reveal for the first time the function of miR-200a in shifting nasopharyngeal carcinoma cell states via a reversible process coined as epithelial-mesenchymal to stem-like transition through differential and specific mechanisms.

Background: The amplified in breast cancer 1 (AIB1) gene has been considered to play an oncogenic role in human cancers, but its clinical/prognostic significance in non-small-cell lung cancer (NSCLC) is still unclear., Patients and Methods: The methods of immunohistochemistry and FISH were utilized to examine protein expression and amplification of AIB1 in 230 informative surgically resected NSCLCs and in 30 samples of normal lung tissues., Results: Overexpression and amplification of AIB1 were found in 48.3% and 8.2% of NSCLCs, respectively. AIB1 overexpression was associated with AIB1 gene amplification and cell proliferation but not related to estrogen receptor (ER)-alpha, ER-beta, progesterone receptor or androgen receptor status. A positive correlation between AIB1 overexpression and an ascending pathologic node stage in lung adenocarcinoma (ADC) was observed (P = 0.043). Univariate survival analysis demonstrated a significant association of AIB1 overexpression with shortened patient survival, especially for those with stage III disease (P < 0.001). Importantly, AIB1 expression was evaluated as the most significant predictor for survival in multivariate analysis (hazards ratio = 2.069, P < 0.001)., Conclusion: Overexpression of AIB1 might provide a selective advantage for lymph node metastasis of lung ADC and serve as a useful biomarker for poor prognosis for NSCLC patients.

Growing evidence suggests that myeloid-derived suppressor cells (MDSCs), which have been named "immature myeloid cells" or "myeloid suppressor cells" (MSCs), play a critical role during the progression of cancer in tumor-bearing mice and cancer patients. As their name implies, these cells are derived from bone marrow and have a tremendous potential to suppress immune responses. Recent studies indicated that these cells also have a crucial role in tumor progression. MDSCs can directly incorporate into tumor endothelium. They secret many pro-angiogenic factors as well. In addition, they play an essential role in cancer invasion and metastasis through inducing the production of matrix metalloproteinases (MMPs), chemoattractants and creating a pre-metastatic environment. Increasing evidence supports the idea that cancer stem cells (CSCs) are responsible for tumorigenesis, resistance to therapies, invasion and metastasis. Here, we hypothesize that CSCs may "hijack" MDSCs for use as alternative niche cells, leading to the maintenance of stemness and enhanced chemo- and radio-therapy resistance. The countermeasure that directly targets to MDSCs may be useful for against angiogenesis and preventing cancer from invasion and metastasis. Therefore, the study of MDSCs is important to understand tumor progression and to enhance the therapeutic efficacy against cancer., (Copyright 2010 Institute of Genetics and Developmental Biology and the Genetics Society of China. Published by Elsevier Ltd. All rights reserved.)

An unusual case of intraparenchymal myofibromatosis of the brain occurring in a 29-year-old woman is described. Preoperative CT and MRI examinations revealed two well-circumscribed nodular masses localized in the wall of the left lateral ventricle and right temporal lobe, respectively. Both masses were completely resected, and the patient remains disease-free 2 years post-surgery. Histopathologically, the lesions were characterized by stratification. From outer to inner, there was a reactive glial component, lamellated well-differentiated muscle-like cells, densely compact collagen fibers and cellular tumor with nodular and hemangiopericytoma-like patterns, respectively. The myofibroblastic nature of this tumor was verified by immunohistochemical staining and ultrastructural analysis. Intraparenchymal myofibromatosis may be confused with, and should be distinguished from, meningioma, myopericytoma, solitary fibrous tumor, leiomyoma and inflammatory myofibroblastic tumor for accurate diagnosis and optimal treatment.

Background: The G-protein-coupled formylpeptide receptor (FPR) that mediates chemotaxis of phagocytic leucocytes induced by bacterial and host-derived chemotactic peptides is selectively expressed by highly malignant human gliomas and contributes to tumour growth and angiogenesis. As invasion of surrounding normal tissues is one of the important features of tumour malignancy, we investigated the function of FPR in the invasive behaviour of human glioblastoma cells., Methods: Cells (FPR(+) and FPR(-)) were isolated by single-cell cloning from a human glioblastoma cell line U-87MG. The FPR expression was assayed by flow cytometry and reverse transcription PCR. The function of FPR was investigated by chemotaxis and calcium flux induced by FPR agonist fMLF. Tumour cell motility was assayed by a wound-healing model in vitro. The growth and invasive phenotype were observed with subcutaneous implantation of tumour cells in nude mice. Over-expression of FPR in FPR(-) cells was performed by transfection of a plasmid vector-containing human FPR gene., Results: One of the glioma clones F9 that expressed high level of FPR showed a more 'motile' phenotype in vitro as compared with a clone G3 without FPR expression. Although F9 and G3 clones both formed subcutaneous tumours in nude mice, only F9 tumours invaded surrounding mouse connective tissues. Over-expression of FPR in G3 clone (G3F) increased the cell motility in vitro and the capacity of the cells to form more rapidly growing and invasive tumours in nude mice. We further found that, in addition to supernatant of necrotic tumour cells, foetal calf serum and human serum used in culture media contained FPR agonist activity and increased the motility of FPR-expressing glioblastoma cells., Conclusion: The expression of FPR is responsible for increased motility of human glioblastoma cells and their formation of highly invasive tumours.

Nasopharyngeal carcinoma (NPC), a highly metastatic and invasive malignant tumor originating from the nasopharynx, is widely prevalent in Southeast Asia, the Middle East and North Africa. Although viral, dietary and genetic factors have been implicated in NPC, the molecular basis of its pathogenesis is not well defined. Based on a recent microRNA (miRNA) microarray study showing miR-200 downregulation in NPC, we further investigated the role of miR-200a in NPC carcinogenesis. We found that the endogenous miR-200a expression level increases with the degree of differentiation in a panel of NPC cell lines, namely undifferentiated C666-1, high-differentiated CNE-1, and low-differentiated CNE-2 and HNE1 cells. By a series of gain-of-function and loss-of-function studies, we showed that over-expression of miR-200a inhibits C666-1 cell growth, migration and invasion, whereas its knock-down stimulates these processes in CNE-1 cells. In addition, we further identified ZEB2 and CTNNB1 as the functional downstream targets of miR-200a. Interestingly, knock-down of ZEB2 solely impeded NPC cell migration and invasion, whereas CTNNB1 suppression only inhibited NPC cell growth, suggesting that the inhibitory effects of miR-200a on NPC cell growth, migration and invasion are mediated by distinct targets and pathways. Our results reveal the important role of miR-200a as a regulatory factor of NPC carcinogenesis and a potential candidate for miRNA-based therapy against NPC., (Copyright 2009 Elsevier Inc. All rights reserved.)

Enrichment of cancer stem cells for studies of carcinogenesis remains a difficult issue. We hypothesized that the unique features of cancer stem cells (CSCs) may allow formation of their colonies in vitro with distinct morphology. We therefore investigated the possibility to use morphological diversity of colonies to identify and enrich CSCs from cultured malignant human glioma cells. We found that a small proportion of the cells from a human glioma cell line U251 formed tight and round-shaped colonies in culture. Most cells in such colonies were capable of self-renewal, generating tumor spheres and differentiating into lineages with markers for neurons, astrocytes and oligodendrocytes. In addition, several neural stem cell-related genes were highly expressed by tumor cells in those tight colonies. Our results thus demonstrate a novel approach to the identification and enrichment of CSCs based on unique morphology of their colonies formed in vitro.

Endothelial progenitor cells (EPCs) are important initiators of vasculogenesis in the process of tumor neovascularization. However, it is unclear how circulating EPCs contribute to the formation of tumor microvessels. In this study, we isolated CD34(+)/CD133(+) cells from human umbilical cord blood (HUCB) and obtained EPCs with the capacities of forming colonies, uptaking acetylated low-density lipoprotein (ac-LDL), binding lectins and expressing vascular endothelial growth factor (VEGF) receptor 2 (VEGFR-2, KDR), CD31 and von Willebrand factor (vWF). These EPCs were actively proliferative and migratory, and could formed capillary-like tubules in response to VEGF. When injected into mice bearing subcutaneously implanted human malignant glioma, EPCs specifically accumulated at the sites of tumors and differentiated into mature endothelial cells (ECs), which accounted for 18% ECs of the tumor microvessels. The incorporation of circulating EPCs into tumor vessel walls significantly affected the morphology and structure of the vasculature. Our results suggest that circulating EPCs constitute important components of tumor microvessel network and contribute to tumor microvascular architecture phenotype heterogeneity.

Gliomas are the most common and lethal tumor type in the brain. The present study investigated the effect of oligomer procyanidins (F2) (F2, degree of polymerization 2-15), a natural fraction isolated from grape seeds on the biological behavior of glioblastoma cells. We found that F2 significantly inhibited the glioblastoma growth, with little cytotoxicity on normal cells, induced G2/M arrest and decreased mitochondrial membrane potential in U-87 cells. It also induced a non-apoptotic cell death phenotype resembling paraptosis in U-87 cells. In addition, it was found for the first time that F2 in non-cytotoxic concentrations selectively inhibited U-87 cell chemotaxis mediated by a G-protein coupled receptor formyl peptide receptor FPR, which is implicated in tumor cell invasion and metastasis. Further experiments indicated that F2 inhibited fMLF-induced U-87 cell calcium mobilization and MAP kinases ERK1/2 phosphorylation. Moreover, F2 attenuated the glioblastoma FPR expression, a new molecular target for glioma therapeutics, which has been shown to play important roles in glioma cells chemotaxis, proliferation and angiogenesis in addition to its promotion to tumor progression, but did not affect FPR mRNA expression in U-87 cells. Taken together, our results suggest that F2 may be a promising candidate for the development of novel anti-tumor therapeutics.

G-protein-coupled formylpeptide receptor (FPR) has recently been found to be functionally expressed in gliomas and are probably involved in their malignant biological behavior. In an attempt to explore the therapeutic significance of FPRs, we used wild-type human glioblastoma cells (U87), the corresponding FPR short-interfering RNA transfected (siRNA U87) cells, and mock-transfected U87 cells (mock U87) to establish xenografts in mice brains. Compared to wild-type and mock transfected cells, siRNA U87 cells formed smaller and more well-differentiated xenografts with fewer mitotic figures and more glial filaments within their cytoplasm. The density of microvessels, which presented as a nearly normal morphous, was also decreased significantly in FPR knockdown cells. Moreover, fewer invasive foci could be observed in the xenografts derived from siRNA U87 cells, which also showed a poor migratory capacity in vitro. We suggest that decreased VEGF and MMP-2/-9 expression might be a possible mechanism for the decreasing angiogenic potential and invasive capability of U87 cells after FPR knockdown. Functional FPR might be essential for sustaining the growth and aggressive phenotype of gliomas, and could therefore be a potential therapeutic target.

Cancer stem cells (CSCs) have multiple potentials in carcinogenesis and tumor progression. However, it is rather difficult to enrich and amplify CSCs either from tumor cell lines or even primary tumor tissues. Therefore, establishing new methodologies for isolation and enrichment based on the functional properties of CSCs is of great importance for studies on CSCs. According to the findings that CSCs possess more infiltrative capability as compared with their differentiated descendants, we propose a novel strategy based on heterogeneity of cancer cell invasiveness for isolation and enrichment of CSCs from committed cancer cell population. In addition, we hypothesize that existence of CSCs might be the real root of tumor invasion and metastasis.

The lipoxygenase inhibitor nordihydroguaiaretic acid, a natural product purified from Larrea divaricata and Guaiacum officinale, showed a marked capacity to induce differentiation of various human malignant tumor cell lines. Nordy, a derivative of nordihydroguaiaretic acid, has been shown to effectively inhibit the growth of malignant human tumors transplanted in mice. However, it is unknown whether Nordy plays an important role in inducing the differentiation effects of human papillomavirus (HPV)-16 subgenes-immortalized human endocervical cells H8. In this study, we showed that Nordy arrested H8 cells in the G0/G1 phase, promoted cell differentiation in morphology, downregulated the expression of HPV-16 E6 mRNA and nuclear antigen Ki67, and inhibited telomerase activity on HPV-16 subgenes-immortalized human endocervical cells H8. Taken together, Nordy could suppress proliferation by inhibiting proliferation-related events and promote cell differentiation in H8 cell. The results suggested that Nordy might be a potential tumor therapeutic agent.

Glioma stem cells (GSCs), or stem cell-like glioma cells, isolated from malignant glioma cell lines, were capable of producing vascular endothelial growth factor (VEGF). However, the exact role of such tumour cells in angiogenesis remains unknown. In this study, we isolated a small proportion of CD133+ GSCs from the human glioblastoma cell line U87 and found that these GSCs possessed multipotent differentiation potential and released high levels of VEGF as compared with CD133(-) tumour cells. The CD133+ GSCs also formed larger xenograft tumours that contained higher VEGF immunoreactivity and denser microvessels. Moreover, GSCs expressed a functional G protein-coupled formylpeptide receptor FPR, which was activated by a chemotactic peptide ligand, N-formylmethionyl-leucyl-phenylalanine (fMLF), to mediate calcium flux and the production of VEGF by GSCs. Our results indicate that FPR expressed by human GSCs may play an important role in glioma angiogenesis., (Copyright (c) 2008 Pathological Society of Great Britain and Ireland. Published by John Wiley & Sons, Ltd.)

A variety of malignant cancers have been found to contain a subpopulation of stem cell-like tumor cells, or cancer stem cells (CSCs). However, the existence of CSCs in U87, a most commonly used glioma cell line, is still controversial. In this study, we demonstrate that U87 cell line contained a fraction of tumor cells that could form tumor spheres and were enriched by progressively increasing the concentration of serum-free neural stem cell medium with or without low dose vincristine. These cells possessed the ability of self-renewal and multipotency, the defined characteristics of CSCs. Moreover, the tumors formed by the secondary spheres displayed typical histological features of human glioblastoma, including cellular pleomorphism, pseudopalisades surrounding necrosis, hyperchromatic nuclei, high density of microvessels and invasion to the brain parenchyma. These results indicate that gradually increasing the concentration of serum-free neural stem cell culture medium with or without vincristine is a simple and effective method for isolation of CSCs to study the initiation and progression of human glioblastoma.

Objective: To investigate the therapeutic effects of Enalaprilat on the myocardial kinetics in rats at early stage of severe scald., Methods: Eighty-four SD rats were inflicted with 30% TBSA full-thickness scald, and randomly divided into scald (S, with intraperitoneal injection of isotonic saline according to Parkland formula, n=30), L (n=30), M (n=12) and H (n=12) groups. The rats in L,M,H groups were intraperitoneally injected with 1,2,4 mg/kg Enalaprilat. Other 6 healthy rats were enrolled into study as control (C group). The myocardial kinetic parameters including left ventricular systolic pressure (LVSP), left ventricular end diastolic pressure (LVEDP), +/- dp/dt max and the levels of A II in myocardium were observed at 1,3,6,12 and 24 post scald hour (PBH) in L and S groups,and at 6,12 PBH in M and H groups. The above indices in C group were also examined., Results: The levels of LVSP, LVEDP, +/- dp/dt max in C group were higher than those in other groups during 3-24 PBH (P < 0.05 or P < 0.01), while those in L,M,H groups were obviously higher than those in S group (P < 0.05 or P < 0.01). The level of +/- dp/dt max in H group at 6,12 PBH were obviously lower than those in L and M groups. The level of A II in S group at 1 PBH was (53.0 +/- 2.6) pg/200 mg, which was significantly higher than thatin C group [(14.8 +/- 0.7) pg/200 mg, P < 0.05 or P < 0.01]; it peaked at6 PBH and lowered afterwards, and they were significantly higher than that in C group at 24 PBH (P < 0.01). The levels of A II in L group during 3-24 PBH were obviously higher than those in C group (P < 0.01), which were also lower than those in S group. The level of A II in S group was significantly higher than in L,M,H groups at 6 PBH [(145.2 +/- 14.5) pg/200 mg. vs. (65.1 +/- 0.9) pg/200 mg, (53.6 +/- 1.1) pg/200 mg, (34.2 +/- 0.9) pg/200 mg, respectively, P < 0.01]., Conclusion: Myocardium can be obviously damaged at early stage after severe scald,cardiac function is impaired. Enalaprilat injection (especially at low dose) can significantly ameliorate the myocardial kinetics indices, and it seems to exert a protective effect on cardiac function.

Aiming at the technical difficulties such as the stability and water balance in the ecological rehabilitation of river flinty slope, a gravel mulch technology was proposed, with the effects of different gravel mulch treatments on the soil anti-erosion capacity, soil water retention property, and plant growth investigated by anti-erosion and pot experiments. The results showed that mulching with the gravels 1.5-2 cm in size could obviously enhance the soil anti-erosion capacity, soil water retention property and plant biomass, but no obvious differences were observed between the mulch thickness of 5 cm and 8 cm. It was indicated that mulching with the gravels 1.5-2 cm in size and 5 cm in thickness was an effective and economical technology for the ecological rehabilitation of river flinty slope.

Nordy is a chirally synthesized compound of a natural lipoxygenase inhibitor nordihydroguaiaretic acid. In this study, we found that Nordy inhibited the growth of human glioma cell lines in vitro and their tumorigenicity in mice. In addition, Nordy promoted differentiation of highly malignant human glioma cells. Investigation into the mechanistic basis of Nordy activities revealed that it altered the pattern of protein expression profiles in tumor cells. By using 2-DE, we found that in human glioma cell lines, at least six proteins were down-regulated after Nordy treatment, while four proteins were elevated in the same cells. Among the six down-regulated proteins, microsequencing with MALDI TOF MS confirmed the identity of five: proliferation-associated gene A (PAG-A), alternative splicing factor-3 (ASF-3), beta-galactoside binding lectin, eukaryotic translation initiation factor 5A (eIF-5A), and coffilin-1 (nonmuscle). Four up-regulated proteins were GST-pi, glyceraldehyde-3-phosphate dehydrogenase, alpha-enolase, and cyclophilin. All these proteins have been reported to participate in key cellular functions including proliferation, metabolism, differentiation, apoptosis, and gene transcription. Our results suggest that Nordy may constitute a promising drug lead for the development of novel antitumor agents targeting proteins that control tumor cell function at multiple levels.

This study is to investigate whether the synthesized chiral compound Nordy has influence on the function of endothelial progenitor cells (EPCs) from human umbilical cord blood induced by vascular endothelial growth factor (VEGF). EPCs were isolated from human umbilical cord blood by density gradient centrifugation. After cultured for 7 -10 days, EPCs were prepared for detecting effect of Nordy on proliferation, migration and tubule-forming activity in Matrigel induced by VEGF. Incubation of EPCs with 100 micromol L(-1) Nordy for 24 h initially inhibited the proliferative capacity of EPCs induced by VEGF (P <0.05). Moreover, 25 -50 micromol L(-1) Nordy also exhibited inhibitory effect at 48 -72 h. In addition, 25 - 100 micromol L(-1) Nordy impaired EPCs migratory and tubule-forming capacity in vitro (P < 0.05). Nordy could inhibit in EPCs the functions of proliferation, migration and tubulogenesis induced by VEGF in vitro, which might be a possible mechanism of its anti-EPCs effects.

Urban stormwater pollution is recognized as one of the major causes of water quality deterioration in the receiving water bodies. Starting in May 2006, a study was conducted to characterize water quality from representative land uses types in Zhenjiang. By analyzing the varieties of suspended solids (SS) and nutrients, the results show that the 5-40 microm size class dominates in all the samples, and urban stormwater pollution mainly results from commercial area where concentrations of pollutants are so high that concentration of SS reaches 978 mg/L. Concentrations of pollutants in initial flush are very high, and they descend sharply along with the time to reach the stable stage. The main phases of pollutants discharge are dissolved nitrogens, particle phosphorus and SS. Analysis of correlation of SS and other pollutants finds that SS are the absorbed carrier of nutrients from urban stormwater runoff, and removal of SS is the effective way to control urban stormwater pollution in Zhenjiang.

Activation of the formylpeptide receptor (FPR), a G-protein-coupled receptor, by its chemotactic peptide ligand N-formylmethionyl-leucyl-phenylalanine (fMLF) promotes the directional migration and survival of human glioblastoma cells. fMLF also stimulates glioblastoma cells to produce biologically active VEGF, an important angiogenic factor involved in tumor progression. In this study, we examined the capacity of FPR to regulate the production of another angiogenic factor, the chemokine IL-8 (CXCL8), in addition to its demonstrated ability to induce VEGF secretion by malignant glioma cells. We showed that the human glioblastoma cell line U87 secreted considerable levels of IL-8 (CXCL8) upon stimulation by the FPR agonist peptide fMLF. Tumor cells transfected with small interference (si)RNA targeting FPR failed to produce IL-8 as well as VEGF in response to fMLF. Glioblastoma cells bearing FPR siRNA exhibited reduced rate of tumorigenicity in nude mice and tumors formed by such tumor cells showed less active angiogenesis and lower level expression of both IL-8 and VEGF. These results suggest that FPR plays an important role in the angiogenesis of human malignant gliomas through increasing the production of angiogenic factors by FPR positive tumor cells.

Objective: To investigate vasculogenic potential of endothelial progenitor cells (EPCs) derived from human umbilical cord blood and their contribution to the neovascularization of malignant glioma in vivo., Methods: EPCs were isolated from human umbilical cord blood by density gradient centrifugation. After 7-10 days of culture, EPCs were investigated for CD34 and VEGFR-2 expression by direct immunofluoresent staining. The proliferative activity, migratory capability and forming capillary-like tubules were also monitored after stimulation with VEGF(50 mg/L) in vitro. Moreover, EPCs were administered into tumor-bearing mice, and the tumor and mouse organs were examined under confocal laser scanning microscope to visualize the distribution and localization of transplanted EPCs. In order to quantity the incorporation of EPCs into tumor vessels, cryosections of the tumor tissue were double-labelled with antihuman CD31 and anti-mouse CD31., Results: After 7 to 10 days of culture, EPCs assumed cobblestone-like monolayer growth pattern with nearly complete confluence, and expressed CD34 and VEGFR-2. Significant proliferative activity, increased migratory capability and forming capillary-like tubules were observed when stimulated with VEGF. The transplanted EPCs in vivo specifically homed to solid tumor tissue and incorporated into the tumor's endothelium. Quantitative analysis revealed that human EPCs contributed significantly to tumor neovascularization by incorporation into tumor vasculature (18.68 +/- 1.32)% of the total vessels., Conclusion: EPCs possess the potential to form neovascular network in tumor and play a role in the phenotypical heterogeneity of tumor microvascular architecture.

Background & Objective: Tumor stem cells (TSCs) possess the ability of chemoresistance. This study was to isolate and identify TSCs from human glioma cell line U87., Methods: After U87 cells were grown as monolayer attached at the bottom of flasks in the medium containing serum, neural stem cell culture medium containing 5 ng/ml vincristine was added to obtain the first generation of tumorosphere. Then, the first generation of tumorosphere was dissociated into single cell suspension and seeded in the neural stem cell culture medium to obtain the second generation of tumorosphere. The morphology of the first and the second generations of tumorosphere was observed by light and scanning electron microscopy, respectively. The expression of nestin, glial fibrillary acidic protein (GFAP), beta-tubulin III and myelin basic protein (MBP) in the second generation of tumorosphere and differentiated cells were determined by immunofluorescence assay and confocal laser scanning microscopy., Results: The first and second generations of tumorosphere were obtained successfully. Cells inside the second generation of tumorosphere with a few protrusions attached with each other tightly. The expression of nestin, a stem cell marker, was detected in the second generation of tumorosphere. The expression of nestin, GFAP, beta-tubulin III, MBP was detected in the differentiated cells derived from the second generation of tumorosphere., Conclusion: Tumor stem cells with the capacity of self-renewal and multipotency exist in U87 cells. Selective culture of tumor cells with treatment of vincristine might be a simple and practical method for isolation of TSCs.

A small population of stem cell-like precursors in solid tumors are linked to histological composition, progression, angiogenesis, metastasis, recurrence and drug resistance of a variety of malignant tumors. Oligoastrocytoma is the most common brain mixed glioma composed of mixed cells of oligodendroglial and astrocytic phenotypes. Identification and characterization of stem cell-like precursors in oligoastrocytoma may shed light on the oncogenesis of this unique type of tumor and assist in the design of novel therapeutic strategy. Here, tumor stem cell-like precursors were identified from primary human anaplastic oligoastrocytomas by labeling of the tumor sections with nestin and CD133. Tumor cells were cultured in vitro in stem cell medium with growth factors and the capacity of the surviving stem cell-like precursors to form tumor spheres was tested. The tumor spheres were further injected subcutaneously into nude mice to observe the contribution of stem cell-like precursors to histological composition and tumor progression. We found that primary human oligoastrocytoma tissues contained nestin+/CD133+ stem cell-like precursors. These cells differentiated into tumor cells with both oligodendroglial and astrocytic characteristics and formed tumor spheres in vitro, which upon implantation in nude mice, grew into tumor nodules containing nestin+/CD133+ cells at levels higher than in the primary tumor tissues. This study revealed for the first time that anaplastic human oligoastrocytomas contained stem cell-like precursors, which exhibit neural stem cell properties with tumorigenicity. These stem cell-like precursors may be responsible for the oligodendroglial and astrocytic components of human oligoastrocytoma and should be considered as therapeutic targets.

Objective: CXCR4 is implicated in the growth, metastasis, and angiogenesis of malignant tumors. We investigated the potential role of CXCR4 in human gliomas., Methods: The expression of CXCR4 messenger ribonucleic acid and protein by human glioma cell lines was examined by reverse-transcriptase polymerase chain reaction and immunocytochemistry analysis. Tumor cell chemotaxis and production of vascular endothelial growth factor induced by the CXCR4 ligand SDF-1beta were measured. Xenograft models were used for evaluation of glioma cell tumorigenesis. CXCR4 expression by xenografted tumors and primary human glioma specimens were evaluated for CXCR4 protein expression. The relationship between CXCR4 expression and patient survival was analyzed. A synthetic lipoxygenase inhibitor, Nordy, was tested for its effects on glioma cell expression and function of CXCR4, as well as on glioma cell tumorigenicity., Results: CXCR4 expression correlated directly with the degree of malignancy of the human glioma cell lines and primary tumors. Activation of CXCR4 induced tumor cell chemotaxis and increased production of vascular endothelial growth factor. Glioma cells expressing higher levels of CXCR4 formed more rapidly growing and lethal tumors in nude mice. Primary human glioma specimens expressing CXCR4 contained high-density microvessels. Patients with CXCR4-positive gliomas had poorer prognosis after surgery. The lipoxygenase inhibitor Nordy diminished CXCR4 expression by glioma cell lines in vitro and reduced their tumorigenicity in nude mice., Conclusion: The level of CXCR4 expression seems to correlate with the degree of malignancy of human gliomas and may contribute to their rapid growth.

Nordy is a chiral compound synthesized based on the structure of a natural lipoxygenase (LO) inhibitor nordihydroguaiaretic acid (NDGA) from plants. The aim of the present study is to investigate the effect of Nordy on malignant human glioma cell responses to chemoattractants and growth promoting signals. We found that Nordy, in a non-cytotoxic concentration range, potently inhibited the chemotaxis and calcium flux of a human glioblastoma cell line U87 induced by a formylpeptide receptor (FPR) agonist, formyl-methionyl-leucyl-phenylalanine (fMLF) and epidermal growth factor (EGF). U87 cells treated by Nordy also showed a significantly impaired proliferation and expression of mRNA for vascular endothelial growth factor (VEGF) induced by fMLF. The chemotactic and proliferation responses of Nordy treated U87 cells to EGF were concomitantly diminished. Further experiments revealed that Nordy did not significantly affect FPR gene expression in U87 cells, but attenuated the activation of a plethora of signaling molecules including ERK1/2, p38, JNK, and Akt when the cells were stimulated by fMLF. EGF-induced EGF receptor phosphorylation was also inhibited in Nordy-treated U87 cells. Moreover, Nordy significantly reduced the tumorigenicity of U87 cells in nude mice. Our results suggest that Nordy is capable of inhibiting glioma cell responses to signals that promote cell motility, growth and production of VEGF. Thus, Nordy may constitute a molecular basis for the development of novel anti-cancer drugs.

Objective: To explore effects of nordy on biological behaviors of human malignant glioblastoma cell line U87MG in vitro and transplanted tumor in vivo, and to identify the differential proteome upon Nordy induced differentiation., Methods: Glioblastoma U87MG cells were induced to differentiate by synthetic lipoxygenase inhibitor, Nordy. The drug was also given via peritoneal injection to nude mice (27 mg/kg body weight) bearing orthotopic transplanted tumors of U87MG cells in the brain. The tumor volumes and GFAP expression were measured. Total proteins of U87MG cells after Nordy treatment were analysed by two-dimensional gel electrophoresis. PDQuest 7.1 computer software was used to compare protein profiles of the treated cells with that of untreated control. Differentially expressed proteins were then selected and characterized by matrix assisted laser desorption ionization-time of flight-mass spectrometry. The functional aspects of these proteins were analyzed by bioinformatics., Results: Nordy suppressed both the proliferation of U87MG cells in vitro and the tumor growth of orthotopic transplanted tumors in vivo (P < 0.01). The differentially expressed proteins induced by Nordy included proliferation-associated gene A, alternative splicing factor ASF-3, eukaryotic translation initiation factor 5A, coffilin 1 (non-muscle), beta galactoside binding lectin, glyceraldehyde-3-phosphate dehydrogenase, enolase 1 and an unknown protein., Conclusions: Nordy promotes the differentiation of glioblastoma cells, by which it may serve as a therapeutic agent. Various proteins identified during Nordy-induced differentiation are involved in the cell proliferation, metabolism, differentiation, apoptosis and gene transcription.

The chemokine receptor CXCR4 plays an important role in tumor growth, angiogenesis and metastasis. Our previous studies showed that Nordy, a synthetic chiral compound of nordihydroguaiaretic acid, inhibited the growth and angiogenesis of various malignant tumors. In this study we examined the capacity of Nordy to regulate CXCR4-mediated production of angiogenic factors by human glioblastoma cells. We found that Nordy potently inhibited CXCR4 ligand SDF-1-induced production of IL-8 and vascular endothelial cell growth factor, two important angiogenic factors implicated in the progression of malignant tumors. Further study revealed that the effect of Nordy was attributable to its down-regulation of the expression of functional CXCR4 in glioblastoma cells. These results suggest that the anti-cancer activity of Nordy is due, at least in part, to its suppression of the chemokine receptor CXCR4 thus reducing the production of angiogenic factors by tumor cells.

Background & Objective: Chemoattractant receptors participate in essential pathophysiologic processes, including inflammation, wound healing, human immunodeficiency virus infection, and most interestingly in the progression of malignant tumor. This study was to explore the functional expression of formylpeptide receptor (FPR) in human glioblastoma cell line U87., Methods: The expression of FPR in U87 and FPR small interfering RNA (siRNA)-transfected U87 cells (FPR-siRNA-U87 cells) was detected with indirect immunofluorescent staining by confocal laser scanning microscopy. FPR was activated by its ligand formy-Met-Leu-Phe (fMLF). Cell proliferation was assessed by MTT assay. The production of vascular endothelial growth factor (VEGF) and interleukin-8 (IL-8) was measured by ELISA, and IL-8 mRNA was assessed by reverse transcription-polymerase chain reaction (RT-PCR)., Results: FPR was expressed on U87 cells, but not on FPR-siRNA-U87 cells. After activation of FPR by fMLF, the proliferation of U87 cells was enhanced markedly (P<0.05), but that of FPR-siRNA-U87 cells had no obvious change (P>0.05). fMLF (100 nmol/L) elicited a time-dependent increase in the secretion of IL-8 and VEGF. When stimulated with 100 nmol/L fMLF for 36 h, the protein levels of VEGF and IL-8 were significantly higher in stimulated U87 cells than in control cells [(3.13+/-0.23) ng/ml vs. (2.55+/-0.25) ng/ml, P<0.05û (7.54+/-0.53) ng/ml vs. (4.02+/-0.09) ng/ml, P<0.05], but those in FPR-siRNA-U87 cells had no obvious change; the mRNA level of IL-8 was significantly increased in U87 cells, but not changed in FPR-siRNA-U87 cells., Conclusion: Activating FPR can promote the proliferation of U87 cells and enhance the production of angiogenic factors.

Objective: To isolate, culture and identify glioma stem cells from human malignant glioma cell line U87, and investigate the changes of pro-angiogenic factors production by glioma stem cells followed by activation of CXCR4 and observe their tumorigenesis as well as the expression of vascular endothelial growth factor when implanted into nude mice., Methods: The ratio of CD133 positive cells was detected by flow cytometry. Magnetic separation of CD133 positive cells was carried out on the magnetic cell sorting system (MACS). Expression of nestin, glial fibrillary acidic protein (GFAP) and CXCR4 on tumorspheres was detected by indirect immunofluorescence under confocal laser scanning microscopy. The functional activation of CXCR4 was assessed by calcium mobilization experiments. ELISA was used to detect the production of vascular endothelial growth factor (VEGF) and interleukin-8 (IL-8) in conditioned medium. Glioma stem cells were implanted into nude mice to assess their tumorigenesis ability and the expression of VEGF., Results: The ratio of CD133 positive cells with stem cell property was 0.5% in U87 cells. Activation of CXCR4 on glioma stem cells induced calcium mobilization and increased VEGF and IL-8 protein secretion. CD133 positive cells secreted more VEGF and IL-8 than their negative counterparts in vitro. Tumors derived from CD133 positive cells grew more rapidly and expressed elevated level of VEGF than their negative counterparts., Conclusions: There are a small fraction of glioma stem cells in human glioblastoma cell line U87. Expressing functional CXCR4 and secreting more pro-angiogenic factors may be involved in tumor angiogenesis mediated by glioma stem cells.

Nordy is a synthesized chrial compound. To investigate the effects of nordy (25 - 100 micromol x L(-1)) on the function of formylpeptide receptor (FPR) of malignant human glioma cells, human glioblastoma cell line U87 was used to detect its proliferation, migration, calcium mobilization, vascular endothelial growth factor (VEGF) mRNA and protein levels after activation of FPR by its agonist N-formyl-methionyl-leucyl-phenylalanine (fMLF). Cell proliferation, migration ability, VEGF mRNA, VEGF protein and calcium mobilization were evaluated by cell counting, chemotaxis assay, RT-PCR, ELISA and spectrometry. Nordy (50 - 100 micromol x L(-1)) potently inhibited the proliferation, migration and calcium mobilization of U87 cells induced by fMLF (P < 0.05). Moreover, 100 micromol x L(-1) nordy showed a significantly impaired VEGF mRNA expression and protein secretion induced by fMLF (P < 0.05). Nordy could inhibit FPR functioning in glioma cell proliferation, migration and angiogenesis, which might be a possible mechanism of its anti-cancer effects.

Objective: To explore the effect of Nordy, a synthesized lipoxygenase Inhibitor, on the expression of formylpeptide receptor (FPR) and its functional activities after activation in human malignant glioma cells., Methods: Human malignant glioma cells of the line U87 were cultured and divided into 3 groups: N-formyl-methionyl-leucyl-phenylalanine (fMLF) group, added with 100 nmol/L fMLF; Nordy group, added with 100 micromol/L Nordy for 12 h and then added with 100 nmol/L fMLF; and control group. The expression of FPR I the U87 cells was detected with indirect immunofluorescence staining and confocal laser scanning microscopy. The effect of Nordy on the U87 cell proliferation induced by fMLF was assayed by MTT method. RT-PCR was used to detect the mRNA expression of vascular endothelial growth factor (VEGF) and interleukin-8 (IL-8) in the U87 cells. ELISA was used to detect protein secretion of VEGF and IL-8 in the supernatant., Results: FPR was expressed on the cytoplasmic membrane of U87 cells. The mean FPR absorbance of the Nordy group was significantly lower than that of the fMLF group. The U87 cell proliferation of the Nordy group was significantly inhibited in comparison with the other 2 groups. The mRNA expression of VEGF and mRNA expression of IL-8 in the U87 cells activated by fMLF were significantly higher than those of the control group (both P<0.05), and Nordy significantly attenuated the mRNA expression of VEGF and IL-8 at different time points (all P<0.05). ELISA showed that the VEGF and IL-8 protein levels in the supernatant were 4.14 ng/ml+/-0.28 ng/ml and 4.03 ng/ml+/-0.59 ng/ml respectively, and increased to 6.46 ng/ml+/-0.33 ng/ml and 7.54 ng/ml+/-0.73 ng/ml respectively after stimulation of fMLF, however, the VEGF and IL-8 protein levels in the supernatant of the Nordy group were 3.59 ng/ml+/-0.33 ng/ml and 3.13 ng/ml+/-0.48 ng/ml respectively., Conclusion: Nordy inhibits the FPR expression and the effects in promoting the cell proliferation and mRNA expression and protein secretion of VEGF and IL-8 after the activation of FPR in malignant human glioma cells, showing that Nordy has the effects of inhibition of tumor growth and angiogenesis.

We recently found that formylpeptide receptor (FPR), a G-protein-coupled receptor that mediates chemotaxis of phagocytic leukocytes induced by bacterial peptide N-formyl-methionyl-leucyl-phenylalanine, is expressed by malignant human glioma cells and promotes tumor growth and angiogenesis. In this study, we examined the effect of Nordy, a novel chiral lipoxygenase inhibitor which was synthesized based on the structure of a natural nordihydroguaiaretic acid, on the expression of FPR by human glioblastoma cells. We found that FPR was expressed at the protein level by highly malignant human glioma cell lines U87 and BT325, and a rat glioma cell line C6. The expression level of FPR was correlated with the degree of the malignancy of tumor cells. The poorly differentiated glioma cell line U87 expressed the highest level of FPR. In U87 glioma cells, the expression of FPR was attenuated at the protein level by Nordy treatment for 48 (P<0.05). Nordy did not affect FPR mRNA expression in U87 cells. In addition, Nordy treatment seemed to promote glioma cell differentiation, as evidenced by their reduced expression of vimentin and increased expression of GFAP. Our results suggest that Nordy was capable of reducing the level of malignancy of glioma cells.

Vascular endothelial cells (ECs) that initiate tumor angiogenesis may acquire distinct properties after conditioning in tumor microenvironment as compared to ECs in non-malignant tissues. Thus far, most in vitro studies of angiogenesis used ECs isolated from normal tissues, which may not fully represent the nature of ECs in tumor vasculature. In this study, glioma-derived microvascular ECs (GDMEC) were purified from human glioma tissues by incubating with magnetic beads coated with anti-CD105 antibody and highly pure (98%) preparations of GDMEC were obtained. These cells exhibited typical EC phenotype, and proliferated rapidly in culture. Interestingly, GDMEC expressed higher levels of VEGF receptors, flt-1 and flk-1, as compared to an established human EC cell line ECV304 and primary human umbilical vascular EC (HUVEC). Functionally, GDMEC were capable of forming intercellular junctions and tubule-like structures (TLS) of various sizes. Stimulation by VEGF further promoted TLS formation with diverse tubular walls by GDMEC. In contrast, TLS formed by ECV304 and HUVEC showed significantly different features. We further observed that Nordy, a synthetic lipoxygenase inhibitor, potently inhibited TLS formation by GDMEC. The results suggest that isolation of highly pure ECs derived from tumor tissues is more appropriate for studies of tumor angiogenesis and for test of potential anti-cancer therapeutic targets.

Numerous studies have showed that chemokine receptors, such as CXCR4, contribute to the growth and metastasis of a variety of malignant tumors. In this study, we investigated the role of CXCR4 in the production of angiogenic factor, vascular endothelial growth factor (VEGF), in various human glioma cells from astrocytic origin. The expression of CXCR4 mRNA and protein in three glioma cell lines, U87-MG, SHG-44, and CHG-5, was determined by RT-PCR and immunocytochemistry, respectively. The malignancies of three gliomas were evaluated by expression of glial fibrillary acidic protein and vimentin, the differentiation markers of astrocytic cells. The role of functional CXCR4 in tumor cell migration was studied with chemotaxis assay. Ca2+ mobilization and VEGF production were measured in the cells after stimulation with CXCR4 ligand, SDF1beta. The results showed that the levels of functional CXCR4 expression at both mRNA and protein levels by several human glioma cell lines were correlated with the degree of differentiation of the tumor cells. Activation of CXCR4 induced glioma cell chemotaxis and could trigger the increase of intracellular [Ca2+]i. Such an activation could result in the increased production of VEGF by the stimulated tumor cells. Our results suggest that CXCR4 may contribute to the high level of VEGF produced by malignant glioma cells and thus constitute a therapeutic target for antiangiogenesis strategy.

Objective: To compare the tubulogenesis capability of malignant glioma-derived microvessel endothelial cells (GDMEC) from human brain with that of ECV304 cells in a three dimentional model and to explore the significance of GDMEC in the study on angiogenesis., Methods: The GDMEC were isolated from malignant gliomas of human brain and purified by selective binding to the monoclonal antibody against CD105 bound to the magnetic MACS MicroBeads. GDMEC and endothelial-like cell line ECV304 were compared with their capabilities of formatting tubule-like structure (TLS) in the three dimentional collagen matrix, with or without inducement by various concentration of vascular endothelial growth factor (VEGF)., Results: The obtained GDMEC had a high purification (98%) and could be successfully cultured in vitro. GDMECs formed more TLS than ECV304 cells of the same number and at the same time points. VEGF could induce rapid formation of TLS in a dose-dependent manner, however, ECV304 cells were less response to VEGF stimulation., Conclusions: GDMEC could maintain their endothelial characteristics and potential capability of angiogenesis. They were more response to VEGF than ECV304, therefore, more suitable for in vitro studies on tumor angiogenesis.

Objective: To investigate the relationship between beta-catenin and matrix metalloproteinase-7 (MMP-7) expression and development/biologic behavior of human colorectal cancer., Methods: Immunohistochemical study for beta-catenin and MMP-7 was carried out on colorectal adenoma-carcinoma tissue microarrays and results analyzed., Results: The nuclear beta-catenin expression rate was 35.9% in adenoma with malignant transformation, significantly higher than that in adenoma (16.7%) and carcinoma (19.7%) (both P < 0.05). The cytoplasmic and nuclear beta-catenin expression rate in adenoma with severe dysplasia was significantly higher than that in adenoma with mild dysplasia (both P < 0.05). The nuclear beta-catenin expression rate in adenocarcinomas of the ulcerative type, with lymph node metastasis and in the late tumor stages were all significantly higher than that in adenocarcinomas of the polypoid type, with negative lymph node and in the early tumor stages (P < 0.05 or P < 0.01). The MMP-7 expression rate in adenocarcinoma (69.2%) was significantly higher than that in normal colorectal mucosa (15.0%), adenoma (35.0%) and adenoma with malignant transformation (46.2%, P < 0.05 or P < 0.01). The MMP-7 expression rate in ulcerative type adenocarcinoma with lymph node metastasis and in late tumor stages was significantly higher than that in polypoid type adenocarcinoma with negative lymph node and in early tumor stages (all P < 0.05). The cytoplasmic and nuclear beta-catenin expression was thus in positive correlation with the expression of MMP-7 (both P < 0.01)., Conclusions: The cytoplasmic and nuclear beta-catenin expression, probably an early event, was related to the development of colorectal cancer. beta-catenin may enhance the degradative function of the target gene MMP-7 through nuclear translocation and may further facilitate local invasion and metastasis by the colorectal cancer cells.