Xiaotan Sanjie decoction attenuates tumor angiogenesis by manipulating Notch-1-regulated proliferation of gastric cancer stem-like cells

AIM: To determine the underlying mechanisms of action and influence of Xiaotan Sanjie (XTSJ) decoction on gastric cancer stem-like cells (GCSCs).

Nestin in gastrointestinal and other cancers: Effects on cells and tumor angiogenesis

Nestin is a class Ⅵ intermediate filament protein that was originally described as a neuronal stem cell marker during central nervous system (CNS) development, and is currently widely used in that capacity. Nestin is also expressed in non-neuronal immature or progenitor cells in normal tissues. Under pathological conditions, nestin is expressed in repair processes in the CNS, muscle, liver, and infarcted myocardium. Furthermore, increased nestin expression has been reported in various tumor cells, including CNS tumors, gastrointestinal stromal tumors, pancreatic cancer, prostate cancer, breast cancer, malignant melanoma, dermatofibrosarcoma protuberances, and thyroid tumors. Nestin is reported to correlate with aggressive growth, metastasis, and poor prognosis in some tumors; however, the roles of nestin in cancer cells have not been well characterized. Furthermore, nestin is more specifically expressed in proliferating small-sized tumor vessels in glioblastoma and gastric, colorectal, and prostate cancers than are other tumor vessel markers. These findings indicate that nestin may be a marker for newly synthesized tumor vessels and a therapeutic target for tumor angiogenesis. It has received a lot of attention recently as a cancer stem cell marker in various cancer cells including brain tumors, malignant rhabdoid tumors, and uterine, cervical, prostate, bladder, head and neck, ovarian, testicular, and pancreatic cancers. The purpose of this review is to clarify the roles of nestin in cancer cells and in tumor angiogenesis, and to examine the association between nestin and cancer stem cells. Nestin has the potential to serve as a molecular target for cancers with nestin-positive cancer cells and nestin-positive tumor vasculature.

Numerical simulation of solid tumor angiogenesis with Endostatin treatment:a combined analysis of inhibiting effect of anti-angiogenic factor and micro mechanical environment of extracellular matrix

To investigate the influence of anti-angiogenesis drug Endostatin on solid tumor angiogenesis, a mathematical model of tumor angiogenesis was developed with combined influences of local extra-cellular matrix mechanical environment, and the inhibiting effects of Angiostatin and Endostatin. Simulation results show that Angiostatin and Endostatin can effectively inhibit the process of tumor angiogenesis, and decrease the number of blood vessels in the tumor. The present model could be used as a valid theoretical method in the investigation of anti-angiogenic therapy of tumors.

Inhibition of tumor angiogenesis by TTF1 from extract of herbal medicine

AIM:To study the inhibition of tumor angiogenesis by 5,2,4'-trihydroxy-6,7,5'-trimethoxyflavone(TTF1) isolated from an extract of herbal medicine Sorbaria sorbifolia.METHODS:Angiogenic activity was assayed using the chick embryo chorioallantoic membrane(CAM) method.Microvessel density(MVD) was determined by staining tissue sections immunohistochemically for CD34 using the Weidner capillary counting method.The mRNA and protein levels of vascular endothelial growth factor(VEGF),vascular endothelialgrowth factor receptor 2(VEGFR2,Flk-1/KDR),basic fibroblast growth factor(bFGF),cyclo-oxygenase(COX)-2 and hypoxia-inducible factor(HIF)-1α were detected by quantitative real-time polymerase chain reaction and Western blotting analysis.RESULTS:The TTF1 inhibition rates for CAM were 30.8%,38.2% and 47.5% with treatment concentrations of 25,50 and 100 μg/embryo × 5 d,respectively.The inhibitory rates for tumor size were 43.8%,49.4% and 59.6% at TTF1 treatment concentrations of 5,10,and 20 μmol/kg,respectively.The average MVD was 14.2,11.2 and 8.5 at treatment concentrations of 5 μmol/kg,10 μmol/kg and 20 μmol/kg TTF1,respectively.The mRNA and protein levels of VEGF,KDR,bFGF,COX-2 and HIF-1α in mice treated with TTF1 were significantly decreased.CONCLUSION:TTF1 can inhibit tumor angiogenesis,and the mechanism may be associated with the down-regulation of VEGF,KDR,bFGF,HIF-1α and COX-2.

THE CLINICAL SIGNIFICANCE OF TUMOR ANGIOGENESIS AND INVASIVENESS-RELATED GENE EXPRESSIONS IN GASTRIC CANCER

Objective: To investigate the correlation among tumor angiogenesis, expressions of p53, nm23-I1, CD44v6, c-erbB-2 proteins and biological behavior and clinical outcome of gastric cancer. Methods: The intratumoral microvessel density (MVD) and expressions of p53, nm23-H1, CD44v6, c-erbB-2 were analyzed semiquantitively by immunohistochemical staining (S-P) of 59 paraffin-embedded gastric tumor specimens that were radically resected at the Department of surgery, Beijing Institute for Cancer Research, between January 1990 and December 1992. The median follow-up period was 75 month (range: 60~96 months). The significdance of these indicators was analyzed retrospectively. Results: MVD for tumors with lymph node metastasis and vascular invasion was significantly higher than those without (P=0.0168 and 0.0176, respectively). The levels of p53, CD44v6, c-erbB-2 expression were significantly higher in the groups of lymph node metastasis, serosal infiltration and vascular invasion than in those without. All differences reached the statistically significant levels (P<0.01~<0.05). The low expression of nm23-H1 was negatively correlated with lymph node metastasis, serosal infiltration and vascular invasion (P<0.01; <0.05 and <0.01, respectively). Univariate analysis showed that the overall survival of patients with higher MVD, or overexpressions of p53, CD44v6, c-erbB-2, or low expression of nm23-H1 were significantly worse than those with opposite conditions (P=0.0214, 0.0062, 0.0045, 0.0159, and 0.0162, respectively). Multivariate analysis showed that expression of p53 in this series was an independent prognostic indicator. Conclusion: The data suggested that the above-mentioned factors might be helpful in evaluating the metastatic potential of gastric cancer and making more effective assessment of prognosis for individual patient. Further study with larger samples and prospective investigation of these results would be worthwhile.

Exploration on Effect and Mechanism of Suiqing Pill (髓清丸) on Tumor Angiogenesis in Nude Mouse B16 Melanoma Model

Objective: To study the effect of Suiqing Pill (SQP, 髓清丸),a TCM compound drug that can activate blood circulation and get rid of blood stasis, on angiogenesis in tumor and its possible mechanism. Method: BALB/c nude mice were inoculated with melanoma cell line B16 at right armpit subcutaneously to establish cancer spontaneous metastasis model. Levels of microvessel density (MVD), vascular endothelial growth factor (VEGF) and protein expression of matrix metalloproteinase-2 (MMP-2) in tumor tissues were observed and compared. Results: Strong expression of anti-Factor Ⅷ (FⅧ) related antigen and plentiful tumor angiogenesis were seen in model control animals, while in the high-dose and low-dose SQP treated model mice, MVD was inhibited by 33. 5% and 22. 6% respectively ( P<0. 01, P<0. 05). The strong positive protein expression of VEGF and MMP-2 in model control also reduced in the SQP treated groups. Conclusion: SQP could inhibit tumor angiogenesis, protein expression of VEGF and MMP-2 in nude mice B16 melanoma models.

Numerical simulation of tumor angiogenesis under the effect of Endostatin:considering mechanical environment in matrix and inhibiting effect of anti-angiogenic factor

To investigate tumor angiogenesis under the influence of Endostatin,mathematical modeling and numerical simulation of tumor angiogenesis are performed,with the mechanical environment in matrix,the inhibiting effects of Angiostatin and Endostatin into consideration.The

Construction of curcumin-loaded micelles and evaluation of the anti-tumor effect based on angiogenesis

Objective:Inhibition of tumor angiogenesis has become a new targeted tumor therapy.In this study,we established a micellar carrier with a tumor neovascularization-targeting effect modified by the neovascularization-targeting peptide NGR.Methods:The targeted polymer poly(ethylene glycol)-b-poly(lactide-co-glycolide)(PEG-PLGA)modified with Asn–Gly–Arg(NGR)peptide was prepared and characterized by 1H nuclear magnetic resonance and Fourier-transform infrared spectrometry.NGR-PEG-PLGA was used to construct curcumin(Cur)-loaded micelles by the solvent evaporation method.The physicochemical properties of the micelles were also investigated.Additionally,we evaluated the antitumor efficacy of the polymer micelles(PM)using in vitro cytology experiments and in vivo animal studies.Results:The particle size of Cur-NGR-PM was 139.70±2.51 nm,and the drug-loading capacity was 14.37±0.06%.In vitro cytological evaluation showed that NGR-modified micelles showed higher cellular uptake through receptor-mediated endocytosis pathways than did unmodified micelles,leading to the apoptosis of tumor cells.Then,in vivo antitumor experiments showed that the modified micelles significantly inhibited tumor growth and were safe.Conclusions:NGR-modified micelles significantly optimized the therapeutic efficacy of Cur.This strategy offers a viable avenue for cancer treatment.

Impaired tumor angiogenesis and VEGF- induced pathway in endothelial CD146 knockout mice

CD146 is a newly identified endothelial biomarker that has been implicated in angiogenesis. Though in vitro angio- genic function of CD146 has been extensively reported, in vivo evidence is still lacking. To address this issue, we generated endothelial-specific CD146 knockout （CD146 EC-Ko） mice using the Tg（Tek-cre） system. Surprisingly, these mice did not exhibit any apparent morphological defects in the development of normal retinal vasculature. To evaluate the role of CD146 in pathological angiogenesis, a xenograft tumor model was used. We found that both tumor volume and vascular density were significantly lower in CD146Ec-KO mice when compared to WT littermates. Additionally, the ability for sprouting, migration and tube formation in response to VEGF treatment was impaired in endothelial cells （ECs）of CD146Ec-Ko mice. Mechanistic studies further confirmed that VEGF- induced VEGFR-2 phosphorylation and AKT/p38 MAPKs/ NF-KB activation were inhibited in these CD146-null ECs, which might present the underlying cause for the observed inhibition of tumor angiogenesis in CD146Ec-Ko mice. These results suggest that CD146 plays a redundant role in physiological angiogenic processes, but becomes essential during pathological angiogenesis as observed in tumorigenesis.

Regulation of tumor angiogenesis by the microtubule-binding protein CLIP-170

Angiogenesis,the expansion of preexisting blood vessels,is a complex process required for tumor growth and metastasis.Although current antiangiogenic strategies have shown promising results in several cancer types,identification of additional antiangiogenic targets is required to improve the therapeutic response.Herein,we show that the microtubule-binding protein CLIP-170(cytoplasmic linker protein of 170 kDa)is highly expressed in breast tumor samples and correlates positively with blood vessel density.Depletion of CLIP-170 signifi cantly impaired vascular endothelial tube formation and sprouting in vitro and inhibited breast tumor growth in mice by decreasing tumor vascularization.Our data further show that CLIP-170 is important for the migration but not the proliferation of vascular endothelial cells.In addition,CLIP-170 promotes the polarization of endothelial cells in response to the angiogenic stimulus.These fi ndings thus demonstrate a critical role for CLIP-170 in tumor angiogenesis and suggest its potential as a novel antiangiogenic target.

CANSTATIN, A ENDOGENOUS INHIBITOR OF ANGIOGENESIS AND TUMOR GROWTH

Canstatin is a novel inhibitor of angiogenesis and tumor growth, derived from the C-terminal globular non-collageneous (NCl) domain of the a2 chain of type IV collagen. It inhibits endothelial cell proliferation and migration in a dose-dependent manner, and induces endothelial cell apoptosis. In vivo experiments show that canstatin significantly inhibits solid tumor growth. The canstatin mediated inhibition of tumor is related to apoptosis. Canstatin- induced apoptosis is associated with phosphatidylinositol 3-kinase/Akt inhibition and is dependend upon signaling events transduced trough membrane death receptor.

Two-dimensional discrete mathematical model of tumor-induced angiogenesis

A 2D discrete mathematical model of a nine-point finite difference scheme is built to simulate tumor-induced angiogenesis. Nine motion directions of an individual endothelial cell and two parent vessels are extended in the present model. The process of tumor-induced angiogenesis is performed by coupling random motility, chemotaxis, and haptotaxis of endothelial cell in different mechanical environments inside and outside the tumor. The results show that nearly realistic tumor microvascular networks with neoplastic pathophysiological characteristics can be generated from the present model. Moreover, the theoretical capillary networks generated in numerical simulations of the discrete model may provide useful information for further clinical research.

An experimental research into endostatin microbubble combined with focused ultrasound for anti-tumor angiogenesis in colon cancer

Objective:to evaluate the therapeutic effect of targeted endostatin-loaded microbubbles,combined with improved,focused,directional ultrasound radiation for inhibition of subcutaneous translocation in situ colon tumor angiogenesis in colon cancer.Methods:65 BALB/c nude mice with subcutaneous translocation in situ colon tumors were randomly divided into five groups.Group A was the control group,without any treatments.In Group B,the mouse was treated with ultrasonic radiation.In Group C,the mouse was treated with ultrasonic radiation combined with empty SonoVue microbubbles.In Group D,the mouse was treated with ultrasonic radiation combined with empty Targestar-SA microbubbles.In Group E,the mouse was treated with ultrasonic radiation combined with endostatin microbubbles.The tumor size was measured before and 1,14,and 28 days after irradiation.The peak intensity(PI),regional blood volume(RBV)and regional blood flow(RBF)were recorded using contrast-enhanced ultrasound.The tumor tissue was removed for pathological examination;the tumor necrosis area and microvascular density(MVD)were evaluated by immunohistochemistry.Results:Tumors in Groups C,D and E were significantly smaller than in Groups A and B at 28 days after irradiation,with Group E the smallest.PI,RBF and RBV of Groups C,D,and E were significantly decreased 28 days after radiation with Group E the lowest,and significantly lower than Groups A and B(all P<0.05).The tumor tissue necrosis area of Group E was clearly greater while MVD was obviously lower than the other groups(all P<0.01)at 28 days after treatment.Conclusion:The targeted endostatin microbubbles,combined with focused,directional ultrasound radiation can damage tumor microvasculature of subcutaneous colon translocation in situ colon cancer,as well as inhibit the tumor angiogenesis.

Glucocorticoids suppress tumor angiogenesis and growth of prostate cancer

Hormone-refractory prostate cancer ( HRPC) sometimes is responsive to treatment with glucocorticoids, such as prednisolone, hydrocortisone and dexamethasone, but the underlying mechanisms are not well established. In a recent paper (Clin Cancer Res, 2006, 12:3003-3009), Yano et al. Hypothesized and confirmed that the therapeutic effect of glucocorticoids on HRPC is attributed to inhibition of angiogenesis. A prostate cancer cell line DU145 that expresses glucocorticoid receptor was used to study the effect of dexamethasone (Dex) on the expres-

Study on vasculogenic mimicry in malignant esophageal stromal tumors

AIM: To investigate whether malignant esophageal stromal tumors contain PAS-positive patterned matrix-associated vascular channels, which are lined by tumor cells, but not vascular endothelial cells. That is vasculogenic mimicry (VM) independent of tumor angiogenesis. METHODS: Thirty-six tissue samples of malignant esophageal stromal tumors were analyzed. Tissue sections were stained for Vascular endothelial growth factor (VEGF), CD31 and periodic acid Schiff (PAS). The level of VEGF, the microvascular density (MVD) and the vasculogenic mimicry density (VMD) were determined. RESULTS: PAS-positive patterned matrix-associated vascular channels were detected in 33.3% (12/36) of tumor samples. Within these patterned channels, red blood cells were found. The level of VEGF and the MVD in tumors containing patterned channels were significantly higher than those in tumors not containing patterned channels (P < 0.05). At the same time, the malignant degree of tumors was higher, the proportions of tumors containing patterned channels were not only more, but also in the each kind of tumors containing patterned channels. CONCLUSION: In malignant esophageal stromal tumors, a VM mechanism causes some tumor cells to deform themselves and secrete extracellular matrix; thus, PAS-positive patterned matrix-associated vascular channels appear and supplying blood to the tumors to sustain their growth and metastasis.

An isoflavone derivative potently inhibits the angiogenesis and progression of triple-negative breast cancer by targeting the MTA2/SerRS/VEGFA pathway

Objective:Angiogenesis plays a vital role in tumor growth and metastasis.Here,we aimed to find novel efficient antiangiogenic molecules targeting vascular endothelial growth factor A(VEGFA)at the transcriptional level to treat triple-negative breast cancer(TNBC).Methods:We used a cell-based seryl tRNA synthetase(SerRS)promoter-driven dual-luciferase reporter system to screen an in-house library of 384 naturally occurring small molecules and their derivatives to find candidate molecules that could upregulate the expression of SerRS,a potent transcriptional repressor of VEGFA.The levels of SerRS and VEGFA were examined by quantitative RT-PCR(qRT-PCR),western blotting,and/or ELISAs in TNBC cells after candidate molecule administration.Zebrafish,the Matrigel plug angiogenesis assay in mice,the TNBC allograft,and xenograft mouse models were used to evaluate thein vivoanti-angiogenic and anti-cancer activities.Furthermore,the potential direct targets of the candidates were identified by proteomics and biochemical studies.Results:We found the most active compound was 3-(4-methoxyphenyl)quinolin-4(1H)-one(MEQ),an isoflavone derivative.In TNBC cells,MEQ treatment resulted in increased SerRS mRNA(P<0.001)and protein levels and downregulated VEGFA production.Both the vascular development of zebrafish and Matrigel plug angiogenesis in mice were inhibited by MEQ.MEQ also suppressed the angiogenesis in TNBC allografts and xenografts in mice,resulting in inhibited tumor growth and prolonged overall survival(P<0.05).Finally,we found that MEQ regulated SerRS transcription by interacting with MTA2(Metastasis Associated 1 Family Member 2).Conclusions:Our findings suggested that the MTA2/SerRS/VEGFA axis is a drug-treatable anti-angiogenic target,and MEQ is a promising anti-tumor molecule that merits further investigation for clinical applications.

Effect of non-anticoagulant N-desulfated heparin on expression of vascular endothelial growth factor, angiogenesis and metastasis of orthotopic implantation of human gastric carcinoma

AIM： To investigate the effect of N-desulfated heparin on tumor metastasis and angiogenesis, and expression of vascular endothelial growth factor （VEGF） of orthotopic implantation of human gastric carcinoma in male severe combined immune deficiency （SCID） mice. METHODS： Human gastric cancer SGC-7901 cells were orthotopically implanted into the stomach of SC/D mice. The mice were randomly divided into normal saline group and N-desulfated heparin group. One week after operation, the mice in N-desulfated heparin group reo ceived i.v. injections of N-desulfated heparin （Shanghai Institute of Cell Biology, Chinese Academy of Sciences, 10 mg/kg.d） twice weekly for 3 wk. The mice in normal saline group received i.v. injections of normal saline （100 μL） twice weekly for 3 wk. The mice were sacrificed six weeks after implantation. Tumor metastasis was evaluo ated histologically for metastasis under microscope. Intratumoral microvessel density （MVD） and VEGF expression were evaluated immuohistochemically. VEGF mRNA expression in gastric tissue of SC/D mice was detected by real time PCR. RESULTS： The tumor metastasis rate was 80% in normal saline group and 20% in N-desulfated heparin group （P 〈 0.05）. MVD was 8.0 ± 3.1 in normal saline group and 4.3 ± 1.8 in N-desulfated heparin group （P 〈 0.05）. VEGF positive immunostaining was found in cytoplasm of cancer cells. The rate of VEGF positive expression was higher in normal saline group than in N-desulfated hepa- rin treated group （90% vs 20%, P 〈 0.05）. VEGF mRNA expression was significantly inhibited by N-desulfated heparin and was higher in normal saline group than in N-desulfated heparin group （Ct value 19.51 ± 1.01 vs 22.55± 1.36, P 〈 0.05）. N-desulfated heparin significantly inhibited the expression of VEGF mRNA in cancer cells. No bleeding occurred in N-desulfated heparin group. CONCLUSION： N-desulfated heparin can inhibit metastasis of gastric cancer by suppressing tumor VEGF expression and tumor angiogenesis, but has no obvious anticoagulant activity.

Tea polyphenols inhibit the growth and angiogenesis of breast cancer xenografts in a mouse model

Objective:To investigate the anti-angiogenic effect of tea polyphenols(TPS)on breast cancer and normal tissues in a mouse model.Methods:Breast cancer was successfully implanted into 48 BALB/c mice,which were then randomly divided into a TP oral gavage group,a TP local injection group,a ginsenoside Rg3 group,and a model control group according to a random number table.The tumor inhibitory rates of each group were calculated,while microvessel density(MVD)and the expression of vascular endothelial growth factor(VEGF),basic fibroblast growth factor(bFGF),and tissue inhibitor of metalloproteinase(TIMP-2)were detected by immunohistochemistry.Results:TPs could inhibit the growth of breast cancer xenografts in the mouse model.The tumor inhibition rates of the TP oral gavage and TP local injection groups were 37.43%and 40.94%,respectively.Compared with the model control group,MVD and VEGF and bFGF expression was downregulated(all P<.05),whereas TIMP-2 expression was elevated in the TP oral gavage and TP local injection groups(P=.015 and P=.032).TPs showed no significant effect on MVD and VEGF and TIMP-2 expression in the heart,brain,and kidney of the mouse model.Conclusion:TPs can restrict the growth of breast cancer by specifically inhibiting the angiogenesis of breast tumor tissue while having little effect on the normal tissue of important organs including the heart,brain,and kidney.

Research progress on the role of cancer-associated fibroblasts in tumorigenesis and development

Tumor microenvironment plays a very important role in the growth,invasion and metastasis of tumor cells.The tumor interstitial microenvironment is an important part of the tumor microenvironment,which includes two parts:the non-cellular and cellular components of the tumor interstitium,specifically including the extracellular matrix,blood vessels,and interstitial cells.Among them,activated interstitial fibroblasts,namely cancer-associated fibroblasts(CAFs),are the main components of tumor interstitial cells,which are most closely related to tumor interstitial fibrosis and tumor progress,and are expected to become a new target for cancer treatment.

Anti-cancer and anti-angiogenic effects of curcumin and tetrahydrocurcumin on implanted hepatocellular carcinoma in nude mice

AIM: To determine the effect of tetrahydrocurcumin (THC) on tumor angiogenesis compared with curcumin (CUR) by using both in vitro and in vivo models of human hepatocellular carcinoma cell line (HepG2). METHODS: The 3-(4,5-dimethylthiazol-2-yl)-2, 5-diphenyl-tetrazolium bromide (MTT) assay was used for testing the anti-proliferating activities of CUR and THC. In male BALB/c nude mice, 2 × 106 human HepG2 cells were inoculated onto a dorsal skin-fold chamber. One day after HepG2 inoculation, the experimental groups were fed oral daily with CUR or THC (300 mg/kg or 3000 mg/kg). On d 7, 14 and 21, the tumor microvasculature was observed using fluorescence videomicroscopy and capillary vascularity (CV) was measured. RESULTS: Pathological angiogenic features including microvascular dilatation, tortuosity, and hyper-permeability were observed. CUR and THC could attenuate these pathologic features. In HepG2-groups, the CV were significantly increased on d 7 (52.43%), 14 (69.17%), and 21 (74.08%), as compared to controls (33.04%,P < 0.001). Treatment with CUR and THC resulted in significant decrease in the CV (P < 0.005 and P < 0.001, respectively). In particular, the anti-angiogenic effects of CUR and THC were dose-dependent manner. However, the beneficial effect of THC treatment than CUR was observed, in particular, from the 21 d CV (44.96% and 52.86%, P < 0.05). CONCLUSION: THC expressed its anti-angiogenesis without any cytotoxic activities to HepG2 cells even at the highest doses. It is suggested that anti-angiogenic properties of CUR and THC represent a common potential mechanism for their anti-cancer actions.