Anti-tumor Angiogenesis Research Articles

Fisetin and polymeric micelles encapsulating fisetin exhibit potent cytotoxic effects towards ovarian cancer cells

BackgroundThe anti-tumor activities of Natural compounds and their derivatives are of great interest to pharmaceutical industries. Fisetin is one of prospective natural compounds in this regard but unfortunately with poor hydrophilicity.MethodsThe effects of unmodified and modified fisetin in cultured ovarian cancer cells were compared by transmission electronmicroscopy to determine apoptotic bodies, MTT assay to quantitate cell numbers, and fluorescence activated cell sorting analyse of various markers to determine the apoptotic state. In addition, the efficacy of fisetin and fisetin-micelles in vivo was determined by using immunocompromised mice. Apoptosis was measured by established markers using both western blot analysis and immunochemistry. Angiogenesis in a xenograft mouse model carring SKOV3 cells was evaluated by color Doppler ultrasound and immunohistochemistry.ResultMultiple lines of evidence indicated that fisetin and fisetin micelles induce apoptosis in ovarian cancer cells in a dose-dependent manner. Histological analysis, terminal deoxynucleotidyltransferase-mediated nick-end labeling assay, western blot, immunohistochemical detection and microvessel density detection demonstrated that fisetin and fisetin micelles induced increased tumor apoptosis, proliferation suppression and antiangiogenesis activities.ConclusionAs far as we know, the present study is the first time to demonstrate the potency of both fisetin and fisetin micelles inducing apoptosis in ovarian cancer cells. Further studies will be needed to validate the therapeutic potential of fisetin and fisetin micelles in ovarian cancer treatment.

Abstract B095: Antitumor activity of lenvatinib mesilate (LEN) via angiogenesis inhibition and tumor FGF signaling pathway in the human hepatocellular carcinoma models

Abstract Background: LEN is an oral multi-targeted tyrosine kinase inhibitor that selectively inhibits vascular endothelial growth factor receptor (VEGFR)1-3, fibroblast growth factor receptor (FGFR)1-4, platelet-derived growth factor receptor α, RET, and KIT. LEN was noninferior to sorafenib (SOR) in overall survival (OS) in a phase 3 study in patients with unresectable hepatocellular carcinoma (HCC) (medians: LEN, 13.6 vs SOR, 12.3 months; hazard ratio: 0.92; 95% confidence interval, 0.79-1.06) and statistically superior to SOR in progression-free survival (PFS), time to progression (TTP), and objective response rate (ORR). We previously reported that LEN showed antitumor activity in multiple HCC xenograft models including patient-derived xenograft (PDx) models. Here, we investigated the mode of action of LEN in preclinical human HCC models to examine antitumor angiogenesis activity in vivo and the effects on FGF signaling pathways both in vitro and in vivo in the preclinical HCC models. Methods: Antiproliferative activity of LEN and SOR was examined using human HCC cells (Hep 3B2.1-7, HuH-7, SNU398, and PLC/PRF/5) in vitro, and antitumor and anti-angiogenesis activity were examined using these HCC cell lines and PDx models. The effect on the FGF signaling pathway was evaluated by determining the phosphorylation of FRS2, a direct substrate of FGFRs, with immunoblotting analysis in both cultured cells and tumor xenografts. Anti-angiogenesis activity was evaluated by determining microvessel density (MVD) within tumor xenografts by immunohistochemical analysis using anti-CD31 antibody. Result: LEN decreased the phosphorylation of FRS2 in FGF19-overexpressing Hep3B2.1-7 and HuH-7 cells at 0.03 to 3 μmol/L in a concentration-dependent manner. LEN showed selective antiproliferative activity against these cells with half-maximal inhibitory concentration (IC50) values of 0.23 and 0.42 μmol/L, respectively, and those concentrations were consistent with ones for inhibitions of the phosphorylation of FRS2. In tumor xenografts, LEN also decreased the phosphorylation of FRS2 by the single administration of LEN between 3 and 30 mg/kg. SNU398 cells are known to have a relatively weak dependence on the FGF signaling pathway without FGF19 expression. LEN inhibited in vitro proliferation of SNU398 cells, and the phosphorylation of FRS2 both in vitro and in vivo at higher doses than in Hep 3B2.1-7 and HuH-7 cells. SOR did not selectively inhibit in vitro proliferation or the phosphorylation of FRS2 in HCC cells with FGF activation in vitro and in vivo. In HCC PDx and PLC/PRF/5 xenograft models, LEN significantly decreased the MVD in a dose-dependent manner between 3 and 30 mg/kg, and it was correlated with tumor growth inhibition in each models. SOR showed clear anti-angiogenesis activity at 30 mg/kg, but not at 10 mg/kg. Conclusion: These results suggest that inhibition of the FGF signaling pathway via FGFR with LEN affects proliferation of HCC cells with activation of the FGF signaling pathway, and the potent anti-angiogenic activity underlies the antitumor activity of LEN in preclinical HCC xenograft models. Citation Format: Taisuke Hoshi, Masahiro Matsuki, Yuji Yamamoto, Yukinori Minoshima, Junji Matsui, Yasuhiro Funahashi. Antitumor activity of lenvatinib mesilate (LEN) via angiogenesis inhibition and tumor FGF signaling pathway in the human hepatocellular carcinoma models [abstract]. In: Proceedings of the AACR-NCI-EORTC International Conference: Molecular Targets and Cancer Therapeutics; 2017 Oct 26-30; Philadelphia, PA. Philadelphia (PA): AACR; Mol Cancer Ther 2018;17(1 Suppl):Abstract nr B095.

Selective delivery of PLXDC1 small interfering RNA to endothelial cells for anti-angiogenesis tumor therapy using CD44-targeted chitosan nanoparticles for epithelial ovarian cancer

Angiogenesis plays an essential role in the growth and metastasis of tumor cells, and the modulation of angiogenesis can be an effective approach for cancer therapy. We focused on silencing the angiogenic gene PLXDC1 as an important factor for anti-angiogenesis tumor therapy. Herein, we developed PLXDC1 small interfering siRNA (siRNA)-incorporated chitosan nanoparticle (CH-NP/siRNA) coated with hyaluronic acid (HA) to target the CD44 receptor on tumor endothelial cells. This study aimed to improve targeted delivery and enhance therapeutic efficacy for tumor anti-angiogenesis. The HA-CH-NP/siRNA was 200 ± 10 nm in size with a zeta potential of 26.4 mV. The loading efficiency of siRNA to the HA-CH-NP/siRNA was up to 60%. The selective binding of HA-CH-NP/siRNA to CD44-positive tumor endothelial cells increased by 2.1-fold compared with that of the CD44 nontargeted CH-NP/siRNA. PLXDC1 silencing by the HA-CH-NP/siRNA significantly inhibited tumor growth in A2780 tumor-bearing mice compared with that in the control group (p < .01), and mRNA expression of PLXDC1 was significantly reduced in the HA-CH-NP/siRNA-treated group. Furthermore, treatment with HA-CH-NP/siRNA resulted in significant inhibition of cell proliferation (p < .001), reduced microvessel density (p < .001), and increased cell apoptosis (p < .001). This study demonstrates that HA-CH-NP/siRNA is a highly selective delivery platform for siRNA, and has broad potential to be used in anti-angiogenesis tumor therapy.

COX‑2 inhibition in the endothelium induces glucose metabolism normalization and impairs tumor progression.

Previous antitumor angiogenesis strategies have focused on targeting angiogenic signals. Encouragingly, the metabolism of tumor endothelial cells (TECs) has gained attention as a therapeutic target in recent years. There is consensus that, in terms of antitumor angiogenesis, the promotion of tumor vascular regression and normalization of the remaining blood vessels are equally important. Presently, tumor vessel normalization (TVN) is an emerging antitumor treatment. The present study focused on the normalization of TEC metabolism. The results demonstrated that TECs have a hyperglycolytic metabolism. Parixibox, a cyclooxygenase-2 (COX-2) blocker, successively reduces the expression of vascular endothelial growth factor (VEGF) in the tumor microenvironment. VEGF further influences the expression of 6-Phosphofructo-2-Kinase/Fructose-2,6-Biphosphatase 3, a key glycolysis gene. Pharmacological blockade of COX-2 restored the glucose metabolism level (particularly glycolysis) in TECs, which may be an important basic process in TVN. Therefore, COX-2, which acts on abnormal tumor vessels, is expected to become a novel target for tumor treatment.

Inhibitory effect of migration-inducing gene-7-shRNA recombinant retrovirus combined with endostatin on growth and metastasis of hepatoma xenograft

Objective: To investigate the inhibitory effect of migration-inducing gene-7(Mig-7)interfered with retrovirus-mediated RNA(shRNA)combined with recombinant human endostatin(ES)on the growth and metastasis of subcutaneous xenograft of human hepatoma cells in nude mice. Methods: Two Mig-7-mRNA oligonucleotide sequences(Mig-7-shRNA-1 and Mig-7-shRNA-2)and one sequence as a negative control(Mig-7-shRNA-N)were designed. The specific Mig-7-shRNA recombinant retrovirus expression vector plasmid was constructed and used for the transfection of human hepatoma MHCC-97H cells with high expression of Mig-7. The subcutaneous xenograft tumor model of human hepatocellular carcinoma(HCC)in nude mice was established, and according to the condition of transfection and administration, the nude mice were divided into pSIREN-M1 group, pSIREN-MN group, ES group, and pSIREN-M1+ES group. The xenograft tumor volume, mass, and metastasis were compared between groups. Immunohistochemistry was used to observe the formation of vasculogenic mimicry(VM)in xenograft tumor and the difference in tumor microvascular density(MVD), and Western blot was used to measure the expression of Mig-7 and vascular endothelial growth factor(VEGF)in each group. A one-way analysis of variance was used for comparison between groups, and the Fisher's exact test was used for comparison of continuous data between groups. Results: Compared with the pSIREN-MN group, the pSIREN-M1 group had significantly lower xenograft tumor volume, mass, and metastasis rate, Mig-7 expression, and formation of VM(P < 0.05), as well as significantly higher VEGF expression and MVD(P < 0.05). Compared with the pSIREN-MN group, the ES group had significantly lower xenograft tumor volume, mass, and metastasis rate, VEGF expression, and MVD(P < 0.05), as well as significantly higher Mig-7 expression and formation of VM(P < 0.05). Compared with the pSIREN-M1 group and the ES group, the pSIREN-M1+ES group had significantly lower xenograft tumor volume, mass, and metastasis rate, Mig-7 expression, formation of VM, VEGF expression, and MVD(P < 0.05). Conclusion: Mig-7-shRNA recombinant retrovirus combined with ES has a better inhibitory effect on the growth and metastasis of HCC xenograft tumor than Mig-7-shRNA recombinant retrovirus or ES alone. The anti-tumor angiogenesis therapy alone, which targets vascular endothelial cells in vivo, has a limited effect, since it may promote the formation of VM.

Research progress of thalidomide in usual non-hematologic malignancies

Thalidomide has anti-tumor effects such as anti-tumor angiogenesis, improvement of immune function and cachexia of patients with advanced tumors, which has been effectively used in elderly patients with castration-resistant prostate cancer, chemotherapy resistant advanced colorectal cancer, advanced hepatocellular carcinoma and metastatic breast cancer. As the in-depth study of the anti-tumor mechanism of thalidomide, more and more clinical researches have focused on the function of thalidomide on non-hematological malignancies, especially it has obtained some achivements in the postoperative adjuvant therapy for high-risk colorectal cancer and primary hepatocellular carcinoma and the salvage therapy for metastatic triple-negative breast cancer and refractory gynecological tumor. Key words: Neoplasms; Therapeutic uses; Thalidomide

Significance of mast cells in non-neoplastic and neoplastic lesions of uterine cervix

Background: Mast cells are involved in multiple biological events. The significance of mast cells in non-neoplastic and neoplastic lesions of cervix has been studied with conflicting results. Its presence in tumor has been described as evidence of host immunologic antitumor response, angiogenesis and tumor invasion. Aims/Objectives: To study mast cell density in various cervical lesions using Toluidine blue stain; To compare the sections studied with conventional Toluidine blue staining and Toluidine blue staining by reducing the pH. Methodology: Cervical biopsy/hysterectomy specimens from archives of Department of Pathology were considered for the study. The sections were studied for histomorphology and mast cell density. The mast cell density was assessed by Toluidine blue staining by conventional method and another method by reducing pH using weak HCL. The stained slides were reviewed for mast cell density under 10 high power field and statistically analysed. Results: Total of 100 cases were studied. Normal cervix 7 cases with mean age of 44.29 and mast cell density (mean) of 45.43. Chronic cervicitis and polypoidal Endocervicitis were 26 and 28 cases, mean age of 45.38 years and 39.14 years and mean mast cell count of 48.38 and 66.96 respectively. Intraepithelial lesions and malignancy were 23 and 16 cases, mean age of 43.56 years and 52.26 years and mean mast cell of 34.47and 34.6 respectively. Maximum number of mast cells was seen in polypoidal Endocervicitis and least number in Squamous cell carcinoma of cervix. Conclusion: The role of mast cell differs in inflammatory and neoplastic lesions of cervix. Mast cells has active role in inflammatory lesions.

Identification and verification of transgelin-2 as a potential biomarker of tumor-derived lung-cancer endothelial cells by comparative proteomics

To investigate heterogeneity of endothelial cells (ECs) in the tumor microenvironment and biomarkers for antitumor angiogenesis therapy, high-purity (>98%) normal (NECs) and tumor-derived CD105+ ECs (TECs) were purified from a mouse Lewis lung carcinoma model bearing 0.5cm tumors by immunomagnetic separation. Proteomics analysis revealed that 48 proteins (28 upregulated and 20 downregulated) were differentially regulated by at least 1.5-fold in TECs, and that these proteins were involved in metabolism, energy pathways, protein folding, cell growth and/or functioned as structural constituents of the cytoskeleton. Upregulation of heat shock protein 60 (Hspd1) and transgelin-2 (Tagln2) was revealed in TECs, and by immunohistochemistry (IHC) in paired tissues from 30 consecutive lung cancer (LC) patients. Higher expression levels of Hspd1, Tagln2 were detected in microvascular ECs of paratumor and tumor tissues than in paired normal counterparts. Stronger Tagln2 staining was associated with clinical stage, tumor size, and histological neural invasion. Higher Hspd1 (area under the curve [AUC], 0.82) and lower Tagln2 (AUC, 0.90) levels were detected in LC patient sera. Pearson correlation analysis revealed a positive correlation between serum Hspd1 and Tagln2 levels. In conclusion, higher Tagln2 levels were associated with tumor development, lymph node metastasis, and neural invasion in LC and may thus serve as a potential biomarker of tumor angiogenesis. SignificanceHigh-purity endothelial cells (normal and tumor derived) were prepared to characterize ECs heterogeneity in the tumor microenvironment and to explore biomarkers of early stages of tumor development by proteomics. Candidate proteins Hspd1 and Tagln2, were further verification in the sera and tumor tissues of lung cancer patients. Moreover, higher Tagln2 was significantly associated with clinical tumor development, metastasis, and neural invasion. All these results indicated a crucial role for Tagln2 in TECs for tumor development and metastasis.

Effects of Mfn2 Gene Overexpression on EGFR Expression and Angiogenesis of a Heterologous Graft Model for Human Breast Infiltrating Duct Carcinoma in Nude Mice

Objective: To investigate the antitumor angiogenesis activity of Mfn2 gene on a heterologous graft model for human breast infiltrating duct carcinoma in nude mice. To investigate the relationship between Mfn2 gene and Epidermal Growth Factor Receptor (EGFR). Methods: Human breast cancer MCF-7 cells steady infected with Mfn2 gene had been constructed. Western blotting was used to detect the overexpression of Mfn2 protein. Then the MCF-7 cells were injected subcutaneously into the breast pad of nude mice. Tumorigenicity and the growth of transplanted tumor in nude mice were observed. Growth curves were plotted based on mean tumor volume in each experimental group at the indicated time points. Meanwhile, the negative control group (NC group) which MCF-7 cells steady infected with PEGFP-N1 was set up. The expression of CD34 (reflect the microvessel density of tumor) and EGFR were detected by immunocytochemistry. Results: The tumor volume of the experimental group was smaller than the negative control group.The expression of CD34 of the experimental group was lower than the negative control group. The experimental group expressed low level of EGFR. Conclusion: Mfn2 significantly inhibits the growth of human breast cancer xenograft in nude mice. Downregulation of EGFR expression and decrease the potential of angiogenesis may be the potential mechanism.

Anti-tumor angiogenesis effect of a new compound: B-9-3 through interference with VEGFR2 signaling.

B-9-3, a derivative of harmine, was first synthesized in our laboratory. We have reported that B-9-3 has an anti-proliferative effect against human lung cancer cells via induction of apoptosis and inhibition of cell migration. In the present study, we first studied that the anti-tumor angiogenesis effect and the molecular mechanism of B-9-3-induced tumor vascular degrade and mortify in lung cancer. In vitro, the results showed that B-9-3 selectively inhibited the proliferation of endothelial cells IC50 = 6.16μg/ml) and vascular fibroblasts (IC50 = 12.59μg/ml) and induced regression of tumor cells of the following: Lewis lung carcinoma (LLC), Mouse fore-stomach carcinoma (MFC), Human ovarian cancer (SK-OV-3), and prostate cancer (22RV1). Moreover, B-9-3 could significantly increase the apoptosis rate (80.95%) of vascular endothelial cells, while inhibiting migration of endothelial cells, capillary tube formation of endothelial cells, neovascularization of the rat thoracic aorta ring, and the angiogenesis of chick chorioallantoic membrane (CAM) predominantly through blocking VEGFR2 signaling pathway. In vivo, we investigated the anti-tumor rate and the signal transduction mechanism of B-9-3 by LCC-bearing C57BL/6 mice. The data showed that the tumor inhibition ratio of high dose (20mg/kg) of B-9-3 was 72.9%, and quantification of CD34 marker indicated a marked reduction in the number of neovessels after B-9-3 treatment as compared with control group (66.87%). Remarkably, using IHC and q-RT-PCR, we found that downregulation of the expression of VEGFR2, VEGF-A, and TGFβ1 in tumor confers enhancement to the angiogenesis effect of B-9-3. These data suggest that the angiogenesis inhibitor B-9-3 selectively induces apoptosis of endothelial cells, in part through disruption of VEGF-A/VEGFR2 signaling.

Recent highlights of experimental research for inhibiting tumor growth by using Chinese medicine.

To give an overview of contemporary experimental research using Chinese medicine (CM) for the treatment of cancer. As an integral part of mainstream medicine in the People's Republic of China, CM emphasizes improvements in holistic physical condition instead of merely killing tumor cells, which is consistent with the current medical model that advocates patient-oriented treatment. Great progress has been made in experimental research, and the principle aspects include anti-tumor angiogenesis, inducing apoptosis and differentiation, reversing multidrug resistance, and improving immune function. As a current hot topic in cancer research, tumor microenvironment (TME) highlights the mutual and interdependent interaction between tumor cells and their surrounding tissues, and the CM treatment concept bears a striking resemblance to it. To date, primary points of TME include extracellular matrix remodeling, inflammation, hypoxia, and angiogenesis, but trials using CM with a focus on TME are rare. Despite considerable recent development, experimental research on CM for solving cancer issues appears insufficient. Greater efforts in this field are urgently needed.

Immunotherapy of tumors with human telomerase reverse transcriptase immortalized human umbilical vein endothelial cells.

Human umbilical endothelial cells (HUVECs) have been proven to be effective in tumor anti-angiogenesis but the mechanism remained to be further demonstrated. The restricted ability of HUVECs to proliferate in vitro also limits their application on a large scale. In the present study, we immortalized HUVECs with hTERT genes by lentiviral infection and explored the antitumor immunity of hTERT-expressing HUVECs (HUVEC-TERTs). Results showed that HUVEC-TERTs maintained high telomere activity and expressed CD31, VEGFR-II and integrin α5. Passage-30 HUVEC-TERTs were able to form vascular tubes in vitro without showing signs of senescence. In vivo HUVEC-TERTs elicited antitumor immunity in mouse LL2 and CT26 models protectively and therapeutically. Both humoral and cellular immunity participated in the tumor anti-angiogenesis as HUVEC-neutralizing sera antibodies and HUVEC-specific CTL were detected. The subsets of activated spleen T lymphocytes included both CD4(+) T cells and CD8(+) T cells. Moreover, MDSCs and Tregs were decreased while T lymphocytes were aggregated in the tumor microenvironment. Collectively, the present study is the first to confirm the antitumor immunity of hTERT-immortalized HUVECs. Both anti-angiogenesis and tumor microenvironmental regulation participated in the antitumor activity. Transducing hTERT genes might be a new strategy to allow HUVECs to be applied on a large scale in cancer immunotherapy.

Main Anti-tumor Angiogenesis Agents Isolated From Chinese Herbal Medicines.

With a long history of clinical use, Chinese herbal medicine (CHM) is emerging as a noticeable choice for its multi-level, multi-target and coordinated intervention effects against tumor. Recently, many agents from CHM have shown powerful anti-angiogenic activities against tumor. In this review, we discussed the anti-tumor angiogenic activities of 6 kinds of agents from CHM (sulfated polysaccharides/glycopeptides, flavonoids, artemisinin, arsenic trioxide, ginsenoside, and tanshinone). The underlying pharmacological mechanisms of cancer angiogenesis inhibition by these agents are also gradually shown to us. Sulfated polysaccharides/glycopeptides and flavonoids may have synergistic effects with targeted anti-angiogenic drugs mainly targeting VEGF pathway by inhibiting bFGF and HIF-1α pathway, respectively. It is interesting that artemisinin and arsenic trioxide, two famous natural products worldwide, also have antitumor activity at least in part via angiogenesis inhibition. In addition, some natural products that are widely used for patients with cancer, such as ginseng and danshen, act as double-edged swords for tumor angiogenesis. Our review is aimed at providing an understanding of anti-angiogenic compounds from CHM and we propose that these breakthrough findings may have important implications for targeted-angiogenesis therapy and modernization of CHM.

Main anti-tumor angiogenesis agents isolated from Chinese herbal medicines

Main anti-tumor angiogenesis agents isolated from Chinese herbal medicines

Carboxymethyl chitosan represses tumor angiogenesis in vitro and in vivo

Carboxymethyl chitosan (CMCS), with potent water solubility, biocompatibility, and non-toxicity, has emerged as a promising candidate for biomedical applications. In this study, the anti-tumor angiogenesis effects of CMCS were evaluated in vitro and in vivo. Our results showed that CMCS could inhibit the 2-dimensional and 3-dimensional migration of human umbilical vein endothelial cells (HUVECs) in vitro. CMCS significantly inhibited the growth of mouse hepatocarcinoma 22 tissues and could promote tumor cell necrosis as suggested by pathological observations. The CD34 expression in H22 tumor tissue, the levels of vascular endothelial growth factor and tissue inhibitor of metalloproteinase 1 in serum was regulated by CMCS treatment. CMCS could significantly improve thymus index, spleen index, tumor necrosis factor α and interferon γ level. In a conclusion, CMCS possessed potent anti-tumor effects by inhibiting tumor angiogenesis, stimulating immune functions. Our date provide more foundation for application of CMCS in biomedicine or biomaterials for targeted anticancer drugs delivery.

Saw Palmetto Extract Inhibits Metastasis and Antiangiogenesis through STAT3 Signal Pathway in Glioma Cell.

Signal transducer and activator of transcription factor 3 (STAT3) plays an important role in the proliferation and angiogenesis in human glioma. Previous research indicated that saw palmetto extract markedly inhibited the proliferation of human glioma cells through STAT3 signal pathway. But its effect on tumor metastasis and antiangiogenesis is not clear. This study is to further clear the impact of saw palmetto extract on glioma cell metastasis, antiangiogenesis, and its mechanism. TUNEL assay indicated that the apoptotic cells in the saw palmetto treated group are higher than that in the control group (p < 0.05). The apoptosis related protein is detected and the results revealed that saw palmetto extract inhibits the proliferation of human glioma. Meanwhile pSTAT3 is lower in the experimental group and CD34 is also inhibited in the saw palmetto treated group. This means that saw palmetto extract could inhibit the angiogenesis in glioma. We found that saw palmetto extract was an important phytotherapeutic drug against the human glioma through STAT3 signal pathway. Saw palmetto extract may be useful as an adjunctive therapeutic agent for treatment of individuals with glioma and other types of cancer in which STAT3 signaling is activated.

Preparation and antitumor effect of a toxin-linked conjugate targeting vascular endothelial growth factor receptor and urokinase plasminogen activator.

The aberrant signaling activation of vascular endothelial growth factor receptor (VEGFR) and urokinase plasminogen activator (uPA) is a common characteristic of many tumors, including lung cancer. Accordingly, VEGFR and uPA have emerged as attractive targets for tumor. KDR (Flk-1/VEGFR-2), a member of the VEGFR family, has been recognized as an important target for antiangiogenesis in tumor. In this study, a recombinant immunotoxin was produced to specifically target KDR-expressing tumor vascular endothelial cells and uPA-expressing tumor cells and mediate antitumor angiogenesis and antitumor effect. Based on its potent inhibitory effect on protein synthesis, Luffin-beta (Lβ) ribosome-inactivating protein was selected as part of a recombinant fusion protein, a single-chain variable fragment against KDR (KDRscFv)-uPA cleavage site (uPAcs)-Lβ-KDEL (named as KPLK). The KDRscFv-uPAcs-Lβ-KDEL (KPLK) contained a single-chain variable fragment (scFv) against KDR, uPAcs, Lβ, and the retention signal for endoplasmic reticulum proteins KDEL (Lys-Asp-Glu-Leu). The KPLK-expressing vector was expressed in Escherichia coli, and the KPLK protein was isolated with nickel affinity chromatography and gel filtration chromatography. Sodium dodecyl sulfate-polyacrylamide gel electrophoresis test demonstrated KPLK was effectively expressed. Result of invitro cell viability assay on non-small cell lung cancer (NSCLC) H460 cell line (uPA-positive cell) revealed that KPLK significantly inhibited cell proliferation, induced apoptosis, and accumulated cells in S and G2/M phases, but the normal cell line (human submandibular gland cell) was unaffected. These effects were enhanced when uPA was added to digest KPLK to release Lβ. For invivo assay of KPLK, subcutaneous xenograft tumor model of nude mice were established with H460 cells. Growth of solid tumors was significantly inhibited in animals treated with KPLK up to 21 days, tumor weights were decreased, and the expression of angiogenesis marker CD31 was downregulated; meanwhile, the apoptosis-related protein casspase-3 was upregulated. These results suggested that the recombinant KPLK may have therapeutic applications on tumors, especially uPA-overexpressing ones.

MiR-7-5p is frequently downregulated in glioblastoma microvasculature and inhibits vascular endothelial cell proliferation by targeting RAF1.

The aberrant expression of microRNAs (miRNAs) is always associated with tumor development and progression. Microvascular proliferation is one of the unique pathologic features of glioblastoma (GBM) . In this study, the microvasculature from GBM or normal brain tissue derived from neurosurgeries was purified and total RNA was isolated from purified microvasculature. The difference of miRNA expression profiles between glioblastoma microvasculature and normal brain capillaries was investigated. It was found that miR-7-5p in GBM microvessels was significantly reduced compared with that in normal brain capillaries. In the in vitro experiments, overexpression of miR-7-5p significantly inhibited human umbilical vein endothelial cell proliferation. Forced expression of miR-7-5p in human umbilical vein endothelial cells in vitro significantly reduced the protein level of RAF1 and repressed the activity of the luciferase, a reporter vector carrying the 3'-untranslated region of RAF1. These findings indicate that RAF1 is one of the miR-7-5p target genes. Furthermore, a significant inverse correlation between miR-7-5p expression and RAF1 protein level in GBM microvasculature was found. These data suggest that miR-7-5p functions as a tumor suppressor gene to regulate GBM microvascular endothelial cell proliferation potentially by targeting the RAF1 oncogene, implicating an important role for miR-7-5p in the pathogenesis of GBM. It may serve as a guide for the antitumor angiogenesis drug development.

Antiangiogenic cancer drug using the zebrafish model.

The process of de novo vessel formation, called angiogenesis, is essential for tumor progression and spreading. Targeting of molecular pathways involved in such tumor angiogenetic processes by using specific drugs or inhibitors is important for developing new anticancer therapies. Drug discovery remains to be the main focus for biomedical research and represents the essence of antiangiogenesis cancer research. To pursue these molecular and pharmacological goals, researchers need to use animal models that facilitate the elucidation of tumor angiogenesis mechanisms and the testing of antiangiogenic therapies. The past few years have seen the zebrafish system emerge as a valid model organism to study developmental angiogenesis and, more recently, as an alternative vertebrate model for cancer research. In this review, we will discuss why the zebrafish model system has the advantage of being a vertebrate model equipped with easy and powerful transgenesis as well as imaging tools to investigate not only physiological angiogenesis but also tumor angiogenesis. We will also highlight the potential of zebrafish for identifying antitumor angiogenesis drugs to block tumor development and progression. We foresee the zebrafish model as an important system that can possibly complement well-established mouse models in cancer research to generate novel insights into the molecular mechanism of the tumor angiogenesis.

The R0 resection rate after neoadjuvant bevacizumab (Bev) plus DOF versus DOF in local advanced gastric carcinoma (LAGC) and its association with circulating tumor cell (CTC).

4043 Background: Local advanced gastric carcinoma (LAGC) is suggested to be potentially cured by R0 resection, and neoadjuvant chemotherapy can increase the R0 resection rate but not enough. Bevacizumab (Bev), an anti-tumor angiogenesis monoclonal antibody, combined with chemotherapy has been shown effective in advanced GC. In addition, CTC has been suggested as an indicator of the anti-tumor drugs’ efficacy. Therefore, in this study, we plan to evaluate the efficacy and safety of neoadjuvant Bev plus docetaxel/oxaliplatin/5-FU/CF (DOF) versus DOF in mainly gastric antrum LAGC, and to investigate whether CTC is an effectiveness indicator. Methods: 80 patients diagnosed as IIIb-IIIc GC have been enrolled and randomly assigned (1:1) to receive neoadjuvant Bev (5 mg/kg, d1) plus DOF (docetaxel, 75 mg/m2, iv, d1; oxaliplatin, 85 mg/m2, iv, d1; 5-FU, iv infusion 600 mg/m2 and iv injection 400mg/m2, d1-2; CF, 200 mg/m2, d1 and d2) or DOF each 3-week, up to 2-4 cycles preoperation, and another 2-4 cycles postope...