Inhibition of tumor angiogenesis and growth by nanoparticle-mediated p53 gene therapy in mice

Mutation of the p53 tumor suppressor gene, the most common genetic alteration in human cancers, results in more aggressive disease and increased resistance to conventional therapies. Aggressiveness may be related to the increased angiogenic activity of cancer cells containing mutant p53. To restore wild-type p53 function in cancer cells, we developed polymeric nanoparticles (NPs) for p53 gene delivery. Previous in vitro and in vivo studies demonstrated the ability of these NPs to provide sustained intracellular release of DNA, thus sustained gene transfection and decreased tumor cell proliferation. We investigated in vivo mechanisms involved in NP-mediated p53 tumor inhibition, with focus on angiogenesis. We hypothesize that sustained p53 gene delivery will help decrease tumor angiogenic activity and thus reduce tumor growth and improve animal survival. Xenografts of p53 mutant tumors were treated with a single intratumoral injection of p53 gene-loaded NPs (p53NPs). We observed intratumoral p53 gene expression corresponding to tumor growth inhibition, over 5 weeks. Treated tumors showed upregulation of thrombospondin-1, a potent antiangiogenic factor, and a decrease in microvessel density vs controls (saline, p53 DNA alone, and control NPs). Greater levels of apoptosis were also observed in p53NP-treated tumors. Overall, this led to significantly improved survival in p53NP-treated animals. NP-mediated p53 gene delivery slowed cancer progression and improved survival in an in vivo cancer model. One mechanism by which this was accomplished was disruption of tumor angiogenesis. We conclude that the NP-mediated sustained tumor p53 gene therapy can effectively be used for tumor growth inhibition.     

Inhibition of breast tumor growth and angiogenesis by a medicinal herb: Ocimum gratissimum

Ocimum sp. is a traditionally used medicinal herb, which shows anti-oxidant, anti-carcinogenic, radio-protective and free radical scavenging properties. So far no detailed studies have been reported on its effects on human cancers. Thus, we analyzed its effects on human breast cancer utilizing in vitro and in vivo methodologies. Aqueous extracts were prepared from the mature leaves of Ocimum gratissimum (OG) cultivated devoid of pesticides. Tumor progression and angiogenesis related processes like chemotaxis, proliferation, apoptosis, 3D growth and morphogenesis, angiogenesis and tumor growth were studied in the presence or absence of the extract, and in some experiments a comparison was made with purified commercially available eugenol, apigenin and ursolic acid. Aqueous OG leaf extract inhibits proliferation, migration, anchorage independent growth, 3D growth and morphogenesis and induction of COX-2 protein in breast cancer cells. A comparative analysis with eugenol, apigenin and ursolic acid showed that the inhibitory effects on chemotaxis and 3D morphogenesis of breast cancer cells were specific to OG extract. In addition, OG extracts reduced tumor size and neoangiogenesis in a MCF10 DCIS.com xenograft model of human DCIS. This is the first detailed report showing that OG leaf extract may be of value as a breast cancer preventive and therapeutic agent and might be considered as additional additive in the arsenal of components aimed at combating breast cancer progression and metastasis.     

EphB4 overexpression in B16 melanoma cells affects arterial-venous patterning in tumor angiogenesis

EphB4 receptor and its ligand ephrinB2 play an important role in vascular development during embryogenesis. In blood vessels, ephrinB2 is expressed in arterial endothelial cells (EC) and mesenchymal supporting cells, whereas EphB4 is only expressed in venous ECs. Previously, we reported that OP9 stromal cells, which support the development of both arterial and venous ECs, in which EphB4 was overexpressed, could inhibit ephrinB2-positive (ephrinB2+) EC development in an embryonic tissue organ culture system. Although the EphB4 receptor is expressed in a variety of tumor cells, its exact function in regulating tumor progression has not been clearly shown. Here we found that overexpression of EphB4 in B16 melanoma cells suppressed tumor growth in a s.c. transplantation tumor model. Histologic examination of these tumors revealed that EphB4 overexpression in B16 cells selectively suppressed arterial ephrinB2+ EC development. By coculturing ephrinB2-expressing SV40-transformed mouse ECs (SVEC) with EphB4-overexpressing B16 cells, we found that EphB4 induced the apoptosis of SVECs. However, ephrinB2 did not induce the apoptosis of EphB4-overexpressing B16 cells. Based on results from these experiments, we concluded that EphB4 overexpression in B16 tumor cells suppresses the survival of arterial ECs in tumors by a reverse signaling via ephrinB2.     

Inhibition of tumor cell invasion and angiogenesis by motuporamines

Tissue invasion is an important determinant of angiogenesis and metastasis and constitutes an attractive target for cancer therapy. We have developed an assay to identify agents that inhibit invasion by mechanisms other than inhibition of cell attachment or cytotoxicity. A screen of marine sponge extracts identified motuporamines as micromolar inhibitors of invasion of basement membrane gels by MDA-231 breast carcinoma, PC-3 prostate carcinoma, and U-87 and U-251 glioma cells. Motuporamine C inhibits cell migration in monolayer cultures and impairs actin-mediated membrane ruffling at the leading edge of lamellae. Motuporamine C also reduces beta1-integrin activation, raising the possibility that it interferes with "inside-out" signaling to integrins. In addition, motuporamine C inhibits angiogenesis in an in vitro sprouting assay with human endothelial cells and an in vivo chick chorioallantoic membrane assay. The motuporamines show little or no toxicity or inhibition of cell proliferation, and they are structurally simple and easy to synthesize, making them attractive drug candidates.     

Inhibition of tumor angiogenesis by hexuronyl hexosaminoglycan sulfate

The efficacy of heparin (HEP), the heparin analogue hexuronyl hexosaminoglycan sulfate (HHS), and hydrocortisone (HC) was studied in inhibiting the growth of four morphologically distinct pancreatic adenocarcinoma lines (CBP, LHP2, LSP3, and Pour-LVG) in hamsters. Animals were inoculated with LD100 doses of one of the four tumor lines and were randomly allocated to groups of five animals, which received in their drinking water either: HEP (1000 U/ml) alone, HHS (10 mg/ml) alone, HC (0.5 mg/ml) alone, HEP plus HC, HHS plus HC, or no additives (control). Tumors were measured, growth rates calculated, and nonparametric statistical comparisons made among the median growth rates of all of the treatment groups. All four tumors were tested in the rabbit cornea assay for their ability to induce angiogenesis. Extracts of tumors from control animals as well as from animals treated with HHS plus HC were prepared for quantitative testing in vitro by endothelial cell migration assay. All four tumor lines caused angiogenesis as measured in the rabbit cornea assay. A reduction in median tumor growth rates was observed in animals treated with HHS plus HC bearing the CBP, Pour-LVG, and LSP3 tumors. Similarly, in vitro capillary endothelial cell migration was decreased by HHS plus HC treatment in animals bearing CBP, Pour-LVG, and LSP3 tumors. Animals bearing the LHP2 tumor showed no effect of HHS plus HC treatment on tumor growth rate and no effect on endothelial cell migration. HEP alone, HHS alone, HC alone, and HEP plus HC showed no effect on tumor growth rate in any of the four tumors tested.     

Inhibition of metastasis,angiogenesis, and tumor growth by Chinese herbal cocktail Tien-Hsien Liquid

Background  

Advanced cancer is a multifactorial disease that demands treatments targeting multiple cellular pathways. Chinese herbal cocktail which contains various phytochemicals may target multiple dys-regulated pathways in cancer cells and thus may provide an alternative/complementary way to treat cancers. Previously we reported that the Chinese herbal cocktail Tien-Hsien Liguid (THL) can specifically induce apoptosis in various cancer cells and have immuno-modulating activity. In this study, we further evaluated the anti-metastatic, anti-angiogenic and anti-tumor activities of THL with a series of in vitro and in vivo experiments.     

Inhibition of bladder carcinoma angiogenesis, stromal support, and tumor growth by halofuginone.

We have previously demonstrated that halofuginone, a widely used alkaloid coccidiostat, is a potent inhibitor of collagen alpha1(I) and matrix metalloproteinase 2 gene expression. Halofuginone also suppresses extracellular matrix deposition and cell proliferation. We investigated the effect of halofuginone on transplantable and chemically induced mouse bladder carcinoma. In both systems, oral administration of halofuginone resulted in a profound anticancerous effect, even when the treatment was initiated at advanced stages of tumor development. Although halofuginone failed to prevent proliferative preneoplastic alterations in the bladder epithelium, it inhibited further progression of the chemically induced tumor into a malignant invasive stage. Histological examination and in situ analysis of the tumor tissue revealed a marked decrease in blood vessel density and in both collagen alpha1(I) and H19 gene expression. H19 is regarded as an early marker of bladder carcinoma. The antiangiogenic effect of halofuginone was also demonstrated by inhibition of microvessel formation in vitro. We attribute the profound antitumoral effect of halofuginone to its combined inhibition of the tumor stromal support, vascularization, invasiveness, and cell proliferation.     

Inhibition of angiogenesis and tumor growth by a synthetic laminin peptide, CDPGYIGSR-NH2.

A laminin-derived synthetic peptide, Cys-Asp-Pro-Gly-Tyr-Ile-Gly-Ser-Arg-NH2 (CDPGYIGSR-H2), containing an active site for cell binding inhibited both angiogenesis and solid tumor growth. It potently suppressed both embryonic angiogenesis of the chick chorioallantoic membrane and migration of vascular endothelial cells induced by a tumor-conditioned medium but neither the in vitro proliferation of endothelial cells nor that of tumor cells. Additionally, in in vivo tests, CDPGYIGSR-NH2 markedly inhibited both the growth of s.c. solid tumor of Sarcoma 180 and that of Lewis lung carcinoma (3LL) in the lungs. On the contrary, ascitic tumor growth of Sarcoma 180 was not affected by this peptide, even though the same cell source was used. It was concluded that solid tumor growth inhibition by CDPGYIGSR-NH2 was due not a direct effect on cell growth but to antiangiogenic effect mediated by the inhibition of endothelial cell migration.     

Inhibition of tumor angiogenesis by a single-chain antibody directed against vascular endothelial growth factor

Monoclonal antibody (Ab) directed against the vascular endothelial growth factor, one of the major inducers of angiogenesis, can inhibit tumor growth in mice. Treatment of cancer patients with monoclonal Ab requires large-scale production of the clean Ab and frequent application of the Ab. This might be improved by using single-chain Ab fragments (scFvs), which can be produced in large quantities in bacteria and are attractive for gene therapeutic approaches. Here we describe anti-vascular endothelial growth factor scFvs derived from a human phage-display library able to block the vascularization of the chorioallantoic membrane of chick embryos and reduce the growth of s.c. tumors in nude mice. This work opens the way to develop gene therapy-based strategies using a scFv to treat angiogenesis-dependent diseases.     

Inhibition of colon tumor progression and angiogenesis by the Ink4a/Arf locus

The Ink4a/Arf locus is frequently methylated in colon carcinoma and other common human cancers, suggesting that the locus may play a broad, as yet poorly defined,role inhibiting tumor progression. We examined the influence of the locus in mice with multiple intestinal neoplasia (Min). Colon tumors in 3-month-old Min mice that were null for the Ink4a/Arf locus (-/-) were moderately larger than in Ink4a/Arf-wild-type (+/+) animals (P = 0.032). More strikingly, one-half of the -/- colon tumors were grossly red in color, whereas most of the +/+ tumors were white (P = 0.0025). This color difference remained statistically significant after normalizing for tumor area (P = 0.016). On histological analysis, -/- colon tumors displayed more RBCs near the tumor surface, twice the number of functional vessels, and features of carcinoma in situ not found in +/+ tumors. Biochemical analyses showed that red tumors had higher hemoglobin and vascular endothelial growth factor (VEGF) content than white tumors. Surprisingly, the small intestinal tumor burden was actually lower in -/- animals, and none of these tumors were red, underscoring the importance of tissue context in the function of the locus. These results provide direct evidence that the Ink4a/Arf locus inhibits colon tumor progression. The enhanced vascularity of the -/- tumors is particularly significant in light of the clinical importance of this property in the detection, recurrence, and therapy of colon tumors.     

Inhibition of tumor growth through suppression of angiogenesis by brain-specific angiogenesis inhibitor 1 gene transfer in murine renal cell carcinoma

This study was designed to elucidate the therapeutic effect of transfering the brain-specific angiogenesis inhibitor 1 (BAI1) gene to a mouse renal cell carcinoma cell line (Renca). Female BALB/c mice were inoculated subcutaneously with wild-type Renca (Renca/Wild) cells or Renca cells transfected with the BAI-1 (Renca/BAI-1) or LacZ (Renca/LacZ) gene. Tumor growth was observed every other day from 3 to 35 days after implantation. Moreover, the intratumoral injection of the adenovirus vector containing the gene encoding BAI1 was conducted at two-day intervals from 11 to 31 days after implantation of the Renca/Wild or Renca/BAI1 tumor. Tumor blood flow was measured by colorimetric angiogenesis assay (CAA). The concentration of the vascular endothelial growth factor (VEGF) in the cell culture supernatants was determined by enzyme-linked immunoassay. The size of the Renca/BAI1 tumor was significantly (p<0.01) suppressed compared to the Renca/Wild and Renca/LacZ tumors 21 days after tumor implantation. The injection of the BAI1 viral vector at 2-day intervals significantly inhibited the growth of both the Renca/Wild and Renca/BAI1 tumors. The blood volume measured by CAA and microvessel density was significantly lower in the Renca/BAI1 than in the Renca/Wild and Renca/LacZ tumors (p<0.01 and p<0.05, respectively). A significant (p<0.01) reduction in VEGF concentration in the supernatant was demonstrated in the Renca/BAI1 compared with the Renca/Wild and Renca/LacZ cell cultures. These observations suggest that the transfer of the BAI1 gene to Renca can suppress the tumor growth via the inhibition of angiogenesis. The down-regulation of VEGF production in tumor cells contributes to this anti-tumor effect.     

Inhibition of tumor growth and angiogenesis by water extract of Gac fruit (Momordica cochinchinensis Spreng)

The antitumor activity of the crude water extract from Gac fruit (Momordica cochinchinensis) was investigated in vivo and in vitro. A water extract prepared from 0.75 and 0.25 mg dry weight of Gac fruit per gram body weight was given daily to Balb/c mice (n=15/group). The water extract inhibited the growth of the colon 26-20 adenocarcinoma cell line, transplanted in Balb/c mice, reducing wet tumor weight by 23.6%. Histological and immunohistochemical results indicated that Gac water extract reduced the density of blood vessels around the carcinoma. The water extract also produced a marked suppression of cell proliferation in colon 26-20 and HepG2 cells. Cell cycle analysis demonstrated a significant accumulation of cells in the S phase by water extract. Immunoblotting showed that cyclin A, Cdk2, p27waf1/Kip1 were down-regulated, whereas the protein level of p21waf1/Cip1 was not decreased. Treatment of colon 26-20 cells with Gac extract induced necrosis rather than apoptosis. The antitumor component was confirmed as a protein with molecular weight of 35 kDa, retained in the water-soluble high molecular weight fraction. Thus, the bioactive antitumor compound in Gac extract is a protein, which is distinct from lycopene, another compound in Gac fruit with potential antitumor activity.     

Inhibition of Met/HGF receptor and angiogenesis by NK4 leads to suppression of tumor growth and migration in malignant pleural mesothelioma

NK4 exhibits two distinct biological actions: antagonistic inhibition of hepatocyte growth factor (HGF) through binding to the Met/HGF receptor, and antiangiogenic action through binding to perlecan. Here, the anti‐tumor effect of NK4 on malignant pleural mesothelioma was investigated. Of the 7 human malignant mesothelioma cell lines (ACC‐Meso‐1, ACC‐Meso‐4, EHMES‐1, EHMES‐10, H28, H2052 and JMN‐1B), only EHMES‐10 cells formed subcutaneous tumors when implanted into mice. For EHMES‐10 cells, HGF facilitated invasion of the cells in collagen gel, whereas NK4 and neutralizing anti‐HGF antibody suppressed the HGF‐induced invasion. In addition, NK4 but not anti‐HGF antibody suppressed proliferation of EHMES‐10 cells in collagen, suggesting that the suppression by NK4 was independent of the HGF‐Met pathway. In the subcutaneous tumor model, recombinant adenovirus‐mediated intratumoral expression of NK4 inhibited tumor growth, while the invasive characteristic of tumor cells was not observed. Analysis of Met receptor tyrosine phosphorylation, proliferation, apoptosis and blood vessels in the tumor tissues indicated that the inhibitory effect of NK4 expression might be primarily caused by the inhibition of tumor angiogenesis. In all the 7 mesothelioma lines, HGF stimulated Met tyrosine phosphorylation, and this was associated with enhanced cell migration. HGF‐dependent Met activation and migration were inhibited by NK4. Since malignant pleural mesothelioma represents an aggressive neoplasm characterized by extensive invasive growth, suppression of invasive growth has therapeutic value. Thus, the simultaneous inhibition of the HGF‐Met pathway and angiogenesis by NK4 for treatment of malignant pleural mesothelioma is significant, particularly to attenuate migration and invasive growth.     

Inhibition of tumor angiogenesis by Brahma Rasayana (BR)

The current therapy for prostate cancer includes radical prostatectomy, radiation therapy and hormonal ablation. Chemotherapy also provides beneficial results for some patients with advanced prostate cancer but with several harmful side effects. Hence there is a need to identify and develop alternate therapies, which can reduce the disease progression with minimal or few side effects. Earlier studies from our laboratory have shown that a Polyherbal mixture, Brahma Rasayna (BR) rich in anti-oxidant principles has a potential to be an anti-tumor agent. BR treatment of MAT-LyLu cell inoculated Copenhagen rats resulted in a decrease of palpable tumor incidence, delay in tumor occurrence and lower mean tumor volumes. Also, a significant reduction in tumor weight and lung metastasis was observed in BR treated animals in comparison to untreated controls. In the present study, we focused to examine the effect of BR on angiogenesis and regulation of molecular markers involved in angiogenesis using in-vivo and in-vitro models. BR treatment showed a significant reduction in Factor VIII expression compared to control indicating reduced angiogenesis. BR treated tumor specimens showed a decrease in the pro-angiogenic factors like VEGF, MMP-9 and MMP-2. Methanolic extract of BR was found to inhibit the proliferation, tube formation, cell migration and attachment of HUVEC on matrigel in a dose dependant manner. These findings suggest the possible mechanism(s) of action of BR in the reduction of tumor growth and metastatic spread.     

Inhibition of angiogenesis and vascular leakiness by angiopoietin-related protein 4

Angiopoietins and angiopoietin-related proteins (ARPs) have been shown to regulate angiogenesis, a process essential for various neovascular diseases including tumors. Here, we identify ARP4/fasting-induced adipose factor/peroxisome proliferator-activated receptor gamma angiopoietin-related as a novel antiangiogenic modulatory factor. We hypothesized that ARP4 may regulate angiogenesis. In vitro experiments using purified recombinant ARP4 protein revealed that ARP4 markedly inhibited the proliferation, chemotaxis, and tubule formation of endothelial cells. Moreover, using corneal neovascularization and Miles permeability assays, we found that both vascular endothelial growth factor-induced in vivo angiogenesis and vascular leakiness were significantly inhibited by the addition of ARP4. Finally, we found remarkable suppression of tumor growth within the dermal layer associated with decreased numbers of invading blood vessels in transgenic mice that express ARP4 in the skin driven by the keratinocyte promoter. These findings demonstrate that ARP4 functions as a novel antiangiogenic modulatory factor and indicate a potential therapeutic effect of ARP4 in neoplastic diseases.     

EphB4及其配体EphrinB2在食管鳞癌中表达及其与血管生成的关系

  目的  探讨EphB4受体及其配体EphrinB2在食管鳞癌组织中的表达及其与血管生成的关系。  方法  应用免疫组织化学Envision plus法检测60例食管鳞癌和10例癌旁组织中EphB4受体及其配体EphrinB2的表达和VEGF的表达, 采用逆转录多聚酶链反应（RT-PCR）法检测60例食管鳞癌和10例癌旁食管组织中EphB4、EphrinB2和VEGF mRNA的相对含量, 并分析EphB4、EphrinB2和VEGF的表达的相关性。  结果  食管鳞癌组织中EphB4、EphrinB2、VEGF mRNA和蛋白的表达均高于癌旁正常食管黏膜, 差异有统计学意义（P < 0.05）。EphB4 mRNA和EphrinB2 mRNA表达正相关（r=0.541, P < 0.05）, EphB4 mRNA、EphrinB2 mRNA均和VEGF mRNA表达正相关（r=0.642, r=0.582;P均 < 0.05）。  结论  EphB4和EphrinB2在食管鳞癌中高表达, 与血管生成密切相关。      

Aquaporins in tumor growth and angiogenesis

The aquaporins (AQPs) are a family of transmembrane water channel proteins widely distributed and play a major role in transcellular and transepithelial water movement. Moreover, recent evidence indicates that AQPs may be involved in cell migration and angiogenesis. This review article summarizes literature data concerning the involvement of AQPs in tumor growth, angiogenesis and metastatic process and suggest a potential therapeutic approach by antagonizing their biological activity.     

Inhibition of tumor metastasis by sodium caffeate and its effect on angiogenesis

OBJECTIVE: Sodium caffeate (SC), the sodium salt of caffeic acid, was synthesized in our laboratory. We studied the antimetastatic effect induced by SC and its inhibition of tumor angiogenesis using various in vitro and in vivo metastasis assays. METHODS: The in vivo inhibitory effect of SC on metastasis and angiogenesis was examined in the Lewis lung carcinoma pulmonary metastasis model and chicken chorioallantoic membrane (CAM) model, respectively. MTT assay and flow cytometry were used to measure the inhibition by SC of the proliferation of transformed human umbilical vein endothelial cells (ECV304) and the apoptosis induced by SC in ECV304 cells, respectively. A cell attachment assay was used to evaluate inhibition by SC of the adhesion activity of human high metastatic giant cell carcinoma of the lung (PG) cells. A cell invasion assay was used to evaluate the effect of SC on the ability of PG cells to cross tissue barriers. Inhibition by SC of gelatinase secretion in PG cells was determined by zymography. RESULTS: In vivo results showed that SC (1 g/kg i.p. for 14 days) inhibited pulmonary metastasis at a rate of 55%. There were no differences in animal weights among the groups. The angiogenesis of CAM was inhibited by SC (200 microg/egg) at a rate of 70%. In vitro studies showed that SC inhibited the proliferation of ECV304 cells by inducing apoptosis. SC also reduced the adhesion and invasion ability of PG cells and inhibited the secretion of MMP-2 and MMP-9 in PG cells. CONCLUSION: SC might be a potential antimetastatic agent with an antiangiogenic effect.     

Inhibition of angiogenesis and murine tumour growth by laminarin sulphate.

LAM S5 is a polysulphated derivative of the glucan laminarian that inhibits basic fibroblast growth factor (bFGF) binding and the bFGF-stimulated proliferation of fetal bovine heart endothelial (FBHE) cells. This report demonstrates that LAM S5 has anti-angiogenic activity, as shown by inhibition of tubule formation by endothelial cells cultured on Matrigel and inhibition of vascularisation of the chick chorioallantoic membrane. In addition, LAM S5 caused a tumour growth delay of the murine RIF-1 tumour of 2.6 days (P = 0.01).