Safety and whole-body antioxidant potential of a novel anthocyanin-rich formulation of edible berries

Edible berry extracts rich in anthocyanins possess a broad spectrum of therapeutic, pharmacologic and anti-carcinogenic properties. Six berry extracts (wild blueberry, bilberry, cranberry, elderberry, raspberry seeds and strawberry), singly and in combination, were studied in our laboratories for antioxidant efficacy, cytotoxic potential, cellular uptake and anti-angiogenic properties. Combinations of edible berry extracts were evaluated to develop a synergistic formula, OptiBerry, which exhibited high oxygen radical absorbance capacity (ORAC) value, low cytotoxicity and superior anti-angiogenic properties compared to the other combinations tested. The current study sought to determine the broad spectrum safety and antioxidant potential of OptiBerry in vivo. Acute oral LD₅₀ of OptiBerry was greater than 5 g/kg in rats. Acute dermal LD₅₀ of OptiBerry was greater than 2 g/kg. No changes in the body weight or adverse effects were observed following necropsy. Primary skin and eye irritation studies were conducted in New Zealand albino rabbits. OptiBerry was classified as slightly irritating to the skin (primary skin irritation index 0.3) and minimally irritating to the eye (maximum mean total score 6.0). The antioxidant potential of OptiBerry was investigated in rats and mice by assessing GSH redox status in tissues as well as by a unique state-of-the-art electron paramagnetic resonance (EPR) imaging of whole-body redox status. A clinically relevant hyperbaric oxygen (HBO) exposure system (2 atm, 2 h) was employed to study the antioxidant properties of OptiBerry. OptiBerry feeding (8 weeks) significantly prevented HBO-induced GSH oxidation in the lung and liver of vitamin E-deficient Sprague Dawley rats. Furthermore, OptiBerry-fed mice, when exposed to HBO, demonstrated significant protection in whole-body HBO-induced oxidation compared to the unfed controls by EPR imaging. Taken together, these results indicate that OptiBerry is reasonably safe and possess antioxidant properties.     

Anti-angiogenic Property of Edible Berries

Recent studies show that edible berries may have potent chemopreventive properties. Anti-angiogenic approaches to prevent and treat cancer represent a priority area in investigative tumor biology. Vascular endothelial growth factor (VEGF) plays a crucial role for the vascularization of tumors. The vasculature in adult skin remains normally quiescent. However, skin retains the capacity for brisk initiation of angiogenesis during inflammatory skin diseases such as psoriasis and skin cancers. We sought to test the effects of multiple berry extracts on inducible VEGF expression by human HaCaT keratinocytes. Six berry extracts (wild blueberry, bilberry, cranberry, elderberry, raspberry seed, and strawberry) and a grape seed proanthocyanidin extract (GSPE) were studied. The extracts and uptake of their constituents by HaCaT were studied using a multi-channel HPLC-CoulArray approach. Antioxidant activity of the extracts was determined by ORAC. Cranberry, elderberry and raspberry seed samples were observed to possess comparable ORAC values. The antioxidant capacity of these samples was significantly lower than that of the other samples studied. The ORAC values of strawberry powder and GSPE were higher than cranberry, elderberry or raspberry seed but significantly lower than the other samples studied. Wild bilberry and blueberry extracts possessed the highest ORAC values. Each of the berry samples studied significantly inhibited both H 2 O 2 as well as TNF &#102 induced VEGF expression by the human keratinocytes. This effect was not shared by other antioxidants such as &#102 -tocopherol or GSPE but was commonly shared by pure flavonoids. Matrigel assay using human dermal microvascular endothelial cells showed that edible berries impair angiogenesis.     

Anti-angiogenic property of edible berries

Recent studies show that edible berries may have potent chemopreventive properties. Anti-angiogenic approaches to prevent and treat cancer represent a priority area in investigative tumor biology. Vascular endothelial growth factor (VEGF) plays a crucial role for the vascularization of tumors. The vasculature in adult skin remains normally quiescent. However, skin retains the capacity for brisk initiation of angiogenesis during inflammatory skin diseases such as psoriasis and skin cancers. We sought to test the effects of multiple berry extracts on inducible VEGF expression by human HaCaT keratinocytes. Six berry extracts (wild blueberry, bilberry, cranberry, elderberry, raspberry seed, and strawberry) and a grape seed proanthocyanidin extract (GSPE) were studied. The extracts and uptake of their constituents by HaCaT were studied using a multi-channel HPLC-CoulArray approach. Antioxidant activity of the extracts was determined by ORAC. Cranberry, elderberry and raspberry seed samples were observed to possess comparable ORAC values. The antioxidant capacity of these samples was significantly lower than that of the other samples studied. The ORAC values of strawberry powder and GSPE were higher than cranberry, elderberry or raspberry seed but significantly lower than the other samples studied. Wild bilberry and blueberry extracts possessed the highest ORAC values. Each of the berry samples studied significantly inhibited both H2O2 as well as TNF alpha induced VEGF expression by the human keratinocytes. This effect was not shared by other antioxidants such as alpha-tocopherol or GSPE but was commonly shared by pure flavonoids. Matrigel assay using human dermal microvascular endothelial cells showed that edible berries impair angiogenesis.     

Inhibition of Helicobacter pylori in vitro by various berry extracts,with enhanced susceptibility to clarithromycin

The objective of this study was to evaluate the effects of various berry extracts, with and without clarithromycin on Helicobacter pylori. Resistance to clarithromycin by H. pylori has been reported, leading to interest in alternatives/adjuncts to therapy with clarithromycin. H. pylori American type culture collection (ATCC) strain 49503 was grown, cell suspensions were made in PBS and diluted 10-fold. One hundred microL of the suspension was then incubated for 18 h with extracts of raspberry, strawberry, cranberry, elderberry, blueberry, bilberry, and OptiBerry, a blend of the six berries, at 0.25-1% concentrations. Serially diluted cell suspensions were exposed for 1 h to clarithromycin at 15 microg/ml. Ten microl of bacterial samples from the 10(-7) dilution tube were plated and incubated for 18 h and the number of colonies were counted. Growth of H. pylori was confirmed by the CLO test. All berry extracts significantly (p < 0.05) inhibited H. pylori, compared with controls, and also increased susceptibility of H. pylori to clarithromycin, with OptiBerry demonstrating maximal effects.     

Anti-Cancer Activity of an Osthole Derivative,NBM-T-BMX-OS01: Targeting Vascular Endothelial Growth Factor Receptor Signaling and Angiogenesis

Angiogenesis occurs during tissue growth, development and wound healing. It is also required for tumor progression and represents a rational target for therapeutic intervention. NBM-T-BMX-OS01 (BMX), derived from the semisynthesis of osthole, an active ingredient isolated from Chinese herb Cnidium monnieri (L.) Cuss., was recently shown to enhance learning and memory in rats. In this study, we characterized the anti-angiogenic activities of NBM-T-BMX-OS01 (BMX) in an effort to develop novel inhibitors to suppress angiogenesis and tumor growth. BMX inhibited vascular endothelial growth factor (VEGF)-induced proliferation, migration and endothelial tube formation in human umbilical endothelial cells (HUVECs). BMX also attenuated VEGF-induced microvessel sprouting from aortic rings ex vivo and reduced HCT116 colorectal cancer cells-induced angiogenesis in vivo. Moreover, BMX inhibited the phosphorylation of VEGFR2, FAK, Akt and ERK in HUVECs exposed to VEGF. BMX was also shown to inhibit HCT116 cell proliferation and to suppress the growth of subcutaneous xenografts of HCT116 cells in vivo. Taken together, this study provides evidence that BMX modulates vascular endothelial cell remodeling and leads to the inhibition of tumor angiogenesis. These results also support the role of BMX as a potential drug candidate and warrant the clinical development in the treatment of cancer.     

Streptochlorin, a marine natural product, inhibits NF-kappaB activation and suppresses angiogenesis in vitro

Angiogenesis is an essential step in tumor progress and metastasis. Accordingly, small molecules that inhibit angiogenesis would appear to be a promising way to cure angiogenesis-related diseases, including cancer. In the present study, we report that streptochlorin, a small molecule from marine actinomycete, exhibits a potent antiangiogenic activity. The compound potently inhibited endothelial cell invasion and tube formation stimulated with vascular endothelial cell growth factor (VEGF) at low micromolar concentrations where it showed no cytotoxicity to the cells. In addition, streptochlorin inhibited TNF-alpha-induced NF-kappaB activation in the newly developed cell-based reporter gene assay. These data demonstrate that streptochlorin is a new inhibitor of NF-kappaB activation and can be a basis for the development of novel anti-angiogenic agents.     

Mathematical Modeling of Cellular Cross-Talk Between Endothelial and Tumor Cells Highlights Counterintuitive Effects of VEGF-Targeted Therapies

Tumor growth and progression are critically dependent on the establishment of a vascular support system. This is often accomplished via the expression of pro-angiogenic growth factors, including members of the vascular endothelial growth factor (VEGF) family of ligands. VEGF ligands are overexpressed in a wide variety of solid tumors and therefore have inspired optimism that inhibition of the different axes of the VEGF pathway—alone or in combination—would represent powerful anti-angiogenic therapies for most cancer types. When considering treatments that target VEGF and its receptors, it is difficult to tease out the differential anti-angiogenic and anti-tumor effects of all combinations experimentally because tumor cells and vascular endothelial cells are engaged in a dynamic cross-talk that impacts key aspects of tumorigenesis, independent of angiogenesis. Here we develop a mathematical model that connects intracellular signaling responsible for both endothelial and tumor cell proliferation and death to population-level cancer growth and angiogenesis. We use this model to investigate the effect of bidirectional communication between endothelial cells and tumor cells on treatments targeting VEGF and its receptors both in vitro and in vivo. Our results underscore the fact that in vitro therapeutic outcomes do not always translate to the in vivo situation. For example, our model predicts that certain therapeutic combinations result in antagonism in vivo that is not observed in vitro. Mathematical modeling in this direction can shed light on the mechanisms behind experimental observations that manipulating VEGF and its receptors is successful in some cases but disappointing in others.     

Modeling the effects of vasculature evolution on early brain tumor growth

Mathematical modeling of both tumor growth and angiogenesis have been active areas of research for the past several decades. Such models can be classified into one of two categories: those that analyze the remodeling of the vasculature while ignoring changes in the tumor mass, and those that predict tumor expansion in the presence of a non-evolving vasculature. However, it is well accepted that vasculature remodeling and tumor growth strongly depend on one another. For this reason, we have developed a two-dimensional hybrid cellular automaton model of early brain tumor growth that couples the remodeling of the microvasculature with the evolution of the tumor mass. A system of reaction-diffusion equations has been developed to track the concentration of vascular endothelial growth factor (VEGF), Ang-1, Ang-2, their receptors and their complexes in space and time. The properties of the vasculature and hence of each cell are determined by the relative concentrations of these key angiogenic factors. The model exhibits an angiogenic switch consistent with experimental observations on the upregulation of angiogenesis. Particularly, we show that if the pathways that produce and respond to VEGF and the angiopoietins are properly functioning, angiogenesis is initiated and a tumor can grow to a macroscopic size. However, if the VEGF pathway is inhibited, angiogenesis does not occur and tumor growth is thwarted beyond 1-2mm in size. Furthermore, we show that tumor expansion can occur in well-vascularized environments even when angiogenesis is inhibited, suggesting that anti-angiogenic therapies may not be sufficient to eliminate a population of actively dividing malignant cells.     

ADAMTS1/METH1 inhibits endothelial cell proliferation by direct binding and sequestration of VEGF165

ADAMTS1 is a metalloprotease previously shown to inhibit angiogenesis in a variety of in vitro and in vivo assays. In the present study, we demonstrate that ADAMTS1 significantly blocks VEGFR2 phosphorylation with consequent suppression of endothelial cell proliferation. The effect on VEGFR2 function was due to direct binding and sequestration of VEGF165 by ADAMTS1. Binding was confirmed by co-immunoprecipitation and cross-linking analysis. Inhibition of VEGF function was reversible, as active VEGF could be recovered from the complex. The interaction required the heparin-binding domain of the growth factor, because VEGF121 failed to bind to ADAMTS1. Structure/function analysis with independent ADAMTS1 domains indicated that binding to VEGF165 was mediated by the carboxyl-terminal (CT) region. ADAMTS1 and VEGF165 were also found in association in tumor extracts. These findings provide a mechanism for the anti-angiogenic activity of ADAMTS1 and describe a novel modulator of VEGF bioavailability.     

R-(-)-β-O-methylsynephrine, a natural product, inhibits VEGF-induced angiogenesis in vitro and in vivo

R-(-)-β-O-methylsynephrine (OMe-Syn) is an active compound isolated from a plant of the Rutaceae family. We conducted cell proliferation assays on various cell lines and found that OMe-Syn more strongly inhibited the growth of human umbilical vein endothelial cells (HUVECs) than that of other normal and cancer cell lines tested. In angiogenesis assays, it inhibited vascular endothelial growth factor (VEGF)-induced invasion and tube formation of HUVECs with no toxicity. The anti-angiogenic activity of OMe-Syn was also validated in vivo using the chorioallantonic membrane (CAM) assay in growing chick embryos. Expression of the growth factors VEGF, hepatocyte growth factor, and basic fibroblast growth factor was suppressed by OMe-Syn in a dose-dependent manner. Taken together, our results indicate that this compound could be a novel basis for a small molecule targeting angiogenesis.     

Anti-angiogenic activity of the recombinant kringle domain of urokinase and its specific entry into endothelial cells

Urokinase plasminogen activator (uPA) belongs to a family of proteins that contains kringle domain and plays an important role in inflammation, tissue remodeling, angiogenesis, and tumor metastasis by pericellular plasminogen activation. Kringle domains of plasminogen have been shown to demonstrate anti-angiogenic and anti-tumor activities. Here, we report our investigation of the kringle domain of uPA for anti-angiogenic activity and a possible cellular mechanism of action. The recombinant kringle domain of uPA (Asp(45)-Lys(135)) (UK1) inhibited endothelial cell proliferation stimulated by basic fibroblast growth factor, vascular endothelial growth factor (VEGF), or epidermal growth factor. It also inhibited migration of endothelial cells induced by VEGF or uPA, and in vivo angiogenesis on the chick chorioallantoic membrane. It did not block plasminogen activation by activated uPA in clot lysis and chromogenic substrate assays. Neither binding of UK1 to immobilized uPA receptor nor competitive inhibition of uPA binding were confirmed by real-time interaction analysis. However, internalization of UK1 followed by translocation from cytosol to nucleus was determined to be specific to endothelial cells. It also elicited a transient increase of Ca(2+) flux of more than 2-fold within 2 min of exposure in an endothelial cell-specific manner. These results suggest that the kringle domain of uPA exhibits anti-angiogenic activity and that its anti-angiogenic activity may occur through a different mechanism from inhibition of uPA-uPA receptor interaction or uPA proteolytic activity and may be associated with endothelial-cell specific internalization not mediated by the uPA receptor.     

GABARBP down-regulates HIF-1α expression through the VEGFR-2 and PI3K/mTOR/4E-BP1 pathways

Human γ-aminobutyrate type A (GABA_A) receptor-binding protein (GABARBP), a tumor suppressor protein with apoptotic function, can be inhibited in response to angiogenesis through the PI3K/Akt signaling cascades. Here, we investigated whether GABARBP over-expression could regulate vascular endothelial growth factor (VEGF)/hypoxia-inducible factor-1α (HIF-1α) expression and angiogenic activity in a carcinoma model system. GABARBP dramatically inhibited VEGF-induced endothelial cell proliferation, migration, and tube formation, as well as VEGFR-2 phosphorylation in vitro. At the same time, GABARBP exposed potent anti-angiogenic activity and remarkably down-regulated the levels of VEGF and HIF-1α protein expression, key components for angiogenesis. In addressing its biological molecular mechanism, GABARBP was found to effectively inhibit the phosphorylation of down-stream PI3K components, such as PDK1, Akt, mTOR, TSC-2, p70S6K, and 4E-BP1 by directly binding with VEGFR-2. In contrast, p38/JNK phosphorylation was not suppressed by GABARBP. These findings disclose a novel function of GABARBP in suppressing VEGF and HIF-1α protein expression, which is important for tumor angiogenesis and tumor growth. Thus, our data strongly provides novel biological mechanistic insights into the regulatory function of GABARBP in ovarian tumor progression, and the important of pre-clinical certification of GABARBP as a potential angiogenesis agent targeting ovarian tumorigenesis.     

BRN-103, a novel nicotinamide derivative, inhibits VEGF-induced angiogenesis and proliferation in human umbilical vein endothelial cells

Anti-angiogenesis is regarded as an effective strategy for cancer treatment, and vascular endothelial growth factor (VEGF) plays a key role in the regulations of angiogenesis and vasculogenesis. In the present study, the authors synthesized five novel nicotinamide derivatives which structurally mimic the receptor tyrosine kinase inhibitor sunitinib and evaluated their anti-angiogenic effects. Transwell migration assays revealed that 2-(1-benzylpiperidin-4-yl) amino-N-(3-chlorophenyl) nicotinamide (BRN-103), among the five derivatives most potently inhibited VEGF-induced human umbilical vein endothelial cells (HUVECs). In addition, BRN-103 dose-dependently inhibited VEGF-induced migration, proliferation, and capillary-like tube formation of HUVECs and vessel sprouting from mouse aortic rings. To understand the molecular mechanisms responsible for these activities, the authors examined the effect of BRN-103 on VEGF signaling pathways in HUVECs. BRN-103 was found to suppress the VEGF-induced phosphorylation of VEGF receptor 2 (VEGR2) and the activations of AKT and eNOS. Taken together, these results suggest that BRN-103 inhibits VEGF-mediated angiogenesis signaling in human endothelial cells.     

A natural small molecule voacangine inhibits angiogenesis both in vitro and in vivo

Angiogenesis, the formation of new blood vessels from pre-existing ones, plays a critical role in normal and pathological phenotypes, including solid tumor growth and metastasis. Accordingly, the development of new anti-angiogenic agents is considered an efficient strategy for the treatment of cancer and other human diseases linked with angiogenesis. We have identified voacangine, isolated from Voacanga africana, as a novel anti-angiogenic agent. Voacangine inhibits the proliferation of HUVECs at an IC(50) of 18 μM with no cytotoxic effects. Voacangine significantly suppressed in vitro angiogenesis, such as VEGF-induced tube formation and chemoinvasion. Moreover, the compound inhibits in vivo angiogenesis in the chorioallantoic membrane at non-toxic doses. In addition, voacangine decreased the expression levels of hypoxia inducible factor-1α and its target gene, VEGF, in a dose-dependent manner. Taken together, these results suggest that the naturally occurring compound, voacangine, is a novel anti-angiogenic compound.     

VEGF111b,a new member of VEGFxxxb isoforms and induced by mitomycin C,inhibits angiogenesis

Vascular endothelial growth factor (VEGF-A) stimulating angiogenesis is required for tumor growth and progression. The conventional VEGF-A isoforms have been considered as pro-angiogenic factors. Another family of VEGF-A isoforms generated by alternative splicing, termed VEGFxxxb isoforms, has anti-angiogenic property, exemplified by VEGF165b. Here, we identify a new number of VEGFxxx family-VEGF111b induced by mitomycin C, although not detected in mitomycin C-unexposed ovarian cancer cells. SKOV3 cells were transfected with pcDNA_3.1 empty vector, pcDNA_3.1-VEGF111b or pcDNA_3.1-VEGF165b to collect conditioned mediums respectively. VEGF111b overexpression inhibits proliferation, migration and tube formation of endothelial cell by inhibiting VEGF-R2 phosphorylation and its downstream signaling, similar to VEGF165b but slightly lower than VEGF165b. The anti-angiogenic property depends on the six amino acids of exon 8b of the VEGFxxxb isoforms. Our results show that VEGF111b is a novel potent anti-angiogenic agent that can target the VEGF-R2 and its signaling pathway to inhibit ovarian tumor growth.