Evidence of ß-blockers drug repurposing for the treatment of triple negative breast cancer: A systematic review.

Triple negative breast cancer (TNBC) is a particularly aggressive subtype of breast cancer (BC) for which limited therapeutic options are available. Recently, ß-blockers (BBs) have been suggested to have favorable effects in the treatment of BC. The aim of this systematic review was to collect evidence from preclinical and clinical studies concerning the scientific evidence for the repurposing of BBs in TNBC treatment. PubMed database was searched to retrieve studies of interest published up to 30/01/2018. All preclinical studies using TNBC in vitro and in vivo models and assessing the effect of any molecule with sympatholytic or sympathomimetic activity on adrenergic receptors were included. Clinical studies concerning BBs were considered eligible. The Newcastle-Ottawa scale was used for the quality assessment of clinical studies. A total of 614 study references were retrieved. Forty-six preclinical studies were included. In in vitro studies, propranolol, a non-selective BB, significantly decreased proliferation, migration and invasion of TNBC cells. Consistently, in in vivo studies, propranolol inhibited metastasis, angiogenesis and tumor growth. Clinical studies, reporting evidence from a total of four distinct retrospective observational cohort studies, showed a beneficial effect of BBs in TNBC treatment. The overall quality of the clinical evidence collected was low. Preclinical evidence collected in this systematic review are in line with the results reported in the clinical studies retrieved, pointing towards a beneficial effect of BB in the treatment of TNBC. However, given the overall low quality of available evidence, no definite conclusion may be drawn.

ALDH2 Activity Reduces Mitochondrial Oxygen Reserve Capacity in Endothelial Cells and Induces Senescence Properties.

Endothelial cells (ECs) are dynamic cells that turn from growth into senescence, the latter being associated with cellular dysfunction, altered metabolism, and age-related cardiovascular diseases. Aldehyde dehydrogenase 2 (ALDH2) is a mitochondrial enzyme metabolizing acetaldehyde and other toxic aldehydes, such as 4-hydroxynonenal (4-HNE). In conditions in which lipid peroxidation products and reactive oxygen species (ROS) are accumulated, ECs become dysfunctional and significantly contribute to the progression of vascular-dependent diseases. The aim of the present study has been to investigate whether inhibition of ALDH2 alters endothelial functions together with the impairment of bioenergetic functions, accelerating the acquisition of a senescent phenotype. HUVECs transfected with siRNA targeting ALDH2 or treated with daidzin, an ALDH2 inhibitor, were used in this study. We observed an alteration in cell morphology associated with endothelial dysfunctions. Loss of ALDH2 reduced cell proliferation and migration and increased paracellular permeability. To assess bioenergetic function in intact ECs, extracellular flux analysis was carried out to establish oxygen consumption rates (OCR). We observed a decrease in mitochondrial respiration and reserve capacity that coincided with SA-ß-Gal accumulation and an increase in p21 and p53 expression in siALDH2 or daidzin-treated HUVECs. Treatment with N-acetyl-L-cysteine (NAC) reduced endothelial dysfunctions mediated by siALDH2, indicating that oxidative stress downstream to siALDH2 plays an instrumental role. Our results highlight that ALDH2 impairment accelerates the acquisition of a premature senescent phenotype, a change likely to be associated with the observed reduction of mitochondrial respiration and reserve capacity.

Characterization of zofenoprilat as an inducer of functional angiogenesis through increased H2 S availability.

BACKGROUND AND PURPOSE: Hydrogen sulfide (H2 S), an endogenous volatile mediator with pleiotropic functions, promotes vasorelaxation, exerts anti-inflammatory actions and regulates angiogenesis. Previously, the SH-containing angiotensin-converting enzyme inhibitor (ACEI), zofenopril, was identified as being effective in preserving endothelial function and inducing angiogenesis among ACEIs. Based on the H2 S donor property of its active metabolite zofenoprilat, the objective of this study was to evaluate whether zofenoprilat-induced angiogenesis was due to increased H2 S availability. EXPERIMENTAL APPROACH: HUVECs were used for in vitro studies of angiogenesis, whereas the Matrigel plug assay was used for in vivo assessments. KEY RESULTS: Zofenoprilat-treated HUVECs showed an increase in all functional features of the angiogenic process in vitro. As zofenoprilat induced the expression of CSE (cystathionine-γ-lyase) and the continuous production of H2 S, CSE inhibition or silencing blocked the ability of zofenoprilat to induce angiogenesis, both in vitro and in vivo. The molecular mechanisms underlying H2 S/zofenoprilat-induced angiogenesis were dependent on Akt, eNOS and ERK1/2 cascades. ATP-sensitive potassium (KATP ) channels, the molecular target that mediates part of the vascular functions of H2 S, were shown to be involved in the upstream activation of Akt and ERK1/2. Moreover, the up-regulation of fibroblast growth factor-2 was dependent on CSE-derived H2 S response to H2 S and KATP activation. CONCLUSIONS AND IMPLICATIONS: Zofenoprilat induced a constant production of H2 S that stimulated the angiogenic process through a KATP channel/Akt/eNOS/ERK1/2 pathway. Thus, zofenopril can be considered as a pro-angiogenic drug acting through H2 S release and production, useful in cardiovascular pathologies where vascular functions need to be re-established and functional angiogenesis induced.

EGFR signaling upregulates expression of microsomal prostaglandin E synthase-1 in cancer cells leading to enhanced tumorigenicity.

In this report we describe the contribution of prostaglandin E(2) (PGE(2)) derived from the inducible microsomal PGE-synthase type-1 (mPGES-1) to the epidermal growth factor receptor (EGFR) oncogenic drive in tumor epithelial cells and in tumor-bearing mice. EGFR stimulation upregulated expression of mPGES-1 in HT-29, A431 and A549 cancer cells. Egr-1, a transcription factor induced by EGF, mediated this response. The Egr-1 rise provoked the overexpression of mPGES-1 messenger and protein, and enhanced PGE(2) formation. These changes were suppressed either by silencing Egr-1, or by upstream blockade of EGFR or ERK1/2 signals. Further, in a clonogenic assay on tumor cells, EGF induced a florid tumorigenic phenotype, which regressed when mPGES-1 was silenced or knocked down. EGF-induced mPGES-1 overexpression in epithelial cell reduced E-cadherin expression, whereas enhancing that of vimentin, suggesting an incipient mesenchymal phenotype. Additionally, inhibiting the EGFR in mice bearing the A431 tumor, the mPGES-1 expression and the tumor growth, exhibited a parallel decline. In conclusion, these findings provide novel evidence that a tight cooperation between the EGF/EGFR and mPGES-1 leads to a significant tumorigenic gain in epithelial cells, and provide clues for controlling the vicious association.

A nanoscale fluorocarbon coating on PET surfaces improves the adhesion and growth of cultured coronary endothelial cells.

Plasma deposition was applied to deposit smooth and nanostructured fluorocarbon coatings on polyethylene terephthalate substrates, with the aim to obtain surfaces with identical chemical composition but different roughness to improve the endothelialization process on PET surfaces. We found that increased roughness was associated with enhanced endothelial cell response, as shown by the ability of cells to grow and adhere to nanostructures. We also observed specific interaction of filopodia protruding from the cell membrane with individual nanostructures, leading to increased cell attachment, spreading and cell viability. Among the modified surfaces, one termed PET-tfl90 emerged as the one capable of best sustaining the formation of a confluent monolayer of endothelial cells. In conclusion, PET modified by nanostructured fluorocarbon film represents an improved graft material, over conventional PET, for endothelial cell adhesion and growth.

TAT-BH4 counteracts Abeta toxicity on capillary endothelium.

Oxidative stress is one of the factor contributing to blood brain barrier degeneration. This phenomenon is observed during pathological conditions such as Alzheimer's disease or cerebral amyloid angiopathy in which brain haemorrhages are very frequent. Both diseases are characterized by beta amyloid peptide deposition either in neurons or in vessels. Oxidative stress leads to impairment of mitochondrial functions and apoptotic cell death subsequent to caspases activation. In this paper we demonstrate that BH4 domain of Bcl-xl administrated to endothelial cells as the conjugated form with TAT peptide, reverts Abeta-induced apoptotic cell death by activating a survival programme which is Akt/endothelial nitric oxide synthase dependent.

Soluble N-cadherin fragment promotes angiogenesis.

Endothelial cells express two dependent intercellular adhesion molecules: vascular endothelial (VE)-cadherin, specific for endothelial cells, and N-cadherin, also present in neuronal, lens, skeletal and heart muscle cells, osteoblasts, pericytes and fibroblasts. While there exists a vast amount of evidence that VE-cadherin promotes angiogenesis, the role of N-cadherin still remains to be elucidated. We found that a soluble 90-kDa fragment N-cadherin promotes angiogenesis in the rabbit cornea assay and in the chorioallantoic assay when cleaved enzymatically from the extracellular domain of N-cadherin. Soluble N-cadherin stimulates migration of endothelial cells in the wound healing assay and stimulates phosphorylation of extracellular regulated kinase. In vitro experiments with PD173074 and knock-down of N-cadherin and fibroblast growth factor (FGF)-receptor, showed that the pro-angiogenic effect of soluble N-cadherin is N-cadherin- and FGF-receptor-dependent. Our results suggest that soluble N-cadherin stimulates migration of endothelial cells through the FGF-receptor.

FGF-2 overexpression opposes the beta amyloid toxic injuries to the vascular endothelium.

Recent evidences suggest that Abeta peptides modulate endothelial cell (EC) functions. At low concentrations, Abeta1-40 enhances the pro-angiogenic activity of FGF-2, whereas deposition of excess Abeta causes EC dysfunction and cerebral amyloid angiopathy (CAA). We investigated whether FGF-2 attenuates EC dysfunction caused by pathological Abeta levels. We studied Abeta1-40 on EC survival, as well as on signals responsible of their angiogenic phenotype. At 5-50 microM Abeta1-40 reduced EC population, caused apoptosis, downregulated FGF-2 production, inhibited FGF-2 binding to heparin, and FGFR1 phosphorylation. Toxic effects were owing to lack of FGF-2 stimulation, as EC overexpressing FGF-2 displayed extraordinary resistance to Abeta1-40 injuries. The FGF-2 mechanism responsible for reversing damages, involves the downstream enhancement of Akt, a pathway independent of eNOS activation. In conclusion, we demonstrate that FGF-2 protects EC from the effects of excess Abeta1-40, suggesting that it may attenuate the consequences of Abeta deposition in pathologies as CAA.

Exogenous BH4/Bcl-2 peptide reverts coronary endothelial cell apoptosis induced by oxidative stress.

BACKGROUND: Vascular endothelium undergoes apoptosis when exposed to reactive oxygen species (ROS), including hydrogen peroxide and superoxide radicals. ROS are believed to be the cause of damage to small vessels during ischemia-reperfusion injury and of arterial damage during atherosclerosis. Hydrogen peroxide-induced apoptosis is mediated through the inhibition of Bcl-xl activity and caspase-3 and caspase-9 activation. The BH4 domain of the Bcl-2 family members is responsible for their antiapoptotic activity. The BH4 domains of Bcl-2 and Bcl-xl inhibit cytochrome c release and the loss of mitochondrial membrane potential. METHODS AND RESULTS: The purpose of this project was to study the antiapoptotic effect of cell-permeant derivative of Bcl-2 (BH4 peptide) on endothelial cells exposed to stress conditions. BH4 peptide was conjugated to the cell-permeable peptide TAT and was applied to endothelial cells under conditions of serum starvation and hydrogen peroxide treatment. TAT-BH4 reduced caspase-3 activity and prevented apoptotic cell death. CONCLUSION: Our results indicate that TAT-BH4 peptide can protect endothelial cells from ROS-induced apoptosis.

Antiangiogenic properties of selected ruthenium(III) complexes that are nitric oxide scavengers.

The nitric oxide synthase (NOS) pathway has been clearly demonstrated to regulate angiogenesis. Increased levels of NO correlate with tumour growth and spreading in different experimental and human cancers. Drugs interfering with the NOS pathway may be useful in angiogenesis-dependent tumours. The aim of this study was to pharmacologically characterise certain ruthenium-based compounds, namely NAMI-A, KP1339, and RuEDTA, as potential NO scavengers to be used as antiangiogenic/antitumour agents. NAMI-A, KP1339 and RuEDTA were able to bind tightly and inactivate free NO in solution. Formation of ruthenium-NO adducts was documented by electronic absorption, FT-IR spectroscopy and (1)H-NMR. Pretreatment of rabbit aorta rings with NAMI-A, KP1339 or RuEDTA reduced endothelium-dependent vasorelaxation elicited by acetylcholine. This effect was reversed by 8-Br-cGMP. The key steps of angiogenesis, endothelial cell proliferation and migration stimulated by vascular endothelial growth factor (VEGF) or NO donor drugs, were blocked by NAMI-A, KP1339 and RuEDTA, these compounds being devoid of any cytotoxic activity. When tested in vivo, NAMI-A inhibited angiogenesis induced by VEGF. It is likely that the antitumour properties previously observed for ruthenium-based NO scavengers, such as NAMI-A, are related to their NO-related antiangiogenic properties.

Cell-mediated delivery of fibroblast growth factor-2 and vascular endothelial growth factor onto the chick chorioallantoic membrane: endothelial fenestration and angiogenesis.

Fibroblast growth factor-2 (FGF2) and vascular endothelial growth factor (VEGF) exert their angiogenic activity by interacting with endothelial cells in a distinct manner. In this study, we investigated the morphological features of endothelial cells of the chick embryo chorioallantoic membrane (CAM) microvasculature after stimulation with FGF2 or VEGF. In order to provide a continuous delivery of the growth factor, we utilized a recently developed gelatin sponge/CAM assay in which a limited number of FGF2- or VEGF-transfected cells were adsorbed onto gelatin sponges and applied on the top of the CAM on day 8 of development. Their angiogenic activity was compared to that exerted by a single bolus of the corresponding growth factor. All the angiogenic stimuli induced a comparable vasoproliferative response, as demonstrated by the appearance of similar numbers of immature blood vessels within the sponge on day 12. No angiogenic response was observed in CAMs implanted with the corresponding parental cell lines or vehicle. Electron microscopy demonstrated that VEGF-overexpressing cells modified the phenotype of the endothelium of the blood vessels at the boundary between the implant and the surrounding CAM mesenchyme. The endothelial lining of 30% of these vessels showed segmental attenuations, was frequently interrupted and became fenestrated, mimicking what is observed in tumor vasculature. In contrast, the vessels consisted of continuous endothelium sealed by tight junctions in all the other experimental conditions. These results indicate that FGF2 and VEGF interact with endothelial cells of the CAM in a distinct manner. Both growth factors induce a potent angiogenic response, but only VEGF delivered in a continuous manner by its transfectants can modify the phenotype of the otherwise quiescent endothelium of CAM blood microvessels. The gelatin sponge/CAM assay may constitute a new model to study the mechanisms leading to endothelial fenestration in tumor growth.

Nitric oxide modulates the angiogenic phenotype of middle-T transformed endothelial cells.

The role of nitric oxide (NO) in the induction of angiogenesis was evaluated in a murine heart endothelioma cell line (H.end.FB) carrying the mT oncogene. Two clonal derivatives of H.end.FB, H80 and H73, exhibiting different NO synthase (NOS) activities were selected and used in the study. The relationship among NOS activity and tumor cell behaviour (growth, and angiogenic capacity) and the molecular control of gene expression were investigated. H.end.FB and H80 on one side and H73 on the other side exhibited the highest and lowest NOS activity, respectively. Cell growth was inversely correlated to the amount of NO produced by the cell lines. Conversely, in the avascular rabbit cornea assay, H.end.FB and H80 cells were strongly angiogenic, while H73 were poorly angiogenic, indicating that the ability of the cells to induce neovascularization was associated with the extent of NO produced. Consistently, systemic administration to rabbits of the NOS inhibitor N(w)-nitro-L-arginine methyl ester (L-NAME) significantly reduced the angiogenicity of H.end.FB cells. RT-PCR evidenced that H.end.FB expressed mRNA for TGF-beta1 and all VEGF isoforms, VEGF165 being predominantly expressed. NOS inhibition reduced the basal expression of VEGF isoforms, while it markedly potentiated TGF-beta1 expression. These results indicate that the endogenous production of NO in tumor cells can serve as an autocrine/paracrine signalling mechanism of progression, by controlling angiogenic factor/modulator expression.

ANG II potentiates mitogenic effect of norepinephrine in vascular muscle cells: role of FGF-2.

We examined the possible cooperation between norepinephrine (NE) and ANG II on proliferation of cultured vascular smooth muscle cells (VSMCs) and the involved cellular mechanisms. Nanomolar NE concentrations stimulated VSMC proliferation through a prazosin-sensitive effect. The pretreatment of cells with 100 nM ANG II for 24 h significantly potentiated the NE-induced VSMC proliferation; this potentiating effect of ANG II was blocked by losartan but was unaffected by the AT(2) receptor antagonist PD-123177. ANG II pretreatment also potentiated the increase in inositol phosphate turnover and upregulated the cell expression of fibroblast growth factor (FGF-2) induced by NE. Anti-FGF-2 neutralizing antibodies prevented the potentiating effect of ANG II on NE-induced cell growth. Both ANG II and NE stimulated extracellular signal-related kinase (ERK1) activation, but an ANG II potentiation of the effect of NE on ERK1 activity was not detectable. Moreover, ANG II significantly increased protein synthesis but did not potentiate the hypertrophic effect of NE. These findings demonstrate that ANG II and NE cooperate in promoting VSMC growth and that FGF-2 upregulation is involved in this effect.

Corneal assay for angiogenesis.

In order to develop angiogenic and antiangiogenic strategies, concerted efforts have been made to provide animal models for more quantitative analysis of in vivo angiogenesis. In vivo techniques consist of the cornea pocket and iris implant in the eye, the rabbit ear chamber, the dorsal skinfold chamber, the cranial window, the hamster cheek pouch window, the sponge implant assay, the fibrin clots, the sodium alginate beads and the Matrigel plugs, the rat mesenteric window, the chick embryo chorioallantoic membrane and the air sac in mice and rats (1). In this chapter we will discuss the avascular cornea assay, and the advantages and disadvantages of using this assay in different species. The cornea assay is based on the placement of an angiogenic inducer (tumor tissue, cell suspension, growth factor) into a corneal pocket in order to evoke vascular outgrowth from the peripherally located limbal vasculature. In comparison to other in vivo assays, this assay has the advantage of measuring only new blood vessels, because the cornea is initially avascular.

I-309 binds to and activates endothelial cell functions and acts as an angiogenic molecule in vivo.

Several chemokines have been shown to act as angiogenic molecules or to modulate the activity of growth factors such as fibroblast growth factor 2 (FGF-2) and vascular endothelial growth factor (VEGF). The detection of the CC chemokine receptor (CCR) 8 message in human umbilical vein endothelial cells (HUVECs) by reverse transcription- polymerase chain reaction (RT-PCR) and RNase protection assay (RPA), prompted us to investigate the potential role exerted by the CC chemokine I-309, a known ligand of such receptor, in both in vitro and in vivo angiogenesis assays. We show here that I-309 binds to endothelial cells, stimulates chemotaxis and invasion of these cells, and enhances HUVEC differentiation into capillary-like structures in an in vitro Matrigel assay. Furthermore, I-309 is an inducer of angiogenesis in vivo in both the rabbit cornea and the chick chorioallantoic membrane assay (CAM).

Cu(II) and Zn(II) complexes with hyaluronic acid and its sulphated derivative. Effect on the motility of vascular endothelial cells.

With the aim of improving the compatibility of biomaterials to be used for the construction of cardiovascular prosthesis, we have designed bioactive macromolecules resulting from chemical modifications of hyaluronic acid (Hyal). The stability constants of Cu(II) and Zn(II) complexes with the sulphated derivative of hyaluronic acid (HyalS3.5) were evaluated. Two different complexes have been found for each metal ion, CuL, Cu(OH)2L and ZnL, Zn(OH)2L (L means the disaccharide unit of the ligands) in aqueous solution at 37 degrees C. The dihydroxo Cu(II) complex was present in high percentage at pH=7.4. On the contrary, the Zn(II) ion was present with a relatively low percentage of both complexes. The ability to stimulate endothelial cell adhesion and migration was evaluated for Hyal, HyalS3.5 and their complexes with Cu(II) and Zn(II) ions. The results revealed that Hyal and [Cu(OH)2HyalS3.5](4.5)- induced cell adhesion, while [ZnHyalS3.5](2.5)- and [Zn(OH)2HyalS3.5](4.5)- inhibited the process. The chemotactic activity of increasing concentrations of the above complexes was also evaluated, demonstrating that [Cu(OH)2HyalS3.5](4.5)- complex at 1 microM concentration was the most active in inducing cell migration. These results have been also strengthened by analysing adherent cell migration in agarose. In conclusion, sulphated hyaluronic acid coordinated to Cu(II) seems to be a promising matrix molecule for the construction of cardiovascular prosthesis.

Abolished angiogenicity and tumorigenicity of Burkitt lymphoma by interleukin-10.

Because of its immunosuppressive properties, interleukin-10 (IL-10) is thought to play an important role in a number of human disease states, including inflammation, autoimmunity, and transplant rejection. In this study, we demonstrate that introduction of human or viral IL-10 genes into Burkitt's lymphoma cells markedly reduced their ability to grow as subcutaneous (sc) tumors in SCID mice. In vivo assays for angiogenesis revealed an inhibition of the angiogenic capacity of the IL-10-transfected lines. Recombinant human IL-10 abolished and viral IL-10 reduced vascular endothelial growth factor (VEGF)-165-induced neovascularization. Furthermore, IL-10 blocked the VEGF- and fibroblast growth factor (FGF)-2-induced proliferation of microvascular endothelial cells in vitro. The current observations suggest a direct role for IL-10 in the prevention of angiogenesis in human lymphoid malignancies.