Low‐dose angiostatic tyrosine kinase inhibitors improve photodynamic therapy for cancer: lack of vascular normalization

Photodynamic therapy (PDT) is an effective clinical treatment for a number of different cancers. PDT can induce hypoxia and inflammation, pro‐angiogenic side effects, which may counteract its angio‐occlusive mechanism. The combination of PDT with anti‐angiogenic drugs offers a possibility for improved anti‐tumour outcome. We used two tumour models to test the effects of the clinically approved angiostatic tyrosine kinase inhibitors sunitinib, sorafenib and axitinib in combination with PDT, and compared these results with the effects of bevacizumab, the anti‐VEGF antibody, for the improvement of PDT. Best results were obtained from the combination of PDT and low‐dose axitinib or sorafenib. Molecular analysis by PCR revealed that PDT in combination with axitinib suppressed VEGFR‐2 expression in tumour vasculature. Treatment with bevacizumab, although effective as monotherapy, did not improve PDT outcome. In order to test for tumour vessel normalization effects, axitinib was also applied prior to PDT. The absence of improved PDT outcome in these experiments, as well as the lack of increased oxygenation in axitinib‐treated tumours, suggests that vascular normalization did not occur. The current data imply that there is a future for certain anti‐angiogenic agents to further improve the efficacy of photodynamic anti‐cancer therapy.     

The β3‐integrin endothelial adhesome regulates microtubule‐dependent cell migration

Integrin β3 is seen as a key anti‐angiogenic target for cancer treatment due to its expression on neovasculature, but the role it plays in the process is complex; whether it is pro‐ or anti‐angiogenic depends on the context in which it is expressed. To understand precisely β3's role in regulating integrin adhesion complexes in endothelial cells, we characterised, by mass spectrometry, the β3‐dependent adhesome. We show that depletion of β3‐integrin in this cell type leads to changes in microtubule behaviour that control cell migration. β3‐integrin regulates microtubule stability in endothelial cells through Rcc2/Anxa2‐driven control of active Rac1 localisation. Our findings reveal that angiogenic processes, both in vitro and in vivo, are more sensitive to microtubule targeting agents when β3‐integrin levels are reduced.     

Pro‐angiogenic potential of human chorion‐derived stem cells: in vitro and in vivo evaluation

Human chorion‐derived stem cells (hCDSC) were previously shown to demonstrate multipotent properties with promising angiogenic characteristics in monolayer‐cell culture system. In our study, we investigated the angiogenic capability of hCDSC in 3‐dimensional (3D) in vitro and in vivo angiogenic models for the purpose of future application in the treatment of ischaemic diseases. Human CDSC were evaluated for angiogenic and endogenic genes expressions by quantitative PCR. Growth factors secretions were quantified using ELISA. In vitro and in vivo vascular formations were evaluated by histological analysis and confocal microscopic imaging. PECAM‐1⁺ and vWF⁺ vascular‐like structures were observed in both in vitro and in vivo angiogenesis models. High secretions of VEGF and bFGF by hCDSC with increased expressions of angiogenic and endogenic genes suggested the possible angiogenic promoting mechanisms by hCDSC. The cooperation of hCDSC with HUVECS to generate vessel‐like structures in our systems is an indication that there will be positive interactions of hCDSC with existing endothelial cells when injected into ischaemic tissues. Hence, hCDSC is suggested as the novel approach in the future treatment of ischaemic diseases.     

Characterization of a novel angiogenic model based on stable, fluorescently labelled endothelial cell lines amenable to scale-up for high content screening

Background. Blood vessel formation is important for many physiological and pathological processes and is therefore a critical target for drug development. Inhibiting angiogenesis to starve a tumour or promoting ‘normalization’ of tumour vasculature in order to facilitate delivery of anticancer drugs are both areas of active research. Recapitulation of vessel formation by human cells in vitro allows the investigation of cell—cell and cell—matrix interactions in a controlled environment and is therefore a crucial step in developing HCS (high content screening) and HTS (high throughput screening) assays to search for modulators of blood vessel formation. HUVECs (human umbilical‐vein endothelial cells) exemplify primary cells used in angiogenesis assays. However, primary cells have significant limitations that include phenotypic decay and/or senescence by six to eight passages in culture, making stable integration of fluorescent markers and large‐scale expansion for HTS problematic. To overcome these limitations for HTS, we developed a novel angiogenic model system that employs stable fluorescent endothelial cell lines based on immortalized HMECs (human microvascular endothelial cell). We then evaluated HMEC cultures, both alone and co‐cultured with an EMC (epicardial mesothelial cell) line that contributes vascular smooth muscle cells, to determine the suitability for HTS or HCS. Results. The endothelial and epicardial lines were engineered to express a panel of nuclear‐ and cytoplasm‐localized fluorescent proteins to be mixed and matched to suit particular experimental goals. HMECs retained their angiogenic potential and stably expressed fluorescent proteins for at least 13 passages after transduction. Within 8 h after plating on Matrigel, the cells migrated and coalesced into networks of vessel‐like structures. If co‐cultured with EMCs, the branches formed cylindrical‐shaped structures of HMECs surrounded by EMC derivatives reminiscent of vessels. Network formation measurements revealed responsiveness to media composition and control compounds. Conclusions. HMEC‐based lines retain most of the angiogenic features of primary endothelial cells and yet possess long‐term stability and ease of culture, making them intriguing candidates for large‐scale primary HCS and HTS (of ～10000–1000000 molecules). Furthermore, inclusion of EMCs demonstrates the feasibility of using epicardial‐derived cells, which normally contribute to smooth muscle, to model large vessel formation. In summary, the immortalized fluorescent HMEC and EMC lines and straightforward culture conditions will enable assay development for HCS of angiogenesis.     

Cell proliferation and cell cycle alterations in oesophageal p53‐mutated cancer cells treated with cisplatin in combination with photodynamic therapy

Objectives: The major goal of anti‐cancer therapies is selective destruction of tumour cells with minimum side effects on normal cells. Towards this aim, combination of different therapeutic modalities has been evaluated for improving control of neoplastic diseases and quality of life for the patient. Photodynamic therapy (PDT) is a procedure for treatment of various types of cancer, but its combination with other established treatments has not been evaluated in detail. We have used KYSE‐510 cells from a human oesophageal carcinoma as an in vitro model to investigate whether cisplatin (CDDP) could be combined with PDT to increase cell death with respect to single treatments. Materials and methods: p53‐mutated KYSE‐510 cells were treated with CDDP alone or in combination with PDT. Analyses of cell viability, cell cycle progression and apoptosis induction were carried out at specific times after treatments. Results: Decrease in cell viability, cell cycle arrest at the G₂/M‐ and S‐phases boundary, and apoptosis induction were observed after single and combined treatments. Conclusions: Our results show that low CDDP doses (0.25–1 μm ) induce cell mortality and cell cycle perturbation, which were more evident when given in combination with PDT, but in contrast to work of other authors no synergistic activity was found. Apoptosis occurred via intrinsic pathways in treated cells, although it did not represent the predominant mode of cell death.     

Design of novel artemisinin‐like derivatives with cytotoxic and anti‐angiogenic properties

Artemisinins are plant products with a wide range of medicinal applications. Most prominently, artesunate is a well tolerated and effective drug for treating malaria, but is also active against several protozoal and schistosomal infections, and additionally exhibits anti‐angiogenic, anti‐tumorigenic and anti‐viral properties. The array of activities of the artemisinins, and the recent emergence of malaria resistance to artesunate, prompted us to synthesize and evaluate several novel artemisinin‐like derivatives. Sixteen distinct derivatives were therefore synthesized and the in vitro cytotoxic effects of each were tested with different cell lines. The in vivo anti‐angiogenic properties were evaluated using a zebrafish embryo model. We herein report the identification of several novel artemisinin‐like compounds that are easily synthesized, stable at room temperature, may overcome drug‐resistance pathways and are more active in vitro and in vivo than the commonly used artesunate. These promising findings raise the hopes of identifying safer and more effective strategies to treat a range of infections and cancer.     

CXCR2 Inhibition Combined with Sorafenib Improved Antitumor and Antiangiogenic Response in Preclinical Models of Ovarian Cancer

Antiangiogenic therapy is important for the treatment of gynecological cancer. However, the therapeutic benefit derived from these treatments is transient, predominantly due to the selective activation of compensatory proangiogenic pathways that lead to rapid development of resistance. We aimed to identify and target potential alternative signaling to anti-vascular endothelial growth factor (VEGF) therapy, with a view toward developing a combination of antiangiogenic agents to provide extended therapeutic benefits. We developed a preclinical in vivo phenotypic resistance model of ovarian cancer resistant to antiangiogenic therapy. We measured dynamic changes in secreted chemokines and angiogenic signaling in tumors and plasma in response to anti-VEGF treatment, as tumors advanced from the initial responsive phase to progressive disease. In tumors that progressed following sorafenib treatment, gene and protein expression levels of proangiogenic CXC chemokines and their receptors were significantly elevated, compared with responsive tumors. The chemokine (C-X-C motif) ligand 8 (CXCL8), also known as interleukin-8 (IL-8) increase was time-dependent and coincided with the dynamics of tumor progression. We used SB225002, a pharmacological inhibitor of chemokine (C-X-C motif) receptor 2 (CXCR2), to disrupt the CXC chemokine-mediated functions of ovarian cancer cells in in vitro assays of cell growth inhibition, spheroid formation, and cell migration. The combination of CXCR2 inhibitor with sorafenib led to a synergistic inhibition of cell growth in vitro, and further stabilized tumor progression following sorafenib in vivo. Our results suggest that CXCR2-mediated chemokines may represent an important compensatory pathway that promotes resistance to antiangiogenic therapy in ovarian cancer. Thus, simultaneous blockage of this proangiogenic cytokine pathway using CXCR2 inhibitors and the VEGF receptor (VEGFR) pathway could improve the outcomes of antiangiogenic therapy.     

3,4‐dimethoxystilbene,a resveratrol derivative with anti‐angiogenic effect,induces both macroautophagy and apoptosis in endothelial cells

Angiogenesis plays an important role in many pathological processes. Identification of novel anti‐angiogenic agents will provide new insights into the mechanisms for angiogenesis as well as potential lead compounds for developing new drugs. In the present study, a series of resveratrol methylated derivatives have been synthesized and screened. We found trans‐3,4‐dimethoxystilbene (3,4‐DMS) with the fullest potential to develop as an anti‐angiogenic agent. In vitro and in vivo analyses suggested that 3,4‐DMS could effectively inhibit endothelial cell proliferation, migration, tube formation, and endogenous neovascularization. Our results showed that 3,4‐DMS exerted its anti‐angiogenic effect likely through induction of endothelial cell apoptosis via a pathway involving p53, Bax, cytochrome c, and caspase proteases. Moreover, 3,4‐DMS also induced macroautophagy in endothelial cells through activation of AMPK and the downstream inhibition of mTOR signaling pathway. Further studies indicated that intracellular calcium ([Ca²⁺]_i) might bridge the 3,4‐DMS‐induced apoptosis and macroautophagy through modulating reactive oxygen species (ROS) levels in endothelial cells. Combination of 3,4‐DMS with inhibitor of autophagy, such as 3‐methyladenine (3‐MA) and autophagy‐related gene (ATG) 5 small interfering RNA (siRNA), potentiated the pro‐apoptotic and anti‐angiogenic effects of 3,4‐DMS. Our study provides a novel angiogenic inhibitor and a useful tool in exploring the molecular mechanisms for the crosstalk between apoptosis and macroautophagy in endothelial cells. 3,4‐DMS could be served as a potential lead compound for developing a class of new drugs targeting angiogenesis‐related diseases. J. Cell. Biochem. 114: 697–707, 2013. © 2012 Wiley Periodicals, Inc.     

Diallyl trisulfide inhibits migration,invasion and angiogenesis of human colon cancer HT‐29 cells and umbilical vein endothelial cells,and suppresses murine xenograft tumour growth

Angiogenesis inhibitors are beneficial for the prevention and treatment of angiogenesis‐dependent diseases including cancer. We examined the cytotoxic, anti‐metastatic, anti‐cancer and anti‐angiogenic effects of diallyl trisulfide (DATS). In HT29 cells, DATS inhibited migration and invasion through the inhibition of focal adhesion kinase (FAK), extracellular signal‐regulated kinase, c‐Jun N‐terminal kinase and p38 which was associated with inhibition of matrix metalloproteinases‐2, ‐7 and ‐9 and VEGF. In human umbilical vein endothelial cells (HUVEC), DATS inhibited the migration and angiogenesis through FAK, Src and Ras. DATS also inhibited the secretion of VEGF. The capillary‐like tube structure formation and migration by HUVEC was inhibited by DATS. The chicken egg chorioallantoic membrane (CAM) assay indicated that DATS treatment inhibited ex‐vivo angiogenesis. We investigated the anti‐tumour effects of DATS against human colon cancer xenografts in BALB/c^nu/nu mice and its anti‐angiogenic activity in vivo. In this in‐vivo study, DATS also inhibited the tumour growth, tumour weight and angiogenesis (decreased the levels of haemoglobin) in HT29 cells. In conclusion, the present results suggest that the inhibition of angiogenesis may be an important mechanism in colon cancer chemotherapy by DATS.     

Endogenous and induced angiogenic characteristics of human chorion‐derived stem cells

Cell‐based therapy using stem cells has emerged as one of the pro‐angiogenic methods to enhance blood vessel growth and sprouting in ischaemic conditions. This study investigated the endogenous and induced angiogenic characteristics of hCDSC (human chorion‐derived stem cell) using QPCR (quantitative PCR) method, immunocytochemistry and fibrin‐matrigel migration assay. The results showed that cultured hCDSC endogenously expressed angiogenic–endogenic‐associated genes (VEGF, bFGF, PGF, HGF, Ang‐1, PECAM‐1, eNOS, Ve‐cad, CD34, VEGFR‐2 and vWF), with significant increase in mRNA levels of PGF, HGF, Ang‐1, eNOS, VEGFR‐2 and vWF following induction by bFGF (basic fibroblast growth factor) and VEGF (vascular endothelial growth factor). These enhanced angiogenic properties suggest that induced hCDSC provides a stronger angiogenic effect for the treatment of ischaemia. After angiogenic induction, hCDSC showed no reduction in the expression of the stemness genes, but had significantly higher levels of mRNA of Oct‐4, Nanog (3), FZD9, ABCG‐2 and BST‐1. The induced cells were positive for PECAM‐1 (platelet/endothelial cell adhesion molecule 1) and vWF (von Willebrand factor) with immunocytochemistry staining. hCDSC also showed endothelial migration behaviour when cultured in fibrin‐matrigel construct and were capable of forming vessels in vivo after implanting into nude mice. These data suggest that hCDSC could be the cells of choice in the cell‐based therapy for pro‐angiogenic purpose.     

Pro‐inflammatory angiogenesis is mediated by p38 MAP kinase

Chronic inflammation is tightly linked to diseases associated with endothelial dysfunction including aberrant angiogenesis. To better understand the endothelial role in pro‐inflammatory angiogenesis, we analyzed signaling pathways in continuously activated endothelial cells, which were either chronically exposed to soluble TNF or the reactive oxygen species (ROS) generating H₂O₂, or express active transmembrane TNF. Testing in an in vitro capillary sprout formation assay, continuous endothelial activation increased angiogenesis dependent on activation of p38 MAP kinase, NADPH oxidase, and matrix metalloproteinases (MMP). p38 MAP kinase‐ and MMP‐9‐dependent angiogenesis in our assay system may be part of a positive feed forward autocrine loop because continuously activated endothelial cells displayed up‐regulated ROS production and subsequent endothelial TNF expression. The pro‐angiogenic role of the p38 MAP kinase in continuously activated endothelial cells was in stark contrast to the anti‐angiogenic activity of the p38 MAP kinase in unstimulated control endothelial cells. In vivo, using an experimental prostate tumor, pharmacological inhibition of p38 MAP kinase demonstrated a significant reduction in tumor growth and in vessel density, suggesting a pro‐angiogenic role of the p38 MAP kinase in pathological angiogenesis in vivo. In conclusion, our results suggest that continuous activation of endothelial cells can cause a switch of the p38 MAP kinase from anti‐angiogenic to pro‐angiogenic activities in conditions which link oxidative stress and autocrine TNF production. J. Cell. Physiol. 226: 800–808, 2011. © 2010 Wiley‐Liss, Inc.     

Effects on in vitro and in vivo angiogenesis induced by small peptides carrying adhesion sequences

It is well known that tumor growth is strictly dependent on neo‐vessel formation inside the tumor mass and that cell adhesion is required to allow EC proliferation and migration inside the tumor. In this work, we have evaluated the in vitro and in vivo effects on angiogenesis of some peptides, originally designed to promote cell adhesion on biomaterials, containing RGD motif mediating cell adhesion via integrin receptors [RGD, GRGDSPK, and (GRGDSP)₄K] or the heparin‐binding sequence of human vitronectin that interacts with HSPGs [HVP(351–359)]. Cell adhesion, proliferation, migration, and capillary‐like tube formation in Matrigel were determined on HUVECs, whereas the effects on in vivo angiogenesis were evaluated using the CAM assay. (GRGDSP)₄K linear sequence inhibited cell adhesion, decreased cell proliferation, migration and morphogenesis in Matrigel, and induced anti‐angiogenic responses on CAM at higher degree than that determined after incubation with RGD or GRGDSPK. Moreover, it counteracted both in vitro and in vivo the pro‐angiogenic effects induced by the Fibroblast growth factor (FGF‐2). On the other hand, HVP was not able to affect cell adhesion and appeared less effective than (GRGDSP)₄K. Our data indicate that the activity of RGD‐containing peptides is related to their adhesive properties, and their effects are modulated by the number of cell adhesion motifs and the aminoacidic residues next to these sequences. The anti‐angiogenic properties of (GRGDSP)₄K seem to depend on its interaction with integrins, whereas the effects of HVP may be partially due to an impairment of HSPGs/FGF‐2. Copyright © 2010 European Peptide Society and John Wiley & Sons, Ltd.     

Downregulation of developmentally regulated endothelial cell locus-1 inhibits the growth of colon cancer

Developmentally regulated endothelial cell locus-1 (Del1) is an embryonic angiogenic factor expressed in early embryonic endothelial cells, but recently has been found to be expressed in some forms of cancers including colon and breast cancers, and melanoma, and human cancer cell lines. Overexpression of Del1 accelerates tumor growth by enhancing vascular formation, implying Del1 may be a potential target for anti-angiogenic cancer therapy. The study aims to investigate whether downregulation of Del1 could inhibit the growth of tumors established in nude Balb/c mice by subcutaneous implantation of human LS-174T colon cancer cells. The shRNA expression vectors targeting human Del1, and vascular endothelial growth factor (VEGF) were constructed. Gene transfection of Del1-shRNA downregulated expression of Del1 in LS-174T cells in vivo and in vitro, but did not alter the proliferative or survival properties of cells in vitro. Gene transfection of VEGF-shRNA downregulated expression of both VEGF and Del1 in LS-174T cells in vivo and in vitro. Both Del1-shRNA and VEGF-shRNA gene therapies exhibited anti-tumor activities and they also showed a synergistic effect in suppressing growth of colon tumors by anti-angiogenesis and anti-proliferation. Although further investigation to clarify the mechanisms explaining the role of Del1 in tumor growth, and the interaction between VEGF and Del1, is required, the results indicate that downregulation of Del1 presents a potent therapeutic strategy to combat colon cancer.     

Polyethylenimine/small interfering RNA‐mediated knockdown of vascular endothelial growth factor in vivo exerts anti‐tumor effects synergistically with Bevacizumab

Background

RNA interference is a powerful method for the knockdown of pathologically relevant genes. The in vivo delivery of siRNAs, preferably through systemic, nonviral administration, poses the major challenge in the therapeutic application of RNAi. Small interfering RNA (siRNA) complexation with polyethylenimines (PEI) may represent a promising strategy for siRNA‐based therapies and, recently, the novel branched PEI F25‐LMW has been introduced in vitro. Vascular endothelial growth factor (VEGF) is frequently overexpressed in tumors and promotes tumor growth, angiogenesis and metastasis and thus represents an attractive target gene in tumor therapy.

Methods

In subcutaneous tumor xenograft mouse models, we established the therapeutic efficacy and safety of PEI F25‐LMW/siRNA‐mediated knockdown of VEGF. In biodistribution and siRNA quantification studies, we optimized administration strategies and, employing chemically modified siRNAs, compared the anti‐tumorigenic efficacies of: (i) PEI/siRNA‐mediated VEGF targeting; (ii) treatment with the monoclonal anti‐VEGF antibody Bevacizumab (Avastin®); and (iii) a combination of both.

Results

Efficient siRNA delivery is observed upon systemic administration, with the biodistribution being dependent on the mode of injection. Toxicity studies reveal no hepatotoxicity, proinflammatory cytokine induction or other side‐effects of PEI F25‐LMW/siRNA complexes or polyethylenimine, and tumor analyses show efficient VEGF knockdown upon siRNA delivery, leading to reduced tumor cell proliferation and angiogenesis. The determination of anti‐tumor effects reveals that, in pancreas carcinoma xenografts, single treatment with PEI/siRNA complexes or Bevacizumab is already highly efficacious, whereas, in prostate carcinoma, synergistic effects of both treatments are observed.

Conclusions

PEI F25‐LMW/siRNA complexes, which can be stored frozen as opposed to many other carriers, represent an efficient, safe and promising avenue in anti‐tumor therapy, and PEI/siRNA‐mediated, therapeutic VEGF knockdown exerts anti‐tumor effects. Copyright © 2010 John Wiley & Sons, Ltd.     

Anti‐angiogenic effects of the blue‐green alga Arthrospira platensis on pancreatic cancer

Arthrospira platensis, a blue‐green alga, is a popular nutraceutical substance having potent antioxidant properties with potential anti‐carcinogenic activities. The aim of our study was to assess the possible anti‐angiogenic effects of A platensis in an experimental model of pancreatic cancer. The effects of an A platensis extract were investigated on human pancreatic cancer cells (PA‐TU‐8902) and immortalized endothelial‐like cells (Ea.hy926). PA‐TU‐8902 pancreatic tumours xenografted to athymic mice were also examined. In vitro migration and invasiveness assays were performed on the tested cells. Multiple angiogenic factors and signalling pathways were analysed in the epithelial, endothelial and cancer cells, and tumour tissue. The A platensis extract exerted inhibitory effects on both migration and invasion of pancreatic cancer as well as endothelial‐like cells. Tumours of mice treated with A platensis exhibited much lesser degrees of vascularization as measured by CD31 immunostaining (P = .004). Surprisingly, the VEGF‐A mRNA and protein expressions were up‐regulated in pancreatic cancer cells. A platensis inhibited ERK activation upstream of Raf and suppressed the expression of ERK‐regulated proteins. Treatment of pancreatic cancer with A platensis was associated with suppressive effects on migration and invasiveness with various anti‐angiogenic features, which might account for the anticancer effects of this blue‐green alga.     

Trichostatin A,a histone deacetylase inhibitor suppresses NADPH Oxidase 4‐Derived Redox Signalling and Angiogenesis

Histone deacetylase (HDAC) inhibitors are known to suppress abnormal development of blood vessels. Angiogenic activity in endothelial cells depends upon NADPH oxidase 4 (Nox4)‐dependent redox signalling. We set out to study whether the HDAC inhibitor trichostatin A (TSA) affects Nox4 expression and angiogenesis. Nox4 expression was measured by real time PCR and Western blot analysis in endothelial cells. Hydrogen peroxide (H₂O₂) was measured by amplex^® red assay in endothelial cells. Nox4 was knocked down by Nox4 shRNA. In vitro angiogenic activities such migration and tubulogenesis were assessed using wound healing and Matrigel assays, respectively. In vivo angiogenic activity was assessed using subcutaneous sponge assay in C57Bl/6 and Nox4‐deficient mice. Trichostatin A reduced Nox4 expression in a time‐ and concentration‐dependent manner. Both TSA and Nox4 silencing decreased Nox4 protein and H₂O₂. Mechanistically, TSA reduced expression of Nox4 via ubiquitination of p300‐ histone acetyltransferase (p300‐HAT). Thus, blocking of the ubiquitination pathway using an inhibitor of ubiquitin‐activating enzyme E1 (PYR‐41) prevented TSA inhibition of Nox4 expression. Trichostatin A also reduced migration and tube formation, and these effects were not observed in Nox4‐deficient endothelial cells. Finally, transforming growth factor beta1 (TGFβ1) enhanced angiogenesis in sponge model in C57BL/6 mice. This response to TGFβ1 was substantially reduced in Nox4‐deficient mice. Similarly intraperitoneal infusion of TSA (1 mg/kg) also suppressed TGFβ1‐induced angiogenesis in C57BL/6 mice. Trichostatin A reduces Nox4 expression and angiogenesis via inhibition of the p300‐HAT‐dependent pathway. This mechanism might be exploited to prevent aberrant angiogenesis in diabetic retinopathy, complicated vascular tumours and malformations.     

Nexavar/Stivarga and Viagra Interact to Kill Tumor Cells

We determined whether the multi‐kinase inhibitor sorafenib or its derivative regorafenib interacted with phosphodiesterase 5 (PDE5) inhibitors such as Viagra (sildenafil) to kill tumor cells. PDE5 and PDGFRα/β were over‐expressed in liver tumors compared to normal liver tissue. In multiple cell types in vitro sorafenib/regorafenib and PDE5 inhibitors interacted in a greater than additive fashion to cause tumor cell death, regardless of whether cells were grown in 10 or 100% human serum. Knock down of PDE5 or of PDGFRα/β recapitulated the effects of the individual drugs. The drug combination increased ROS/RNS levels that were causal in cell killing. Inhibition of CD95/FADD/caspase 8 signaling suppressed drug combination toxicity. Knock down of ULK‐1, Beclin1, or ATG5 suppressed drug combination lethality. The drug combination inactivated ERK, AKT, p70 S6K, and mTOR and activated JNK. The drug combination also reduced mTOR protein expression. Activation of ERK or AKT was modestly protective whereas re‐expression of an activated mTOR protein or inhibition of JNK signaling almost abolished drug combination toxicity. Sildenafil and sorafenib/regorafenib interacted in vivo to suppress xenograft tumor growth using liver and colon cancer cells. From multiplex assays on tumor tissue and plasma, we discovered that increased FGF levels and ERBB1 and AKT phosphorylation were biomarkers that were directly associated with lower levels of cell killing by ‘rafenib + sildenafil. Our data are now being translated into the clinic for further determination as to whether this drug combination is a useful anti‐tumor therapy for solid tumor patients. J. Cell. Physiol. 230: 2281–2298, 2015. © 2015 The Authors. Journal of Cellular Physiology Published by Wiley Periodicals, Inc.     

Reprogramming of myeloid angiogenic cells by Bartonella henselae leads to microenvironmental regulation of pathological angiogenesis

The contribution of myeloid cells to tumour microenvironments is a decisive factor in cancer progression. Tumour‐associated macrophages (TAMs) mediate tumour invasion and angiogenesis through matrix remodelling, immune modulation and release of pro‐angiogenic cytokines. Nothing is known about how pathogenic bacteria affect myeloid cells in these processes. Here we show that Bartonella henselae, a bacterial pathogen causing vasculoproliferative diseases (bacillary angiomatosis), reprogrammes human myeloid angiogenic cells (MACs), a pro‐angiogenic subset of circulating progenitor cells, towards a TAM‐like phenotype with increased pro‐angiogenic capacity. B. henselae infection resulted in inhibition of cell death, activation of angiogenic cellular programmes and induction of M2 macrophage polarization. MACs infected with B. henselae incorporated into endothelial sprouts and increased angiogenic growth. Infected MACs developed a vascular mimicry phenotype in vitro, and expression of B. henselae adhesin A was essential in inducing these angiogenic effects. Secretome analysis revealed that increased pro‐angiogenic activities were associated with the creation of a tumour‐like microenvironment dominated by angiogenic inflammatory cytokines and matrix remodelling compounds. Our results demonstrate that manipulation of myeloid cells by pathogenic bacteria can contribute to microenvironmental regulation of pathological tissue growth and suggest parallels underlying both bacterial infections and cancer.     

Phosphatidylinositol 4‐kinase III beta is the target of oxoglaucine and pachypodol (Ro 09‐0179) for their anti‐poliovirus activities,and is located at upstream of the target step of brefeldin A

In recent years, phosphatidylinositol 4‐kinase III beta (PI4KB) has emerged as a conserved target of anti‐picornavirus compounds. In the present study, PI4KB was identified as the direct target of the plant‐derived anti‐picornavirus compounds, oxoglaucine and pachypodol (also known as Ro 09‐0179). PI4KB was also identified as the target via which pachypodol interferes with brefeldin A (BFA)‐induced Golgi disassembly in non‐infected cells. Oxysterol‐binding protein (OSBP) inhibitor also has interfering activity against BFA. It seems that this interference is not essential for the anti‐poliovirus (PV) activities of BFA and PI4KB/OSBP inhibitors. BFA inhibited early to late phase PV replication (0 to 6 hr postinfection) as well as PI4KB inhibitor, but with some delay compared to guanidine hydrochloride treatment. In contrast with PI4KB/OSBP inhibitors, BFA inhibited viral nascent RNA synthesis, suggesting that BFA targets some step of viral RNA synthesis located downstream of the PI4KB/OSBP pathway in PV replication. Our results suggest that PI4KB is a major target of anti‐picornavirus compounds identified in vitro for their anti‐picornavirus activities and for some uncharacterized biological phenomena caused by these compounds, and that BFA and PI4KB/OSBP inhibitors synergistically repress PV replication by targeting distinct steps in viral RNA replication.