Rania Harfouche

Roles of reactive oxygen species in angiopoietin-1/tie-2 receptor signaling

In this study we identified the involvement of reactive oxygen species (ROS) in signaling and biological effects of the angiopoietin‐1 (Ang‐1)/tie‐2 receptor pathway. Exposure of human umbilical vein endothelial cells to Ang‐1 (50 ng/ml) induced rapid and transient production of ROS, particularly superoxide anions. ROS production was attenuated by preincubation with a peptide (gp91ds‐tat) that inhibits the association of the gp91phox subunit with the p47phox subunit of NADPH oxidase and by the expression of a dominant‐negative form of Rac‐1 (Rac1N17). These results suggest that ROS production in response to Ang‐1 exposure originates mainly from a Rac‐1‐dependent NADPH oxidase. Overexpression of antioxidants (superoxide dismutase and catalase) and Rac1N17, as well as preincubation with selective inhibitors of NADPH oxidase augmented basal p38 phosphorylation, inhibited Ang‐1‐induced PAK‐1 phosphorylation and potentiated Ang‐1‐induced Erk1/2 phosphorylation but had no influence on AKT and SAPK/JNK phosphorylation by Ang‐1. Exposure to Ang‐1 (100 ng/ml) for 5 h induced a threefold increase in endothelial cell migration, a response that was strongly inhibited by overexpression of antioxidants, Rac1N17, and selective NADPH oxidase inhibitors. We conclude that activation of tie‐2 receptors by Ang‐1 triggers the production of ROS through activation of NADPH oxidase and that ROS generation by Ang‐1 promotes endothelial cell migration while negatively regulating Erk1/2 phosphorylation.

Nanoparticle-mediated targeting of MAPK signaling predisposes tumor to chemotherapy

The MAPK signal transduction cascade is dysregulated in a majority of human tumors. Here we report that a nanoparticle-mediated targeting of this pathway can optimize cancer chemotherapy. We engineered nanoparticles from a unique hexadentate-polyD,L-lactic acid-co-glycolic acid polymer chemically conjugated to PD98059, a selective MAPK inhibitor. The nanoparticles are taken up by cancer cells through endocytosis and demonstrate sustained release of the active agent, resulting in the inhibition of phosphorylation of downstream extracellular signal regulated kinase. We demonstrate that nanoparticle-mediated targeting of MAPK inhibits the proliferation of melanoma and lung carcinoma cells and induces apoptosis in vitro. Administration of the PD98059-nanoparticles in melanoma-bearing mice inhibits tumor growth and enhances the antitumor efficacy of cisplatin chemotherapy. Our study shows the nanoparticle-mediated delivery of signal transduction inhibitors can emerge as a unique paradigm in cancer chemotherapy.

Angiopoietin-1 activates both anti- and proapoptotic mitogen-activated protein kinases

In this study, we identified whether mitogen‐activated protein kinases (MAPKs) mediate the effects of angiopoietin‐1 (Ang‐1) on endothelial cell apoptosis. Exposure of human umbilical vein endothelial cells to Ang‐1 (300 ng/ml) evoked within 15–30 min a 15‐fold and a 5‐fold increase in phosphorylation of ERK1/2 and p38 MAPKs, respectively. Inhibitors of the PI‐3 kinase pathway attenuated Ang‐1‐induced ERK1/2 phosphorylation at a level up‐stream from Raf and MEK1/2, but these inhibitors augmented Ang‐1‐induced p38 phosphorylation. When serum and growth supplements were withdrawn, the percentage of endothelial apoptosis tripled over 24 h compared with control cells. The presence of Ang‐1 (300 ng/ml) significantly attenuated endothelial cell apoptosis and inhibited caspase‐9, ‐7, and ‐3 activation. These antiapoptotic effects were augmented when a p38 inhibitor was combined with Ang‐1, whereas inhibition of ERK1/2 eliminated the antiapoptotic properties of Ang‐1. We conclude that both anti‐ (ERK1/2) and pro‐ (p38) apoptotic members of MAPKs are simultaneously activated by Ang‐1 in endothelial cells and that activation of ERK1/2 by Ang‐1 is mediated through the PI‐3 kinase pathway. The strong antiapoptotic effects of the ERK and the PI‐3 kinase pathways mask the proapoptotic function of p38 MAPKs resulting in net attenuation of apoptosis by Ang‐1.

Shape effect of carbon nanovectors on angiogenesis.

Physically diverse carbon nanostructures are increasingly being studied for potential applications in cancer chemotherapy. However, limited knowledge exists on the effect of their shape in tuning the biological outcomes when used as nanovectors for drug delivery. In this study, we evaluated the effect of doxorubicin-conjugated single walled carbon nanotubes (CNT-Dox) and doxorubicin-conjugated spherical polyhydroxylated fullerenes or fullerenols (Ful-Dox) on angiogenesis. We report that CNTs exert a pro-angiogenic effect in vitro and in vivo. In contrast, the fullerenols or doxorubicin-conjugated fullerenols exerted a dramatically opposite antiangiogenic activity in zebrafish and murine tumor angiogenesis models. Dissecting the angiogenic phenotype into discrete cellular steps revealed that fullerenols inhibited endothelial cell proliferation, while CNTs attenuated the cytotoxic effect of doxorubicin on the endothelial cells. Interestingly, CNT promoted endothelial tubulogenesis, a late step during angiogenesis. Further, mechanistic studies revealed that CNTs, but not fullerenols, induced integrin clustering and activated focal adhesion kinase and downstream phosphoinositide-3-kinase (PI3K) signaling in endothelial cells, which can explain the distinct angiogenic outcomes. The results of the study highlight the function of physical parameters of nanoparticles in determining their activity in biological settings.

Nanoparticle-mediated targeting of phosphatidylinositol-3-kinase signaling inhibits angiogenesis

ObjectiveDysregulation of the phosphatidylinositol-3-kinase (PI3K) signaling pathway is a hallmark of human cancer, occurring in a majority of tumors. Activation of this pathway is critical for transformation and also for the angiogenic switch, which is a key step for tumor progression. The objective of this study was to engineer a PI3K inhibitor-loaded biodegradable nanoparticle and to evaluate its efficacy.Methods and resultsHere we report that a nanoparticle-enabled targeting of the PI3K pathway results in inhibition of downstream Akt phosphorylation, leading to inhibition of proliferation and induction of apoptosis of B16/F10 melanoma. It, however, failed to exert a similar activity on MDA-MB-231 breast cancer cells, resulting from reduced internalization and processing of nanoparticles in this cell line. Excitingly, the nanoparticle-enabled targeting of the PI3K pathway resulted in inhibition of endothelial cell proliferation and tubulogenesis, two key steps in tumor angiogenesis. Furthermore, it inhibited both B16/F10- and MDA-MB-231-induced angiogenesis in a zebrafish tumor xenotransplant model.ConclusionOur study, for the first time, shows that targeting of the PI3K pathway using nanoparticles can offer an attractive strategy for inhibiting tumor angiogenesis.

Coupling growth-factor engineering with nanotechnology for therapeutic angiogenesis

Therapeutic angiogenesis is an emerging paradigm for the management of ischemic pathologies. Proangiogenic Therapy is limited, however, by the current inability to deliver angiogenic factors in a sustained manner at the site of pathology. In this study, we investigated a unique nonglycosylated active fragment of hepatocyte growth factor/scatter factor, 1K1, which acts as a potent angiogenic agent in vitro and in a zebrafish embryo and a murine matrigel implant model. Furthermore, we demonstrate that nanoformulating 1K1 for sustained release temporally alters downstream signaling through the mitogen activated protein kinase pathway, and amplifies the angiogenic outcome. Merging protein engineering and nanotechnology offers exciting possibilities for the treatment of ischemic disease, and furthermore allows the selective targeting of downstream signaling pathways, which translates into discrete phenotypes.

Mechanistic Studies of Gemcitabine-Loaded Nanoplatforms in Resistant Pancreatic Cancer Cells

BackgroundPancreatic cancer remains the deadliest of all cancers, with a mortality rate of 91%. Gemcitabine is considered the gold chemotherapeutic standard, but only marginally improves life-span due to its chemical instability and low cell penetrance. A new paradigm to improve Gemcitabine’s therapeutic index is to administer it in nanoparticles, which favour its delivery to cells when under 500 nm in diameter. Although promising, this approach still suffers from major limitations, as the choice of nanovector used as well as its effects on Gemcitabine intracellular trafficking inside pancreatic cancer cells remain unknown. A proper elucidation of these mechanisms would allow for the elaboration of better strategies to engineer more potent Gemcitabine nanotherapeutics against pancreatic cancer.MethodsGemcitabine was encapsulated in two types of commonly used nanovectors, namely poly(lactic-co-glycolic acid) (PLGA) and cholesterol-based liposomes, and their physico-chemical parameters assessed in vitro. Their mechanisms of action in human pancreatic cells were compared with those of the free drug, and with each others, using cytotoxity, apoptosis and ultrastructural analyses.ResultsPhysico-chemical analyses of both drugs showed high loading efficiencies and sizes of less than 200 nm, as assessed by dynamic light scattering (DLS) and transmission electron microscopy (TEM), with a drug release profile of at least one week. These profiles translated to significant cytotoxicity and apoptosis, as well as distinct intracellular trafficking mechanisms, which were most pronounced in the case of PLGem showing significant mitochondrial, cytosolic and endoplasmic reticulum stresses.ConclusionsOur study demonstrates how the choice of nanovector affects the mechanisms of drug action and is a crucial determinant of Gemcitabine intracellular trafficking and potency in pancreatic cancer settings.

PEGylated liposomal Gemcitabine: Insights into a potential breast cancer therapeutic

PurposeNanoencapsulation of chemotherapeutics is an established method to target breast tumors and has been shown to enhance the efficacy of therapy in various animal models. During the past two decades, the nucleoside analog Gemcitabine has been under investigation to treat both recalcitrant and localized breast cancer, often in combination with other chemotherapeutics. In this study, we investigated the chemotherapeutic efficacy of a novel Gemcitabine-encapsulated liposome previously formulated by our group, GemPo, on both sensitive (4T1) and recalcitrant (MDA-MB-231) breast cancer cell lines.MethodsGemcitabine free drug and liposomal Gemcitabine were compared both in vitro and in vivo using breast cancer models.ResultsWe demonstrated that GemPo differently hindered the growth, survival and migration of breast cancer cells, according to their drug sensitivities. Specifically, whereas GemPo was a more potent cytotoxic and apoptotic agent in sensitive breast cancer cells, it more potently inhibited cell migration in the resistant cell line. However, GemPo still acted as a more potent inhibitor of migration, in comparison with free Gemcitabine, irrespective of cell sensitivity. Administration of GemPo in a 4T1-bearing mouse model inhibited tumor growth while increasing mice survival, as compared with free Gemcitabine and a vehicle control. Interestingly, the inclusion of a mitotic inhibitor, Paclitaxel, synergized only with free Gemcitabine in this model, yet was as effective as GemPo alone. However, inclusion of Paclitaxel with GemPo significantly improved mouse survival.ConclusionsOur study is the first to demonstrate the pleiotropic effects of Gemcitabine and Gemcitabine-loaded nanoparticles in breast cancer, and opens the door for a novel treatment for breast cancer patients.

Proteomic and scanning electron microscopic analysis of submandibular sialoliths

ObjectivesSeveral theories have been proposed regarding the genesis of sialoliths, including the organic core theory, which suggests epithelial or bacterial etiology originating in the central core. Our aim was to use novel methodologies to analyze central areas (the core) of calculi from sialolithiasis patients.Materials and methodsThe structures of the halves of six submandibular salivary stones were analyzed by scanning electron microscopy (SEM). After structural analysis, from the other six halves, samples from the central parts of the core and peripheral parts of the core were digested with trypsin and analyzed by matrix-assisted laser desorption ionization–time of flight mass spectrometry. The peptide mass fingerprints were compared with the results of in silico digestion.ResultsSEM analysis of the sialoliths showed that organic structures (collagen/fibrous-like structures, bacterial fragments) were visible only outside of the core in the concentric layers of external areas, but not in the core area. The mass spectrometry (MS)/MS post-source decay experiments were completed from the four, most intense signals observed in the MS spectrum and human defensin was proven to be present in three of the examined samples, originated from the peripheral region of three cores.ConclusionsAlthough proteomic analysis demonstrated defensin protein in the peripheral region of the core in three sialoliths, SEM failed to prove organic structures in the core.Clinical relevanceNew investigation modalities still cannot prove organic structures in the core, henceforward challenging the organic core theory.

Glycomic Signature of Mouse Embryonic Stem Cells During Differentiation

Background: The glycome has emerged as a key regulator of cell fate, partly through its ability to potentiate the action of numerous signaling pathways. We recently demonstrated that a sulfated component of the glycome plays a critical role in promoting the differentiation of embryonic stem cell (ESC)-derived embryoid bodies by modulating downstream growth factors, such as the insulin-like growth factor (IGF) signaling axis. However, the exact components of the glycome which promote ESC differentiation versus stemness remain uncharacterized, due to the lack of a rapid, simple and easily quantifiable methodology. As a proof-of-concept in this study, we utilized a custom-made glycoarray in combination with bioinformatics and molecular biology tools in order to uncover novel glyco-signatures underlying ESC differention in an embryoid body model. A better elucidation of the glycomic transcriptomal signature underlying ESC differentiation would allow us to better manipulate these cells towards a desired lineage. Method: We used a custom-designed Affymetrix microarray, the Glycogene-chip, to screen the transcriptome of differentiating embryoid bodies versus that of undifferentiated ESC. In conjunction with gene ontology, pathway analyses, real-time PCR and immunoblotting, we validated the involvement of the IGF family, and furthermore, uncovered novel differentially regulated genes belonging to the glycoprotein (Angiopoietin-1 and Angiopoietin-like members), sulfotransferase, sulfatase and glycosyltransferase families. Conclusion: These results suggest that the Glycogene-chip, in conjunction with the embryoid body model, provides a fast and reliable tool to uncover novel glycomic signatures that are critical to maitain ESC stemness versus differentiation. In turn, this will allow us to understand the mechanisms governing ESC fate, bringing us one step closer towards finding a new paradigm for the regenerative medicine field.