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The palladium-catalyzed uncaging of alloc-protected amines in living cells has been extensively stud- ied mostly in the form of nanostructures or mesostructures. However, the scarcity of kinetic and mechanistic studies of discrete palladium complexes for deallylation reactions has hindered progress. Herein, we report the development of a series of discrete palladium complexes bearing acetanilide lig- ands, which exhibit a good balance between reactivity and stability in living cells. We investigated the catalytic activity and cytotoxicity of these complexes in SiHa cells and found that acetanilide[tri(2- furyl)phosphine]palladium(II) triflate showed promising results. Under physiological conditions, this pal- ladium complex exhibited a second-order reaction rate of 30 M −1 s −1 , and liquid chromatography-mass spectrometry (LC-MS) studies suggested the possibility of a tandem Heck/Tsuji-Trost mechanism. Our re- sults demonstrate the potential of using these discrete palladium complexes for bioorthogonal chemistry studies in living cells.

The pandemic's impacts on European electricity markets have been enormous, especially in countries with abundant near-zero marginal cost of production like France. This article provides an in-depth quantitative study of the impacts of the crisis on the French electricity sector. During the lockdown episode, France has experienced unparalleled demand reductions (-11.5%) and energy price falls (-40%) resulting in revenue losses of 1.2 billion € (-45%) for market participants. This paper argues that the observed market outcomes during the crisis are somehow indicative of outcomes in a future with abundant renewable power, where prices will fall in a more sustainable way.

The management of locally advanced rectal cancer has passed a long way of developments, where total mesorectal excision and preoperative radiotherapy are crucial to secure clinical outcome. These and other aspects of multidisciplinary strategies are in-depth summarized in the literature, while our mini-review pursues a different goal. From an ethical and medical standpoint, we witness a delayed implementation of novel therapies given the cost/time consuming process of organizing randomized trials that would bridge an already excellent local control in cT3-4 node-positive disease with long-term survival. This unfortunate separation of clinical research and medical care provides a strong motivation to repurpose known pharmaceuticals that suit for treatment intensification with a focus on distant control. In the framework of on-going phase II-III IG/IMRT-SIB trials, we came across an intriguing translational observation that the ratio of circulating (protumor) myeloid-derived suppressor cells to (antitumor) central memory CD8+ T cells is drastically increased, a possible mechanism of tumor immuno-escape and spread. This finding prompts that restoring the CD45RO memory T-cell pool could be a part of integrated adjuvant interventions. Therefore, the immunocorrective potentials of modified IL-2 and the anti-diabetic drug metformin are thoroughly discussed in the context of tumor immunobiology, mTOR pathways and revised Warburg effect.

Tumor acidosis promotes disease progression through a stimulation of fatty acid (FA) metabolism in cancer cells. Instead of blocking the use of FAs by acidic cancer cells, we examined whether excess uptake of specific FAs could lead to antitumor effects. We found that n-3 but also remarkably n-6 polyunsaturated FA (PUFA) selectively induced ferroptosis in cancer cells under ambient acidosis. Upon exceeding buffering capacity of triglyceride storage into lipid droplets, n-3 and n-6 PUFA peroxidation led to cytotoxic effects in proportion to the number of double bonds and even more so in the presence of diacylglycerol acyltransferase inhibitors (DGATi). Finally, an n-3 long-chain PUFA-rich diet significantly delayed mouse tumor growth when compared with a monounsaturated FA-rich diet, an effect further accentuated by administration of DGATi or ferroptosis inducers. These data point out dietary PUFA as a selective adjuvant antitumor modality that may efficiently complement pharmacological approaches.

Warburg's hypothesis that cancer cells take up a lot of glucose in the presence of ambient oxygen but convert pyruvate into lactate due to impaired mitochondrial function led to the misconception that cancer cells rely on glycolysis as their major source of energy. Most recent 13C-based metabolomic studies, including in cancer patients, indicate that cancer cells may also fully oxidize glucose. In addition to glucose-derived pyruvate, lactate, fatty acids and amino acids supply substrates to the TCA cycle to sustain mitochondrial metabolism. Here, we discuss how the metabolic flexibility afforded by these multiple mitochondrial inputs allows cancer cells to adapt according to the availability of the different fuels and the microenvironmental conditions such as hypoxia and acidosis. In particular, we focused on the role of the TCA cycle in interconnecting numerous metabolic routes in order to highlight metabolic vulnerabilities that represent attractive targets for a new generation of anticancer drugs.

The aim of this work is to develop the first validated HPLC-UV method quantification in blood serum for a new endoplasmic reticulum (ER)-specific benzophenazine photosensitizer (OR-141). A fast solid phase extraction (SPE) cleaning sample procedure was achieved on C18 encapped (ec) SPE cartridges and the separation was performed on a RP-18e column (5μM) using an isocratic elution with methanol. The method has been fully validated according to accuracy profiles based on total error and tolerance intervals. Calibration was performed in the matrix and trueness (

Bioenergetic preferences of cancer cells foster tumor acidosis that in turn leads to dramatic reduction in glycolysis and glucose-derived acetyl-coenzyme A (acetyl-CoA). Here, we show that the main source of this critical two-carbon intermediate becomes fatty acid (FA) oxidation in acidic pH-adapted cancer cells. FA-derived acetyl-CoA not only fuels the tricarboxylic acid (TCA) cycle and supports tumor cell respiration under acidosis, but also contributes to non-enzymatic mitochondrial protein hyperacetylation, thereby restraining complex I activity and ROS production. Also, while oxidative metabolism of glutamine supports the canonical TCA cycle in acidic conditions, reductive carboxylation of glutamine-derived α-ketoglutarate sustains FA synthesis. Concomitance of FA oxidation and synthesis is enabled upon sirtuin-mediated histone deacetylation and consecutive downregulation of acetyl-CoA carboxylase ACC2 making mitochondrial fatty acyl-CoA degradation compatible with cytosolic lipogenesis. Perturbations of these regulatory processes lead to tumor growth inhibitory effects further identifying FA metabolism as a critical determinant of tumor cell proliferation under acidosis.

Crosstalk between cancer-associated fibroblasts (CAFs) and colorectal cancer cells promotes tumor growth and contributes to chemoresistance. In this study, we assessed the sensitivity of a primary CAF cell line, CT5.3hTERT, to standard-of-care and alternative cytotoxic treatments. Paclitaxel (PTX) and acriflavine (ACF) were identified as the most promising molecules to inhibit CAF development. To allow the translational use of both drugs, we developed lipid nanocapsule (LNC) formulations for PTX and ACF. Finally, we mixed CAFs and tumor cell lines in a cocultured spheroid, and the effect of both drugs was investigated by histological analyses. We demonstrated CAF inhibition by LNC-ACF and whole tumor inhibition by LNC-PTX. Altogether, we proposed a new strategy to reduce CAF populations in the colorectal microenvironment that should be tested in vivo.

Integrin-targeted nanoparticles are promising for the delivery of small interfering RNA (siRNA) to tumor cells or tumor endothelium in cancer therapy aiming at silencing genes essential for tumor growth. However, during the process of optimizing and realizing their full potential, it is pertinent to gain a basic mechanistic understanding of the bottlenecks existing for nanoparticle-mediated intracellular delivery. We designed αvβ3 integrin-targeted nanoparticles by coupling arginine-glycine-aspartate (RGD) or RGD peptidomimetic (RGDp) ligands to the surface of poly(ethylene glycol) (PEG) grafted chitosan-poly(ethylene imine) hybrid nanoparticles. The amount of intracellular siRNA delivered by αvβ3-targeted versus non-targeted nanoparticles was quantified in the human non-small cell lung carcinoma cell line H1299 expressing enhanced green fluorescent protein (EGFP) using a stem-loop reverse transcription quantitative polymerase chain reaction (RT-qPCR) approach. Data demonstrated that the internalization of αvβ3-targeted nanoparticles was highly dependent on the surface concentration of the ligand. Above a certain threshold concentration, the use of targeted nanoparticles provided a two-fold increase in the number of siRNA copies/cell, subsequently resulting in as much as 90% silencing of EGFP at well-tolerated carrier concentrations. In contrast, non-targeted nanoparticles mediated low levels of gene silencing, despite relatively high intracellular siRNA concentrations, indicating that these nanoparticles might end up in late endosomes or lysosomes without releasing their cargo to the cell cytoplasm. Thus, the silencing efficiency of the chitosan-based nanoparticles is strongly dependent on the uptake and the intracellular trafficking in H1299 EGFP cells, which is critical information towards a more complete understanding of the delivery mechanism that can facilitate the future design of efficient siRNA delivery systems.

The purpose of this study was to develop vitamin E-based micelles loaded with Doxorubicin (DOX) (DOX-TOS-TPGS), taking advantages of the anti-cancer activity of vitamin E derivatives: Tocopherol succinate (TOS) and D- α-tocopherol polyethylene2000 succinate (TPGS). Therefore, we developed micelles consisting in a mixture of TOS (as solubilizer) and TPGS2000 (as stabilizer) (1:1). DOX-TOS-TPGS micelles exhibited a size of 78nm and a ζ potential of -7mV. High drug loading (40% w/w) was achieved. The critical micellar concentration was determined at 14μg/ml. In vitro, after 24h, DOX-TOS- TPGS micelles exhibited higher cytotoxicity than free-DOX (IC50 on MCF-7 cells, at 24h, 58.15 vs 4.89μg/ml). In vivo anti-tumor efficacy, performed on two tumor models (CT26 and MCF-7), demonstrated a 100% long-term survival of mice (- bearing MCF-7 tumors) when treated with DOX-TOS-TPGS compared to DOX-free (mean survival time=32 days). Interestingly, the survival time of mice treated with unloaded TOS-TPGS micelles was similar to DOX-free, suggesting the anti-cancer activity of vitamin E derivatives. Based on these results, it can be concluded that the formulations developed in this work may be considered as an effective DOX delivery system for cancer chemotherapy.

Stem cells from the apical papilla (SCAP) are a population of mesenchymal stem cells likely involved in regenerative endodontic procedures and have potential use as therapeutic agents in other tissues. In these situations, SCAP are exposed to hypoxic conditions either within a root canal devoid of an adequate blood supply or in a scaffold material immediately after implantation. However, the effect of hypoxia on SCAP proliferation and differentiation is largely unknown. Therefore, the objective of this study was to evaluate the effect of hypoxia on the fate of SCAP.SCAP were cultured under normoxia (21% O2) or hypoxia (1% O2) in basal or differentiation media. Cellular proliferation, gene expression, differentiation, and protein secretion were analyzed by live imaging, quantitative reverse-transcriptase polymerase chain reaction, cellular staining, and enzyme-linked immunosorbent assay, respectively.Hypoxia had no effect on SCAP proliferation, but it evoked the up-regulation of genes specific for osteogenic differentiation (runt-related transcription factor 2, alkaline phosphatase, and transforming growth factor-β1), neuronal differentiation ( 2'-3'-cyclic nucleotide 3' phosphodiesterase, SNAIL, neuronspecific enolase, glial cell-derived neurotrophic factor and neurotrophin 3), and angiogenesis (vascular endothelial growth factor A and B). Hypoxia also increased the sustained production of VEGFa by SCAP. Moreover, hypoxia augmented the neuronal differentiation of SCAP in the presence of differentiation exogenous factors as detected by the up-regulation of NSE, VEGFB, and GDNF and the expression of neuronal markers (PanF and NeuN).This study shows that hypoxia induces spontaneous differentiation of SCAP into osteogenic and neurogenic lineages while maintaining the release of the proangiogenic factor VEGFa. This highlights the potential of SCAP to promote pulp-dentin regeneration. Moreover, SCAP may represent potential therapeutic agents for neurodegenerative conditions because of their robust differentiation potential.

The thyroid is a highly vascularized endocrine gland, displaying a characteristic epithelial organization in closed spheres, called follicles. Here we investigate how endothelial cells are recruited into the developing thyroid and if they control glandular organization as well as thyrocytes and C-cells differentiation. We show that endothelial cells closely surround, and then invade the expanding thyroid epithelial cell mass to become closely associated with nascent polarized follicles. This close and sustained endothelial:epithelial interaction depends on epithelial production of the angiogenic factor, Vascular Endothelial Growth Factor-A (VEGF-A), as its thyroid-specific genetic inactivation reduced the endothelial cell pool of the thyroid by >90%. Vegfa KO also displayed decreased C-cells differentiation and impaired organization of the epithelial cell mass into follicles. We developed an ex vivo model of thyroid explants that faithfully mimicks bilobation of the thyroid anlagen, endothelial and C-cells invasion, folliculogenesis and differentiation. Treatment of thyroid explants at e12.5 with a VEGFR2 inhibitor ablated the endothelial pool and reproduced ex vivo folliculogenesis defects observed in conditional Vegfa KO. In the absence of any blood supply, rescue by embryonic endothelial progenitor cells restored folliculogenesis, accelerated lumen expansion and stimulated calcitonin expression by C-cells. In conclusion, our data demonstrate that, in developing mouse thyroid, epithelial production of VEGF-A is necessary for endothelial cells recruitment and expansion. In turn, endothelial cells control epithelial reorganization in follicles and C-cells differentiation.

Background: This phase II study assessed the safety and efficacy of everolimus, an oral mammalian target of rapamycin inhibitor in advanced transitional carcinoma cell (TCC) after failure of platinum-based therapy. Patients and methods: Thirty-seven patients with advanced TCC received everolimus 10 mg/day until progressive disease (PD) or unacceptable toxicity. The primary end point was the disease control rate (DCR), defined as either stable disease (SD), partial response (PR), or complete response at 8 weeks. Angiogenesis-related proteins were detected in plasma and changes during everolimus treatment were analyzed. PTEN expression and PIK3CA mutations were correlated to disease control. Results: Two confirmed PR and eight SD were observed, resulting in a DCR of 27% at 8 weeks. Everolimus was well tolerated. Compared with patients with noncontrolled disease, we observed in patients with controlled disease a significant higher baseline level of angiopoietin-1 and a significant early plasma decrease in angiopoietin-1, endoglin,and platelet-derived growth factor-AB. PTEN loss was observed only in patients with PD. Conclusions: Everolimus showed clinical activity in advanced TCC. The profile of the plasma angiogenesis-related proteins suggested a role of the everolimus antiangiogenic properties in disease control. PTEN loss might be associated with everolimus resistance.

The tripeptide Leu-Asp-Val (LDV) is known to bind α(4)β(1) integrin in leukemia cells. Here we have synthesized a LDV peptidomimetic equipped with a biotin-conjugated spacer-arm. Compound 9 acts as an inhibitor of the α(4)β(1) integrin in an adhesion assay using fluorescently labeled, α(4)β(1) integrin-expressing leukemia CCRF-CEM cells. Furthermore, when bound to neutravidin-coated plates, compound 9 could capture CCRF-CEM cells. Such biotin-conjugated LDV peptidomimetic may thus represent a novel tool for biotechnological applications using avidin interaction for leukapheresis or leukemia cell targeting.

An approach using physical vapor deposition technology to produce nanoparticles (NPs) containing radioactive atoms and the methodology to transfer them in pure water is investigated. NPs are synthesized by magnetron sputtering at high pressure and radioactive atoms are loaded on magnetron cathodes prior to sputtering. The technique was tested for gold cathode loaded with Co and Au. Linked to biological vector molecules, the nanoparticles can be used to enhance diagnostic sensitivity in medical imaging or to treat cancer.Sizes and morphologies of the NPs were analyzed by electron microscopy, UV-Visible spectroscopy and atomic absorption spectroscopy. Results show well dispersed NPs with sizes varying between 5 and 10. nm. Activities of these NPs were measured with a CAPINTEC well counter and a High Purity Germanium detector system. Centrifugation analyses also demonstrate that the choice of the activated metal which can be alloyed with NPs plays an important role in the synthesis. This was confirmed by the Au-Co phase diagram that shows that cobalt cannot be included efficiently in the gold NPs conversely to gold. © 2011 Elsevier B.V.

Introduction Tumours are not only composed of malignant cells. Indeed, blood vessels, composed of endothelial cells, are required for tumour growth. Interestingly, the induction of angiogenesis requires not only endothelial cells but also immune cells. Among them, tumor-associated macrophages (TAMs) make up to 50% of the tumour immune infiltrate. These TAMs are strong angiogenesis inducer and are in part responsible of the tumour inflammation. Two kinds of macrophages are detected in tumours: pro-inflammatory and anti-tumoral M1 macrophages and anti-inflammatory and pro-tumoral M2 macrophages. Hypoxia is another key feature of tumour microenvironment which induces angiogenesis and enhances tumour inflammation and metastasis. Two types of hypoxia occur in tumour: chronic hypoxia, which impacts cells too far from the blood vessels, and cycling hypoxia (CyH), which causes intermittent oxygenation of malignant and non-malignant cells. We have studied the impact of CyH on macrophage polarisation and activity. Secondly, the impact of CyH on the communication between macrophages and endothelial cells was investigated. Then, the impact of this dialogue on tumour growth and metastasis will be studied. Material and methods THP-1 monocytes are differentiated into M0 macrophages by PMA. M0 macrophages are then polarised into M1 macrophages with LPS and IFNg (24 hour incubation) or into M2 macrophages with IL-4 and IL-13 (48 hour incubation). After exposing these macrophages to normoxia, chronic hypoxia or CyH, the expression and secretion of pro-inflammatory factors were studied by RT-qPCR and ELISA, respectively. To investigate the communication between macrophages and endothelial cells under CyH, endothelial cells were incubated with CyH-exposed macrophage conditioned-medium and the expression of endothelial cell activation markers was analysed by RT-qPCR. Results and discussions CyH induced a pro-inflammatory phenotype in M0 macrophages and enhanced the pro-inflammatory phenotype of M1 macrophages. Indeed, an increased expression of TNFa, IL-6 and IL-1β was observed. These effects depended on NF-kB activation since IKK inhibition prevented these effects. The conditioned-media of M0, M1 and M2 macrophages exposed to CyH induced endothelial cell activation as observed by an increased IL-6, IL-8 and ICAM-1 expression. Conclusion CyH induced a pro-inflammatory phenotype in M0 and M1 macrophages via NF-kB activation. The M0, M1 and M2 macrophages exposed to cycling hypoxia induced endothelial cell activation by a secreted molecule that needs to be identified.

OBJECTIVE: To characterize the human ovarian xenograft revascularization process. DESIGN: Prospective experimental study. SETTING: Gynecology research unit in a university hospital. PATIENT(S): Ovarian biopsies were obtained from 12 women aged 22-35 years. INTERVENTION(S): Frozen-thawed human ovarian fragments were intraperitoneally grafted into nude mice. MAIN OUTCOME MEASURE(S): Graft perfusion was evaluated by Hoechst 33342 uptake. Murine and human vascularization was analyzed by CD31 and von Willebrand factor double immunostaining. RESULT(S): On day 3, some murine neovessels and perfused areas were located at the periphery of the fragments. Nonperfused native human vessels were present in the fragments. From day 5, perfused areas and murine endothelial areas progressively increased. Host angiogenesis initiated ovarian graft reperfusion: murine neovessels penetrated from the periphery and were colocalized with perfused areas. By day 10, the increase in perfusion and murine vascularization was significant. The center of the fragments was perfused and a significant increase was observed in human vasculature. CONCLUSION(S): Host and graft vessels contributed sequentially to graft revascularization: murine angiogenesis initiated reperfusion from day 5 and, by day 10, human angiogenesis was shown to participate in graft revascularization. Host and graft angiogenesis are potential targets to reduce the avascular period after grafting.

The oxygen consumption rate of tumor cells affects tumor oxygenation and response to therapies. Highly sensitive methods for determining cellular oxygen consumption are, therefore, needed to identify treatments that can modulate this parameter. We compared the performances of three different methods for measuring cellular oxygen consumption: electron paramagnetic resonance (EPR) oximetry, the Clark electrode, and the MitoXpress fluorescent assay. To compare the assays, we used K562 cells in the presence of rotenone and hydrocortisone, compounds that are known to inhibit the mitochondrial electron transport chain to different extents. The EPR method was the only one that could identify both rotenone and hydrocortisone as inhibitors of tumor cell oxygen consumption. The Clark electrode and the fluorescence assay demonstrated a significant decrease in cellular oxygen consumption after administration of the most potent inhibitor (rotenone) but failed to show any significant effect of hydrocortisone. EPR oximetry is, therefore, the most sensitive method for identifying inhibitors of oxygen consumption on cell assays, whereas the Clark electrode offers the unique opportunity to add external compounds during experiments and still shows great sensitivity in studying enzyme and chemical reactions that consume oxygen (non-cell assays). Finally, the MitoXpress fluorescent assay has the advantage of a high-sample throughput and low bulk requirements but at the cost of a lower sensitivity.

Paclitaxel (PTX)-loaded PEGylated PLGA based nanoparticles (NP) have been previously described as more effective in vitro and in vivo than Taxol(R). The aim of this study was to test the hypothesis that our PEGylated PLGA-based nanoparticles grafted with the RGD peptide or RGD-peptidomimetic (RGDp) would target the tumor endothelium and would further enhance the anti-tumor efficacy of PTX. The ligands were grafted on the PEG chain of PCL-b-PEG included in the nanoparticles. We observed in vitro that RGD-grafted nanoparticles were more associated to Human Umbilical Vein Endothelial cells (HUVEC) by binding to alpha(v)beta(3) integrin than non-targeted nanoparticles. Doxorubicin was also used to confirm the findings observed for PTX. In vivo, we demonstrated the targeting of RGD and RGDp-grafted nanoparticles to tumor vessels as well as the effective retardation of TLT tumor growth and prolonged survival times of mice treated by paclitaxel-loaded RGD-nanoparticles when compared to non-targeted nanoparticles. Hence, the targeting of anti-cancer drug to tumor endothelium by RGD-labeled NP is a promising approach.

Tumor cells fuel their metabolism with glucose and glutamine to meet the bioenergetic and biosynthetic demands of proliferation. Hypoxia and oncogenic mutations drive glycolysis, with the pyruvate to lactate conversion being promoted by increased expression of lactate dehydrogenase A and inactivation of pyruvate dehydrogenase. The NAD(+) pool is consecutively regenerated and supports the high glycolytic flux required to produce anabolic intermediates. Glutaminolysis provides metabolic intermediates such as alpha-ketoglutarate to feed and thereby maintain the tricarboxylic acid cycle as a biosynthetic hub. Glycolysis and glutaminolysis share the capacity to generate NADPH, from the pentose phosphate pathway and through the malate conversion into pyruvate, respectively. Both pathways ultimately lead to the secretion of lactate. More than a waste product, lactate was recently identified as a major energy fuel in tumors. Lactate produced by hypoxic tumor cells may indeed diffuse and be taken up by oxygenated tumor cells. Preferential utilization of lactate for oxidative metabolism spares glucose which may in turn reach hypoxic tumor cells. Monocarboxylate transporter 1 regulates the entry of lactate into oxidative tumor cells. Its inhibition favors the switch from lactate-fuelled respiration to glycolysis and consecutively kills hypoxic tumor cells from glucose starvation. Combination with radiotherapy renders remaining cells more sensitive to irradiation, emphasizing how interference with tumor cell metabolism may complement current anticancer modalities.

Hypoxia and blood flow heterogeneities are characteristics of solid tumours and are major obstacles for therapy. Exploiting the biology of nitric oxide (NO), a small radical with multiple functions, is particularly attractive to circumvent these sources of resistance and to sensitise tumour to cytotoxic treatments such as radiotherapy and chemotherapy. Indeed, while NO mediates angiogenic effects, NO may also promote tumour perfusion, drug delivery and oxygenation. Different strategies to deliver NO to tumours and pertaining to the FECS-EJC award laureate's work are reviewed, with a focus on their therapeutic potential. The development of techniques to monitor how and to which extent NO delivery influences the phenotype of a given tumour in a given patient is also discussed.

The heat shock protein 90 (Hsp90) plays a crucial role in the stability of several proteins that are essential for malignant transformation. Hsp90 is therefore an interesting therapeutic target for cancer therapy. In this paper, we investigated whether an oxidative stress generated during ascorbate-driven menadione redox cycling (ascorbate/menadione), affects Hsp90 leading to the degradation of some critical proteins and cell death. Unlike 17-AAG, which inhibits Hsp90 but enhances Hsp70 levels, ascorbate/menadione-treated cells present an additional Hsp90 protein band of about 70kDa as shown by Western blot analysis, suggesting Hsp90 cleavage. This Hsp90 cleavage seems to be a selective phenomenon since it was observed in a large panel of cancer cell lines but not in non-transformed cells. Antibodies raised against either the N-terminus or the C-terminus domains of Hsp90 suggest that the site of cleavage should be located at its N-terminal part. Furthermore, antibodies raised against either the alpha- or the beta-Hsp90 isoform show that Hsp90beta is cleaved while the alpha isoform is down-regulated. We have further shown that different Hsp90 client proteins like Bcr-Abl (a chimerical protein expressed in K562 leukemia cells), RIP and Akt, were degraded when K562 cells were exposed to an oxidative stress. Both Hsp90 cleavage and Bcr-Abl degradation were observed by incubating K562 cells with another H(2)O(2)-generating system (glucose/glucose oxidase) and by incubating KU812 cells (another leukemia cell line) with ascorbate/menadione. Due to the major role of Hsp90 in stabilizing oncogenic and mutated proteins, these results may have potential clinical applications.

Due to its surface properties, especially the presence of amine groups, plasma polymerized allylamine (PPAA) is involved in a large range of applications. Most of them are related to biology and biochemistry. In this work, we demonstrate a quick and simple way to produce PPAA thin films by conventional DC magnetron sputtering. To the best of our knowledge, this is the first method not involving radiofrequency (RF) or microwave discharges. In this paper, we show preliminary characterizations on the produced films (bulk analysis) carried out by ion beam techniques. The Hydrogen content in the whole layers was measured by ERD. The surface composition was probed by XPS on pentafluorobenzaldehyde (PFBA) derivatized films: the maximum concentration of amine groups is 1.6%. (C) 2008 Elsevier B.V. All rights reserved.

Expansion of the thyroid microvasculature is the earliest event during goiter formation, always occurring before thyrocyte proliferation; however, the precise mechanisms governing this physiological angiogenesis are not well understood. Using reverse transcriptase-polymerase chain reaction and immunohistochemistry to measure gene expression and laser Doppler to measure blood flow in an animal model of goitrogenesis, we show that thyroid angiogenesis occurred into two successive phases. The first phase lasted a week and involved vascular activation; this process was thyroid-stimulating hormone (TSH)-independent and was directly triggered by expression of vascular endothelial growth factor (VEGF) by thyrocytes as soon as the intracellular iodine content decreased. This early reaction was followed by an increase in thyroid blood flow and endothelial cell proliferation, both of which were mediated by VEGF and inhibited by VEGF-blocking antibodies. The second, angiogenic, phase was TSH-dependent and was activated as TSH levels increased. This phase involved substantial up-regulation of the major proangiogenic factors VEGF-A, fibroblast growth factor-2, angiopoietin 1, and NG2 as well as their receptors Flk-1/VEGFR2, Flt-1/VEGFR1, and Tie-2. In conclusion, goiter-associated angiogenesis promotes thyroid adaptation to iodine deficiency. Specifically, as soon as the iodine supply is limited, thyrocytes produce proangiogenic signals that elicit early TSH-independent microvascular activation; if iodine deficiency persists, TSH plasma levels increase, triggering the second angiogenic phase that supports thyrocyte proliferation.

Vascularized tumors are exposed to intermittent hypoxia, that is, hypoxia followed by periods of reoxygenation. Abnormal structure and dysfunction of tumor blood vessels are responsible for these conditions. These repeated short periods of hypoxia concern tumor cells as well as endothelial cells. However, the effects of intermittent hypoxia are poorly understood. The aim of this study was to investigate the effects of intermittent hypoxia on endothelial cells and particularly on HIF-1α, a central actor in adaptive response to hypoxia. For that, endothelial cells were exposed to four repeated cycles of 1-h hypoxia followed by 30 min of reoxygenation. We showed that repeated cycles of hypoxia/reoxygenation induced a modification in HIF-lα phosphorylation pattern: a progressive increase in HIF-1α phosphorylated form was observed during the hypoxic periods. Activation of p42/p44, Akt and PKA was observed in parallel. PKA was shown to be involved in the phosphorylation of HIF-lα under intermittent hypoxia, while p42/p44 and Akt were not. As HIF-1 activity is often associated with enhanced cell survival, a better knowledge of the effects of intermittent hypoxia on endothelial cells and the highlight of particular mechanisms induced by intermittent hypoxia are essential to understand the behavior of endothelial cells during neo-angiogenesis. © 2007 Elsevier B.V. All rights reserved.

The identification of a critical role of tumour stroma in the regulation of tumour interstitial fluid pressure and the simultaneous discovery of the impact of anti-angiogenic drugs on tumour hemodynamics have provided new potential for improving tumour delivery of anti-cancer drugs. Here, we review the most recent studies investigating how tumour-associated fibroblasts and macrophages as well as the extracellular matrix itself may be targeted to facilitate delivery of both low-molecular weight drugs and macromolecules. In addition, we summarize the current understanding of the use of vasoactive compounds, radiotherapy and vascular-disrupting agents as potential adjuvants to maximize tumour delivery of anti-cancer drugs. The impact of these strategies on the diffusive and convective modes of drug transport is discussed in the light of Fick's and Starling's laws. Finally, we discuss how transcytosis through caveolae may also be exploited to optimize the selective delivery of conventional chemotherapy to the subendothelial tumour cell compartment.

Objectives We investigated whether retroinfusion of liposomal endothelial nitric oxide synthase (eNOS) S1177D complementary deoxyribonucleic acid (cDNA) would affect neovascularization and function of the ischemic myocardium. Background Recently, we demonstrated the feasibility of liposomal eNOS cDNA transfection via retroinfusion in a model of acute myocardial ischemia/reperfusion. In the present study, we used this approach to target a phosphomimetic eNOS construct (eNOS S1177D) into chronic ischemic myocardium in a pig model of hibernation. Methods Pigs (n = 6/group) were subjected to percutaneous implantation of a reduction stent graft into the left anterior descending artery (LAD), inducing total occlusion within 28 days. At day 28, retroinfusion of saline solution containing liposomal green fluorescent protein or eNOS S1177D cDNA (1.5 mg/animal, 2 x 10 min) was performed. Furthermore, L-nitroarginine-methylester (L-NAME) was applied orally from day 28, where indicated. At day 28 and day 49, fluorescent microspheres were injected into the left atrium for perfusion analysis. Regional functional reserve (at atrial pacing 140/min) was assessed at day 49 by subendocardial segment shortening (SES) (sonomicrometry, percent of ramus circumflexus region). Results The eNOS S1177D overexpression increased endothelial cell proliferation as well as capillary and collateral growth at day 49. Concomitantly, eNOS S1177D overexpression enhanced regional myocardial perfusion from 62 +/- 4% (control) to 77 +/- 3% of circumflex coronary artery-perfused myocardium, unless L-NAME was co-applied (69 +/- 5%). Similarly, eNOS S1177D cDNA improved functional reserve of the LAD (33 +/- 5% vs. 7 +/- 3% of circumflex coronary artery-perfused myocardium), except for L-NAME coapplication (13 +/- 6%). Conclusions Retroinfusion of eNOS S1177D cDNA induces neovascularization via endothelial cell proliferation and collateral growth. The resulting gain of perfusion enables an improved functional reserve of the hibernating myocardium.

Caveolae are plasmalemmal invaginations formed by the sequestration of cholesterol and glycosphingolipids with self-associating molecules named caveolins, resulting in a platform for the assembly of signaling complexes at the surface of the cell. The enrichment of the endothelial nitric oxide synthase in caveolae and its direct interaction with caveolin both account for the exquisite regulation of nitric oxide production in cardiovascular tissues. Dissection of the angiogenic signaling cascade downstream vascular endothelial growth factor recently led to recognition that although the former enables the compartmentation of endothelial nitric oxide synthase and optimizes the process leading to its activation, the latter maintains the enzyme in its inactivated state in the absence of stimulation. Alteration in caveolin abundance or subcellular location may lead endothelial cells or cardiac myocytes to favor one mode of regulation over the other and thereby alter the subtle equilibrium governing nitric oxide production in these cells.

The endothelial isoform of nitric oxide synthase (eNOS) is dually acylated and thereby targeted to signal-transducing microdomains termed caveolae. In endothelial cells, eNOS interacts with caveolin-1, which represses eNOS enzyme activity. In cardiac myocytes, eNOS associates with the muscle-specific caveolin-3 isoform, but whether this interaction affects NO production and regulates myocyte function is unknown. We isolated neonatal cardiac myocytes from mutant mice with targeted disruption of the eNOS gene and transfected them with wild-type (WT) eNOS or myristoylation-deficient (myr-) eNOS mutant cDNA. In myocytes expressing WT eNOS, the muscarinic cholinergic agonist carbachol completely abrogated the spontaneous beating rate and induced a 4-fold elevation of the cGMP level. By contrast, in the myr- eNOS myocytes, carbachol failed to exert its negative chronotropic effect and to increase cGMP levels. We then used a reversible permeabilization protocol to load intact neonatal rat myocytes with an oligopeptide corresponding to the caveolin-3 scaffolding domain. This peptide completely and specifically inhibited the carbachol-induced negative chronotropic effect and the accompanying cGMP elevation. Thus, our results suggest that acylated eNOS may couple muscarinic receptor activation to heart rate control and indicate a key role for eNOS/caveolin interactions in the cholinergic modulation of cardiac myocyte function.

Nitric oxide production in the vascular endothelium is promoted by diverse agonists that transiently increase intracellular Ca2+ concentration and activate the endothelial nitric-oxide synthase (eNOS), a Ca2+/calmodulin-dependent enzyme. eNOS is acylated by the fatty acids myristate and palmitate and is targeted thereby to plasmalemmal signal-transducing domains termed caveolae. eNOS enzyme activity is markedly attenuated by its interactions with caveolin, the structural scaffolding protein of caveolae. We have discovered that in living cells, the eNOS-caveolin heteromeric complex undergoes cycles of dissociation and re-association modulated by Ca2+-mobilizing agonists. Calcium ionophore A23187 and the muscarinic cholinergic agonist carbachol both promote the dissociation of eNOS from caveolin in cultured cells, associated with translocation of eNOS from caveolae. As [Ca2+]i returns to basal levels, eNOS re-associates with caveolin, and the inhibited enzyme complex is then restored to caveolae, a process accelerated by palmitoylation of the enzyme. These data establish an eNOS-caveolin regulatory cycle, wherein enzyme activation is modulated by reversible protein-protein interactions controlled by Ca2+/calmodulin and by enzyme palmitoylation. Alterations in this cycle are likely to have an important influence on nitric oxide-dependent signaling in the vascular wall.

Nitric oxide is synthesized in diverse mammalian tissues by a family of calmodulin-dependent nitric oxide synthases. The endothelial isoform of nitric oxide synthase (eNOS) is targeted to the specialized signal-transducing membrane domains termed plasmalemmal caveolae. Caveolin, the principal structural protein in caveolae, interacts with eNOS and leads to enzyme inhibition in a reversible process modulated by Ca2+-calmodulin (Michel, J. B., Feron, O., Sacks, D., and Michel, T. (1997) J. Biol. Chem. 272, 15583-15586). Caveolin also interacts with other structurally distinct signaling proteins via a specific region identified within the caveolin sequence (amino acids 82-101) that appears to subserve the role of a "scaffolding domain." We now report that the co-immunoprecipitation of eNOS with caveolin is completely and specifically blocked by an oligopeptide corresponding to the caveolin scaffolding domain. Peptides corresponding to this domain markedly inhibit nitric oxide synthase activity in endothelial membranes and interact directly with the enzyme to inhibit activity of purified recombinant eNOS expressed in Escherichia coli. The inhibition of purified eNOS by the caveolin scaffolding domain peptide is competitive and completely reversed by Ca2+-calmodulin. These studies establish that caveolin, via its scaffolding domain, directly forms an inhibitory complex with eNOS and suggest that caveolin inhibits eNOS by abrogating the enzyme's activation by calmodulin.

In cardiac myocytes, as well as specialized conduction and pacemaker cells, agonist binding to muscarinic acetylcholine receptors (mAchRs) results in the activation of several signal transduction cascades including the endothelial isoform of nitric-oxide synthase (eNOS) expressed in these cells. Recent evidence indicates that, as in endothelial cells, eNOS in cardiac myocytes is localized to plasmalemma caveolae, specialized lipid microdomains that contain caveolin-3, a muscle-specific isoform of the scaffolding protein caveolin. In this report, using a detergent-free method for isolation of sarcolemmal caveolae from primary cultures of adult rat ventricular myocytes, we demonstrated that the muscarinic cholinergic agonist carbachol promotes the translocation of mAchR into low density gradient fractions containing most myocyte caveolin-3 and eNOS. Following isopycnic centrifugation, the different gradient fractions were exposed to the muscarinic radioligand [3H]quinuclidinyl benzilate (QNB), and binding was determined after membrane filtration or immunoprecipitation. In a direct radioligand binding assay, we found that [3H]QNB binding can be detected in caveolin-enriched fractions only when cardiac myocytes have been previously exposed to carbachol. Furthermore, most of this [3H]QNB binding can be specifically immunoprecipitated by an antibody to the m2 mAchR, indicating that the translocation of this receptor subtype is responsible for the [3H]QNB binding detected in the low density fractions. Moreover, the [3H]QNB binding could be quantitatively immunoprecipitated from the light membrane fractions with a caveolin-3 antibody (but not a control IgG1 antibody), confirming that the m2 mAchR is targeted to caveolae after carbachol treatment. Importantly, atropine, a muscarinic cholinergic antagonist, did not induce translocation of m2 mAchR to caveolae and prevented receptor translocation in response to the agonist carbachol. Thus, dynamic targeting of sarcolemmal m2 mAchR to caveolae following agonist binding may be essential to initiate specific downstream signaling cascades in these cells.

The endothelial nitric-oxide synthase (eNOS) is a key determinant of vascular homeostasis. Like all known nitric-oxide synthases, eNOS enzyme activity is dependent on Ca2+-calmodulin. eNOS is dynamically targeted to specialized cell surface signal-transducing domains termed plasmalemmal caveolae and interacts with caveolin, an integral membrane protein that comprises a key structural component of caveolae. We have previously reported that the association between eNOS and caveolin is quantitative and tissue-specific (Feron, O., Belhassen, L., Kobzick, L., Smith, T. W., Kelly, R. A., and Michel, T. (1996) J. Biol. Chem. 271, 22810-22814). We now report that in endothelial cells the interaction between eNOS and caveolin is importantly regulated by Ca2+-calmodulin. Addition of calmodulin disrupts the heteromeric complex formed between eNOS and caveolin in a Ca2+-dependent fashion. In addition, overexpression of caveolin markedly attenuates eNOS enzyme activity, but this inhibition is reversed by purified calmodulin. Caveolin overexpression does not affect the activity of the other NOS isoforms, suggesting eNOS-specific inhibition of NO synthase by caveolin. We propose a model of reciprocal regulation of eNOS in endothelial cells wherein the inhibitory eNOS-caveolin complex is disrupted by binding of Ca2+-calmodulin to eNOS, leading to enzyme activation. These findings may have broad implications for the regulation of Ca2+-dependent signal transduction in plasmalemmal caveolae.

The endothelial isoform of nitric oxide synthase (eNOS) modulates cardiac myocyte function and is expressed in the particulate subcellular fraction. We have previously shown that eNOS is targeted to plasmalemmal caveolae in endothelial cells. Caveolae, specialized domains of the plasma membrane, may serve to sequester signaling proteins; a family of transmembrane proteins, the caveolins, form a key structural component of these microdomains. Caveolae in cardiac tissues contain the muscle-specific isoform caveolin-3, and caveolae in endothelial cells contain the widely expressed isoform caveolin-1, which shares limited sequence identity with caveolin-3. Our immunohistochemical analyses of rat cardiac muscle used isoform-specific caveolin antibodies to reveal prominent caveolin-3 staining in myocyte sarcolemmal membranes and at intercalated discs, whereas caveolin-1 staining was prominent in the vascular endothelium. Caveolin or eNOS antibodies were utilized to immunoprecipitate cardiac myocyte or cultured aortic endothelial cell lysates, which then were analyzed in immunoblots. In endothelial cells, we found that eNOS is quantitatively immunoprecipitated by antibodies to caveolin-1. In cardiac myocyte lysates, nearly all the eNOS is immunoprecipitated instead by antibodies to caveolin-3 and, conversely, eNOS antiserum immunoprecipitated primarily caveolin-3. These studies establish expression of eNOS in cardiac myocyte caveolae and document tissue-specific and quantitative associations of eNOS with caveolin. These findings may have important implications for the regulation of eNOS by caveolin isoforms and by other signaling proteins targeted to caveolae.

Background Mesenchymal stem cells (MSCs) are widely used for cell therapy, particularly for the treatment of ischemic heart disease. Mechanisms underlying control of their proliferation capacity, which is known to be inversely correlated to their differentiation abilities, have not been fully characterized. The AMP-activated protein kinase (AMPK) controls several cell functions including cell proliferation. Objective We hypothesized that AMPK plays a crucial role in the control of MSC proliferation. Methods MSCs isolated from WT or AMPKα1 KO adult mouse bone marrow were stimulated with the specific AMPK activator, A769662. Protein phosphorylation state was determined by immunoblotting, cell viability was quantified by FACS after AnnexinV and Propidium Iodide staining, cell proliferation was quantified by EdU incorporation and gene expression was analyzed by real-time PCR. Results MSCs isolated from murine bone marrow exclusively expressed the AMPKa1 catalytic subunit. A769662 was able to induce a robust and sustained AMPK activation in WT MSCs but not in AMPKa1 KO MSCs. This A769662-dependent AMPK activation blocks WT MSC proliferation but not in KO MSCs without affecting their viability. Moreover, AMPKa1 deletion induces an increase of basal MSC proliferation. Among AMPK downstream targets proposed to regulate cell proliferation, we showed that neither the p70 ribosomal S6 protein kinase/eukaryotic elongation factor 2-dependent protein synthesis pathway nor p21 was involved, whereas p27 expression was increased by A-769662. Silencing p27 expression prevented the A-769662- dependent inhibition of MSC proliferation. Conclusions These results suggest that activating AMPK promotes a principal prerequisite for cell differentiation, the arrest of MSC proliferation via p27 regulation.

observed decreased p16. Detailed analysis showed that HLX1 affects INK4a expression at the mRNA level and HLX1 binds to the INK4a promoter as observed by ChIP. We also observed increased levels of the repressive H3K27me3 mark and recruitment of PRC2 components at the Ink4a/Arf locus correlating with HLX1 expression. Co-immunoprecipitation studies showed that HLX1 associates with the PRC2, in particular with Suz12. RNAi studies showed that the repression of p16 by HLX1 is dependent of PRCs. In an attempt to understand if the repression of HLX1 was a property shared by other homeobox genes, we tested 20 homeoboxcontaining genes and identified that multiple homeobox can also repress p16. Conclusions: We identified the homeobox protein HLX1 as a novel p16 repressor. HLX1 binds to the INK4a promoter region and recruits Polycomb repressive complexes. Multiple homeobox proteins can also regulate p16 expression, which implies a conserved role for this family oftranscription factors in regulating the Ink4a/Arf locus, highlighting its potential physiological relevance for both senescence and carcinogenesis.

Early detection of tumor-specific autoantibodies (auto-Abs) has the potential to be used for cancer screening and diagnosis. Whether auto-Ab may be useful to track metastatic progression or response to treatment is, however, largely unknown. To address these issues, the serological proteome was analyzed in an invasive but treatment-responsive mouse tumor model. Among 40 serum-reactive proteins identified by multiplex analysis, we chose to focus on glucose-regulated protein 78 (GRP78), a chaperone protein involved in the endoplasmic reticulum stress response. We first validated GRP78 as a protein overexpressed and mislocalized in tumor cells. We then documented that an increase in GRP78 auto-Ab titer preceded the detection of a palpable tumor mass, correlated with metastatic progression, and was influenced by the onset of tumor neovascularization. We also found that chemotherapy and radiotherapy, both leading to inhibition of tumor growth, oppositely influenced the anti-GRP78 immune response. Whereas radiation increased the concentration of GRP78 auto-Ab by three-fold, the auto-Ab titer was reduced in response to bolus or metronomic administration of cyclophosphamide. Finally, we established a decrease in auto-Ab-producing B lymphocytes in response to chemotherapy and the overexpression of GRP78 together with a strong immunoglobulin response in irradiated tumors. In conclusion, we identified GRP78 auto-Ab as an early marker of tumor and metastatic progressions. However, the multiple influences of anticancer treatments on the humoral immune system calls for caution when exploiting such auto-Ab as markers of the tumor response. © 2010 Neoplasia Press, Inc.