Acidosis-induced metabolic reprogramming in tumor cells enhances the anti-proliferative activity of the PDK inhibitor dichloroacetate.

Altered metabolic pathways in cancer such as exacerbated glycolytic flux and increased glutamine metabolism are promising targets for anti-cancer therapies. While commonly observed in glycolytic tumors, extracellular acidosis has never been considered as a potential modulator of anti-metabolic drug activity such as dichloroacetate (DCA). Using cancer cells from various origins selected for their ability to proliferate under acidic conditions, we found that DCA exerts greater inhibitory effects on the growth of these acid-adapted cells than in parental cells. Moreover, daily DCA administration to mice led to a significant decrease in tumor growth from acid-adapted cells but not from parental cells. 13C-tracer studies revealed that DCA induced a double metabolic shift, diminishing glycolysis and increasing intracellular glutamine in acid-adapted cells. As a consequence, DCA reduced the pentose phosphate pathway activity more extensively and increased apoptosis in acid-adapted cells. Finally, the combination of DCA with a glutaminase inhibitor significantly enhanced the cytotoxic effects of DCA. Overall, the interplay between acidosis and DCA exposure leads to metabolic reprogramming that considerably alters cellular fitness.

Cancer heterogeneity is not compatible with one unique cancer cell metabolic map.

The Warburg effect and its accompanying metabolic features (anaplerosis, cataplerosis) are presented in textbooks and reviews as a hallmark (general characteristic): the metabolic map of cancer. On the other hand, research articles on specific tumors since a few years emphasize various biological features of different cancers, different cells in a cancer and the dynamic heterogeneity of these cells. We have analysed the research literature of the subject and show the generality of a dynamic, evolving biological and metabolic, spatial and temporal heterogeneity of individual cancers. We conclude that there is no one metabolic map of cancer but several and describe the two extremes of a panel from the hypoxic to the normoxic state. The implications for the significance of general 'omic' studies, and on therapeutic conclusions drawn from them and for the diagnostic use of fractional biopsies is discussed.

A new ER-specific photosensitizer unravels (1)O2-driven protein oxidation and inhibition of deubiquitinases as a generic mechanism for cancer PDT.

Photosensitizers (PS) are ideally devoid of any activity in the absence of photoactivation, and rely on molecular oxygen for the formation of singlet oxygen ((1)O2) to produce cellular damage. Off-targets and tumor hypoxia therefore represent obstacles for the use of PS for cancer photodynamic therapy. Herein, we describe the characterization of OR141, a benzophenazine compound identified through a phenotypic screening for its capacity to be strictly activated by light and to kill a large variety of tumor cells under both normoxia and hypoxia. This new class of PS unraveled an unsuspected common mechanism of action for PS that involves the combined inhibition of the mammalian target of rapamycin (mTOR) signaling pathway and proteasomal deubiquitinases (DUBs) USP14 and UCH37. Oxidation of mTOR and other endoplasmic reticulum (ER)-associated proteins drives the early formation of high molecular weight (MW) complexes of multimeric proteins, the concomitant blockade of DUBs preventing their degradation and precipitating cell death. Furthermore, we validated the antitumor effects of OR141 in vivo and documented its highly selective accumulation in the ER, further increasing the ER stress resulting from (1)O2 generation upon light activation.

Monte Carlo Calculation of Radioimmunotherapy with (90)Y-, (177)Lu-, (131)I-, (124)I-, and (188)Re-Nanoobjects: Choice of the Best Radionuclide for Solid Tumour Treatment by Using TCP and NTCP Concepts.

Radioimmunotherapy has shown that the use of monoclonal antibodies combined with a radioisotope like (131)I or (90)Y still remains ineffective for solid and radioresistant tumour treatment. Previous simulations have revealed that an increase in the number of (90)Y labelled to each antibody or nanoobject could be a solution to improve treatment output. It now seems important to assess the treatment output and toxicity when radionuclides such as (90)Y, (177)Lu, (131)I, (124)I, and (188)Re are used. Tumour control probability (TCP) and normal tissue complication probability (NTCP) curves versus the number of radionuclides per nanoobject were computed with MCNPX to evaluate treatment efficacy for solid tumours and to predict the incidence of surrounding side effects. Analyses were carried out for two solid tumour sizes of 0.5 and 1.0 cm radius and for nanoobject (i.e., a radiolabelled antibody) distributed uniformly or nonuniformly throughout a solid tumour (e.g., Non-small-cell-lung cancer (NSCLC)). (90)Y and (188)Re are the best candidates for solid tumour treatment when only one radionuclide is coupled to one carrier. Furthermore, regardless of the radionuclide properties, high values of TCP can be reached without toxicity if the number of radionuclides per nanoobject increases.

Dynamic contrast-enhanced MRI in mouse tumors at 11.7 T: comparison of three contrast agents with different molecular weights to assess the early effects of combretastatin A4.

Dynamic contrast-enhanced (DCE)-MRI is useful to assess the early effects of drugs acting on tumor vasculature, namely anti-angiogenic and vascular disrupting agents. Ultra-high-field MRI allows higher-resolution scanning for DCE-MRI while maintaining an adequate signal-to-noise ratio. However, increases in susceptibility effects, combined with decreases in longitudinal relaxivity of gadolinium-based contrast agents (GdCAs), make DCE-MRI more challenging at high field. The aim of this work was to explore the feasibility of using DCE-MRI at 11.7 T to assess the tumor hemodynamics of mice. Three GdCAs possessing different molecular weights (gadoterate: 560 Da, 0.29 mmol Gd/kg; p846: 3.5 kDa, 0.10 mmol Gd/kg; and p792: 6.47 kDa, 0.15 mmol Gd/kg) were compared to see the influence of the molecular weight in the highlight of the biologic effects induced by combretastatin A4 (CA4). Mice bearing transplantable liver tumor (TLT) hepatocarcinoma were divided into two groups (n = 5-6 per group and per GdCA): a treated group receiving 100 mg/kg CA4, and a control group receiving vehicle. The mice were imaged at 11.7 T with a T1 -weighted FLASH sequence 2 h after the treatment. Individual arterial input functions (AIFs) were computed using phase imaging. These AIFs were used in the Extended Tofts Model to determine K(trans) and vp values. A separate immunohistochemistry study was performed to assess the vascular perfusion and the vascular density. Phase imaging was used successfully to measure the AIF for the three GdCAs. In control groups, an inverse relationship between the molecular weight of the GdCA and K(trans) and vp values was observed. K(trans) was significantly decreased in the treated group compared with the control group for each GdCA. DCE-MRI at 11.7 T is feasible to assess tumor hemodynamics in mice. With K(trans) , the three GdCAs were able to track the early vascular effects induced by CA4 treatment.

Glucose deprivation increases monocarboxylate transporter 1 (MCT1) expression and MCT1-dependent tumor cell migration.

The glycolytic end-product lactate is a pleiotropic tumor growth-promoting factor. Its activities primarily depend on its uptake, a process facilitated by the lactate-proton symporter monocarboxylate transporter 1 (MCT1). Therefore, targeting the transporter or its chaperon protein CD147/basigin, itself involved in the aggressive malignant phenotype, is an attractive therapeutic option for cancer, but basic information is still lacking regarding the regulation of the expression, interaction and activities of both proteins. In this study, we found that glucose deprivation dose-dependently upregulates MCT1 and CD147 protein expression and their interaction in oxidative tumor cells. While this posttranslational induction could be recapitulated using glycolysis inhibition, hypoxia, oxidative phosphorylation (OXPHOS) inhibitor rotenone or hydrogen peroxide, it was blocked with alternative oxidative substrates and specific antioxidants, pointing out at a mitochondrial control. Indeed, we found that the stabilization of MCT1 and CD147 proteins upon glucose removal depends on mitochondrial impairment and the associated generation of reactive oxygen species. When glucose was a limited resource (a situation occurring naturally or during the treatment of many tumors), MCT1-CD147 heterocomplexes accumulated, including in cell protrusions of the plasma membrane. It endowed oxidative tumor cells with increased migratory capacities towards glucose. Migration increased in cells overexpressing MCT1 and CD147, but it was inhibited in glucose-starved cells provided with an alternative oxidative fuel, treated with an antioxidant, lacking MCT1 expression, or submitted to pharmacological MCT1 inhibition. While our study identifies the mitochondrion as a glucose sensor promoting tumor cell migration, MCT1 is also revealed as a transducer of this response, providing a new rationale for the use of MCT1 inhibitors in cancer.

Tumour hypoxia determines the potential of combining mTOR and autophagy inhibitors to treat mammary tumours.

BACKGROUND: Hypoxia can activate autophagy, a self-digest adaptive process that maintains cell turnover. Mammalian target of rapamycin (mTOR) inhibitors are used to treat cancer but also stimulate autophagy. METHODS: Human mammary cancer cells and derived xenografts were used to examine whether hypoxia could exacerbate autophagy-mediated resistance to the mTOR inhibitor rapamycin. RESULTS: Rapamycin exerted potent antitumour effects in MCF-7 and MDA-MB-231 mammary tumours through a marked inhibition of angiogenesis, but the autophagy inhibitor chloroquine (CQ) failed to further sensitise tumours to mTOR inhibition. Rapamycin treatment actually led to tumour reoxygenation, thereby preventing the development of autophagy. Chloroquine alone, however, blocked the growth of MCF-7 tumours and in vitro blunted the hypoxia-induced component of autophagy in these cells. Finally, when initiating CQ treatment in large, hypoxic tumours, a robust antitumour effect could be observed, which also further increased the antiproliferative effects of rapamycin. CONCLUSION: The mTOR inhibitor rapamycin significantly contributes to tumour growth inhibition and normalisation of the tumour vasculature through potent antiangiogenic effects. The resulting reduction in hypoxia accounts for a lack of sensitisation by the autophagy inhibitor CQ, except if the tumours are already at an advanced stage, and thus largely hypoxic at the initiation of the combination of rapamycin and CQ treatment.

PTEN deficiency is associated with reduced sensitivity to mTOR inhibitor in human bladder cancer through the unhampered feedback loop driving PI3K/Akt activation.

BACKGROUND: Preclinical studies have shown that PTEN loss enhances sensitivity to mammalian target of Rapamycin (mTOR) inhibitors because of facilitated PI3K (phosphatidylinositol-3 kinase)/Akt activation and consecutive stimulation of the mTOR pathway. In patients with advanced transitional cell carcinoma (TCC) treated with the mTOR inhibitor everolimus, PTEN loss was, however, associated with resistance to treatment. METHODS: Transitional cell carcinoma specimens, human bladder cancer cells and derived mouse xenografts were used to evaluate how the PTEN status influences the activity of mTOR inhibitors. RESULTS: Transitional cell carcinoma patients with a shorter progression-free survival under everolimus exhibited PTEN deficiency and increased Akt activation. Moreover, PTEN-deficient bladder cancer cells were less sensitive to rapamycin than cells expressing wild-type PTEN, and rapamycin strikingly induced Akt activation in the absence of functional PTEN. Inhibition of Akt activation by the PI3K inhibitor wortmannin interrupted this rapamycin-induced feedback loop, thereby enhancing the antiproliferative effects of the mTOR inhibitor both in vitro and in vivo. CONCLUSION: Facilitation of Akt activation upon PTEN loss can have a more prominent role in driving the feedback loop in response to mTOR inhibition than in promoting the mTOR pathway. These data support the use of both PI3K and mTOR inhibitors to treat urothelial carcinoma, in particular in the absence of functional PTEN.

Endothelial cell metabolism and tumour angiogenesis: glucose and glutamine as essential fuels and lactate as the driving force.

Angiogenic endothelial cells and tumour cells can survive under hypoxic conditions and even proliferate and migrate in a low-oxygen environment. In both cell types, high rates of glycolysis (i.e. conversion of glucose to lactate) and glutaminolysis provide most of the required biosynthetic intermediates and energy to support sprouting and cell division without coupling to oxidative phosphorylation. This metabolic preference is observed under hypoxic conditions, but also in situations in which oxygen is present. In the case of tumour cells, this is known as the Warburg effect and is largely governed by oncogenes. In endothelial cells lining tumour blood vessels, the option of respiration-independent metabolism allows the neovasculature to resist the hostile environment of fluctuating oxygen tension (ranging from severe hypoxia to quasi-normal levels of oxygen). In addition, accumulation in tumours of lactate, the end-product of glycolysis, largely contributes to the angiogenic phenotype through inhibition of prolyl hydroxylase 2 and the activation of HIF1α and NFκB. Activation of the latter in a hypoxia-independent manner leads to the increased production of interleukin-8/CXCL8 which drives the autocrine stimulation of endothelial cell proliferation and maturation of neovessels. In conclusion, the addiction of proliferating endothelial cells for glucose and glutamine as fuels and the driving force of lactate to promote angiogenesis provide novel potential treatment options without the disadvantages of conventional anti-angiogenic drugs.

Gut microbiota-derived propionate reduces cancer cell proliferation in the liver.

BACKGROUND: Metabolites released by the gut microbiota may influence host metabolism and immunity. We have tested the hypothesis that inulin-type fructans (ITF), by promoting microbial production of short-chain fatty acids (SCFA), influence cancer cell proliferation outside the gut. METHODS: Mice transplanted with Bcr-Abl-transfected BaF3 cells, received ITF in their drinking water. Gut microbiota was analysed by 16S rDNA polymerase chain reaction (PCR)-denaturing gradient gel electrophoresis (DGGE) and qPCR. Serum Short-chain fatty acids were quantified by UHPLC-MS. Cell proliferation was evaluated in vivo, by molecular biology and histology, and in vitro. RESULTS: Inulin-type fructans treatment reduces hepatic BaF3 cell infiltration, lessens inflammation and increases portal propionate concentration. In vitro, propionate reduces BaF3 cell growth through a cAMP level-dependent pathway. Furthermore, the activation of free fatty acid receptor 2 (FFA2), a Gi/Gq-protein-coupled receptor also known as GPR43 and that binds propionate, lessens the proliferation of BaF3 and other human cancer cell lines. CONCLUSION: We show for the first time that the fermentation of nutrients such as ITF into propionate can counteract malignant cell proliferation in the liver tissue. Our results support the interest of FFA2 activation as a new strategy for cancer therapeutics. This study highlights the importance of research focusing on gut microbes-host interactions for managing systemic and severe diseases such as leukaemia.

Electron paramagnetic resonance as a sensitive tool to assess the iron oxide content in cells for MRI cell labeling studies.

MRI cell tracking is a promising technique to track various cell types (stem cells, tumor cells, etc.) in living animals. Usually, cells are incubated with iron oxides (T(2) contrast agent) in order to take up the particles before being injected in vivo. Iron oxide quantification is important in such studies for validating the labeling protocols and assessing the dilution of the particles with cell proliferation. We here propose to implement electron paramagnetic resonance (EPR) as a very sensitive method to quantify iron oxide concentration in cells. Iron oxide particles exhibit a unique EPR spectrum, which directly reflects the number of particles in a sample. In order to compare EPR with existing methods (Perls's Prussian blue reaction, ICP-MS and fluorimetry), we labeled tumor cells (melanoma and renal adenocarcinoma cell lines) and fibroblasts with fluorescent iron oxide particles, and determined the limits of detection of the different techniques. We show that EPR is a very sensitive technique and is specific for iron oxide quantification as measurements are not affected by endogenous iron. As a consequence, EPR is well adapted to perform ex vivo analysis of tissues after cell tracking experiments in order to confirm MRI results.

Phase II study of everolimus in patients with locally advanced or metastatic transitional cell carcinoma of the urothelial tract: clinical activity, molecular response, and biomarkers.

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.

Multimodal assessment of early tumor response to chemotherapy: comparison between diffusion-weighted MRI, 1H-MR spectroscopy of choline and USPIO particles targeted at cell death.

The aim of this study was to determine the value of different magnetic resonance (MR) protocols to assess early tumor response to chemotherapy. We used a murine tumor model (TLT) presenting different degrees of response to three different cytotoxic agents. As shown in survival curves, cyclophosphamide (CP) was the most efficient drug followed by 5-fluorouracil (5-FU), whereas the etoposide treatment had little impact on TLT tumors. Three different MR protocols were used at 9.4 Tesla 24 h post-treatment: diffusion-weighted (DW)-MRI, choline measurement by (1) H MRS, and contrast-enhanced MRI using ultrasmall iron oxide nanoparticles (USPIO) targeted at phosphatidylserine. Accumulation of contrast agent in apoptotic tumors was monitored by T(2) -weighted images and quantified by EPR spectroscopy. Necrosis and apoptosis were assessed by histology. Large variations were observed in the measurement of choline peak areas and could not be directly correlated to tumor response. Although the targeted USPIO particles were able to significantly differentiate between the efficiency of each cytotoxic agent and best correlated with survival endpoint, they present the main disadvantage of non-specific tumor accumulation, which could be problematic when transferring the method to the clinic. DW-MRI presents a better compromise by combining longitudinal studies with a high dynamic range; however, DW-MRI was unable to show any significant effect for 5-FU. This study illustrates the need for multimodal imaging in assessing tumor response to treatment to compensate for individual limitations.

Iron oxide particles covered with hexapeptides targeted at phosphatidylserine as MR biomarkers of tumor cell death.

The aim of the study was to evaluate the ability of a new MR contrast agent to detect cell death as a biomarker of the efficacy of anti-cancer treatment. The phosphatidylserine-targeted hexapeptide (E3) was coupled to pegylated ultrasmall iron oxide nanoparticles (USPIO) that can be detected by magnetic resonance imaging (MRI) and by electron paramagnetic resonance (EPR). USPIO binding to staurosporine-treated TLT (transplantable liver tumor) cells, evaluated by X-Band EPR, indicated twice as much binding of USPIO grafted with the E3 peptide, compared with USPIO grafted with a scrambled peptide or ungrafted USPIO. In vivo experiments were carried out using TLT cells implanted intramuscularly into NMRI mice, and tumor cell death was induced by irradiation. After intravenous injection of the different types of USPIO, the accumulation of contrast agent was evaluated ex vivo by X-band EPR, in vivo by L-band EPR and by T(2)-weighted MRI. In irradiated tumors there was greater accumulation of the targeted USPIO particles compared with control particles or compared with the targeted particles in untreated tissues. In conclusion, phosphatidylserine-targeting of USPIO particles can detect dying tissues. This molecular targeted system should be evaluated further as a potential biomarker of tumor response to treatment.

Radioimmunotherapy with radioactive nanoparticles: biological doses and treatment efficiency for vascularized tumors with or without a central hypoxic area.

PURPOSE: Radioactive atoms attached to monoclonal antibodies are used in radioimmunotherapy to treat cancer while limiting radiation to healthy tissues. One limitation of this method is that only one radioactive atom is linked to each antibody and the deposited dose is often insufficient to eradicate solid and radioresistant tumors. In a previous study, simulations with the Monte Carlo N-Particle eXtended code showed that physical doses up to 50 Gy can be delivered inside tumors by replacing the single radionuclide by a radioactive nanoparticle of 5 nm diameter containing hundreds of radioactive atoms. However, tumoral and normal tissues are not equally sensitive to radiation, and previous works did not take account the biological effects such as cellular repair processes or the presence of less radiosensitive cells such as hypoxic cells. METHODS: The idea is to adapt the linear-quadratic expression to the tumor model and to determine biological effective doses (BEDs) delivered through and around a tumor. This BED is then incorporated into a Poisson formula to determine the shell control probability (SCP) which predicts the cell cluster-killing efficiency at different distances "r" from the center of the tumor. BED and SCP models are used to analyze the advantages of injecting radioactive nanoparticles instead of a single radionuclide per vector in radioimmunotherapy. RESULTS: Calculations of BED and SCP for different distances r from the center of a solid tumor, using the non-small-cell lung cancer as an example, were investigated for 90Y2O3 nanoparticles. With a total activity of about 3.5 and 20 MBq for tumor radii of 0.5 and 1.0 cm, respectively, results show that a very high BED is deposited in the well oxygenated part of the spherical carcinoma. CONCLUSIONS: For either small or large solid tumors, BED and SCP calculations highlight the important benefit in replacing the single beta-emitter 90Y attached to each antibody by a 90Y2O3 nanoparticle.

The transcription factor GATA-1 is overexpressed in breast carcinomas and contributes to survivin upregulation via a promoter polymorphism.

Expression of survivin, a member of the inhibitor of apoptosis protein family, is elevated in human cancers and considered as a new therapeutic target. Mechanism upregulating survivin expression in tumour cells is poorly understood. In this study, we show that breast cancer patients harbouring a polymorphism G235A in the survivin promoter present a higher level of survivin expression. This polymorphism creates a binding site for the transcription factor GATA-1 inducing a second GATA-1-binding site in survivin promoter. At the mRNA level, GATA-1 was present in breast carcinomas and adjacent normal tissues, whereas the protein was only detected in carcinomas by western blot and immunohistochemistry. Transfection of wild-type and different constitutively active GATA-1 mutants (serine 26, 178 or 310) showed that only phospho-serine 26 GATA-1 was able to increase survivin expression. This increase was higher in G235A than in G235G cell lines. Phospho-serine 26 GATA-1 bound directly survivin promoter, with a stronger interaction in G235A than in G235G polymorphism indicating that both GATA-1-binding sites are functional. These data identify GATA-1 as a key feature in tumour aggressiveness by enhancing survivin expression and delineate its targeting as a possible new therapeutic strategy in breast carcinomas.

eNOS activation by physical forces: from short-term regulation of contraction to chronic remodeling of cardiovascular tissues.

Nitric oxide production in response to flow-dependent shear forces applied on the surface of endothelial cells is a fundamental mechanism of regulation of vascular tone, peripheral resistance, and tissue perfusion. This implicates the concerted action of multiple upstream "mechanosensing" molecules reversibly assembled in signalosomes recruiting endothelial nitric oxide synthase (eNOS) in specific subcellular locales, e.g., plasmalemmal caveolae. Subsequent short- and long-term increases in activity and expression of eNOS translate this mechanical stimulus into enhanced NO production and bioactivity through a complex transcriptional and posttranslational regulation of the enzyme, including by shear-stress responsive transcription factors, oxidant stress-dependent regulation of transcript stability, eNOS regulatory phosphorylations, and protein-protein interactions. Notably, eNOS expressed in cardiac myocytes is amenable to a similar regulation in response to stretching of cardiac muscle cells and in part mediates the length-dependent increase in cardiac contraction force. In addition to short-term regulation of contractile tone, eNOS mediates key aspects of cardiac and vascular remodeling, e.g., by orchestrating the mobilization, recruitment, migration, and differentiation of cardiac and vascular progenitor cells, in part by regulating the stabilization and transcriptional activity of hypoxia inducible factor in normoxia and hypoxia. The continuum of the influence of eNOS in cardiovascular biology explains its growing implication in mechanosensitive aspects of integrated physiology, such as the control of blood pressure variability or the modulation of cardiac remodeling in situations of hemodynamic overload.

Caveolae, caveolin and control of vascular tone: nitric oxide (NO) and endothelium derived hyperpolarizing factor (EDHF) regulation.

Endothelium plays a crucial role in the regulation of cardiovascular homeostasis through the release of vasoactive factors. Nitric oxide (NO) and endothelium-derived hyperpolarizing factors (EDHF) are the two major actors controlling the vasomotor tone. The endothelial nitric oxide synthase (eNOS) was reported in the mid 90ies to be under the control of caveolin, the structural protein of caveolae. Nowadays, a large body of evidence has confirmed that the caveolin/eNOS interaction was needed to prevent inadequate NO production under basal conditions but also to facilitate the integration of extracellular stimuli to intracellular NO signals. Compartmentation of key actors in the EDHF signaling pathway is now also proposed to take place into caveolae. Accordingly, caveolin-deficient animals revealed both an unopposed NO production promoting vessel dilation and a lack of EDHF-driven vasorelaxation. The transient receptor potential (TRP) channels are the link between caveolae and EDHF. Different TRP channels involved in the capacitative calcium entry were found to directly interact with caveolin-1 in endothelial cells. TRPC1 and TRPC4 form a complex with the endoplasmic reticulum IP3 receptor thereby optimizing calcium signaling. EDHF-driven vasodilation was documented to be altered in a TRPV4-deficient mouse model. The close vicinity between TRPV4 and SKCa channels in caveolae together with the gap-junctions subunits connexins support a role of these microdomains in the generation and propagation of EDHF to vascular smooth muscle cells. In conclusion, caveolae and caveolin are important control points in the control of blood pressure by the endothelium. This also highlights how any alteration in the caveolae integrity or caveolin abundance may lead to and/or exacerbate endothelial dysfunction and associated cardiovascular diseases.

Role of caveolar compartmentation in endothelium-derived hyperpolarizing factor-mediated relaxation: Ca2+ signals and gap junction function are regulated by caveolin in endothelial cells.

BACKGROUND: In endothelial cells, caveolin-1, the structural protein of caveolae, acts as a scaffolding protein to cluster lipids and signaling molecules within caveolae and, in some instances, regulates the activity of proteins targeted to caveolae. Specifically, different putative mediators of the endothelium-derived hyperpolarizing factor (EDHF)-mediated relaxation are located in caveolae and/or regulated by the structural protein caveolin-1, such as potassium channels, calcium regulatory proteins, and connexin 43, a molecular component of gap junctions. METHODS AND RESULTS: Comparing relaxation in vessels from caveolin-1 knockout mice and their wild-type littermates, we observed a complete absence of EDHF-mediated vasodilation in isolated mesenteric arteries from caveolin-1 knockout mice. The absence of caveolin-1 is associated with an impairment of calcium homeostasis in endothelial cells, notably, a decreased activity of Ca2+-permeable TRPV4 cation channels that participate in nitric oxide- and EDHF-mediated relaxation. Moreover, morphological characterization of caveolin-1 knockout and wild-type arteries showed fewer gap junctions in vessels from knockout animals associated with a lower expression of connexins 37, 40, and 43 and altered myoendothelial communication. Finally, we showed that TRPV4 channels and connexins colocalize with caveolin-1 in the caveolar compartment of the plasma membrane. CONCLUSIONS: We demonstrated that expression of caveolin-1 is required for EDHF-related relaxation by modulating membrane location and activity of TRPV4 channels and connexins, which are both implicated at different steps in the EDHF-signaling pathway.

Radioimmunotherapy with radioactive nanoparticles: first results of dosimetry for vascularized and necrosed solid tumors.

Radioimmunotherapy uses monoclonal antibodies that are still labeled with only one radioactive atom. The aim of this paper is to assess, by means of MCNPX simulations, the doses delivered around and throughout a solid tumor when the radioactive atom linked to each antibody is replaced by a 5 nm diameter nanoparticle composed of numerous radionuclides. A new model for a spherical vascularized tumor has been developed in which the antibody distributions inside the tumor can be uniform or heterogeneous. It is also possible to simulate a central necrotic core inside the tumor where the concentration of radiolabeled antibodies is assumed to be zero. Dosimetry calculations have been performed for the beta-emitting radionuclide (90)Y2O3. Preliminary results show that the irregularity of vasculature and the presence of a necrotic core have a noticeable influence on the deposited dose profiles. Moreover, with a total activity of 5 and 34 MBq for tumor radii of 0.5 and 1.0 cm, respectively, viable tumor cells can receive doses of up to 50 Gy, even if high nonuniformity of the total activity is observed in the tumor. These simulations still require accurate information about antibody characteristics and necrosis sizes but clearly confirm that the use of monoclonal antibodies conjugated to nanoparticles could lead to a considerable enhancement of treatment efficacy against cancer.