Single and combined impacts of irradiation and surgery on lymphatic vasculature and fibrosis associated to secondary lymphedema.

Lymphedema (LD) refers to a condition of lymphatic dysfunction associated with excessive fluid accumulation, fibroadipose tissue deposition and swelling. In industrialized countries, LD development mainly results from a local disruption of the lymphatic network by an infection or cancer-related surgery (secondary LD). In the absence of efficient therapy, animal models are needed to decipher the cellular and molecular mechanisms underlying LD and test putative drugs. In this study, we optimized and characterized a murine model of LD that combines an irradiation of the mice hind limb and a radical surgery (lymph node resection associated to lymphatic vessel ligation). We investigated the respective roles of irradiation and surgery in LD formation by comparing their impacts, alone or in combination (with different intervention sequences), on eight different features of the pathology: swelling (paw thickness), indocyanine green (ICG) clearance, lymphatic vasculature remodeling, epidermal and dermal thickening, adipocyte accumulation, inflammatory cell infiltration and collagen deposition. This study supports the importance of radiation prior to surgery to experimentally induce a rapid, severe and sustained tissue remodeling harboring the different hallmarks of LD. We provide the first experimental evidence for an excessive deposition of periostin (POSTN) and tenascin-C (TNC) in LD. Through a computerized method of digital image quantification, we established the spatial map of lymphatic expansion, as well as collagen, POSTN and TNC deposition in papillary and reticular dermis of lymphedematous skins. This mouse model is available to study the patho-physiology of LD and test potential therapeutic targets.

[Palliation by radiation is also our business !] / La radiothérapie palliative, c'est aussi notre rayon !

Radiotherapy (RT), both with a curative and a palliative intent, is one of the cornerstones of oncological treatments. A variety of symptoms linked to cancer can be relieved with RT (such as pain, bleeding, compression exerted by a tumour lesion…). Very often, palliative RT is proposed when other medical treatments (painkillers, morphine…) are no longer efficient, or the patient does not tolerate them anymore. Palliative RT is an integral part of the global supportive oncological care. Indeed, patients' wishes and prognosis are taken into account in each and every step of the treatment pathway. Every treatment deserves an individualized approach and benefits from the best available techniques.

La radiothérapie, à la fois à visée curative et palliative, est l'un des piliers des traitements oncologiques. Une multitude de symptômes liés au cancer (douleurs, saignements, diverses conséquences liées à une compression exercée par une lésion tumorale…) peuvent être soulagés grâce à une radiothérapie palliative (RTP). Bien souvent, la RTP est proposée lorsque les traitements médicamenteux dits «classiques¼ ne font plus suffisamment effet ou si le patient ne les tolère plus (antidouleurs, morphine…). La RTP fait partie intégrante des soins oncologiques de support. En effet, le pronostic du patient, ainsi que ses souhaits, sont pris en compte à chacune des étapes qui constituent le trajet de soins, y compris en RTP. Ainsi, chaque traitement est individualisé et bénéficie des meilleures techniques disponibles.

Assessment of the functional impact of germline BRCA1/2 variants located in non-coding regions in families with breast and/or ovarian cancer predisposition.

PURPOSE: The molecular mechanism of breast and/or ovarian cancer susceptibility remains unclear in the majority of patients. While germline mutations in the regulatory non-coding regions of BRCA1 and BRCA2 genes have been described, screening has generally been limited to coding regions. The aim of this study was to evaluate the contribution of BRCA1/2 non-coding variants. METHODS: Four BRCA1/2 non-coding regions were screened using high-resolution melting analysis/Sanger sequencing or next-generation sequencing on DNA extracted from index cases with breast and ovarian cancer predisposition (3926 for BRCA1 and 3910 for BRCA2). The impact of a set of variants on BRCA1/2 gene regulation was evaluated by site-directed mutagenesis, transfection, followed by Luciferase gene reporter assay. RESULTS: We identified a total of 117 variants and tested twelve BRCA1 and 8 BRCA2 variants mapping to promoter and intronic regions. We highlighted two neighboring BRCA1 promoter variants (c.-130del; c.-125C > T) and one BRCA2 promoter variants (c.-296C > T) inhibiting significantly the promoter activity. In the functional assays, a regulating region within the intron 12 was found with the same enhancing impact as within the intron 2. Furthermore, the variants c.81-3980A > G and c.4186-2022C > T suppress the positive effect of the introns 2 and 12, respectively, on the BRCA1 promoter activity. We also found some variants inducing the promoter activities. CONCLUSION: In this study, we highlighted some variants among many, modulating negatively the promoter activity of BRCA1 or 2 and thus having a potential impact on the risk of developing cancer. This selection makes it possible to conduct future validation studies on a limited number of variants.

Clinical value of R-spondins in triple-negative and metaplastic breast cancers.

BACKGROUND: RSPO ligands, activators of the Wnt/ß-catenin pathway, are overexpressed in different cancers. The objective of this study was to investigate the role of RSPOs in breast cancer (BC). METHODS: Expression of RSPO and markers of various cancer pathways were measured in breast tumours and cell lines by qRT-PCR. The effect of RSPO on the Wnt/ß-catenin pathway activity was determined by luciferase assay, western blotting, and qRT-PCR. The effect of RSPO2 inhibition on proliferation was determined by using RSPO2 siRNAs. The effect of IWR-1, an inhibitor of the Wnt/ß-catenin pathway, was examined on the growth of an RSPO2-positive patient-derived xenograft (PDX) model of metaplastic triple-negative BC. RESULTS: We detected RSPO2 and RSPO4 overexpression levels in BC, particularly in triple-negative BC (TNBC), metaplastic BC, and triple-negative cell lines. Various mechanisms could account for this overexpression: presence of fusion transcripts involving RSPO, and amplification or hypomethylation of RSPO genes. Patients with RSPO2-overexpressing tumours have a poorer metastasis-free survival (P=3.6 × 10-4). RSPO2 and RSPO4 stimulate Wnt/ß-catenin pathway activity. Inhibition of RSPO expression in a TN cell line inhibits cell growth, and IWR-1 significantly inhibits the growth of an RSPO2-overexpressing PDX. CONCLUSIONS: RSPO overexpression could therefore be a new prognostic biomarker and therapeutic target for TNBC.

Effects of high frequency ultrasound irradiation on incorporation of SiO2 particles within polypyrrole films.

This paper deals with the effect of ultrasound on polypyrrole/SiO2 composite film elaboration through various steps (particle dispersion, electrosynthesis). Experiments were carried out on stainless steel in phosphoric acid solution. An efficient method for dispersion of SiO2 particles prior to electropolymerization, based on low frequency irradiation (20kHz), was proposed. It was shown that mechanical effects of high frequency ultrasound (i.e. mass transfer improvement) led to enhancement of electropolymerization kinetics. Scanning electron microscopy imaging and glow discharge optical emission spectroscopy revealed localization of SiO2 particles in the outer region of the films as well as better incorporation of particles under high frequency ultrasound irradiation. Finally, anticorrosion behavior of formed films was investigated in sodium chloride solution by Open Circuit Potential and anodic polarization methods. The results showed that polypyrrole/SiO2 films elaborated under ultrasound irradiation exhibit the best protective performances.

[Functional imaging and radiotherapy]. / Utilisation de l'imagerie fonctionnelle en radiothérapie.

Medical imaging plays a crucial role in the diagnosis, staging and therapeutic strategy of oncologic patients. The development of medical imaging over the last decade has allowed significant progresses in radiotherapy. Indeed, medical imaging is now considered the corner stone of radiotherapy. The main challenge for the radiation oncologist consists in the tumour identification with a view to irradiate the tumour at a curative dose while avoiding healthy tissues. To achieve these goals, the radiotherapist daily uses anatomical imaging such as computed tomography (CT) or magnetic resonance imaging (MRI). Since several years now, the development of functional imaging such as positron emission tomography (PET) combined with CT or functional MRI has opened new perspectives in the management of oncologic diseases. Indeed, these imaging techniques offer new information on tumour metabolism that may be taken into account to plan the radiotherapy treatment. This article illustrates the different imaging techniques used in radiotherapy and the role of functional imaging for establishing new therapeutic strategies in radiation oncology.

Genome-wide gene expression profiling to predict resistance to anthracyclines in breast cancer patients.

Validated biomarkers predictive of response/resistance to anthracyclines in breast cancer are currently lacking. The neoadjuvant Trial of Principle (TOP) study, in which patients with estrogen receptor (ER)-negative tumors were treated with anthracycline (epirubicin) monotherapy, was specifically designed to evaluate the predictive value of topoisomerase II-alpha (TOP2A) and develop a gene expression signature to identify those patients who do not benefit from anthracyclines. Here we describe in details the contents and quality controls for the gene expression and clinical data associated with the study published by Desmedt and colleagues in the Journal of Clinical Oncology in 2011 (Desmedt et al., 2011). We also provide R code to easily access the data and perform the quality controls and basic analyses relevant to this dataset.

[Cyberknife and benign pathologies]. / Cyberknife et pathologies bénignes.

Conventional radiotherapy is known to be an effective treatment approach even for "benign" pathologies. However, this kind of treatment yields a high potential for side effects. The Cyberknife, a robotic stereotactic radiotherapy device, enables to offset a large proportion of the disadvantages encountered with conventional radiotherapy essentially through the high precision of dose administration and sparing of healthy tissues. Therefore, it seems to be a treatment of choice in the approach of some benign intracranial diseases. We review published data on indications and outcome of Cyberknife for intracranial "non-malignant" disease.

Electrosynthesis and characterization of conducting polypyrrole elaborated under high frequency ultrasound irradiation.

The effects of high frequency ultrasound (500kHz) on pyrrole electropolymerization in sodium perchlorate aqueous medium have been investigated. Cyclic voltametry studies showed that there is no influence on pyrrole oxidation potential. Scanning Electron Microscopy (SEM) imaging, and mechanical and optical profiling, revealed thinner, denser and more homogeneous surface structure for polypyrrole films elaborated under ultrasound irradiation. This is attributed to cavitation bubble asymmetric collapse close to the interface, which should induce changes in the nucleation-growth mechanism during the first polymerization stage. An increase of approximately 27% in doping level for sonicated films was revealed by X-ray Photoelectron Spectroscopy (XPS) analyses.

Doping properties of PEDOT films electrosynthesized under high frequency ultrasound irradiation.

The present study presents the use of high frequency ultrasound (500 kHz, 25 W) for 3,4-ethylenedioxythiophene (EDOT) electropolymerization in aqueous medium in order to investigate its effects on conducting polymer properties. It was shown that mass transfer increases under ultrasound irradiation which improved electropolymerization and the comparison with experiments carried out at the same mass transfer level (4.6 × 10(-5) ms(-1)) puts in evidence that stirring effect is not the only phenomenon induced by ultrasound during electrodeposition. PEDOT films elaborated under ultrasonication present increased doping levels revealed by X-ray Photoelectron Spectroscopy (XPS) analysis, especially in the case of thick films (measured by mechanical probe), thanks to better incorporation of counter ions within polymer matrix as another consequence of mass transport improvement under ultrasound and probably film heating by wave absorption for the highest thicknesses. A dilation of the film under sonication leading to an increase in film thickness was also highlighted. Finally, a refining of the surface structure was also observed via SEM imaging.

Inhibition of the Wnt/beta-catenin pathway by the WWOX tumor suppressor protein.

The WWOX gene encodes a candidate tumor suppressor protein (WWOX) implicated in a variety of human diseases such as cancer. To better understand the molecular mechanisms of WWOX action, we investigated novel partners of this protein. Using the two-hybrid system and a coimmunoprecipitation assay, we observed a physical association between WWOX and the Dishevelled protein (Dvl) family signaling elements involved in the Wnt/beta-catenin pathway. We found that enforced WWOX expression inhibited, and inhibition of endogenous WWOX expression stimulated the transcriptional activity of the Wnt/beta-catenin pathway. Inhibition of endogenous WWOX expression also enhanced the effect of Wnt-3a on beta-catenin stability. Moreover, we observed the sequestration of Dvl-2 wild type and Dvl-2NESm, a mutated form of Dvl-2 predominantly localized in the nucleus, in the cytoplasm compartment by WWOX. Our results indicate that WWOX is a novel inhibitor of the Wnt/beta-catenin pathway. WWOX would act, at least in part, by preventing the nuclear import of the Dvl proteins.

Effect of ultrasounds on the electrochemical synthesis of polypyrrole, application to the adhesion and growth of biological cells.

In this study, a new way to synthesize polypyrrole films is presented. This original way consists in the electropolymerization of polypyrrole under high frequency ultrasonic irradiation on conductive fluorine-doped tin oxide surfaces. The polypyrrole films obtained are then compared, in terms of chemical structure and morphology, to polypyrrole films synthesized by standard electrochemical methodology. Next, these polymer films are tested as an alternative to biomaterials that are commonly used as cell culture substrates. Thus, the adhesion and growth of osteoblastics cells and microbial cells on polymer-modified surfaces are investigated by using qualitative observation and quantitative tests. These studies proved the non-toxicity of the polymer films for osteoblastic and microbial cells but also a different behaviour of osteoblastic cells and microbial cells with polypyrrole films.

Biological conversion of a water-soluble prodrug of cyclosporine A.

UNIL088 is a water-soluble prodrug of cyclosporine A (CsA) designed for topical ocular delivery. The pro-moiety is grafted via an ester function to CsA and the solubilizing group is a phosphate ion. The aim of this study was to elucidate the conversion mechanisms by which UNIL088 generates CsA. UNIL088 was incubated in rabbit tears at physiological temperature to study its enzymatic and chemical conversion, respectively. Metabolites and intermediates were identified using a quadrupole-time of flight (QqTOF) mass spectrometer, which allowed biotransformation pathways to be deduced. Conversion is activated by the chemical or enzymatic hydrolysis of the terminal ester function of the pro-moiety, leading to the phospho-serine-sarcosine-cyclosporine A that spontaneously converts into CsA. In addition to the main biotransformation pathway, a secondary reaction involved hydrolysis of the phosphate ester group of the pro-moiety, probably by phosphatases present in tears.

Evaluation of a topical cyclosporine A prodrug on corneal graft rejection in rats.

PURPOSE: To assess the efficacy of a topical cyclosporine A (CsA), water-soluble prodrug, for promoting the survival of allogenic rat corneal grafts after penetrating keratoplasty (PKP). METHODS: Corneas of Brown-Norway rats (donors) were transplanted to Lewis rats (recipients). Transplanted rats were divided in three treatment groups: group I (PBS) and group II (0.26% Debio088) received drops five times per day. Group III received a daily intramuscular CsA injection (10 mg/kg/day). Blood CsA concentrations were measured on days 2 and 14. On day 4, 10, 13 after PKP, grafts were scored for corneal transparency, edema and extent of neovascularization. An opacity score of greater than or equal to 3 was considered as a nonreversible graft rejection process. On day 14, the experimental eyes were processed for histology. RESULTS: On day 13, 12 of the 18 corneal transplants (67%) in group I showed irreversible graft rejection. Three of 18 transplants (19%) in group II and 5 of 16 transplants (28%) in group III showed irreversible graft rejection (p=0.013/p=0.019, OR=0.14/0.06 versus vehicle). Each mean clinical score for edema, opacity, and neovessels in group II were significantly lower than those of the grafts in group I (respectively p=0.010, p=0.013, p=0.024) and III except for neovessels (respectively p=0.002, p=0.001, p=0.057). Histology confirmed the clinical results. The mean CsA blood levels for groups II and III were, respectively 54+/-141 mug/l and 755+/-319 mug/l on day 2 and 14+/-34 mug/l and 1318+/-463 mug/l on day 14. CONCLUSIONS: Debio088 CsA prodrug drops given five times daily are as effective as intramuscular injection of 10 mg/kg/day for the prevention of acute corneal graft rejection in rats.

A novel self-microemulsifying formulation of paclitaxel for oral administration to patients with advanced cancer.

To explore the parmacokinetics, safety and tolerability of paclitaxel after oral administration of SMEOF#3, a novel Self-Microemulsifying Oily Formulation, in combination with cyclosporin A (CsA) in patients with advanced cancer. Seven patients were enrolled and randomly assigned to receive oral paclitaxel (SMEOF#3) 160 mg+CsA 700 mg on day 1, followed by oral paclitaxel (Taxol) 160 mg+CsA 700 mg on day 8 (group I) or vice versa (group II). Patients received paclitaxel (Taxol) 160 mg as 3-h infusion on day 15. The median (range) area under the plasma concentration-time curve of paclitaxel was 2.06 (1.15-3.47) microg h ml(-1) and 1.97 (0.58-3.22) microg h ml(-1) after oral administration of SMEOF#3 and Taxol, respectively, and 4.69 (3.90-6.09) microg h ml(-1) after intravenous Taxol. Oral SMEOF#3 resulted in a lower median T(max) of 2.0 (0.5-2.0) h than orally applied Taxol (T(max)=4.0 (0.8-6.1) h, P=0.02). The median apparent bioavailability of paclitaxel was 40 (19-83)% and 55 (9-70)% for the oral SMEOF#3 and oral Taxol formulation, respectively. Oral paclitaxel administered as SMEOF#3 or Taxol was safe and well tolerated by the patients. Remarkably, the SMEOF#3 formulation resulted in a significantly lower T(max) than orally applied Taxol, probably due to the excipients in the SMEOF#3 formulation resulting in a higher absorption rate of paclitaxel.

A novel water-soluble cyclosporine A prodrug: ocular tolerance and in vivo kinetics.

The purpose of this study is to demonstrate that a novel water-soluble prodrug of cyclosporine A (CsA) intended for topical ocular administration, does not induce eye irritation in a rabbit model and is able to generate therapeutic concentrations of CsA in the precorneal area immediately after administration. The eye irritancy of the prodrug and CsA control solution was assessed by the Draize test and by confocal laser ophthalmoscopy (CLSO). Residence time and tear concentrations of prodrug and CsA in the rabbit eye were assessed by HPLC. The Draize test showed an excellent tolerance for the prodrug solution while the reference CsA oil solution induced lachrymation and irritation. The CLSO-measured corneal lesions, subsequent to treatment with the prodrug and reference solutions, were 3% and 9%, respectively. The prodrug transformed rapidly, leading to relatively stable CsA concentrations in tears with a maximal concentration of 94 microg ml(-1) over the observation period. This study demonstrated that the prodrug solution was well tolerated and that clinically significant CsA tear concentrations were achieved. UNIL088 is a promising molecule in the treatment of immune-related disorders of the eye.

Conversion of cyclosporine A prodrugs in human tears vs rabbits tears.

The aim of this study was to evaluate the rate and mechanism of conversion of two water-soluble prodrugs of cyclosporine A (CsA) intended for topical delivery to the eye. The new molecules were designed according to the double prodrug concept: a solubilizing moiety was grafted onto CsA via an ester function, which could be hydrolysed via a two-step process (enzymatic and chemical). Prodrug solutions were prepared extemporaneously in an isotonic and neutral aqueous medium compatible with ophthalmic use. The rates of conversion into the parent molecule were determined by incubating the prodrugs in fresh rabbit or human tears or in a phosphate buffer solution (PBS) at pH 7.4. Both prodrugs were converted into CsA within the first minute in the presence of rabbit tears with rate constants of k=5.9x10(-3)min(-1) and k=3.8x10(-3)min(-1), respectively, for UNIL088 and UNIL089, whereas chemical conversion in PBS was negligible (k=0.5x10(-3)min(-1) for both molecules). Incubation of UNIL088 in human tears showed a significantly high conversion rate. It is concluded that the developed double prodrugs underwent a bioconversion in physiological media and thus represent promising candidates for topical delivery of CsA to the eye.

Ethanol induces higher BEC in CB1 cannabinoid receptor knockout mice while decreasing ethanol preference.

AIMS: Previous studies have shown that CB(1) cannabinoid receptors are involved in the behavioural effects induced by chronic ethanol administration in Wistar rats by using SR 141716, a CB(1) cannabinoid receptor antagonist. These studies have now been extended to investigate the effect of acute and chronic alcoholization on blood ethanol concentration (BEC) and ethanol preference in CB(1) knockout (-/-) mice. METHODS: BEC was monitored for a period of 8 h in both CB(1)(-/-) male mice and CB(1) male wild-type (+/+) mice, which had received an acute i.p. injection of ethanol in 1, 3 or 5 g/kg doses. Ethanol preference was assayed in both groups of male mice in non-forced ethanol administration and forced chronic pulmonary alcohol administration for 14 and 39 days, respectively. RESULTS: After an acute intraperitoneal ethanol injection of 5 g/kg, CB(1)(-/-) mice showed a significant higher BEC during the ethanol elimination stage than the CB(1)(+/+) mice. However, those in the 1 and 3 g/kg groups showed no significant difference. A 2-3 fold increase in BEC was observed in CB(1)(-/-) mice on days 10 and 11 after commencement of forced chronic pulmonary alcoholization in comparison with CB(1)(+/+) mice, although comparable BEC values were assayed in both groups on day 12. In addition, these CB(1)(-/-) mice showed a significantly lower preference for ethanol than CB(1)(+/+) mice. CONCLUSIONS: The studies on CB(1)(-/-) and CB(1)(+/+) mice have clearly confirmed the involvement of CB(1) receptor on ethanol induced behavioural effects and also revealed that CB(1) receptors may be implicated in ethanol absorption/distribution, particularly after administration of high ethanol doses.

Gene regulation by phorbol 12-myristate 13-acetate in MCF-7 and MDA-MB-231, two breast cancer cell lines exhibiting highly different phenotypes.

We have examined the effects of the protein kinase C (PKC)-activator phorbol 12-myristate 13-acetate (PMA) on gene expression in two breast cancer cell (BCC) lines exhibiting highly different phenotypes. These are the estrogen receptor alpha (ERalpha)-positive, weakly invasive, luminal epithelial-like MCF-7 and the ERalpha-negative, highly invasive, fibroblast-like MDA-MB-231. They express constitutively low and high PKC activities, respectively. After a 24-h exposition to 100 nM PMA, the number of genes showing an altered expression at the 2-fold change level was much higher in MCF-7 (n=435) than in MDA-MB-231 (n=18) BCC. Four of these genes, namely CDC2, CENPA, NR4A1 and MMP10, were altered in the same way in both cell lines. Two genes were regulated in an opposite way: ID1 and EVA1. Many of the genes down-regulated in MCF-7 BCC appeared to be preferentially expressed in the G1, S, and/or G2 phases of the cell cycle. The ERalpha gene, ESR1, and other genes associated to the ERalpha-positive, luminal epithelial-like BCC phenotype were down-regulated, while a series of genes related to a more aggressive, fibroblast-like BCC phenotype were up-regulated. Other altered genes were notably linked to cell architecture, supporting profound effects of PMA on cell morphology and motility, as well as on the interactions between BCC and their neighboring proteins. Of note, all the modulated genes involved in proteolysis and its control were up-regulated. In summary, PMA effects suggest that PKC activation may induce, to some extent, a more fibroblast-like phenotype in the ERalpha-positive, luminal epithelial-like MCF-7 BCC, and significantly modulate the interactions of these cells with their environment.

Taurine concentration in the brain and in the plasma following intraperitoneal injections.

Summary. The effect of different taurine doses (0.050, 0.125, 0.250, 0.500 and 1.000 g/kg) administered intraperitoneally to Wistar rats was studied in both the plasma and the hippocampal microdialysate content. The samples were analyzed by reverse phased HPLC for the microdialysate samples and by HPLC with ion-exchange post-column derivatization (ninhydrin) for the plasma samples. In both plasma and microdialysate, we observed a dose dependent increase of taurine concentration. The AUC curves obtained from both microdialysate and plasma samples showed that the increase of taurine concentrations were linear. The mean ratio between AUC's microdialysate and plasma was 1.63+/-0.21 showing thus an unbalance between plasma and brain taurine content; a mechanism which enhance taurine transfer from the plasma to the brain was assumed.