Transcriptomic Study on Human Skin Samples: Identification of Two Subclasses of Actinic Keratoses.

Actinic keratoses (AKs) are sun-damaged skin areas that affect 20% of the European adult population and more than 50% of people aged 70 years and over. There are currently no clinical or histological features allowing us to identify to which clinical class (i.e., regression or progression) an AK belongs. A transcriptomic approach seems to be a robust tool for AK characterization, but there is a need for additional studies, including more patients and elucidating the molecular signature of an AK. In this context, the present study, including the largest number of patients to date, is the first aiming at identifying biological features to objectively distinguish different AK signatures. We highlight two distinct molecular profiles: AKs featuring a molecular profile similar to squamous cell carcinomas (SCCs), which are called "lesional AKs" (AK_Ls), and AKs featuring a molecular profile similar to normal skin tissue, which are called "non-lesional AKs" (AK_NLs). The molecular profiles of both AK subclasses were studied, and 316 differentially expressed genes (DEGs) were identified between the two classes. The 103 upregulated genes in AK_L were related to the inflammatory response. Interestingly, downregulated genes were associated with keratinization. Finally, based on a connectivity map approach, our data highlight that the VEGF pathway could be a promising therapeutic target for high-risk lesions.

A new algorithm for a better characterization and timing of the anti-VEGF vascular effect named "normalization".

Antiangiogenics are widely used in cancer treatment in combination with chemotherapy and radiotherapy for their vascular effects. Antiangiogenics are supposed to induce morphological and functional changes in the chaotic tumor vasculature that would help enhance the therapeutic efficacy of chemotherapy and radiotherapy through the amelioration of the drug delivery or the oxygenation in the tumor, respectively. However, finding the best treatment sequence is not an easy task to achieve and no consensus has yet been established because of the lack of knowledge regarding when and for how long the vascular network is ameliorated. The aim of this work was to develop a dedicated image processing algorithm able to analyze the vascular structures on optical microscopy images of the vascular network and to follow its fine modifications in vivo, over time. We applied this algorithm to follow the evolution of the vascular parameters (vascularized tissue surface, branches, sprouts and length), in response or not to anti-VEGF therapy (10 mg/kg/day) and determine precisely whether there is really a vascular "normalization" with anti-VEGF therapy in comparison with the parameters extracted from healthy vascular networks. We found that for this determination, the choice of region of interest to analyze is critical as it is important to compare only microcirculation areas and avoid areas with arteriole-venule-capillary hierarchy. The algorithm analysis allowed us to define a vascular "normalization" in treated tumors, between 8 and 12 days of bevacizumab treatment that was confirmed by standard immunohistochemical analysis, microvascular permeability assessment and immunohistological blood perfusion assessment.

PRKDC mutations associated with immunodeficiency, granuloma, and autoimmune regulator-dependent autoimmunity.

BACKGROUND: PRKDC encodes for DNA-dependent protein kinase catalytic subunit (DNA-PKcs), a kinase that forms part of a complex (DNA-dependent protein kinase [DNA-PK]) crucial for DNA double-strand break repair and V(D)J recombination. In mice DNA-PK also interacts with the transcription factor autoimmune regulator (AIRE) to promote central T-cell tolerance. OBJECTIVE: We sought to understand the causes of an inflammatory disease with granuloma and autoimmunity associated with decreasing T- and B-cell counts over time that had been diagnosed in 2 unrelated patients. METHODS: Genetic, molecular, and functional analyses were performed to characterize an inflammatory disease evocative of a combined immunodeficiency. RESULTS: We identified PRKDC mutations in both patients. These patients exhibited a defect in DNA double-strand break repair and V(D)J recombination. Whole-blood mRNA analysis revealed a strong interferon signature. On activation, memory T cells displayed a skewed cytokine response typical of TH2 and TH1 but not TH17. Moreover, mutated DNA-PKcs did not promote AIRE-dependent transcription of peripheral tissue antigens in vitro. The latter defect correlated in vivo with production of anti-calcium-sensing receptor autoantibodies, which are typically found in AIRE-deficient patients. In addition, 9 months after bone marrow transplantation, patient 1 had Hashimoto thyroiditis, suggesting that organ-specific autoimmunity might be linked to nonhematopoietic cells, such as AIRE-expressing thymic epithelial cells. CONCLUSION: Deficiency of DNA-PKcs, a key AIRE partner, can present as an inflammatory disease with organ-specific autoimmunity, suggesting a role for DNA-PKcs in regulating autoimmune responses and maintaining AIRE-dependent tolerance in human subjects.

Multifunctional ultrasmall nanoplatforms for vascular-targeted interstitial photodynamic therapy of brain tumors guided by real-time MRI.

Photodynamic therapy (PDT) for brain tumors appears to be complementary to conventional treatments. A number of studies show the major role of the vascular effect in the tumor eradication by PDT. For interstitial PDT (iPDT) of brain tumors guided by real-time imaging, multifunctional nanoparticles consisting of a surface-localized tumor vasculature targeting neuropilin-1 (NRP-1) peptide and encapsulated photosensitizer and magnetic resonance imaging (MRI) contrast agents, have been designed. Nanoplatforms confer photosensitivity to cells and demonstrate a molecular affinity to NRP-1. Intravenous injection into rats bearing intracranial glioma exhibited a dynamic contrast-enhanced MRI for angiogenic endothelial cells lining the neovessels mainly located in the peripheral tumor. By using MRI completed by NRP-1 protein expression of the tumor and brain adjacent to tumor tissues, we checked the selectivity of the nanoparticles. This study represents the first in vivo proof of concept of closed-head iPDT guided by real-time MRI using targeted ultrasmall nanoplatforms. From the clinical editor: The authors constructed tumor vascular peptide targeting multifunctional silica-based nanoparticles, with encapsulated gadolinium oxide as MRI contrast agent and chlorin as a photosensitizer, as a proof of concept novel treatment for glioblastoma in an animal model.

FVIIIra, CD15, and tryptase performance in the diagnosis of skin stab wound vitality in forensic pathology.

The timing of skin wounds is one of the most challenging problems in forensic pathology. In the first minutes or hours after infliction, histological examination fails to determine whether a wound was sustained before or after death. The aim of this study was to evaluate the use of three immunohistochemical markers (FVIIIra, CD15, and tryptase) for the interpretation of the timing of cutaneous stab wounds. We evaluated these markers in intravital wounds from autopsy cases (n = 12) and surgical specimens (n = 58). As controls, we used normal skin samples from autopsies (n = 8) and an original ex vivo surgical human model of recent postmortem wounds (n = 24). We found overexpression of FVIIIra in 100 % of vital wounds, but also in 53 % of the controls. The number of CD15-positive cells was higher in wound margins than in internal controls (p < 0.0001) and was significantly correlated with the time interval between incision and devascularization (p = 0.0005; minimal time for positivity, 9 min). Using the anti-tryptase antibody, we found that the mast cell degranulation rate was higher in wound margins (p < 0.0001) and correlated with the time interval (minimal time, 1 min). The sensitivity and specificity for the diagnosis of vitality were respectively 100 and 47 % for FVIIIra, 47 and 100 % for CD15, and 60 and 100 % for tryptase. The inter-observer agreement coefficients were 0.68 for FVIIIra, 0.90 for CD15, and 0.46 for tryptase. Finally, we demonstrated that these markers were not reliable in putrefied or desiccated specimens. In conclusion, CD15 and tryptase, but not FVIIIra, may be useful markers for differentiating recent antemortem from postmortem injuries.

[The markers of wound vitality in forensic pathology]. / Les marqueurs de vitalité des blessures en pathologie médicolégale.

Skin wounds datation is one of the most challenging problems in forensic pathology. The vitality of a recent wound cannot be affirmed when no inflammatory cell is visible. There are in the literature numerous studies about wound vitality, looking for markers involved in coagulation or inflammation, using various methods such as enzymology, molecular biology or immunohistochemistry. In this update, we first introduce some methodological principles to respect. Then, we review the main studies available in the literature. We insist on immunohistochemistry, which seems to be the more valuable method, given its easiness to perform and the possibility to analyze the localization of the molecules of interest. Some markers are promising, such as TNFα, IL-6, IL-1ß, TGFα or TGFß1. Before using them in daily practice, these first results need to be confirmed with other studies, driven by independent teams and integrating multiple controls. Most notably, the antibodies have to be tested in numerous post-mortem wounds. Indeed, there is a critical risk of overexpression in post-mortem wounds, and some interesting markers have been secondary invalidated because of post-mortem false positivity (e.g. fibronectin, P-selectin). Finally, optimal sensibility and specificity values would be probably reached by combining several markers, validated with large groups of pre- and post-mortem wounds.

The role of platelets and von Willebrand factor in the procoagulant phenotype of inflammatory bowel disease.

OBJECTIVE: Although the risk for thrombosis is well documented for inflammatory bowel disease (IBD) patients, the underlying pathological mechanism seems to be different from other thrombotic conditions. Deciphering the actors responsible for the increased risk of thrombosis in IBD would help to improve management of this frequent complication. DESIGN: We studied the interplay between platelets, coagulation, and von Willebrand factor (VWF) in 193 IBD patients and in experimental models (acute and chronic) of colitis in wild-type and VWF-deficient mice. RESULTS: We found a platelet-dependent increase in thrombin generation in IBD patients and in our mouse model of colitis. Agglutinated platelets were present in the blood of patients and mice. Interestingly, we observed not only a significant increase in total VWF antigen, but we were able to detect the presence of active VWF (VWF in its platelet-binding conformation; 3.2±2.7µg/ml) in the plasma of 30% of all IBD patients. In healthy controls, active VWF levels were below 0.3µg/ml. This led us to further explore experimental colitis in VWF-deficient mice and we observed that these mice were protected against the procoagulant state triggered by the colitis. Unexpectedly, these mice also manifested a significant worsening of colitis severity both in acute and chronic models. CONCLUSION: Platelets and VWF (including its active form) appear to be central players in the procoagulant phenotype in IBD. We observed that the role of VWF in hemostasis differs from its role in colic tissue healing, potentially opening new therapeutic avenues for a life-threatening complication in IBD patients.

Involvement of the TGFß pathway in the regulation of α5 ß1 integrins by caveolin-1 in human glioblastoma.

Caveolin-1 plays a crucial role in the development of cancer and its progression. We previously reported that glioblastoma cells expressing low levels of caveolin-1 exerted a more aggressive phenotype than cells expressing high levels. Such phenotype was due to the induction of α(5) ß(1) integrin subsequent to the depletion of caveolin-1. Caveolin-1 was identified as a transcriptional repressor of α(5) ß(1) integrin. The current study was designed to identify in vitro, the molecular mechanisms by which caveolin-1 controls α(5) ß(1) integrin expression and to determine if a negative correlation between caveolin-1 and α(5) ß(1) integrins also exists in biopsies and xenografted human brain tumors. We showed that depletion of caveolin-1 lead to the activation of the TGFß/TGFßRI/Smad2 pathway which in turn induced the expression of α(5) ß(1) integrins. We showed that cells expressing the lowest levels of caveolin-1 but the highest levels of α(5) ß(1) integrins and TGFßRI were the most sensitive to a α(5) ß(1) integrin antagonist and a TGFßRI inhibitor. Screening human glioma biopsies and human glioblastoma xenografts, we isolated subgroups with either low levels of caveolin-1 but high levels of α(5) ß(1) integrin and TGFßRI or high levels of caveolin-1 but low levels of α(5) ß(1) integrin and TGFßRI. In conclusion, caveolin-1 controls α(5) ß(1) integrin expression through the TGFß/TGFßRI/Smad2 pathway. The status of caveolin-1/α(5) ß(1) integrins/TGFßRI might be a useful marker of the tumor evolution/prognosis as well as a predictor of anti-TGFß or anti-α(5) ß(1) integrin therapies.

The VWF/LRP4/αVß3-axis represents a novel pathway regulating proliferation of human vascular smooth muscle cells.

AIMS: Von Willebrand factor (VWF) is a plasma glycoprotein involved in primary haemostasis, while also having additional roles beyond haemostasis namely in cancer, inflammation, angiogenesis, and potentially in vascular smooth muscle cell (VSMC) proliferation. Here, we addressed how VWF modulates VSMC proliferation and investigated the underlying molecular pathways and the in vivo pathophysiological relevance. METHODS AND RESULTS: VWF induced proliferation of human aortic VSMCs and also promoted VSMC migration. Treatment of cells with a siRNA against αv integrin or the RGT-peptide blocking αvß3 signalling abolished proliferation. However, VWF did not bind to αvß3 on VSMCs through its RGD-motif. Rather, we identified the VWF A2 domain as the region mediating binding to the cells. We hypothesized the involvement of a member of the LDL-related receptor protein (LRP) family due to their known ability to act as co-receptors. Using the universal LRP-inhibitor receptor-associated protein, we confirmed LRP-mediated VSMC proliferation. siRNA experiments and confocal fluorescence microscopy identified LRP4 as the VWF-counterreceptor on VSMCs. Also co-localization between αvß3 and LRP4 was observed via proximity ligation analysis and immuno-precipitation experiments. The pathophysiological relevance of our data was supported by VWF-deficient mice having significantly reduced hyperplasia in carotid artery ligation and artery femoral denudation models. In wild-type mice, infiltration of VWF in intimal regions enriched in proliferating VSMCs was found. Interestingly, also analysis of human atherosclerotic lesions showed abundant VWF accumulation in VSMC-proliferating rich intimal areas. CONCLUSION: VWF mediates VSMC proliferation through a mechanism involving A2 domain binding to the LRP4 receptor and integrin αvß3 signalling. Our findings provide new insights into the mechanisms that drive physiological repair and pathological hyperplasia of the arterial vessel wall. In addition, the VWF/LRP4-axis may represent a novel therapeutic target to modulate VSMC proliferation.

Recombinant activated factor VII attenuates major arterial bleeding in noncoagulopathic rabbits.

BACKGROUND AND OBJECTIVE: Recombinant activated factor VII (rFVIIa), which is used off-label as an adjuvant therapy for uncontrolled and life-threatening bleeding, might also attenuate intractable bleeding related to macrovascular arterial lesions. Here we evaluated the efficacy of rFVIIa in sealing a large arterial wound in haemostatically competent rabbits. METHODS: Sixty male New Zealand rabbits were randomly divided into vehicle control and 80 and 200 µg kg⁻¹ rFVIIa groups (n = 20 animals each). A standardized wound of the isolated right carotid artery was made in all rabbits with an 18-G catheter. Bleeding, which was limited by mild compression, was assessed every minute. At 5 min, an intravenous bolus of vehicle or human rFVIIa was given and the animals were further observed for 1 h. Efficacy was assessed from the bleeding duration and blood mass lost. Statistical significance was defined as P less than 0.05. All investigators were blinded to the treatment the animals received. RESULTS: The bleeding duration and blood mass lost were significantly reduced in both rFVIIa dosage groups as compared with the vehicle control group. For the vehicle, 80 and 200 µg kg⁻¹ rFVIIa groups, the median bleeding durations were 56 min (range 7-60 min), 15 min (range 5-60 min) and 10 min (range 5-60 min), respectively; and the median blood mass losses were 22.5 g (range 1-58 g), 12 g (range 0-36 g) and 5 g (range 0-31 g), respectively. The prothrombin time was shorter in the rFVIIa groups. Visual inspection of the carotid artery and microscopic analysis of the liver and kidney revealed neither gross thrombi nor entrapped microthrombi in any rabbit. CONCLUSION: Recombinant FVIIa at 80 or 200 µg kg⁻¹ promoted the sealing of a large and slightly compressed arterial wound in rabbits. These results suggest a potential role for the drug in the management of massive bleeds due to an arterial lesion when surgical intervention is not immediately and readily available. Safety should remain a matter of concern.

Neuropilin-1 targeting photosensitization-induced early stages of thrombosis via tissue factor release.

PURPOSE: This article characterizes the vascular effects following vascular-targeted photodynamic therapy with a photosensitizer which actively targets endothelial cells. METHODS: This strategy was considered by coupling a chlorin to a heptapeptide targeting neuropilin-1 in human malignant glioma-bearing nude mice. A laser Doppler microvascular perfusion monitor was used to monitor microvascular blood perfusion in tumor tissue. Endothelial cells' ultra structural integrity was observed by transmission electron microscopy. The consequences of photosensitization on tumor vessels, tissue factor expression, fibrinogen consumption, and thrombogenic effects were studied by immunohistochemical staining. RESULTS: Treatment of glioma-bearing mice with the conjugate showed a statistically significant tumor growth delay. Vascular effect was characterized by a decrease in tumor tissue blood flow at about 50% baseline during treatment not related to variations in temperature. This vascular shutdown was mediated by tumor blood vessels' congestion. A pro-thrombotic behavior of targeted endothelial cells in the absence of ultra structural changes led to the induction of tissue factor expression from the earliest times post-treatment. Expression of tissue factor-initiated thrombi formation was also related to an increase in fibrinogen consumption. CONCLUSION: Using a peptide-conjugated photosensitizer targeting neuropilin-1, induction of tissue factor expression immediately post-treatment, led to the establishment of thrombogenic effects within the vessel lumen.

Assessment of apoptosis by immunohistochemistry to active caspase-3, active caspase-7, or cleaved PARP in monolayer cells and spheroid and subcutaneous xenografts of human carcinoma.

Immunohistochemistry to active caspase-3, recently recommended for apoptosis detection, is inappropriate to detect apoptosis involving caspase-7. Cleavage of poly-ADP-ribose polymerase 1 (PARP-1), a major substrate of both caspases, is a valuable marker of apoptosis. Apoptosis evaluation induced in vitro either by paclitaxel or by photodynamic treatment (PDT) with Foscan in HT29 or KB monolayer cells and HT29 spheroids yielded a close percentage of labeled cells whatever the antibody used, whereas in control specimens, cleaved PARP (c-PARP) immunostaining failed to detect apoptosis as efficiently as active caspase-3 or -7 immunostaining. Studies in MDA-MB231 monolayer cells and HT29 xenografts either subjected or not subjected to Foscan-PDT resulted in a significant higher number of active caspase-3-labeled cells, although immunofluorescence analysis showed c-PARP and active caspase-3 perfectly colocalized in tumors. A restricted expression of c-PARP was obvious in the greater part of caspase-3 expressing cells from control tumor, whereas photosensitized tumors showed a higher number of cells expressing large fluorescent spots from both active caspase-3 and c-PARP. These results support the assumption that c-PARP expression was dependent on treatment-induced apoptosis. The absence of caspase-7 activation in some caspase-3-expressing cells undergoing Foscan-PDT shows the relevance of using antibodies that can discriminate caspase-dependent apoptotic pathways.

Increased expression of matrilin-3 not only in osteoarthritic articular cartilage but also in cartilage-forming tumors, and down-regulation of SOX9 via epidermal growth factor domain 1-dependent signaling.

OBJECTIVE: To identify regulators of the cartilaginous phenotype, on the basis of their differential expression in human conventional chondrogenic tumors compared with articular cartilage. METHODS: Differential proteomics analysis revealed matrilin-3 (MATN3) as a candidate regulator of the cartilaginous phenotype. Its capacity to modulate gene expression was investigated in human HCS-2/8 chondrosarcoma cells and transfected chondrocytes, using cell culture fractionation, reverse transcription-polymerase chain reaction, and Western blot analyses. RESULTS: Increased expression of the cartilage-specific matrix protein MATN3 was specifically observed in enchondromas and conventional chondrosarcomas. A substantial fraction of MATN3 was found in cytoplasmic structures of tumor cells, as demonstrated by immunohistochemistry. Analyses of intracellular MATN3 revealed that it corresponded to an imperfectly maturated MATN3 polypeptide, both in HCS-2/8 human chondrosarcoma cells and in transfected human chondrocytes. Moderately increased expression of MATN3 resulted in its intracellular retention. Antibody-mediated blockade of soluble, extracellular MATN3 in HCS-2/8 cell cultures resulted in increased expression of MATN3 and the chondrogenic transcription factor SOX9. Conversely, increased ectopic expression of MATN3 resulted in decreased expression of MATN3 and SOX9 in primary chondrocytes, while a mutant MATN3 lacking its first epidermal growth factor (EGF)-like domain failed to down-regulate SOX9. CONCLUSION: Aberrant expression and processing of MATN3 are hallmarks of conventional cartilaginous neoplasms. A particular step in the maturation of MATN3 limits its processing through the secretion machinery, resulting in its intracellular accumulation upon increased expression. Soluble, secreted MATN3, however, down-regulates SOX9 at the messenger RNA and protein levels. The first EGF-like domain of MATN3 is a critical determinant of its regulatory activity toward SOX9. These activities of MATN3 suggest that its increased expression in osteoarthritis might contribute to the degeneration of articular cartilage.

[Detection of apoptosis in vivo: comparison of different methods in histological sections of subcutaneous xenografts of HT29 human colon adenocarcinoma]. / Quantification de l'apoptose in vivo: comparaison de méthodes de détection sur coupes histologiques de tumeurs xénogreffées issus de la lignée cellulaire humaine d'adénocarcinome colique HT-29.

UNLABELLED: Apoptosis detection in histological section is very important, but usual methods (TUNEL and morphologic changes) are controversial. Immunohistochemical stains of activated proteins during apoptosis improve detection of cell death in tissue sections. Activated caspase-3 and cleaved cytokeratin-18 are more and more used. OBJECTIVE: This study compared immunohistochemical markers (activated caspase-3, cleaved cytokeratin-18 and two antibodies not yet evaluated: activated caspase-7 and cleaved PARP-1), cellular morphology and TUNEL. MATERIAL AND METHODS: Tumour xenografts of the human colon cancer cell line HT29 were used, treated by photodynamic therapy, to obtain large numbers of cells undergoing apoptosis. Apoptotic cells were quantified and apoptotic indices were determined for each marker. RESULTS: Comparison of apoptosis index indicated statistically best sensibility with activated-caspase-3 and cleaved-cytokeratin-19. CONCLUSION: Immunohistochemistry for activated caspase-3 and cleaved cytkeratin-18 appear to be an easy, sensitive, and reliable method for detecting and quantifying apoptosis in this model. There are therefore recommended for the detection and quantification of apoptosis in tissue sections. Other markers as cleaved PARP-1 and activated caspase-7 can be an interessant solution: advantages and inconvenience for each methods are exposed.

[Glyoxal: a possible polyvalent substitute for formaldehyde in pathology?]. / Le glyoxal : un possible substitut polyvalent du formaldéhyde en anatomie pathologique ?

The quest for formaldehyde substitutes is motivated by two fundamental developments: the OSHA regulation standard declaring it hazardous and advocating its substitution with less dangerous chemicals and the fact that formalin is a poor preserver of nucleic acids. Among the non-alcoholic formalin substitute, glyoxal has been hailed as the best alternative. In this work, we showed that glyoxal-containing fixatives are not plausible polyvalent substitution options.

Measurement of hypoxia using invasive oxygen-sensitive electrode, pimonidazole binding and 18F-FDG uptake in anaemic or erythropoietin-treated mice bearing human glioma xenografts.

Relationship between haemoglobin levels and tumour oxygenation has been already reported. The purpose of this work was to compare in human malignant glioma-bearing mice the sensitivity of two well established techniques of tumour hypoxia assessment, especially their ability to detect expected weak variations of tumour oxygenation status associated to haemoglobin level modifications. The relationship between tumour hypoxia and glucose metabolism was also investigated. Experiments were performed on a human malignant glioma (GBM Nan1) xenografted into nude mice. Twenty-four hours after tumour implantation, animals were randomized into three groups: 'Anaemia' for mice subjected to repeated blood samplings, 'Control', and 'rHuEPO' for mice receiving recombinant human erythropoietin. Once the tumours reached a volume of 300+/-100 mm(3), tumour hypoxia was assessed both using the pO(2)-Histograph, Eppendorftrade mark and the pimonidazole binding assay. Glucose metabolism was evaluated by (18)F-FDG autoradiography and compared with the pimonidazole binding distribution pattern. Repeated blood samplings significantly reduced mean haemoglobin levels (10.9+/-2.0 g/dl), inducing chronic anaemia in mice, while daily administration of rHuEPO led to increase of haemoglobin levels (15.8+/-2.0 g/dl). Oxygenation status evaluated by a microelectrode was worsened in anaemic mice (mean pO(2) in tumour = 6.9+/-0.8 mmHg) and improved in rHuEPO-treated animals (mean pO(2)in tumour = 11.4+/-1.2 mmHg). No correlation was observed between the oxygen-sensitive probe and pimonidazole labelling results: both techniques give different but complementary information about tumour hypoxia. Areas of high pimonidazole binding and areas of high (18)F-FDG uptake superimposed well. Present results confirm that modification of haemoglobin levels leads to alteration of tumour oxygenation status. These variations were detectable using the oxygen-sensitive electrode but not the pimonidazole binding assay. The strong correlation between pimonidazole labelling and (18)F-FDG uptake suggests a positive relationship between hypoxia and increased glucose metabolism in this tumour model.

Proteasome inhibition by bortezomib does not translate into efficacy on two malignant glioma xenografts.

Bortezomib and other proteasome inhibitors have demonstrated an interesting antitumor activity against glioma cell lines. The present study aimed to evaluate the cytotoxic potential of bortezomib in vivo on two human malignant glioma xenografts using doses relevant to clinical practice. The TCG3 and U87 malignant glioma xenografts were heterotopically implanted onto nude mice. Bortezomib effects were evaluated using the three different doses of 0.25, 0.45 and 0.90 mg/kg. Proteasome chymotrypsin-like activity was measured by a fluorimetric method. Analysis of the cell cycle distribution was performed after propidium iodide staining. The apoptotic rate and proliferative index were determined by an immunohistochemical detection of cleaved caspase-3 and Ki-67, respectively. Our data showed that bortezomib induced a dose-dependent inhibition of proteasome chymotrypsin-like activity in the two glioma models. Maximal inhibition was achieved 24 h after drug injection and was approximately 30% of basal proteasome activity. However, this effect did not induce any increase in the apoptotic rate and did not modify cell cycle distribution. At the maximal dose tested (0.90 mg/kg), bortezomib did not show any growth delay as compared to untreated tumors, in either of the xenograft models. In conclusion, our study is the first to demonstrate that bortezomib, at a clinically relevant dose, did not have any effect on the apoptosis and proliferation of malignant gliomas in vivo. These results contrast with the promising preclinical data obtained in vitro with this drug and emphasize the importance of performing preclinical studies on animal models, in conditions close to clinical settings.

Expression of PPARgamma is reduced by medium supplementation with L-glutamine in human colorectal Caco-2 cells.

Peroxisome proliferator-activated receptor-gamma (PPARgamma) belongs to the nuclear hormone receptor family. This receptor is implicated in colon cell differentiation and in colon cancer. Receptor activation by specific agonists has been shown to protect against colon cancer progression. PPARgamma protein content within cells is modulated by several mechanisms, including proteasome degradation, activation of Wnt signalling pathways and presence of fermentation products such as butyrate. Herein, we investigated the impact of L-glutamine on PPARgamma expression during the differentiation of Caco-2 cells grown in medium containing dialyzed fetal calf serum supplemented or not with L-glutamine. Using RT-PCR and Western blotting, we demonstrated that PPARgamma expression was decreased when L-glutamine was added to the medium. Using immunohistochemistry, we demonstrated that PPARgamma immunostaining was mainly found in cytoplasm when cells were cultured with L-glutamine while it was found in nuclei and cytoplasm when cells were grown without the addition of L-glutamine. Supershift retardation assays demonstrated a decrease of PPARgamma binding onto consensus peroxisome proliferator response element. We concluded that L-glutamine modulated PPARgamma expression in Caco-2 cells.