Effect of Zr3Fe addition on hydrogen storage behaviour of Ti2CrV alloys.

In this work, the hydrogen storage behavior of Ti2CrV + X wt.% Zr3Fe, where X = 2, 4, 6, 8 and 10 was investigated. The synthesis of all samples was carried out through arc-melting, followed by comprehensive characterization using X-ray diffraction, scanning electron microscopy, and energy-dispersive spectroscopy. The pure-Ti2CrV as-cast sample presented a single-phase microstructure. However, the addition of the Zr3Fe led to a remarkable transformation, resulting in the appearance of a Zr-rich secondary phase. It was found that the first hydrogenation is improved with the addition of at least 6 wt% of Zr3Fe, avoiding any preheating of the sample. These samples achieved their maximum capacity in approximately 10 min at room temperature. The maximum capacity recorded was 4.2 wt% H for the sample with X = 6 wt% Zr3Fe, while for X = 8 and 10 wt% Zr3Fe, the capacity recorded was 4.1 wt% and 4.0 wt%, respectively.

Microstructure and First Hydrogenation Properties of TiHfZrNb1-xV1+x Alloy for x = 0, 0.1, 0.2, 0.4, 0.6 and 1.

The effect of the substitution of Nb by V on the microstructure and hydrogen storage properties of TiHfZrNb1-xV1+x alloys (x = 0.1, 0.2, 0.4, 0.6 and 1) was investigated. For x = 0, the alloy was pure BCC and upon the substitution of niobium by vanadium, the BCC was progressively replaced by HCP and FCC phases. For x = 0.6, a C15 phase was also present and becomes the main phase for x = 1. The substitution greatly enhanced the first hydrogenation and makes it possible at room temperature under 20 bars of hydrogen. The capacity of all substituted alloys was around 2 wt.%.

Enhancement of First Hydrogenation of Ti1V0.9Cr1.1 BCC Alloy by Cold Rolling and Ball Milling.

In this study, we evaluated the effects of a mechanical treatment by cold rolling (CR) and ball milling (BM) on the first hydrogenation of Ti1V0.9Cr1.1 alloy. The as-cast alloy has a body-centered cubic (BCC) crystal structure, and the first hydrogenation at room temperature under 20 bars of hydrogen is practically impossible. However, the samples mechanically activated by CR or BM readily absorbed hydrogen. The sample cold-rolled for one pass exhibited faster kinetics than the sample ball-milled for five minutes, but both samples reached the same storage capacity of 3.6 wt % hydrogen. Increasing the amount of rolling or the milling time decreased the hydrogen capacity. CR is considered the best and most efficient method for the activation of Ti1V0.9Cr1.1 alloy.

Mechanochemistry of Metal Hydrides: Recent Advances.

This paper is a collection of selected contributions of the 1st International Workshop on Mechanochemistry of Metal Hydrides that was held in Oslo in May 2018. In this paper, the recent developments in the use of mechanochemistry to synthesize and modify metal hydrides are reviewed. A special emphasis is made on new techniques beside the traditional way of ball milling. High energy milling, ball milling under hydrogen reactive gas, cryomilling and severe plastic deformation techniques such as High-Pressure Torsion (HPT), Surface Mechanical Attrition Treatment (SMAT) and cold rolling are discussed. The new characterization method of in-situ X-ray diffraction during milling is described.

Microstructure Optimization of Mg-Alloys by the ECAP Process Including Numerical Simulation, SPD Treatments, Characterization, and Hydrogen Sorption Properties.

Both numerical simulation and hardness measurements were used to determine the mechanical and microstructural behavior of AZ31 bulk samples when submitted to the Equal Channel Angular Pressing (ECAP) technique. Billets of this representative of Mg-rich alloys were submitted to different numbers of passes for various ECAP modes (anisotropic A, isotropic BC). The strain distribution, the grain size refinement, and the micro-hardness were used as indicators to quantify the effectiveness of the different processing routes. Structural characterizations at different scales were achieved using Scanning Electron Microscopy (SEM), micro-analysis, metallography, Small Angle Neutron Scattering SANS, X-Ray Diffraction (XRD), and texture determination. The grain and crystallite size distribution and orientation as well as defect impacts were determined. Anelastic Spectroscopy (AS) on mechanically deformed samples have shown that the temperature of ECAP differentiate the fragile to ductile regime. MgH2 consolidated powders were checked for using AS to detect potential hydrogen motions and interaction with host metal atoms. After further optimization, the different mechanically-treated samples were submitted to hydrogenation/dehydrogenation (H/D) cycles, which shows that, for a few passes, the BC mode is better than the A one, as supported by theoretical and experimental microstructure analyses. Accordingly, the hydrogen uptake and (H/D) reactions were correlated with the optimized microstructure peculiarities and interpreted in terms of Johnson-Avrami- Mehl-Kolmogorov (JAMK) and Jander models, successively.

Endothelial microRNAs regulating the NF-κB pathway and cell adhesion molecules during inflammation.

The surface of endothelial cells is covered with cell adhesion molecules, including E-selectin, intercellular adhesion molecule 1 (ICAM-1), and vascular cell adhesion molecule 1 (VCAM- 1) , that mediate the adhesion and extravasation of leukocytes and play pivotal roles in inflammatory response. microRNAs (miRNAs) regulate the expression of these important cell adhesion molecules through two distinct major mechanisms, namely via modulating the proinflammatory NF-κB pathway, which controls their transcription, and via directly targeting them. The present review highlights the role of various miRNAs in controlling the expression of E-selectin, ICAM-1, and VCAM-1: a type of regulation that can be harnessed for therapeutic prevention of inflammation-associated diseases such as atherosclerosis and sepsis. The roles of secreted miRNAs as paracrine regulators, and cell adhesion molecule-based miRNA delivery are also addressed.-Zhong, L., Simard, M. J., Huot, J. Endothelial microRNAs regulating the NF-κB pathway and cell adhesion molecules during inflammation.

p38 activation induces production of miR-146a and miR-31 to repress E-selectin expression and inhibit transendothelial migration of colon cancer cells.

Extravasation of circulating cancer cells determines their metastatic potential. This process is initiated by the adhesion of cancer cells to vascular endothelial cells through specific interactions between endothelial adhesion receptors such as E-selectin and their ligands on cancer cells. In the present study, we show that miR-146a and miR-181b impede the expression of E-selectin by repressing the activity of its transcription factor NF-κB, thereby impairing the metastatic potentials of colon cancer cells by decreasing their adhesion to, and migration through, the endothelium. Among the two microRNAs, only miR-146a is activated by IL-1ß, through the activation of p38, ERK and JNK MAP kinases, as well as their downstream transcription factors GATA2, c-Fos and c-Jun. Inhibiting p38 MAP kinase increases NF-κB activity, at least partially via miR-146a. Inhibiting p38 also increases the expression of E-selectin at the post-transcriptional level via decreasing miR-31, which targets E-selectin mRNA and also depends on p38 for its expression. In response to IL-1ß, p38 MAP kinase hence represses the expression of E-selectin at the transcriptional and the post-transcriptional levels, via miR-146a and miR-31, respectively. These results highlight novel mechanisms by which p38 downregulates the expression of E-selectin through different microRNAs following inflammatory stimuli associated to cancer progression.

The p38 pathway, a major pleiotropic cascade that transduces stress and metastatic signals in endothelial cells.

By gating the traffic of molecules and cells across the vessel wall, endothelial cells play a central role in regulating cardiovascular functions and systemic homeostasis and in modulating pathophysiological processes such as inflammation and immunity. Accordingly, the loss of endothelial cell integrity is associated with pathological disorders that include atherosclerosis and cancer. The p38 mitogen-activated protein kinase (MAPK) cascades are major signaling pathways that regulate several functions of endothelial cells in response to exogenous and endogenous stimuli including growth factors, stress and cytokines. The p38 MAPK family contains four isoforms p38α, p38ß, p38γ and p38δ that are encoded by four different genes. They are all widely expressed although to different levels in almost all human tissues. p38α/MAPK14, that is ubiquitously expressed is the prototype member of the family and is referred here as p38. It regulates the production of inflammatory mediators, and controls cell proliferation, differentiation, migration and survival. Its activation in endothelial cells leads to actin remodeling, angiogenesis, DNA damage response and thereby has major impact on cardiovascular homeostasis, and on cancer progression. In this manuscript, we review the biology of p38 in regulating endothelial functions especially in response to oxidative stress and during the metastatic process.

p38 and JNK pathways control E-selectin-dependent extravasation of colon cancer cells by modulating miR-31 transcription.

Extravasation of circulating cancer cells is a key event of metastatic dissemination that is initiated by the adhesion of cancer cells to vascular endothelial cells. It requires the interaction between adhesion receptors such as E-selectin present on endothelial cells and their ligands on cancer cells. Notably, E-selectin influences the metastatic potential of breast, bladder, gastric, pancreatic, and colorectal carcinoma as well as of leukemia and lymphoma. Here, we show that E-selectin expression induced by the pro-inflammatory cytokine IL-1ß is directly and negatively regulated by miR-31. The transcription of miR-31 is activated by IL-1ß. This activation depends on p38 and JNK MAP kinases, and their downstream transcription factors GATA2, c-Fos and c-Jun. The miR-31-mediated repression of E-selectin impairs the metastatic potential of colon cancer cells by decreasing their adhesion to, and migration through, the endothelium. These results highlight for the first time that microRNA mediates E-selectin-dependent extravasation of colon cancer cells.

Oxidative stress disassembles the p38/NPM/PP2A complex, which leads to modulation of nucleophosmin-mediated signaling to DNA damage response.

Oxidative stress is a leading cause of endothelial dysfunction. The p38 MAPK pathway plays a determinant role in allowing cells to cope with oxidative stress and is tightly regulated by a balanced interaction between p38 protein and its interacting partners. By using a proteomic approach, we identified nucleophosmin (NPM) as a new partner of p38 in HUVECs. Coimmunoprecipitation and microscopic analyses confirmed the existence of a cytosolic nucleophosmin (NPM)/p38 interaction in basal condition. Oxidative stress, which was generated by exposure to 500 µM H2O2, induces a rapid dephosphorylation of NPM at T199 that depends on phosphatase PP2A, another partner of the NPM/p38 complex. Blocking PP2A activity leads to accumulation of NPM-pT199 and to an increased association of NPM with p38. Concomitantly to its dephosphorylation, oxidative stress promotes translocation of NPM to the nucleus to affect the DNA damage response. Dephosphorylated NPM impairs the signaling of oxidative stress-induced DNA damage via inhibition of the phosphorylation of ataxia-telangiectasia mutated and DNA-dependent protein kinase catalytic subunit. Overall, these results suggest that the p38/NPM/PP2A complex acts as a dynamic sensor, allowing endothelial cells to react rapidly to acute oxidative stress.-Guillonneau, M., Paris, F., Dutoit, S., Estephan, H., Bénéteau, E., Huot, J., Corre, I. Oxidative stress disassembles the p38/NPM/PP2A complex, which leads to modulation of nucleophosmin-mediated signaling to DNA damage response.

Enhancement of the initial hydrogenation of Mg by ball milling with alkali metal amides MNH2 (M = Li or Na).

The introduction of 4 wt% of MNH2 (M = Li, Na) and other additives (Li, MgH2, NaCl, and NaBr) into pure Mg by ball milling greatly enhances the first hydrogenation (activation). Under 2 MPa of H2 at 608 K, the best activation performance is achieved with the NaNH2 additive.

MicroRNA-196a promotes an oncogenic effect in head and neck cancer cells by suppressing annexin A1 and enhancing radioresistance.

Radiotherapy is a major treatment modality for head and neck squamous cell carcinoma (HNSCC). Up to 50% of patients with locally advanced disease relapse after radical treatment and there is therefore a need to develop predictive bomarkers for clinical use that allow the selection of patients who are likely to respond. MicroRNA (miRNA) expression profiling of a panel of HNSCC tumours with and without recurrent disease after surgery and radiotherapy detected miR-196a as one of the highest upregulated miRNAs in the poor prognostic group. To further study the role of miR-196a, its expression was determined in eight head and neck cancer cell lines. Overexpression of miR-196a in HNSCC cells, with low endogenous miR-196a expression, significantly increased cell proliferation, migration and invasion, and induced epithelial to mesenchymal transition. Conversely, miR-196a knockdown in cells with high endogenous expression levels significantly reduced oncogenic behaviour. Importantly, overexpression of miR-196a increased radioresistance of cells as measured by gamma H2AX staining and MTT survival assay. Annexin A1 (ANXA1), a known target of miR-196a, was found to be directly modulated by miR-196a as measured by luciferase assay and confirmed by Western blot analysis. ANXA1 knockdown in HNSCC exhibited similar phenotypic effects to miR-196a overexpression, suggesting the oncogenic effect of miR-196a may at least be partly regulated through suppression of ANXA1. In conclusion, this study identifies miR-196a as a potential important biomarker of prognosis and response of HNSCC to radiotherapy. Furthermore, our data suggest that miR-196a and/or its target gene ANXA1 could represent important therapeutic targets in HNSCC.

Hydrogenation Properties of TiFe Doped with Zirconium.

The goal of this study was to optimize the activation behaviour of hydrogen storage alloy TiFe. We found that the addition of a small amount of Zr in TiFe alloy greatly reduces the hydrogenation activation time. Two different procedural synthesis methods were applied: co-melt, where the TiFe was melted and afterward re-melted with the addition of Zr, and single-melt, where Ti, Fe and Zr were melted together in one single operation. The co-melted sample absorbed hydrogen at its maximum capacity in less than three hours without any pre-treatment. The single-melted alloy absorbed its maximum capacity in less than seven hours, also without pre-treatment. The reason for discrepancies between co-melt and single-melt alloys was found to be the different microstructure. The effect of air exposure was also investigated. We found that the air-exposed samples had the same maximum capacity as the argon protected samples but with a slightly longer incubation time, which is probably due to the presence of a dense surface oxide layer. Scanning electron microscopy revealed the presence of a rich Zr intergranular phase in the TiFe matrix, which is responsible for the enhanced hydrogenation properties of these Zr-doped TiFe alloys.

In-Vitro and Ex-Vivo Investigations of the Microtubule Binding Drug Targetin on Angiogenesis.

BACKGROUND: Intervention aimed at disrupting or inhibiting newly formed vascular network is highly desired to attenuate the progression of angiogenesis-dependent diseases. In cancer, this is tightly associated with the generation of VEGF by hypoxia inducible factor-1α following its activation by hypoxia. In light of the multiple cellular roles played by microtubules and their involvement in the processing of the hypoxia inducible factor-1α transcript, modulation of microtubule dynamics is emerging as a logical approach to suppress tumor reliance on angiogenesis. Targetin is a novel noscapinoid that interferes with microtubule dynamicity and inhibits the growth of cell lines from many types of cancers. METHODS AND RESULTS: Utilizing in-vitro and ex-vivo angiogenic models, we discovered the vascular disrupting and anti-angiogenic properties of Targetin. Targetin disrupted pre-assembled capillary-like networks of human endothelial cells by severing cell-cell junctions, inhibiting endothelial cell proliferation and metabolic activity in the presence and absence of vascular endothelial growth factor (VEGF) and basic fibroblast growth factor (bFGF). Furthermore, we show that Targetin significantly inhibits the formation of neovasculature network sprouting from rat aortic explants stimulated with proangiogenic stimuli, namely VEGF or bFGF. CONCLUSION: We conclude that Targetin is a potential clinically promising anti-angiogenic agent for the treatment of many diseases including cancers.

Development of a tridimensional microvascularized human skin substitute to study melanoma biology.

Cutaneous malignant melanomas represent an important clinical problem because they are highly invasive, they can metastasize to distant sites and are typically resistant to available therapy. The precise molecular determinants responsible for melanoma progression and chemo-resistance are not yet known, in part due to lack of pertinent experimental models that mimic human melanoma progression. Accordingly, we developed a complex human microvascularized reconstructed skin substitute in which the organized three-dimensional (3D) architecture of the native skin is reproduced. Human melanoma cell lines derived from primary and metastatic sites were added to this 3D model. Our results demonstrate that histological features and behavior of melanoma cells applied in our skin substitute model are specific to their site of origin. In particular, the ability of melanoma cells to cross the dermal-epidermal junction correlates with their metastatic potential. In addition, a potent angiogenic effect was detected for an aggressive metastatic cell line that produces VEGF. The presence of a microvascular network within this model will allow studying a crucial step of the metastatic process. We conclude that such an in vitro human tumor microvascularized reconstructed skin substitute promises to be a versatile and efficient model to investigate skin cancer progression and to screen new anticancer drugs to improve currents clinical treatments.

Regulation of endothelial permeability and transendothelial migration of cancer cells by tropomyosin-1 phosphorylation.

BACKGROUND: Loss of endothelial cell integrity and selective permeability barrier is an early event in the sequence of oxidant-mediated injury and may result in atherosclerosis, hypertension and facilitation of transendothelial migration of cancer cells during metastasis. We already reported that endothelial cell integrity is tightly regulated by the balanced co-activation of p38 and ERK pathways. In particular, we showed that phosphorylation of tropomyosin-1 (tropomyosin alpha-1 chain = Tm1) at Ser283 by DAP kinase, downstream of the ERK pathway might be a key event required to maintain the integrity and normal functions of the endothelium in response to oxidative stress. METHODS: Endothelial permeability was assayed by monitoring the passage of Dextran-FITC through a tight monolayer of HUVECs grown to confluence in Boyden chambers. Actin and Tm1 dynamics and distribution were evaluated by immunofluorescence. We modulated the expression of Tm1 by siRNA and lentiviral-mediated expression of wild type and mutated forms of Tm1 insensitive to the siRNA. Transendothelial migration of HT-29 colon cancer cells was monitored in Boyden chambers similarly as for permeability. RESULTS: We provide evidence indicating that Tm1 phosphorylation at Ser283 is essential to regulate endothelial permeability under oxidative stress by modulating actin dynamics. Moreover, the transendothelial migration of colon cancer cells is also regulated by the phosphorylation of Tm1 at Ser283. CONCLUSION: Our finding strongly support the role for the phosphorylation of endothelial Tm1 at Ser283 to prevent endothelial barrier dysfunction associated with oxidative stress injury.

Annexin-1-mediated endothelial cell migration and angiogenesis are regulated by vascular endothelial growth factor (VEGF)-induced inhibition of miR-196a expression.

Endothelial cell migration induced in response to vascular endothelial growth factor (VEGF) is an essential step of angiogenesis. It depends in part on the activation of the p38/MAPKAP kinase-2/LIMK1/annexin-A1 (ANXA1) signaling axis. In the present study, we obtained evidence indicating that miR-196a specifically binds to the 3'-UTR region of ANXA1 mRNA to repress its expression. In accordance with the role of ANXA1 in cell migration and angiogenesis, the ectopic expression of miR-196a is associated with decreased cell migration in wound closure assays, and the inhibitory effect of miR-196a is rescued by overexpressing ANXA1. This finding highlights the fact that ANXA1 is a required mediator of VEGF-induced cell migration. miR-196a also reduces the formation of lamellipodia in response to VEGF suggesting that ANXA1 regulates cell migration by securing the formation of lamellipodia at the leading edge of the cell. Additionally, in line with the fact that cell migration is an essential step of angiogenesis, the ectopic expression of miR-196a impairs the formation of capillary-like structures in a tissue-engineered model of angiogenesis. Here again, the effect of miR-196a is rescued by overexpressing ANXA1. Moreover, the presence of miR-196a impairs the VEGF-induced in vivo neo-vascularization in the Matrigel Plug assay. Interestingly, VEGF reduces the expression of miR-196a, which is associated with an increased level of ANXA1. Similarly, the inhibition of miR-196a with an antagomir results in an increased level of ANXA1. We conclude that the VEGF-induced decrease of miR-196a expression may participate to the angiogenic switch by maintaining the expression of ANXA1 to levels required to enable p38-ANXA1-dependent endothelial cell migration and angiogenesis in response to VEGF.

miR-20a represses endothelial cell migration by targeting MKK3 and inhibiting p38 MAP kinase activation in response to VEGF.

Endothelial cell migration induced in response to vascular endothelial growth factor (VEGF) is a crucial step of angiogenesis and it depends on the activation of the p38 MAP-kinase pathway downstream of VEGFR2. In this study, we investigated the role of microRNAs (miRNAs) in regulating these processes. We found that the VEGF-induced p38 activation and cell migration are modulated by overexpression of Argonaute 2, a key protein in the functioning of miRNAs. Thereafter, we found that miR-20a expression is increased by VEGF and that its ectopic expression inhibits VEGF-induced actin remodeling and cell migration. Moreover, the expression of miR-20a impairs the formation of branched capillaries in a tissue-engineered model of angiogenesis. In addition, the lentivirus-mediated expression of miR-20a precursor (pmiR-20a) is associated with a decrease in the VEGF-induced activation of p38. In contrast, these processes are increased by inhibiting miR-20a with a specific antagomir. Interestingly, miR-20a does not modulate VEGFR2 or p38 protein expression level. miR-20a does not affect either the expression of other known actors of the p38 MAP kinase pathway except MKK3. Indeed, by using quantitative PCR and Western Blot analysis, we found that pmiR-20a decreases the expression of MKK3 and we obtained evidence indicating that miR-20a specifically binds to the 3'UTR region of MKK3 mRNA. In accordance, the VEGF-induced activation of p38 and cell migration are impaired when the MKK3 expression is knocked down by siRNA. We conclude that miR-20a acts in a feedback loop to repress the expression of MKK3 and to negatively regulate the p38 pathway-mediated VEGF-induced endothelial cell migration and angiogenesis.

17beta-hydroxysteroid dehydrogenase type 1 modulates breast cancer protein profile and impacts cell migration.

INTRODUCTION: Human 17beta-hydroxysteroid dehydrogenase type 1 (17ß-HSD1) is a steroid-converting enzyme that has long been known to play critical roles in estradiol synthesis and more recently in dihydrotestosterone (DHT) inactivation, showing a dual function that promotes breast cancer cell proliferation. Previously, we reported the first observation of the influence of the enzyme on endogenous estrogen-responsive gene expression. Here, we demonstrate the impact of 17ß-HSD1 expression on the breast cancer cell proteome and investigate its role in cell migration. METHODS: 17ß-HSD1 was stably transfected in MCF7 cells and the proteome of the generated cells overexpressing 17ß-HSD1 (MCF7-17ßHSD1 cells) was compared to that of the wild type MCF7 cells. Proteomics study was performed using two-dimensional gel electrophoresis followed by mass spectrometry analysis of differentially expressed protein spots. Reverse transcription quantitative real-time PCR (RT-qPCR) was used to investigate the transcription of individual gene. The effect of 17ß-HSD1 on MCF7 cell migration was verified by a wound-healing assay. RESULTS: Proteomic data demonstrate that the expression of more than 59 proteins is modulated following 17ß-HSD1 overexpression. 17ß-HSD1 regulates the expression of important genes and proteins that are relevant to cell growth control, such as BRCA2 and CDKN1A interacting protein (BCCIP) and proliferating cell nuclear antigen (PCNA) which are down- and upregulated in MCF7-17ßHSD1 cells, respectively. RT-qPCR data reveal that 17ß-HSD1 increases the mRNA levels of estrogen receptors (ER) alpha and beta by 171 and 120%, respectively, while decreasing that of the androgen receptor by 64%. Interestingly, 17ß-HSD1 increases the mRNA transcript (by 3.6 times) and the protein expression of the metastasis suppressor gene nm23-H1 and the expression of the two enzymes are closely correlated. We have further shown that 17ß-HSD1 expression is associated with an increase of MCF7 cell migration. CONCLUSIONS: In addition to the regulation of important genes, we have demonstrated for the first time that 17ß-HSD1 increases breast cancer cell migration, in spite of its positive regulation of the antimetastatic gene NM23. This is also correlated to its stimulation of breast cancer cell growth, further confirming its targeting in ER positive breast cancer. The novel findings in this study suggest several directions for future research on the contribution of 17ß-HSD1 to breast cancer progression and related treatment.

Survival advantages conferred to colon cancer cells by E-selectin-induced activation of the PI3K-NFκB survival axis downstream of Death receptor-3.

BACKGROUND: Extravasation of circulating cancer cells is a key event of metastatic dissemination that is initiated by the adhesion of cancer cells to endothelial cells. It requires interactions between adhesion receptors on endothelial cells and their counter-receptors on cancer cells. Notably, E-selectin, a major endothelial adhesion receptor, interacts with Death receptor-3 present on metastatic colon carcinoma cells. This interaction confers metastatic properties to colon cancer cells by promoting the adhesion of cancer cells to endothelial cells and triggering the activation of the pro-migratory p38 and pro-survival ERK pathways in the cancer cells. In the present study, we investigated further the mechanisms by which the E-selectin-activated pathways downstream of DR3 confer a survival advantage to colon cancer cells. METHODS: Cell survival has been ascertained by using the WST-1 assay and by evaluating the activation of the PI3 kinase/NFκB survival axis. Apoptosis has been assayed by determining DNA fragmentation by Hoechst staining and by measuring cleavage of caspases-8 and -3. DR3 isoforms have been identified by PCR. For more precise quantification, targeted PCR reactions were carried out, and the amplified products were analyzed by automated chip-based microcapillary electrophoresis on an Agilent 2100 Bioanalyzer instrument. RESULTS: Interaction between DR3-expressing HT29 colon carcinoma cells and E-selectin induces the activation of the PI3K/Akt pathway. Moreover, p65/RelA, the anti-apoptotic subunit of NFκB, is rapidly translocated to the nucleus in response to E-selectin. This translocation is impaired by the PI3K inhibitor LY294002. Furthermore, inhibition of the PI3K/Akt pathway increases the cleavage of caspase 8 in colon cancer cells treated with E-selectin and this effect is still further increased when both ERK and PI3K pathways are concomitantly inhibited. Intriguingly, metastatic colon cancer cell lines such as HT29 and SW620 express higher levels of a splice variant of DR3 that has no trans-membrane domain and no death domain. CONCLUSION: Colon cancer cells acquire an increased capacity to survive via the activation of the PI3K/NFκB pathway following the stimulation of DR3 by E-selectin. Generation of a DR3 splice variant devoid of death domain can further contribute to protect against apoptosis.