[Show abstract][Hide abstract] ABSTRACT: The present work was aimed at studying the antioxidative activity and hepatoprotective effects of methanolic extract (ME) of Hammada scoparia leaves against ethanol-induced liver injury in male rats. The animals were treated daily with 35 % ethanol solution (4 g kg(-1) day(-1)) during 4 weeks. This treatment led to an increase in the lipid peroxidation, a decrease in antioxidative enzymes (catalase, superoxide dismutase, and glutathione peroxidase) in liver, and a considerable increase in the serum levels of aspartate and alanine aminotransferase and alkaline phospahatase. However, treatment with ME protects efficiently the hepatic function of alcoholic rats by the considerable decrease in aminotransferase contents in serum of ethanol-treated rats. The glycogen synthase kinase-3 β was inhibited after ME administration, which leads to an enhancement of glutathione peroxidase activity in the liver and a decrease in lipid peroxidation rate by 76 %. These biochemical changes were consistent with histopathological observations, suggesting marked hepatoprotective effect of ME. These results strongly suggest that treatment with methanolic extract normalizes various biochemical parameters and protects the liver against ethanol induced oxidative damage in rats.
Journal of physiology and biochemistry 08/2012; · 1.65 Impact Factor
[Show abstract][Hide abstract] ABSTRACT: Focal adhesion kinase (FAK) activity contributes to many advanced cancer phenotypes, but little is known about its role in human acute myeloid leukemia (AML). Here, we show that FAK splice variants are abnormally expressed in the primitive leukemic cells of poor prognosis AML patients. In the CD34(+) 38(-) 123(+) long-term culture-initiating cell-enriched leukemic cells of these patients, FAK upregulates expression of Frizzled-4 and phosphorylates Pyk2 to enable the required association of Pyk2 with the Wnt5a/Frizzled-4/LRP5 endocytosis complex and downstream activation of β-catenin, thereby replacing the Wnt3a-controlled canonical pathway used by normal hematopoietic stem cells. Transduction of primitive normal human hematopoietic cells with FAK splice variants induces a marked increase in their clonogenic activity and signaling via the Wnt5a-controlled canonical pathway. Targeting FAK or β-catenin efficiently eradicates primitive leukemic cells in vitro suggesting that FAK could be a useful therapeutic target for improved treatment of poor prognosis AML cases.
[Show abstract][Hide abstract] ABSTRACT: Therapeutic resistance of acute myeloid leukemia stem cells, enriched in the CD34(+)38(-)123(+) progenitor population, is supported by extrinsic factors such as the bone marrow niche. Here, we report that when adherent onto fibronectin or osteoblast components, CD34(+)38(-)123(+) progenitors survive through an integrin-dependent activation of glycogen synthase kinase 3β (GSK3β) by serine 9-dephosphorylation. Strikingly, GSK3β-mediated survival was restricted to leukemic progenitors from female patients. GSK3β inhibition restored sensitivity to etoposide, and impaired the clonogenic capacities of adherent leukemic progenitors from female patients. In leukemic progenitors from female but not male patients, the scaffolding protein RACK1, activated downstream of α(5)β(1)-integrin engagement, was specifically upregulated and controlled GSK3β activation through the phosphatase protein phosphatase 2A (PP2A). In a mirrored manner, survival of adherent progenitors (CD34(+)38(-)) from male but not female healthy donors was partially dependent on this pathway. We conclude that the GSK3β-dependent survival pathway might be sex-specific in normal immature population and flip-flopped upon leukemogenesis. Taken together, our results strengthen GSK3β as a promising target for leukemic stem cell therapy and reveal gender differences as a new parameter in anti-leukemia therapy.
[Show abstract][Hide abstract] ABSTRACT: In search for compounds able to reduce cell adhesion-mediated drug resistance (CAM-DR), we studied effects of Hammada scoparia extracts on leukemic cells adherent or in suspension. We show that H. scoparia flavonoidic fraction and its compound rutin induce apoptosis specifically in adherent leukemic cells and abolish CAM-DR. Importantly, rutin inhibited survival of adherent leukemic progenitors (CD34(+)38(-)123(+)) but spared normal progenitors (CD34(+)38(-)). The pro-apoptotic effects of rutin were correlated with a decrease of active GSK3β and inhibitors of GSK3β reproduced rutin-induced cytotoxicity. This study uncovers the potential of H. scoparia flavonoids and rutin to overcome CAM-DR in acute myeloid leukemia.
Leukemia research 01/2011; 35(8):1093-101. · 2.36 Impact Factor
[Show abstract][Hide abstract] ABSTRACT: Cell survival mediated by integrin engagement has been implicated in cell adhesion-mediated drug resistance. We have recently demonstrated that the activation of glycogen synthase kinase 3 beta (GSK3beta) is a new pathway supporting the chemoresistance of leukemic cells adhered to fibronectin.
We show here that in conditions of serum starvation, the fibronectin receptor alpha(5)beta(1) integrin, but not alpha(4)beta(1), induced activation of GSK3beta through Ser-9 dephosphorylation in adherent U937 cells. The GSK3beta-dependent survival pathway occurred in adherent leukemic cells from patients but not in the HL-60 and KG1 cell lines. In adhesion, activated GSK3beta was found in the cytosol/plasma membrane compartment and was co-immunoprecipitated with alpha(5) integrin, the phosphatase PP2A and the scaffolding protein RACK1. PP2A and its regulatory subunit B' regulated the Ser-9 phosphorylation of GSK3beta. In adherent leukemic cells, alpha(5)beta(1) integrin but not alpha(4)beta(1) upregulated the resistance to TNFalpha-induced apoptosis. Both extrinsic and intrinsic apoptotic pathways were under the control of alpha(5)beta(1) and GSK3beta.
Our data show that, upon serum starvation, alpha(5)beta(1) integrin engagement could regulate specific pro-survival functions through the activation of GSK3beta.
PLoS ONE 01/2010; 5(3):e9807. · 3.53 Impact Factor
[Show abstract][Hide abstract] ABSTRACT: Defining the impact on health of exposure to a low-dose pesticide mixture via food intake is a topical question since epidemiological studies suggest that this may increase the risk of pathologies and particularly haematopoietic malignancies. Here we investigated on the haematopoietic system of mice, the effect of a mixture of six pesticides frequently ingested through the intake of fruits and vegetables produced in France (alachlor, captan, diazinon, endosulfan, maneb, mancozeb). The mixture was administered repeatedly by gavage to mice for 4 weeks at levels derived from the human Acceptable Daily Intake (ADI) level adapted to the mean weight of mice. Using a NMR-based metabonomic approach, we show that this treatment led to specific gender-linked variations in the level of hepatic metabolites involved in oxidative stress and in the regulation of glucose metabolism, indicating a metabolic signature for this repeated administration. Interestingly, exposure to the low-dose pesticide mixture induced significant changes in the blood cell counts with modifications in the clonogenic and differentiating capacities of haematopoietic progenitors showing abnormalities in the granulocytic and monocytic lineages in female and male mice, respectively. From a molecular point of view, the changes induced by the pesticide treatment correlated with modifications of the PI 3-kinase/Akt signalling pathway, the tyrosine kinase Pyk2 and the c-Myc transcription factor, which are involved in the balance between self-renewal and differentiation of haematopoietic stem cells. Our results point to a significant effect of a very low dose of a mixture of commonly used pesticides on mice metabolism and haematopoietic system with major differences between males and females.
[Show abstract][Hide abstract] ABSTRACT: The cellular level of the CDC25A phosphatase is tightly regulated during both the normal and genotoxic-perturbed cell cycle. Here, we describe a caspase-dependent cleavage of this protein at residue D223 in non-genotoxic apoptotic conditions. This specific proteolysis generates a catalytically active C-terminal fragment that localizes to the nuclear compartment. Accumulation of this active CDC25A fragment leads to reduced inhibitory phosphorylation of the CDC25A substrate cyclin-dependent kinase 2 (CDK2) on Tyr15. Moreover, CDK2 was found stably associated with this fragment, as well as with an ectopically expressed CDC25A224-525 truncation mutant that mimicks the cleavage product. Ectopic expression of this mutant induced CDK2 Tyr15 dephosphorylation, whereas its catalytically inactive version did not. Finally, this 224-525 mutant initiated apoptosis when transfected into HeLa cells, whereas its catalytic inactive form did not. Altogether, this study demonstrates for the first time that caspase-dependent cleavage of CDC25A is a central step linking CDK2 activation with non-genotoxic apoptotic induction.
Cell death and differentiation 11/2008; 16(2):208-18. · 8.24 Impact Factor
[Show abstract][Hide abstract] ABSTRACT: Anaplastic lymphoma kinase (ALK) is a receptor tyrosine kinase, initially discovered as part of the NPM-ALK fusion protein, resulting from the t(2;5) translocation that is frequently associated with anaplastic large-cell lymphomas. The native ALK protein is normally expressed in the developing and, at a weaker level, adult nervous system. We recently demonstrated that the oncogenic, constitutively kinase-activated NPM-ALK protein was antiapoptotic when expressed in Jurkat lymphoblastic cells treated with cytotoxic drugs. In contrast, we now show that Jurkat cells overexpressing the wild-type ALK receptor are more sensitive to doxorubicin-induced apoptosis than parental cells. Moreover, the ALK protein is cleaved during apoptosis in a caspase-dependent manner. Mutation of aspartic residues to asparagine allowed us to map the caspase cleavage site in the juxtamembrane region of ALK. In order to assess the role of ALK in neural cell-derived tissue, we transiently expressed ALK in the 13.S.1.24 rat neuroblast immortalized cell line. ALK expression led to apoptotic cell death of the neuroblasts. ALK ligation by specific activating antibodies decreased ALK-facilitated apoptosis in both lymphoid and neuronal cell lines. Moreover, ALK transfection reduced the survival of primary cultures of cortical neurons. Thus, ALK has a proapoptotic activity in the absence of ligand, whereas it is antiapoptotic in the presence of its ligand and when the kinase is intrinsically activated. These properties place ALK in the growing family of dependence receptors.
Molecular and Cellular Biology 09/2006; 26(16):6209-22. · 5.37 Impact Factor
[Show abstract][Hide abstract] ABSTRACT: The effects of cell adhesion on leukemia cell proliferation remain poorly documented and somehow controversial. In this work, we investigated the effect of adhesion to fibronectin on the proliferation of acute myeloid leukemia (AML) cell lines (U937 and KG1a) and CD34+ normal or leukemic primary cells. We observed an increased rate of proliferation of AML cells when adhered to fibronectin, concomitant with accelerated S-phase entry and accumulation of CDC25A. Conversely, normal CD34+ cell proliferation was decreased by adhesion to fibronectin with a concomitant drop in CDC25A expression. Importantly, we showed that both small interfering RNA (siRNA)-mediated CDC25A down-regulation and a recently developed CDC25 pharmacologic inhibitor impaired this adhesion-dependent proliferation, establishing a functional link between CDC25A accumulation and adhesion-dependent proliferation in leukemic cells. CDC25A accumulation was found only slightly dependent on transcriptional regulation and essentially due to modifications of the proteasomal degradation of the protein as shown using proteasome inhibitors and reverse transcription-PCR. Interestingly, CDC25A regulation was Chk1 dependent in these cells as suggested by siRNA-mediated down-regulation of this protein. Finally, we identified activation of the phosphatidylinositol 3-kinase/Akt pathway as an adhesion-dependent regulation mechanism of CDC25A protein expression. Altogether, our data show that in leukemic cells adhesion to fibronectin increases CDC25A expression through proteasome- and Chk1-dependent mechanisms, resulting in enhanced proliferation. They also suggest that these adhesion-dependent proliferation properties of hematopoietic cells may be modified during leukemogenesis.
Cancer Research 08/2006; 66(14):7128-35. · 8.65 Impact Factor
[Show abstract][Hide abstract] ABSTRACT: Relapses following chemotherapy are a major hindrance to patients' survival in acute myeloid leukemia (AML). To investigate the role of the hematopoietic niche in the chemoresistance of leukemic cells, we examined two pathways: one mediated by adhesion molecules/integrins, and the other by soluble factors of the morphogen Wnt pathway. In our study, both the adhesion of leukemic blasts to fibronectin and the addition of Wnt antagonists induced, independently, resistance of AML cells to daunorubicin in a cell survival assay. Using pharmacological inhibitors and siRNA, we showed that both resistance pathways required the activity of the glycogen synthase kinase 3beta (GSK3beta). Moreover, the AML cell protection downstream of GSK3beta was mediated by NF-kappaB. A link between the adhesion and the Wnt pathway was found, as adhesion of U937 on human osteoblasts, a component of the hematopoietic niche, triggered the secretion of the Wnt antagonist sFRP-1 and supported resistance to daunorubicin. The osteoblast-conditioned medium could also confer chemoresistance to U937 cells cultured in suspension, and this cell protective effect was abrogated after depletion of sFRP-1. In the context of this potential double in vivo resistance, modulators of the common signal GSK3beta and of its target NF-kappaB could represent important novel therapeutic tools.
[Show abstract][Hide abstract] ABSTRACT: Activation of the Wnt/β-catenin pathway has recently been shown to be crucial to the establishment of leukemic stem cells in chronic myeloid leukemia. We sought to determine whether β-catenin was correlated to clonogenic capacity also in the acute myeloid leukemia (AML) setting. Eighty-two patients were retrospectively evaluated for β-catenin expression by Western blot. β-Catenin was expressed (although at various protein levels) in 61% of patients, and was undetectable in the remaining cases. In our cohort, β-catenin expression was correlated with the clonogenic proliferation of AML-colony forming cells (AML-CFC or CFU-L) in methylcellulose in the presence of 5637-conditioned medium, and more strikingly with self-renewing of leukemic cells, as assessed in vitro by a re-plating assay. In survival analyses, β-catenin appeared as a new independent prognostic factor predicting poor event-free survival and shortened overall survival (both with P
[Show abstract][Hide abstract] ABSTRACT: The microenvironment is now considered as an important source of potential therapeutic targets in diverse pathologies. In cardiovascular diseases and in cancer, common processes involving stromal remodeling, cell invasion, and angiogenesis can promote progression of the pathology. At each step of the pathogenesis, cell adhesion needs to be modulated to allow adaptation of cell survival/motility/proliferation functions to the microenvironment. Among adhesion receptors, integrins, responsible for cell/matrix or cell/cell interactions, play a key role in the cellular responses. Moreover, their engagement conditions the sensitivity to apoptosis induced by therapeutic drugs. Targeting of the extracellular side of integrins in order to modulate their adhesive functions is under development and has reached clinical indications. However, improvement of oral availability and of cell signaling control is required in the future. Targeting of the extracellular or the intracellular key proteins involved in integrin-dependent signaling pathway seems promising. Yet, although some common key enzyme inhibitors are under development, a better knowledge of the specificity of integrin activation and interaction with partners upon pathogenesis is of major importance in envisaging the antagonism of integrin-linked signals as a therapeutic tool alone or in association with other therapies.
Current Pharmaceutical Design 02/2005; 11(16):2119-34. · 3.31 Impact Factor
[Show abstract][Hide abstract] ABSTRACT: The phosphoinositide metabolism that is highly controlled by a set of kinases, phosphatases and phospholipases leads to the production of several second messengers playing critical roles in intracellular signal transduction mechanisms. Recent discoveries have unraveled unexpected roles for the three phosphatidylinositol monophosphates, PtdIns(3)P, PtdIns(4)P and PtdIns(5)P, that appear now as important lipid messengers able to specifically interact with proteins. The formation of functionally distinct and independently regulated pools of phosphatidylinositol monophosphates probably contributes to the specificity of the interactions with their targets. The relative enrichment of organelles in a particular species of phosphoinositides (i.e. PtdIns(3)P in endosomes, PtdIns(4)P in Golgi and PtdIns(4,5)P2 in plasma membrane) suggests the notion of lipid-defined organelle identity. PtdIns(3)P is now clearly involved in vesicular trafficking by interaction with a set of FYVE domain-containing proteins both in yeast and in mammals. PtdIns(4)P, which until now was only considered as a precursor for PtdIns(4,5)P2, appears as a regulator on its own, by recruiting a set of proteins to the trans-Golgi network. PtdIns(5)P, the most recently discovered inositol lipid, is also emerging as a potentially important signaling molecule.
[Show abstract][Hide abstract] ABSTRACT: Majority of anaplastic large-cell lymphomas (ALCLs) are associated with the t(2;5)(p23;q35) translocation, fusing the NPM (nucleophosmin) and ALK (anaplastic lymphoma kinase) genes (NPM-ALK). Recent studies demonstrated that ALK may also be involved in variant translocations, namely, t(1;2)(q25;p23), t(2;3)(p23;q21), t(2;17)(p23;q23) and inv(2)(p23q35), which create the TPM3-ALK, TFG-ALK5, CLTC-ALK, and ATIC-ALK fusion genes, respectively. Although overexpression of NPM-ALK has previously been shown to transform fibroblasts, the transforming potential of variant X-ALK proteins has not been precisely investigated. We stably transfected the cDNAs coding for NPM-ALK, TPM3-ALK, TFG-ALK, CLTC-ALK or ATIC-ALK into nonmalignant NIH3T3 cells. All X-ALK variants are tyrosine phosphorylated and their subcellular distribution was in agreement with that observed in tumors. Moreover, our results show that the in vitro transforming capacity of NIH3T3-transfected cells are in relation to the level of X-ALK fusion proteins excepted for TPM3-ALK for which there is an inverse correlation. The differences between the five X-ALK variants with regard to proliferation rate, colony formation in soft agar, invasion, migration through the endothelial barrier and tumorigenicity seem to be due to differential activation of various signaling pathways such as PI3-kinase/AKT. These findings may have clinical implications in the pathogenesis and prognosis of ALK-positive ALCLs.
[Show abstract][Hide abstract] ABSTRACT: Focal adhesion kinase (FAK) is a nonreceptor tyrosine kinase playing an important role in cell motility and survival. However, very little is known about FAK in normal and leukemic myeloid cells. In this study, FAK protein expression and mRNA were detected in 25 of 60 cases (42%) of acute myeloid leukemia (AML). Whereas FAK was expressed in 46% of CD34+ AML cells, it was not detected in normal purified CD34+ cells. Conversely, the FAK homologue proline-rich tyrosine kinase 2 (PYK2) was found to be expressed both in normal and leukemic myeloid cells. When expressed, FAK displayed phosphorylation on Tyr-397, an important step for its activation. Moreover, FAK expression was correlated with the phosphorylation of PYK2 on Tyr-881, a critical site for the PYK2 function in cell migration. FAK+ AML cells displayed significantly higher migration capacities and resistance to daunorubicin, compared with FAK- cells. The implication of FAK in both cell motility and drug resistance was demonstrated by small interfering RNA experiments with the FAK-positive KG1 cell line. However, adhesion on fibronectin efficiently protected FAK- AML cells from daunorubicin-mediated killing, suggesting that cellular adhesion mediated-drug resistance is not mediated by FAK. Finally, in a retrospective cohort of 60 AML patients, FAK expression was significantly correlated with high blast cell count, early death, and shorter survival rate. Altogether, this study shows that FAK is aberrantly expressed and activated in about half of the cases of AML and suggests that FAK may contribute to the regulation of AML cell transit from the marrow to blood compartment and that it may influence clinical outcome.
Cancer Research 06/2004; 64(9):3191-7. · 8.65 Impact Factor
[Show abstract][Hide abstract] ABSTRACT: The involvement of phosphoinositide 3-kinase C2alpha in vascular smooth muscle cell migration was investigated. Products of phosphoinositide 3-kinase, phosphatidylinositol-3-phosphate, and phosphatidylinositol-3,4-bis-phosphate were increased upon smooth muscle cell migration but their synthesis was affected only partially by phosphoinositide 3-kinase inhibitors, wortmannin and LY-294002. Using specific antibody, we showed that the wortmannin/LY-294002 poorly sensitive phosphoinositide 3-kinase C2alpha is expressed in smooth muscle cells. Measurement of phosphoinositide 3-kinase C2alpha activity in vitro, after immunoprecipitation, clearly demonstrated its activation upon smooth muscle cell migration. Moreover, for the first time, phosphoinositide 3-kinase C2alpha was found to be differentially regulated by alpha(v)beta(3) and alpha(v)beta(5) integrin engagement. Finally, we have identified two new potential phosphoinositide 3-kinase C2alpha-binding proteins, p70 and p110, which both may be tyrosine phosphorylated. Thus, phosphoinositide 3-kinase C2alpha might represent a new regulatory pathway of cell migration downstream of integrin engagement.
Biochemical and Biophysical Research Communications 10/2002; 297(2):261-6. · 2.28 Impact Factor
[Show abstract][Hide abstract] ABSTRACT: The involvement of phosphoinositide 3-kinase C2α in vascular smooth muscle cell migration was investigated. Products of phosphoinositide 3-kinase, phosphatidylinositol-3-phosphate, and phosphatidylinositol-3,4-bis-phosphate were increased upon smooth muscle cell migration but their synthesis was affected only partially by phosphoinositide 3-kinase inhibitors, wortmannin and LY-294002. Using specific antibody, we showed that the wortmannin/LY-294002 poorly sensitive phosphoinositide 3-kinase C2α is expressed in smooth muscle cells. Measurement of phosphoinositide 3-kinase C2α activity in vitro, after immunoprecipitation, clearly demonstrated its activation upon smooth muscle cell migration. Moreover, for the first time, phosphoinositide 3-kinase C2α was found to be differentially regulated by αvβ3 and αvβ5 integrin engagement. Finally, we have identified two new potential phosphoinositide 3-kinase C2α-binding proteins, p70 and p110, which both may be tyrosine phosphorylated. Thus, phosphoinositide 3-kinase C2α might represent a new regulatory pathway of cell migration downstream of integrin engagement.
Biochemical and Biophysical Research Communications. 09/2002;
[Show abstract][Hide abstract] ABSTRACT: Fibrinogen deposition and smooth muscle cell migration are important causes of atherosclerosis and angiogenesis. Involvement of calpains in vascular smooth muscle cell adhesion onto fibrinogen was investigated. Using calpain inhibitors, we showed that activation of calpains was required for smooth muscle cell spreading. An increase of 32P-labeled phosphatidic acid and phosphatidylinositol-3,4-bisphosphate, respective products of phospholipase C and phosphoinositide 3-kinase activities, was measured in adherent cells. Addition of the calpain inhibitor calpeptin strongly decreased phosphatidic acid and phosphatidylinositol-3,4-bisphosphate. However, smooth muscle cell spreading was prevented by the phospholipase C inhibitor U-73122, but poorly modified by phosphoinositide 3-kinase inhibitors wortmannin and LY-294002. Moreover, PLC was found to act upstream of the PI 3-kinase IA isoform. Thus, our data provide the first evidence that calpains are required for smooth muscle cell spreading. Further, phospholipase C activation is pointed as a key step of cell-spreading regulation by calpains.
Biochemical and Biophysical Research Communications 12/2001; · 2.28 Impact Factor
[Show abstract][Hide abstract] ABSTRACT: Smooth muscle cell migration is a key step of atherosclerosis and angiogenesis. We demonstrate that alpha(V)beta(3) and alpha(V)beta(5) integrins synergistically regulate smooth muscle cell migration onto vitronectin. Using an original haptotactic cell migration assay, we measured a strong stimulation of phosphoinositide metabolism in migrating vascular smooth muscle cells. Phosphatidic acid production and phosphoinositide 3-kinase IA activation were triggered only upon alpha(V)beta(3) engagement. Blockade of alpha(V)beta(3) engagement or phospholipase C activity resulted in a strong inhibition of smooth muscle cell spreading on vitronectin. By contrast, blockade of alpha(V)beta(5) reinforced elongation and polarization of cell shape. Moreover, Pyk2-associated tyrosine kinase and phosphoinositide 4-kinase activities measured in Pyk2 immunoprecipitates were stimulated upon cell migration. Blockade of either alpha(V)beta(3) or alpha(V)beta(5) function, as well as inhibition of phospholipase C activity, decreased both Pyk2-associated activities. We demonstrated that the Pyk2-associated phosphoinositide 4-kinase corresponded to the beta isoform. Our data point to the metabolism of phosphoinositides as a regulatory pathway for the differential roles played by alpha(V)beta(3) and alpha(V)beta(5) upon cell migration and identify the Pyk2-associated phosphoinositide 4-kinase beta as a common target for both integrins.
Journal of Biological Chemistry 12/2001; 276(45):41832-40. · 4.65 Impact Factor