Abstract: Signal transducers and activators of transcription (STAT) proteins were described as a family of latent cytosolic transcription factors whose activation is dependent on phosphorylation via growth factor- and cytokine-membrane receptors including interferon and interleukin, or by non-receptor intracellular tyrosine kinases, including Src. A vast majority of natural substances are capable of modulating mitogenic signals, cell survival, apoptosis, cell cycle regulation, angiogenesis as well as processes involved in metastasis development. The inhibition of STAT3 phosphorylation by natural and dietary compounds leads to decreased protein expression of STAT3 targets essentially involved in regulation of the cell cycle and apoptotic cell death. This review details the cell signaling pathways involving STAT transcription factors as well as the corresponding compounds from nature able to interfere with this regulatory system in human cancer.
Abstract: As a histone deacetylase inhibitor, valproic acid (VPA) is a candidate for anticancer therapy. Besides, VPA exhibits various mechanisms of action and its effects on the molecular basis of hematopoiesis remain unclear. To study the effects of VPA on the hematopoietic system, we performed microarray analysis using K562 cells treated with 1mM VPA over a 72h time course. The association between gene ontology (GO) terms and the lists of differentially expressed genes was tested using the Bioconductor package GOstats. Enrichment analysis for cellular differentiation pathways was performed based on manually curated gene lists. Results from microarray analysis were confirmed by studying cell differentiation features at the molecular and cellular levels using other hematopoietic cell lines as well as hematopoietic stem/progenitor CD34(+) cells. Microarray analysis revealed 3440 modulated genes in the presence of VPA. Genes involved in the granulo-monocytic differentiation pathway were up-regulated while genes of the erythroid pathway were down-regulated. This was confirmed by analyzing erythrocytic and myeloid membrane markers and lineage-related gene expression in HEL, MEG01, HL60 as well as CD34(+) cells. Moreover, GATA-1 and its co-factors (FOG1, SP1) were down-regulated, while myelopoiesis activator PU.1 was up-regulated, in agreement with an inhibition of erythropoiesis. Our functional profiling and cell phenotyping approach demonstrates that VPA is able to alter hematopoietic homeostasis by modifying the cell population balance in the myeloid compartment. This may lead to a potential failure of erythropoiesis in patients with cancer or chronic inflammatory diseases having a well-described propensity to anemia.
Abstract: Erythropoietin (EPO) is a glycoprotein that is mainly produced in the adult kidney, and it was initially highlighted for its action on the hematopoietic system. Moreover, EPO is also expressed in several non-hematopoietic tissues, where it plays a role in the protection from apoptosis and inflammation due to hypoxia, toxicity or injury. These protective effects are mainly known and studied in cardioprotection and neuroprotection but are also reported in retina degeneration, auditory injury and pancreatic-related diseases. The tissue protective effect of EPO is mainly mediated through the interaction with the heterodimeric receptor EPOR/βcR. Human recombinant EPO (HuREPO), which has been developed to treat anemia, is not adequate for tissue protection. The low affinity of the alternative receptor for EPO involves the injection of excessive concentration of erythropoiesis-stimulating agents (ESAs), implicating side effects due to the cross-talk with hematopoietic activity. For these reasons, EPO derivatives with less affinity for the EPO homodimeric receptor are under development. In this review, we provide an overview of the erythroid and non-erythroid functions of EPO by detailing the molecular mechanisms activated by the binding of EPO to its receptors in different tissues.
Abstract: Cardiac steroids are used to treat various diseases including congestive heart failure and cancer. The aim of this study was to investigate the anti-leukemic activity of UNBS1450, a hemi-synthetic cardenolide belonging to the cardiac steroid glycoside family. Here, we report that, at low nanomolar concentrations, UNBS1450 induces apoptotic cell death. Subsequently, we have investigated the molecular mechanisms leading to apoptosis activation. Our results show that UNBS1450 inhibits NF-κB transactivation and triggers apoptosis by cleavage of pro-caspases 8, 9 and 3/7, by decreasing expression of anti-apoptotic Mcl-1 and by recruitment of pro-apoptotic Bak and Bax protein eventually resulting in cell death.
Abstract: Many physiological perturbations can cause anemia. In cancer patients, activation of the immune system leads to the production of proinflammatory cytokines including tumor necrosis factor alpha (TNFα), that have been shown to inhibit red-cell production via poorly understood mechanisms. Treatment of anemia by human recombinant erythropoietin (EPO) is strongly suspected to induce tumor growth. This study focuses on the mechanisms involved in TNFα-mediated inhibition of erythropoiesis. CD34(+) hematopoietic stem/progenitor cells (HSPCs) were isolated from human cord blood. Erythropoiesis was achieved in vitro by stimulating cells with EPO. We show that TNFα clearly affected erythroid development, as assessed by May-Grünwald/Giemsa staining, flow cytometry analysis and fluorescent microscopy. The amount of hemoglobin-producing cells as well as the expression of GATA-1 target erythro-specific genes (EPO receptor, glycophorin A and globins) was found decreased after TNFα treatment of HSPC. In correlation, TNFα induced the expression of the transcription factors GATA-2 and PU.1, described as inhibitors of erythropoiesis. In this regard, TNFα promoted the formation of the GATA-1/PU.1 complex that has been reported to block the transcriptional activity of GATA-1. Our results clearly demonstrate that TNFα prevents EPO-mediated erythropoiesis of HSPC as an early event, by directly affecting erythroid cell development.
Abstract: In this study, we investigated the biological effects of heteronemin, a marine sesterterpene isolated from the sponge Hyrtios sp. on chronic myelogenous leukemia cells. To gain further insight into the molecular mechanisms triggered by this compound, we initially performed DNA microarray profiling and determined which genes respond to heteronemin stimulation in TNFalpha-treated cells and which genes display an interaction effect between heteronemin and TNFalpha. Within the differentially regulated genes, we found that heteronemin was affecting cellular processes including cell cycle, apoptosis, mitogen-activated protein kinases (MAPKs) pathway and the nuclear factor kappaB (NF-kappaB) signaling cascade. We confirmed in silico experiments regarding NF-kappaB inhibition by reporter gene analysis, electrophoretic mobility shift analysis and I-kappaB degradation. In order to assess the underlying molecular mechanisms, we determined that heteronemin inhibits both trypsin and chymotrypsin-like proteasome activity at an IC(50) of 0.4 microM. Concomitant to the inhibition of the NF-kappaB pathway, we also observed a reduction in cellular viability. Heteronemin induces apoptosis as shown by annexin V-FITC/propidium iodide-staining, nuclear morphology analysis, pro-caspase-3, -8 and -9 and poly(ADP-ribose) polymerase (PARP) cleavage as well as truncation of Bid. Altogether, results show that this compound has potential as anti-inflammatory and anti-cancer agent.
Abstract: Valproic acid (VPA), a branched short-chain fatty acid, is widely used as an antiepileptic drug and a mood stabilizer. Antiepileptic properties have been attributed to inhibition of Gamma Amino Butyrate (GABA) transaminobutyrate and of ion channels. VPA was recently classified among the Histone Deacetylase Inhibitors, acting directly at the level of gene transcription by inhibiting histone deacetylation and making transcription sites more accessible. VPA is a widely used drug, particularly for children suffering from epilepsy. Due to the increasing number of clinical trials involving VPA, and interesting results obtained, this molecule will be implicated in an increasing number of therapies. However side effects of VPA are substantially described in the literature whereas they are poorly discussed in articles focusing on its therapeutic use. This paper aims to give an overview of the different clinical-trials involving VPA and its side effects encountered during treatment as well as its molecular properties.
Abstract: Buthionine sulfoximine (BSO) is a well-known inhibitor of glutathione synthesis, producing slow glutathione (GSH) depletion and oxidative stress; some "responder" cells avoid BSO-induced death by trans-activating the prosurvival protein Bcl-2. Here we show that BSO activates a noncanonical, inhibitory NF-kappaB- and p65-independent NF-kappaB pathway via a multistep process leading to the up-regulation of Bcl-2. The slow BSO-induced GSH depletion allows separation of two redox-related phases, namely, early thiol disequilibrium and late frank oxidative stress; each phase contributes to the progressive activation of a p50-p50 homodimer. The early phase, coinciding with substantial thiol depletion, produces a cytosolic preparative complex, consisting of p50 and its interactor Bcl-3 linked by interprotein disulfide bridges. The late phase, coinciding with reactive oxygen species production, is responsible, probably via p38 activation, for nuclear targeting of the complex and trans-activation of Bcl-2.
Abstract: Anemia of cancer and chronic inflammatory diseases is a frequent complication affecting quality of life. For cancer patients it represents a particularly bad prognostic. Low level of erythropoietin is considered as one of the causes of anemia in these pathologies. The deficiency in erythropoietin production results from pro-inflammatory cytokines effect. However, few data is available concerning molecular mechanisms involved in cytokine-mediated anemia. Some recent publications have demonstrated the direct effect of pro-inflammatory cytokines on cell differentiation towards erythroid pathway, without erythropoietin defect. This suggested that pro-inflammatory cytokine-mediated signaling pathways affect erythropoietin activity. They could interfere with erythropoietin-mediated signaling pathways, inducing early apoptosis and perturbing the expression and regulation of specific transcription factors involved in the control of erythroid differentiation. In this review we summarize the effect of tumor necrosis factor (TNF)alpha, TNF-related apoptosis-inducing ligand (TRAIL), and interferon (IFN)-gamma on erythropoiesis with a particular interest for molecular feature.
Abstract: Erythropoiesis is considered as a multistep and tightly regulated process under the control of a series of cytokines including erythropoietin (Epo). Epo activates specific signaling pathways and leads to activation of key transcription factors such as GATA-1, in order to ensure erythroid differentiation. Deregulation leads to a decreased number of red blood cells, a hemoglobin deficiency, thus a limited oxygen-carrying capacity in the blood. Anemia represents a frequent complication in various diseases such as cancer or inflammatory diseases. It reduces both quality of life and prognosis in patients. Tumor necrosis factor alpha (TNFalpha) was described to be involved in the pathogenesis of inflammation and cancer related anemia. Blood transfusions and erythroid stimulating agents (ESAs) including human recombinant Epo (rhuEpo) are currently used as efficient treatments. Moreover, the recently described conflicting effects of ESAs in distinct studies require further investigations on the molecular mechanisms involved in TNFalpha-caused anemia. The present study aims to evaluate the current knowledge and the importance of the effect of the proinflammatory cytokine TNFalpha on erythropoiesis in inflammatory and malignant conditions.
Abstract: The hematopoietic transcription factor GATA-1 regulates the expression of several genes associated with differentiation of erythroid cells. We show here the inhibitory effect of tumor necrosis factor alpha (TNFalpha), a proinflammatory cytokine, on hemoglobinization and erythroid transcription factor GATA-1 expression in erythroleukemia (HEL) as well as in chronic myelogenous leukemia (K562) cells, which were induced to differentiate towards the erythroid lineage after aclacinomycin (Acla), doxorubicin (Dox) or hemin (HM) treatment. As a result, we observed i) a decreased expression of Friend of GATA-1 (FOG-1), an essential cofactor of GATA-1 transcription factor, ii) a downregulation of GATA-1 by proteasomal degradation and iii) a reduced acetylation level of GATA-1 in HM-induced K562 cells after TNF treatment. As a result, these modifications i) decreased the level of GATA-1/FOG-1 complex, ii) unsettled the GATA-1/GATA-2 balance, iii) reduced GATA-1 transcriptional activity and iv) inhibited erythroid marker gene expression (glycophorin A, erythropoietin receptor, gamma-globin) independently of the cell line or the inducer used. These data provided new insights into the role of GATA-1 regulation in TNFalpha-mediated inhibition of erythroid differentiation in erythroleukemia.
Abstract: The proinflammatory cytokine tumor necrosis factor alpha (TNFalpha) has been linked to inflammation- and cancer-related anemia, which reduces both quality of life and prognosis of patients. The aim of this study was to reveal molecular mechanisms linked to the inhibition of erythroid differentiation by TNFalpha. In this study, we showed that the inhibition of erythropoietin (Epo)-mediated differentiation by TNFalpha lead to a downregulation of hemoglobin synthesis and was correlated to a modulation of key erythroid transcription factors. Thus, a reverse of the transcription factor GATA-1/GATA-2 balance normally present during erythropoiesis, as well as a downregulation of the cofactor of GATA-1, friend of GATA-1 (FOG-1), and the coregulating transcription factor nuclear factor erythroid 2 (NF-E2) was observed after TNFalpha treatment. Moreover, we showed a reduction of GATA-1/FOG-1 interaction due to a reduced transcription of GATA-1 and a proteasome-dependent FOG-1 degradation after TNFalpha treatment. These changes led to an inhibition of erythroid gene expression including Epo receptor (EpoR), alpha- and gamma-globin, erythroid-associated factor (ERAF), hydroxymethylbilane synthetase (HMBS), and glycophorin A (GPA). An analysis of distinct signaling pathway activations then revealed an activation of p38 by TNF, as well as a corresponding involvement of this mitogen-activated protein kinase (MAPK) in the cytokine-dependent inhibition of erythroid differentiation. Indeed the p38 inhibitor, SB203580, abrogated the inhibitory effect of TNFalpha on the major erythroid transcription factor GATA-1 as well as erythroid marker expression in Epo-induced TF-1 cells. Overall, these data contribute to a better understanding of cytokine-dependent anemia, by giving first hints about key erythroid transcription factor modulations after TNFalpha treatment as well as an involvement of p38 in the inhibition of erythroid differentiation.
Abstract: Constitutive tyrosine kinase activity of the breakpoint cluster region (Bcr)-Abl fusion protein is characteristic of chronic myelogenous leukemia (CML). As resistance against Imatinib a Bcr-abl inhibitor used in CML, was described, Heat shock protein (Hsp90) became an alternative target as inhibition of Bcr-Abl-Hsp90 complex leads to proliferation arrest. Here, we used natural product Radicicol (Rad), a macrocyclic antifungal, as an Hsp90 inhibitor to investigate the effect of Bcr-Abl inactivation on erythroid gene expression and subsequently on the transcription factors involved in their regulation. We showed that all erythroid genes studied were over-expressed after Rad treatment while Bcr-Abl expression was inhibited. Specific transcription factor NF-E2 was induced in Rad-treated cells as well as GATA-1 cofactors Friend of GATA (FOG)1 and SP1, whereas PU.1 was downregulated. Moreover, p38 mitogen activated protein kinase (MAPK) inhibition prevented Rad-mediated differentiation of K562 in correlation with decreased gamma-globin expression and suppression of Rad-mediated inhibition of PU.1. In conclusion, our results show that Radicicol leads to Bcr-Abl inactivation via Hsp90 inhibition inducing reactivation of the erythroid program in K562 cells.
Abstract: The inducible transcription factor nuclear factor kappaB (NF-kappaB) plays a central role in the regulation of immune, inflammatory and carcinogenic responses. While normal activation of NF-kappaB is required for cell survival and immunity, its deregulated expression is a characteristic of inflammatory and infectious diseases. In this study, we investigated the molecular mechanisms induced by lactones and chalcones isolated from Fijian kava (Piper methysticum) used in traditional medicine against urinary tract infections and asthma. In order to understand underlying regulatory mechanisms, inhibition of both NF-kappaB-driven reporter gene expression and TNFalpha-induced binding of NF-kappaB to a consensus response element was achieved at concentrations of 320 microM (flavokavain A), 175 microM (flavokavain B) and 870 microM (kavain and dihydrokavain). Moreover, kavain and flavokavains A and B treatment led to inhibition of both inhibitor of kappaB (IkappaB) degradation and subsequent translocation of p50 and p65 NF-kappaB subunits from the cytoplasm to the nucleus as shown by Western blot analysis. Additionally, kinase selectivity screening demonstrates that flavokavain A, but not kavain, nor flavokavain B, inhibits the IkappaB kinase (IKK) as well as PRAK (p38-regulated/activated kinase), MAPKAP-K3 (MAPK-activated protein kinase 3), DYRK1A (dual-specificity tyrosine-phosporylated and regulated kinase 1A) and Aurora B. Altogether, these results give a first insight into anti-inflammatory mechanisms triggered by traditionally used chemopreventive kava compounds.
Abstract: Up-regulation of tumor necrosis factor alpha (TNFalpha) is linked to solid tumors as well as to hematologic disorders including different forms of anemia and multiple myeloma. This cytokine was shown to contribute to inhibition of erythroid maturation mechanisms which are characterized by the expression of specific genes regulated by GATA-1 and NF-E2 transcription factors. Here, we assessed the inhibiting effect of TNFalpha on erythroid differentiation using K562 cells which can be chemically induced to differentiate towards the erythroid pathway by aclacinomycin A, an anthracyclin. Results show that induced hemoglobinization of K562 cells as well as gamma-globin and erythropoietin receptor gene expression are decreased by TNFalpha via the inhibition of GATA-1 at its mRNA and protein expression level. Additionally, both constitutive and induced binding activity of GATA-1 is abolished and induced activation of a GATA-1 driven luciferase reporter construct is inhibited. Altogether, our results provide insight into the molecular mechanisms of inflammation-induced inhibition of erythroid differentiation.
Abstract: Over-expression of glutathione S-transferase P1 is related to chemotherapeutic drug resistance as well as to differentiation of human erythroleukemia cells. In opposition to previously described differentiating inducers which enhance the GST-resistance phenotype, time- and concentration-dependent activation of both erythroid and megakaryocytic differentiation pathways by butyric acid progressively diminished GSTP1 mRNA expression. GSTP1 mRNA expression decreased by 25% (p<0.01) and 64% (p<0.01) in 1mM and 2mM butyric acid-differentiated K562 cells, respectively. These results were associated to both a reduction of GATA-1 binding activity to the GSTP1 promoter and to a posttranscriptional destabilization of GSTP1 mRNA in a concentration dependent manner. Indeed, GSTP1 mRNA half-life decreased from 43.8 to 36.2 h and 12.6 h in 1mM- and 2mM-treated cells, respectively.
Abstract: Chemoprevention is a promising anti-cancer approach with reduced secondary effects in comparison to classical chemotherapy. Curcumin, one of the most studied chemopreventive agents, is a natural compound extracted from Curcuma longa L. that allows suppression, retardation or inversion of carcinogenesis. Curcumin is also described as an anti-tumoral, anti-oxidant and anti-inflammatory agent capable of inducing apoptosis in numerous cellular systems. In this review, we describe both properties and mode of action of curcumin on carcinogenesis, gene expression mechanisms and drug metabolism.
Abstract: Glutathione S-transferases (GSTs) play an important role in the protection of cells against xenobiotics and lipid hydroperoxides generated by oxidative stress. In human, the GSTP1-1 expression is commonly increased in many tumors and involved in the development of antineoplastic drug resistance. Reactive oxygen species are released at inflammation sites and oxidative stress conditions enhance the expression of genes encoding antioxidant enzymes such as GSTs. Here we investigated the regulation of the GSTP1-1 gene expression in the K562 cell line by nuclear factor kappaB (NF-kappaB) and the pro-inflammatory cytokine tumor necrosis factor alpha (TNFalpha). By studying GSTP1-1 mRNA expression and NF-kappaB/GSTP1-1 promoter interactions, we showed the implication of NF-kappaB in the GSTP1-1 gene expression and we described a new specific TNFalpha-inducible NF-kappaB binding site upstream of the minimal promoter. Moreover, TNFalpha treatment as well as cotransfection of NF-kappaB signaling pathway intermediates induced an activation of the GSTP1-1 gene promoter in K562 cells. Site-directed mutagenesis of the NF-kappaB site strongly inhibited TNFalpha- and NF-kappaBp65-induced promoter activation. Altogether, we showed that a sequence located at -323/-314 within the GSTP1-1 promoter bound NF-kappaB p50/65 and p65/p65 dimers and that this kappaB site was involved in the regulation of the gene by TNFalpha.
Abstract: Glutathione S-transferase P1-1 (GSTP1-1) is a phase II drug metabolism enzyme implicated in carcinogenesis and development of resistance to anti-cancer drugs. It was previously shown that both activating protein 1 (AP-1) and nuclear factor kappaB (NF-kappaB) are involved in its regulation. In the present study we examined the inhibitory effect of several chemopreventive agents on the tumor necrosis factor (TNF) alpha- or 12-O-tetradecanoylphorbol 13 acetate (TPA)-induced promoter activity of GSTP1-1, as demonstrated by transient transfection experiments in K562 and U937 leukemia cells. Our results provide evidence for a differential effect of chemopreventive agents such as beta-lapachone, emodin, sanguinarine and capsaicin, which significantly inhibit reporter gene expression as well as TNFalpha- and TPA-induced binding of AP-1 and NF-kappaB, whereas trans-anethole and silymarin do not produce any inhibitory effect. Our results demonstrate the ability of selected chemopreventive agents to decrease GSTP1-1 gene expression mechanisms and could thus contribute to reduce the incidence of glutathione related drug resistance in human leukemia.
Abstract: Glutathione S-transferases (GST) are involved in cellular protection against xenobiotics, oxidative stress as well as in resistance against chemotherapeutic compounds such as doxorubicin. Levels of human placental type GSTP1-1 are known to be increased in many tumors and hematopoietic diseases. In this work, we compare transcriptional mechanisms in cells that express or not GSTP1-1. Transient transfection assays are used to show that different GST-promoter reporter constructs generate cell-type specific levels of luciferase activity. In expressing cells, transcriptional activity is strongly dependent on AP-1 binding elements within the -65 to -75 bp region of the GSTP1 gene as shown by site-directed mutagenesis. Electrophoretic mobility shift assays show that DNA binding activity is exclusively observed in GSTP1-1-expressing cells and is increased after stimulation with hydrogen peroxide, TPA, tert-butylhydroquinone and doxorubicin. Non-expressing cells present neither constitutive nor inducible AP-1 binding. Taken together, our results provide evidence for the induction of the GSTP1 gene via AP-1 binding activity in leukemia cells and contribute to a better understanding of the molecular events regulating genes involved in drug resistance mechanisms.
Abstract: GSTP1-1 gene expression mechanisms were investigated in hemin-induced erythroid differentiation of K562 cells. Hemoglobin production during differentiation was followed by a significant increase in GSTP1-1 mRNA (1.7-fold, P < 0.01) and protein (1.2-fold, P < 0.01) after 4 days of induction. This increase in mRNA production was not due to transcriptional up-regulation by GATA-1 previously shown to regulate GSTP1-1 during erythroid and megakaryocytic differentiation. Moreover, a drastic decrease in differentiation-specific GATA-1 mRNA expression was correlated to a reduction in GATA-1 promoter binding activity. Neither AP-1 nor NF-kappaB transcription factor binding activities could provide an explanation to the GSTP1-1 mRNA overexpression in hemin-treated cells. GSTP1-1 mRNA stability analysis using actinomycin D as an inhibitor of mRNA neosynthesis showed that mRNA half-life was doubled in hemin-induced erythroid differentiation of K562 cells. These results allow us to add stabilization of GSTP1-1 mRNA as a novel regulatory mechanism during hemin-mediated differentiation of K562 cells.
Abstract: GATA-1 is the founding member of the GATA family of transcription factors. GATA-1 and GATA family member GATA-2 are expressed in erythroid and megakaryocytic lineages, in which they play a crucial role in cell maturation and differentiation. GATA-1 regulates the transcription of many specific and nonspecific erythroid genes by binding to DNA at the consensus sequence WGATAR, which is recognized by all of the GATA family of transcription factors. However, it was identified in eosinophilic cells and also in Sertoli cells in testis. Its activity depends on close cooperation with a functional network of cofactors, among them Friend of GATA, PU.1, and CBP/p300. The GATA-1 protein structure has been well described and includes two zinc fingers that are directly involved in the interaction with DNA and other proteins in vivo. GATA-1 mutations in the zinc fingers can cause deregulation of required interactions and lead to severe dysfunction in the hematopoietic system.
Abstract: To investigate the stability of curcumin in physiological media, the absorption variation of a curcumin solution was measured in 0.1% and 10% FCS. Under daylight conditions, curcumin degraded very rapidly in 0.1% FCS and was found to be more stable in higher serum concentrations. Under dark conditions, almost no decomposition could be observed after 2 h, whether the measurements were performed in 0.1% or 10% FCS. Furthermore, depending on the medium concentration, differential glutathione S-transferase P1-1 mRNA expression could be observed in K562 cells after incubation with curcumin. Indeed, incubation in 0.1% FCS led to a decrease of mRNA expression, whereas incubation in 10% FCS induced an increase of mRNA production.
Abstract: Apoptosis is a type of cell death that has been observed and studied for more than a century. The process of apoptosis was described as "programmed cell death" in 1964, and the term apoptosis, from a Greek word meaning "to fall away from" and describing the fall of dead leaves from trees in autumn, was only coined in 1972. During the last 30 years, this type of cell death has been extensively investigated and the molecular mechanisms underlying this cell suicide well characterized. Apoptosis is a physiological phenomenon necessary to tissue and body genesis and homeostasis, but defects in its regulation may cause numerous diseases, including cancer. Investigating the mechanisms of apoptosis is thus important to discover new cellular regulators that could be potential targets for new death-inducing drugs.
Abstract: Curcumin presents strong antioxidant and anticancer properties. However, molecular mechanisms leading to curcumin-induced cell death are poorly understood. The effect of curcumin was compared in two different leukemia cell lines: K562 and Jurkat. Cell death was induced in both cell lines, and apoptosis pathways were investigated by Western blot analysis. Decreases in pro-caspase 8 and 9 levels were observed. BH(3) interacting domain death agonist (Bid) was also cleaved. Jurkat cells appeared to be more sensitive to curcumin, and apoptosis takes place earlier.
Abstract: Glutathione S-transferase P1-1 (GSTP1-1) conjugates glutathione to electrophilic compounds and its expression is correlated to chemotherapeutic drug resistance. Results show that GSTP1-1 mRNA as well as protein expressions are increased during Aclarubicin (Acla)- and Doxorubicin (Dox)-induced erythroid differentiation of human K562 cells. In contrast, during megakaryocytic differentiation by 12-O-tetradecanoyl phorbol 13-acetate (TPA), GSTP1-1 expression decreased at both mRNA and protein levels. In order to clarify the molecular mechanisms leading to these variations, we identified a GATA sequence located at -1208 relative to the transcriptional start site of the GSTP1-1 promoter. By gel shift, competition, and supershift analyses we show here the specificity of the GATA-1 binding regulated by both anthracyclines and TPA. Altogether, these results demonstrate for the first time the implication of GATA-1 in differentiation-specific variations of GSTP1-1 expression.
Abstract: Expression of glutathione S-transferase P1-1 (GSTP1-1) is correlated to carcinogenesis and resistance of cancer cells against chemotherapeutic agents. Curcumin, a natural compound extracted from Curcuma longa, has shown strong antioxidant and anticancer properties and also the ability to regulate a wide variety of genes that require activating protein 1 and nuclear factor kappaB (NF-kappaB) activation. In the present study, we examined the inhibitory effect of curcumin on the expression of GSTP1-1 mRNA as well as protein, and we correlated this inhibition with the apoptotic effect of curcumin on K562 leukemia cells. Curcumin efficiently inhibited the tumour necrosis factor alpha- and phorbol ester-induced binding of AP-1 and NF-kappaB transcription factors to sites located on the GSTP1-1 gene promoter. TNFalpha-induced GSTP1-1 promoter activity was also inhibited by curcumin as shown by reporter gene assay. In parallel, curcumin induced pro-caspases 8 and 9 as well as poly ADP ribose polymerase cleavage and thus leading to apoptosis in K562 cells. Our results overall add a novel role for curcumin as this chemoprotective compound could contribute to induce apoptosis by its ability to inhibit the GSTP1-1 expression at the level of transcription.
Abstract: Overexpression of the glutathione S-transferase P1 (GSTP1) gene is related to drug resistance in human cancer cells. However, the mechanisms of the transcriptional activation of this gene remain unclear. In this study, we examined the molecular mechanisms underlying phorbol ester mediated gene regulation using human K562 leukemia cells as a model. Promoter deletion analyses revealed that the activator protein-1 (AP-1) transcription factor site was crucial for 12-O-tetradecanoyl phorbol 13-acetate (TPA)-mediated GSTP1 gene transcription. Electrophoretic mobility shift assays and transient transfection analysis demonstrated that both DNA binding and transactivation activities of AP-1 were induced by TPA. By supershift analysis, we identified transcription factors c-jun and fra-1 as well as NF-E2p45 as components of the induced binding complex. These results show for the first time that the phorbol ester TPA is involved in the molecular mechanism(s) mediating the activation of the GSTP1 promoter in a human leukemia model.
Abstract: To study the relationship between methylation and the transcriptional activity of the minimal promoter of the glutathione S-transferase GSTP1 gene encoding glutathione S-transferase P1-1, GSTP1 mRNA levels as well as basal promoter activity were compared in human leukemia cell lines. The K562 erythroleukemia cell line presented a strong GSTP1 promoter activity, as measured in transient transfection assays using a luciferase reporter plasmid, and correlated with a high mRNA whereas in Raji cells no mRNA was expressed. In order to establish a relationship between the expression and the methylation status, we used in vitro bisulfite sequencing which indicated that both methylated and unmethylated GSTP1 promoter alleles coexisted in K562 cells, whereas Raji lymphoma cells showed a nearly uniform hypermethylation of the promoter region. To determine the impact of methylation, we used in vitro SssI methylation of the minimal GSTP1 promoter, which led to the silencing of the promoter activity in transient transfection assays in expressing K562 as well as in non-expressing Raji cells. These data are in good agreement with previously obtained results and indicate that methylation of CpG sites of the basal promoter is an essential mechanism in the control of GSTP1 gene expression in human leukemia.
Abstract: Induction of specific gene expression may provide an alternative or a support to conventional cytotoxic chemotherapy of cancer, as well as to therapy for sickle cell diseases. In this respect, pharmacological induction of expression of the endogenous gamma-globin gene is a realistic approach to therapy of beta-globin disorders. Erythroid differentiation and inhibition of proliferation of the human CML K562 cell line was induced by guanosine 5'-triphosphate (GTP). The hemoglobin production in cells was correlated to an increase in alpha- and gamma-globin mRNA expression. At the transcriptional level, we showed that both the expression of the major erythroid transcription factor GATA-1 (protein and mRNA) and its binding capacity to the gamma-globin gene promoter was transiently increased. Moreover, GTP moderately stimulated the gamma-globin gene promoter after 48 h of treatment. At the post-transcriptional level, GTP treatment led to a drastic increase of the gamma-globin mRNA half-life. This stabilizing effect of GTP was mediated via the 3'-untranslated region (3'-UTR) of the gamma-globin mRNA. In conclusion, mechanism of GTP-mediated differentiation of K562 cells is linked to an early activation of gamma-globin gene transcription followed by a stabilization of its mRNA.
Abstract: Tumor cells, and particularly leukemic cells, can be considered as maturation-arrested cells which have escaped some normal control and continue to proliferate. This maturation arrest can be reversed by differentiation agents such as antitumor drugs currently used in conventional cytotoxic chemotherapy. In this respect, anthracyclines have been shown to trigger the differentiation of leukemic and solid tumor cells, but the molecular mechanisms by which such drugs lead to the differentiating phenotype are still poorly understood. Using human leukemic multipotent K562 cells, we have demonstrated that subtoxic concentrations of aclacinomycin (ACLA) and doxorubicin (DOX) preferentially stimulate the hemoglobinic pathway (globins and heme synthesis) and the expression of mRNAs of globins and of porphobilinogen deaminase (PBGD). However, our results indicate that both drugs exert this differentiating effect along distinct regulatory pathways. Indeed, only ACLA and not DOX induces the expression of erythropoietin receptor (EpoR) mRNAs and of membrane EpoR, as well as an overexpression of the erythroid transcription factors GATA-1 and NF-E2 known to play a central role in erythroid gene regulation. Similarly, using transfection assays, ACLA but not DOX activates the regulatory regions (promoters and enhancers) of GATA-1, EpoR, PBGD, epsilon- and gamma-globin genes. Finally, results of run-on assays indicate that ACLA induces an enhancement of the transcription rate of these erythroid genes whereas DOX preferentially increases stability of GATA-1, NF-E2 and PBGD mRNAs. In conclusion, ACLA mainly acts at the transcriptional level via specific activation of erythroid regulatory regions whereas DOX rather acts at the posttranscriptional level by increasing the half-lives of erythroid mRNAs.
Abstract: Aclacinomycin (ACLA) and doxorubicin (DOX) were used at subtoxic concentrations to induce erythroid differentiation in the human leukemic cell line K562. Cell hemoglobinization was accompanied by the increased expression of genes encoding gamma-globin and porphobilinogen deaminase (PBGD), an enzyme of heme synthesis. By using run-on assays, ACLA was shown to induce an enhancement of the transcription of erythroid genes, including gamma-globin, PBGD, erythropoietin receptor, and GATA-1 transcription factor. In contrast, in DOX-treated cells, the transcription rate of these genes was unchanged in comparison with control cells. In addition, inhibition of mRNA synthesis with actinomycin D indicated that DOX induced an increased stability of PBGD and GATA-1 mRNAs, whereas ACLA did not affect the half-lives of these mRNAs. Because the increase in erythroid mRNA steady-state level in anthracycline-treated cells was inhibited by cycloheximide, this suggests that transcriptional activation in ACLA-treated cells and mRNA stabilization in DOX-treated cells were dependent on de novo protein synthesis. Finally, GATA-1 protein level was shown to be increased in ACLA-treated but not in DOX-treated cells. These two anthracyclines, although closely related in their structures, appeared to act as differentiation inducers by distinct mechanisms. Indeed, erythroid gene expression was demonstrated to be regulated transcriptionally by ACLA and mainly posttranscriptionally by DOX.
Abstract: Human erythroleukemic K 562 cells were induced to were induced to differentiate along the erythroid lineage by anthracycline antitumor drugs, such as aclacinomycin (ACLA) and doxorubicin (DOX). Subsequent stimulation of heme and globin synthesis led to a differential quantitative expression of hemoglobins. Gower 1 (epsilon2, zeta2) was the major type for ACLA and X (epsilon2, gamma2) for DOX. Although ACLA and DOX increased both the expression of gamma-globin and porphobilinogen deaminase mRNAs, striking differences were observed in the expression of erythropoietin receptor mRNAs and in erythroid transcription factors GATA-1 and NF-E2, known to play a key role in erythroid gene regulation. Indeed, ACLA induces an increase either in the binding capacity of GATA-1 and NF-E2 or in the accumulation of erythropoietin receptor, GATA-1 and NF-E2 transcripts. In contrast, their expression with DOX was not significantly modified compared to uninduced cells, except for a slight decrease in NF-E2 expression on day 3. In conclusion, these data show that: 1. increased expression of erythroid transcription factors and erythroid genes are associated only with ACLA treatment, and 2. although cytotoxicity of both ACLA and DOX is certainly dependent on DNA intercalation, regulation of differentiation processes by these two drugs involves distinct mechanisms.
Abstract: Anthracycline antitumor drugs, particularly aclacinomycin (ACM) have been shown to be potent inducers of erythroid differentiation in human leukemic K562 cells. Here we report that such an event is associated with an overexpression of the erythroid-specific transcription factors GATA-1 and NFE-2. Using the electrophoretic mobility shift assay, during differentiation over 3 days of culture, we have observed an increase in the binding either of GATA-1 to the promoter of the gamma-globin gene (region -201 to -156) or NFE-2 to the promotor of the porphobilinogen deaminase gene (region -170 to -142). Both events were paralleled by a recruitment of hemoglobinized cells and a stimulation of heme synthesis. Enhanced binding capacity of GATA-1 was confirmed by an increase in its mRNAs. Moreover, GATA-1 and NFE-2 overexpression has been shown to be specific of the differentiating effect of the drug and not of its growth inhibitory effect. In contrast, no change was observed in the binding of the ubiquitous factors OTF-1 and AP-1, except on day 3, where AP-1 decreased. Although ACM is a DNA-intercalating agent, it did not directly affect transcription factors binding to their cis-sequences as assessed by the preincubation of the oligonucleotides probes with increasing concentrations of ACM. Taken together, these results strongly suggest that ACM could exert their erythroid-differentiating activity by modulating the expression of transcription factors which specifically regulate the transcription of erythroid genes.
