Effect of curcumin treatment on protein phosphorylation in K562 cells.

Deregulation of signaling pathways is a common feature observed in human cancers and other diseases. Therefore, there is a strong need for compounds that are able to modulate or inactivate upregulated signaling events. Natural compounds extracted from plants have long been used and still present a dynamic domain in the research of new therapeutic tools. Among those molecules, curcumin was already described for its antioxidative, anti-inflammatory, and antiseptic properties. Many actions of curcumin target proteins and kinases implicated in the signaling pathways. However, the effects described depend on the treatment conditions used, as well as the cell line studied, and these features vary strongly from one study to the other. During this work, we evaluated the effect of one curcumin treatment (20 muM, 48 h) on the phosphorylation of a number of proteins and kinases in the human chronic myelogenous leukemia cell line K562. These results allow to compare the results obtained in one condition on various proteins.

Curcumin regulates signal transducer and activator of transcription (STAT) expression in K562 cells.

Signal transducers and activators of transcription (STATs) play important roles in numerous cellular events as for example differentiation, inflammation or immune response. Furthermore, constitutive STAT activation can be observed in a high number of tumors. In our hands, curcumin treatment induced a decrease of nuclear STAT3, -5a and -5b, without affecting neither STAT1, nor the phosphorylation state of STAT1, -3 or -5 in the K562 cell line. Most interestingly, the decrease of nuclear STAT5a and -5b after curcumin treatment was accompanied by an increase of truncated STAT5 isoforms, indicating that curcumin is able to induce the cleavage of STAT5 into its dominant negative variants lacking the STAT5 C-terminal region. Interferon (IFN)-beta and -gamma treatment induced IFN-stimulated responsive element (ISRE) transcriptional activity, which was efficiently inhibited by curcumin pre-treatment. In parallel, IFN-gamma treatment induced an increase of the amount of nuclear STAT1 and -3, as well as their phosphorylated isoforms. Again, curcumin pre-treatment inhibited these increases. Finally, curcumin treatment inhibited Jak2 mRNA expression as well as cyclin D1 and v-src gene expression in K562 chronic leukaemia cells.

Inhibition of TNFalpha-induced activation of nuclear factor kappaB by kava (Piper methysticum) derivatives.

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.

Transcriptional and post-transcriptional regulation of glutathione S-transferase P1 expression during butyric acid-induced differentiation of K562 cells.

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.

Tumor necrosis factor alpha inhibits aclacinomycin A-induced erythroid differentiation of K562 cells via GATA-1.

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.

Chemopreventive and therapeutic effects of curcumin.

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.

Photoreaction of [Ru(hat)2phen]2+ with guanosine-5'-monophosphate and DNA: formation of new types of photoadducts.

[Ru(hat)2phen]2+ (HAT=1,4,5,8,9,12-hexaazatriphenylene, phen=1,10-phenanthroline) interacts with a good affinity with polynucleotides and DNA by intercalation, despite the presence of a second voluminous ancillary HAT ligand. It photoreacts with guanosine-5'-monophosphate (GMP). From HPLC, ESMS and NMR analyses, it can be concluded that this complex forms photoadducts with GMP. In contrast to the photoadducts isolated with Ru-TAP complexes (TAP=1,4,5,8-tetraazaphenanthrene), the photoadducts with [Ru(hat)2phen]2+ contain a covalent link between the oxygen atom of the guanine unit and a HAT ligand. Formation of oxidised photoadducts and compounds resulting from the addition of two GMP entities to the complex are also detected as side products. In the presence of oligo- and polynucleotides, [Ru(hat)2phen]2+ yields photoadducts when guanine bases are present.

Expression of glutathione S-transferase P1-1 in leukemic cells is regulated by inducible AP-1 binding.

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.

Effect of chemopreventive agents on glutathione S-transferase P1-1 gene expression mechanisms via activating protein 1 and nuclear factor kappaB inhibition.

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.

A beginner's guide to NF-kappaB signaling pathways.

Nuclear factor kappaB (NF-kappaB) belongs to a family of heterodimeric transcription factors that play a key role in inflammatory and stress responses as well as in tumor cell resistance to apoptosis. These effects are due to the NF-kappaB-dependent transcription of many proinflammatory and antiapoptotic genes, whose products ensure various cell responses to environmental conditions. The signal transduction pathways leading to NF-kappaB activation are well characterized, and the different steps implicated in these pathways involve proteins that could constitute targets for NF-kappaB inhibition. Several inhibitors aiming to prevent NF-kappaB activity and thus the transcription of target genes are studied, and a few compounds seem particularly promising. We try here to summarize the advantages that can issue from various studies on NF-kappaB.

Curcumin stability and its effect on glutathione S-transferase P1-1 mRNA expression in K562 cells.

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.