Inhibition of heme synthesis decreases transferrin receptor expression in mouse erythroleukemia cells.

The coordination of transferrin receptor (TfR) expression and heme synthesis was investigated in mouse erythroleukemia (MEL) cells of line 707 treated with heme synthesis inhibitors or in a variant line Fw genetically deficient in heme synthesis. Cells of line 707 were induced for differentiation by 5 mM hexamethylene bisacetamide (HMBA). TfR expression increased in the course of induction, as judged by increased TfR mRNA synthesis, increased cytoplasmic TfR mRNA level, and by the increased number of cellular 125I-Tf binding sites. Addition of 0.1 mM succinylacetone (SA) decreased cellular TfR to the level comparable with the uninduced cells. The decrease was reverted by the iron chelator desferrioxamine (DFO) but not by exogenous hemin. In short-term (1-2 hours) incubation, SA inhibited 59Fe incorporation from transferrin into heme, whereas total cellular 59Fe uptake was increased. A decrease in TfR mRNA synthesis was apparent after 2 hours of SA treatment. Conversely, glutathione peroxidase mRNA synthesis, previously shown to be inducible by iron, was increased by SA treatment. Cells of heme deficient line Fw did not increase the number of Tf binding sites after the induction of differentiation by 5 mM sodium butyrate. SA had no effect on TfR expression in Fw cells. The results suggest that the depletion of cellular non-heme iron due to the increase in heme synthesis maintains a high level of transferrin receptor expression in differentiating erythroid cells even after the cessation of cell division.     

Dual signaling functions of the hybrid sensor kinase RpfC of Xanthomonas campestris involve either phosphorelay or receiver domain-protein interaction

The hybrid sensor kinase RpfC positively regulates the expression of a range of virulent genes and negatively modulates the synthesis of the quorum sensing signal diffusible signal factor (DSF) in Xanthomonas campestris. Three conserved amino acid residues of RpfC implicated in phosphorelay (His(198) in the histidine kinase domain, Asp(512) in the receiver domain, and His(657) in the histidine phosphotransfer domain) were essential for activation of the production of extracellular enzymes and extracellular polysaccharide (EPS) virulence factors but were not essential for repression of DSF biosynthesis. Domain deletion and subsequent in trans expression analysis revealed that the receiver domain of RpfC alone was sufficient to repress DSF overproduction in an rpfC deletion mutant. Further deletion and alanine scanning mutagenesis analyses identified a peptide of 107 amino acids and three amino acid residues (Gln(496), Glu(504), and Ile(552)) involved in modulating DSF production. Co-immunoprecipitation and far Western blot analyses suggested an interaction between the receiver domain and RpfF, the enzyme involved in DSF biosynthesis. These data support a model in which RpfC modulates two different functions (virulence factor synthesis and DSF synthesis) by utilization of a conserved phosphorelay system and a novel domain-specific protein-protein interaction mechanism, respectively. This latter mechanism represents an added dimension to conventional two-component signaling paradigms.     

The oviduct produces erythropoietin in an estrogen- and oxygen-dependent manner

Previously, we showed that erythropoietin (Epo) is produced in the mouse uterus, where Epo is indispensable for estrogen (E(2))-dependent angiogenesis. Expression of uterine Epo mRNA is stimulated by E(2) and hypoxia. The hypoxic induction requires the presence of E(2). In the present study, we examined other female reproductive organs in the mouse with respect to Epo mRNA expression and its stimuli (E(2) and hypoxia)-induced changes. Although Epo mRNA expression was seen in the ovary and oviduct, the E(2)-induced stimulation of Epo mRNA was found only in the oviduct. The E(2)-induced stimulation in the oviduct was transient and rapidly downregulated. Epo mRNA expression in the oviduct was hypoxia inducible, in both the presence and the absence of E(2). E(2)-dependent production of Epo and its mRNA expression were also found by use of cultured oviducts. The E(2) action is probably mediated through the E(2) receptor, and de novo protein synthesis is not required for E(2) induction of Epo mRNA. In the oviduct, the ampulla and isthmus regions produce Epo.     

Astroglial cytoprotection by erythropoietin pre-conditioning: implications for ischemic and degenerative CNS disorders

Erythropoietin (Epo) is a glycoprotein secreted by the kidney in response to hypoxia that stimulates erythropoiesis through interaction with cell surface Epo receptors. Pre-treatment with Epo has been shown to protect neurons in models of ischemic injury. The mechanism responsible for this neuroprotection and the effects of Epo on astroglial and other non-neuronal cell populations remain unknown. In the present study, we determined whether Epo pre-treatment protects neonatal rat astrocytes from apoptotic cell death resulting from treatment with nitric oxide, staurosporine (STS) and arsenic trioxide and possible mechanisms mediating Epo-related cytoprotection. Epo (5-20 U/mL) significantly attenuated multiple hallmarks of apoptotic cell death in astroglia exposed to nitric oxide and STS but not arsenic trioxide. Epo (20 U/mL) induced mild oxidative stress as shown by increases in heme oxygenase (HO)-1 mRNA and protein expression that could be suppressed by antioxidant coadministration. Moreover, coincubation with tin-mesoporphyrin, a competitive inhibitor of HO activity, abrogated the cytoprotective effects of Epo (20 U/mL) in the face of STS treatment. Thus, induction of the ho-1 gene may contribute to the glioprotection accruing from high-dose Epo exposure. Epo may augment astroglial resistance to certain chemical stressors by oxidative stress-dependent and -independent mechanisms.     

Anti-proliferative activity of disulfiram through regulation of the AKT-FOXO axis: A proteomic study of molecular targets

Due to its potent anti-tumor activity, well-investigated pharmacokinetic properties and safety profile, disulfiram (DSF) has emerged as a promising candidate for drug repurposing in cancer therapy. Although several molecular mechanisms have been proposed for its anti-cancer effects, the precise underlying mechanisms remain unclear. In the present study, we showed that DSF inhibited proliferation of cancer cells by inducing reactive oxygen species (ROS) production, a G1 cell cycle arrest and autophagy. Moreover, DSF triggered apoptosis via suppression of the anti-apoptotic protein survivin.To elucidate the mechanisms for the anti-proliferative activities of DSF, we applied a 2-DE combined with MALDI-TOF-MS/MS analysis to identify differentially expressed proteins in breast cancer cells upon treatment with DSF. Nine differentially expressed proteins were identified among which, three candidates including calmodulin (CaM), peroxiredoxin 1 (PRDX1) and collagen type I alpha 1 (COL1A1) are involved in the regulation of the AKT signaling pathway. The results of western blot analysis confirmed that DSF inhibited p-AKT, suggesting that DSF induces its anti-tumor effects via AKT blockade. Moreover, we found that DSF increased the mRNA levels of FOXO1, FOXO3 and FOXO4, and upregulated the expression of their target genes involved in G1 cell cycle arrest, apoptosis and autophagy. Finally, DSF potentiated the anti-proliferative effects of well-known chemotherapeutic agents such as arsenic trioxide (ATO), doxorubicin, paclitaxel and cisplatin. Altogether, these findings provide mechanistic insights into the anti-growth activities of DSF.     

Dihydroartemisinin Inhibits Glucose Uptake and Cooperates with Glycolysis Inhibitor to Induce Apoptosis in Non-Small Cell Lung Carcinoma Cells

Despite recent advances in the therapy of non-small cell lung cancer (NSCLC), the chemotherapy efficacy against NSCLC is still unsatisfactory. Previous studies show the herbal antimalarial drug dihydroartemisinin (DHA) displays cytotoxic to multiple human tumors. Here, we showed that DHA decreased cell viability and colony formation, induced apoptosis in A549 and PC-9 cells. Additionally, we first revealed DHA inhibited glucose uptake in NSCLC cells. Moreover, glycolytic metabolism was attenuated by DHA, including inhibition of ATP and lactate production. Consequently, we demonstrated that the phosphorylated forms of both S6 ribosomal protein and mechanistic target of rapamycin (mTOR), and GLUT1 levels were abrogated by DHA treatment in NSCLC cells. Furthermore, the upregulation of mTOR activation by high expressed Rheb increased the level of glycolytic metabolism and cell viability inhibited by DHA. These results suggested that DHA-suppressed glycolytic metabolism might be associated with mTOR activation and GLUT1 expression. Besides, we showed GLUT1 overexpression significantly attenuated DHA-triggered NSCLC cells apoptosis. Notably, DHA synergized with 2-Deoxy-D-glucose (2DG, a glycolysis inhibitor) to reduce cell viability and increase cell apoptosis in A549 and PC-9 cells. However, the combination of the two compounds displayed minimal toxicity to WI-38 cells, a normal lung fibroblast cell line. More importantly, 2DG synergistically potentiated DHA-induced activation of caspase-9, -8 and -3, as well as the levels of both cytochrome c and AIF of cytoplasm. However, 2DG failed to increase the reactive oxygen species (ROS) levels elicited by DHA. Overall, the data shown above indicated DHA plus 2DG induced apoptosis was involved in both extrinsic and intrinsic apoptosis pathways in NSCLC cells.     

Docosahexaenoic acid induces proteasome-dependent degradation of estrogen receptor α and inhibits the downstream signaling target in MCF-7 breast cancer cells

About two thirds of breast cancers in women are hormone-dependent and require estrogen for growth, its effects being mainly mediated through estrogen receptor α (ERα). Docosahexaenoic acid (DHA, 22:6n-3) and arachidonic acid (AA, 20:4n-6) have opposite effects on carcinogenesis, with DHA suppressing and AA promoting tumor growth both in vitro and in vivo. However, the mechanism is not clear. Here, we examined whether the effect is mediated through changes in ERα distribution. MCF-7 cells, an ERα-positive human breast cancer cell line, was cultured in estrogen-free medium containing 0, 10 or 60 μM DHA or AA, then were stimulated with estradiol. DHA supplementation resulted in down-regulation of ERα expression (particularly in the extranuclear fraction), a reduction in phosphorylated MAPK, a decrease in cyclin D1 levels and an inhibition in cell viability. In contrast, AA had no such effects. The DHA-induced decrease in ERα expression resulted from proteasome-dependent degradation and not from decreased ERα mRNA expression. We propose that breast cancer cell proliferation is inhibited by DHA through proteasome-dependent degradation of ERα, reduced cyclin D1 expression and inhibition of MAPK signaling.     

Cloning and characterization of a proliferation-associated cytokine-inducible protein, CIP29

We identified a novel erythropoietin (Epo)-induced protein (CIP29) in lysates of human UT-7/Epo leukemia cells using two-dimensional gel analysis and cloned its full-length cDNA. CIP29 contains 210 amino acids with a predicted MW of 24 kDa, and has a N-terminal SAP DNA-binding motif. CIP29 expression was higher in cancer and fetal tissues than in normal adult tissues. CIP29 mRNA expression is cytokine regulated in hematopoietic cells, being up-regulated by Epo in UT7/Epo cells, and by thrombopoietin (Tpo), FLT3 ligand (FL) and stem cell factor (SCF) in primary human CD34(+) cells. Up-regulation of CIP29 in UT7/Epo cells by Epo was associated with cell cycle progression but not with antiapoptosis. Epo withdrawal reduced CIP29 expression concomitant with cell cycle arrest. Overexpression of CIP29-GFP in HEK293 cells enhances cell cycle progression. CIP29 appears to be a new cytokine regulated protein involved in normal and cancer cell proliferation.     

Dihydroartemisinin increases gemcitabine therapeutic efficacy in ovarian cancer by inducing reactive oxygen species

Ovarian cancer is the major cause of death in women gynecological malignancy and gemcitabine (GEM) is commonly used in related chemotherapy. However, more than 90% GEM is catalyzed into an inactive metabolite 2′-deoxy-2′,2′-difluorouridine by stromal and cellular cytidine deaminase (CDA). Dihydroartemisinin (DHA), which possesses an intramolecular endoperoxide bridge, could be activated by heme or ferrous iron to produce reactive oxygen species (ROS). The excess ROS generation will excite expression of heme oxygenase-1 and suppress CDA expression. Under low CDA expression, the inactivation of GEM is decreased in turn to exert excellent therapeutic efficiency. Herein, we first studied the ROS generation by DHA in vitro with A2780 cells by means of flow cytometry and confocal laser scanning microscopy. Furthermore, cytotoxicity assay in vitro showed that DHA + GEM had synergistic effect, with molar ratio of DHA and GEM at 10. Eventually, in A2780 ovarian cancer xenograft tumor model, DHA + GEM exhibited significant antitumor efficiency with lower blood toxicity than GEM alone. Noteworthy, the combination treatment group completely eliminated the tumors on day 14.     

Dihydroartemisinin induces apoptosis in human gastric cancer cell line BGC-823 through activation of JNK1/2 and p38 MAPK signaling pathways

Dihydroartemisinin (DHA), a semi-synthetic derivative of artemisinin, is associated with a broad range of biological properties including antitumor activity. However, the effect of DHA on gastric cancer has not been clearly clarified. The aim of this study was to investigate the role and mechanism of DHA in human gastric cancer cell line BGC-823. Cell viability was assessed by MTT assay. Cell apoptosis was analyzed with flow cytometry. The expressions of extracellular signal-regulated kinase (ERK), c-Jun N-terminal kinase (JNK), p38 mitogen-activated protein kinase (p38 MAPK) and their phosphorylated forms as well as apoptosis related proteins were examined by western blot analysis. The results demonstrated that DHA inhibited cell viability of BGC-823 cells in a dose- and time-dependent manner. DHA treatment upregulated the expression of Bax, cleaved caspase-3 and -9, and degraded form of PARP, and downregulated the Bcl-2 expression and Bcl-2/Bax ratio. Meanwhile, DHA increased the phosphorylation of ERK1/2, JNK1/2 and p38 MAPK. Synthetic inhibitors of JNK1/2 or p38 MAPK kinase activity, but not inhibitor of ERK1/2, significantly abolished the DHA-induced activation of caspase-3 and -9. In vivo tumor-suppression assay further indicated that DHA displayed significant inhibitory effect on BGC-823 xenografts in tumor growth. These results indicate that DHA induces apoptosis of BGC-823 cells through JNK1/2 and p38 MAPK signaling pathways and DHA could serve as a potential additional chemotherapeutic agent for treatment of gastric cancer.     

Diverse effects of ethanol on the pathway of inducible prostaglandin E2 production in macrophages.

The effects of ethanol on inducible prostaglandin production in RAW macrophages were investigated. Indomethacin (1 microM) or cycloheximide (1 microM) abolished prostaglandin E2 (PGE2) production induced by lipopolysaccharide (LPS, 1 microg/ml). Ethanol at concentrations from 100 mM to 600 mM concentration-dependently inhibited inducible PGE2 production, while ethanol only at higher concentrations (400 mM or more) showed cytotoxity to the cells. Cyclooxygenase-2 (COX-2) activity, estimated by transformation of exogenous arachidonic acid into PGE2, was not affected by ethanol (100-400 mM). LPS-induced expression of COX-2 mRNA was inhibited by ethanol (50-400 mM). On the other hand, protein expression of COX-2 by LPS was significantly increased by ethanol (100-400 mM). Ethanol alone at concentrations up to 600 mM did not induce expression of COX-2 protein. In a medium containing arachidonic acid (1 microM), ethanol at a low concentration (100 mM) did not significantly affect LPS-induced PGE2 production. These results suggest that ethanol shows diverse effects on the pathway of inducible PGE2 production in macrophages. Finally, ethanol may suppress utilization of arachidonic acid, resulting in reduction of inducible PGE2 production. Further study is needed to elucidate the mechanism of dissociation of ethanol effects on protein and mRNA expression.     

Downregulation of COX-2 and iNOS by amentoflavone and quercetin in A549 human lung adenocarcinoma cell line

Flavonoids are natural polyphenolic compounds ubiquitously present in the plant kingdom. They are reported to exhibit numerous beneficial health effects. In the present study, we demonstrate the potential effects of different flavonoids on cytokines mediated cyclooxygenase-2 and inducible nitric oxide synthase expression and activities in A549 cell line using quercetin, amentoflavone and flavanone. Our data revealed that quercetin, at 50 micro M concentration inhibited PGE(2) biosynthesis by A549 very strongly with little effect on COX-2 mRNA and protein expression. Unlike quercetin, amentoflavone inhibited both PGE(2) biosynthesis and COX-2 mRNA and protein expression strongly. In another set of experiment, quercetin inhibited iNOS protein expression completely without affecting iNOS mRNA expression. In contrast, amentoflavone although exerted no inhibitory effect on iNOS mRNA expression, did inhibit weakly iNOS protein expression. Flavanone had no inhibitory effect on either enzyme at the same concentration. Taken together, our data indicated that amentoflavone and quercetin differentially exerted supression of PGE(2) biosynthesis via downregulation of COX-2/iNOS expression.     

Docosahexaenoic acid inhibition of inflammation is partially via cross-talk between Nrf2/heme oxygenase 1 and IKK/NF-κB pathways

We examined the underlying mechanisms involved in n-3 docosahexaenoic acid (DHA) inhibition of inflammation in EA.hy926 cells. The present results demonstrated that pretreatment with DHA (50 and 100 μM) inhibited tumor necrosis factor-alpha (TNF-α)-induced intercellular adhesion molecule 1 (ICAM-1) protein, mRNA expression and promoter activity. In addition, TNF-α-stimulated inhibitory kappa B (IκB) kinase (IKK) phosphorylation, IκB phosphorylation and degradation, p65 nuclear translocation, and nuclear factor-κB (NF-κB) and DNA binding activity were attenuated by pretreatment with DHA. DHA triggered early-stage and transient reactive oxygen species (ROS) generation and significantly increased the protein expression of heme oxygenase 1 (HO-1), induced nuclear factor erythroid 2-related factor 2 (Nrf2) translocation to the nucleus and up-regulated antioxidant response element (ARE)-luciferase reporter activity. Moreover, DHA inhibited Nrf2 ubiquitination and proteasome activity. DHA activated Akt, p38 and ERK1/2 phosphorylation, and specific inhibitors of respective pathways attenuated DHA-induced Nrf2 nuclear translocation and HO-1 expression. Transfection with HO-1 siRNA knocked down HO-1 expression and partially reversed the DHA-mediated inhibition of TNF-α-induced p65 nuclear translocation and ICAM-1 expression. Importantly, we show for the first time that HO-1 plays a down-regulatory role in NF-κB nuclear translocation, and inhibition of Nrf2 ubiquitination and proteasome activity are involved in increased cellular Nrf2 level by DHA. In this study, we show that HO-1 plays a down-regulatory role in NF-κB nuclear translocation and that the protective effect of DHA against inflammation is partially via up-regulation of Nrf2-mediated HO-1 expression and inhibition of IKK/NF-κB signaling pathway.