Licochalcone A Potently Inhibits Tumor Necrosis Factor ␣- Induced Nuclear Factor-B Activation through the Direct Inhibition of IB Kinase Complex Activation

ABSTRACT Glycyrrhiza inflata has been used as a traditional medicine with anti-inflammatory activity; however, its mechanism has not been fully understood. Licochalcone A is a major and biogenetically characteristic chalcone isolated from G. inflata. Here, we found that licochalcone A strongly inhibited tumor necrosis (TNF)-␣-induced nuclear localization, DNA binding activity, and the transcriptional activity of nuclear factor-B (NF-B). Whereas licochalcone A had no effect on the recruitment of receptor-interacting protein 1 and IB kinase ␤ (IKK␤) to TNF receptor I by TNF-␣, it significantly inhibited TNF-␣-induced IB kinase complex (IKK) activation and inhibitor of nuclear factor-B degradation. It is interesting that we found that the cysteine residue at position 179 of IKK␤ is essential for licochalcone A-induced IKK inhibition, because licochalcone A failed to affect the kinase activity of the IKK␤ (C179A) mutant. In contrast, a structurally related compound, echinatin, failed to inhibit TNF-␣-induced IKK activation and NF-B activation, suggesting that the 1,1-dimethy-2-propenyl group in licochalcone A is important for the inhibition of NF-B. In addition, TNF-␣-induced expression of inflammatory cytokines CCL2/ monocyte chemotactic protein-1and CXCL1/KC was clearly inhibited by licochalcone A but not echinatin. Taken together, licochalcone A might contribute to the potent anti-inflammatory effect of G. inflata through the inhibition of IKK activation

EBP2, a novel NPM‐ALK‐interacting protein in the nucleolus, contributes to the proliferation of ALCL cells by regulating tumor suppressor p53

The oncogenic fusion protein nucleophosmin‐anaplastic lymphoma kinase (NPM‐ALK), found in anaplastic large‐cell lymphoma (ALCL), localizes to the cytosol, nucleoplasm, and nucleolus. However, the relationship between its localization and transforming activity remains unclear. We herein demonstrated that NPM‐ALK localized to the nucleolus by binding to nucleophosmin 1 (NPM1), a nucleolar protein that exhibits shuttling activity between the nucleolus and cytoplasm, in a manner that was dependent on its kinase activity. In the nucleolus, NPM‐ALK interacted with Epstein–Barr virus nuclear antigen 1‐binding protein 2 (EBP2), which is involved in rRNA biosynthesis. Moreover, enforced expression of NPM‐ALK induced tyrosine phosphorylation of EBP2. Knockdown of EBP2 promoted the activation of the tumor suppressor p53, leading to G0/G1‐phase cell cycle arrest in Ba/F3 cells transformed by NPM‐ALK and ALCL patient‐derived Ki‐JK cells, but not ALCL patient‐derived SUDH‐L1 cells harboring p53 gene mutation. In Ba/F3 cells transformed by NPM‐ALK and Ki‐JK cells, p53 activation induced by knockdown of EBP2 was significantly inhibited by Akt inhibitor GDC‐0068, mTORC1 inhibitor rapamycin, and knockdown of Raptor, an essential component of mTORC1. These results suggest that the knockdown of EBP2 triggered p53 activation through the Akt‐mTORC1 pathway in NPM‐ALK‐positive cells. Collectively, the present results revealed the critical repressive mechanism of p53 activity by EBP2 and provide a novel therapeutic strategy for the treatment of ALCL

Jak2 FERM Domain Interaction with the Erythropoietin Receptor Regulates Jak2 Kinase Activity▿

Janus kinases are essential for signal transduction by a variety of cytokine receptors and when inappropriately activated can cause hematopoietic disorders and oncogenesis. Consequently, it can be predicted that the interaction of the kinases with receptors and the events required for activation are highly controlled. In a screen to identify phosphorylation events regulating Jak2 activity in EpoR signaling, we identified a mutant (Jak2-Y613E) which has the property of being constitutively activated, as well as an inactivating mutation (Y766E). Although no evidence was obtained to indicate that either site is phosphorylated in signaling, the consequences of the Y613E mutation are similar to those observed with recently described activating mutations in Jak2 (Jak2-V617F and Jak2-L611S). However, unlike the V617F or L611S mutant, the Y613E mutant requires the presence of the receptor but not Epo stimulation for activation and downstream signaling. The properties of the Jak2-Y613E mutant suggest that under normal conditions, Jak2 that is not associated with a receptor is locked into an inactive state and receptor binding through the FERM domain relieves steric constraints, allowing the potential to be activated with receptor engagement

Treatment with ODC inhibitor, DFMO, significantly inhibited tumorigenesis induced by JAK2 (V617F).

<p>Ba/F3 cells were infected with empty virus (-) and retrovirus encoding JAK2 V617F mutant and EpoR. Transduced Ba/F3 cells were s.c. injected into nude mice (1×10⁷ cells/mice) and then given either standard water (H₂O) or water containing 1% DFMO for 10 days. (A) Nude mice were photographed 16 days post-inoculation. Arrows indicate tumors in nude mice. (B, C) Sixteen days post-inoculation, three mice were sacrificed. Morphological changes of the spleen and liver are shown in the photograph. The weights of the tumor, liver, and spleen were measured and plotted in the graph. * and ** indicate significant differences p<0.05 and p<0.01, respectively. (D) Sixteen days after inoculation, sections of the liver were stained with H&E (magnification: ×100). (E) Mouse survival was monitored daily for 30 days post-inoculation (n = 10).</p

c-Myc T58A mutant conferred growth-factor independence on Ba/F3 cells.

<p>(A) Schematic diagram of wild-type (c-Myc), T58A and In373 mutants of c-Myc. (B-H) Ba/F3 cells were infected with empty virus (−) or retrovirus encoding wild-type c-Myc (c-Myc) or two c-Myc mutants (T58A, In373). (B) Transduced cells were incubated without IL-3 (2 ng/mL) for 6 hr and whole cell lysates were immunoblotted (IB) with anti-c-Myc antibody or anti-β-actin antibody. (C, D) Transduced Ba/F3 cells were cultured with/without IL-3 (2 ng/mL) for 12 hr. (C) ODC mRNA was analyzed by quantitative real-time PCR. GAPDH mRNA was analyzed as an internal control. Data are the mean ± S.D. of the relative expression levels in three independent experiments. (D) Whole cell lysates were immunoblotted (IB) with anti-ODC antibody or anti-β-actin antibody. (E) Viable transduced Ba/F3 cells and VF/EpoR cells were counted in the presence and absence of IL-3 (2 ng/mL) for 3 days. Data are the mean ± S.D. of the relative expression levels in three independent experiments. (F) Transduced Ba/F3 cells were left untreated or stimulated with IL-3 (2 ng/mL) for 24 hr. The viability of these cells was determined by the trypan blue exclusion method. Results are the mean ± S.D. of three independent experiments. (G) Transduced cells were cultured without IL-3 (2 ng/mL) for 24 hr. Cells were then fixed, treated with propidium iodide (PI) and subjected to FACS analysis. (H) Transduced Ba/F3 cells were cultured with/without IL-3 (2 ng/mL) for 24 hr. DNA was isolated from cells and subjected to agarose gel electrophoresis.</p

List of Ba/F3-derived cell lines analyzed in this study.

a<p><a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0052844#pone-0052844-g002" target="_blank">Fig. 2A</a> shows structures of c-Myc mutants, T58A and In373.</p>b<p>c-Myc expression was tested under the absence of Epo stimulation.</p>c<p>ND means “Not Detected”.</p>d<p>Tumorigenesis was tested by the transplantation of cells into nude mice.</p

JAK2 (V617F) induced expression of c-Myc through STAT5 activation.

<p>(A–E) Ba/F3 cell lines were infected with empty virus (−), retroviruses encoding wild-type JAK2 c-HA (WT), JAK2 mutant c-HA (V617F) and EpoR c-Flag as indicated in each figure. (A) WT/EpoR cells and VF/EpoR cells were cultured without Epo for 12 hr, and then total RNAs were prepared from each cell line. The enhancement of gene expression induced by JAK2 (V617F) was determined by DNA array. The ratio of the altered gene expressions in VF/EpoR cells was calculated by dividing the amount of each gene in VF/EpoR cells by their amount in WT/EpoR cells. (B, C) A series of Ba/F3 cells expressing the described genes was cultured untreated or stimulated with Epo (5 U/mL) for 12 hr. (D, E) A series of Ba/F3 cells expressing the indicated genes were cultured in the absence of Epo, and treated with DMSO (0.1%) or AG490 (30 µM) for 12 hr. (F) HEL cells were treated with DMSO (0.1%) or AG490 (10, 20, 30 µM) for 24 hr. (G, H) VF/EpoR cells were infected with retrovirus harboring shRNA against firefly luciferase (control) or STAT5. (I, J) Ba/F3 cells were infected with empty virus (−), retrovirus encoding wild-type STAT5 (WT) or the constitutively active mutant of STAT5 (1*6). The cells were cultured without Epo for 12 hr. (B, E, H, J) The mRNA expression of c-Myc and ODC was analyzed by quantitative real-time PCR. GAPDH mRNA was analyzed as an internal control. Data are the mean ± S.D. of the relative expression levels in three experiments. (C, D, F, G, I) Whole cell lysates were immunoblotted (IB) with anti-c-Myc antibody, anti-ODC antibody, anti-HA antibody, anti-EpoR antibody, anti-phospho-JAK2 antibody (Y1007/1008), anti-phospho-STAT5 antibody (Y694), anti-STAT5 antibody or anti-β-actin antibody.</p

Inhibition of GSK-3β inhibited downregulation of c-Myc and apoptosis.

<p>(A) Transduced Ba/F3 cells were treated with DMSO (0.1%) or the indicated concentrations of GSK-3β inhibitor in the absence of IL-3 for 18 hr. The viability of these cells was determined by the trypan blue exclusion method. Results are the mean ± S.D. of three independent experiments. (B-E) Transduced Ba/F3 cells were treated with DMSO (0.1%) or GSK-3β inhibitor (0.1 µM) for 18 hr. (B) Whole cell lysates were immunoblotted (IB) with anti-phospho-c-Myc antibody (Thr58), anti-c-Myc antibody or anti-β-actin antibody. (C) The expression amounts of c-Myc and its mutants were normalized with the protein amount of β-actin, and the quantified ratios of c-Myc and its mutants (T58A, In373) are shown in the graph. Results are the mean ± S.D. of three independent experiments. (D) Total RNA was prepared and mRNA of ectopic c-Myc and its mutants (T58A, In373) was detected by quantitative real time-PCR. GAPDH mRNA was analyzed as an internal control. Data are the mean ± S.D. of the relative expression levels in three independent experiments. (E) DNA was isolated from cells and subjected to agarose gel electrophoresis. (F, G) Ba/F3 cell lines were infected with empty virus (−), retrovirus encoding JAK2 mutant c-HA (V617F) and retroviruses encoding wild-type EpoR c-Flag (WT) or EpoR mutant c-Flag (Y479F). Transduced Ba/F3 cells were incubated without IL-3 for 12 hr. (F) Whole cell lysates were immunoblotted (IB) with anti-phospho-JAK2 antibody (Y1007/1008), anti-phospho-GSK-3β antibody (S9), anti-GSK-3β antibody, anti-c-Myc antibody, anti-Bcl-XL antibody, anti-HA antibody, anti-Flag antibody or anti-β-actin antibody. (G) c-Myc mRNA and Bcl-XL mRNA were analyzed by quantitative real-time PCR. GAPDH mRNA was evaluated as an internal control. Data are the mean ± S.D. of the relative expression levels in three independent experiments.</p