Chie Ishikawa

Molecular characterization of Legionella pneumophila-induced interleukin-8 expression in T cells

BackgroundLegionella pneumophila is the causative agent of human Legionnaires disease. During infection, the bacterium invades macrophages and lung epithelial cells, and replicates intracellularly. However, little is known about its interaction with T cells. We investigated the ability of L. pneumophila to infect and stimulate the production of interleukin-8 (IL-8) in T cells. The objective of this study was to assess whether L. pneumophila interferes with the immune system by interacting and infecting T cells.ResultsWild-type L. pneumophila and flagellin-deficient Legionella, but not L. pneumophila lacking a functional type IV secretion system Dot/Icm, replicated in T cells. On the other hand, wild-type L. pneumophila and Dot/Icm-deficient Legionella, but not flagellin-deficient Legionella or heat-killed Legionella induced IL-8 expression. L. pneumophila activated an IL-8 promoter through the NF-κB and AP-1 binding regions. Wild-type L. pneumophila but not flagellin-deficient Legionella activated NF-κB, p38 mitogen-activated protein kinase (MAPK), Jun N-terminal kinase (JNK), and transforming growth factor β-associated kinase 1 (TAK1). Transfection of dominant negative mutants of IκBα, IκB kinase, NF-κB-inducing kinase, TAK1, MyD88, and p38 MAPK inhibited L. pneumophila-induced IL-8 activation. Inhibitors of NF-κB, p38 MAPK, and JNK blocked L. pneumophila-induced IL-8 expression. In addition, c-Jun, JunD, cyclic AMP response element binding protein, and activating transcription factor 1, which are substrates of p38 MAPK and JNK, bound to the AP-1 site of the IL-8 promoter.ConclusionsTaken together, L. pneumophila induced a flagellin-dependent activation of TAK1, p38 MAPK, and JNK, as well as NF-κB and AP-1, which resulted in IL-8 production in human T cells, presumably contributing to the immune response in Legionnaires disease.

Antiadult T‐cell leukemia effects of brown algae fucoxanthin and its deacetylated product, fucoxanthinol

Adult T‐cell leukemia (ATL) is a fatal malignancy of T lymphocytes caused by human T‐cell leukemia virus type 1 (HTLV‐1) infection and remains incurable. Carotenoids are a family of natural pigments and have several biological functions. Among carotenoids, fucoxanthin is known to have antitumorigenic activity, but the precise mechanism of action is not elucidated. We evaluated the anti‐ATL effects of fucoxanthin and its metabolite, fucoxanthinol. Both carotenoids inhibited cell viability of HTLV‐1‐infected T‐cell lines and ATL cells, and fucoxanthinol was approximately twice more potent than fucoxanthin. In contrast, other carotenoids, β‐carotene and astaxanthin, had mild inhibitory effects on HTLV‐1‐infected T‐cell lines. Importantly, uninfected cell lines and normal peripheral blood mononuclear cells were resistant to fucoxanthin and fucoxanthinol. Both carotenoids induced cell cycle arrest during G1 phase by reducing the expression of cyclin D1, cyclin D2, CDK4 and CDK6, and inducing the expression of GADD45α, and induced apoptosis by reducing the expression of Bcl‐2, XIAP, cIAP2 and survivin. The induced apoptosis was associated with activation of caspase‐3, ‐8 and ‐9. Fucoxanthin and fucoxanthinol also suppressed IκBα phosphorylation and JunD expression, resulting in inactivation of nuclear factor‐κB and activator protein‐1. Mice with severe combined immunodeficiency harboring tumors induced by inoculation of HTLV‐1‐infected T cells responded to treatment with fucoxanthinol with suppression of tumor growth, showed extensive tissue distribution of fucoxanthinol, and the presence of therapeutically effective serum concentrations of fucoxanthinol. Our preclinical data suggest that fucoxanthin and fucoxanthinol could be potentially useful therapeutic agents for patients with ATL.

Fucoxanthin and its deacetylated product, fucoxanthinol, induce apoptosis of primary effusion lymphomas

Primary effusion lymphoma (PEL) is a rare type of non-Hodgkins lymphoma caused by human herpesvirus 8. Conventional chemotherapy has limited effect on PEL, and the prognosis is poor. Carotenoids are a family of natural pigments and have several biological functions. We evaluated the anti-PEL effects of carotenoid, fucoxanthin (FX) and its metabolite, fucoxanthinol (FXOH). Treatment of PEL cells with FX or FXOH induced cell cycle arrest during G₁ phase and caspase-dependent apoptosis. FX and FXOH treatment silenced NF-κB, AP-1 and Akt activation, in conjunction with down-regulation of anti-apoptotic proteins and cell cycle regulators. Importantly, proteasome degradation was responsible for the low levels of proteins after FXOH treatment. In animal studies, treatment with FX reduced the growth of PEL-cell tumors. The results provide the rationale for future clinical use of FX and FXOH for the treatment of PEL.

Effects of hippuristanol, an inhibitor of eIF4A, on adult T-cell leukemia

We evaluated the anti-adult T-cell leukemia (ATL) effects of hippuristanol, an eukaryotic translation initiation inhibitor from the coral Isis hippuris. Hippuristanol inhibited proliferation of HTLV-1-infected T-cell lines and ATL cells, but not normal peripheral blood mononuclear cells. It induced cell cycle arrest during G₁ phase by reducing the expression of cyclin D1, cyclin D2, CDK4 and CDK6, and induced apoptosis by reducing the expression of Bcl-x(L), c-IAP2, XIAP and c-FLIP. The induced apoptosis was associated with activation of caspase-3, -8 and -9. Hippuristanol also suppressed IkappaBalpha phosphorylation and depleted IKKalpha, IKKgamma, JunB and JunD, resulting in inactivation of NF-kappaB and AP-1. It also suppressed carbonic anhydrase type II expression. In addition to its in vitro effects, hippuristanol suppressed tumor growth in mice with severe combined immunodeficiency harboring tumors induced by inoculation of HTLV-1-infected T cells. These preclinical data suggest that hippuristanol could be a potentially useful therapeutic agent for patients with ATL.

The tumor suppressor gene WWOX links the canonical and noncanonical NF-κB pathways in HTLV-I Tax-mediated tumorigenesis

Both the canonical and noncanonical nuclear factor κB (NF-κB) pathways have been linked to tumorigenesis. However, it remains unknown whether and how the 2 signaling pathways cooperate during tumorigenesis. We report that inhibition of the noncanonical NF-κB pathway significantly delays tumorigenesis mediated by the viral oncoprotein Tax. One function of noncanonical NF-κB activation was to repress expression of the WWOX tumor suppressor gene. Notably, WWOX specifically inhibited Tax-induced activation of the canonical, but not the noncanonical NF-κB pathway. Mechanistic studies indicated that WWOX blocked Tax-induced inhibitors of κB kinaseα (IKKα) recruitment to RelA and subsequent RelA phosphorylation at S536. In contrast, WWOX Y33R, a mutant unable to block the IKKα recruitment and RelA phosphorylation, lost the ability to inhibit Tax-mediated tumorigenesis. These data provide one important mechanism by which Tax coordinates the 2 NF-κB pathways for tumorigenesis. These data also suggest a novel role of WWOX in NF-κB regulation and viral tumorigenesis.

LBH589, a deacetylase inhibitor, induces apoptosis in adult T-cell leukemia/lymphoma cells via activation of a novel RAIDD-caspase-2 pathway

Adult T-cell leukemia/lymphoma (ATLL), an aggressive neoplasm etiologically associated with human T-lymphotropic virus type-1 (HTLV-1), is resistant to treatment. In this study, we examined the effects of a new inhibitor of deacetylase enzymes, LBH589, on ATLL cells. LBH589 effectively induced apoptosis in ATLL-related cell lines and primary ATLL cells and reduced the size of tumors inoculated in SCID mice. Analyses, including with a DNA microarray, revealed that neither death receptors nor p53 pathways contributed to the apoptosis. Instead, LBH589 activated an intrinsic pathway through the activation of caspase-2. Furthermore, small interfering RNA experiments targeting caspase-2, caspase-9, RAIDD, p53-induced protein with a death domain (PIDD) and RIPK1 (RIP) indicated that activation of RAIDD is crucial and an event initiating this pathway. In addition, LBH589 caused a marked decrease in levels of factors involved in ATLL cell proliferation and invasion such as CCR4, IL-2R and HTLV-1 HBZ-SI, a spliced form of the HTLV-1 basic zipper factor HBZ. In conclusion, we showed that LBH589 is a strong inducer of apoptosis in ATLL cells and uncovered a novel apoptotic pathway initiated by activation of RAIDD.

Phosphorylated c-Jun and Fra-1 induce matrix metalloproteinase-1 and thereby regulate invasion activity of 143B osteosarcoma cells

Osteosarcoma is the most common primary malignancy of bone and patients often develop pulmonary metastases. Despite the advances in surgical and medical management, the mechanisms underlying human osteosarcoma progression and metastasis remain to be elucidated. Gene expression profiles were compared by the cDNA microarray technique between two different human osteosarcoma sublines, MNNG/HOS and 143B, which differ greatly in spontaneous pulmonary metastatic potential. Here we report an enhanced expression of matrix metalloproteinase (MMP)-1 in the highly metastatic human osteosarcoma cell line 143B. Moreover, the in vitro invasion activity of 143B cells was MMP-1-dependent. The activator protein (AP)-1 binding site in the MMP-1 gene promoter was required for the constitutive expression of MMP-1 in 143B cells. Two AP-1 components, c-Jun and Fra-1, were phosphorylated, and bound to the AP-1 binding site of the MMP-1 promoter in 143B cells. Activated c-Jun and Fra-1 were essential for MMP-1 gene expression in 143B cells. Mitogen-activated protein kinase pathways including the c-Jun NH(2)-terminal kinase and the extracellular signal-regulated kinase activate c-Jun and Fra-1 and thereby regulate c-Jun/Fra-1 mediated events, establishing the mitogen-activated protein kinase/AP-1/MMP-1 axis as important in 143B cells. These data suggest that MMP-1 plays a central role in osteosarcoma invasion. Accordingly, MMP-1 might be a biomarker and therapeutic target for invasive osteosarcomas and pulmonary metastases.

Targeting Bcl-2 family proteins in adult T-cell leukemia/lymphoma: in vitro and in vivo effects of the novel Bcl-2 family inhibitor ABT-737.

Adult T-cell leukemia/lymphoma (ATLL) is a peripheral T-cell malignancy caused by human T-lymphotropic virus type I (HTLV-1). ABT-737, a small molecule inhibitor of Bcl-2, Bcl-X(L), and Bcl-w, significantly induced apoptosis in HTLV-1 infected T-cell lines as well as in fresh ATLL cells, and synergistically enhanced the cytotoxicity and apoptosis induced by conventional cytotoxic drugs. Moreover, ABT-737 significantly inhibited the in vivo tumor growth of an ATLL mouse model. These results suggest that the use of an agent targeting anti-apoptotic bcl-2 family proteins, either alone or in combination with other conventional drugs, represents a novel promising approach for ATLL.

Honokiol induces cell cycle arrest and apoptosis via inhibition of survival signals in adult T-cell leukemia

BACKGROUND Honokiol, a naturally occurring biphenyl, possesses anti-neoplastic properties. We investigated activities of honokiol against adult T-cell leukemia (ATL) associated with human T-cell leukemia virus type 1 (HTLV-1). METHODS Cell viability was assessed using colorimetric assay. Propidium iodide staining was performed to determine cell cycle phase. Apoptotic effects were evaluated by 7A6 detection and caspases activity. Expressions of cell cycle- and apoptosis-associated proteins were analyzed by Western blot. We investigated the efficacy of honokiol in mice harboring tumors of HTLV-1-infected T-cell origin. RESULTS Honokiol exhibited cytotoxic activity against HTLV-1-infected T-cell lines and ATL cells. We identified two different effects of honokiol on HTLV-1-infected T-cell lines: cell cycle inhibition and induction of apoptosis. Honokiol induced G1 cell cycle arrest by reducing the expression of cyclins D1, D2, E, CDK2, CDK4, CDK6 and c-Myc, while apoptosis was induced via reduced expression of cIAP-2, XIAP and survivin. The induced apoptosis was also associated with activation of caspases-3 and -9. In addition, honokiol suppressed the phosphorylation of IκBα, IKKα, IKKβ, STAT3, STAT5 and Akt, down-regulated JunB and JunD, and inhibited DNA binding of NF-κB, AP-1, STAT3 and STAT5. These effects resulted in the inactivation of survival signals including NF-κB, AP-1, STATs and Akt. Honokiol was highly effective against ATL in mice CONCLUSIONS Our data suggested that honokiol is a systemically available, non-toxic inhibitor of ATL cell growth that should be examined for potential clinical application. GENERAL SIGNIFICANCE Our findings provide a rationale for clinical evaluation of honokiol for the management of ATL.

A modified version of galectin-9 suppresses cell growth and induces apoptosis of human T-cell leukemia virus type I-infected T-cell lines.

Retraction: The following article has been retracted through agreement between the first author and several coauthors, the journal Editor‐in‐Chief, Peter Lichter, and John Wiley & Sons, Ltd.: Okudaira T, Hirashima M, Ishikawa C, Makishi S, Tomita M, Matsuda T, Kawakami H, Taira N, Ohshiro K, Masuda M, Takasu N, Mori N. (2007). A modified version of galectin‐9 suppresses cell growth and induces apoptosis of human T‐cell leukemia virus type I‐infected T‐cell lines. Int J Cancer; 120 (May (10)): 2251‐2261, published online on 2 FEB 2007 in Wiley Online Library (www.onlinelibrary.wiley.com). After an investigation the retraction has been agreed due to inappropriate duplication of images and overlap with other published work.