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Objectives: We aimed to determine the role of dihydroorotate dehydrogenase (DHODH) in pathogenesis of adult T-cell leukemia (ATL) caused by human T-cell leukemia virus type 1 (HTLV-1) and the effects of its inhibition on the de novo pyrimidine biosynthesis pathway., Methods: Cell proliferation, viability, cycle, and apoptosis were analyzed using WST-8 assays, flow cytometry, and Hoechst 33342 staining. To elucidate the molecular mechanisms involved in the anti-ATL effects of DHODH knockdown and inhibition, RT-PCR and immunoblotting were conducted., Results: HTLV-1-infected T-cell lines aberrantly expressed DHODH. Viral infection and the oncoprotein, Tax, enhanced DHODH expression, while knockdown of DHODH decreased HTLV-1-infected T-cell growth. In addition, BAY2402234, a DHODH inhibitor, exerted an anti-proliferative effect, which was reversed by uridine supplementation. BAY2402234 induced DNA damage and S phase arrest by downregulating c-Myc, CDK2, and cyclin A and upregulating p53 and cyclin E. It also induced caspase-mediated apoptosis by the upregulation of pro-apoptotic and downregulation of anti-apoptotic proteins. Furthermore, BAY2402234 induced caspase-independent ferroptosis and necroptosis. It decreased phosphorylation of IKK, IκBα, PTEN, Akt, and its downstream targets, suggesting that inhibition of NF-κB and Akt signaling is involved in its anti-ATL action., Conclusion: These findings highlight DHODH as a potential therapeutic target for treating ATL., (© 2024 John Wiley & Sons A/S. Published by John Wiley & Sons Ltd.)

Patients with adult T-cell leukemia (ATL), which is caused by human T-cell leukemia virus type 1 (HTLV-1), show poor prognosis because of drug resistance. Heat shock protein (HSP) 90 is reportedly essential for ATL cell survival as it regulates important signaling pathways, thereby making HSP90 inhibitors new therapeutic candidates for ATL. However, HSP90 inhibition increases the expression of other HSPs, suggesting that HSPs may contribute to drug resistance. The heat shock factor 1 (HSF1) transcription factor is the primary regulator of the expression of HSPs. Furthermore, targeting HSF1 disrupts the HSP90 chaperone function. Herein, we demonstrated that HSF1 is overexpressed in HTLV-1-infected T cells. HSF1 knockdown inhibited the proliferation of HTLV-1-infected T cells. HSF1 inhibitor KRIBB11 reduced the expression and phosphorylation of HSF1, downregulated HSP70 and HSP27 expression, and suppressed Akt, nuclear factor-κB, and AP-1 signals. KRIBB11 treatment induced DNA damage, upregulated p53 and p21, and reduced the expression of cyclin D2/E, CDK2/4, c-Myc, MDM2, and β-catenin, thereby preventing retinoblastoma protein phosphorylation and inhibiting G1-S cell cycle progression. KRIBB11 also induced caspase-mediated apoptosis concomitant with the suppression of Bcl-xL, Mcl-1, XIAP, c-IAP1/2, and survivin expression. KRIBB11 inhibited HSP70 and HSP90 upregulation through treatment with AUY922, an HSP90 inhibitor, and enhanced the cytotoxic effect of AUY922, suggesting a salvage role of HSF1-dependent HSP induction in response to drug treatment. Finally, treatment of mice with KRIBB11 reduced ATL tumor growth. Therefore, this study provides a strong rationale to target HSF1 and validates the anti-ATL activity of KRIBB11., (© 2023. The Author(s), under exclusive licence to Springer Science+Business Media, LLC, part of Springer Nature.)

Since treatment options for adult T cell leukemia (ATL) associated with human T cell leukemia virus type 1 (HTLV-1) fail to obtain long-term response, novel therapies targeting ATL-dysregulated pathways are necessary. Dysregulated nuclear import and export machinery is common in malignancies. This study aimed to investigate the potential of exportin-1 (XPO1), which mediates nuclear export of cargos, as a target in ATL. RT-PCR and western blotting were performed to determine XPO1 expression. We evaluated XPO1's effects on cell proliferation and viability through WST-8 assays, cell cycle and apoptosis via Hoechst 33342 staining and flow cytometry, and intracellular signaling cascades using western blotting. XPO1 expression was upregulated in HTLV-1-infected T cells. XPO1 knockdown reduced cell proliferation. XPO1 inhibitor KPT-330 also reduced proliferation, increased DNA damage, and induced G1 cell cycle arrest and caspase-dependent apoptosis. KPT-330 downregulated cell cycle regulators (CDK2/4/6, cyclin D2, c-Myc and phosphorylated pRb) and anti-apoptotic proteins (XIAP, c-IAP1/2, survivin and Mcl-1), and upregulated p53, p21 and Bak. KPT-330 suppressed XPO1 and increased the nuclear localization of cargos (NF-κB RelA and its negative regulator IκBα, protein phosphatase 2A and its inhibitor SET, p53 and its negative regulator MDM2, p21, p27, FOXO1 and pRb). KPT-330 treatment resulted in the abrogation of aberrant pathways (NF-κB, Akt and STAT3/5) simultaneously through the activation of tumor suppressor proteins and inhibition of oncogenes and proliferative/survival factors. These findings encourage investigating the use of KPT-330 in clinical trials targeting ATL., (© 2022. The Author(s), under exclusive licence to Springer Science+Business Media, LLC, part of Springer Nature.)

Human T cell leukemia virus type 1 (HTLV-1) is responsible for adult T cell leukemia (ATL); however, molecular and cellular mechanisms underlying HTLV-1-induced leukemogenesis are unclear. BCL6 oncogene is involved in cancer progression and a preferred target of anti-cancer treatments. Here, we aimed to evaluate BCL6 expression and the effects of BCL6 inhibitor (FX1) on HTLV-1-infected T cell lines. BCL6 expression was higher in HTLV-1-infected T cell lines than that in uninfected T cell lines. BCL6 was localized mostly in the nucleus. The virus oncoprotein Tax induced BCL6 mRNA expression in T cells, whereas BCL6 knockdown reduced HTLV-1-infected T cell proliferation; thus, confirmed the association of BCL6 with cancer progression. Further, FX1 efficiently inhibited the cell growth and survival of HTLV-1-infected T cell lines in a dose- and time-dependent manner. The decreased levels of cell cycle regulatory proteins (phosphorylated retinoblastoma protein, cyclin-dependent kinase 4, cyclin D2 and c-Myc) and the increased levels of BCL6 target proteins (p21, p27 and p53) showed that FX1 arrested cell cycle at the G1 phase. Apoptosis was induced concomitantly with Bak upregulation and downregulation of survivin, Bcl-xL and Mcl-1, as well as with the activation of caspase-3, -8, -9 and poly(ADP-ribose) polymerase. FX1 also inhibited NF-κB and Akt signaling pathways. These events were because of the induction of the activity of cell cycle checkpoint proteins and relief of direct repression of the targets of cell cycle checkpoint proteins. Thus, BCL6 might be considered a novel target for ATL treatment., (© 2021. The Author(s), under exclusive licence to Springer Science+Business Media, LLC, part of Springer Nature.)

There is no cure for adult T cell leukemia/lymphoma (ATLL) associated with human T cell leukemia virus type 1 (HTLV-1), and novel targeted strategies are needed. NF-κB and AP-1 are crucial for ATLL, and both are transported to the nucleus by an importin (IPO)α/β heterodimeric complex to activate target genes. In this study, we aimed to elucidate the function of IPOβ1 in ATLL. The expression of IPOβ1 was analyzed by western blotting and RT-PCR. Cell growth, viability, cell cycle, apoptosis and intracellular signaling cascades were examined by the water-soluble tetrazolium-8 assay, flow cytometry and western blotting. Xenograft tumors in severe combined immune deficient mice were used to evaluate the growth of ATLL cells in vivo. IPOβ1 was upregulated in HTLV-1-infected T cell lines. Further, IPOβ1 knockdown or the IPOβ1 inhibitor importazole and the IPOα/β1 inhibitor ivermectin reduced HTLV-1-infected T cell proliferation. However, the effect of inhibitors on uninfected T cells was less pronounced. Further, in HTLV-1-infected T cell lines, inhibitors suppressed NF-κB and AP-1 nuclear transport and DNA binding, induced apoptosis and poly (ADP-ribose) polymerase cleavage, and activated caspase-3, caspase-8 and caspase-9. Inhibitors also mediated G 1 cell cycle arrest. Moreover, the expression of NF-κB- and AP-1-target proteins involved in cell cycle and apoptosis was reduced. In vivo, the IPOα/β1 inhibitor ivermectin decreased ATLL tumor burden without side effects. IPOβ1 mediated NF-κB and AP-1 translocation into ATLL cell nuclei, thereby regulating cell growth and survival, which provides new insights for targeted ATLL therapies. Thus, ivermectin, an anti-strongyloidiasis medication, could be a potent anti-ATLL agent.

Objectives: T-cell receptor (TCR) signaling-induced activation of NF-κB requires assembly of the CARD11-BCL10-MALT-1 complex and IκB kinase (IKK). Gain-of-function alterations in this component of the TCR/NF-κB pathway are associated with the development of HTLV-1-driven adult T-cell leukemia (ATL). We aimed to determine whether inhibition of MALT-1-mediated NF-κB activation could have anti-ATL activity., Methods: RT-PCR, immunoblotting, and electrophoretic mobility shift assays were performed to assess expression levels of MALT-1 and the intracellular signaling cascades. Cell proliferation, cell cycle progression, and apoptotic events were examined using WST-8 assays, flow cytometry, and Hoechst 33342 staining., Results: MALT-1 expression was upregulated in ATL-derived T-cell lines compared to that in normal PBMCs and uninfected or HTLV-1-transformed T-cell lines. Targeting MALT-1 with siRNA decreased cell proliferation. A MALT-1 inhibitor (MI-2) suppressed cleavage of the MALT-1-target protein, CYLD, and inhibited proliferation via G1 phase arrest. MI-2 induced apoptosis through caspase-3/8/9 activation and inhibited the phosphorylation of IKKα/β and IκBα, resulting in the accumulation of IκBα and suppression of NF-κB-DNA binding. Additionally, MI-2 inhibited the expression of apoptosis- and cell cycle-related proteins regulated by NF-κB., Conclusions: MALT-1 plays an important regulatory role in NF-κB signaling during ATL-genesis, and targeting MALT-1 is a promising therapeutic strategy for this disease., (© 2020 John Wiley & Sons A/S. Published by John Wiley & Sons Ltd.)

Adult T-cell leukemia/lymphoma (ATLL) is an aggressive disease caused by infection with human T-cell leukemia virus type 1 (HTLV-1). Successful treatment is limited by resistance to chemotherapies. Therefore, there is an urgent need to develop novel effective strategies. Artesunate (ART), a widely used antimalarial compound, has been shown to exert cytotoxicity. Here, we aimed to assess the anti-ATLL activities of ART and to elucidate the possible molecular mechanisms involved in this effect. Compared with uninfected T cells, HTLV-1-infected T-cell lines were sensitive to ART-induced cytotoxicity. ART caused cell cycle arrest at G 1 and/or G 2 /M phases, which was associated with decreased expression of cyclin dependent kinase 1/2/4/6, cyclin B1/D2/E and c-Myc, and increased expression of p21. ART-induced apoptosis corresponded to activation of caspase-8/9/3; decreased expression of Bcl-xL, Bcl-2, myeloid cell leukemia-1, survivin, X-linked inhibitor of apoptosis protein and cellular inhibitor of apoptosis 1/2; and increased expression of Bak. ART increased intracellular reactive oxygen species and activation of the DNA damage marker γ-H2AX. Moreover, ART-induced cytotoxicity was partly reversed by treatment with a reactive oxygen species scavenger, iron chelator, and necroptosis or ferroptosis inhibitor, suggesting the involvement of caspase-dependent and -independent lethal pathways. These effects were correlated with inhibition of nuclear factor-κB and activator protein-1 signaling through dephosphorylation of IκBα, IκB kinase (IKK) α and IKKβ, and decreased expression of JunB and JunD. Importantly, intraperitoneal injection with ART lowered tumor burden in an ATLL murine model. These preclinical results provide a rationale for evaluating the efficacy of ART in patients with ATLL., Competing Interests: Declaration of competing interest The authors declare that they have no competing interests., (Copyright © 2020 Elsevier B.V. All rights reserved.)

Adult T‑cell leukemia/lymphoma (ATLL) constitutes an aggressive malignancy caused by human T‑cell leukemia virus type 1 (HTLV‑1) that is resistant to available chemotherapeutics. The constitutive activation of Janus kinase (JAK)-signal transducer and activator of transcription (STAT) signaling is an important feature of ATLL, and spleen tyrosine kinase (SYK) is overexpressed in HTLV‑1-transformed T‑cell lines. In this study, we evaluated the effects of SYK- (PRT060318) or JAK- (JAK inhibitor 1) selective inhibitors and the dual SYK/JAK inhibitor, cerdulatinib, on the viability of HTLV‑1-transformed and ATLL-derived T‑cell lines. Cell proliferation, viability, cell cycle, apoptosis and intracellular signaling cascades were analyzed by the water-soluble tetrazolium-8 assay, flow cytometry and western blot analysis. HTLV‑1-infected T‑cell lines were sensitive to both SYK-selective and pan-JAK inhibitors, whereas cerdulatinib more potently suppressed cell proliferation and reduced cell viability than either of these agents alone. By contrast, the cytotoxic effects of cerdulatinib on uninfected T‑cell lines and peripheral blood mononuclear cells from a healthy donor were less pronounced. Cerdulatinib induced cell cycle arrest in the G2/M phase, which was associated with a decreased cyclin-dependent kinase 1 and cyclin B1, and an increased p21 and p27 expression. Hoechst staining revealed chromatin condensation and nuclear fragmentation in the cells treated with cerdulatinib, and an increased fraction of apoptotic APO2.7-stained cells was detected by flow cytometry. This corresponded to the activation of caspase-8, -9 and -3, and decreased levels of the anti-apoptotic factors, Bcl-xL, survivin, X-linked inhibitor of apoptosis (XIAP) and c‑FLIP. The cerdulatinib-induced decrease in cell viability was partly reversed by the caspase inhibitor, z‑VAD‑FMK. These anti-ATLL effects were associated with the suppression of SYK and JAK/STAT signaling, along with that of the downstream factors, AKT, ERK, activator protein‑1 and nuclear factor-κB. Finally, oral dosing with cerdulatinib lowered the tumor burden in a murine model of ATLL. Thus, our findings indicate that the simultaneous inhibition of therapeutically relevant targets, such as SYK and JAK is a more effective approach than single-agent therapy for the treatment of ATLL.

Adult T‑cell leukemia/lymphoma (ATLL) is a disorder involving human T-cell leukemia virus type 1 (HTLV‑1)-infected T‑cells characterized by increased clonal neoplastic proliferation. PDZ-binding kinase (PBK) [also known as T‑lymphokine-activated killer cell-originated protein kinase (TOPK)] is a serine/threonine kinase expressed in proliferative cells and is phosphorylated during mitosis. In this study, the expression and phosphorylation of PBK/TOPK were examined by western blot analysis and RT‑PCR. We found that PBK/TOPK was upregulated and phosphorylated in HTLV‑1-transformed T‑cell lines and ATLL‑derived T‑cell lines. Notably, CDK1/cyclin B1, which phosphorylates PBK/TOPK, was overexpressed in these cells. HTLV‑1 infection upregulated PBK/TOPK expression in peripheral blood mononuclear cells (PBMCs) in co-culture assays. The potent PBK/TOPK inhibitors, HI‑TOPK‑032, and fucoidan from brown algae, decreased the proliferation and viability of these cell lines in a dose‑dependent manner. By contrast, the effect of HI‑TOPK‑032 on PBMCs was less pronounced. Treatment with HI‑TOPK‑032 resulted in G1 cell cycle arrest, and decreased CDK6 expression and pRb phosphorylation, which are critical determinants of progression through the G1 phase. In addition, HI‑TOPK‑032 induced apoptosis, as evidenced by morphological changes, the cleavage of poly(ADP-ribose) polymerase with the activation of caspase‑3, -8 and -9, and an increase in the sub‑G1 cell population and APO2.7-positive cells. Moreover, HI‑TOPK‑032 inhibited the expression of cellular inhibitor of apoptosis 2 (c-IAP2), X-linked inhibitor of apoptosis protein (XIAP), survivin and myeloid cell leukemia‑1 (Mcl‑1), and induced the expression of Bak and interferon-induced protein with tetratricopeptide repeats (IFIT)1, 2 and 3. It is noteworthy that the use of this inhibitor led to the inhibition of the phosphorylation of IκB kinase (IKK)α, IKKβ, IκBα, phosphatase and tensin homolog (PTEN) and Akt, and to the decreased protein expression of JunB and JunD, suggesting that PBK/TOPK affects the nuclear factor-κB, Akt and activator protein‑1 signaling pathways. In vivo, the administration of HI‑TOPK‑032 suppressed tumor growth in an ATLL xenograft model. Thus, on the whole, this study on the identification and functional analysis of PBK/TOPK suggests that this kinase is a promising molecular target for ATLL treatment.

Background: Human T-cell leukemia virus type 1 (HTLV-1) causes adult T-cell leukemia (ATL). Viral Tax protein plays a major role in ATL development. Pim family of serine/threonine kinases is composed of Pim-1, -2, and -3. The potential of Pim family as a target in ATL was analyzed., Methods: RT-PCR and Western blotting were used to determine the expression of Pim kinases, Tax, and intracellular signal molecules. Knockdown of Pim-3 and RelA was performed using small interfering RNA. The effects on cell proliferation, viability, cell cycle, and apoptosis were analyzed by WST-8, propidium iodide, and APO2.7 assay. NF-κB DNA binding activity was investigated by electrophoretic mobility shift assay., Results: Pim-3 expression was restricted to HTLV-1-infected T-cell lines. Tax induced Pim-3 expression through NF-κB. Knockdown of Pim-3 showed growth inhibition of HTLV-1-infected T cells. NJC97-NH, a novel inhibitor of the Pim-1/3 kinases, inhibited cell viability. NJC97-NH induced G2/M cell cycle arrest associated with downregulation of cyclin A and cyclin B1 expression, as well as apoptosis accompanied with downregulation of XIAP and Mcl-1 expression through inhibition of NF-κB pathway, mediated through decrease in IκBα and RelA phosphorylation., Conclusion: Pim-3 is a potentially suitable target for the development of novel therapeutic agents against ATL., (© 2017 John Wiley & Sons A/S. Published by John Wiley & Sons Ltd.)

Human T-cell leukemia virus type 1 (HTLV-1) is the causative agent of adult T-cell leukemia/lymphoma (ATLL) but there is no effective treatment for HTLV-1-associated diseases. Herein, we determined the effect of butein, a bioactive plant polyphenol, on cell growth, apoptosis and signaling pathways in HTLV-1-infected T-cell lines and on tumor growth in SCID mice. Treatment with butein caused a decrease in viability of HTLV-1-infected T-cell lines. T cells cultured with butein showed obvious apoptosis morphology, and cleavage of poly(ADP-ribose) polymerase with activation of caspase-3, -8 and -9. Pretreatment of cells with caspase inhibitor partially blocked butein-induced inhibition of cell viability. Butein also resulted in cell cycle arrest at G1 phase. Butein markedly downregulated the protein expression levels of CDK4, CDK6, cyclin D1, cyclin D2, cyclin E, survivin, XIAP, c-IAP2 and phospho-pRb. Butein also inhibited i) total and phospho-protein levels of IκB kinase (IKK)α and IKKβ, ii) degradation and phosphorylation of IκBα, iii) JunB and JunD, iv) total and phospho-protein levels of Akt, v) phosphorylation of RelA, vi) heat shock protein 90, and vii) DNA-binding activity of NF-κB and AP-1. In mice harboring ATLL xenograft tumors, butein caused a significant inhibition of tumor growth and reduced serum levels of soluble interleukin-2 receptor α chain and soluble cluster of differentiation 30. Considered together, the results indicated that butein has antiproliferative and proapoptotic properties through the suppression of NF-κB, AP-1 and Akt signaling in HTLV-1-infected T cells, both in vitro and in vivo, suggesting its therapeutic potential against HTLV-1-associated diseases including ATLL.

Protein kinase C (PKC)-δ is a member of the PKC family. It has been implicated in tumor suppression as well as survival of various cancers. The aggressive malignancy of T lymphocytes known as adult T-cell leukemia (ATL) is associated with human T-cell leukemia virus type 1 (HTLV-1) infection. In this study, we show that HTLV-1-infected T cells are characterized by phosphorylation and nuclear translocation of PKC-δ. Expression of HTLV-1 regulatory protein Tax increased PKC-δ phosphorylation. Blockade of PKC-δ by rottlerin suppressed PKC-δ phosphorylation and inhibited cell viability in HTLV-1-infected T-cell lines and primary ATL cells. Rottlerin induced cell cycle arrest at the G1 phase and caspase-mediated apoptosis of HTLV-1-infected T cells. Rottlerin downregulated the expression of proteins involved in G1/S cell cycle transition, cyclin D2, CDK4 and 6, and c-Myc, resulting in dephosphorylation of retinoblastoma protein (pRb). Furthermore, rottlerin reduced the expression of important anti-apoptotic proteins (e.g., survivin, XIAP, Bcl-xL and c-FLIP) and Bcl-2 phosphorylation, and activated the pro-apoptotic protein Bax. Our results showed that permanent activation of nuclear factor-κB (NF-κB) by HTLV-1 Tax allows infected cells to escape cell cycle arrest and apoptosis and that PKC-δ mediates Tax-induced activation of NF-κB. Based on these findings, new therapies designed to target PKC-δ could be potentially useful in the treatment of ATL.

The aim of this study was to establish the role of serine/threonine protein phosphatase 2A (PP2A) in the survival of leukemic cells from patients with adult T cell leukemia (ATL), associated with human T cell leukemia virus type 1 (HTLV-1). In HTLV-1-infected T cell lines and ATL cells, okadaic acid (OkA), a potent PP2A inhibitor, induced decrease in cell viability and G₁ cell cycle arrest by decreasing the expression levels of cyclin D2, cyclin-dependent kinase 4 and cyclin-dependent kinase 6, phosphorylation of pRb, and upregulation of p21, p27 and GADD45α. OkA-induced apoptosis was also due to the suppression of expression of Bcl-2, Bcl-x(L) and XIAP, and the activation of caspases-3, -8 and -9, and caspase-3 downstream mammalian STE20-like kinase 1 and H2AX. OkA inhibited nuclear factor-kappa B DNA binding and activated mitogen-activated protein (MAP) kinases. Other new PP2A-specific inhibitors, cytostatin and rubratoxin A, also induced decrease in cell viability through caspase-dependent mechanism. MAP kinase inhibitors confirmed the role of p38 MAP kinase in PP2A inhibitors-induced apoptosis. OkA resulted in the generation of reactive oxygen species, and exogenous antioxidant prevented activation of the indicated caspases. Finally, PP2A knockdown inhibited cell growth. The results showed that PP2A inhibition caused reactive oxygen species generation and affected distinct signaling pathways, resulting in the activation of H2AX and subsequent apoptotic cell death. These results suggest that PP2A is a potentially useful target in the treatment of ATL.

Human T-cell leukemia virus type 1 (HTLV-1) infection is associated with the development of adult T-cell leukemia (ATL) and various inflammatory diseases. CD69 is a marker of early activation of lymphocytes. We investigated the effects of HTLV-1 infection on the expression of CD69. The CD69 gene was upregulated in all viral protein Tax-expressing HTLV-1-transformed T-cell lines, except MT-2 and peripheral blood mononuclear cells from patients with ATL compared with uninfected T-cell line, Tax-negative ATL-derived T-cell lines and normal peripheral blood mononuclear cells. Flow cytometric analysis and immunohistochemical analysis confirmed the enhanced expression of CD69 in HTLV-1-transformed T-cell lines and in ATL cells in lymph nodes and skin lesions, and its absence in MT-2 and peripheral blood mononuclear cells. CD69 expression was induced following infection of human T-cell line with HTLV-1, and specifically by Tax. Tax transcriptionally activated CD69 gene through both nuclear factor-κB and cyclic adenosine 3',5'-monophosphate response element-binding protein signaling pathways. Detailed analysis of the CD69 promoter indicated that the Tax-induced expression of CD69 was regulated by multiple cis-acting elements and by the interplay of transcription factors of the nuclear factor-κB, early growth response and cyclic adenosine 3',5'-monophosphate response element-binding protein families. The lack of CD69 expression in MT-2 is due to epigenetic mechanism involving deacetylation, but not methylation. We conclude that CD69 is a Tax-regulated gene, and its regulation by Tax may play a role in cellular activation and HTLV-1-induced disease pathogenesis., (Copyright © 2013 Elsevier B.V. All rights reserved.)

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., (Copyright © 2012 Elsevier B.V. All rights reserved.)

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., (Copyright © 2011 Elsevier Ireland Ltd. All rights reserved.)

We evaluated the effects of Bidens pilosa, a plant found in tropical and subtropical regions, and investigated the molecular pathways responsible for the anti-adult T-cell leukemia (ATL) effect. Water extracts of B. pilosa had growth suppressive effects on human T-cell leukemia virus type 1 (HTLV-1)-infected T-cell lines and ATL cells. B. pilosa extracts arrested cells in G1 cell cycle and induced apoptosis of HTLV-1-infected T-cell lines. B. pilosa extracts inhibited also the phosphorylation of IκB kinase β and IκBα, and NF-κB-DNA binding, in conjunction with reduction of expression of proteins involved in G1/S cell cycle transition and suppression of apoptosis. Reactive oxygen species played a role in B. pilosa-mediated suppression of NF-κB activity. B. pilosa extracts also inhibited the expression of JunB and JunD, resulting in suppression of AP-1-DNA binding. In animals harboring tumors of HTLV-1-infected T-cell origin, treatment with B. pilosa extracts suppressed tumor growth. Our results suggest that B. pilosa is a potentially useful medicinal plant for treatment of ATL.

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.

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., (Copyright © 2011 Elsevier Inc. All rights reserved.)

Adult T-cell leukemia (ATL) is a T-cell malignancy associated with human T-cell leukemia virus type 1 (HTLV-1). Mutations of tumor suppressor genes have been described in ATL. Although Tax, a product of HTLV-1, is associated with cellular genetic aberrations, the mechanisms of such association are not fully clear. Activation-induced cytidine deaminase (AID) is involved in somatic DNA alterations of the immunoglobulin gene for amplification of immune diversity. However, inappropriate expression of AID acts as a genomic mutator that contributes to tumorigenesis. To gain insight into the molecular mechanism underlying the emergence of somatic mutations in various genes during leukemogenesis, we examined the expression of AID. HTLV-1-infected T-cell lines and ATL cells expressed high levels of AID compared with uninfected T-cell lines and normal peripheral blood mononuclear cells (PBMCs). Immunohistochemistry showed AID-positive ATL cells in lymph nodes and skin lesions. Infection of a human T-cell line and normal PBMCs with HTLV-1 induced AID expression. Tax transcriptionally activated AID gene through both the nuclear factor-kappaB subunit p50 and cyclic adenosine 3',5'-monophosphate response element-binding protein signaling pathways. p50, which lacks a transactivation domain, interacted with the transcriptional coactivator Bcl-3 in HTLV-1-infected T cells. Thus, activation of p50/Bcl-3 complexes in T cells in response to Tax might explain the constitutive expression of AID in HTLV-1-infected T cells. The constitutive expression of AID in ATL cells can be speculated to result from mutations induced by the Tax-activated AID and/or other Tax-associated mutagenic mechanisms during the pre-leukemic stage, which cause functional modification within the AID promoter or in any of its cellular regulatory activator proteins.

Caveolin-1 is implicated in the regulation of signal pathways. Adult T-cell leukemia (ATL) is a T-cell malignancy causatively associated with human T-cell leukemia virus type 1 (HTLV-1). To determine the role of caveolin-1 in leukemogenesis, we examined caveolin-1 expression levels in HTLV-1-infected T-cell lines and ATL cells. These cells expressed high levels of caveolin-1 compared with uninfected T-cell lines and normal peripheral blood mononuclear cells (PBMCs). Caveolin-1-positive ATL cells were detected in ATL lymph nodes and skin lesions, and caveolin-1 was also detected in the plasma of patients with ATL. Infection of a human T-cell line, an epithelial cell line, and normal PBMCs with HTLV-1 induced caveolin-1 expression. The viral protein Tax transcriptionally activated caveolin-1 gene through nuclear factor-kappaB and cAMP response element binding protein signal pathways. HTLV-1-infected T-cell lines, and ATL cells are known to be resistant to transforming growth factor beta (TGF-beta)-induced growth inhibition. Caveolin-1 was colocalized with TGF-beta type I receptor in HTLV-1-infected T-cell lines and suppressed TGF-beta signaling. Caveolin-1 knockdown in an HTLV-1-infected T-cell line exhibited susceptibility to TGF-beta. Thus, we describe a new function for Tax, repression of TGF-beta signaling through caveolin-1 expression, which may play a critical role in ATL leukemogenesis.

Human T-cell leukemia virus type 1 (HTLV-1) is the etiologic agent for adult T-cell leukemia (ATL). Aurora A, a mitotic checkpoint protein, is overexpressed in human cancer cells. The cell cycle-dependent turnover of Aurora A is regulated by E3 ubiquitin ligases such as checkpoint with fork head-associated and ring finger (CHFR). Here, we found overexpression of Aurora A protein in HTLV-1-infected T-cell lines and primary ATL cells. The expression of CHFR mRNA was reduced in these cells by abnormal methylation of CHFR promoter region. Knockdown of Aurora A using small interfering RNA suppressed the growth of HTLV-1-infected T-cell line. Transfection of Aurora A expression plasmid enhanced Tax-induced nuclear factor-kappaB (NF-kappaB) reporter activity. Transfection of CHFR expression plasmid into an HTLV-1-infected T-cell line reduced cell growth, Aurora A protein level and constitutive NF-kappaB reporter activity. Aurora kinase inhibitor suppressed the growth and survival of HTLV-1-infected T-cell lines and primary ATL cells. It also reduced constitutive NF-kappaB activity in an HTLV-1-infected T-cell line by reducing IkappaB kinase beta phosphorylation and the expression of antiapoptotic protein survivin. Our results suggested that loss of CHFR expression resulted to accumulation of Aurora A, which increased NF-kappaB activity. These findings highlight the critical role of Aurora A in HTLV-1-infected T cells, making this molecule a potentially suitable target for future therapies for ATL.

Background: Adult T-cell leukemia/lymphoma (ATLL) is a malignancy derived from T cells infected with human T-cell leukemia virus type 1 (HTLV-1), and it is known to be resistant to standard anticancer therapies. Indole-3-carbinol (I3C), a naturally occurring component of Brassica vegetables such as cabbage, broccoli and Brussels sprout, is a promising chemopreventive agent as it is reported to possess antimutagenic, antitumorigenic and antiestrogenic properties in experimental studies. The aim of this study was to determine the potential anti-ATLL effects of I3C both in vitro and in vivo., Results: In the in vitro study, I3C inhibited cell viability of HTLV-1-infected T-cell lines and ATLL cells in a dose-dependent manner. Importantly, I3C did not exert any inhibitory effect on uninfected T-cell lines and normal peripheral blood mononuclear cells. I3C prevented the G1/S transition by reducing the expression of cyclin D1, cyclin D2, Cdk4 and Cdk6, and induced apoptosis by reducing the expression of XIAP, survivin and Bcl-2, and by upregulating the expression of Bak. The induced apoptosis was associated with activation of caspase-3, -8 and -9, and poly(ADP-ribose) polymerase cleavage. I3C also suppressed IkappaBalpha phosphorylation and JunD expression, resulting in inactivation of NF-kappaB and AP-1. Inoculation of HTLV-1-infected T cells in mice with severe combined immunodeficiency resulted in tumor growth. The latter was inhibited by treatment with I3C (50 mg/kg/day orally), but not the vehicle control., Conclusion: Our preclinical data suggest that I3C could be potentially a useful chemotherapeutic agent for patients with ATLL.

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, beta-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 G(1) phase by reducing the expression of cyclin D1, cyclin D2, CDK4 and CDK6, and inducing the expression of GADD45alpha, 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 IkappaBalpha phosphorylation and JunD expression, resulting in inactivation of nuclear factor-kappaB 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., ((c) 2008 Wiley-Liss, Inc.)

Clinical trials for treatment of adult T-cell leukemia (ATL) caused by human T-cell leukemia virus type I (HTLV-I) using all-trans-retinoic acid (ATRA) have shown satisfactory therapeutic responses, although efficacies were limited. Recently, many synthetic retinoids have been developed and among them, a novel synthetic retinoid, Am80 (Tamibarotene) is an RARalpha- and RARbeta-specific retinoid expected to overcome ATRA resistance. The present study examined the inhibitory effects of Am80 on HTLV-I-infected T-cell lines and ATL cells. Am80 had negligible growth inhibition of peripheral blood mononuclear cells but marked growth inhibition of both HTLV-I-infected T-cell lines and ATL cells. Am80 arrested cells in the G1 phase of the cell cycle and induced apoptosis in HTLV-I-infected T-cell lines. It inhibited also the phosphorylation of IkappaBalpha and NF-kappaB-DNA binding, in conjunction with reduction of expression of proteins involved in the G1/S cell cycle transition and apoptosis. Am80 also inhibited the expression of JunD, resulting in suppression of AP-1-DNA binding. Furthermore, severe combined immunodeficient mice with tumors induced by subcutaneous inoculation of HTLV-I-infected T cells, responded to Am80 treatment with partial regression of tumors and no side-effects. These findings demonstrate that Am80 is a potential inhibitor of NF-kappaB and AP-1, and is a potentially useful therapeutic agent against ATL.

ATL is a fatal malignancy of T lymphocytes caused by HTLV-I infection and remains incurable. Galectins are a family of animal lectins that function both extracellularly (by interacting with cell surface and extracellular matrix glycoproteins and glycolipids) and intracellularly (by interacting with cytoplasmic and nuclear proteins) to modulate signaling pathways. We found that protease-resistant galectin-9 by modification of its linker peptide, hG9NC(null), prevented cell growth of HTLV-I-infected T-cell lines and primary ATL cells. The suppression of cell growth was inhibited by lactose, but not by sucrose, indicating that beta-galactoside binding is essential for hG9NC(null)-induced cell growth suppression. hG9NC(null) induced cell cycle arrest by reducing the expression of cyclin D1, cyclin D2, cyclin B1, Cdk1, Cdk4, Cdk6, Cdc25C and c-Myc, and apoptosis by reducing the expression of XIAP, c-IAP2 and survivin. Most of these genes are regulated by NF-kappaB, which plays a critical role in oncogenesis by HTLV-I. hG9NC(null) suppressed IkappaBalpha phosphorylation, resulting in suppression of NF-kappaB. Most importantly, treatment with hG9NC(null) (6.7 mg/kg injected intraperitoneally every day) reduced tumor formation from an HTLV-I-infected T-cell line when these cells were inoculated subcutaneously into SCID mice. Our results suggest that hG9NC(null) could be a suitable agent for the management of ATL., ((c) 2007 Wiley-Liss, Inc.)

The molecular chaperone Hsp90 is involved in the stabilization and conformational maturation of many signaling proteins that are deregulated in cancers. The geldanamycin derivative 17-AAG is currently tested in clinical trials and known to inhibit the function of Hsp90 and promote the proteasomal degradation of its misfolded client proteins. ATL is a fatal malignancy of T lymphocytes caused by HTLV-I infection and remains incurable. Since Hsp90 is overexpressed in HTLV-I-infected T-cell lines and primary ATL cells, we analyzed the effects of 17-AAG on cell survival, apoptosis and expression of signal transduction proteins. HTLV-I-infected T-cell lines and primary ATL cells were significantly more sensitive to 17-AAG in cell survival assays than normal PBMCs. 17-AAG induced the inhibition of cell cycle and apoptosis. These effects could be mediated by inactivation of NF-kappaB, AP-1 and PI3K/Akt pathways, as well as reduction of expression of proteins involved in the G1-S cell cycle transition and apoptosis. Proteasome inhibition interfered with 17-AAG-mediated signaling proteins depletion. Collectively, our results indicate that 17-AAG suppresses ATL cell survival through, at least in part, destabilization of several client proteins and suggest that 17-AAG is a potentially useful chemotherapeutic agent for ATL., ((c) 2007 Wiley-Liss, Inc.)

Adult T-cell leukemia (ATL) is caused by human T-cell leukemia virus type I (HTLV-I) and remains incurable. NIK-333, a novel synthetic retinoid, prevents the recurrence of human hepatoma after surgical resection of primary tumors. We explored the effects of NIK-333 on HTLV-I-infected T-cell lines and ATL cells. NIK-333 inhibited cell proliferation, induced G1 arrest, and resulted in massive apoptosis in all tested HTLV-I-infected T-cell lines and ATL cells, whereas little effect was observed on normal peripheral blood mononuclear cells. NIK-333 treatment decreases the levels of cyclin D1, cyclin D2, cIAP2, and XIAP proteins. Further analysis showed that NIK-333 inactivated nuclear factor-kappaB in HTLV-I-infected T-cell lines. In animal studies, treatment with NIK-333 (100 mg/kg given orally every other day) produced partial inhibition of growth of tumors of a HTLV-I-infected T-cell line transplanted s.c. in severe combined immunodeficient mice. Our results indicate that NIK-333 is a potentially useful therapeutic agent for patients with ATL.