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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.)

Adult T-cell leukemia (ATL), caused by HTLV-1, is the most lethal hematological malignancy. NEDD8-activating enzyme (NAE) is a component of the NEDD8 conjunction pathway that regulates cullin-RING ubiquitin ligase (CRL) activity. HTLV-1-infected T cells expressed higher levels of NAE catalytic subunit UBA3 than normal peripheral blood mononuclear cells. NAE1 knockdown inhibited proliferation of HTLV-1-infected T cells. The NAE1 inhibitor MLN4924 suppressed neddylation of cullin and inhibited the CRL-mediated turnover of tumor suppressor proteins. MLN4924 inhibited proliferation of HTLV-1-infected T cells by inducing DNA damage, leading to S phase arrest and caspase-dependent apoptosis. S phase arrest was associated with CDK2 and cyclin A downregulation. MLN4924-induced apoptosis was mediated by the upregulation of pro-apoptotic and downregulation of anti-apoptotic proteins. Furthermore, MLN4924 inhibited NF-κB, AP-1, and Akt signaling pathways and activated JNK. Therefore, neddylation inhibition is an attractive strategy for ATL therapy. Our findings support the use of MLN4924 in ATL clinical trials.

Patients with adult T-cell leukemia (ATL), caused by the human T-cell leukemia virus type 1 (HTLV-1), exhibit poor prognosis owing to drug resistance. Pimozide is a dopamine D2 receptor antagonist and antipsychotic shown to exhibit anticancer activity. Herein, we investigated whether pimozide exerts anti-ATL effects and explored the mechanisms underlying these effects. Pimozide inhibited cell growth and survival in HTLV-1-infected T cells but not in the uninfected T cells. The dopamine D2 receptor subfamily mRNA expression levels in HTLV-1-infected T cells were high. Pimozide induced G1 cell cycle arrest concomitant with the upregulation of p21/p27/p53, and suppression of cyclin D2/E, cyclin-dependent kinase 2/4/6 and c-Myc expression, and pRb phosphorylation. Pimozide also induced apoptosis by activating caspases, upregulating pro-apoptotic proteins and downregulating anti-apoptotic proteins. Additionally, it promoted reactive oxygen species (ROS) generation and increased the expression of the endoplasmic reticulum stress marker activating transcription factor 4 and the DNA damage-inducible protein GADD45α and the phosphorylation of the DNA damage marker H2AX. Furthermore, pimozide-induced cytotoxicity was partially inhibited by a ROS scavenger, and pan-caspase and necroptosis inhibitors, indicating the involvement of caspase-dependent and -independent lethal pathways. The activities of the nuclear factor-κB, Akt, STAT3/5 and AP-1 signaling pathways were inhibited via the dephosphorylation of IκBα, IκB kinase α/β, Akt and STAT3/5, in addition to reduced JunB and JunD expression in HTLV-1-infected T cells. Pimozide also exhibited potent anti-ATL activity in the xenograft mouse model. These findings demonstrated the efficacy of pimozide as a potential therapeutic agent for ATL., (Copyright © 2021 Elsevier B.V. All rights reserved.)

Human T-cell leukemia virus type 1 (HTLV-1) causes either adult T-cell leukemia (ATL) or chronic inflammatory disorders such as HTLV-1-associated myelopathy/tropical spastic paraparesis. These diseases are not curable as yet; therefore new agents for treatment and prevention are needed. Carotenoids are natural plant compounds with anti-carcinogenic activities. Peridinin is one of the most abundant carotenoids found in nature. Based on a series of past experiments, here we investigated the effects of peridinin extracted from Okinawan coral Isis hippuris on the proliferation and survival of HTLV-1-infected T-cell lines. The results of water-soluble tetrazolium-8 assay indicated that peridinin dose-dependently inhibits cell proliferation and viability of HTLV-1-infected T-cell lines. Flow cytometry showed that low concentration of peridinin induced cell cycle arrest at G1 phase, while higher concentration induced apoptosis. Peridinin caused cleavage of caspase-3, -8 and -9. Peridinin significantly reduced the expression of G1 cell cycle regulators, including cyclin D1, cyclin D2, CDK4, CDK6 and c-Myc, and anti-apoptotic proteins, including survivin, XIAP and Bcl-2, in a dose-dependent manner. Peridinin suppressed DNA binding of NF-κB. Peridinin inhibited phosphorylation of IκBα, RelA, Akt and p70 S6 kinase, and reduced protein expression level of 3-phosphoinositide-dependent protein kinase 1. Thus, peridinin exerts its anti-proliferative and pro-apoptotic effects by suppressing NF-κB and Akt signaling in HTLV-1-infected T cells. Peridinin also reduced tumor growth in mice harboring ATL xenograft tumors. The results suggested that peridinin is a potentially suitable therapeutic agent against HTLV-1-associated diseases.

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.

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.)

Primary effusion lymphoma (PEL) is a rare type of non-Hodgkin's 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., (Copyright © 2010 Elsevier Ireland Ltd. All rights reserved.)

Legionella pneumophila is the causative agent of human Legionnaires' disease. L. pneumophila has been shown to induce apoptosis of T-cells and this may be important pathologically and clinically. The present study has determined the molecular mechanisms underlying L. pneumophila-induced apoptosis, which were unclear. Wild-type L. pneumophila and flagellin-deficient Legionella, but not L. pneumophila lacking a functional type IV secretion system Dot/Icm, replicated in T-cells. However, apoptosis was efficiently induced in T-cells only by wild-type L. pneumophila, and not flagellin-deficient or Dot/Icm-deficient Legionella. Induction of apoptosis involved activation of the initiator caspase 9 and effector caspase 3. Infection with L. pneumophila inhibited phosphorylation of Akt (also known as protein kinase B) and the Akt substrate GSK3beta (glycogen synthase kinase 3beta), and reduced the levels of beta-catenin, a transcriptional activator regulated by GSK3beta. It also caused the activation of the pro-apoptotic protein Bax and inhibited the expression of the anti-apoptotic protein XIAP (X-linked inhibitor of apoptosis) via inhibition of the Akt pathway. In conclusion, L. pneumophila induces mitochondria-mediated T-cell apoptosis through inhibition of the Akt/GSK3beta signalling pathway.

The identification of galectin-9 as a ligand for T-cell immunoglobulin- and mucin-domain-containing molecule-3 (Tim-3), expressed on T-helper type-1 (Th1) cells, has established the Tim-3-galectin-9 pathway as a regulator of Th1 immunity. Whereas there is compelling evidence for the effects of galectin-9 on T-cell fate, limited information is available on the impact of galectin-9 on B lymphocytes. We found that protease-resistant galectin-9, hG9NC(null), but not galectin-1 or -8, prevented cell growth of malignant B cells, such as Burkitt lymphoma (BL) and Hodgkin lymphoma (HL). beta-galactoside binding was essential for galectin-9-induced cell growth suppression. hG9NC(null) induced cell cycle arrest by reducing the expression of cyclin D1, D2, B1, Cdk4, Cdc25C and c-Myc, and apoptosis by reducing the expression of XIAP, c-IAP2 and survivin. Most of the genes that encode these proteins are regulated by nuclear factor-kappaB (NF-kappaB), and constitutive activation of NF-kappaBeta is a common characteristic of both types of malignancies. hG9NC(null) inhibited IkappaBalpha phosphorylation, resulting in suppression of NF-kappaB. AP-1 has also been implicated in the control of cell survival. hG9NC(null) inhibited the expression of JunD, resulting in the suppression of AP-1. Our results suggest that hG9NC(null) is a potentially suitable agent for the management of BL and HL.

Background: Legionella pneumophila pneumonia often exacerbates acute lung injury (ALI) and acute respiratory distress syndrome (ARDS). Apoptosis of alveolar epithelial cells is considered to play an important role in the pathogenesis of ALI and ARDS. In this study, we investigated the precise mechanism by which A549 alveolar epithelial cells induced by L. pneumophila undergo apoptosis. We also studied the effect of methyl prednisolone on apoptosis in these cells., Methods: Nuclear deoxyribonucleic acid (DNA) fragmentation and caspase activation in L. pneumophila-infected A549 alveolar epithelial cells were assessed using the terminal deoxyribonucleotidyl transferase-mediated triphosphate (dUTP)-biotin nick end labeling method (TUNEL method) and colorimetric caspase activity assays. The virulent L. pneumophila strain AA100jm and the avirulent dotO mutant were used and compared in this study. In addition, we investigated whether methyl prednisolone has any influence on nuclear DNA fragmentation and caspase activation in A549 alveolar epithelial cells infected with L. pneumophila., Results: The virulent strain of L. pneumophila grew within A549 alveolar epithelial cells and induced subsequent cell death in a dose-dependent manner. The avirulent strain dotO mutant showed no such effect. The virulent strains of L. pneumophila induced DNA fragmentation (shown by TUNEL staining) and activation of caspases 3, 8, 9, and 1 in A549 cells, while the avirulent strain did not. High-mobility group box 1 (HMGB1) protein was released from A549 cells infected with virulent Legionella. Methyl prednisolone (53.4 muM) did not influence the intracellular growth of L. pneumophila within alveolar epithelial cells, but affected DNA fragmentation and caspase activation of infected A549 cells., Conclusion: Infection of A549 alveolar epithelial cells with L. pneumophila caused programmed cell death, activation of various caspases, and release of HMGB1. The dot/icm system, a major virulence factor of L. pneumophila, is involved in the effects we measured in alveolar epithelial cells. Methyl prednisolone may modulate the interaction of Legionella and these cells.

Citrin is a mitochondrial aspartate-glutamate carrier primarily expressed in liver. Adult-onset type II citrullinemia is caused by mutations in the SLC25A13 gene that encodes for citrin, and patients with this condition do not express citrin. We found apoptotic hepatocytes in one such patient. This finding prompted us to investigate the role of citrin in hepatocyte survival. Knockdown of citrin by a vector-based short-hairpin RNA technique reduced cell viability and induced apoptosis of a hepatocellular carcinoma cell line, Hep3B cells. Caspase-3/7 and caspase-9 were activated, and PARP was cleaved. Citrin knockdown also increased the expression of Bax and Bak, and reduced expression of Bcl-xL and Bcl-2. These alterations resulted in the release of cytochrome c from the mitochondria. Our results indicated that citrin downregulation induces apoptosis of hepatocytes through the mitochondrial death pathway, highlighting the importance of citrin in survival of hepatocytes and maintenance of liver function.

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.)

Purpose: To characterize the time course of apoptosis and degeneration in a transgenic mouse model of retinal degeneration based on truncated mutant HRG4; to investigate the nature of binding of the mutant HRG4 to its target, ADP-ribosylation factor-like (ARL)2; to study its effects on the downstream molecules Binder-of-ARL2 (BART) and adenine nucleotide transporter (ANT)-1 and on the induction of apoptosis., Methods: Saturation binding, microscopic morphometric, Western blot, immunofluorescence, and TUNEL analyses were used., Results: Increased apoptosis did not occur until 20 months in the transgenic retina, consistent with the delayed-onset degeneration in this model. The truncated HRG4 protein exhibited approximately threefold greater affinity for ARL2 than the wild-type HRG4, likely resulting in nonfunctional sequestration of ARL2. A significant decrease in ARL2 was present by 20 months, accompanied by a 50% decrease in ANT-1 in the photoreceptor synaptic mitochondria, with evidence of mitochondrial dysfunction. Preapoptotic degeneration in the photoreceptor synapse was demonstrated with cytochrome c release and caspase 3 activation within the synapse-without evidence of TUNEL-positive apoptosis in the photoreceptor cell body-indicating an initial event in the synapse leading to apoptosis. Caspase 3 was activated in the accompanying secondary neuron, consistent with transsynaptic degeneration., Conclusions: The results support a novel mechanism of retinal degeneration in which preapoptotic degeneration starts in the photoreceptor synapse because of a deficiency in ANT-1 and spreads to the secondary neuron transsynaptically, followed by apoptosis and degeneration in the cell body of the photoreceptor.

Adult T-cell leukemia (ATL), an aggressive malignancy of CD4+ T cells associated with human T-cell leukemia virus type I (HTLV-I) infection, carries a very poor prognosis because of the resistance of leukemic cells to any conventional regimen, including chemotherapy. We examined the effect of ritonavir, an HIV protease inhibitor, on HTLV-I-infected T-cell lines and primary ATL cells and found that it induced apoptosis and inhibited transcriptional activation of NF-kappaB in these cells. Furthermore, ritonavir inhibited expression of Bcl-xL, survivin, c-Myc, and cyclin D2, the targets of NF-kappaB. In nonobese diabetic/severe combined immunodeficient (NOD/SCID)/gammacnull (NOG) mice, ritonavir very efficiently prevented tumor growth and leukemic infiltration in various organs of NOG mice at the same dose used for treatment of patients with AIDS. Our data indicate that ritonavir has potent anti-NF-kappaB and antitumor effects and might be clinically applicable for treatment of ATL. These results would provide a new concept and novel platform for new drug development of leukemia and solid cancer as well.

Objectives: Members of the tumor necrosis factor family are potent inducers of apoptosis in sensitive cells and may be suitable for novel anti-cancer therapies aimed at inducing apoptosis via the activation of receptors with the death domain on malignant cells. We characterized the sensitivity of Burkitt's lymphoma (BL) cell lines to tumor necrosis factor-related apoptosis-inducing ligand (TRAIL) and anti-Fas agonist, and investigated the mechanism of resistance of BL cell lines to TRAIL and Fas apoptotic pathways., Methods: Epstein-Barr virus (EBV) status in BL cell lines was determined by PCR. The extent of apoptosis following exposure to TRAIL and anti-Fas agonist was measured by 7A6 antigen staining. Expression of TRAIL receptors and Fas was determined by flow cytometry and reverse transcriptase-PCR. Western blot analyses were used to determine the expression of proapoptotic and antiapoptotic proteins. NF-kappaB activity was evaluated by electrophoretic mobility shift assay., Results: The sensitivity of BL cell lines to anti-Fas agonist depended on the expression of Fas. In contrast, the expression of TRAIL receptors did not correlate with the sensitivity to TRAIL-induced apoptosis. Interestingly, EBV-infected BL cell lines which showed constitutive levels of NF-kappaB activation, were TRAIL-resistant. NF-kappaB inhibitors reversed the resistance to TRAIL-induced apoptosis., Conclusions: Our results suggest that activation of NF-kappaB by EBV infection plays an important role in resistance of BL cell lines to TRAIL-induced apoptosis, and that NF-kappaB inhibitors may be useful adjuncts in clinical use of TRAIL against BL.

Although hepatic injury is reported in cases with dengue haemorrhagic fever and dengue shock syndrome, its mechanism remains poorly understood. Several findings suggest that dengue virus (DEN) induces apoptosis of hepatocytes in vivo. In this work, DEN type 2 (DEN-2) strain NGC was shown to induce apoptosis in the hepatic cell line HepG2, and infection of HepG2 cells was found to induce Apo2 ligand (Apo2L, also known as tumour necrosis factor-related apoptosis-inducing ligand or TRAIL) expression. Furthermore, Apo2L/TRAIL induced apoptosis in HepG2 cells, which expressed the Apo2L/TRAIL receptor DR5/TRAIL-R2 on their surface. Analysis of the Apo2L/TRAIL promoter revealed that this gene was activated by DEN-2 infection, whose responsive element was overlapping NF-kappaB- and Sp1-binding sites located at nt -75 to -65. The proteasome inhibitor N-acetyl-L-leucinyl-L-leucinyl-L-norleucinal (LLnL) inhibited Apo2L/TRAIL mRNA expression, and LLnL and anti-Apo2L/TRAIL antibody inhibited DEN-2-induced apoptosis. It was proposed that DEN infection promotes apoptosis partly through the induction of Apo2L/TRAIL expression.

Adult T-cell leukemia (ATL) is caused by human T-cell leukemia virus type 1 (HTLV-1) and remains incurable. The highest endemic area of HTLV-1 carriers in Japan is located in Okinawa, and novel treatments are urgently needed in this area. We extracted fucoidan, a sulfated polysaccharide, from the brown seaweed Cladosiphon okamuranus Tokida cultivated in Okinawa, Japan and examined its tumor-suppression activity against ATL. Fucoidan significantly inhibited the growth of peripheral blood mononuclear cells of ATL patients and HTLV-1-infected T-cell lines but not that of normal peripheral blood mononuclear cells. Fucoidan induced apoptosis of HTLV-1-infected T-cell lines mediated through downregulation of cellular inhibitor of apoptosis protein-2 and survivin and G1 phase accumulation through the downregulation of cyclin D2, c-myc, and hyperphosphorylated form of the retinoblastoma tumor suppressor protein. Further analysis showed that fucoidan inactivated NF-kappaB and activator protein-1 and inhibited NF-kappaB-inducible chemokine, C-C chemokine ligand 5 (regulated on activation, normal T expressed and secreted) production, and homotypic cell-cell adhesion of HTLV-1-infected T-cell lines. In vivo use of fucoidan resulted in partial inhibition of growth of tumors of an HTLV-1-infected T-cell line transplanted subcutaneously in severe combined immune deficient mice. Our results indicate that fucoidan is a potentially useful therapeutic agent for patients with ATL.

Inhibition of histone deacetylase (HDAC) activity induces growth arrest, differentiation, and, in certain cell types, apoptosis. FR901228, FK228, or depsipeptide, is an HDAC inhibitor effective in T-cell lymphomas. Adult T-cell leukemia (ATL) is caused by human T-cell leukemia virus type 1 (HTLV-1) and remains incurable. We examined whether FR901228 is effective for treatment of ATL by assessing its ability to induce apoptosis of HTLV-1-infected T-cell lines and primary leukemic cells from ATL patients. FR901228 induced apoptosis of Tax-expressing and -unexpressing HTLV-1-infected T-cell lines and selective apoptosis of primary ATL cells, especially those of patients with acute ATL. FR901228 also efficiently reduced the DNA binding of NF-kappaB and AP-1 in HTLV-1-infected T-cell lines and primary ATL cells and down-regulated the expression of Bcl-x(L) and cyclin D2, regulated by NF-kappaB. Although the viral protein Tax is an activator of NF-kappaB and AP-1, FR901228-induced apoptosis was not associated with reduced expression of Tax. In vivo use of FR901228 partly inhibited the growth of tumors of HTLV-1-infected T cells transplanted subcutaneously in SCID mice. Our results indicated that FR901228 could induce apoptosis of these cells and suppress the expression of NF-kappaB and AP-1 and suggest that FR901228 could be therapeutically effective in ATL.

Human T-cell leukemia virus type I (HTLV-I) is the causative agent of an aggressive form of leukemia designated adult T-cell leukemia (ATL). We have previously demonstrated that all T-cell lines infected with HTLV-I and primary leukemic cells from ATL patients display constitutively high activity of transcription factor NF-kappaB. In this study we showed that Bay 11-7082, an inhibitor of NF-kappaB, induced apoptosis of HTLV-I-infected T-cell lines but only negligible apoptosis of HTLV-I-negative T cells. Bay 11-7082 rapidly and efficiently reduced the DNA binding of NF-kappaB in HTLV-I-infected T-cell lines and down-regulated the expression of the antiapoptotic gene, Bcl-x(L), regulated by NF-kappaB, whereas it had little effect on the DNA binding of another transcription factor, AP-1. Although the viral protein Tax is an activator of NF-kappaB, Bay 11-7082-induced apoptosis of HTLV-I-infected cells was not associated with reduced expression of Tax. Furthermore, Bay 11-7082- induced apoptosis of primary ATL cells was more prominent than that of normal peripheral blood mononuclear cells, and apoptosis of these cells was also associated with down-regulation of NF-kappaB activity. Our results indicate that NF-kappaB plays a crucial role in the pathogenesis and survival of HTLV-I-infected leukemic cells and that it is a suitable target for the prevention and treatment of ATL.

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. [ABSTRACT FROM AUTHOR]

Summary: 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. [ABSTRACT FROM AUTHOR]

Summary: 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 G1 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. [ABSTRACT FROM AUTHOR]

Abstract: We studied the effects of AZD1152, an Aurora B kinase inhibitor, on Burkitt''s lymphoma (BL) and Hodgkin''s lymphoma (HL) in human tissues and cell cultures and in a murine xenograft model of lymphoma. Aurora kinase A and B levels were assessed by RT-PCR and immunohistochemistry. They were aberrantly expressed in BL and HL cell lines, and in lymph nodes from patients with BL and HL. Next, activation of the Aurora B promoter was detected by reporter gene assays. The promoter activity of Aurora B kinase was high in BL cell lines and the Aurora B promoter contained a positive regulatory region between −74 and −104 from the transcription initiation site. AZD1152-hQPA had antiproliferative effects in the BL and HL cell lines studied; inhibited the phosphorylation of histone H3 and retinoblastoma proteins, and resulted in cells with >4N DNA content. AZD1152-hQPA induced caspase-dependent apoptosis of some cell lines, demonstrated by loss of mitochondrial membrane potential, activation of caspase-9, followed by activation of caspase-3. This effect was accompanied by the inhibition of survivin expression. In vivo efficacy was determined in NOD/SCID/γcnull mice implanted with the Ramos human BL cell line. AZD1152 had anti-tumour effects in this murine xenograft model. There preclinical data suggest that the inhibition of Aurora B kinase is a potentially useful therapeutic strategy in BL and HL. [Copyright &y& Elsevier]

The Akt signaling pathway is important for survival and growth of cancer cells. In the present paper we show that the Akt signaling pathway is constitutively activated in human T-cell leukemia virus type I (HTLV-I)-infected T-cell lines and in primary adult T-cell leukemia (ATL) cells. Curcumin, a natural compound present in turmeric, has been studied vigorously as a potent chemopreventive agent for cancer therapy because of its inhibitory effect on proliferation and induction of apoptosis in several tumor cell lines. We investigated the effect of curcumin on Akt activity in HTLV-I-infected T-cell lines and primary ATL cells. Phosphorylated PDK1 is an activator of Akt by phosphorylating Akt. Curcumin reduced phosphorylation of PDK1 and inhibited constitutive activation of Akt. Curcumin activated glycogen synthase kinase (GSK)-3β, a downstream target of Akt kinase, by inhibiting phosphorylation of this protein. Curcumin reduced the expression of cell cycle regulators, cyclin D1 and c-Myc proteins, which are both degraded by activated GSK-3β. Our results suggest that activation of the Akt signaling pathway plays an important role in ATL cell survival, and that curcumin may have anti-ATL properties mediated, at least in part, by inhibiting Akt activity. We propose that Akt-targeting agents could be useful for the treatment of ATL. In this regard, curcumin is a potentially promising compound for the treatment of ATL. ( Cancer Sci 2006; 97: 322 – 327) [ABSTRACT FROM AUTHOR]

Abstract: Lactacystin (LC) is a specific inhibitor of the proteasome, and has recently been shown to induce apoptosis in certain cell lines. In the present study, we established Fas-resistant adult T-cell leukemia (ATL) cell subclones RSO4 and RST1 from their parental Fas-sensitive cell lines SO4 and ST1, and examined whether LC can overcome Fas resistance. LC completely inhibited proteasome function as determined by a peptidyl-MCA substrate (LLVY-MCA and LLE-MCA), and induced apoptosis in these cell lines irrespective of Fas sensitivity at low concentrations (∼ 10 μM). LC induced the activation of caspase 3 (CPP32/Yama) and caspase 6 proteases in an identical manner to Fas-mediated apoptosis. Moreover, LC induced the activation of caspase 8 (FLICE) protease, which is the initiator of the Fas-mediated apoptotic cascade. Synthesized proteasome inhibitory peptide MG-115 (ZLLnV-CHO) also induced apoptosis in these cell lines. These results indicated that proteasome inhibitors overcome Fas-resistance by bypassing the proximal part of the Fas signal. Inhibition of the proteasome function may be a new strategy for the treatment of ATL. [ABSTRACT FROM AUTHOR]

Abstract: 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-XL, 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 &y& Elsevier]

Abstract: Activation of the activator protein 1 (AP-1) plays a critical role in oncogenesis by human T-cell leukemia virus type 1 (HTLV-1), the etiologic agent of adult T-cell leukemia (ATL), and is required for maintenance of the malignant phenotype. Curcumin (diferuloylmethane), the major pigment of the spice turmeric, has anti-tumor activity; however, the effect of curcumin against ATL has not been elucidated. In this study, we examined the effects of curcumin on AP-1 activity in HTLV-1-infected T-cell lines. Curcumin suppressed the constitutive AP-1 DNA-binding and transcriptional activity in HTLV-1-infected T-cell line. Curcumin also inhibited HTLV-1 Tax-induced AP-1 transcriptional activity. JunD was detectable as a major component of the AP-1–DNA complex in HTLV-1-infected T-cell lines using the supershift assay. The expression of JunD was suppressed by curcumin treatment. Curcumin inhibited the growth of HTLV-1-infected T-cell lines by inducing cell cycle arrest followed by apoptosis. Our results suggest that suppression of the constitutively active AP-1 by curcumin is due to, at least in-part, reducing the expression of JunD by curcumin. Inhibition of AP-1 activity by curcumin may be one of the mechanisms responsible for the anti-ATL effect of curcumin. We propose that curcumin is a potentially promising compound for the treatment of ATL. [Copyright &y& Elsevier]

Adult T-cell leukemia (ATL), a CD4+-T-cell malignancy caused by human T-cell leukemia virus type 1 (HTLV-1), is difficult to cure, and novel treatments are urgently needed. Apo2 ligand (Apo2L; also tumor necrosis factor-related apoptosis-inducing ligand [TRAIL]) has been implicated in antitumor therapy. We found that HTLV-1-infected T-cell lines and primary ATL cells were more resistant to Apo2L-induced apoptosis than uninfected cells. Interestingly, HTLV-1-infected T-cell lines and primary ATL cells constitutively expressed Apo2L mRNA. Inducible expression of the viral oncoprotein Tax in a T-cell line up-regulated Apo2L mRNA. Analysis of the Apo2L promoter revealed that this gene is activated by Tax via the activation of NF-κB. The sensitivity to Apo2L was not correlated with expression levels of Apo2L receptors, intracellular regulators of apoptosis (FLICE-inhibitory protein and active Akt). NF-κB plays a crucial role in the pathogenesis and survival of ATL cells. The resistance to Apo2L-induced apoptosis was reversed by N-acetyl-L-leucinyl-L-leucinyl-L-norleucinal (LLnL), an NF-κB inhibitor. LLnL significantly induced the Apo2L receptors DR4 and DR5. Our results suggest that the constitutive activation of NF-κB is essential for Apo2L gene induction and protection against Apo2L-induced apoptosis and that suppression of NF-κB may be a useful adjunct in clinical use of Apo2L against ATL. [ABSTRACT FROM AUTHOR]