Tomohiro Kozako

Oversulfated fucoidan inhibits the basic fibroblast growth factor-induced tube formation by human umbilical vein endothelial cells: its possible mechanism of action.

We have previously demonstrated that chemically oversulfated fucoidan (OSF) but not native fucoidan (NF) effectively suppresses the tube structure formation by human umbilical vein endothelial cells (HUVEC) on the basement membrane preparation, Matrigel. In this study, using more defined systems where basic fibroblast growth factor (bFGF) induces the tube formation by HUVEC on collagen gel, we investigated the mechanism responsible for the inhibition of angiogenesis by OSF in vitro. Unlike NF and desulfated fucoidan (desF), OSF potently inhibited the bFGF-induced HUVEC migration and tube formation. ELISA for tissue-type plasminogen activator (t-PA) and plasminogen activator inhibitor-1 (PAI-1) in the culture media indicated that OSF increased the bFGF-induced release of PAI-1 antigen, but not of t-PA antigen. Analyses of the binding of bFGF to HUVEC surfaces and the following protein tyrosine phosphorylation revealed that OSF could promote the cell binding and autophosphorylation of 140 and 160 kDa receptors. In heparitinase-treated HUVEC, contrarily, the bFGF binding and PAI-1 release were decreased by OSF. These results suggest that OSF is a highly sulfated unique polysaccharide that can promote the binding of bFGF to the heparan sulfate molecules required for binding to the high affinity receptors with tyrosine kinase activity. The resultant increase in PAI-1 release may play a key role for the prevention of cell migration accompanied by matrix proteolysis.

Fasting promotes the expression of SIRT1, an NAD+-dependent protein deacetylase, via activation of PPARα in mice

Calorie restriction (CR) extends lifespans in a wide variety of species. CR induces an increase in the NAD+/NADH ratio in cells and results in activation of SIRT1, an NAD+-dependent protein deacetylase that is thought to be a metabolic master switch linked to the modulation of lifespans. CR also affects the expression of peroxisome proliferator-activated receptors (PPARs). The three subtypes, PPARα, PPARγ, and PPARβ/δ, are expressed in multiple organs. They regulate different physiological functions such as energy metabolism, insulin action and inflammation, and apparently act as important regulators of longevity and aging. SIRT1 has been reported to repress the PPARγ by docking with its co-factors and to promote fat mobilization. However, the correlation between SIRT1 and other PPARs is not fully understood. CR initially induces a fasting-like response. In this study, we investigated how SIRT1 and PPARα correlate in the fasting-induced anti-aging pathways. A 24-h fasting in mice increased mRNA and protein expression of both SIRT1 and PPARα in the livers, where the NAD+ levels increased with increasing nicotinamide phosphoribosyltransferase (NAMPT) activity in the NAD+ salvage pathway. Treatment of Hepa1-6 cells in a low glucose medium conditions with NAD+ or NADH showed that the mRNA expression of both SIRT1 and PPARα can be enhanced by addition of NAD+, and decreased by increasing NADH levels. The cell experiments using SIRT1 antagonists and a PPARα agonist suggested that PPARα is a key molecule located upstream from SIRT1, and has a role in regulating SIRT1 gene expression in fasting-induced anti-aging pathways.

Reduced Frequency, Diversity, and Function of Human T Cell Leukemia Virus Type 1-Specific CD8+ T Cell in Adult T Cell Leukemia Patients

Human T cell lymphotropic virus type 1 (HTLV-1)-specific CTL are thought to be immune effectors that reduce the risk of adult T cell leukemia (ATL). However, in vivo conditions of anti-HTLV-1 CTL before and after ATL development have yet to be determined. To characterize anti-HTLV-1 CTL in asymptomatic HTLV-1 carriers (AC) and ATL patients, we analyzed the frequency and diversity of HTLV-1-specific CD8+ T cells in PBMC of 35 AC and 32 ATL patients using 16 distinct epitopes of HTLV-1 Tax or Env/HLA tetramers along with intracellular cytolytic effector molecules (IFN-γ, perforin, and granzyme B). Overall frequency of subjects possessing Tax-specific CD8+ T cells was significantly lower in ATL than AC (53 vs 90%; p = 0.001), whereas the difference in Env-specific CD8+ T cells was not statistically significant. AC possessed Tax11–19/HLA-A*0201-specific tetramer+ cells by 90% and Tax301–309/HLA-A*2402-specific tetramer+ cells by 92%. Some AC recognized more than one epitope. In contrast, ATL recognized only Tax11–19 with HLA-A*0201 and Tax301–309 with HLA-A*2402 at frequencies of 30 and 55%. There were also significant differences in percentage of cells binding Tax11–19/HLA-A*0201 and Tax301–309/HLA-A*2402 tetramers between AC and ATL. Anti-HTLV-1 Tax CD8+ T cells in AC and ATL produced IFN-γ in response to Tax. In contrast, perforin and granzyme B expression in anti-HTLV-1 CD8+ T cells of ATL was significant lower than that of AC. Frequency of Tax-specific CD8+ T cells in AC was related to proviral load in HLA-A*0201. These results suggest that decreased frequency, diversity, and function of anti-HTLV-1 Tax CD8+ T cell clones may be one of the risks of ATL development.

High expression of the longevity gene product SIRT1 and apoptosis induction by sirtinol in adult T-cell leukemia cells

Adult T‐cell leukemia‐lymphoma (ATL) is an aggressive peripheral T‐cell neoplasm that develops after long‐term infection with human T‐cell leukemia virus (HTLV‐1). SIRT1, a nicotinamide adenine dinucleotide+‐dependent histone/protein deacetylase, plays a crucial role in various physiological processes, such as aging, metabolism, neurogenesis and apoptosis, owing to its ability to deacetylate numerous substrates, such as histone and NF‐κB, which is implicated as an exacerbation factor in ATL. Here, we assessed how SIRT1 is regulated in primary ATL cells and leukemic cell lines. SIRT1 expression in ATL patients was significantly higher than that in healthy controls, especially in the acute type. Sirtinol, a SIRT1 inhibitor, induced significant growth inhibition or apoptosis in cells from ATL patients and leukemic cell lines, especially HTLV‐1‐related cell lines. Sirtinol‐induced apoptosis was mediated by activation of the caspase family and degradation of SIRT1 in the nucleus. Furthermore, SIRT1 knockdown by SIRT1‐specific small interfering RNA caused apoptosis via activation of caspase‐3 and PARP in MT‐2 cells, HTLV‐1‐related cell line. These results suggest that SIRT1 is a crucial antiapoptotic molecule in ATL cells and that SIRT1 inhibitors may be useful therapeutic agents for leukemia, especially in patients with ATL.

Anti-apoptotic roles of plasminogen activator inhibitor-1 as a neurotrophic factor in the central nervous system

Plasminogen activator inhibitor-1 (PAI-1), a member of the serpin gene family, is the primary inhibitor of urokinase-type and tissue-type PAs. PAI-1 plays an important role in the process of peripheral tissue remodeling and fibrinolysis through the regulation of PA activity. This serpin is also produced in brain tissues and may regulate the neural protease sequence in the central nervous system (CNS), as it does in peripheral tissues. In fact, PAI-1 mRNA is up-regulated in mouse brain after stroke. The serpin activity of PAI-1 helps to prevent tissue-type PA-induced neuron death. However, we have previously found that PAI-1 has a novel biological function in the CNS: the contribution to survival of neurites on neurons. In neuronally differentiated rat pheochromocytoma (PC-12) cells, a deficiency of PAI-1 in vitro caused a significant reduction in Bcl-2 and Bcl-X(L) mRNAs and an increase in Bcl-X(S) and Bax mRNAs. The change in the balance between mRNA expressions of the anti- and pro-apoptotic Bcl-2 family proteins promoted the apoptotic sequence: caspase-3 activation, cytochrome c release from mitochondria and DNA fragmentation. Our results indicate that PAI-1 has an anti-apoptotic role in neurons. PAI-1 prevented the disintegration of the formed neuronal networks by maintaining or promoting neuroprotective signaling through the MAPK/ERK pathway, suggesting that the neuroprotective effect of PAI-1 is independent of its action as a protease inhibitor. This review discusses the neuroprotective effects of PAI-1 in vitro, together with the relevant data from other laboratories. Special emphasis is placed on its action on PC-12 cells.

HTLV-1 provirus load in peripheral blood lymphocytes of HTLV-1 carriers is diminished by green tea drinking

Human T‐cell lymphotropic virus type 1 (HTLV‐1) is causatively associated with adult T‐cell leukemia (ATL) and HTLV‐1‐associated myelopathy/tropical spastic paraparesis (HAM/TSP). Since a high level of HTLV‐1 provirus load in circulating lymphocytes is thought to be a risk for ATL and HAM/TSP, diminution of HTLV‐1 provirus load in the circulation may prevent these intractable diseases. Our previous study (Jpn J Cancer Res 2000; 91: 34–40) demonstrated that green tea polyphenols inhibit in vitro growth of ATL cells, as well as HTLV‐1‐infected T‐cells. The present study aimed to investigate the in vivo effect of green tea polyphenols on HTLV‐1 provirus load in peripheral blood lymphocytes on HTLV‐1 carriers. We recruited 83 asymptomatic HTLV‐1 carriers to examine HTLV‐1 provirus DNA with or without administration of capsulated green tea extract powder. Thirty‐seven subjects were followed up for 5 months by measuring HTLV‐1 provirus load after daily intake of 9 capsules of green tea extract powder per day (equivalent to 10 cups of regular green tea), and 46 subjects lived ad libitum without intake of any green tea capsule. The real‐time PCR quantification of HTLV‐1 DNA revealed a wide range of variation of HTLV‐1 provirus load among asymptomatic HTLV‐1 carriers (0.2‐200.2 copies of HTLV‐1 provirus load per 1000 peripheral blood lymphocytes). Daily intake of the capsulated green tea for 5 months significantly diminished the HTLV‐1 provirus load as compared with the controls (P=0.031). These results suggest that green tea drinking suppresses proliferation of HTLV‐1‐infected lymphocytes in vivo.

Anticancer Agents Targeted to Sirtuins

Sirtuins are nicotinamide adenine dinucleotide+-dependent deacetylases of which there are seven isoforms (SIRT1–7). Sirtuin activity is linked to gene expression, lifespan extension, neurodegeneration, and age-related disorders. Numerous studies have suggested that sirtuins could be of great significance with regard to both antiaging and tumorigenesis, depending on its targets in specific signaling pathways or in specific cancers. Recent studies have identified small chemical compounds that modulate sirtuins, and these modulators have enabled a greater understanding of the biological function and molecular mechanisms of sirtuins. This review highlights the possibility of sirtuins, especially SIRT1 and SIRT2, for cancer therapy targets, and focuses on the therapeutic potential of sirtuin modulators both in cancer prevention and treatment.

Target epitopes of HTLV-1 recognized by class I MHC-restricted cytotoxic T lymphocytes in patients with myelopathy and spastic paraparesis and infected patients with autoimmune disorders†

Human T‐cell lymphotropic virus type I (HTLV‐1) causes adult T‐cell leukemia/lymphoma and HTLV‐1‐associated myelopathy/tropical spastic paraparesis (HAM/TSP). The different patterns of clinical diseases are thought to be linked to immunogenetic host factors. A variety of autoimmune diseases, such as Sjögrens syndrome, have been reported in persons infected with HTLV‐1, although the precise relationship between these disorders and HTLV‐1 infection remains unknown. There is no report on the repertoire of HTLV‐1‐specific CD8+ T‐cells in HAM/TSP patients or carriers with autoimmune diseases, both characterized by an abnormal immune state. In this study, to characterize HTLV‐1‐specific CD8+ T‐cells in asymptomatic HTLV‐1 carriers, HAM/TSP patients and carriers with autoimmune diseases, we examined the frequency and diversity of HTLV‐1‐specific CD8+ T‐cells using HTLV‐1 tetramers. HTLV‐1 Env‐specific CD8+ T‐cells were significantly more frequent in HAM/TSP and carriers with autoimmune diseases compared with asymptomatic HTLV‐1 carriers, while the frequency of HTLV‐1 Tax‐specific CD8+ T‐cells was not significantly different among them. CD8+ cells binding to HTLV‐1 Tax tetramers in carriers with autoimmune diseases were significantly reduced compared with HAM/TSP patients. This study demonstrates the importance of CD8+ T‐cells recognizing HTLV‐1 Env‐tetramers in HAM/TSP patients and carriers with autoimmune diseases, thereby suggesting that the diversity, frequency and repertoire of HTLV‐1 Env‐specific CD8+ T‐cell clones may be related to the hyperimmune response in HAM/TSP and carriers with autoimmune diseases, although different immunological mechanisms may mediate the hyperimmunity in these conditions. J. Med. Virol. 83:501–509, 2011.

Oligomannose‐coated liposomes efficiently induce human T‐cell leukemia virus‐1‐specific cytotoxic T lymphocytes without adjuvant

Human T‐cell leukemia virus‐1 (HTLV‐1) causes adult T‐cell leukemia/lymphoma, which is an aggressive peripheral T‐cell neoplasm. Insufficient T‐cell response to HTLV‐1 is a potential risk factor in adult T‐cell leukemia/lymphoma. Efficient induction of antigen‐specific cytotoxic T lymphocytes is important for immunological suppression of virus‐infected cell proliferation and oncogenesis, but efficient induction of antigen‐specific cytotoxic T lymphocytes has evaded strategies utilizing poorly immunogenic free synthetic peptides. Here, we examined the efficient induction of an HTLV‐1‐specific CD8+ T‐cell response by oligomannose‐coated liposomes (OMLs) encapsulating the human leukocyte antigen (HLA)‐A*0201‐restricted HTLV‐1 Tax‐epitope (OML/Tax). Immunization of HLA‐A*0201 transgenic mice with OML/Tax induced an HTLV‐1‐specific gamma‐interferon reaction, whereas immunization with epitope peptide alone induced no reaction. Upon exposure of dendritic cells to OML/Tax, the levels of CD86, major histocompatibility complex class I, HLA‐A02 and major histocompatibility complex class II expression were increased. In addition, our results showed that HTLV‐1‐specific CD8+ T cells can be efficiently induced by OML/Tax from HTLV‐1 carriers compared with epitope peptide alone, and these HTLV‐1‐specific CD8+ T cells were able to lyse cells presenting the peptide. These results suggest that OML/Tax is capable of inducing antigen‐specific cellular immune responses without adjuvants and may be useful as an effective vaccine carrier for prophylaxis in tumors and infectious diseases by substituting the epitope peptide.

Programmed death-1 (PD-1)/PD-1 ligand pathway-mediated immune responses against human T-lymphotropic virus type 1 (HTLV-1) in HTLV-1-associated myelopathy/tropical spastic paraparesis and carriers with autoimmune disorders.

Human T-lymphotropic virus-1 (HTLV-1) causes HTLV-1-associated myelopathy/tropical spastic paraparesis (HAM/TSP) and adult T-cell leukemia-lymphoma in individuals with dysfunctional immune responses. In this study, to characterize the HTLV-1-specific cytotoxic T lymphocyte (CTL) populations in asymptomatic HTLV-1 carriers (ACs), HAM/TSP patients, and carriers with autoimmune disorders (CAIDs), we examined the role of programmed death-1 and its ligand (PD-1/PD-L1) in HTLV-1-specific CTL functions using an HTLV-1 Tax/HLA-A*0201 tetramer and an HTLV-1 Tax/HLA-A*2402 tetramer. Interestingly, the percentage of HTLV-1 Tax301-309/HLA-A*2402 tetramer(+)CD8(+) cells expressing PD-1 in ACs was significantly higher than the percentage of HTLV-1 Tax11-19/HLA-A*0201 tetramer(+)CD8(+) cells expressing PD-1. PD-1 expression was significantly downregulated on HTLV-1-specific CTLs in HAM/TSP compared with ACs. PD-L1 expression was observed in a small proportion of unstimulated lymphocytes from ACs and was greater in ACs than in HAM/TSP and CAIDs after short-term culture. Furthermore, CTL degranulation was impaired in HAM/TSP, whereas anti-PD-L1 blockade significantly increased CTL function in ACs. Downregulation of PD-1 on HTLV-1-specific CTLs and loss of PD-L1 expression in HAM/TSP and CAIDs, along with impaired function of HTLV-1-specific CTLs in HAM/TSP, may underlie the apparently dysfunctional immune response against HTLV-1.