Yukihiro Akao

Possible dominant-negative mutation of the SHIP gene in acute myeloid leukemia

The SH2 domain-containing inositol 5′-phosphatase (SHIP) is crucial in hematopoietic development. To evaluate the possible tumor suppressor role of the SHIP gene in myeloid leukemogenesis, we examined primary leukemia cells from 30 acute myeloid leukemia (AML) patients, together with eight myeloid leukemia cell lines. A somatic mutation at codon 684, replacing Val with Glu, was detected in one patient, lying within the signature motif 2, which is the phosphatase active site. The results of an in vitro inositol 5′-phosphatase assay revealed that the mutation reduced catalytic activity of SHIP. Leukemia cells with the mutation showed enhanced Akt phosphorylation following IL-3 stimulation. K562 cells transfected with the mutated SHIP-V684E cDNA showed a growth advantage even at lower serum concentrations and resistance to apoptosis induced by serum deprivation and exposure to etoposide. These results suggest a possible role of the mutated SHIP gene in the development of acute leukemia and chemotherapy resistance through the deregulation of the phosphatidylinositol-3,4,5-triphosphate (PI(3,4,5)P3)/Akt signaling pathway. This is the first report of a mutation in the SHIP gene in any given human cancer, and indicates the need for more attention to be paid to this gene with respect to cancer pathogenesis.

MicroRNA-143 functions as a tumor suppressor in human bladder cancer T24 cells.

MicroRNA (miR)-143 and -145 were down-regulated in human bladder cancer T24 cells. The enforced expression of miR-143 induced growth-suppression in T24 cells through down-regulation of ERK5 and Akt expression at translational level, and chemically-modified synthetic miR-143 (miR-143/BP) exhibited a greater growth inhibitory effect than wild-type miR-143. In addition, the synthetic miR-143/BP induced apoptotic cell death in some of the transfected cells. Furthermore, co-treatment with the synthetic miR-143/BP and cisplatin showed the additive growth-suppressing effect on T24 cells. These findings suggest that the chemically-modified synthetic miR-143 functions as a tumor suppressor in T24 cells by targeting ERK5 and/or Akt.

Anti-oncogenic microRNA-203 induces senescence by targeting E2F3 protein in human melanoma cells.

Background: MicroRNA-203 is down-regulated, and its exogenous expression inhibits cell growth in human melanoma cells. Results: MicroRNA-203 induced senescence by cell cycle arrest through targeting E2F3. Conclusion: MicroRNA-203 is a novel senescence-associated microRNA in melanoma cells. Significance: This study has revealed the relationship between senescence and carcinogenesis in melanoma cells with respect to dysregulation of anti-oncogenic microRNA-203. MicroRNAs regulate gene expression by repressing translation or directing sequence-specific degradation of their complementary mRNA. We recently reported that miR-203 is down-regulated, and its exogenous expression inhibits cell growth in canine oral malignant melanoma tissue specimens as well as in canine and human malignant melanoma cells. A microRNA target database predicted E2F3 and ZBP-89 as putative targets of microRNA-203 (miR-203). The expression levels of E2F3a, E2F3b, and ZBP-89 were markedly up-regulated in human malignant melanoma Mewo cells compared with those in human epidermal melanocytes. miR-203 significantly suppressed the luciferase activity of reporter plasmids containing the 3′-UTR sequence of either E2F3 or ZBP-89 complementary to miR-203. The ectopic expression of miR-203 in melanoma cells reduced the levels of E2F3a, E2F3b, and ZBP-89 protein expression. At the same time, miR-203 induced cell cycle arrest and senescence phenotypes, such as elevated expression of hypophosphorylated retinoblastoma and other markers for senescence. Silencing of E2F3, but not of ZBP-89, inhibited cell growth and induced cell cycle arrest and senescence. These results demonstrate a novel role for miR-203 as a tumor suppressor acting by inducing senescence in melanoma cells.

α-Mangostin extracted from the pericarp of the mangosteen (Garcinia mangostana Linn) reduces tumor growth and lymph node metastasis in an immunocompetent xenograft model of metastatic mammary cancer carrying a p53 mutation

AbstractBackgroundThe mangosteen fruit has a long history of medicinal use in Chinese and Ayurvedic medicine. Recently, the compound α-mangostin, which is isolated from the pericarp of the fruit, was shown to induce cell death in various types of cancer cells in in vitro studies. This led us to investigate the antitumor growth and antimetastatic activities of α-mangostin in an immunocompetent xenograft model of mouse metastatic mammary cancer having a p53 mutation that induces a metastatic spectrum similar to that seen in human breast cancers.MethodsMammary tumors, induced by inoculation of BALB/c mice syngeneic with metastatic BJMC3879luc2 cells, were subsequently treated with α-mangostin at 0, 10 and 20 mg/kg/day using mini-osmotic pumps and histopathologically examined. To investigate the mechanisms of antitumor ability by α-mangostin, in vitro studies were also conducted.ResultsNot only were in vivo survival rates significantly higher in the 20 mg/kg/day α-mangostin group versus controls, but both tumor volume and the multiplicity of lymph node metastases were significantly suppressed. Apoptotic levels were significantly increased in the mammary tumors of mice receiving 20 mg/kg/day and were associated with increased expression of active caspase-3 and -9. Other significant effects noted at this dose level were decreased microvessel density and lower numbers of dilated lymphatic vessels containing intraluminal tumor cells in mammary carcinoma tissues. In vitro, α-mangostin induced mitochondria-mediated apoptosis and G1-phase arrest and S-phase suppression in the cell cycle. Since activation by Akt phosphorylation plays a central role in a variety of oncogenic processes, including cell proliferation, anti-apoptotic cell death, angiogenesis and metastasis, we also investigated alterations in Akt phosphorylation induced by α-mangostin treatment both in vitro and in vivo. Quantitative analysis and immunohistochemistry showed that α-mangostin significantly decreased the levels of phospho-Akt-threonine 308 (Thr308), but not serine 473 (Ser473), in both mammary carcinoma cell cultures and mammary carcinoma tissues in vivo.ConclusionsSince lymph node involvement is the most important prognostic factor in breast cancer patients, the antimetastatic activity of α-mangostin as detected in mammary cancers carrying a p53 mutation in the present study may have specific clinical applications. In addition, α-mangostin may have chemopreventive benefits and/or prove useful as an adjuvant therapy, or as a complementary alternative medicine in the treatment of breast cancer.

Replacement treatment with microRNA-143 and -145 induces synergistic inhibition of the growth of human bladder cancer cells by regulating PI3K/Akt and MAPK signaling pathways

We recently reported that both microRNA (miR)-143 and -145 are downregulated in human bladder cancer T24 cells and that miR-143 targets ERK5. In this study, we assessed the anti-tumor effects of combination treatment with miR-143 and -145 on bladder cancer cell lines T24, SNK57, and NKB1, in which the expression levels of miR-143 and -145 are downregulated. The ectopic expression of both miR-143 and -145 led to a significantly synergistic growth inhibition of T24 and NKB1 cells, but not that of SNK57 cells with the levels of miR-143 and -145 higher than those in T24 and NKB1 cells. The MAPK signaling pathway in NKB1 cells and both PI3K/Akt and MAPK signaling pathways in T24 cells were synergistically repressed by the co-treatment with miR-143 and -145. We newly elucidated that miR-143 targeted akt and that miR-145 targeted integrin-linked kinase (ilk) in T24 cells based on the results of a luciferase activity assay. Silencing of ilk significantly inhibited the growth of all the bladder cancer cells tested. Also, the level of phosphorylated ERK1/2 in T24 cells and that of phosphorylated Akt in SNK57 and NKB1 cells were decreased by ilk silencing. This study has provided novel important evidence with regard to the functions of anti-oncogenic miR-143 and -145 and also suggests the possible use of miR-143 and -145 for combination replacement therapy in cancers in which both miRNAs are downregulated.

Colorectal cancer cell-derived microvesicles containing microRNA-1246 promote angiogenesis by activating Smad 1/5/8 signaling elicited by PML down-regulation in endothelial cells

Emerging studies on circulating microRNAs (miRNAs) or microvesicles (MVs) have shown the potential of them to be novel biomarkers and therapeutic targets for cancer. However, the biological roles of these miRNAs and MVs have not been validated yet. To determine the biological significance of MVs, we used human colorectal cancer cells as the MV donor and endothelial cells (HUVECs) as the MV recipient and demonstrated the transfer of colorectal cancer cell-derived MVs (CRC-MVs) to HUVECs and evaluated the roles of these MVs and their cargo in tumor angiogenesis. Consequently, the incubation of HUVECs with CRC-MVs promoted the proliferation, migration, and tube formation activities of these cells. Among the cargoes shuttled by the MVs, miR-1246 and TGF-β were considered to be responsible for the pro-angiogenic function of MVs by activating Smad 1/5/8 signaling in the HUVECs. These results suggest that colorectal cancer cells secreted MVs to contribute to tumor angiogenesis.

Mitochondria in neurodegenerative disorders: regulation of the redox state and death signaling leading to neuronal death and survival

In Parkinson’s disease, impaired function of mitochondrial complex I is involved in selective degeneration of dopamine neurons in the substantia nigra. Mitochondria are now considered to play an active role in neuronal death process through activating “intrinsic” apoptotic signaling, in addition to production of reactive oxygen species. This paper presents our recent findings on new functions of mitochondria in regulation of their redox state and function through reversible “S-glutathionylation”, a mixed disulfide binding between sulfhydryl groups of GSH and protein cysteine in complex I subunits. Type A monoamine oxidase (MAO-A) localized at the mitochondrial outer membrane is a binding site of neurotoxins leading to apoptosis. Rasagiline and (−)deprenyl, type B MAO inhibitors of propagylamine-derivatives, bind to MAO-A to protect neuronal cells against apoptosis through induction of pro-survival Bcl-2 and neurotrophic factors. This review discusses the new role of mitochondria in regulation of neuronal cell death of neurodegenerative disorders.

Cell Type-specific Localization of Sphingosine Kinase 1a in Human Tissues

Cell type-specific localization of sphingosine kinase 1a (SPHK1a) in tissues was analyzed with a rabbit polyclonal antibody against the 16 C-terminal amino acids derived from the recently reported mouse cDNA sequence of SPHK1a. This antibody (anti-SPHK1a antibody) can react specifically with SPHK1a of mouse, rat, and human tissues. Utilizing its crossreactivity to human SPHK1a, the cell-specific localization of SPHK1a in human tissues was histochemically examined. Strong positive staining for SPHK1a was observed in the white matter in the cerebrum and cerebellum, the red nucleus and cerebral peduncle in the midbrain, the uriniferous tubules in the kidney, the endothelial cells in vessels of various organs, and in megakaryocytes and platelets. The lining cells of sinusoids in the liver and splenic cords in the spleen showed moderate staining. Columnar epithelia in the intestine and Leydigs cells in the testis showed weak staining patterns. In addition, TPA-treated HEL cells, a human leukemia cell line, showed a megakaryocytic phenotype accompanied with increases in immunostaining of both SPHK1a and SPHK enzyme activity, suggesting that SPHK1a may be a novel marker of megakaryocytic differentiation and that this antibody is also useful for in vitro study of differentiation models.

Anti-cancer effects of naturally occurring compounds through modulation of signal transduction and miRNA expression in human colon cancer cells

Much evidence indicates that various naturally occurring compounds have an anti-cancer effect, but the detailed mechanisms are not well understood. In this study, we selected anti-cancer phytochemicals such as epigallocatechin-3-gallate (EGCG), resveratrol (RES) and α-mangostin (α-M), all of which are well-characterized chemopreventive agents. We sought to elucidate the mechanism of their anti-cancer effects and the synergistic effects obtained by combined treatment with the anti-cancer drug 5-fluorouracil (5-FU) in three human colon cancer cell lines. The numbers of viable cells were consistently decreased by the treatment with EGCG, RES or α-M at more than 10 μM in all three cell lines tested. All compounds mainly induced apoptosis and suppressed the PI3K/Akt signaling pathway. Additionally, α-M, which had the greatest PI3K/Akt-suppressing activity, also suppressed MAP kinase (MAPK)/Erk1/2 signaling. Importantly, the combination treatment with RES and 5-FU induced a remarkably synergistic enhancement of growth inhibition and apoptosis through the additional suppression of the MAPK/Erk1/2 signaling pathway in colon cancer DLD-1 cells. Interestingly, RES increased the intracellular expression level of miR-34a, which down-regulated the target gene E2F3 and its downstream Sirt1, resulting in growth inhibition. These findings indicate that these compounds functioned as chemosensitizers when combined with anti-cancer drugs through the modulation of apoptotic and growth-related signaling pathways. Also, RES exerted its anti-cancer activity in part through a newly defined mechanism, i.e., the miR-34a/E2F3/Sirt1 cascade.

MicroRNA-208 modulates BMP-2-stimulated mouse preosteoblast differentiation by directly targeting V-ets erythroblastosis virus E26 oncogene homolog 1.

MicroRNAs (miRs) represent a class of endogenous ∼18–25 nucleotide RNAs that regulate gene expression through translational repression by binding to a target mRNA. These miRs regulate several biological functions, such as cell growth, cell differentiation, carcinogenesis, and so on. In a previous report, we have indicated that miR-141 and -200a act as preosteoblast differentiation modulators. In the present study, using microRNA array and in silico analyses, we found that miR-208 is closely involved in preosteoblast differentiation by partially regulating the expression of Ets1 (V-ets erythroblastosis virus E26 oncogene homolog 1), which transactivates osteopontin, runt-related transcription factor 2, parathyroid hormone-related protein, and type I procollagen. Furthermore, the enforced expression of mature miR-208 in murine preosteoblast in MC3T3-E1 cells or primary osteoblast cells remarkably attenuated BMP-2-induced preosteoblast differentiation. In addition, we determined that Ets1 is a target gene of miR-208 by using a sensor luciferase reporter assay. Taken together, these results suggest that the down-regulation of miR-208 in BMP-2-stimulated osteoblast differentiation is an important part of the regulatory machinery involved in early osteogenesis.