Anti-angiogenic effects of differentiation-inducing factor-1 involving VEGFR-2 expression inhibition independent of the Wnt/β-catenin signaling pathway

<p>Abstract</p> <p>Background</p> <p>Differentiation-inducing factor-1 (DIF-1) is a putative morphogen that induces cell differentiation in <it>Dictyostelium discoideum</it>. DIF-1 inhibits proliferation of various mammalian tumor cells by suppressing the canonical Wnt/β-catenin signaling pathway. To assess the potential of a novel cancer chemotherapy based on the pharmacological effect of DIF-1, we investigated whether DIF-1 exhibits anti-angiogenic effects <it>in vitro </it>and <it>in vivo</it>.</p> <p>Results</p> <p>DIF-1 not only inhibited the proliferation of human umbilical vein endothelial cells (HUVECs) by restricting cell cycle in the G₀/G₁phase and degrading cyclin D1, but also inhibited the ability of HUVECs to form capillaries and migrate. Moreover, DIF-1 suppressed VEGF- and cancer cell-induced neovascularization in Matrigel plugs injected subcutaneously to murine flank. Subsequently, we attempted to identify the mechanism behind the anti-angiogenic effects of DIF-1. We showed that DIF-1 strongly decreased vascular endothelial growth factor receptor-2 (VEGFR-2) expression in HUVECs by inhibiting the promoter activity of human VEGFR-2 gene, though it was not caused by inhibition of the Wnt/β-catenin signaling pathway.</p> <p>Conclusion</p> <p>These results suggested that DIF-1 inhibits angiogenesis both <it>in vitro </it>and <it>in vivo</it>, and reduction of VEGFR-2 expression is involved in the mechanism. A novel anti-cancer drug that inhibits neovascularization and tumor growth may be developed by successful elucidation of the target molecules for DIF-1 in the future.</p

Proteomic Identification of Protein Targets for 15-Deoxy-Δ12,14-Prostaglandin J2 in Neuronal Plasma Membrane

15-deoxy-Δ12,14-prostaglandin J2 (15d-PGJ2) is one of factors contributed to the neurotoxicity of amyloid β (Aβ), a causative protein of Alzheimer's disease. Type 2 receptor for prostaglandin D2 (DP2) and peroxysome-proliferator activated receptorγ (PPARγ) are identified as the membrane receptor and the nuclear receptor for 15d-PGJ2, respectively. Previously, we reported that the cytotoxicity of 15d-PGJ2 was independent of DP2 and PPARγ, and suggested that 15d-PGJ2 induced apoptosis through the novel specific binding sites of 15d-PGJ2 different from DP2 and PPARγ. To relate the cytotoxicity of 15d-PGJ2 to amyloidoses, we performed binding assay [3H]15d-PGJ2 and specified targets for 15d-PGJ2 associated with cytotoxicity. In the various cell lines, there was a close correlation between the susceptibilities to 15d-PGJ2 and fibrillar Aβ. Specific binding sites of [3H]15d-PGJ2 were detected in rat cortical neurons and human bronchial smooth muscle cells. When the binding assay was performed in subcellular fractions of neurons, the specific binding sites of [3H]15d-PGJ2 were detected in plasma membrane, nuclear and cytosol, but not in microsome. A proteomic approach was used to identify protein targets for 15d-PGJ2 in the plasma membrane. By using biotinylated 15d-PGJ2, eleven proteins were identified as biotin-positive spots and classified into three different functional proteins: glycolytic enzymes (Enolase2, pyruvate kinase M1 (PKM1) and glyceraldehyde 3-phosphate dehydrogenase (GAPDH)), molecular chaperones (heat shock protein 8 and T-complex protein 1 subunit α), cytoskeletal proteins (Actin β, F-actin-capping protein, Tubulin β and Internexin α). GAPDH, PKM1 and Tubulin β are Aβ-interacting proteins. Thus, the present study suggested that 15d-PGJ2 plays an important role in amyloidoses not only in the central nervous system but also in the peripheral tissues

ALDH2 Polymorphisms and Clinical Medicine

２型アルデヒド脱水素酵素（aldehyde dehydrogenase 2：ALDH2）は，アルコール（エタノール）の代謝で生ずるアセトアルデヒドを酸化する酵素であり，人が酒に「強い」か「弱い」かは，この酵素の遺伝子多型に大きく依存することがよく知られている．しかし，ALDH2遺伝子多型は，単に飲酒の可否を決定するだけでなく，様々な疾患の発生や薬物の代謝と関連することが，近年明らかとなってきている．たとえば，低活性型の遺伝子型を有する人では，アルコール摂取後に血中や組織中に毒性を持つアセトアルデヒドが蓄積するため，高活性型の遺伝子型を有する人と比較して，消化管や肝臓などの癌化率が増加することが知られている．また2002 年以来，ALDH2がニトログリセリン（glyceryl trinitrate：GTN）を代謝（活性化）し，その薬効発現に関わることがわかってきた．今では，プロドラッグであるGTN をALDH2が還元代謝することで一酸化窒素（NO）が産生され，血管拡張反応が起きると考えられている．しかし，ALDH2遺伝子多型がGTNの血管拡張作用に及ぼす影響を調査する精度の高い研究は行われていなかった．そこで最近我々は，健康成人を対象とした臨床試験を実施し，GTN による血管拡張に要する時間はALDH2活性が低下するほど延長することを示し，ALDH2 がGTN 薬効発現に重要な役割を果たしていることを明らかにした．さらに，ALDH2が心血管保護作用を有する可能性が示唆されている．動物実験において，ALDH2は心筋虚血再還流後の心筋梗塞範囲を減少させることが示され，ALDH2が活性酸素種（ROS）や毒性アルデヒドを減少させることがその機序と考えられている．また，GTNはALDH2阻害作用を有することから，GTN の長期投与はALDH2を抑制することにより心血管障害を悪化させる可能性も考えられる．本稿では，ALDH2 遺伝子多型と臨床医学との関わりについて，最新の知見に基づき概説したい