12 research outputs found
Pharmacologic characterization of TBP1901, a prodrug form of aglycone curcumin, and CRISPR-Cas9 screen for therapeutic targets of aglycone curcumin
プロドラッグ型クルクミン注射製剤の抗腫瘍効果及び治療標的の包括的な解析 --安全性の高い抗がん薬としての開発に期待--. 京都大学プレスリリース. 2022-10-21.Curcumin (aglycone curcumin) has antitumor properties in a variety of malignancies via the alteration of multiple cancer-related biological pathways; however, its clinical application has been hampered due to its poor bioavailability. To overcome this limitation, we have developed a synthesized curcumin β-D-glucuronide sodium salt (TBP1901), a prodrug form of aglycone curcumin. In this study, we aimed to clarify the pharmacologic characteristics of TBP1901. In β-glucuronidase (GUSB)-proficient mice, both curcumin β-D-glucuronide and its active metabolite, aglycone curcumin, were detected in the blood after TBP1901 injection, whereas only curcumin β-D-glucuronide was detected in GUSB-impaired mice, suggesting that GUSB plays a pivotal role in the conversion of TBP1901 into aglycone curcumin in vivo. TBP1901 itself had minimal antitumor effects in vitro, whereas it demonstrated significant antitumor effects in vivo. Genome-wide clustered regularly interspaced short palindromic repeats (CRISPR)-Cas9 screen disclosed the genes associated with NF-κB signaling pathway and mitochondria were among the highest hit. In vitro, aglycone curcumin inhibited NF-kappa B signaling pathways whereas it caused production of reactive oxygen species (ROS). ROS scavenger, N-acetyl-L-cysteine, partially reversed antitumor effects of aglycone curcumin. In summary, TBP1901 can exert antitumor effects as a prodrug of aglycone curcumin through GUSB-dependent activation
22q11欠失症候群モデルマウスの神経発達障害には、マイクロRNAが介在するCxcr4/Cxcl12シグナリングの欠損が寄与する
22q11 deletion syndrome (22q11DS) frequently accompanies psychiatric conditions, some of which are classified as schizophrenia and bipolar disorder in the current diagnostic categorization. However, it remains elusive how the chromosomal microdeletion leads to the mental manifestation at the mechanistic level. Here we show that a 22q11DS mouse model with a deletion of 18 orthologous genes of human 22q11 (Df1/+ mice) has deficits in migration of cortical interneurons and hippocampal dentate precursor cells. Furthermore, Df1/+ mice show functional defects in Chemokine receptor 4/Chemokine ligand 12 (Cxcr4/Cxcl12; Sdf1) signaling, which reportedly underlie interneuron migration. Notably, the defects in interneuron progenitors are rescued by ectopic expression of Dgcr8, one of the genes in 22q11 microdeletion. Furthermore, heterozygous knockout mice for Dgcr8 show similar neurodevelopmental abnormalities as Df1/+ mice. Thus, Dgcr8-mediated regulation of microRNA is likely to underlie Cxcr4/Cxcl12 signaling and associated neurodevelopmental defects. Finally, we observe that expression of CXCL12 is decreased in olfactory neurons from sporadic cases with schizophrenia compared with normal controls. Given the increased risk of 22q11DS in schizophrenia that frequently shows interneuron abnormalities, the overall study suggests that CXCR4/CXCL12 signaling may represent a common downstream mediator in the pathophysiology of schizophrenia and related mental conditions.博士(医学)・乙1331号・平成26年3月17
Identification of a novel protein kinase mediating Akt survival signaling to the ATM protein
We identified a novel human AMP-activated protein kinase (AMPK) family member, designated ARK5, encoding 661 amino acids with an estimated molecular mass of 74 kDa. The putative amino acid sequence reveals 47, 45.8, 42.4, and 55% homology to AMPK-alpha1, AMPK-alpha2, MELK and SNARE respectively, suggesting that it is a new member of the AMPK family. It has a putative Akt phosphorylation motif at amino acids 595600, and Ser(600) was found to be phosphorylated by active Akt resulting in the activation of kinase activity toward the SAMS peptide, a consensus AMPK substrate. During nutrient starvation, ARK5 supported the survival of cells in an Akt-dependent manner. In addition, we also demonstrated that ARK5, when activated by Akt, phosphorylated the ATM protein that is mutated in the human genetic disorder ataxia-telangiectasia and also induced the phosphorylation of p53. On the basis of our current findings, we propose that a novel AMPK family member, ARK5, is the tumor cell survival factor activated by Akt and acts as an ATM kinase under the conditions of nutrient starvation
カイコ主要体液タンパク質遺伝子の発現調節機構に関する研究
Holometabolous insects drastically change their body structures during post-embryonic development and metamorphosis. Their developmental processes are mainly controlled by two insect hormones, juvenile hormone (JH) and ecdysteroid. By contrast with the study for the mode of ecdysteroid action, little is known about the mechanism how JH exerts its function. For the purpose of investigating the JH functions, I focused on the regulatory mechanisms of expression of the major plasma protein gene in the silkworm, Bombyx mori. Plasma proteins termed "SP1" and "30K proteins" are synthesized by the fat body cells of the silkworm, B. mori , in a sex- and stage-specific manner during the larval development. I have successfully established a primary culture of the fat body cells to investigate the regulatory mechanisms of plasma protein gene expression.The primary cultures of fat body cells contained at least two cell types: small, oval cells, and large, spherical cells. The cells adhered and migrated on the culture dish. By the seventh day of cultivation, the cells have clustered to form the fat body-like structures that were maintained for at least three months. Plasma proteins were actively synthesized in the primary cultured cells isolated from the fat body of the final instar larvae only when the cells tightly adhered to the culture dish. Immunocytochemical analysis revealed that only 10-15% of the clustered cells synthesized plasma proteins in our culture system. These results indicate that the primary culture comprises heterogeneous cells which are morphologically and functionally distinct. The patterns of SP1 syntheses in primary cultures faithfully reproduced its sex-dependency in vivo. Subsequently, I established a method for introduction of DNAs into the fat body cells to analyze the regulatory element of 30K protein gene. Chimeric genes containing the 5\u27 sequences (-1668 to +14) of 30K protein 6G1 gene fused to the firefly luciferase gene were introduced into fat body cells using a method of electroporation. Optimum introduction of DNA was obtained at 90 V, 1075 μF, 1 pmol of DNA and 4 x 10^6 cells in 0.5 ml of serum-free Grace\u27s insect medium as an electroporation buffer using a 0.4 cm cuvette. When the constructs were transfected into fat body cells, the luciferase gene was accurately transcribed under the control of the 30K protein 6G1 gene promoter. By contrast, the fusion gene consisting of the 5\u27 upstream region (-1480 to +16) of B. mori LCP30 gene and the luciferase gene did not express in the fat body cells . These results show that the promoter constructs introduced into the fat body cells exhibit their promoter-specific expression. The analysis of 5\u27 upstream sequence of 30K protein 6G1 gene revealed that the enhancer elements are located in the sequence between nucleotide (nt) position -176 and -48 of the 30K protein gene. It was considered that the synthesis of 30K protein might be negatively regulated by JH in vivo. In this thesis, I clearly showed that JH suppressed the 30K protein synthesis by use of the primary culture system of fat body cells . Next, I analyzed the JH-responsive element of 30K protein 6G1 genes using the electroporation method. Analysis using a series of deletion mutants revealed that the JH-responsive elements of 30K protein 6G1 gene are located downstream of nt position -176 with its enhancer elements.東京都立大学, 2000-03-25, 博士(理学), 甲第568号東京都立大
Curcumin β‐D‐glucuronide exhibits anti‐tumor effects on oxaliplatin‐resistant colon cancer with less toxicity in vivo
水溶性プロドラッグ型抗がん剤CMGの治療抵抗性大腸がんに対する抗腫瘍効果を解明 --難治性がん治療薬の開発に期待--. 京都大学プレスリリース. 2020-03-16.The NF‐kappa B (NF‐κB) pathway plays a pivotal role in tumor progression and chemoresistance, and its inhibition has been shown to suppress tumor growth in a variety of preclinical models. Recently, we succeeded in synthesizing a water‐soluble injectable type of curcumin β‐D‐glucuronide (CMG), which is converted into a free‐form of curcumin by β‐glucuronidase in vivo. Herein, we aimed to clarify the efficacy, safety and pharmacokinetics of CMG in a xenograft mouse model. First, we confirmed that the presence of KRAS/TP53 mutations significantly increased the IC50 of oxaliplatin (L‐OHP) and NF‐κB activity in HCT116 cells in vitro. Then, we tested the efficacy of CMG in an HCT116 colon cancer xenograft mice model. CMG demonstrated superior anticancer effects compared to L‐OHP in an L‐OHP‐resistant xenograft model. With regard to safety, significant bodyweight loss, severe myelosuppression and AST/ALT elevation were observed in L‐OHP‐treated mice, whereas none of these toxicity was noted in CMG‐treated mice. The combination of CMG and L‐OHP exhibited additive effects in these xenograft models without increasing toxicity. Pharmacokinetic analysis revealed that high levels of free‐form curcumin were maintained in the tumor tissue after 48 hours following CMG administration, but it was not detected in other major organs, such as the heart, liver and spleen. Immunohistochemistry revealed reduced NF‐κB activity in the tumor tissue extracted from CMG‐treated mice compared with that from control mice. These results indicated that CMG could be a promising anticancer prodrug for treating colon cancer with minimal toxicity