Role of plant homeodomain finger protein 8 in P19 embryonic carcinoma cells revealed by genome editing and specific inhibitor.

Plant homeodomain finger protein 8 (PHF8) is a histone demethylase that regulates the expression of various genes. PHF8 targets repressor histone markers and activates gene expression. Although PHF8 has been involved in X-linked mental retardation and certain types of cancers, the role of PHF8 remains largely unknown, and its relevance to the pathogenesis of these diseases is also uncertain. In the present study, we aimed to clarify the cellular function of PHF8 in P19 cells using Phf8 knockout (KO) cells generated via the CRISPR-Cas9 system and by performing PHF8 specific inhibitor experiments, instead of using PHF8 small interfering RNA transfection. After establishing Phf8 KO cells, we analyzed the effects of PHF8 on neuronal differentiation and cell proliferation. Both PHF8 deficiency and inhibition of its activity did not considerably affect neuronal differentiation, however, they showed an increased trend of promoted neurite outgrowth. Moreover, we found that PHF8 regulated cell proliferation via the MEK/ERK pathway. PHF8 deficiency and activity inhibition reduced the phosphorylation of ERK and MEK. The MEK expression level was associated with PHF8 expression, as revealed by chromatin immunoprecipitation analysis. These results suggested that PHF8 regulates cell proliferation via the MEK/ERK pathway in P19 embryonic carcinoma cells.

Clinical study designs and patient selection methods based on genomic biomarkers: Points-to-consider documents.

Recently, genomic biomarkers have been widely used clinically for prediction of the efficacy and safety of pharmacotherapy and diagnosis and prognosis of pathological conditions. Therefore, genomic biomarkers are anticipated to accelerate not only precision medicine for pharmacotherapy but also development of molecularly targeted drugs. Because the design of clinical studies involving biomarkers may differ from conventional clinical study designs, a concept paper focused on clinical studies and patient selection methods based on genomic biomarkers is desired to prompt innovative drug development. Thus, this concept paper aimed to compile and present current scientific information from the related guidelines regarding application of genomic biomarkers to clinical trials and studies for drug development. We hope that this concept paper will prompt the development of guidelines for biomarker application to drug development by industry, regulatory authorities, the medical profession, and academia.

Serum lipidomics for exploring biomarkers of bortezomib therapy in patients with multiple myeloma.

Although the proteasome inhibitor bortezomib (BTZ) shows excellent efficacy in multiple myeloma (MM), a fraction of patients has a suboptimal or no response to this agent. In addition, BTZ-induced peripheral neuropathy (BiPN), a frequent side-effect of this therapy, limits its use in some patients. This study aimed to explore serum lipid biomarker candidates to predict the response to BTZ and the severity of BiPN. Fifty-nine serum samples were collected from patients with MM prior to receiving BTZ plus low-dose dexamethasone therapy. Serum levels of phospholipids, sphingolipids, neutral lipids, and polyunsaturated fatty acids and their oxidation products were measured by a comprehensive lipidomic study. Overall, 385 lipid metabolites were identified in patients' sera; lower levels of several glycerophospholipids, sphingolipids, and cholesteryl esters were associated with a poor treatment response. Metabolites related to platelet-activating factor biosynthesis and cholesterol metabolism appeared particularly relevant. Furthermore, several lysophosphatidylcholines, phosphatidylcholines, ceramides, neutral lipids, and oxidative fatty acids were significantly increased or decreased in patients with BiPN grades ranging from G0 to G3. Among these compounds, mediators reportedly inducing myelin breakdown and stimulating inflammatory responses were prominent. Although further study is necessary to validate these biomarker candidates, our results contribute to the development of predictive biomarkers for response to BTZ treatment, or ensuing severe BiPN, in patients with MM.

An irreversible inhibitor of peptidyl-prolyl cis/trans isomerase Pin1 and evaluation of cytotoxicity.

Pin1 (protein interacting with never in mitosis A-1) is a member of the peptidyl prolyl isomerase (PPIase) family, and catalyzes cis-trans isomerization of pThr/Ser-Pro amide bonds. Because Pin1 is overexpressed in various cancer cell lines and promotes cell growth, it is considered a target for anticancer agents. Here, we designed and synthesized a covalently binding Pin1 inhibitor (S)-2 to target Pin1's active site. This compound inhibited Pin1 in protease-coupled assay, and formed a covalent bond with Cys113 of Pin1, as determined by ESI-MS. The acetoxymethyl ester of (S)-2, i.e., 6, suppressed cyclin D1 expression in human prostate cancer PC-3 cells, and exhibited cytotoxicity. Pin1-knockdown experiments indicated that a target for the cytotoxicity of 6 is Pin1.

HDAC8 regulates neural differentiation through embryoid body formation in P19 cells.

Histone acetylation and deacetylation correlate with diverse biological phenomena through gene transcription. Histone deacetylases (HDACs) regulate deacetylation of histones and other proteins. However, as a member of the HDAC family, HDAC8 function during neurodevelopment is currently unknown. Therefore, we investigated HDAC8 function during neurodevelopment by examining embryoid body (EB) formation in P19 cells. HDAC8-selective inhibitor (NCC-149) (HDAC8i)-treated cells showed smaller EBs than non-treated cells, as well as reduced expression levels of the neuronal marker, NeuN. Additionally, HDAC8i treatment led to inhibition of cellular proliferation by G2/M phase accumulation and downregulated cyclin A2 and cyclin B1 gene expression. Furthermore, two independent HDAC8 knockout cell lines were established by CRISPR-Cas9, which resulted in smaller EBs, similar to HDAC8i-treated cells. These results suggest that HDAC8 regulates neural differentiation by exerting control of EB formation.

Inhibition of amyloid fibril formation and cytotoxicity by caffeic acid-conjugated amyloid-ß C-terminal peptides.

Amyloid-ß (Aß) deposition and oxidative stress observed in the brains of patients with Alzheimer's disease (AD) are important targets for therapeutic intervention. In this study, we conjugated the antioxidants caffeic acid (CA) and dihydrocaffeic acid (DHCA) to Aß1-42 C-terminal motifs (Aßx-42: x=38, 40) to synthesize CA-Aßx-42 and DHCA-Aßx-42, respectively. Among the compounds, CA-Aß38-42 exhibited potent inhibitory activity against Aß1-42 aggregation and scavenged Aß1-42-induced intracellular oxidative stress. Moreover, CA-Aß38-42 significantly protected human neuroblastoma SH-SY5Y cells against Aß1-42-induced cytotoxicity, with an IC50 of 4µM. These results suggest that CA-Aß38-42 might be a potential lead for the treatment of AD.

Design, synthesis, and evaluation of Trolox-conjugated amyloid-ß C-terminal peptides for therapeutic intervention in an in vitro model of Alzheimer's disease.

Two hallmarks of Alzheimer's disease (AD) observed in the brains of patients with the disease include oxidative injury and deposition of protein aggregates comprised of amyloid-ß (Aß) variants. To inhibit these toxic processes, we synthesized antioxidant-conjugated peptides comprised of Trolox and various C-terminal motifs of Aß variants, TxAßx-n (x=34, 36, 38, 40; n=40, 42, 43). Most of these compounds were found to exhibit anti-aggregation activities. Among them, TxAß36-42 significantly inhibited Aß1-42 aggregation, showed potent antioxidant activity, and protected SH-SY5Y cells from Aß1-42-induced cytotoxicity. Thus, this method represents a promising strategy for developing multifunctional AD therapeutic agents.

(7-Diethylaminocoumarin-4-yl)methyl ester of suberoylanilide hydroxamic acid as a caged inhibitor for photocontrol of histone deacetylase activity.

Histone deacetylases (HDACs) are involved in epigenetic control of the expression of various genes by catalyzing deacetylation of ε-acetylated lysine residues. Here, we report the design, synthesis and evaluation of the (7-diethylaminocoumarin-4-yl)methyl ester of suberoylanilide hydroxamic acid (AC-SAHA) as a caged HDAC inhibitor, which releases the known pan-HDAC inhibitor SAHA upon cleavage of the photolabile (7-diethylaminocoumarin-4-yl)methyl protecting group in response to photoirradiation. A key advantage of AC-SAHA is that the caged derivative itself shows essentially no HDAC-inhibitory activity. Upon photoirradiation, AC-SAHA decomposes to SAHA and a 7-diethylaminocoumarin derivative, together with some minor products. We confirmed that AC-SAHA inhibits HDAC in response to photoirradiation in vitro by means of chemiluminescence assay. AC-SAHA also showed photoinduced inhibition of proliferation of human colon cancer cell line HCT116, as determined by MTT assay. Thus, AC-SAHA should be a useful tool for spatiotemporally controlled inhibition of HDAC activity, as well as a candidate chemotherapeutic reagent for human colon cancer.

Visible Light-Controlled Nitric Oxide Release from Hindered Nitrobenzene Derivatives for Specific Modulation of Mitochondrial Dynamics.

Nitric oxide (NO) is a physiological signaling molecule, whose biological production is precisely regulated at the subcellular level. Here, we describe the design, synthesis, and evaluation of novel mitochondria-targeted NO releasers, Rol-DNB-mor and Rol-DNB-pyr, that are photocontrollable not only in the UV wavelength range but also in the biologically favorable visible wavelength range (530-590 nm). These caged NO compounds consist of a hindered nitrobenzene as the NO-releasing moiety and a rhodamine chromophore. Their NO-release properties were characterized by an electron spin resonance (ESR) spin trapping method and fluorometric analysis using NO probes, and their mitochondrial localization in live cells was confirmed by costaining. Furthermore, we demonstrated visible light control of mitochondrial fragmentation via activation of dynamin-related protein 1 (Drp1) by means of precisely controlled NO delivery into mitochondria of cultured HEK293 cells, utilizing Rol-DNB-pyr.

Identification of SNAIL1 Peptide-Based Irreversible Lysine-Specific Demethylase 1-Selective Inactivators.

Inhibition of lysine-specific demethylase 1 (LSD1), a flavin-dependent histone demethylase, has recently emerged as a new strategy for treating cancer and other diseases. LSD1 interacts physically with SNAIL1, a member of the SNAIL/SCRATCH family of transcription factors. This study describes the discovery of SNAIL1 peptide-based inactivators of LSD1. We designed and prepared SNAIL1 peptides bearing a propargyl amine, hydrazine, or phenylcyclopropane moiety. Among them, peptide 3, bearing hydrazine, displayed the most potent LSD1-inhibitory activity in enzyme assays. Kinetic study and mass spectrometric analysis indicated that peptide 3 is a mechanism-based LSD1 inhibitor. Furthermore, peptides 37 and 38, which consist of cell-membrane-permeable oligoarginine conjugated with peptide 3, induced a dose-dependent increase of dimethylated Lys4 of histone H3 in HeLa cells, suggesting that they are likely to exhibit LSD1-inhibitory activity intracellularly. In addition, peptide 37 decreased the viability of HeLa cells. We believe this new approach for targeting LSD1 provides a basis for development of potent selective inhibitors and biological probes for LSD1.

Peptidyl prolyl isomerase Pin1-inhibitory activity of D-glutamic and D-aspartic acid derivatives bearing a cyclic aliphatic amine moiety.

Pin1 is a peptidyl prolyl isomerase that specifically catalyzes cis-trans isomerization of phosphorylated Thr/Ser-Pro peptide bonds in substrate proteins and peptides. Pin1 is involved in many important cellular processes, including cancer progression, so it is a potential target of cancer therapy. We designed and synthesized a novel series of Pin1 inhibitors based on a glutamic acid or aspartic acid scaffold bearing an aromatic moiety to provide a hydrophobic surface and a cyclic aliphatic amine moiety with affinity for the proline-binding site of Pin1. Glutamic acid derivatives bearing cycloalkylamino and phenylthiazole groups showed potent Pin1-inhibitory activity comparable with that of known inhibitor VER-1. The results indicate that steric interaction of the cyclic alkyl amine moiety with binding site residues plays a key role in enhancing Pin1-inhibitory activity.

A double bond-conjugated dimethylnitrobenzene-type photolabile nitric oxide donor with improved two-photon cross section.

Photocontrollable NO donors enable precise spatiotemporal release of NO under physiological conditions. We designed and synthesized a novel dimethylnitrobenzene-type NO donor, Flu-DNB-DB, which contains a carbon-carbon double bond in place of the amide bond of previously reported Flu-DNB. Flu-DNB-DB releases NO in response to one-photon activation in the blue wavelength region, and shows a greatly increased two-photon cross-section (δu) at 720 nm (Flu-DNB: 0.12 GM, Flu-DNB-DB: 0.98 GM). We show that Flu-DNB-DB enables precisely controlled intracellular release of NO in response to 950 nm pulse laser irradiation for as little as 1s. This near-infrared-light-controllable NO source should be a valuable tool for studies on the biological roles of NO.

Profiling and relative quantification of multiply nitrated and oxidized fatty acids.

The levels of nitro fatty acids (NO2-FA), such as nitroarachidonic, nitrolinoleic, nitrooleic, and dinitrooleic acids, are elevated under various inflammatory conditions, and this results in different anti-inflammatory effects. However, other multiply nitrated and nitro-oxidized FAs have not been studied so far. Owing to the low concentrations in vivo, NO2-FA analytics usually relies on targeted gas chromatography-tandem mass spectrometry (MS/MS) or liquid chromatography-MS/MS, and thus require standard compounds for method development. To overcome this limitation and increase the number and diversity of analytes, we performed in-depth mass spectrometry (MS) profiling of nitration products formed in vitro by incubating fatty acids with NO2BF4, and ONOO(-). The modified fatty acids were used to develop a highly specific and sensitive multiple reaction monitoring LC-MS method for relative quantification of 42 different nitrated and oxidized species representing three different groups: singly nitrated, multiply nitrated, and nitro-oxidized fatty acids. The method was validated in in vitro nitration kinetic studies and in a cellular model of nitrosative stress. NO2-FA were quantified in lipid extracts from 3-morpholinosydnonimine-treated rat primary cardiomyocytes after 15, 30, and 70 min from stress onset. The relatively high levels of dinitrooleic, nitroarachidonic, hydroxynitrodocosapenataenoic, nitrodocosahexaenoic, hydroxynitrodocosahexaenoic, and dinitrodocosahexaenoic acids confirm the presence of multiply nitrated and nitro-oxidized fatty acids in biological systems for the first time. Thus, in vitro nitration was successfully used to establish a targeted LC-MS/MS method that was applied to complex biological samples for quantifying diverse NO2-FA. Graphical Abstract Schematic representation of study design which combined in vitro nitration of different fatty acids, MS/MS characterization and optimization of MRM method for relative quantification, which was applied to follow dynamic of fatty acid nitration in cellular model of SIN-1 treated cardiomyoctes.

NCL1, a highly selective lysine-specific demethylase 1 inhibitor, suppresses prostate cancer without adverse effect.

Herein, we investigated therapeutic potential of a novel histone lysine demethylase 1 (LSD1) inhibitor, NCL1, in prostate cancer. Hormone-sensitive prostate cancer cells, (LNCaP) and castration resistant cancer cells (PC3 and PCai1) were treated with NCL1, and LSD1 expression and cell viability were assessed. Prostate cancer cells showed strong LSD1 expression, and cell viability was decreased by NCL1. ChIP analysis showed that NCL1 induced H3K9me2 accumulation at the promoters of androgen-responsive genes. NCL1 also induced G1 cell cycle arrest and apoptosis. In addition, autophagosomes and autolysosomes were induced by NCL1 treatment in LNCaP. Furthermore, LC3-II expression was significantly increased by NCL1 and chloroquine. In mice injected subcutaneously with PCai1 and intraperitoneally with NCL1, tumor volume was reduced with no adverse effects in NCL1-treated mice. Finally, LSD1 expression in human cancer specimens was significantly higher than that in normal prostate glands. In conclusion, NCL1 effectively suppressed prostate cancer growth without adverse events. We suggest that NCL1 is a potential therapeutic agent for hormone-resistant prostate cancer.

Development of photo-controllable hydrogen sulfide donor applicable in live cells.

Hydrogen sulfide (H2S) has multiple physiological roles, for example, in vasodilation and inflammation. It is a highly reactive gas under ambient conditions, so controllable H2S donors are required for studying its biological functions. Here, we describe the design, synthesis and application of a H2S donor (SPD-2) that utilizes xanthone photochemistry to control H2S release. H2S generation from SPD-2 was completely dependent on UVA-irradiation (325-385nm), as confirmed by methylene blue assay and by the use of a H2S-selective fluorescent probe. SPD-2 was confirmed to provide controlled H2S delivery in live cells, and should be suitable for various biological applications.

Visible light-induced nitric oxide release from a novel nitrobenzene derivative cross-conjugated with a coumarin fluorophore.

Nitric oxide (NO) is a well-known free-radical molecule which is endogenously biosynthesised and shows various functions in mammals. To investigate NO functions, photocontrollable NO donors, compounds which release NO in response to light, are expected to be potentially useful. However, most of the conventional NO donors require harmful ultra-violet light for NO release. In this study, two dimethylnitrobenzene derivatives conjugated with coumarins were designed, synthesized and evaluated as photocontrollable NO donors. The optical properties and efficiency of photo-induced NO release were dependent upon the nature of the conjugation system. One of these compounds, Bhc-DNB (1), showed spatiotemporally well-controlled NO release in cultured cells upon exposure to light in the less-cytotoxic visible wavelength range (400-430 nm).

Histone deacetylase inhibitor valproic acid promotes the differentiation of human induced pluripotent stem cells into hepatocyte-like cells.

In this study, we aimed to elucidate the effects and mechanism of action of valproic acid on hepatic differentiation from human induced pluripotent stem cell-derived hepatic progenitor cells. Human induced pluripotent stem cells were differentiated into endodermal cells in the presence of activin A and then into hepatic progenitor cells using dimethyl sulfoxide. Hepatic progenitor cells were matured in the presence of hepatocyte growth factor, oncostatin M, and dexamethasone with valproic acid that was added during the maturation process. After 25 days of differentiation, cells expressed hepatic marker genes and drug-metabolizing enzymes and exhibited drug-metabolizing enzyme activities. These expression levels and activities were increased by treatment with valproic acid, the timing and duration of which were important parameters to promote differentiation from human induced pluripotent stem cell-derived hepatic progenitor cells into hepatocytes. Valproic acid inhibited histone deacetylase activity during differentiation of human induced pluripotent stem cells, and other histone deacetylase inhibitors also enhanced differentiation into hepatocytes. In conclusion, histone deacetylase inhibitors such as valproic acid can be used to promote hepatic differentiation from human induced pluripotent stem cell-derived hepatic progenitor cells.

Regulation of tissue factor pathway inhibitor-2 (TFPI-2) expression by lysine-specific demethylase 1 and 2 (LSD1 and LSD2).

Tissue factor pathway inhibitor-2 (TFPI-2) is a major inhibitor of extracellular matrix degradation. Decreases in TFPI-2 contribute to malignant tumor cell production, and TFPI-2 is a presumed tumor suppressor. TFPI-2 gene transcription is regulated by two epigenetic mechanisms: DNA methylation of the promoter and K4 methylation of histone 3 (H3). Lysine-specific demethylase 1 (LSD1) and LSD2 demethylate H3K4me2/1. LSD1 has been implicated in TFPI-2 regulation through both epigenetic mechanisms, but the involvement of LSD2 remains unknown. We prepared a monoclonal anti-LSD2 antibody that clearly distinguishes LSD2 from LSD1. Knockdown of LSD1 or LSD2 by siRNAs increased TFPI-2 protein and mRNA. Simultaneous knockdown of both LSD1 and LSD2 showed additive effects. Bisulfite sequencing revealed that CpG sites in the TFPI-2 promoter region were unmethylated. These results indicate that LSD2 also contributes to TFPI-2 regulation through histone modification, and that further studies of the involvement of LSD2 in tumor malignancy are warranted.

Synthesis and radical-scavenging activity of a dimethyl catechin analogue.

Catechin analogue 1 with methyl substituents ortho to the catechol hydroxyl groups was synthesized to improve the antioxidant ability of (+)-catechin. The synthetic scheme involved a solid acid catalyzed Friedel-Crafts coupling of a cinnamyl alcohol derivative to 3,5-dibenzyloxyphenol followed by hydroxylation and then cyclization through an intermediate orthoester. The antioxidative radical scavenging activity of 1 against galvinoxyl radical, an oxyl radical, was found to be 28-fold more potent than (+)-catechin.

Photomanipulation of vasodilation with a blue-light-controllable nitric oxide releaser.

Spatiotemporally controllable nitric oxide (NO)-releasers allow us to analyze the physiological effects of NO, a gaseous mediator that modulates many biological signaling networks, and are also candidate chemotherapeutic agents. We designed and synthesized a blue-light-controllable NO releaser, named NOBL-1, which bears an N-nitrosoaminophenol moiety for NO release tethered to a BODIPY dye moiety for harvesting blue light. Photoinduced electron transfer from N-nitrosoaniline to the antenna moiety upon irradiation with relatively noncytotoxic blue light (470-500 nm) should result in NO release with formation of a stable quinone moiety. NO release from NOBL-1 was confirmed by ESR spin trapping and fluorescence detection. Spatially controlled NO release in cells was observed with DAR-4M AM, a fluorogenic NO probe. We also demonstrated temporally controlled vasodilation of rat aorta ex vivo by blue-light-induced NO release from NOBL-1. This compound should be useful for precise examination of the functions of NO with excellent spatiotemporal control.