Neurotoxic and cardiotoxic effects of N-methyl-1-(naphthalen-2-yl)propan-2-amine (methamnetamine) and 1-phenyl-2-pyrrolidinylpentane (prolintane).

Two synthetic phenylethylamines, N-methyl-1-(naphthalen-2-yl)propan-2-amine (MNA) and 1-phenyl-2-pyrrolidinylpentane (prolintane), are being abused by people seeking hallucinogens for pleasure. These new psychotropic substances may provoke problems because there is no existing information about their toxicity and pharmacological behaviors. Therefore, we evaluated the safety of nerves and cardiovascular systems by determining toxicity after MNA and prolintane drugs administrations to mice and rat. Consequently, side effects such as increased spontaneous motion and body temperature were observed in oral administration of MNA. In addition, both substances reduced motor coordination levels. The IHC tests were conducted to see whether the immune response also shows abnormalities in brain tissue compared to the control group. It has been confirmed that the length of allograft inflammatory factor 1(IBA-1), an immune antibody known as microglia marker, has been shortened. We identified that a problem with the contact between synapses and neurons might be possibly produced. In the assessment of the cardiac toxicity harmfulness, no substances have been confirmed to be toxic to myocardial cells, but at certain concentrations, they have caused the QT prolongation, an indicator of ventricular arrhythmia. In addition, the hERG potassium channel, the biomarker of the QT prolongation, has been checked for inhibition. The results revealed that the possibility of QT prolongation through the hERG channel could not be excluded, and the two substances can be considered toxic that may cause ventricular arrhythmia. In sum, this study demonstrated that the possibility of toxicity in MNA and prolintane compounds might bring many harmful effects on nerves and hearts.

Molecular Mechanisms for Ketone Body Metabolism, Signaling Functions, and Therapeutic Potential in Cancer.

The ketone bodies (KBs) ß-hydroxybutyrate and acetoacetate are important alternative energy sources for glucose during nutrient deprivation. KBs synthesized by hepatic ketogenesis are catabolized to acetyl-CoA through ketolysis in extrahepatic tissues, followed by the tricarboxylic acid cycle and electron transport chain for ATP production. Ketogenesis and ketolysis are regulated by the key rate-limiting enzymes, 3-hydroxy-3-methylglutaryl-CoA synthase 2 and succinyl-CoA:3-oxoacid-CoA transferase, respectively. KBs participate in various cellular processes as signaling molecules. KBs bind to G protein-coupled receptors. The most abundant KB, ß-hydroxybutyrate, regulates gene expression and other cellular functions by inducing post-translational modifications. KBs protect tissues by regulating inflammation and oxidative stress. Recently, interest in KBs has been increasing due to their potential for treatment of various diseases such as neurological and cardiovascular diseases and cancer. Cancer cells reprogram their metabolism to maintain rapid cell growth and proliferation. Dysregulation of KB metabolism also plays a role in tumorigenesis in various types of cancer. Targeting metabolic changes through dietary interventions, including fasting and ketogenic diets, has shown beneficial effects in cancer therapy. Here, we review current knowledge of the molecular mechanisms involved in the regulation of KB metabolism and cellular signaling functions, and the therapeutic potential of KBs and ketogenic diets in cancer.

2-((2-(4-Iodo-2,5-dimethoxyphenyl)ethylamino)methyl)phenol (25I-NBOH) and 2-(((2-(4-chloro-2,5-dimethoxyphenyl)ethyl)amino)methyl)phenol (25C-NBOH) induce adverse effects on the cardiovascular system.

Two new psychoactive substances (NPSs) classified as phenethylamines, namely 2-((2-(4-Iodo-2,5-dimethoxyphenyl)ethylamino)methyl)phenol (25I-NBOH) and 2-(((2-(4-chloro-2,5-dimethoxyphenyl)ethyl)amino)methyl)phenol (25C-NBOH), are being abused by people seeking recreational hallucinogens. These NPSs may cause serious health problems as their adverse effects are not known in most cases. Therefore, in the present study, we evaluated the cardiotoxicity of 25I-NBOH and 25C-NBOH using 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide (MTT) assay, rat electrocardiography (ECG), Langendorff test, and human ether-a-go-go-related gene (hERG) assay. Furthermore, we analyzed the expression levels of p21 CDC42/RAC1-activated kinase 1 (PAK1), which is known to play various roles in the cardiovascular system. In the MTT assay, treatment with 25I-NBOH or 25C-NBOH dramatically decreased viability of H9c2 cardiomyocytes. Meanwhile, these two compounds significantly increased QT intervals and RR intervals in the rat ECG measurement. 25I-NBOH down-regulated the PAK1 protein expression in rat primary cardiomyocytes as well as H9c2 cells. However, 25C-NBOH had no effect on the PAK1 expression in H9c2 cells. In an in-depth study, 25I-NBOH inhibited potassium channels in the hERG assay, but in ex vivo test, the substance did not affect the left ventricular developed pressure (LVDP) and heart rate of the isolated rat hearts. Taken together, these results suggest that both 25I-NBOH and 25C-NBOH may have adverse cardiovascular effect. Further investigation would be needed to determine which factors mainly influence the relationship between PAK1 expression and cardiotoxicity.

In vitro and in vivo estrogenic activity of triclosan.

Triclosan (TCS) is an antibacterial and antifungal agent used in many consumer products and exhibits a chemical structure similar to non-steroidal estrogen, which is known to induce endocrine disruption. Triclosan has been found in human plasma, urine, and breast milk, and the safety of TCS-containing products has been disputed. Although studies attempted to determine the estrogenic activity of TCS, no clear results have emerged. The aim of the present study was to examine estrogenic activity of TCS using an in vitro E-screen assay and an in vivo uterotrophic assay. The in vitro E-screen assay demonstrated that TCS significantly enhanced proliferation of MCF-7 breast cancer cells, although not in a concentration-dependent manner. The in vivo uterotrophic results showed no significant change in the weight of uteri obtained from TCS-administered Sprague-Dawley rats. Further, to understand the estrogenic activity attributed to TCS at the molecular level, gene-expression profiling of uterus samples was performed from both TCS- or estrogen-treated rats and the genes and cellular processes affected by TCS or estrogen were compared. Data demonstrated that both the genes and cellular processes affected by TCS or estrogen were significantly similar, indicating the possibility that TCS-mediated estrogenic activity occurred at the global transcriptome level. In conclusion, in vitro and gene-profiling results suggested that TCS exhibited estrogenic activity.

Honokiol inhibits hepatoma carcinoma cell migration through downregulated Cyclophilin B expression.

Hepatocellular carcinoma (HCC) is the fifth common types of cancer with poor prognosis in the world. Honokiol (HNK), a natural biphenyl compound derived from the magnolia plant, has been reported to exert anticancer effects, but its mechanism has not been elucidated exactly. In the present study, HNK treatment significantly suppressed the migration ability of HepG2 and Hep3B human hepatocellular carcinoma. The treatment reduced the expression levels of the genes associated with cell migration, such as S100A4, MMP-2, MMP-9 and Vimentin. Interestingly, treatment with HNK significantly reduced the expression level of Cyclophilin B (CypB) which stimulates cancer cell migration. However, overexpressed CypB abolished HNK-mediated suppression of cell migration, and reversed the apoptotic effects of HNK. Altogether, we concluded that the suppression of migration activities by HNK was through down-regulated CypB in HCC. These finding suggest that HNK may be a promising candidate for HCC treatment via regulation of CypB.

Non-Polar Myxococcus fulvus KYC4048 Metabolites Exert Anti-Proliferative Effects via Inhibition of Wnt/ß-Catenin Signaling in MCF-7 Breast Cancer Cells.

The Wnt/ß-catenin signaling pathway is involved in breast cancer and Myxococcus fulvus KYC4048 is a myxobacterial strain that can produce a variety of bioactive secondary metabolites. Although a previous study revealed that KYC4048 metabolites exhibit anti-proliferative effects on breast cancer, the biochemical mechanism involved in their effects remains unclear. In the present study, KYC4048 metabolites were separated into polar and non-polar (ethyl acetate and n-hexane) fractions via liquid-liquid extraction. The effects of these polar and non-polar KYC4048 metabolites on the viability of breast cancer cells were then determined by MTT assay. Expression levels of Wnt/ß-catenin pathway proteins were determined by Western blot analysis. Cell cycle and apoptosis were measured via fluorescence-activated cell sorting (FACS). The results revealed that non-polar KYC4048 metabolites induced cell death of breast cancer cells and decreased expression levels of WNT2B, ß-catenin, and Wnt target genes (c-Myc and cyclin D1). Moreover, the n-hexane fraction of non-polar KYC4048 metabolites was found most effective in inducing apoptosis, necrosis, and cell cycle arrest, leading us to conclude that it can induce apoptosis of breast cancer cells through the Wnt/ß-catenin pathway. These findings provide evidence that the n-hexane fraction of non-polar KYC4048 metabolites can be developed as a potential therapeutic agent for breast cancer via inhibition of the Wnt/ß-catenin pathway.

Effective Differentiation of Induced Pluripotent Stem Cells Into Dental Cells.

BACKGROUND: A biotooth is defined as a complete living tooth, made in laboratory cultures from a spontaneous interplay between epithelial and mesenchymal cell-based frontal systems. A good solution to these problems is to use induced pluripotent stem cells (iPSCs). However, no one has yet formulated culture conditions that effectively differentiate iPSCs into dental epithelial and dental mesenchymal cells phenotypes analogous to those present in tooth development. RESULTS: Here, we tried to induce differentiation methods for dental epithelial cells (DEC) and dental mesenchymal cells from iPSCs. For the DEC differentiation, the conditional media of SF2 DEC was adjusted to embryoid body. Moreover, we now report on a new cultivation protocol, supported by transwell membrane cell culture that make it possible to differentiate iPSCs into dental epithelial and mesenchymal cells with abilities to initiate the first stages in de novo tooth formation. CONCLUSIONS: Implementation of technical modifications to the protocol that maximize the number and rate of iPSC differentiation, into mesenchymal and epithelial cell layers, will be the next step toward growing an anatomically accurate biomimetic tooth organ. Developmental Dynamics 248:129-139, 2019. © 2018 Wiley Periodicals, Inc.

Correction to: Licochalcone A induces apoptosis through endoplasmic reticulum stress via a phospholipase Cγ1-, Ca2+-, and reactive oxygen species-dependent pathway in HepG2 human hepatocellular carcinoma cells.

The original version of this article contained mistakes in figures. The western blot data for pro-caspase-3 and cleaved caspase-3 (Fig. 1d), ß-actin (Fig. 1d), PLCγ1 (Fig. 5d), and eIF2α (Fig. 7d) are incorrect. The corrected Figs. 1d, 5d, and 7d are shown below. The corrections do not influence either the validity of the published data or the conclusion described in the article.

Correction to: Apicidin induces endoplasmic reticulum stress- and mitochondrial dysfunction-associated apoptosis via phospholipase Cγ1- and Ca2+-dependent pathway in mouse Neuro-2a neuroblastoma cells.

The original version of this article contained a mistake in the figure. The Ca2 + confocal image for the 2-APB/Apicidin-120 min in Fig. 5d is incorrect. The correction does not influence either the validity of the published data or the conclusion described in the article. The corrected Fig. 5d is given below.

Methoxetamine Induces Cytotoxicity in H9c2 Cells: Possible Role of p21 Protein (Cdc42/Rac)-Activated Kinase 1.

The abuse of new psychoactive substances (NPS) is an emerging social problem. Methoxetamine, one of the NPS, was designed as an alternative to ketamine and it was considered an NPS candidate owing to its high addictive potential. However, cardiotoxicity of the phencyclidine analogue, methoxetamine, has not been extensively evaluated. P21 protein (Cdc42/Rac)-activated kinase 1 (PAK-1) is associated with the drug-induced cardiotoxicity and hypertrophy of cardiomyocytes. In the present study, we investigated the effects of methoxetamine on rat cardiomyocytes and PAK-1. Methoxetamine (at 10 µM) reduced cell viability and PAK-1 mRNA levels in H9c2 cells. Methoxetamine treatment (100 µM) decreased the beating rate of primary cardiomyocytes. However, 100 µM methoxetamine-induced heart rate decline was less than 100 µM PCP- or ketamine-induced heart rate decline. Meanwhile, fingolimod hydrochloride (FTY720, 1 µM), a PAK-1 activator, increased cell viability and inhibited hypertrophy induced by methoxetamine in H9c2 cells. These results suggest that methoxetamine may have harmful effects on the cardiovascular system through the regulation of the expression and function of PAK-1.

Alpha-naphthoflavone induces apoptosis through endoplasmic reticulum stress via c-Src-, ROS-, MAPKs-, and arylhydrocarbon receptor-dependent pathways in HT22 hippocampal neuronal cells.

α-Naphthoflavone (αNF) is a prototype flavone, also known as a modulator of aryl hydrocarbon receptor (AhR). In the present study, we investigated the molecular mechanisms of αNF-induced cytotoxic effects in HT22 mouse hippocampal neuronal cells. αNF induced apoptotic cell death via activation of caspase-12 and -3 and increased expression of endoplasmic reticulum (ER) stress-associated proteins, including C/EBP homologous protein (CHOP). Inhibition of ER stress by treatment with the ER stress inhibitor, salubrinal, or by CHOP siRNA transfection reduced αNF-induced cell death. αNF activated mitogen-activated protein kinases (MAPKs), such as p38, JNK, and ERK, and inhibition of MAPKs reduced αNF-induced CHOP expression and cell death. αNF also induced accumulation of reactive oxygen species (ROS) and an antioxidant, N-acetylcysteine, reduced αNF-induced MAPK phosphorylation, CHOP expression, and cell death. Furthermore, αNF activated c-Src kinase, and inhibition of c-Src by a kinase inhibitor, SU6656, or siRNA transfection reduced αNF-induced ROS accumulation, MAPK activation, CHOP expression, and cell death. Inhibition of AhR by an AhR antagonist, CH223191, and siRNA transfection of AhR and AhR nuclear translocator reduced αNF-induced AhR-responsive luciferase activity, CHOP expression, and cell death. Finally, we found that inhibition of c-Src and MAPKs reduced αNF-induced transcriptional activity of AhR. Taken together, these findings suggest that αNF induces apoptosis through ER stress via c-Src-, ROS-, MAPKs-, and AhR-dependent pathways in HT22 cells.

Correction to: Release of overexpressed CypB activates ERK signaling through CD147 binding for hepatoma cell resistance to oxidative stress.

The original version of this article contained a mistake. The bands for HA Tag and t-ERK in Figs. 2d, 2h, 3d are incorrect. The author informs that these errors had no influence in the scientific content of the paper. The corrected figures (Figs. 2 and 3) are given below.

Effects of Myxococcus fulvus KYC4048 Metabolites on Breast Cancer Cell Death.

Using MCF7 breast cancer cells, we tested the anticancer activity of metabolites from 130 strains of myxobacteria newly isolated in South Korea. Of these, three strains whose metabolites had high anticancer activity and low cell toxicity were selected and identified by their fruiting body morphology, cell morphology, and 16S rRNA sequence. Strains KYC4030 and KYC4048 were determined to be Myxococcus fulvus, whereas strain KYC4081 was identified as Corallococcus coralloides. We found that metabolites of M. fulvus KYC4048 demonstrated no toxicity in normal cells but specifically induced cancer cell death by suppressing the expression of WNT2B. This discovery highlights the value of assessing the metabolic and biomedical potential of myxobacteria, even those that are already known but were isolated from new areas, and the possible use of metabolites from M. fulvus KYC4048 in cancer treatment.

Fluoxetine induces apoptosis through endoplasmic reticulum stress via mitogen-activated protein kinase activation and histone hyperacetylation in SK-N-BE(2)-M17 human neuroblastoma cells.

Fluoxetine (FLX) is an antidepressant drug that belongs to the class of selective serotonin reuptake inhibitors. FLX is known to induce apoptosis in multiple types of cancer cells. In this study, the molecular mechanisms underlying the anti-cancer effects of FLX were investigated in SK-N-BE(2)-M17 human neuroblastoma cells. FLX induced apoptotic cell death, activation of caspase-4, -9, and -3, and expression of endoplasmic reticulum (ER) stress-associated proteins, including C/EBP homologous protein (CHOP). Inhibition of ER stress by treatment with the ER stress inhibitors, salubrinal and 4-phenylbutyric acid or CHOP siRNA transfection reduced FLX-induced cell death. FLX induced phosphorylation of mitogen-activated protein kinases (MAPKs) family, p38, JNK, and ERK, and an upstream kinase apoptosis signal kinase 1 (ASK1). Inhibition of MAPKs and ASK1 reduced FLX-induced cell death and CHOP expression. We then showed that FLX reduced mitochondrial membrane potential (MMP) and ER stress inhibitors as well as MAPK inhibitors ameliorated FLX-induced loss of MMP. Interestingly, FLX induced hyperacetylation of histone H3 and H4, upregulation of p300 histone acetyltransferase (HAT), and downregulation of histone deacetylases (HDACs). Treatment with a HAT inhibitor anacardic acid or p300 HAT siRNA transfection blocked FLX-induced apoptosis in SK-N-BE(2)-M17 cells. However, FLX did not induce histone acetylation and anacardic acid had no protective effect on FLX-induced cell death and CHOP expression in MYCN non-amplified SH-SY5Y human neuroblastoma and MYCN knockdowned SK-N-BE(2)-M17 cells. These findings suggest that FLX induces apoptosis in neuroblastoma through ER stress and mitochondrial dysfunction via the ASK1 and MAPK pathways and through histone hyperacetylation in a MYCN-dependent manner.

Estrogenic activity of zinc pyrithione: an in vivo and in vitro study.

Zinc pyrithione (ZP) is commonly used to prevent dandruff and seborrheic dermatitis. Many consumers are exposed daily to high doses of ZP, causing serious concerns about its toxicity. The reproductive and developmental toxicities were previously reported in pregnant rats. However, the estrogenic activity of ZP at varying degrees of exposure has been rarely studied. Thus, we performed an uterotrophic assay, E-screen assay, and gene expression profiling to assess the estrogenic activity of ZP. For the uterotrophic assay, ZP (2, 10, or 50 mg/kg/d) was subcutaneously administered to ovariectomized rats every day for three days. Uteri were extracted 24 hours after the last dose. Then, wet and blotted uterine weights were measured. For the E-screen essay, MCF-7 cells (a breast cancer cell line) were exposed to 10-9 to 10-6 M of ZP, and cell proliferation was then measured. For the gene expression analysis, changes of gene expression levels in uterine samples taken for the uterotrophic assay were analyzed. In the uterotrophic assay, the concentration of ZP had no significant effect on uterine weight. In the E-screen assay, ZP at any concentration showed no significant increase in MCF-7 cell proliferation, compared to the control group. However, 10-6 M of ZP significantly reduced cell viability. The changes in gene expression slightly differed between the ZP and control groups. The in vivo and in vitro assays, together with gene expression analysis, demonstrated that ZP showed no significant estrogenic activity.