Andrographolide suppresses the malignancy of pancreatic cancer via alleviating DNMT3B-dependent repression of tumor suppressor gene ZNF382.

BACKGROUND: Pancreatic ductal adenocarcinoma (PDAC) is a highly aggressive cancer type that urgently requires effective therapeutic strategies. Andrographolide, a labdane diterpenoid compound abundant in Andrographis paniculata, has anticancer effects against various cancer types, but its anticancer activity and mechanism against PDAC remain largely uncharacterized. PURPOSE: This study explores novel drug target(s) and underlying molecular mechanism of andrographolide against PDAC. STUDY DESIGN AND METHODS: The malignant phenotypes of PDAC cells, PANC-1 and MIA PaCa-2 cells, were measured using MTT, clonogenic assays, and Transwell migration assays. A PDAC xenograft animal model was used to evaluate tumor growth in vivo. Western blot, immunofluorescence and immunohistochemistry were used for measuring protein expression. The TCGA database was analyzed to evaluate promoter methylation status, gene expression, and their relationship with patient survival rates. RT-qPCR was used for detecting mRNA expression. Reporter assays were used for detecting signal transduction pathways. Promoter DNA methylation was determined by sodium bisulfite treatment and methylation-specific PCR (MSP). The biological function and role of specific genes involved in drug effects were measured through gene overexpression. RESULTS: Andrographolide treatment suppressed the proliferation and migration of PDAC cells and impaired tumor growth in vivo. Furthermore, andrographolide induced the mRNA and protein expression of zinc finger protein 382 (ZNF382) in PDAC cells. Overexpression of ZNF382 inhibited malignant phenotypes and cancer-associated signaling pathways (AP-1, NF-κB and ß-catenin) and oncogenes (ZEB-1, STAT-3, STAT-5, and HIF-1α). Overexpression of ZNF382 delayed growth of PANC-1 cells in vivo. ZNF382 mRNA and protein expression was lower in tumor tissues than in adjacent normal tissues of pancreatic cancer patients. Analysis of the TCGA database found the ZNF382 promoter is hypermethylated in primary pancreatic tumors which correlates with its low expression. Furthermore, andrographolide inhibited the expression of DNA methyltransferase 3 beta (DNMT3B) and increased the demethylation of the ZNF382 promoter in PDAC cells. Overexpression of DNMT3B attenuated the andrographolide-suppressed proliferation and migration of PDAC cells. CONCLUSION: Our finding revealed that ZNF382 acts as a tumor suppressor gene in pancreatic cancer and andrographolide restores ZNF382 expression to suppress pancreatic cancer, providing a novel molecular target and a promising therapeutic approach for treating pancreatic cancer.

Characterization of Hair Metabolome in 5xFAD Mice and Patients with Alzheimer's Disease Using Mass Spectrometry-Based Metabolomics.

Hair emerged as a biospecimen for long-term investigation of endogenous metabolic perturbations, reflecting the chemical composition circulating in the blood over the past months. Despite its potential, the use of human hair for metabolomics in Alzheimer's disease (AD) research remains limited. Here, we performed both untargeted and targeted metabolomic approaches to profile the key metabolic pathways in the hair of 5xFAD mice, a widely used AD mouse model. Furthermore, we applied the discovered metabolites to human subjects. Hair samples were collected from 6-month-old 5xFAD mice, a stage marked by widespread accumulation of amyloid plaques in the brain, followed by sample preparation and high-resolution mass spectrometry analysis. Forty-five discriminatory metabolites were discovered in the hair of 6-month-old 5xFAD mice compared to wild-type control mice. Enrichment analysis revealed three key metabolic pathways: arachidonic acid metabolism, sphingolipid metabolism, and alanine, aspartate, and glutamate metabolism. Among these pathways, six metabolites demonstrated significant differences in the hair of 2-month-old 5xFAD mice, a stage prior to the onset of amyloid plaque deposition. These findings suggest their potential involvement in the early stages of AD pathogenesis. When evaluating 45 discriminatory metabolites for distinguishing patients with AD from nondemented controls, a combination of l-valine and arachidonic acid significantly differentiated these two groups, achieving a 0.88 area under the curve. Taken together, these findings highlight the potential of hair metabolomics in identifying disease-specific metabolic alterations and developing biomarkers for improving disease detection and monitoring.

EXOSC5 maintains cancer stem cell activity in endometrial cancer by regulating the NTN4/integrin ß1 signalling axis.

Endometrial carcinoma (EC) is a common type of uterine cancer in developed countries, originating from the uterine epithelium. The incidence rate of EC in Taiwan has doubled from 2005. Cancer stem cells (CSCs) are a subpopulation of cancer cells that have high tumorigenicity and play a crucial role in the malignant processes of cancer. Targeting molecules associated with CSCs is essential for effective cancer treatments. This study delves into the role of Exosome component 5 (EXOSC5) in EC. Data from The Cancer Genome Atlas suggests a correlation between high EXOSC5 mRNA expression and unfavorable EC prognosis. EXOSC5 knockdown diminished EC-CSC self-renewal and reduced expression of key cancer stemness proteins, including c-MYC and SOX2. Intriguingly, this knockdown significantly curtailed tumorigenicity and CSC frequency in EC tumor spheres. A mechanistic examination revealed a reduction in netrin4 (NTN4) levels in EXOSC5-depleted EC cells. Moreover, NTN4 treatment amplified EC cell CSC activity and, when secreted, NTN4 partnered with integrin ß1, subsequently triggering the FAK/SRC axis to elevate c-MYC activity. A clear positive relation between EXOSC5 and NTN4 was evident in 93 EC tissues. In conclusion, EXOSC5 augments NTN4 expression, activating c-MYC via the integrin ß1/FAK/SRC pathway, offering potential avenues for EC diagnosis and treatment.

Disulfiram/Copper Suppresses Cancer Stem Cell Activity in Differentiated Thyroid Cancer Cells by Inhibiting BMI1 Expression.

Differentiated thyroid carcinomas (DTCs), which have papillary and follicular types, are common endocrine malignancies worldwide. Cancer stem cells (CSCs) are a particular type of cancer cells within bulk tumors involved in cancer initiation, drug resistance, and metastasis. Cells with high intracellular aldehyde hydrogenase (ALDH) activity are a population of CSCs in DTCs. Disulfiram (DSF), an ALDH inhibitor used for the treatment of alcoholism, reportedly targets CSCs in various cancers when combined with copper. This study reported for the first time that DSF/copper can inhibit the proliferation of papillary and follicular DTC lines. DSF/copper suppressed thyrosphere formation, indicating the inhibition of CSC activity. Molecular mechanisms of DSF/copper involved downregulating the expression of B lymphoma Mo-MLV insertion region 1 homolog (BMI1) and cell cycle-related proteins, including cyclin B2, cyclin-dependent kinase (CDK) 2, and CDK4, in a dose-dependent manner. BMI1 overexpression diminished the inhibitory effect of DSF/copper in the thyrosphere formation of DTC cells. BMI1 knockdown by RNA interference in DTC cells also suppressed the self-renewal capability. DSF/copper could inhibit the nuclear localization and transcriptional activity of c-Myc and the binding of E2F1 to the BMI1 promoter. Overexpression of c-Myc or E2F1 further abolished the inhibitory effect of DSF/copper on BMI1 expression, suggesting that the suppression of c-Myc and E2F1 by DSF/copper was involved in the downregulation of BMI1 expression. In conclusion, DSF/copper targets CSCs in DTCs by inhibiting c-Myc- or E2F1-mediated BMI1 expression. Therefore, DSF is a potential therapeutic agent for future therapy in DTCs.

Ractopamine at legal residue dosage accelerates atherosclerosis by inducing endothelial dysfunction and promoting macrophage foam cell formation.

Ractopamine, a synthetic ß-adrenoreceptor agonist, is used as an animal feed additive to increase food conversion efficiency and accelerate lean mass accretion in farmed animals. The U.S. Food and Drug Administration claimed that ingesting products containing ractopamine residues at legal dosages might not cause short-term harm to human health. However, the effect of ractopamine on chronic inflammatory diseases and atherosclerosis is unclear. Therefore, we investigated the effects of ractopamine on atherosclerosis and its action mechanism in apolipoprotein E-null (apoe-/-) mice and human endothelial cells (ECs) and macrophages. Daily treatment with ractopamine for four weeks increased the body weight and the weight of brown adipose tissues and gastrocnemius muscles. However, it decreased the weight of white adipose tissues in apoe-/- mice. Additionally, ractopamine exacerbated hyperlipidemia and systemic inflammation, deregulated aortic cholesterol metabolism and inflammation, and accelerated atherosclerosis. In ECs, ractopamine treatment induced endothelial dysfunction and increased monocyte adhesion and transmigration across ECs. In macrophages, ractopamine dysregulated cholesterol metabolism by increasing oxidized low-density lipoprotein (oxLDL) internalization and decreasing reverse cholesterol transporters, increasing oxLDL-induced lipid accumulation. Collectively, our findings revealed that ractopamine induces EC dysfunction and deregulated cholesterol metabolism of macrophages, which ultimately accelerates atherosclerosis progression.

Heat-Killed Lactobacilli Preparations Promote Healing in the Experimental Cutaneous Wounds.

Probiotics are defined as microorganisms with beneficial health effects when consumed by humans, being applied mainly to improve allergic or intestinal diseases. Due to the increasing resistance of pathogens to antibiotics, the abuse of antibiotics becomes inefficient in the skin and in systemic infections, and probiotics may also provide the protective effect for repairing the healing of infected cutaneous wounds. Here we selected two Lactobacillus strains, L. plantarum GMNL-6 and L. paracasei GMNL-653, in heat-killed format to examine the beneficial effect in skin wound repair through the selection by promoting collagen synthesis in Hs68 fibroblast cells. The coverage of gels containing heat-killed GMNL-6 or GMNL-653 on the mouse tail with experimental wounds displayed healing promoting effects with promoting of metalloproteinase-1 expression at the early phase and reduced excessive fibrosis accumulation and deposition in the later tail-skin recovery stage. More importantly, lipoteichoic acid, the major component of Lactobacillus cell wall, from GMNL-6/GMNL-653 could achieve the anti-fibrogenic benefit similar to the heat-killed bacteria cells in the TGF-ß stimulated Hs68 fibroblast cell model. Our study offers a new therapeutic potential of the heat-killed format of Lactobacillus as an alternative approach to treating skin healing disorders.

Tribbles Pseudokinase 3 Contributes to Cancer Stemness of Endometrial Cancer Cells by Regulating ß-Catenin Expression.

Endometrial cancer (EC) is the second most common gynecological malignancy worldwide. Tribbles pseudokinase 3 (TRIB3) is a scaffolding protein that regulates intracellular signal transduction, and its role in tumor development is controversial. Here, we investigated the biological function of TRIB3 in EC. We found that the messenger RNA (mRNA) expression level of TRIB3 was significantly and positively correlated with shorter overall survival of EC patients in The Cancer Genome Atlas database. The protein expression of TRIB3 was found to be significantly increased in EC cancer stem cells (CSCs) enriched by tumorsphere cultivation. Knockdown of TRIB3 in EC cells suppressed tumorsphere formation, the expression of cancer stemness genes, and the in vivo tumorigenesis. The expression of ß-catenin at both the protein and the mRNA levels was downregulated upon TRIB3 silencing. TRIB3 was found to interact with E74 Like ETS transcription factor 4 (ELF4) in the nucleus and bound to ELF4 consensus sites within the catenin beta 1 (CTNNB1) promoter in EC cell lines. These data indicated that TRIB3 may regulate CTNNB1 transcription by enhancing the recruitment of ELF4 to the CTNNB1 promoter. In conclusion, our results suggest that TRIB3 plays an oncogenic role in EC and positively regulates the self-renewal and tumorigenicity of EC-CSCs. Targeting TRIB3 is considered as a potential therapeutic strategy in future EC therapy.

Early Blood Biomarkers Distinguish Inflammation from Neonatal Hypoxic-Ischemia Encephalopathy.

Neonatal hypoxic-ischemic encephalopathy is the most common cause of neurological disability in infancy. Superimposed inflammation may further worsen neurological outcomes. Reliable biomarkers which are both sensitive to hypoxic-ischemia and inflammation are critically needed. We tested plasma osteopontin (OPN) and glial fibrillary astrocytic protein (GFAP) within the reported therapeutic window (90 min after hypoxic-ischemic (HI) injury) in neonatal rats with different HI severity and inflammation. Two different HI severity groups (mild-HI with 75 min hypoxia and severe-HI with 150 min hypoxia) were established. Inflammation-sensitized HI brain injury induced by lipopolysaccharide (LPS) further increased apoptotic neurons and infarct volumes. In HI alone groups, OPN was significantly decreased (p < 0.001) but GFAP was slightly increased (p < 0.05) at 90 min after HI either in mild-HI or severe-HI compared with naïve group. In LPS-sensitized HI groups, both OPN and GFAP were significantly increased either in LPS-mild-HI or LPS-severe-HI groups compared with the naïve group (all p < 0.05). Induced inflammation by LPS exaggerated neonatal HI brain injury. The plasma OPN and GFAP levels may be useful to differentiate HI alone groups from inflammation-sensitized HI groups or naïve group.

NAD(P)H:quinone oxidoreductase 1 determines radiosensitivity of triple negative breast cancer cells and is controlled by long non-coding RNA NEAT1.

Radioresistant cells cause recurrence in patients with breast cancer after they undergo radiation therapy. The molecular mechanisms by which cancer cells obtain radioresistance should be understood to develop radiation-sensitizing agents. Results showed that the protein expression and activity of NAD(P)H:quinone oxidoreductase 1 (NQO1) were upregulated in radioresistant MDA-MB-231 triple-negative breast cancer (TNBC) cells. NQO1 knockdown inhibited the proliferation of NQO1 expressing Hs578t TNBC cells or the radioresistant MDA-MB-231 cells, whereas NOQ1 overexpression increased the survival of MDA-MB-231 cells, which lack of NQO1 expression originally, under irradiation. The cytotoxicity of ß-lapachone, an NQO1-dependent bioactivatable compound, was greater in radioresistant MDA-MB-231 cells than in parental cells. ß-lapachone displayed a radiosensitization effect on Hs578t or radioresistant MBDA-MB-231 cells. The expression of the long noncoding RNA NEAT1 positively regulated the NQO1 expression in radioresistant MDA-MB-231 cells at a translational level rather than at a transcription level. The inhibition of the NEAT1 expression through the CRISPR-Cas9 method increased the sensitivity of radioresistant MDA-MB-231 cells to radiation and decreased their proliferation, the activity of cancer stem cells, and the expression of stemness genes, including BMI1, Oct4, and Sox2. In conclusion, the NQO1 expression in triple-negative breast cancer cells determined their radiosensitivity and was controlled by NEAT1. In addition, NOQ1 bioactivatable compounds displayed potential for application in the development of radiation sensitizers in breast cancer.

Oxygenated Water Inhibits Adipogenesis and Attenuates Hepatic Steatosis in High-Fat Diet-Induced Obese Mice.

The expansion of adipose tissue mass is the primary characteristic of the process of becoming obesity, which causes chronic adipose inflammation and is closely associated with type 2 diabetes mellitus (T2DM). Adipocyte hypertrophy restricts oxygen availability, leading to microenvironmental hypoxia and adipose dysfunction. This study aimed at investigating the effects of oxygenated water (OW) on adipocyte differentiation (adipogenesis) and the metabolic function of mature adipocytes. The effects of OW on adipogenesis and the metabolic function of mature adipocytes were examined. Meanwhile, the in vivo metabolic effects of long-term OW consumption on diet-induced obesity (DIO) mice were investigated. OW inhibited adipogenesis and lipid accumulation through down-regulating critical adipogenic transcription factors and lipogenic enzymes. While body weight, blood and adipose parameters were not significantly improved by long-term OW consumption, transient circulatory triglyceride-lowering and glucose tolerance-improving effects were identified. Notably, hepatic lipid contents were significantly reduced, indicating that the DIO-induced hepatic steatosis was attenuated, despite no improvements in fibrosis and lipid contents in adipose tissue being observed in the OW-drinking DIO mice. The study provides evidence regarding OW's effects on adipogenesis and mature adipocytes, and the corresponding molecular mechanisms. OW exhibits transient triglyceride-lowering and glucose tolerance-improving activity as well as hepatic steatosis-attenuating functions.

The protoapigenone analog WYC0209 targets CD133+ cells: A potential adjuvant agent against cancer stem cells in urothelial cancer therapy.

Urothelial carcinoma (UC) is one of the highest incidence cancers that rank the fourth commonly diagnosed tumors worldwide. The unresectable lesions that are resistant to therapeutic interventions is the major cause leading to death. Previous studies had shown that the resistance and metastatic consequence may arise from cancer stem-like cells population. The phytochemical flavonoids have promised bioactivity and potent anti-carcinogenic effects, and trap great attentions for cancer chemoprevention and/or adjuvant chemotherapy. However, the mechanisms of flavonoids on cancer stemness is still obscured. In this study, we analyzed the biofunctional effects of as-prepared flavonoid derivative-WYC0209 on T24, BFTC905 and BFTC909 human UC cell lines. Our results demonstrated that WYC0209 significantly induced anti-cell viability on UC cells through decreased Akt/NFkB signaling. Moreover, WYC0209 enhanced the cell apoptosis through activated the caspase-3 activity and inactivated Bcl-xL expression. Interestingly, WYC0209 dramatically inhibited the cancer stem cells (CSCs) traits, including attenuation of side population and tumorsphere formation in which were through declined EMT-CSCs markers including MDR1, ABCG2 and BMI-1. We further validated the effects of WYC0209 on several CSC surface markers including CD133, CD44, SOX-2 and Nanog. Our results showed that WYC0209 markedly inhibited CD133 expressions in both transcriptional and translational levels. High expression levels of CD133 was also demonstrated in human upper tract UC specimens. In summary, our study showed that WYC0209 may potentially as an adjuvant agent to against CD133-driven UC CSCs and provide a beneficial strategy to against UC cancer therapeutics resistant.

Astaxanthin Ameliorates Ischemic-Hypoxic-Induced Neurotrophin Receptor p75 Upregulation in the Endothelial Cells of Neonatal Mouse Brains.

Ischemic stroke is a leading cause of human death in present times. Two phases of pathological impact occur during an ischemic stroke, namely, ischemia and reperfusion. Both periods include individual characteristic effects on cell injury and apoptosis. Moreover, these conditions can cause severe cell defects and harm the blood-brain barrier (BBB). Also, the BBB components are the major targets in ischemia-reperfusion injury. The BBB owes its enhanced protective roles to capillary endothelial cells, which maintain BBB permeability. One of the nerve growth factor (NGF) receptors initiating cell signaling, once activated, is the p75 neurotrophin receptor (p75NTR). This receptor is involved in both the survival and apoptosis of neurons. Although many studies have attempted to explain the role of p75NTR in neurons, the mechanisms in endothelial cells remain unclear. Endothelial cells are the first cells to encounter p75NTR stimuli. In this study, we found the upregulated p75NTR expression and reductive expression of tight junction proteins after in vivo and in vitro ischemia-reperfusion injury. Moreover, astaxanthin (AXT), an antioxidant drug, was utilized and was found to reduce p75NTR expression and the number of apoptotic cells. This study verified that p75NTR plays a prominent role in endothelial cell death and provides a novel downstream target for AXT.

Glucose Transporter 3 is Essential for the Survival of Breast Cancer Cells in the Brain.

Breast cancer brain metastasis commonly occurs in one-fourth of breast cancer patients and is associated with poor prognosis. Abnormal glucose metabolism is found to promote cancer metastasis. Moreover, the tumor microenvironment is crucial and plays an active role in the metabolic adaptations and survival of cancer cells. Glucose transporters are overexpressed in cancer cells to increase glucose uptake. The glucose transporter 3 (GLUT3) is a high-affinity glucose transporter that is highly expressed in mammalian neurons. GLUT3 is also overexpressed in several malignant brain tumors. However, the role of GLUT3 in breast cancer brain metastasis remains unknown. The results of the present study demonstrated that GLUT3 is highly overexpressed in brain metastatic breast cancers and mediates glucose metabolic reprogramming. Furthermore, knockdown of cAMP-response element binding protein (CREB) could directly regulate GLUT3 expression in brain metastatic breast cancer cells. Notably, we verified and provided a novel role of GLUT3 in mediating glucose metabolism and assisting breast cancer cells to survive in the brain to promote brain metastasis.

Genetic deletion of soluble epoxide hydrolase delays the progression of Alzheimer's disease.

BACKGROUND: Soluble epoxide hydrolase (sEH) is a bifunctional enzyme with COOH-terminal hydrolase and NH2-terminal lipid phosphatase activities. It is expressed in various cell types in the brain and is involved in the pathogenesis of inflammatory and neurodegenerative diseases. Alzheimer's disease (AD) is a progressive neuroinflammatory and neurodegenerative disease. However, the pathological significance of sEH and underlying molecular mechanism in AD remain unclear. METHODS: To examine the role of sEH in pathogenesis of AD, we used wild-type (WT) mice, soluble epoxide hydrolase deficient (sEH-/-) and two mouse models of AD, including amyloid precursor protein (APP)/presenilin 1 (PS1) transgenic (APP/PS1 Tg) and APP/PS1 Tg/sEH-/- mice. Western blotting analysis and immunohistochemistry assay were performed to evaluate the protein expression. Locomotion, nesting building ability, Y-maze, and Morris water maze tests were conducted to study mouse behavior. The levels of interleukin (IL)-1ß, IL-4, IL-6, and IL-10 and the activities of NF-κB and nuclear factor of activated T cells (NFAT) were measured by commercial assay kits. The quantitative protein level profiling in the brain lysate was analyzed using LC-MS/MS approaches. RESULTS: We demonstrated that the level of sEH was increased in the brain and predominantly appeared in hippocampal astrocytes of APP/PS1 Tg mice. Genetic ablation of sEH in APP/PS1 Tg mice delayed the progression of AD as evidenced by the alleviation in behavior outcomes and Aß plaque deposition. In addition, loss of the function of sEH in APP/PS1 Tg mice increased astrogliosis and the production of astrocyte-derived anti-inflammatory cytokines including IL-1ß, IL-4, and IL-10, as well as the activity of NF-kB and NFAT. Moreover, analysis of gene ontology in the AD brain revealed that important signaling pathways and processes related to AD pathogenesis such as translational regulation, oxidative stress, cytoskeleton reorganization, and small GTPase signal transduction were altered in APP/PS1 Tg/sEH-/- mice compared with APP/PS1 Tg mice. CONCLUSION: Our results suggest that sEH is a crucial regulator in the progression of AD and might be a potential therapeutic target for the treatment of AD.

Tribbles Homolog 3 Involved in Radiation Response of Triple Negative Breast Cancer Cells by Regulating Notch1 Activation.

Breast cancer is the most common cancer for women in Taiwan and post-lumpectomy radiotherapy is one of the therapeutic strategies for this malignancy. Although the 10-year overall survival of breast cancer patients is greatly improved by radiotherapy, the locoregional recurrence is around 10% and triple negative breast cancers (TNBCs) are at a high risk for relapse. The aim of this paper is to understand the mechanisms of radioresistance in breast cancers which may facilitate the development of new treatments in sensitizing breast cancer toward radiation therapy. Tribbles homolog 3 (TRIB3) is a pseudokinase protein and known to function as a protein scaffold within cells. It has been reported that higher TRIB3 expression is a poor prognostic factor in breast cancer patients with radiotherapy. In this study, we investigate the involvement of TRIB3 in the radiation response of TNBC cells. We first found that the expression of TRIB3 and the activation of Notch1, as well as Notch1 target genes, increased in two radioresistant TNBC cells. Knockdown of TRIB3 in radioresistant MDA-MB-231 TNBC cells decreased Notch1 activation, as well as the CD24-CD44⁺ cancer stem cell population, and sensitized cells toward radiation treatment. The inhibitory effects of TRIB3 knockdown in self-renewal or radioresistance could be reversed by forced expression of the Notch intracellular domain. We also observed an inhibition in cell growth and accumulated cells in the G0/G¹ phase in radioresistant MDA-MB-231 cells after knockdown of TRIB3. With immunoprecipitation and mass spectrometry analysis, we found that, BCL2-associated transcription factor 1 (BCLAF1), BCL2 interacting protein 1 (BNIP1), or DEAD-box helicase 5 (DDX5) were the possible TRIB3 interacting proteins and immunoprecipitation data also confirmed that these proteins interacted with TRIB3 in radioresistant MDA-MB-231 cells. In conclusion, the expression of TRIB3 in radioresistant TNBC cells participated in Notch1 activation and targeted TRIB3 expression may be a strategy to sensitize TNBC cells toward radiation therapy.

Genetic Deletion of Soluble Epoxide Hydroxylase Causes Anxiety-Like Behaviors in Mice.

Soluble epoxide hydrolase (sEH), an enzyme with COOH-terminal hydrolase and NH2-terminal lipid phosphatase activities, is expressed in regions of the brain such as the cortex, white matter, hippocampus, substantia nigra, and striatum. sEH is involved in the regulation of cerebrovascular and neuronal function upon pathological insults. However, the physiological significance of sEH and its underlying mechanism in modulating brain function are not fully understood. In this study, we investigated the role of sEH in anxiety and potential underlying mechanisms in mice. Western blot for protein phosphorylation and expression was performed. Immunohistochemical analyses and Nissl and Golgi staining were performed for histological examination. Mouse behaviors were evaluated by open field activity, elevated plus maze, classical fear conditioning, social preference test, and Morris water maze. Our results demonstrated that the expression of sEH was upregulated during postnatal development in wild-type (WT) mice. Genetic deletion of sEH (sEH-/-) in mice resulted in anxiety-like behavior and disrupted social preference. Increased olfactory bulb (OB) size and altered integrity of neurites were observed in sEH-/- mice. In addition, ablation of sEH in mice decreased protein expression of tyrosine hydroxylase and reduced dopamine production in the brain. Moreover, the level of phosphorylated calmodulin kinase II (CaMKII) and glycogen synthase kinase 3 α/ß (GSK3α/ß) was higher in sEH-/- mice than in WT mice. Collectively, these findings suggest that sEH is a key player in neurite outgrowth of neurons, OB development in the brain, and the development of anxiety-like behavior, by regulating the CaMKII-GSK3α/ß signaling pathway.

mGluR5 upregulation and the effects of repeated methamphetamine administration and withdrawal on the rewarding efficacy of ketamine and social interaction.

Repeated, recreational ketamine (KE) or methamphetamine (MA) administration seldom produce neurotoxicity, while combining MA and KE administration have been thought to render changes in neural plasticity and motivational behavior. In this study, we sought to assess whether pre-exposure to multiple MA injections and withdrawal may affect low-dose KE-produced rewarding effects, social interaction behavior and its neurochemical underpinnings. A 10-day MA injections (2 mg/kg/day) and 10-day withdrawal regimen was found to cause reliable behavioral sensitization. While KE (1 mg/kg) induced weak conditioned place preference (CPP), pre-exposure to this MA-withdrawal regimen enhanced such KE CPP magnitude. This MA-withdrawal regimen also caused impairments in the social interaction behavior in the sociability, social novelty test. Compared with the mice undergoing the 10-day saline-withdrawal or MA regimen, mice receiving the 10-day MA-withdrawal regimen exhibited lower dopamine-releasing probability in the nucleus accumbens, inferring the MA-withdrawal regimen-primed preference for KE rewarding effects. Likewise, mice receiving the MA-withdrawal regimen had high expression in mGluR5 protein but unaltered EAAT3, Homer2 expression in hippocampal tissues. Pretreatment with MPEP, an mGluR5 antagonist, prevented the MA-withdrawal regimen-induced increment in the KE CPP magnitude and impairments in social interaction behavior. We, thus, conclude that repeated MA administration and abstinence may enhance KE rewarding effects and produce eminent deficits in social recognition and interest. And these effects correlate with the mGluR5 over-expression and modulation of the KE-stimulating effect on dopamine release.

Hinokitiol up-regulates miR-494-3p to suppress BMI1 expression and inhibits self-renewal of breast cancer stem/progenitor cells.

Hinokitiol (ß-thujaplicin) is a tropolone-related compound that has anti-microbe, anti-inflammation, and anti-tumor effects. Cancer stem/progenitor cells (CSCs) are a subpopulation of cancer cells with tumor initiation, chemoresistant, and metastatic properties and have been considered the important therapeutic target in future cancer therapy. Previous studies reported that hinokitiol exhibits an anti-cancer activity against murine tumor cells through the induction of autophagy. The current research revealed that hinokitiol suppressed the self-renewal capabilities of human breast CSCs (BCSCs) and inhibited the expression of BMI1 at protein level without suppressing its mRNA. Treatment of hinokitiol in mammospheres induced the expression of miR-494-3p and inhibition of miR-494-3p expression in BCSCs. This treatment abolished the suppressive effects of hinokitiol in mammosphere formation and BMI1 expression. BMI1 is a target of miR-494-3p by luciferase-based 3'UTR reporter assay. Overexpression of miR-494-3p in BCSCs caused the down-regulation of BMI1 protein, inhibition of mammosphere forming capability, and suppression of their tumorigenicity. Moreover, miR-494-3p expression was significantly and inversely correlated with patient survival in two independent public database sets. Furthermore, treatment of hinokitiol in vivo suppressed the growth of xenograft human breast tumors as well as the expression of BMI1 and ALDH1A1 in xenograft tumors. In conclusion, these data suggest that hinokitiol targets BCSCs through the miR-494-3p-mediated down-modulation of BMI1 expression.

Hsp90α Mediates BMI1 Expression in Breast Cancer Stem/Progenitor Cells through Facilitating Nuclear Translocation of c-Myc and EZH2.

Heat shock protein 90 (Hsp90) is a molecular chaperone that facilitates the correct folding and functionality of its client protein. Numerous Hsp90-client proteins are involved in cancer development. Thus, Hsp90 inhibitors have potential applications as anti-cancer drugs. We previously discovered that Hsp90α expression increased in breast cancer stem cells (BCSCs), which can initiate tumorigenesis and metastasis and resist treatment. In the present study, we further demonstrated that 17-dimethylaminoethylamino-17-demethoxygeldanamycin (17-DMAG), an inhibitor of Hsp90, could suppress the self-renewal of BCSCs by downregulating B lymphoma Mo-MLV insertion region 1 homolog (BMI1), a polycomb family member with oncogenic activity in breast cancer. Through immunoprecipitation analysis, we found that BMI1 did not interact with Hsp90α and that the downregulation of BMI1 by 17-DMAG was mediated by the inhibition of c-Myc and enhancement of zeste homolog 2 (EZH2) expression. The transcriptional and BMI1 promoter-binding activities of c-Myc in BCSCs were inhibited by 17-DMAG treatment. The overexpression of EZH2 attenuated the inhibitory effect of 17-DMAG on BMI1 and c-Myc expression. Furthermore, Hsp90α could be co-immunoprecipitated with c-Myc and EZH2 and bind to the BMI1 promoter. Treatment with 17-DMAG decreased the nuclear expression of EZH2 and c-Myc but not that of Hsp90α. In conclusion, our data suggested that Hsp90α could positively regulate the self-renewal of BCSCs by facilitating the nuclear translocation of c-Myc and EZH2 to maintain BMI1 expression.