Enhanced IRE1α Phosphorylation/Oligomerization-Triggered XBP1 Splicing Contributes to Parkin-Mediated Prevention of SH-SY5Y Cell Death under Nitrosative Stress.

Mutations in parkin, a neuroprotective protein, are the predominant cause of autosomal recessive juvenile Parkinson's disease. Neuroinflammation-derived nitrosative stress has been implicated in the etiology of the chronic neurodegeneration. However, the interactions between genetic predisposition and nitrosative stress contributing to the degeneration of dopaminergic (DA) neurons remain incompletely understood. Here, we used the SH-SY5Y neuroblastoma cells to investigate the function of parkin and its pathogenic mutants in relation to cell survival under nitric oxide (NO) exposure. The results showed that overexpression of wild-type parkin protected SH-SY5Y cells from NO-induced apoptosis in a reactive oxygen species-dependent manner. Under nitrosative stress conditions, parkin selectively upregulated the inositol-requiring enzyme 1α/X-box binding protein 1 (IRE1α/XBP1) signaling axis, an unfolded protein response signal through the sensor IRE1α, which controls the splicing of XBP1 mRNA. Inhibition of XBP1 mRNA splicing either by pharmacologically inhibiting IRE1α endoribonuclease activity or by genetically knocking down XBP1 interfered with the protective activity of parkin. Furthermore, pathogenic parkin mutants with a defective protective capacity showed a lower ability to activate the IRE1α/XBP1 signaling. Finally, we demonstrated that IRE1α activity augmented by parkin was possibly mediated through interacting with IRE1α to regulate its phosphorylation/oligomerization processes, whereas mutant parkin diminished its binding to and activation of IRE1α. Thus, these results support a direct link between the protective activity of parkin and the IRE1α/XBP1 pathway in response to nitrosative stress, and mutant parkin disrupts this function.

Hispolon Induces Apoptosis, Suppresses Migration and Invasion of Glioblastoma Cells and Inhibits GBM Xenograft Tumor Growth In Vivo.

Hispolon, a polyphenol compound isolated from Phellinus linteus, has been reported to exhibit antioxidant, antiproliferative, and antitumor activities. This study aimed to explore the antitumor effects of hispolon on glioblastoma multiforme (GBM) cells in vitro and in vivo. The results revealed that hispolon significantly inhibited GBM cell proliferation and induced apoptosis through caspase-9 and caspase-3 activation and PARP cleavage. Hispolon also induced cell cycle G2/M phase arrest in GBM cells, as supported by flow cytometry analysis and confirmed by a decrease in cyclin B1, cdc2, and cdc25c protein expressions in a dose- and time-dependent manner. Furthermore, hispolon suppressed the migration and invasion of GBM cells by modulating epithelial-mesenchymal transition (EMT) markers via wound healing, transwell assays, and real-time PCR. Moreover, hispolon significantly reduced tumor growth in DBTRG xenograft mice and activated caspase-3 in hispolon-treated tumors. Thus, our findings revealed that hispolon is a potential candidate for the treatment of GBM.

Peroxiredoxin 5 Silencing Sensitizes Dopaminergic Neuronal Cells to Rotenone via DNA Damage-Triggered ATM/p53/PUMA Signaling-Mediated Apoptosis.

Peroxiredoxins (Prxs) are a family of thioredoxin peroxidases. Accumulating evidence suggests that changes in the expression of Prxs may be involved in neurodegenerative diseases pathology. However, the expression and function of Prxs in Parkinson's disease (PD) remains unclear. Here, we showed that Prx5 was the most downregulated of the six Prx subtypes in dopaminergic (DA) neurons in rotenone-induced cellular and rat models of PD, suggesting possible roles in regulating their survival. Depletion of Prx5 sensitized SH-SY5Y DA neuronal cells to rotenone-induced apoptosis. The extent of mitochondrial membrane potential collapse, cytochrome c release, and caspase activation was increased by Prx5 loss. Furthermore, Prx5 knockdown enhanced the induction of PUMA by rotenone through a p53-dependent mechanism. Using RNA interference approaches, we demonstrated that the p53/PUMA signaling was essential for Prx5 silencing-exacerbated mitochondria-driven apoptosis. Additionally, downregulation of Prx5 augmented rotenone-induced DNA damage manifested as induction of phosphorylated histone H2AX (γ-H2AX) and activation of ataxia telangiectasia mutated (ATM) kinase. The pharmacological inactivation of ATM revealed that ATM was integral to p53 activation by DNA damage. These findings provided a novel link between Prx5 and DNA damage-triggered ATM/p53/PUMA signaling in a rotenone-induced PD model. Thus, Prx5 might play an important role in protection against rotenone-induced DA neurodegeneration.

Phenethyl isothiocyanate alters the gene expression and the levels of protein associated with cell cycle regulation in human glioblastoma GBM 8401 cells.

Glioblastoma is the most common and aggressive primary brain malignancy. Phenethyl isothiocyanate (PEITC), a member of the isothiocyanate family, can induce apoptosis in many human cancer cells. Our previous study disclosed that PEITC induces apoptosis through the extrinsic pathway, dysfunction of mitochondria, reactive oxygen species (ROS)-induced endoplasmic reticulum (ER) stress, and intrinsic (mitochondrial) pathway in human brain glioblastoma multiforme (GBM) 8401 cells. To the best of our knowledge, we first investigated the effects of PEITC on the genetic levels of GBM 8401 cells in vitro. PEITC may induce G0/G1 cell-cycle arrest through affecting the proteins such as cdk2, cyclin E, and p21 in GBM 8401 cells. Many genes associated with cell-cycle regulation of GBM 8401 cells were changed after PEITC treatment: 48 genes were upregulated and 118 were downregulated. The cell-division cycle protein 20 (CDC20), Budding uninhibited by benzimidazole 1 homolog beta (BUB1B), and cyclin B1 were downregulated, and clusterin was upregulated in GBM 8401 cells treated with PEITC. These changes of gene expression can provide the effects of PEITC on the genetic levels and potential biomarkers for glioblastoma. © 2015 Wiley Periodicals, Inc. Environ Toxicol 32: 176-187, 2017.

6-Mercaptopurine attenuates tumor necrosis factor-α production in microglia through Nur77-mediated transrepression and PI3K/Akt/mTOR signaling-mediated translational regulation.

BACKGROUND: The pathogenesis of several neurodegenerative diseases often involves the microglial activation and associated inflammatory processes. Activated microglia release pro-inflammatory factors that may be neurotoxic. 6-Mercaptopurine (6-MP) is a well-established immunosuppressive drug. Common understanding of their immunosuppressive properties is largely limited to peripheral immune cells. However, the effect of 6-MP in the central nervous system, especially in microglia in the context of neuroinflammation is, as yet, unclear. Tumor necrosis factor-α (TNF-α) is a key cytokine of the immune system that initiates and promotes neuroinflammation. The present study aimed to investigate the effect of 6-MP on TNF-α production by microglia to discern the molecular mechanisms of this modulation. METHODS: Lipopolysaccharide (LPS) was used to induce an inflammatory response in cultured primary microglia or murine BV-2 microglial cells. Released TNF-α was measured by enzyme-linked immunosorbent assay (ELISA). Gene expression was determined by real-time reverse transcription polymerase chain reaction (RT-PCR). Signaling molecules were analyzed by western blotting, and activation of NF-κB was measured by ELISA-based DNA binding analysis and luciferase reporter assay. Chromatin immunoprecipitation (ChIP) analysis was performed to examine NF-κB p65 and coactivator p300 enrichments and histone modifications at the endogenous TNF-α promoter. RESULTS: Treatment of LPS-activated microglia with 6-MP significantly attenuated TNF-α production. In 6-MP pretreated microglia, LPS-induced MAPK signaling, IκB-α degradation, NF-κB p65 nuclear translocation, and in vitro p65 DNA binding activity were not impaired. However, 6-MP suppressed transactivation activity of NF-κB and TNF-α promoter by inhibiting phosphorylation and acetylation of p65 on Ser276 and Lys310, respectively. ChIP analyses revealed that 6-MP dampened LPS-induced histone H3 acetylation of chromatin surrounding the TNF-α promoter, ultimately leading to a decrease in p65/coactivator-mediated transcription of TNF-α gene. Furthermore, 6-MP enhanced orphan nuclear receptor Nur77 expression. Using RNA interference approach, we further demonstrated that Nur77 upregulation contribute to 6-MP-mediated inhibitory effect on TNF-α production. Additionally, 6-MP also impeded TNF-α mRNA translation through prevention of LPS-activated PI3K/Akt/mTOR signaling cascades. CONCLUSIONS: These results suggest that 6-MP might have a therapeutic potential in neuroinflammation-related neurodegenerative disorders through downregulation of microglia-mediated inflammatory processes.

PEITC inhibits human brain glioblastoma GBM 8401 cell migration and invasion through the inhibition of uPA, Rho A, and Ras with inhibition of MMP-2, -7 and -9 gene expression.

Glioblastoma is the most aggressive primary brain malignancy, and the efficacy of multimodality treatments remains unsatisfactory. Phenethyl isothiocyanate (PEITC), one member of the isothiocyanate family, was found to inhibit the migration and invasion of many types of human cancer cells. In our previous study, PEITC induced the apoptosis of human brain glioblastoma GBM 8401 cells through the extrinsic and intrinsic signaling pathways. In the present study, we first investigated the effects of PEITC on the migration and invasion of GBM 8401 cells. PEITC decreased the migration of GBM 8401 cells in a dose-dependent manner as determined from scratch wound healing and Transwell migration assays. The percentage of inhibition ranged from 46.89 to 15.75%, and from 27.80 to 7.31% after a 48-h treatment of PEITC as determined from the Transwell migration assay and invasion assay, respectively. The western blot analysis indicated that PEITC decreased the levels of proteins associated with migration and invasion, Ras, uPA, RhoA, GRB2, p-p38, p-JNK, p-ERK, p65, SOS1, MMP-2, MMP-9 and MMP-13, in a dose-dependent manner. Real-time PCR analyses revealed that PEITC reduced the mRNA levels of MMP-2, MMP-7, MMP-9 and RhoA in a dose- and time-dependent manner. PEITC exhibited potent anticancer activities through the inhibition of migration and invasion in the GBM 8401 cells. Our findings elucidate the possible molecular mechanisms and signaling pathways of the anti-metastatic effects of PEITC on human brain glioblastoma cells, and PEITC may be considered as a therapeutic agent.

Epigenetic regulation contributes to urocortin-enhanced midbrain dopaminergic neuron differentiation.

The production of midbrain dopaminergic (mDA) neurons requires precise extrinsic inductive signals and intrinsic transcriptional cascade at a specific time point in development. Urocortin (UCN) is a peptide of the corticotropin-releasing hormone family that mediates various responses to stress. UCN was first cloned from adult rat midbrain. However, the contribution of UCN to the development of mDA neurons is poorly understood. Here, we show that UCN is endogenously expressed in the developing ventral midbrain (VM) and its receptors are exhibited in Nurr1(+) postmitotic mDA precursors and TH(+) neurons, suggesting possible roles in regulating their terminal differentiation. UCN treatment increased DA cell numbers in rat VM precursor cultures by promoting the conversion of Nurr1(+) precursors into DA neurons. Furthermore, neutralization of secreted UCN with anti-UCN antibody resulted in a reduction in the number of DA neurons. UCN induced an abundance of acetylated histone H3 and enhanced late DA regulator Nurr1, Foxa2, and Pitx3 expressions. Using pharmacological and RNA interference approaches, we further demonstrated that histone deacetylase (HDAC) inhibition and late transcriptional factors upregulation contribute to UCN-mediated DA neuron differentiation. Chromatin immunoprecipitation analyses revealed that UCN promoted histone acetylation of chromatin surrounding the TH promoter by directly inhibiting HDAC and releasing of methyl CpG binding protein 2-CoREST-HDAC1 repressor complex from the promoter, ultimately leading to an increase in Nurr1/coactivators-mediated transcription of TH gene. Moreover, UCN treatment in vivo also resulted in increased DA neuron differentiation. These findings suggest that UCN might contribute to regulate late mDA neuron differentiation during VM development.

PEITC induces apoptosis of Human Brain Glioblastoma GBM8401 Cells through the extrinsic- and intrinsic -signaling pathways.

Glioblastoma is the most common and most aggressive primary brain malignancy. The multimodality treatments for this tumor including surgery, radiotherapy, and chemotherapy, are still not completely satisfied. Phenethyl isothiocyanate (PEITC), one member of the isothiocyanate family, has been shown to induce apoptosis in many human cancer cells. In this study, we investigate the pro-apoptotic effects caused by PETIC in human brain glioblastoma multiforme GBM 8401 cells. In our data, PEITC induced the cell morphological changes and decreased the cell viability of GBM8401 cells in a dose- and time-dependent manner. Moreover, the analysis of cell cycle distribution detected by flow cytometry showed that PEITC induced significantly sub-G1 phase (apoptotic population) in GBM 8401 cells. In addition, PEITC promoted the production of reactive oxygen species (ROS) and increase in [Ca2+]I, but decreased the mitochondrial membrane potential (ΔΨm) in treated cells. PEITC also induced caspases activities in GBM 8401 cells. Results from Western blot analysis indicated that PEITC promoted Fas, FasL, FADD, TRAIL, caspase-8, -9, -3, increased the pro-apoptotic protein (Bax, Bid and Bak), and inhibited the anti-apoptotic proteins (Bcl-2 and Bcl-xl) in GBM 8401 cells. Furthermore, PEITC promoted the release of cytochrome c, AIF and Endo G. GADD153, GRP 78, XBP-1 and IRE-1α, Calpain I and II in GBM 8401 cells. PEITC also promoted the expression of associated protein with endoplasmic reticulum (ER) stress. PEITC induces apoptosis through the extrinsic (death receptor) pathway, dysfunction of mitochondria, ROS induced ER stress, intrinsic (mitochondrial) pathway in GBM 8401 cells. The possible molecular mechanisms and signaling pathways of the anti-cancer properties of PEITC for human brain glioblastoma cells were postulated.

TCTP is essential for ß-cell proliferation and mass expansion during development and ß-cell adaptation in response to insulin resistance.

The perinatal period is critical for ß-cell mass establishment, which is characterized by a transient burst in proliferation to increase ß-cell mass in response to the need for glucose homeostasis throughout life. In adulthood, the ability of ß-cells to grow, proliferate, and expand their mass is also characteristic of pathological states of insulin resistance. Translationally controlled tumor-associated protein (TCTP), an evolutionarily highly conserved protein that is implicated in cell growth and proliferation, has been identified as a novel glucose-regulated survival-supporting protein in pancreatic ß-cells. In this study, the enhanced ß-cell proliferation detected both during the perinatal developmental period and in insulin-resistant states in high-fat diet-fed mice was found to parallel the expression of TCTP in pancreatic ß-cells. Specific knockout of TCTP in ß-cells led to increased expression of total and nuclear Forkhead box protein O1 and tumor suppressor protein 53, and decreased expression of p70S6 kinase phosphorylation and cyclin D2 and cyclin-dependent kinase 2. This resulted in decreased ß-cell proliferation and growth, reduced ß-cell mass, and insulin secretion. Together, these effects led to hyperglycemia. These observations suggest that TCTP is essential for ß-cell mass expansion during development and ß-cell adaptation in response to insulin resistance.

Histone deacetylase inhibition mediates urocortin-induced antiproliferation and neuronal differentiation in neural stem cells.

During cortical development, cell proliferation and cell cycle exit are carefully regulated to ensure that the appropriate numbers of cells are produced. Urocortin (UCN) is a member of the corticotrophin releasing hormone (CRH) family of neuropeptides that regulates stress responses. UCN is widely distributed in adult rat brain. However, the expression and function of UCN in embryonic brain is, as yet, unclear. Here, we show that UCN is endogenously expressed in proliferative zones of the developing cerebral cortex and its receptors are exhibited in neural stem cells (NSCs), thus implicating the neuropeptide in cell cycle regulation. Treatment of cultured NSCs or organotypic slice cultures with UCN markedly reduced cell proliferation. Furthermore, blocking of endogenous UCN/CRHRs system either by treatment with CRHRs antagonists or by neutralization of secreted UCN with anti-UCN antibody increased NSCs proliferation. Cell cycle kinetics analysis demonstrated that UCN lengthened the total cell cycle duration via increasing the G1 phase and accelerated cell cycle exit. UCN directly inhibited the histone deacetylase (HDAC) activity and induced a robust increase in histone H3 acetylation levels. Using pharmacological and RNA interference approaches, we further demonstrated that antiproliferative action of UCN appeared to be mediated through a HDAC inhibition-induced p21 upregulation. Moreover, UCN treatment in vitro and in vivo led to an increase in neuronal differentiation of NSCs. These findings suggest that UCN might contribute to regulate NSCs proliferation and differentiation during cortical neurogenesis.

Induction of apoptosis coupled to endoplasmic reticulum stress in human prostate cancer cells by n-butylidenephthalide.

BACKGROUND: N-butylidenephthalide (BP) exhibits antitumor effect in a variety of cancer cell lines. The objective of this study was to obtain additional insights into the mechanisms involved in BP induced cell death in human prostate cancer cells. METHODS/PRINCIPAL FINDINGS: Two human prostate cancer cell lines, PC-3 and LNCaP, were treated with BP, and subsequently evaluated for their viability and cell cycle profiles. BP caused cell cycle arrest and cell death in both cell lines. The G0/G1 phase arrest was correlated with increase levels of CDK inhibitors (p16, p21 and p27) and decrease of the checkpoint proteins. To determine the mechanisms of BP-induced growth arrest and cell death in prostate cancer cell lines, we performed a microarray study to identify alterations in gene expression induced by BP in the LNCaP cells. Several BP-induced genes, including the GADD153/CHOP, an endoplasmic reticulum stress (ER stress)-regulated gene, were identified. BP-induced ER stress was evidenced by increased expression of the downstream molecules GRP78/BiP, IRE1-α and GADD153/CHOP in both cell lines. Blockage of IRE1-α or GADD153/CHOP expression by siRNA significantly reduced BP-induced cell death in LNCaP cells. Furthermore, blockage of JNK1/2 signaling by JNK siRNA resulted in decreased expression of IRE1-α and GADD153/CHOP genes, implicating that BP-induced ER stress may be elicited via JNK1/2 signaling in prostate cancer cells. BP also suppressed LNCaP xenograft tumor growth in NOD-SCID mice. It caused 68% reduction in tumor volume after 18 days of treatment. CONCLUSIONS: Our results suggest that BP can cause G0/G1 phase arrest in prostate cancer cells and its cytotoxicity is mediated by ER stress induction. Thus, BP may serve as an anticancer agent by inducing ER stress in prostate cancer.

Systemic administration of urocortin after intracerebral hemorrhage reduces neurological deficits and neuroinflammation in rats.

BACKGROUND: Intracerebral hemorrhage (ICH) remains a serious clinical problem lacking effective treatment. Urocortin (UCN), a novel anti-inflammatory neuropeptide, protects injured cardiomyocytes and dopaminergic neurons. Our preliminary studies indicate UCN alleviates ICH-induced brain injury when administered intracerebroventricularly (ICV). The present study examines the therapeutic effect of UCN on ICH-induced neurological deficits and neuroinflammation when administered by the more convenient intraperitoneal (i.p.) route. METHODS: ICH was induced in male Sprague-Dawley rats by intrastriatal infusion of bacterial collagenase VII-S or autologous blood. UCN (2.5 or 25 µg/kg) was administered i.p. at 60 minutes post-ICH. Penetration of i.p. administered fluorescently labeled UCN into the striatum was examined by fluorescence microscopy. Neurological deficits were evaluated by modified neurological severity score (mNSS). Brain edema was assessed using the dry/wet method. Blood-brain barrier (BBB) disruption was assessed using the Evans blue assay. Hemorrhagic volume and lesion volume were assessed by Drabkin's method and morphometric assay, respectively. Pro-inflammatory cytokine (TNF-α, IL-1ß, and IL-6) expression was evaluated by enzyme-linked immunosorbent assay (ELISA). Microglial activation and neuronal loss were evaluated by immunohistochemistry. RESULTS: Administration of UCN reduced neurological deficits from 1 to 7 days post-ICH. Surprisingly, although a higher dose (25 µg/kg, i.p.) also reduced the functional deficits associated with ICH, it is significantly less effective than the lower dose (2.5 µg/kg, i.p.). Beneficial results with the low dose of UCN included a reduction in neurological deficits from 1 to 7 days post-ICH, as well as a reduction in brain edema, BBB disruption, lesion volume, microglial activation and neuronal loss 3 days post-ICH, and suppression of TNF-α, IL-1ß, and IL-6 production 1, 3 and 7 days post-ICH. CONCLUSION: Systemic post-ICH treatment with UCN reduces striatal injury and neurological deficits, likely via suppression of microglial activation and inflammatory cytokine production. The low dose of UCN necessary and the clinically amenable peripheral route make UCN a potential candidate for development into a clinical treatment regimen.

Therapeutic benefit of urocortin in rats with intracerebral hemorrhage.

OBJECT: Intracerebral hemorrhage (ICH) accounts for about 15% of all deaths due to stroke. It frequently causes brain edema, leading to an expansion of brain volume that exerts a negative impact on ICH outcomes. The ICH-induced brain edema involves inflammatory mechanisms. The authors' in vitro study shows that urocortin (UCN) exhibits antiinflammatory and neuroprotective effects. Therefore, the neuroprotective effect of UCN on ICH in rats was investigated. METHODS: Intracerebral hemorrhage was induced by an infusion of bacteria collagenase type VII-S or autologous blood into the unilateral striatum of anesthetized rats. At 1 hour after the induction of ICH, UCN (0.05, 0.5, and 5 µg) was infused into the lateral ventricle on the ipsilateral side. The authors examined the injury area, brain water content, blood-brain barrier permeability, and neurological function. RESULTS: The UCN, administered in the ipsilateral lateral ventricle, was able to penetrate into the injured striatum. Posttreatment with UCN reduced the injury area, brain edema, and blood-brain barrier permeability and improved neurological deficits of rats with ICH. CONCLUSIONS: Posttreatment with UCN through improving neurological deficits of rats with ICH dose dependently provided a potential therapeutic agent for patients with ICH or other brain injuries.

Activation of NAG-1 via JNK signaling revealed an isochaihulactone-triggered cell death in human LNCaP prostate cancer cells.

BACKGROUND: We explored the mechanisms of cell death induced by isochaihulactone treatment in LNCaP cells. METHODS: LNCaP cells were treated with isochaihulactone and growth inhibition was assessed. Cell cycle profiles after isochaihulactone treatment were determined by flow cytometry. Expression levels of cell cycle regulatory proteins, caspase 9, caspase 3, and PARP were determined after isochaihulactone treatment. Signaling pathway was verified by inhibitors pre-treatment. Expression levels of early growth response gene 1 (EGR-1) and nonsteroidal anti-inflammatory drug-activated gene 1 (NAG-1) were determined to investigate their role in LNCaP cell death. NAG-1 expression was knocked down by si-NAG-1 siRNA transfection. Rate of cell death and proliferation were obtained by MTT assay. RESULTS: Isochaihulactone caused cell cycle arrest at G2/M phase in LNCaP cells, which was correlated with an increase of p53 and p21 levels and downregulation of the checkpoint proteins cdc25c, cyclin B1, and cdc2. Bcl-2 phosphorylation and caspase activation were also observed. Isochaihulactone induced phosphorylation of c-Jun-N-terminal kinase (JNK), and JNK inhibitor partially reduced isochaihulactone-induced cell death. Isochaihulactone also induced the expressions of EGR-1 and NAG-1. Expression of NAG-1 was reduced by JNK inhibitor, and knocking down of NAG-1 inhibited isochaihulactone-induced cell death. CONCLUSIONS: Isochaihulactone apparently induces G2/M cell cycle arrest via downregulation of cyclin B1 and cdc2, and induces cellular death by upregulation of NAG-1 via JNK activation in LNCaP cells.

Urocortin modulates dopaminergic neuronal survival via inhibition of glycogen synthase kinase-3ß and histone deacetylase.

Urocortin (UCN) is a member of the corticotropin-releasing hormone (CRH) family of neuropeptides that regulates stress responses. Although UCN is principally expressed in dopaminergic neurons in rat substantia nigra (SN), the function of UCN in modulating dopaminergic neuronal survival remains unclear. Using primary mesencephalic cultures, we demonstrated that dopaminergic neurons underwent spontaneous cell death when their age increased in culture. Treatment of mesencephalic cultures with UCN markedly prolonged the survival of dopaminergic neurons, whereas neutralization of UCN with anti-UCN antibody accelerated dopaminergic neurons degeneration. UCN increased intracellular cAMP levels followed by phosphorylating glycogen synthase kinase-3ß (GSK-3ß) on Ser9. Moreover, UCN directly inhibited the histone deacetylase (HDAC) activity and induced a robust increase in histone H3 acetylation levels. Using pharmacological approaches, we further demonstrated that inhibition of GSK-3ß and HDAC contributes to UCN-mediated neuroprotection. These results suggest that dopaminergic neuron-derived UCN might be involved in an autocrine protective signaling mechanism.

The anti-cancer efficacy of curcumin scrutinized through core signaling pathways in glioblastoma.

Curcumin has been verified as an anti-cancer compound via multiple molecular targets. Its effective mechanisms include cell cycle arrest, inducing apoptosis, suppressing oncogenes, and enhancing tumor suppressor genes. The resistance of cells to chemotherapy, however, derives from the variable genetic aberration of cancer cells. Consequently, the core signaling pathways of glioblastoma have been explored to evaluate the efficacy of curcumin in proceeding through mutated genes in those pathways. In this study, the efficacy of curcumin was investigated in DBTRG cells. The cytotoxic ability was detected with MTT assay, and the influence of the cell cycle was checked with flow cytometry. The influence of the core signaling pathways was evaluated by Western blotting through the predominantly mutated proteins which included p53, p21, and cdc2 in the p53 pathway, CDKN2A/p16 and RB in the RB pathway, and EGFR, mTOR, Ras, PTEN, and Akt in the RTK-Ras-PI3K pathway. In addition, the apoptotic effect was determined by apoptosis-associated proteins Bcl-2, Bax, and caspase 3. Curcumin exhibits superior cytotoxicity on glioblastoma in a dose- and time-dependent manner in the MTT assay. In the core signaling pathways of glioblastoma, curcumin either significantly influences the p53 pathway by enhancing p53 and p21 and suppressing cdc2 or significantly inhibits the RB pathway by enhancing CDKN2A/p16 and suppressing phosphorylated RB. In the apoptotic pathway, the Bax and caspase 3 are significantly suppressed by curcumin and the Giemsa stain elucidates apoptotic features of DBTRG cells as well. In conclusion, curcumin appears to be an effective anti-glioblastoma drug through inhibition of the two core signaling pathways and promotion of the apoptotic pathway.

Autonomic regulation of insulin secretion is changed by pentobarbital in mice.

It has been established that insulin secretion is regulated by autonomic nervous homeostasis. In the screen of plasma glucose level, anesthetized animals were widely used. However, effects of anesthetics on blood glucose remain unclear. In the present study, we compared the hypoglycemic action of ginseng that was induced by insulin secretion in mice between conscious and under anesthesia with pentobarbital. The hypoglycemic effect of ginseng was only produced in anesthetized BALB/c mice but not in the conscious mice. Similar results were also observed in C57BL/6 mice. However, the hypoglycemic action of ginseng failed to produce in anesthetized BALB/c mice received streptozotocin to induce type-1 like diabetes showing an insulin-dependent manner. The plasma insulin level in anesthetized BALB/c mice was markedly raised by ginseng but this effect was not observed in conscious mice. Blockade of muscarinic receptors by atropine inhibited ginseng-induced insulin secretion in anesthetized mice. Otherwise, the hypoglycemic action of ginseng was restored in conscious mice treated guanethidine at a sufficient dose to block sympathetic tone. In conclusion, the obtained results suggest that insulin secretion regulated by autonomic nervous homeostasis can be changed by pentobarbital through decrement in sympathetic tone to increase the insulin secretion induced by agent(s) via higher of parasympathetic tone. This finding is suitable to explain the critical hypoglycemia was not observed in subjects received ginseng.

Glycogen synthase kinase-3ß inactivation inhibits tumor necrosis factor-α production in microglia by modulating nuclear factor κB and MLK3/JNK signaling cascades.

BACKGROUND: Deciphering the mechanisms that modulate the inflammatory response induced by microglial activation not only improves our insight into neuroinflammation but also provides avenues for designing novel therapies that could halt inflammation-induced neuronal degeneration. Decreasing glycogen synthase kinase-3ß (GSK-3ß) activity has therapeutic benefits in inflammatory diseases. However, the exact molecular mechanisms underlying GSK-3ß inactivation-mediated suppression of the inflammatory response induced by microglial activation have not been completely clarified. Tumor necrosis factor-α (TNF-α) plays a central role in injury caused by neuroinflammation. We investigated the regulatory effect of GSK-3ß on TNF-α production by microglia to discern the molecular mechanisms of this modulation. METHODS: Lipopolysaccharide (LPS) was used to induce an inflammatory response in cultured primary microglia or murine BV-2 microglial cells. Release of TNF-α was measured by ELISA. Signaling molecules were analyzed by western blotting, and activation of NF-κB and AP-1 was measured by ELISA-based DNA binding analysis and luciferase reporter assay. Protein interaction was examined by coimmunoprecipitation. RESULTS: Inhibition of GSK-3ß by selective GSK-3ß inhibitors or by RNA interference attenuated LPS-induced TNF-α production in cultured microglia. Exploration of the mechanisms by which GSK-3ß positively regulates inflammatory response showed that LPS-induced IκB-α degradation, NF-κBp65 nuclear translocation, and p65 DNA binding activity were not affected by inhibition of GSK-3ß activity. However, GSK-3ß inactivation inhibited transactivation activity of p65 by deacetylating p65 at lysine 310. Furthermore, we also demonstrated a functional interaction between mixed lineage kinase 3 (MLK3) and GSK-3ß during LPS-induced TNF-α production in microglia. The phosphorylated levels of MLK3, MKK4, and JNK were increased upon LPS treatment. Decreasing GSK-3ß activity blocked MLK3 signaling cascades through disruption of MLK3 dimerization-induced autophosphorylation, ultimately leading to a decrease in TNF-α secretion. CONCLUSION: These results suggest that inactivation of GSK-3ß might represent a potential strategy to downregulate microglia-mediated inflammatory processes.

Urocortin modulates inflammatory response and neurotoxicity induced by microglial activation.

Microglia are the major inflammatory cells in the brain. Recent studies have highlighted the reciprocal roles of other brain cells in modulating the microglial inflammatory responses. Urocortin (UCN) is a member of the corticotropin-releasing hormone (CRH) family of neuropeptides that function to regulate stress responses. In the present study, we demonstrated that expression of UCN in rat substantia nigra was found to be localized principally to dopaminergic neurons. In cell culture models, the CRH receptors were expressed in microglia, and CRHR expression was up-regulated by treatment with LPS. Thus, it might be proposed that UCN regulates cellular communication between dopaminergic neurons and microglia. We show that femtomolar concentrations of UCN could inhibit LPS-induced TNF-alpha production in cultured microglia. Investigation of the underlying signaling pathway that mediated the anti-inflammatory effect of UCN the involved PI3K/Akt and glycogen synthase kinase-3beta pathway, but not cAMP pathway. Furthermore, UCN protected dopaminergic neurons against LPS-induced neurotoxicity by inhibiting microglial activation in LPS-treated mesencephalic neuron-glia cultures. These results suggest that endogenous UCN and its receptors might be involved in a complex network of paracrine interaction between dopaminergic neurons and glia.

G-CSF protects dopaminergic neurons from 6-OHDA-induced toxicity via the ERK pathway.

Granulocyte colony-stimulating factor (G-CSF) is known to have various functions such as induction of survival, proliferation and differentiation of hematopoietic cells. Recently, this factor has also been shown to exhibit neuroprotective effects in rat ischemic brain. In the present study, we first demonstrated that both G-CSF and G-CSF receptor were expressed in dopaminergic neurons in the adult substantia nigra and mesencephalic cultures, suggesting that G-CSF might exert its neuroprotective effects in dopaminergic neurons. Pretreatment with G-CSF protected dopaminergic neurons from 6-hydroxydopamine (6-OHDA)-induced neurotoxicity. Investigation of the underlying mechanisms showed that the extracellular-regulated kinase (ERK), but not Janus kinase/signal transducer(s) and activator(s) of transcription (JAK/STAT), was activated following G-CSF treatment. Moreover, G-CSF also increased phosphorylation of Bad, and restored 6-OHDA-induced decrease in Bcl-xL level. The 6-OHDA-caused caspase-3 activation in dopaminergic neurons was inhibited by G-CSF. Inhibition of ERK abrogated G-CSF-mediated Bad phosphorylation, Bcl-xL expression, activated caspase-3 reduction, and the protection of dopaminergic neurons. Taken together, G-CSF prevents dopaminergic neurons from 6-OHDA-induced toxicity via ERK pathway followed by inhibiting the apoptosis-execution process. These results suggest that G-CSF might have a therapeutic potential in Parkinson's disease.