Protection against glutathione depletion-associated oxidative neuronal death by neurotransmitters norepinephrine and dopamine: Protein disulfide isomerase as a mechanistic target for neuroprotection.

Oxidative stress is extensively involved in neurodegeneration. Clinical evidence shows that keeping the mind active through mentally-stimulating physical activities can effectively slow down the progression of neurodegeneration. With increased physical activities, more neurotransmitters would be released in the brain. In the present study, we investigated whether some of the released neurotransmitters might have a beneficial effect against oxidative neurodegeneration in vitro. Glutamate-induced, glutathione depletion-associated oxidative cytotoxicity in HT22 mouse hippocampal neuronal cells was used as an experimental model. We showed that norepinephrine (NE, 50 µM) or dopamine (DA, 50 µM) exerted potent protective effect against glutamate-induced cytotoxicity, but this effect was not observed when other neurotransmitters such as histamine, γ-aminobutyric acid, serotonin, glycine and acetylcholine were tested. In glutamate-treated HT22 cells, both NE and DA significantly suppressed glutathione depletion-associated mitochondrial dysfunction including mitochondrial superoxide accumulation, ATP depletion and mitochondrial AIF release. Moreover, both NE and DA inhibited glutathione depletion-associated MAPKs activation, p53 phosphorylation and GADD45α activation. Molecular docking analysis revealed that NE and DA could bind to protein disulfide isomerase (PDI). In biochemical enzymatic assay in vitro, NE and DA dose-dependently inhibited the reductive activity of PDI. We further revealed that the protective effect of NE and DA against glutamate-induced oxidative cytotoxicity was mediated through inhibition of PDI-catalyzed dimerization of the neuronal nitric oxide synthase. Collectively, the results of this study suggest that NE and DA may have a protective effect against oxidative neurodegeneration through inhibition of protein disulfide isomerase and the subsequent activation of the MAPKs‒p53‒GADD45α oxidative cascade.

Mechanism underlying resveratrol's attenuation of paclitaxel cytotoxicity in human breast cancer cells: Role of the SIRT1-FOXO1-HER3 signaling pathway.

Resveratrol (RES), a dietary phenolic compound, was reported to have cancer chemoprotective and chemotherapeutic effects. Earlier we unexpectedly observed that RES has a growth-enhancing effect in some breast cancer cells and can diminish the susceptibility of MDA-MB-231 and SKBR-3 cells to paclitaxel-induced cell death, but this phenomenon is not observed in MCF-7 cells. The present study seeks to determine the mechanism underlying RES's attenuation of paclitaxel cytotoxicity in cancer cells. It is found that RES reduces the anticancer action of paclitaxel only in the human breast cancer cells that express HER3 protein. Treatment of SKBR-3 cells with RES increases HER3 expression in a dose-dependent manner. The induction of HER3 expression by RES confers resistance of breast cancer cells against paclitaxel cytotoxicity. Furthermore, it is observed that the SIRT1-FOXO1 signaling pathway plays an important role in mediating RES-induced upregulation of HER3 expression. In conclusion, the present study reveals the mechanism for RES-induced resistance against paclitaxel in some human breast cancer cells, and it is suggested that the combined use of RES and paclitaxel is not suitable for treating human breast cancer that expresses HER3 protein.

4-Hydroxyestrone, an Endogenous Estrogen Metabolite, Can Strongly Protect Neuronal Cells Against Oxidative Damage.

Earlier studies showed that endogenous estrogens have neuroprotective effect against oxidative damage. The present study seeks to investigate the protective effect of various endogenous estrogen metabolites against oxidative neurotoxicity in vitro and in vivo. Using immortalized mouse hippocampal neuronal cells as an in vitro model, 4-hydroxyestrone, an estrone metabolite with little estrogenic activity, is found to have the strongest neuroprotective effect against oxidative neurotoxicity among 25 endogenous estrogen metabolites tested, and its protective effect is stronger than 17ß-estradiol. Similarly, 4-Hydroxyestrone also exerts a stronger protective effect than 17ß-estradiol against kanic acid-induced hippocampal oxidative damage in rats. Neuroprotection by 4-hydroxyestrone involves increased cytoplasmic translocation of p53 resulting from SIRT1-mediated deacetylation of p53. Analysis of brain microsomal enzymes shows that estrogen 4-hydroxylation is the main metabolic pathway in the central nervous system. Together, these results show that 4-hydroxyestrone is an endogenous neuroestrogen that can strongly protect against oxidative neuronal damage.

Upregulated cyclin B1/CDK1 mediates apoptosis following 2-methoxyestradiol-induced mitotic catastrophe: Role of Bcl-XL phosphorylation.

2-Methoxyestradiol is an endogenous nonpolar metabolite of 17ß-estradiol with a strong antitubulin activity. Earlier we showed that 2-methoxyestradiol increases the level and activity of cyclin B1/CDK1, which subsequently induces mitotic prometaphase arrest. In the present study, we demonstrate that upregulation of cyclin B1/CDK1 is responsible for the increased phosphorylation of the anti-apoptotic proteins Bcl-2 and Bcl-XL in 2-methoxyestradiol-induced, mitotically-arrested cancer cells. Additional analysis shows that only the increase in phosphorylation of Bcl-XL, but not Bcl-2, is associated with activation of the mitochondrial cell death pathway. We find that MAD2 is an important upstream mediator of the antitubulin function of 2-methoxyestradiol, resulting in activation of the MKK4-JNK1 pathway. JNK1 activation then leads to cyclin B1/CDK1 upregulation, which further increases Bcl-2 and Bcl-XL phosphorylation. Together, these results indicate that cyclin B1/CDK1 upregulation in cancer cells undergoing 2-methoxyestradiol-induced mitotic catastrophe causes apoptosis via Bcl-XL phosphorylation.

A novel PI3K/mTOR dual inhibitor, CMG002, overcomes the chemoresistance in ovarian cancer.

OBJECTIVE: Ovarian cancer is the leading cause of gynecologic-related mortality worldwide. Despite successful initial treatment, overall survival rates are very low because tumors develop resistance to chemotherapeutic drugs. The PI3K/mTOR pathway is a key signaling pathway involved in drug resistance of ovarian cancer cells. The aim of this study was to examine the effect of a newly developed PI3K/mTOR dual inhibitor, CMG002, on chemoresistant ovarian cancer cells. METHODS: We examined the effects of CMG002, and its synergistic effects when combined with paclitaxel or cisplatin, on cell viability, cell cycle arrest, and apoptosis of PTX-resistant SKpac17 or cisplatin-resistant A2780cis ovarian cancer cells in vitro. Western blot analysis was performed to assess expression of PI3K, p-mTOR, p-Akt, p-S6, Bim, and caspase-3. In vivo studies were carried out in a xenograft mouse model, followed by TUNEL and immunohistochemical staining of excised tumor tissue. RESULTS: CMG002 showed marked toxicity against chemoresistant ovarian cancer cells and re-sensitized these cells to chemotherapeutic agents by suppressing cell proliferation and inducing G1 cell cycle arrest and apoptosis. In vivo xenograft studies revealed that treatment with CMG002, either alone or in combination with paclitaxel or cisplatin, led to a marked reduction in tumor growth. CMG002 caused marked suppression of mTOR (Ser2448), Akt (Ser473), Akt (Thr308), and S6 (Ser235/236) phosphorylation, both in vitro and in vivo. CONCLUSION: Taken together, CMG002, a very potent PI3K/mTOR dual inhibitor, induced cytotoxicity in chemoresistant ovarian cancer cells, suggesting that this novel inhibitor might be a new therapeutic strategy for chemoresistant ovarian cancer.

A comparison of patients' and pharmacists' satisfaction with medication counseling provided by community pharmacies: a cross-sectional survey.

BACKGROUND: Medication counseling is a critical component of pharmaceutical care to promote the safe and effective use of medications and to maximize therapeutic outcomes. The assessment of patients' and pharmacists' satisfaction with medication counseling services could be one of the vital parameters for predicting the quality of pharmacy services. No study has measured and compared both patients' and pharmacists' satisfaction with medication counseling. The objectives of this study were to describe and compare patients' and pharmacists' levels of satisfaction with medication counseling services offered by community pharmacists in South Korea. METHODS: This was a descriptive, cross-sectional survey. The online survey was distributed to patients and community pharmacists using a structured questionnaire. The questionnaires consisted of 4 main areas: (1) responders' characteristics (2) current state of medication counseling methods provided by community pharmacies (3) overall satisfaction with medication counseling (4) demand for the development of medication counseling standards. A comparison between patients and pharmacists was made using either a chi-square test or a Fisher's exact test. RESULTS: Between June 13, 2014 and July 15, 2014, a total of 252 patients and 620 pharmacists completed the survey. It was found that 47.3% of pharmacists and 34.0% of patients were satisfied with the current medication counseling service. Pharmacists showed a higher degree of satisfaction with the medication counseling service compared to patients (p <0.05). A major reason for patients not being satisfied with the medication counseling from community pharmacists was the insufficient time spent on counseling (51.2%). The pharmacists' perception of a major barrier to providing appropriate medication counseling for patients was the lack of time (24.3%). Moreover, a substantial number of patients (88%) and pharmacists (73%) supported the development of medication counseling standards to improve community pharmacist counseling services (p < 0.001). CONCLUSIONS: This study showed that both patients and pharmacists have low levels of satisfaction with the current medication counseling service offered by community pharmacists. This study provides baseline data for the development of national guidelines for medication counseling by pharmacists.

Interferon-induced transmembrane protein 1 (IFITM1) overexpression enhances the aggressive phenotype of SUM149 inflammatory breast cancer cells in a signal transducer and activator of transcription 2 (STAT2)-dependent manner.

BACKGROUND: Inflammatory breast cancer (IBC) is a very aggressive and lethal subtype of breast cancer that accounts for about 4 % of all breast cancers diagnosed in the United States. Despite the efforts of several investigators to identify the molecular factors driving the aggressive phenotype of IBC, a great deal is still unknown about the molecular underpinnings of the disease. In the present study, we investigated the role of interferon-induced transmembrane protein 1 (IFITM1), a well-known interferon-stimulated gene (ISG), in promoting the aggressiveness of SUM149 IBC cells. METHODS: Western blot and real-time polymerase chain reaction analyses were performed to assess the protein and messenger RNA (mRNA) levels of IFITM1 and other ISGs in three IBC cell lines: SUM149, MDA-IBC-3, and SUM190. IFITM1 expression and cellular localization were assessed by using immunofluorescence, while the tumorigenic potential was assessed by performing cell migration, invasion, and colony formation assays. Small interfering RNA and short hairpin RNA knockdowns, enzyme-linked immunosorbent assays, and luciferase assays were performed to determine the functional significance of IFITM1 and signal transducers and activators of transcription 1 and 2 (STAT1/2) in SUM149 cells. RESULTS: We found that IFITM1 was constitutively overexpressed at the mRNA and protein levels in triple-negative SUM149 IBC cells, but that it was not expressed in SUM190 and MDA-IBC-3 IBC cells, and that suppression of IFITM1 or blockade of the IFNα signaling pathway significantly reduced the aggressive phenotype of SUM149 cells. Additionally, we found that knockdown of STAT2 abolished IFITM1 expression and IFITM1 promoter activity in SUM149 cells and that loss of STAT2 significantly inhibited the ability of SUM149 cells to proliferate, migrate, invade, and form 2-D colonies. Notably, we found that STAT2-mediated activation of IFITM1 was particularly dependent on the chromatin remodeler brahma-related gene 1 (BRG1), which was significantly elevated in SUM149 cells compared with SUM190 and MDA-IBC-3 cells. CONCLUSIONS: These findings indicate that overexpression of IFITM1 enhances the aggressive phenotype of triple-negative SUM149 IBC cells and that this effect is dependent on STAT2/BRG1 interaction. Further studies are necessary to explore the potential of IFITM1 as a novel therapeutic target and prognostic marker for some subtypes of IBCs.

Mechanism of Ascorbate-Induced Cell Death in Human Pancreatic Cancer Cells: Role of Bcl-2, Beclin 1 and Autophagy.

The present study investigates the anticancer effect of ascorbate in MIA-PaCa-2 human pancreatic cancer cells using both in vitro and in vivo models, with a focus on assessing the role of oxidative stress and autophagy as important mechanistic elements in its anticancer actions. We showed that ascorbate suppresses the growth of human pancreatic cancer cells via the induction of oxidative stress and caspase-independent cell death. Ascorbate induces the formation of autophagosomes and the presence of autophagy inhibitors suppresses ascorbate-induced cell death. These data suggest that the induction of autophagosome formation contributes to ascorbate-induced pancreatic cancer cell death.

Growth inhibition of luteolin on HepG2 cells is induced via p53 and Fas/Fas-ligand besides the TGF-ß pathway.

Flavonoids, a class of natural polyphenolic compounds, inhibit cell cycle progression and induce apoptosis. This study was performed to investigate the antiproliferative effect of luteolin, the flavonoid isolated from Ixeris sonchifolia Hance, and to elucidate the detailed apoptotic mechanism in HCC cells. According to the result of MTT assay luteolin possessed antiproliferative effect, and HepG2 cells were the most sensitive to luteolin. Propidium iodide staining, fluorescence activated cell sorting analysis, western blot analysis and RT-PCR were applied to compare the difference of apoptotic event between the two HCC cell lines, with wild-type p53 (HepG2) or not (Hep3B) based on time and concentration. The treatment of luteolin upregulated the expression levels of transforming growth factor ß1 (TGF­ß1), p21WAF1/CIP1, p27KIP1, Smad4, and Fas in HCC cells. Thus, the expression of p21WAF1/CIP1 was controlled by another factor, such as TGF­ß1 in addition to p53, and notably the key factor might be p21WAF1/CIP1 in the remarkable switch to G1 cell cycle arrest in HepG2 cells rather than p27KIP1. Luteolin induced apoptotic cell death in Hep3B cells while caused G1 arrest in HepG2 cells. Taken together, we conclude that luteolin induces apoptosis from G1 arrest via three signaling pathways of TGF­ß1, p53, and Fas/Fas-ligand in HCC cells.

Targeting interferon response genes sensitizes aromatase inhibitor resistant breast cancer cells to estrogen-induced cell death.

INTRODUCTION: Estrogen deprivation using aromatase inhibitors (AIs) is currently the standard of care for postmenopausal women with hormone receptor-positive breast cancer. Unfortunately, the majority of patients treated with AIs eventually develop resistance, inevitably resulting in patient relapse and, ultimately, death. The mechanism by which resistance occurs is still not completely known, however, recent studies suggest that impaired/defective interferon signaling might play a role. In the present study, we assessed the functional role of IFITM1 and PLSCR1; two well-known interferon response genes in AI resistance. METHODS: Real-time PCR and Western blot analyses were used to assess mRNA and protein levels of IFITM1, PLSCR1, STAT1, STAT2, and IRF-7 in AI-resistant MCF-7:5C breast cancer cells and AI-sensitive MCF-7 and T47D cells. Immunohistochemistry (IHC) staining was performed on tissue microarrays consisting of normal breast tissues, primary breast tumors, and AI-resistant recurrence tumors. Enzyme-linked immunosorbent assay was used to quantitate intracellular IFNα level. Neutralizing antibody was used to block type 1 interferon receptor IFNAR1 signaling. Small interference RNA (siRNA) was used to knockdown IFITM1, PLSCR1, STAT1, STAT2, IRF-7, and IFNα expression. RESULTS: We found that IFITM1 and PLSCR1 were constitutively overexpressed in AI-resistant MCF-7:5C breast cancer cells and AI-resistant tumors and that siRNA knockdown of IFITM1 significantly inhibited the ability of the resistant cells to proliferate, migrate, and invade. Interestingly, suppression of IFITM1 significantly enhanced estradiol-induced cell death in AI-resistant MCF-7:5C cells and markedly increased expression of p21, Bax, and Noxa in these cells. Significantly elevated level of IFNα was detected in AI-resistant MCF-7:5C cells compared to parental MCF-7 cells and suppression of IFNα dramatically reduced IFITM1, PLSCR1, p-STAT1, and p-STAT2 expression in the resistant cells. Lastly, neutralizing antibody against IFNAR1/2 and knockdown of STAT1/STAT2 completely suppressed IFITM1, PLSCR1, p-STAT1, and p-STAT2 expression in the resistant cells, thus confirming the involvement of the canonical IFNα signaling pathway in driving the overexpression of IFITM1 and other interferon-stimulated genes (ISGs) in the resistant cells. CONCLUSION: Overall, these results demonstrate that constitutive overexpression of ISGs enhances the progression of AI-resistant breast cancer and that suppression of IFITM1 and other ISGs sensitizes AI-resistant cells to estrogen-induced cell death.

Role of cyclin B1/Cdc2 in mediating Bcl-XL phosphorylation and apoptotic cell death following nocodazole-induced mitotic arrest.

Treatment of cancer cells with microtubule inhibitors causes mitotic arrest, which subsequently leads to cell death via activation of the intrinsic apoptotic pathway. Mitotically arrested cells typically display increased phosphorylation (i.e., inactivation) of two key anti-apoptotic proteins, Bcl-2 and Bcl-XL , but the mechanisms that regulate their phosphorylation as well as their role in apoptotic cell death following mitotic arrest are still poorly understood at present, which are the focus of this study. We recently showed that cyclin B1 and cell division cycle 2 (Cdc2) proteins are strongly up-regulated in human breast cancer cells following treatment with nocodazole (a prototypical microtubule inhibitor), and their up-regulation plays a critical role in the development of mitotic prometaphase arrest. In this study, we present evidence showing that the up-regulated cyclin B1/Cdc2 complex in nocodazole-treated human breast cancer cells is also responsible for the increased phosphorylation of Bcl-2 and Bcl-XL . However, only the increased phosphorylation of Bcl-XL , but not the phosphorylation of Bcl-2, contributes to subsequent activation of the intrinsic cell death pathway. In addition, evidence is presented to show that mitotic arrest deficient 2 (MAD2) is a key upstream mediator of the up-regulation of cyclin B1/Cdc2 as well as the subsequent increase in phosphorylationof Bcl-2 and Bcl-XL in nocodazole-treated cancer cells. Together, these results reveal that the up-regulated cyclin B1/Cdc2 complex not only mediates prometaphase arrest in nocodazole-treated cells, but also activates the subsequent intrinsic cell death pathway in these cells via increased phosphorylation of Bcl-XL .

PDIp is a major intracellular oestrogen-storage protein that modulates tissue levels of oestrogen in the pancreas.

E(2) (17ß-oestradiol), a female sex hormone, has important biological functions in a woman's body. The pancreas, often considered a non-classical E(2)-targeting organ, is known to be functionally regulated by E(2), but little is known about how oestrogen actions are regulated in this organ. In the present study we report that PDIp (pancreas-specific protein disulfide isomerase), a protein-folding catalyst, can act as a major intracellular E(2) storage protein in a rat model to modulate the pancreatic tissue level, metabolism and action of E(2). The purified endogenous PDIp from both rat and human pancreatic tissues can bind E(2) with a K(d) value of approximately 150 nM. The endogenous PDIp-bound E(2) accounts for over 80% of the total protein-bound E(2) present in rat and human pancreatic tissues, and this binding protects E(2) from metabolic disposition and prolongs its duration of action. Importantly, we showed in ovariectomized female rats that the E(2) level in the pancreas reaches its highest level (9-fold increase over its basal level) at 24-48 h after a single injection of E(2), and even at 96 h its level is still approximately 5-fold higher. In contrast, the E(2) level in the uterus quickly returns to its basal level at 48 h after reaching its maximal level (approximately 2-fold increase) at 24 h. Taken together, these results show for the first time that PDIp is a predominant intracellular oestrogen storage protein in the pancreas, which offers novel mechanistic insights into the accumulation and action of oestrogen inside pancreatic cells.

Rapid generation of mitochondrial superoxide induces mitochondrion-dependent but caspase-independent cell death in hippocampal neuronal cells that morphologically resembles necroptosis.

Studies in recent years have revealed that excess mitochondrial superoxide production is an important etiological factor in neurodegenerative diseases, resulting from oxidative modifications of cellular lipids, proteins, and nucleic acids. Hence, it is important to understand the mechanism by which mitochondrial oxidative stress causes neuronal death. In this study, the immortalized mouse hippocampal neuronal cells (HT22) in culture were used as a model and they were exposed to menadione (also known as vitamin K(3)) to increase intracellular superoxide production. We found that menadione causes preferential accumulation of superoxide in the mitochondria of these cells, along with the rapid development of mitochondrial dysfunction and cellular ATP depletion. Neuronal death induced by menadione is independent of the activation of the MAPK signaling pathways and caspases. The lack of caspase activation is due to the rapid depletion of cellular ATP. It was observed that two ATP-independent mitochondrial nucleases, namely, AIF and Endo G, are released following menadione exposure. Silencing of their expression using specific siRNAs results in transient suppression (for ~12h) of mitochondrial superoxide-induced neuronal death. While suppression of the mitochondrial superoxide dismutase expression markedly sensitizes neuronal cells to mitochondrial superoxide-induced cytotoxicity, its over-expression confers strong protection. Collectively, these findings showed that many of the observed features associated with mitochondrial superoxide-induced cell death, including caspase independency, rapid depletion of ATP level, mitochondrial release of AIF and Endo G, and mitochondrial swelling, are distinctly different from those of apoptosis; instead they resemble some of the known features of necroptosis.

Critical role of cyclin B1/Cdc2 up-regulation in the induction of mitotic prometaphase arrest in human breast cancer cells treated with 2-methoxyestradiol.

Earlier studies showed that 2-methoxyestradiol (2ME(2)), an endogenous nonpolar metabolite of estradiol-17ß, is a strong inducer of G(2)/M cell cycle arrest (based on analysis of cellular DNA content) in human cancer cell lines. The present study sought to investigate the molecular mechanism underlying 2ME(2)-induced cell cycle arrest. We found that 2ME(2) can selectively induce mitotic prometaphase arrest, but not G(2) phase arrest, in cultured MDA-MB-435s and MCF-7 human breast cancer cells. During the induction of prometaphase arrest, there is a time-dependent initial up-regulation of cyclin B1 and Cdc2 proteins, occurring around 12-24h. The strong initial up-regulation of cyclin B1 and Cdc2 matches in timing the 2ME(2)-induced prometaphase arrest. The 2ME(2)-induced prometaphase arrest is abrogated by selective knockdown of cyclin B1 and Cdc2, or by pre-treatment of cells with roscovitine, an inhibitor of cyclin-dependent kinases, or by co-treatment of cells with cycloheximide, a protein synthesis inhibitor that was found to suppress the early up-regulation of cyclin B1 and Cdc2. In addition, we provided evidence showing that MAD2 and JNK1 are important upstream mediators of 2ME(2)-induced up-regulation of cyclin B1 and Cdc2 as well as the subsequent induction of mitotic prometaphase arrest. In conclusion, treatment of human cancer cells with 2ME(2) causes up-regulation of cyclin B1 and Cdc2, which then mediate the induction of mitotic prometaphase arrest.

Role of cyclin B1/Cdc2 up-regulation in the development of mitotic prometaphase arrest in human breast cancer cells treated with nocodazole.

BACKGROUND: During a normal cell cycle, the transition from G2 phase to mitotic phase is triggered by the activation of the cyclin B1-dependent Cdc2 kinase. Here we report our finding that treatment of MCF-7 human breast cancer cells with nocodazole, a prototypic microtubule inhibitor, results in strong up-regulation of cyclin B1 and Cdc2 levels, and their increases are required for the development of mitotic prometaphase arrest and characteristic phenotypes. METHODOLOGY/PRINCIPAL FINDINGS: It was observed that there was a time-dependent early increase in cyclin B1 and Cdc2 protein levels (peaking between 12 and 24 h post treatment), and their levels started to decline after the initial increase. This early up-regulation of cyclin B1 and Cdc2 closely matched in timing the nocodazole-induced mitotic prometaphase arrest. Selective knockdown of cyclin B1or Cdc2 each abrogated nocodazole-induced accumulation of prometaphase cells. The nocodazole-induced prometaphase arrest was also abrogated by pre-treatment of cells with roscovitine, an inhibitor of cyclin-dependent kinases, or with cycloheximide, a protein synthesis inhibitor that was found to suppress cyclin B1 and Cdc2 up-regulation. In addition, we found that MAD2 knockdown abrogated nocodazole-induced accumulation of cyclin B1 and Cdc2 proteins, which was accompanied by an attenuation of nocodazole-induced prometaphase arrest. CONCLUSIONS/SIGNIFICANCE: These observations demonstrate that the strong early up-regulation of cyclin B1 and Cdc2 contributes critically to the rapid and selective accumulation of prometaphase-arrested cells, a phenomenon associated with exposure to microtubule inhibitors.

Critical role of the JNK-p53-GADD45α apoptotic cascade in mediating oxidative cytotoxicity in hippocampal neurons.

BACKGROUND AND PURPOSE: Glutamate-induced oxidative stress plays a critical role in the induction of neuronal cell death in a number of disease states. We sought to determine the role of the c-Jun NH(2) -terminal kinase (JNK)-p53-growth arrest and DNA damage-inducible gene (GADD) 45α apoptotic cascade in mediating glutamate-induced oxidative cytotoxicity in hippocampal neuronal cells. EXPERIMENTAL APPROACH: HT22 cells, a mouse hippocampal neuronal cell line, were treated with glutamate to induce oxidative stress in vitro. Kainic acid-induced oxidative damage to the hippocampus in rats was used as an in vivo model. The signalling molecules along the JNK-p53-GADD45α cascade were probed with various means to determine their contributions to oxidative neurotoxicity. KEY RESULTS: Treatment of HT22 cells with glutamate increased the mRNA and protein levels of GADD45α, and these increases were suppressed by p53 knock-down. Knock-down of either p53 or GADD45α also prevented glutamate-induced cell death. Glutamate-induced p53 activation was preceded by accumulation of reactive oxygen species, and co-treatment with N-acetyl-cysteine prevented glutamate-induced p53 activation and GADD45α expression. Knock-down of MKK4 or JNK, or the presence of SP600125 (a JNK inhibitor), each inhibited glutamate-induced p53 activation and GADD45α expression. In addition, we also confirmed the involvement of GADD45α in mediating kainic acid-induced hippocampal oxidative neurotoxicity in vivo. CONCLUSIONS: AND IMPLICATIONS Activation of the JNK-p53-GADD45α cascade played a critical role in mediating oxidative cytotoxicity in hippocampal neurons. Pharmacological inhibition of this signalling cascade may provide an effective strategy for neuroprotection.

Mechanism for the protective effect of resveratrol against oxidative stress-induced neuronal death.

Oxidative stress can induce cytotoxicity in neurons, which plays an important role in the etiology of neuronal damage and degeneration. This study sought to determine the cellular and biochemical mechanisms underlying resveratrol's protective effect against oxidative neuronal death. Cultured HT22 cells, an immortalized mouse hippocampal neuronal cell line, were used as an in vitro model, and oxidative stress and neurotoxicity were induced in these neuronal cells by exposure to high concentrations of glutamate. Resveratrol strongly protected HT22 cells from glutamate-induced oxidative cell death. Resveratrol's neuroprotective effect was independent of its direct radical scavenging property, but instead was dependent on its ability to selectively induce the expression of mitochondrial superoxide dismutase (SOD2) and, subsequently, reduce mitochondrial oxidative stress and damage. The induction of mitochondrial SOD2 by resveratrol was mediated through the activation of the PI3K/Akt and GSK-3beta/beta-catenin signaling pathways. Taken together, the results of this study show that up-regulation of mitochondrial SOD2 by resveratrol represents an important mechanism for its protection of neuronal cells against oxidative cytotoxicity resulting from mitochondrial oxidative stress.

Induction of a reversible, non-cytotoxic S-phase delay by resveratrol: implications for a mechanism of lifespan prolongation and cancer protection.

BACKGROUND AND PURPOSE: Resveratrol (RES) has been shown to prolong lifespan and prevent cancer formation. At present, the precise cellular mechanisms of RES actions are still not clearly understood, and this is the focus of this study. EXPERIMENTAL APPROACH: Using human hepatocellular carcinoma-derived HepG2 cells as a model, we studied RES-induced changes in cell growth, cell cycle progression and apoptosis. KEY RESULTS: RES at lower concentrations induced a strong but reversible S-phase delay and mild DNA synthesis inhibition, yet without causing apoptotic or necrotic cell death. At high concentrations, RES induced apoptosis, which is mainly mediated by the mitochondrial pathway. Overall, RES was a relatively weak apoptotic agent. Mechanistically, MEK inhibition was identified as an important early signalling event for RES-induced apoptosis. In comparison, activation of CDK2 and checkpoint kinase 2, and inhibition of phosphatidylinositol 3'-kinase/Akt signalling pathway contributed to the induction by RES of a reversible, non-cytotoxic S-phase delay. CONCLUSION AND IMPLICATIONS: It is hypothesized that the induction of a non-cytotoxic S-phase delay may represent a useful mechanistic strategy for lifespan prolongation and cancer prevention. When cell cycles are selectively slowed down in the S phase, it would cumulatively increase the total lifespan of an organism if the total numbers of cell divisions of a given organism are assumed to remain basically constant. Likewise, when cells proceed through the cell cycles at a reduced pace during DNA replication, it may allow cells more time to repair the damaged DNA, and thereby reduce the chances for mutagenesis and tumour initiation.

Mechanism of glutamate-induced neurotoxicity in HT22 mouse hippocampal cells.

Glutamate is an endogenous excitatory neurotransmitter. At high concentrations, it is neurotoxic and contributes to the development of certain neurodegenerative diseases. There is considerable controversy in the literature with regard to whether glutamate-induced cell death in cultured HT22 cells (an immortalized mouse hippocampal cell line) is apoptosis, necrosis, or a new form of cell death. The present study focused on investigating the mechanism of glutamate-induced cell death. We found that glutamate induced, in a time-dependent manner, both necrosis and apoptosis in HT22 cells. At relatively early time points (8-12 h), glutamate induced mostly necrosis, whereas at late time points (16-24 h), it induced mainly apoptosis. Glutamate-induced mitochondrial oxidative stress and dysfunction were crucial early events required for the induction of apoptosis through the release of the mitochondrial apoptosis-inducing factor (AIF), which catalyzed DNA fragmentation (an ATP-independent process). Glutamate-induced cell death proceeded independently of the Bcl-2 family proteins and caspase activation. The lack of caspase activation likely resulted from the lack of intracellular ATP when the mitochondrial functions were rapidly disrupted by the mitochondrial oxidative stress. In addition, it was observed that activation of JNK, p38, and ERK signaling molecules was also involved in the induction of apoptosis by glutamate. In conclusion, glutamate-induced apoptosis is AIF-dependent but caspase-independent, and is accompanied by DNA ladder formation but not chromatin condensation.

Signal pathway of 17beta-estradiol-induced MUC5B expression in human airway epithelial cells.

MUC5B is a major mucin of the human respiratory tract, and it is not clear how MUC5B expression is regulated in various airway diseases. The goal of this study was to determine the mechanisms by which 17beta-estradiol induces MUC5B gene expression in airway epithelial cells. It was found that E2, a sex hormone, stimulates MUC5B gene overexpression by interaction with estrogen receptor alpha (ERalpha) and by acting through extracellular signal-regulated kinase 1/2 (ERK1/2)-mitogen-activated protein kinase (MAPK). Pretreatment with ER antagonist ICI 182,780 blocked both E2-induced ERK1/2-MAPK activation and MUC5B gene expression. It was also found that the activation of p90 ribosomal S 6 protein kinase 1 (RSK1), cAMP-response element-binding protein (CREB), and cAMP-response element (CRE) (-956 region of the MUC5B promoter)-responsive signaling cascades via ERK1/2 MAPK are crucial aspects of the intracellular mechanisms that mediate MUC5B gene expression. Taken together, these studies give additional insights into the molecular mechanism of hormone-induced MUC5B gene expression and enhance our understanding of abnormal mucin secretion in response to hormonal imbalances.