Neuroprotective Effects of Probucol against Rotenone-Induced Toxicity via Suppression of Reactive Oxygen Species Production in SH-SY5Y Cells.

Probucol is a hyperlipidemic drug with antioxidant properties. It has been reported to prevent mitochondrial dysfunction, reduce oxidative stress, and suppress neurotoxicity in neurodegenerative disease models, including Parkinson's disease models. However, the molecular mechanisms underlying the neuroprotective effects of probucol have been not examined yet. Thus, in this study, we investigated whether probucol can alleviate the effects of a mitochondrial complex I inhibitor, rotenone, on a human neuroblastoma cell line (SH-SY5Y). We evaluated the cell viability and cytotoxicity and apoptosis rates of SH-SY5Y cells treated with rotenone and probucol or edaravone, a known free-radical scavenger. Subsequently, mitochondrial membrane potential (MMP) and reactive oxygen species (ROS) levels in the cells were evaluated to determine the effects of probucol on mitochondrial function. We found that rotenone caused cytotoxicity, cell apoptosis, and mitochondrial dysfunction, enhanced ROS generation, and impaired MMP. However, probucol could inhibit this rotenone-induced decrease in cell viability, MMP loss, intracellular ROS generation, and apoptosis. These results suggest that probucol exerts neuroprotective effects via MMP stabilization and the inhibition of ROS generation. Additionally, this effect of probucol was equal to or greater than and more persistent than that of edaravone. Thus, we believe probucol may be a promising drug for the treatment of neurodegenerative diseases, such as Parkinson's and Alzheimer's diseases.

An educational intervention improved knowledge of dietary supplements in college students.

BACKGROUND: We have previously reported on the prevalence of dietary supplements among college students; it was deduced that their intake of supplements increased according to their grade (i.e., 13.1% in the first grade to 20.5% in the sixth grade). We also reported that some students had experienced adverse events in Japan due to their intake of these supplements. However, awareness of dietary supplements among college students remains limited, even among pharmaceutical students. Being appropriately educated about them is important for pharmaceutical students, both for themselves as well as for their future careers as pharmacists. METHODS: We conducted a lecture-based educational intervention about dietary supplements on 328 college students in Japan-184 from pharmaceutical science and 144 from environmental science or food and life science disciplines. The purpose of this study was to evaluate the effects of an educational intervention on college students' understanding of dietary supplements. The intervention involved a lecture that covered the quality of dietary supplements, how they differed from drugs, and a summary of their adverse events. The lecture was evaluated using a 14-question questionnaire. We then compared the pre- and post-intervention responses to the same questionnaire using a Wilcoxon signed-rank test. The questions were assessed using a Likert scale that ranged from "strongly agree" to "strongly disagree"; the latter being the preferred answer. RESULTS: Before the intervention had taken place, the students' understanding of dietary supplements was shown to be deficient. Conversely, post-intervention, their knowledge levels had significantly improved, especially concerning agreement on whether "Dietary supplements are safe because they are just food items". Pre-intervention, 2.7% strongly agreed and 37.5% agreed; post-intervention, 1.2% strongly agreed and 15.6% agreed. On whether "Dietary supplements made from natural ingredients or herbs are safe", at the pre-intervention stage 2.8% strongly agreed and 44.0% agreed and post-intervention, 2.2% strongly agreed and 16.9% agreed. On whether "Dietary supplements made from food items are safe", 4.0% strongly agreed and 43.6% agreed pre-intervention and 0.9% strongly agreed and 16.6% agreed post-intervention. Despite there being a greater number of pharmaceutical students who had a correct understanding of dietary supplements before the intervention, these students still showed improvement after the lecture. CONCLUSION: An intervention in the form of a single educational lecture has the capacity to improve college students' understanding of dietary supplements. It is important for pharmacists to be appropriately educated about dietary supplements when they consult with patients. We will evaluate the long-term effects of the intervention on the alumni (pharmacists) in a subsequent study.

MicroRNA-101 inhibits the expression of Rhes, a striatal-enriched small G-protein, at the post-transcriptional level in vitro.

OBJECTIVE: Ras homolog enriched in striatum (Rhes) is a small GTP-binding protein that is predominantly localized in the striatal region of the brain. Rhes affects various signaling pathways and plays important roles in Huntington's disease development caused by striatal anomalies. However, the mechanism underlying the regulation of Rhes expression is not fully understood. We hypothesized that Rhes expression might be regulated by microRNAs (miRNAs), which are small noncoding RNAs that regulate gene expression by interacting with the 3'-untranslated region (3'UTR) of mRNA. This study therefore investigated the interaction between miRNAs and the Rhes mRNA 3'UTR. RESULTS: The results of luciferase assay showed that miR-101, the miRNA determined to have the highest possibility of interacting with the Rhes mRNA 3'UTR using DIANA-microT, significantly inhibits luciferase activity, suggesting that miR-101 directly targets the Rhes mRNA 3'UTR. Additionally, Rhes protein levels in cultured cells co-transfected with a plasmid containing the complete Rhes cDNA and miR-101 were significantly downregulated by miR-101 as demonstrated by western blot analysis. These results support our hypothesis that Rhes expression is regulated by miRNA and indicate that miR-101 may be a potent modulator of Rhes expression in striatal neurons.

Bergamottin Promotes Adipocyte Differentiation and Inhibits Tumor Necrosis Factor-α-induced Inflammatory Cytokines Induction in 3T3-L1 Cells.

Nowadays, a lot of food ingredients are marketed as dietary supplements for health. Because the effectiveness and mechanisms of these compounds have not been fully characterized, they might have unknown functions. Therefore, we investigated the effect of several food ingredients (Bergamottin, Chrysin, L-Citrulline and ß-Carotene) known as health foods on adipocyte differentiation by using 3T3-L1 preadipocytes. In this study, we found that Bergamottin, a furanocoumarin isolated from grapefruit juice, promotes adipocyte differentiation. In addition, Bergamottin increases the expression of adiponectin, an anti-inflammatory adipokine, and peroxisome proliferator activated receptor γ (PPARγ), a nuclear receptor regulating adipocyte differentiation. Furthermore, the anti-inflammatory activity of Bergamottin was demonstrated by its inhibition of the activation of nuclear factor-κB (NF-κB), an inflammatory transcription factor. Stimulation of mature 3T3-L1 adipocytes by tumor necrosis factor-α (TNF-α) decreased the expression of the endogeneous NF-κB inhibitor, IκBα. Treatment with Bergamottin further decreased the TNF-α-induced change in IκBα expression, suggesting that Bergamottin mediated the inhibition of NF-κB activation. In addition, Bergamottin decreased the TNF-α-induced increase in the mRNA levels of pro-inflammatory adipokines, monocyte chemoattractant protein-1 and interleukin-6. Taken together, our results show that Bergamottin treatment could inhibit inflammatory activity through promoting adipocyte differentiation, which in turn suggests that Bergamottin has the potential to minimize the risk factors of metabolic syndrome.

St. John's wort promotes adipocyte differentiation and modulates NF-κB activation in 3T3-L1 cells.

St. John's wort (SJW), or Hypericum perforatum, is a perennial herb that has been used in the treatment of depression in several countries. Though its therapeutic effect on depression has been extensively studied, its influence on metabolic syndrome is yet to be fully characterized. Therefore, we investigated the effect of SJW extract on adipocyte differentiation and its anti-inflammatory effects by using 3T3-L1 preadipocytes. Oil Red O staining indicated that SJW promotes adipocyte differentiation, while immunoblots indicated that SJW increases the expression of peroxisome proliferator activated receptor γ (PPARγ), a nuclear receptor regulating adipocyte differentiation, and adiponectin, an anti-inflammatory adipokine. Furthermore, the anti-inflammatory activity of SJW was demonstrated by its inhibition of the activation of nuclear factor-κB (NF-κB), an inflammatory transcription factor. Stimulation of mature 3T3-L1 adipocytes by tumor necrosis factor-α (TNF-α) decreased the expression of the NF-κB inhibitor IκBα, and increased its phosphorylation. Treatment with SJW further decreased the TNF-α-induced perturbation in IκBα expression and phosphorylation, which indicated that SJW mediated the inhibition of NF-κB activation. In addition, SJW decreased the TNF-α-induced increase in the mRNA levels of pro-inflammatory adipokines, interleukin-6 (IL-6), and monocyte chemoattractant protein-1 (MCP-1). Collectively, our results indicate that SJW treatment could promote adipocyte differentiation probably through its anti-inflammatory activity, which in turn suggests that SJW has the potential to minimize the risk factors of metabolic syndrome.

Identification of muscle-specific microRNAs in serum of muscular dystrophy animal models: promising novel blood-based markers for muscular dystrophy.

Duchenne muscular dystrophy (DMD) is a lethal X-linked disorder caused by mutations in the dystrophin gene, which encodes a cytoskeletal protein, dystrophin. Creatine kinase (CK) is generally used as a blood-based biomarker for muscular disease including DMD, but it is not always reliable since it is easily affected by stress to the body, such as exercise. Therefore, more reliable biomarkers of muscular dystrophy have long been desired. MicroRNAs (miRNAs) are small, ∼22 nucleotide, noncoding RNAs which play important roles in the regulation of gene expression at the post-transcriptional level. Recently, it has been reported that miRNAs exist in blood. In this study, we hypothesized that the expression levels of specific serum circulating miRNAs may be useful to monitor the pathological progression of muscular diseases, and therefore explored the possibility of these miRNAs as new biomarkers for muscular diseases. To confirm this hypothesis, we quantified the expression levels of miRNAs in serum of the dystrophin-deficient muscular dystrophy mouse model, mdx, and the canine X-linked muscular dystrophy in Japan dog model (CXMD(J)), by real-time PCR. We found that the serum levels of several muscle-specific miRNAs (miR-1, miR-133a and miR-206) are increased in both mdx and CXMD(J). Interestingly, unlike CK levels, expression levels of these miRNAs in mdx serum are little influenced by exercise using treadmill. These results suggest that serum miRNAs are useful and reliable biomarkers for muscular dystrophy.

α-Synuclein Transgenic Drosophila As a Model of Parkinson's Disease and Related Synucleinopathies.

α-Synuclein (α-Syn) is a major component of protein inclusions known as Lewy bodies, which are hallmarks of synucleinopathies such as Parkinson's disease (PD). The α-Syn gene is one of the familial PD-causing genes and is also associated with an increased risk of sporadic PD. Numerous studies using α-Syn expressing transgenic animals have indicated that α-Syn plays a critical role in the common pathogenesis of synucleinopathies. Drosophila melanogaster has several advantages for modeling human neurodegenerative diseases and is widely used for studying their pathomechanisms and therapies. In fact, Drosophila models expressing α-Syn have already been established and proven to replicate several features of human PD. In this paper, we review the current research on synucleinopathies using α-Syn Drosophila models and, moreover, explore the possibilities of these models for comprehensive genetic analyses and large-scale drug screening towards elucidating the molecular pathogenesis and developing therapies for synucleinopathies.

Mechanisms of hybrid oligomer formation in the pathogenesis of combined Alzheimer's and Parkinson's diseases.

BACKGROUND: Misfolding and pathological aggregation of neuronal proteins has been proposed to play a critical role in the pathogenesis of neurodegenerative disorders. Alzheimer's disease (AD) and Parkinson's disease (PD) are frequent neurodegenerative diseases of the aging population. While progressive accumulation of amyloid beta protein (Abeta) oligomers has been identified as one of the central toxic events in AD, accumulation of alpha-synuclein (alpha-syn) resulting in the formation of oligomers and protofibrils has been linked to PD and Lewy body Disease (LBD). We have recently shown that Abeta promotes alpha-syn aggregation and toxic conversion in vivo, suggesting that abnormal interactions between misfolded proteins might contribute to disease pathogenesis. However the molecular characteristics and consequences of these interactions are not completely clear. METHODOLOGY/PRINCIPAL FINDINGS: In order to understand the molecular mechanisms involved in potential Abeta/alpha-syn interactions, immunoblot, molecular modeling, and in vitro studies with alpha-syn and Abeta were performed. We showed in vivo in the brains of patients with AD/PD and in transgenic mice, Abeta and alpha-synuclein co-immunoprecipitate and form complexes. Molecular modeling and simulations showed that Abeta binds alpha-syn monomers, homodimers, and trimers, forming hybrid ring-like pentamers. Interactions occurred between the N-terminus of Abeta and the N-terminus and C-terminus of alpha-syn. Interacting alpha-syn and Abeta dimers that dock on the membrane incorporated additional alpha-syn molecules, leading to the formation of more stable pentamers and hexamers that adopt a ring-like structure. Consistent with the simulations, under in vitro cell-free conditions, Abeta interacted with alpha-syn, forming hybrid pore-like oligomers. Moreover, cells expressing alpha-syn and treated with Abeta displayed increased current amplitudes and calcium influx consistent with the formation of cation channels. CONCLUSION/SIGNIFICANCE: These results support the contention that Abeta directly interacts with alpha-syn and stabilized the formation of hybrid nanopores that alter neuronal activity and might contribute to the mechanisms of neurodegeneration in AD and PD. The broader implications of such hybrid interactions might be important to the pathogenesis of other disorders of protein misfolding.

Alpha-synuclein alters Notch-1 expression and neurogenesis in mouse embryonic stem cells and in the hippocampus of transgenic mice.

Altered expression and mutations in alpha-synuclein (alpha-syn) have been linked to Parkinson's disease (PD) and related disorders. The neurological alterations in PD patients have been associated with degeneration of dopaminergic cells and other neuronal populations. Moreover, recent studies in murine models have shown that alterations in neurogenesis might also contribute to the neurodegenerative phenotype. However, the mechanisms involved and the effects of alpha-syn expression on neurogenesis are not yet clear. To this end, murine embryonic stem (mES) cells were infected with lentiviral (LV) vectors expressing wild-type (WT) and mutant alpha-syn. Compared with mES cells infected with LV-green fluorescent protein (GFP), cells expressing WT and mutant alpha-syn showed reduced proliferation as indicated by lower 5-bromo-2'-deoxyuridine uptake, increased apoptosis, and reduced expression of neuronal markers such as neuron specific enolase and beta-III tubulin. The alterations in neurogenesis in alpha-syn-expressing mES cells were accompanied by a reduction in Notch-1 and Hairy and enhancer of split-5 (Hes-5) mRNA and protein levels. Moreover, levels of total Notch-1 and Notch intracellular domain (NICD) were lower in mES cells expressing WT and mutant alpha-syn compared with GFP controls. The reduced survival of alpha-syn-expressing mES cells was reverted by overexpressing constitutively active NICD. Similarly, in alpha-syn transgenic mice, the alterations in neurogenesis in the hippocampal subgranular zone were accompanied by decreased Notch-1, NICD, and Hes-5 expression. Together, these results suggest that accumulation of alpha-syn might impair survival of NPCs by interfering with the Notch signaling pathway. Similar mechanisms could be at play in PD and Lewy body disease.

Statins reduce neuronal alpha-synuclein aggregation in in vitro models of Parkinson's disease.

Aggregation of alpha-synuclein (alpha-syn) is believed to play a critical role in the pathogenesis of disorders such as dementia with Lewy bodies and Parkinson's disease. The function of alpha-syn remains unclear, although several lines of evidence suggest that alpha-syn is involved in synaptic vesicle trafficking probably via lipid binding. Moreover, interactions with cholesterol and lipids have been shown to be involved in alpha-syn aggregation. In this context, the main objective of this study was to determine if statins--cholesterol synthesis inhibitors--might interfere with alpha-syn accumulation in cellular models. For this purpose, we studied the effects of lovastatin, simvastatin, and pravastatin on the accumulation of alpha-syn in a stably transfected neuronal cell line and in primary human neurons. Statins reduced the levels of alpha-syn accumulation in the detergent insoluble fraction of the transfected cells. This was accompanied by a redistribution of alpha-syn in caveolar fractions, a reduction in oxidized alpha-syn, and enhanced neurite outgrowth. In contrast, supplementation of the media with cholesterol increased alpha-syn aggregation in detergent insoluble fractions of transfected cells and was accompanied by reduced neurite outgrowth. Taken together, these results suggest that regulation of cholesterol levels with cholesterol inhibitors might be a novel approach for the treatment of Parkinson's disease.

The utility of SELENBP1 gene expression as a biomarker for major psychotic disorders: replication in schizophrenia and extension to bipolar disorder with psychosis.

While microarray studies are generating novel insights into the etiology of major psychiatric disorders, the validation of microarray-identified candidate genes and their role in the causality of these disorders has been less often studied. We have previously demonstrated, by microarray, up-regulation of SELENBP1 in the brain and blood of patients with schizophrenia. The main aim of the current study was to validate this finding using quantitative real-time PCR (QPCR) in an independent brain cohort that included patients with bipolar disorder. Our sample consisted of mRNAs from the dorsolateral prefrontal cortex (dlPFC) of 34 schizophrenic patients, 33 bipolar disorder patients (including 20 with psychotic history), and 34 normal control subjects. QPCR was employed to assess gene expression changes, with C(T) values analyzed using an ANCOVA approach. The results demonstrated that SELENBP1 mRNA was upregulated in schizophrenic brains versus controls (P = 0.046) and, in addition, that SELENBP1 gene expression was strongly positively correlated with presence of psychosis across diagnoses (P < 0.001, increased by 12%). Based on these findings, we conclude that elevated SELENBP1 is a possibly consistent feature in the schizophrenic brain and that this finding could underlie some commonalities of psychosis across the boundaries of diagnoses. Future studies should exploit DNA-based methods and molecular investigations on the role of SELENBP1 in order to gain insights into the nature of its influence on schizophrenia and psychotic symptoms.

alpha-Synuclein aggregates interfere with Parkin solubility and distribution: role in the pathogenesis of Parkinson disease.

Parkinson disease (PD) belongs to a heterogeneous group of neurodegenerative disorders with movement alterations, cognitive impairment, and alpha-synuclein accumulation in cortical and subcortical regions. Jointly, these disorders are denominated Lewy body disease. Mutations in the parkin gene are the most common cause of familial parkinsonism, and a growing number of studies have shown that stress factors associated with sporadic PD promote parkin accumulation in the insoluble fraction. alpha-Synuclein and parkin accumulation and mutations in these genes have been associated with familial PD. To investigate whether alpha-synuclein accumulation might be involved in the pathogenesis of these disorders by interfering with parkin solubility, synuclein-transfected neuronal cells were transduced with lentiviral vectors expressing parkin. Challenging neurons with proteasome inhibitors or amyloid-beta resulted in accumulation of insoluble parkin and, to a lesser extent, alpha-tubulin. Similarly to neurons in the brains of patients with Lewy body disease, in co-transduced cells alpha-synuclein and parkin colocalized and co-immunoprecipitated. These effects resulted in decreased parkin and alpha-tubulin ubiquitination, accumulation of insoluble parkin, and cytoskeletal alterations with reduced neurite outgrowth. Taken together, accumulation of alpha-synuclein might contribute to the pathogenesis of PD and other Lewy body diseases by promoting alterations in parkin and tubulin solubility, which in turn might compromise neural function by damaging the neuronal cytoskeleton. These studies provide a new perspective on the potential nature of pathogenic alpha-synuclein and parkin interactions in Parkinson disease.

Plasma levels of DJ-1 as a possible marker for progression of sporadic Parkinson's disease.

DJ-1 is a multifunctional protein whose loss of function by gene mutations may play a causative role for familial Parkinson's disease (PD). A recent study has shown that the expression of this molecule is upregulated in both brains and cerebrospinal fluids (CSF) in various neurological disorders, including sporadic PD, Alzheimer's disease (AD) and stroke, raising a possibility that DJ-1 could be a potential biomarker for these diseases. In this context, the main objective of the present study was to determine if DJ-1 was increased in the plasma of PD patients. For this purpose, blood plasma samples collected from sporadic PD patients, dementia with Lewy bodies (DLB) and healthy age-matched controls were analyzed by immunoblotting and enzyme-linked immunosorbent assay. The results showed that the plasma DJ-1 levels in PD (n=104) were higher than those in control (n=80) (p<0.05). Moreover, the plasma DJ-1 levels in the advanced stage of PD (n=52, Yahr III-IV) were higher than those in the early stage of PD (n=52, Yahr I-II) (p<0.05), demonstrating that the plasma DJ-1 was correlated with the disease severity in PD. Plasma DJ-1 levels were also significantly higher in DLB (n=30) compared with both controls and early stage of PD (p<0.01). Taken together, these results suggest that the plasma DJ-1 could be a useful biomarker for the evaluation of the disease severity in PD and possibly in other Lewy body diseases.

ATP modulates the release of noradrenaline through two different prejunctional receptors on the adrenergic nerves of rat prostate.

1. The effects of adenosine and ATP receptor agonists on the release of endogenous noradrenaline from electrically stimulated (2 Hz, 0.1 msec) rat prostate were examined in order to clarify the pharmacological properties of prejunctional receptors for adenosine and ATP on the adrenergic nerve varicosities in the prostate. Noradrenaline was quantified by HPLC coupled with electrochemical detection techniques. 2. Both adenosine and ATP receptor agonists (1 micromol/L) inhibited noradrenaline release and the relative order of inhibitory effect was N(6)-cyclopentyl-adenosine (CPA) > 5'-N-ethylcarboxamidoadenosine > 2-chloroadenosine > adenosine > 2-methylthio-ATP (2mSATP) > AMP > ATP. 3. The adenosine receptor agonist CPA (1 nmol/L-1 micromol/L) and the ATP receptor agonist 2mSATP (100 nmol/L-100 micromol/L) inhibited the stimulation-induced release of noradrenaline in a concentration-dependent manner. The concentrations of CPA and 2mSATP that produced 50% inhibition of noradrenaline release were 9.6 nmol/L and 1.4 micromol/L, respectively. 4. 1,3-Dipropyl-8-cyclopentylxanthine, an adenosine A(1) receptor antagonist, significantly reduced the inhibitory effects of not only CPA, but also 2mSATP. 5. Suramin, an ATP receptor antagonist, significantly reduced the inhibitory effects of 2mSATP, but not those of CPA. 6. Pyridoxalphosphate-6-azophenyl-2',4'-disulphonic acid, another ATP receptor antagonist, had no effect on the inhibitory action of either agonist. 7. These results suggest that, in the sympathetic nerve terminals of rat prostate, adenosine and ATP induce inhibition of noradrenaline release via the activation of adenosine A(1) and/or xanthine-sensitive ATP receptors, which play an inhibitory regulatory role in adrenergic neurotransmission in the prostate.

Involvement of the paxillin pathway in JB6 Cl41 cell transformation.

Paxillin is a substrate of the Src tyrosine onco-kinase and is involved in cell transformation, cell spreading, migration, and cancer development mediated through the mitogen-activated protein kinase signaling cascades. Here, we showed that paxillin plays a key role in skin cell transformation induced by epidermal growth factor (EGF) or 12-O-tetradecanoylphorbol-13-acetate (TPA). To investigate the mechanism of paxillin's role in cell transformation, we established a paxillin knockdown stably transfected cell line by introducing small interfering RNA-paxillin (si-paxillin). The si-paxillin cells displayed a dramatic suppression of cell proliferation and anchorage-independent cell transformation induced by EGF or TPA compared with si-mock control cells. In si-paxillin cells, decreased activator protein-1 (AP-1)-dependent luciferase activity corresponded with suppressed AP-1 DNA binding activity. Importantly, knockdown of paxillin inhibited EGF- or TPA-induced c-Jun phosphorylation at Ser(63) and Ser(73). Furthermore, total c-Jun protein level was dramatically decreased in si-paxillin cells and was dependent on serum deprivation time. The down-regulation of c-Jun was restored in si-paxillin cells by treatment with the proteasome inhibitor lactacystin but not by the lysosome inhibitor leupeptin. These results clearly provided evidence that paxillin regulates c-Jun protein level and plays a key role in cell transformation most likely through the regulation of c-Jun stability.

Theaflavin-3, 3'-digallate induces epidermal growth factor receptor downregulation.

Black tea is one of the most popular beverages worldwide and especially in Western nations. Theaflavins, a mixture of theaflavin (TF-1), theaflavin-3-gallate (TF-2a), theaflavin-3'-gallate (TF-2b), and theaflavin-3,3'-digallate (TF-3) are the major components of black tea. Among these black tea components, theaflavin is generally considered to be the more effective component for the inhibition of carcinogenesis. Recently, TF-3 has been shown to have an antiproliferative effect on tumor cells, but the mechanism is not clear. In this study, we showed that TF-3-induced internalization and downregulation of the epidermal growth factor receptor (EGFR). These results suggested that TF-3 induces EGFR endocytosis and degradation. We further showed that TF-3 stimulated EGFR ubiquitination and tyrosine kinase activation. Interestingly, TF-3-induced EGFR downregulation is inhibited by the proteasome inhibitor, MG132, but not by the EGFR-specific receptor tyrosine kinase inhibitor, AG1478. Furthermore, pretreatment with TF-3 inhibited EGF-induced EGFR autophosphorylation, ERKs phosphorylation and AP-1 activation in JB6 Cl41 cells. In addition, TF-3 inhibited EGF-induced anchorage-independent cell transformation. Overall, our results indicate that TF-3 might exert chemopreventive effects through the downregulation of the EGFR.

Effects of MAP kinase inhibitors on epidermal growth factor-induced neoplastic transformation of human keratinocytes.

We previously reported data regarding the mechanism of neoplastic transformation in JB6 Cl41 mouse skin epidermal cells. However, experimental in vitro models for studying neoplastic transformation of human cells could provide further insight into the mechanisms of human cancer development. In this study, we have established a neoplastic transformation model with HaCaT cells, a human keratinocyte cell line, and showed the usefulness of this cell line for studying the mechanisms of neoplastic transformation. Epidermal growth factor (EGF) treatment induced a dose-dependent anchorage-independent cell transformation in HaCaT cells. Furthermore, PD98059, a mitogen-activated protein (MAP) kinase/ERK kinase (MEK) inhibitor, or SP600125, c-Jun N-terminal kinase (JNK) inhibitor, decreased cell growth, EGF-induced DNA synthesis and transformation. Unlike observations in the JB6 mouse epidermal cell model, SB203580, a stress-activated protein kinase-2/p38 alpha and beta (p38) inhibitor, increased EGF-induced transformation in HaCaT cells. These results suggest that extracellular-signal regulated kinase (ERK), JNK, or p38 are implicated in EGF-induced neoplastic transformation of human cells.

Phosphorylation of histone H3 at serine 10 is indispensable for neoplastic cell transformation.

Very little is known about the role of histone H3 phosphorylation in malignant transformation and cancer development. Here, we examine the function of H3 phosphorylation in cell transformation in vivo. Introduction of small interfering RNA-H3 into JB6 cells resulted in decreased epidermal growth factor (EGF)-induced cell transformation. In contrast, wild-type histone H3 (H3 WT)-overexpressing cells markedly stimulated EGF-induced cell transformation, whereas the H3 mutant S10A cells suppressed transformation. When H3 WT was overexpressed, EGF induction of c-fos and c-jun promoter activity was significantly increased compared with control cells but not in the H3 mutant S10A or S28A cells. In addition, activator protein-1 activity in H3 WT-overexpressing cells was markedly up-regulated by EGF in contrast to the H3 mutant S10A or S28A cells. These results indicate that the phosphorylation of histone H3 at Ser10 is an essential regulatory mechanism for EGF-induced neoplastic cell transformation.

A novel role for mixed-lineage kinase-like mitogen-activated protein triple kinase alpha in neoplastic cell transformation and tumor development.

Previously, no member of the mixed-lineage kinase (MLK) protein family was known to function as an oncogene. Here, we demonstrate that MLK-like mitogen-activated protein triple kinase (MLTK)-alpha, a member of the MLK family, induced neoplastic cell transformation and tumorigenesis in athymic nude mice. Introduction of small interference RNA (siRNA)-MLTK-alpha into MLTK-alpha-overexpressing cells dramatically suppressed cell transformation. Nuclear accumulation of the pHisG-MLTK-alpha fusion protein was observed after epidermal growth factor or 12-O-tetradecanoylphorbol-13-acetate treatment. Phosphorylation of downstream mitogen-activated protein kinase-targeted transcription factors including c-Myc, Elk-1, c-Jun, and activating transcription factor (ATF) 2 was also differentially enhanced in MLTK-alpha-overexpressing cells exposed to epidermal growth factor or 12-O-tetradecanoylphorbol-13-acetate stimulation compared with cells expressing mock vector or siRNA-MLTK-alpha. Very importantly, MLTK-alpha-overexpressing cells formed fibrosarcomas when injected s.c. into athymic nude mice, whereas almost no tumor formation was observed in mice that received injections of mock or siRNA-MLTK-alpha stably transfected cells. These results are the first to indicate that MLTK-alpha plays a key role in neoplastic cell transformation and cancer development.

Feeding of Ginkgo biloba extract (GBE) enhances gene expression of hepatic cytochrome P-450 and attenuates the hypotensive effect of nicardipine in rats.

Ginkgo biloba extract (GBE) has been used clinically for improving peripheral vascular diseases in France and Germany and is ingested widely as a herbal medicine in some countries. However, accurate information about its safety as an herbal medicine has not been sufficiently established. To address this issue, we examined the effect of GBE on hepatic drug metabolizing enzymes and their influence on hypotensive drug in rats. Male rats were fed either a control diet or diet containing GBE (0.5% w/w) for 4 weeks. The feeding of a GBE diet did not change the serum transaminase activity, but increased the liver weight and the phospholipid concentration in the liver. In addition, the GBE diet markedly increased the content of cytochrome P-450 (CYP), and the activity of glutathione S-transferase in the liver. Furthermore, the GBE diet markedly induced levels of CYP2B1/2, CYP3A1 and CYP3A2 mRNA in the liver. The levels of CYP1A1, CYP1A2, CYP2E1, CYP2C11 and CYP4A1 were unchanged. The feeding of GBE for 4 weeks significantly reduced the hypotensive effect of nicardipine that was reported to be metabolized by CYP3A2 in rats. These findings suggest that GBE reduces the therapeutic potency of the Ca2+ channel blocker, nicardipine, via enhancement of cytochrome P-450 expression.