(E)-2-methoxy-4-(3-(4-methoxyphenyl) prop-1-en-1-yl) Phenol Ameliorates MPTP-Induced Dopaminergic Neurodegeneration by Inhibiting the STAT3 Pathway.

Neuroinflammation is implicated in dopaminergic neurodegeneration. We have previously demonstrated that (E)-2-methoxy-4-(3-(4-methoxyphenyl) prop-1-en-1-yl) phenol (MMPP), a selective signal transducer and activator of transcription 3 (STAT3) inhibitor, has anti-inflammatory properties in several inflammatory disease models. We investigated whether MMPP could protect against 1-methyl-4-phenyl-1,2,3,6-tetrahydropyridine (MPTP)-induced dopaminergic cell loss and behavioral impairment. Imprinting control region (ICR) mice (8 weeks old, n = 10 per group) were administered MMPP (5 mg/kg) in drinking water for 1 month, and injected with MPTP (15 mg/kg, four times with 2 h intervals) during the last 7 days of treatment. MMPP decreased MPTP-induced behavioral impairments in rotarod, pole, and gait tests. We also showed that MMPP ameliorated dopamine depletion in the striatum and inflammatory marker elevation in primary cultured neurons by high-performance liquid chromatography and immunohistochemical analysis. Increased activation of STAT3, p38, and monoamine oxidase B (MAO-B) were observed in the substantia nigra and striatum after MPTP injection, effects that were attenuated by MMPP treatment. Furthermore, MMPP inhibited STAT3 activity and expression of neuroinflammatory proteins, including ionized calcium binding adaptor molecule 1 (Iba1), inducible nitric oxide synthase (iNOS), and glial fibrillary acidic protein (GFAP) in 1-methyl-4-phenylpyridinium (MPP+; 0.5 mM)-treated primary cultured cells. However, mitogen-activated protein kinase (MAPK) inhibitors augmented the activity of MMPP. Collectively, our results suggest that MMPP may be an anti-inflammatory agent that attenuates dopaminergic neurodegeneration and neuroinflammation through MAO-B and MAPK pathway-dependent inhibition of STAT3 activation.

Loss of parkin reduces lung tumor development by blocking p21 degradation.

Several epidemiological studies have demonstrated the reciprocal relationship between the development of cancer and Parkinson's disease (PD). However, the possible mechanisms underlying this relationship remain unclear. To identify this relationship, we first compared lung tumor growth in parkin knockout (KO) mice and wild-type (WT) mice. Parkin KO mice showed decreased lung tumor growth and increased expression of p21, a cell cycle arrester, as compared with WT mice. We also found that parkin interacts with p21, resulting in its degradation; however, parkin KO, knockdown, as well as mutation (R275W or G430D) reduced the degradation of p21. We investigated whether parkin KO increases the association of p21 with proliferating cell nuclear antigen (PCNA) or CDK2 by reducing p21 degradation, and, thus, arresting the cell cycle. The interaction between p21 and PCNA or CDK2 was also enhanced by parkin knockdown, and this increased interaction induced sub G0/G1 arrest, leading to cell death. Therefore, our data indicate that parkin KO reduces the development of lung tumors via cell cycle arrest by blocking the degradation of p21. These findings suggest that PD could be associated with lower lung cancer incidence.

Deficiency of parkin suppresses melanoma tumor development and metastasis through inhibition of MFN2 ubiquitination.

Parkin, a critical gene of Parkinson's disease, is involved in the development of numerous cancers. However, the effect of parkin deficiency on melanoma growth and metastasis has not been reported. We showed that the tumor size and number of surface lung metastases, and expression of tumor growth and metastasis marker proteins were significantly lower in parkin-KO mice than those observed in non-transgenic controls. In an in vitro study, we also showed that parkin siRNA inhibited cell growth and migration of B16F10 and SK-Mel-28 cells. Parkin-specific ubiquitination of mitofusin-2 (MFN2) was decreased in tumors and metastasized lung tissues of parkin-KO mice. Moreover, we showed that parkin directly binds and ubiquitinates MFN2. Knockdown of MFN2 decreased the expression of Bax and apoptotic cell death, but increased that of Bcl2 and apoptotic cancer cell death. However, these effects were reversed by knockdown of parkin. Conversely, inhibitory effects on melanoma growth and migration of parkin siRNA were reversed by MFN2 siRNA. These data indicate that melanoma development was inhibited in parkin-KO mice through maintaining of MFN2 level by inhibition of ubiquitinating ability of parkin.

Genetic and phenotypic characterization of the novel mouse substrain C57BL/6N Korl with increased body weight.

In inbred mouse lines, there is generally little genetic difference between individuals. This small genetic variability facilitates carrying out research on minute changes of various traits and the gene pool. Also, characterizing the diversity and detecting selective genetic and phenotypic signatures are crucial to understanding the genomic basis of a population and to identify specific patterns of evolutionary change. In this study, we investigated the underlying genetic profiles of a newly developed mouse strain, C57BL/6NKorl (Korl), established through sibling mating over 30 generations. To analyse the distinctive genomic features of Korl mice, we used whole-genome sequencing from six samples, which were compared to those of other C57BL/6N-based mouse strains. Korl strain-specific polymorphisms were identified and signatures of a selective sweep were detected. In particular, the candidate genes related to the increased body weight of the Korl strain were identified. Establishment of the genetic profile of Korl mice can provide insight into the inbreeding-induced changes to the gene pool, and help to establish this strain as a useful model for practical and targeted research purposes.

Comparison of commonly used ICR stocks and the characterization of Korl:ICR.

Mouse is a commonly used animal in life science studies and is classified as outbred if genetically diverse and inbred if genetically homogeneous. Outbred mouse stocks, are used in toxicology, oncology, infection and pharmacology research. The National Institute of Food and Drug Safety Evaluation (NIFDS; former the Korea National Institute of Health) have bred ICR mice for more than 50 years. We investigated to provide users with information and promote accountability to the Korl:ICR. To secure the indigenous data, biological characteristics of Korl:ICR were identified by comparing with other ICR stocks. This domestic ICR stock was denominated as 'Korl:ICR'. Phylogenetic analysis using SNPs indicated that the population stratification of the Korl:ICR was allocated different area with other ICR. In addition, we measured litter size, body weight, body length, various organ weight, hematology and clinical blood chemistry of the Korl:ICR compared to other ICR. Otherwise, there are no significant differences among the biological phenotypes of Korl:ICR and other ICR. These results suggest that as a genetically indigenous source colony, the Korl:ICR is seperated (or independent) stock with other ICR. Also, we confirmed that there is no difference among the Korl:ICR and other ICR on biological phenotypes. Therefore, the Korl:ICR source colony might be a new stock in distinction from other ICR, it is a good milestone in securing ownership of the national laboratory animal resource. The NIFDS expects that the Korl:ICR mice will be useful animal resource for our domestic researchers.

Piperlongumine attenuates experimental autoimmune encephalomyelitis through inhibition of NF-kappaB activity.

Multiple sclerosis (MS) is a chronic, autoimmune and neurodegenerative disease in which demyelination sporadically and repeatedly occurs in the central nervous system (CNS). The activity of nuclear factor kappa B (NF-κB), a family of transcription factors, was increased in the cerebrospinal fluid (CSF) and/or the serum and brain and/or spinal cord of MS patients than in a healthy donors. In our study, we investigated whether piperlongumine (PL), which is known to have inhibitory effect on activity of NF-κB, can alleviate an experimental autoimmune encephalomyelitis (EAE). The mice were immunized with myelin oligodendrocyte glycoprotein 35-55 (MOG35-55), and then we injected PL (1.5mg/kg/day or 3.0mg/kg/day) into the mice intraperitoneally on every second day from days 2 to 28. For in vitro study, we treated PL (0.5, 1 and 2.5µM) to RAW 264.7 and Jurkat cells with each stimulator. We observed that the paralytic severity and neuropathology of EAE in PL-treated group were decreased compared with the EAE group. PL showed a suppressed effect on demyelination, immune cells infiltration, astrocytes/microglials activation, level of inflammatory cytokines and proteins as well as NF-κB activity. Production of inflammatory cytokines and proteins as well as translocation of NF-κB into nucleus by treatment stimulators in RAW 264.7 and Jurkat cells were reduced by PL. Moreover, treatment of NF-κB inhibitor further inhibited production of inflammatory cytokines and proteins. These results suggest that PL can mitigate MOG-induced EAE symptoms and activation of macrophages and T cells by inhibiting NF-κB signaling. Therefore, PL could be useful for the treatment for MS.

Comparison of the response using ICR mice derived from three different sources to ethanol/hydrochloric acid-induced gastric injury.

Animal models for gastric ulcers produced by physical, pharmacological and surgical methods have been widely employed to evaluate therapeutic drugs and investigate the mechanism of action of this disease. ICR mice were selected to produce this model, even though several mice and rats have been widely used in studies of gastric ulcers. To compare the responses of ICR mice obtained from three different sources to gastric ulcer inducers, alterations in gastric injury, histopathological structure, and inflammation were measured in Korl:ICR (Korea NIFDS source), A:ICR (USA source) and B:ICR (Japan source) treated with three concentrations of ethanol (EtOH) (50, 70, and 90%) in 150 mM hydrochloric acid (HCl) solution. Firstly, the stomach lesion index gradually increased as the EtOH concentration increased in three ICR groups. Moreover, a significant increase in the level of mucosal injury, edema and the number of inflammatory cells was similarly detected in the EtOH/HCl treated group compared with the vehicle treated group in three ICR groups. Furthermore, the number of infiltrated mast cells and IL-1ß expression were very similar in the ICR group derived from three different sources, although some differences in IL-1ß expression were detected. Especially, the level of IL-1ß mRNA in 50 and 90EtOH/HCl treated group was higher in Korl:ICR and A:ICR than B:ICR. Overall, the results of this study suggest that Korl:ICR, A:ICR and B:ICR derived from different sources have an overall similar response to gastric ulcer induced by EtOH/HCl administration, although there were some differences in the magnitude of their responses.

(E)-4-(3-(3,5-dimethoxyphenyl)allyl)-2-methoxyphenol inhibits growth of colon tumors in mice.

In our previous study, we found that (E)-2,4-bis(p-hydroxyphenyl)-2-butenal showed anti-cancer effect, but it showed lack of stability and drug likeness. We have prepared several (E)-2,4-bis(p-hydroxyphenyl)-2-butenal analogues by Heck reaction. We selected two compounds which showed significant inhibitory effect of colon cancer cell growth. Thus, we evaluated the anti-cancer effects and possible mechanisms of one compound (E)-4-(3-(3,5-dimethoxyphenyl)allyl)-2-methoxyphenol in vitro and in vivo. In this study, we found that (E)-4-(3-(3,5-dimethoxyphenyl)allyl)-2-methoxyphenol induced apoptotic cell death in a dose dependent manner (0-15 µg/ml) through activation of Fas and death receptor (DR) 3 in HCT116 and SW480 colon cancer cell lines. Moreover, the combination treatment with (E)-4-(3-(3,5-dimethoxyphenyl)allyl)-2-methoxyphenol and nuclear factor κB (NF-κB) inhibitor, phenylarsine oxide (0.1 µM) or signal transducer and activator of transcription 3 (STAT3) inhibitor, Stattic (50 µM) increased the expression of Fas and DR3 more significantly. In addition, (E)-4-(3-(3,5-dimethoxyphenyl)allyl)-2-methoxyphenol suppressed the DNA binding activity of both STAT3 and NF-κB. Knock down of STAT3 or NF-κB p50 subunit by STAT3 small interfering RNA (siRNA) or p50 siRNA magnified (E)-4-(3-(3,5-dimethoxyphenyl)allyl)-2-methoxyphenol-induced inhibitory effect on colon cancer cell growth. Besides, the expression of Fas and DR3 was increased in STAT3 siRNA or p50 siRNA transfected cells. Moreover, docking model and pull-down assay showed that (E)-4-(3-(3,5-dimethoxyphenyl)allyl)-2-methoxyphenol directly bound to STAT3 and NF-κB p50 subunit. Furthermore, (E)-4-(3-(3,5-dimethoxyphenyl)allyl)-2-methoxyphenol inhibited colon tumor growth in a dose dependent manner (2.5 mg/kg-5 mg/kg) in mice. Therefore, these findings indicated that (E)-4-(3-(3,5-dimethoxyphenyl)allyl)-2-methoxyphenol may be a promising anti-cancer agent for colon cancer with more advanced research.

Exposure-outcome analysis in depressed patients treated with paroxetine using population pharmacokinetics.

PURPOSE: This study investigated population pharmacokinetics of paroxetine, and then performed an integrated analysis of exposure and clinical outcome using population pharmacokinetic parameter estimates in depressed patients treated with paroxetine. PATIENTS AND METHODS: A total of 271 therapeutic drug monitoring (TDM) data were retrospectively collected from 127 psychiatric outpatients. A population nonlinear mixed-effects modeling approach was used to describe serum concentrations of paroxetine. For 83 patients with major depressive disorder, the treatment response rate and the incidence of adverse drug reaction (ADR) were characterized by logistic regression using daily dose or area under the concentration-time curve (AUC) estimated from the final model as a potential exposure predictor. RESULTS: One compartment model was developed. The apparent clearance of paroxetine was affected by age as well as daily dose administered at steady-state. Overall treatment response rate was 72%, and the incidence of ADR was 30%. The logistic regression showed that exposure predictors were not associated with treatment response or ADR in the range of dose commonly used in routine practice. However, the incidence of ADR increased with the increase of daily dose or AUC for the patients with multiple concentrations. CONCLUSION: In depressed patients treated with paroxetine, TDM may be of limited value for individualization of treatment.

The influences of CYP2D6 genotypes and drug interactions on the pharmacokinetics of venlafaxine: exploring predictive biomarkers for treatment outcomes.

RATIONALE: Two biomarkers: concentration ratio of O-desmethylvenlafaxine/venlafaxine and concentration sum of venlafaxine + O-desmethylvenlafaxine were adopted to indicate venlafaxine responses, but neither is validated. OBJECTIVES: To evaluate the ability of two biomarkers in reflecting venlafaxine pharmacokinetic variations, and to further examine their relationship with venlafaxine treatment outcomes. METHODS: Two well-defined influencing factors: CYP2D6 genotypes and drug interactions were enriched into a three-period crossover study to produce venlafaxine pharmacokinetic variations: In each period, healthy CYP2D6 extensive metabolizers (EM group; n = 12) and CYP2D6*10/*10 intermediate metabolizers (IM group; n = 12) were pretreated with clarithromycin (CYP3A4 inhibitor), or nothing (control), or clarithromycin + paroxetine (CYP3A4 + CYP2D6 inhibitors), before administration of a single-dose of 75 mg venlafaxine. Both biomarkers were evaluated (1) for their relationship with the influencing factors in healthy volunteers and (2) for their relationships with the venlafaxine responses/adverse events reported in two patient studies. RESULTS: Significant venlafaxine pharmacokinetic variations were observed between the EM and IM groups (geometric mean ratio [95 % CI] of area under the curve, 3.0 [1.8-5.1] in the control period), and between the control and clarithromycin + paroxetine periods (4.1 [3.5-4.7] and 2.0 [1.7-2.4] in the EM and IM group, respectively). O-Desmethylvenlafaxine/venlafaxine was superior to venlafaxine + O-desmethylvenlafaxine to reflect the influencing factors. In the patient studies, O-desmethylvenlafaxine/venlafaxine > 4 showed high precision in predicting venlafaxine responders/partial-responders (92 %) and patients without venlafaxine-related adverse events (88 %); the O-desmethylvenlafaxine/venlafaxine < 4 and venlafaxine + O-desmethylvenlafaxine > 400 ng/ml combination showed higher precision (100 %) than O-desmethylvenlafaxine/venlafaxine < 4 alone (65 %) in predicting venlafaxine non-responders. CONCLUSION: We propose using O-desmethylvenlafaxine/venlafaxine for CYP2D6 phenotyping, and O-desmethylvenlafaxine/venlafaxine with venlafaxine + O-desmethylvenlafaxine for predicting venlafaxine treatment outcomes in future prospective studies.

Comprehensive variant screening of the UGT gene family.

PURPOSE: UGT1A1, UGT2B7, and UGT2B15 are well-known pharmacogenes that belong to the uridine diphosphate glucuronyltransferase gene family. For personalized drug treatment, it is important to study differences in the frequency of core markers across various ethnic groups. Accordingly, we screened single nucleotide polymorphisms (SNPs) of these three genes and analyzed differences in their frequency among five ethnic groups, as well as attempted to predict the function of novel SNPs. MATERIALS AND METHODS: We directly sequenced 288 subjects consisting of 96 Korean, 48 Japanese, 48 Han Chinese, 48 African American, and 48 European American subjects. Subsequently, we analyzed genetic variability, linkage disequilibrium (LD) structures and ethnic differences for each gene. We also conducted in silico analysis to predict the function of novel SNPs. RESULTS: A total of 87 SNPs were detected, with seven pharmacogenetic core SNPs and 31 novel SNPs. We observed that the frequencies of UGT1A1 *6 (rs4148323), UGT1A1 *60 (rs4124874), UGT1A1 *93 (rs10929302), UGT2B7 *2 (rs7439366), a part of UGT2B7 *3 (rs12233719), and UGT2B15 *2 (rs1902023) were different between Asian and other ethnic groups. Additional in silico analysis results showed that two novel promoter SNPs of UGT1A1 -690G>A and -689A>C were found to potentially change transcription factor binding sites. Moreover, 673G>A (UGT2B7), 2552T>C, and 23269C>T (both SNPs from UGT2B15) changed amino acid properties, which could cause structural deformation. CONCLUSION: Findings from the present study would be valuable for further studies on pharmacogenetic studies of personalized medicine and drug response.

Screening of dihydropyrimidine dehydrogenase genetic variants by direct sequencing in different ethnic groups.

Dihydropyrimidine dehydrogenase (DPYD) is an enzyme that regulates the rate-limiting step in pyrimidine metabolism, especially catabolism of fluorouracil, a chemotherapeutic agent for cancer. In order to determine the genetic distribution of DPYD, we directly sequenced 288 subjects from five ethnic groups (96 Koreans, 48 Japanese, 48 Han Chinese, 48 African Americans, and 48 European Americans). As a result, 56 polymorphisms were observed, including 6 core polymorphisms and 18 novel polymorphisms. Allele frequencies were nearly the same across the Asian populations, Korean, Han Chinese and Japanese, whereas several SNPs showed different genetic distributions between Asians and other ethnic populations (African American and European American). Additional in silico analysis was performed to predict the function of novel SNPs. One nonsynonymous SNP (+199381A > G, Asn151Asp) was predicted to change its polarity of amino acid (Asn, neutral to Asp, negative). These findings would be valuable for further research, including pharmacogenetic and drug responses studies.

Frequency of CYP2C9 alleles in Koreans and their effects on losartan pharmacokinetics.

AIM: CYP2C9 enzyme metabolizes numerous clinically important drugs. The aim of this study is to investigate the frequencies of CYP2C9 genotypes and the effects of selected alleles on losartan pharmacokinetics in a large sample of the Korean population. METHODS: The CYP2C9 gene was genotyped in 1796 healthy Korean subjects. CYP2C9 alleles (CYP2C9*1, *2, *3 and *13 alleles) were measured using polymerase chain reaction-restriction fragment length polymorphism (PCR-RFLP) assay and direct sequencing assay. The enzymatic activity of each CYP2C9 genotype was evaluated using losartan as the substrate. RESULTS: The frequencies of CYP2C9*1, *3 and *13 allele were 0.952 (95% confidence interval 0.945-0.959), 0.044 (95% CI 0.037-0.051) and 0.005 (95% CI 0.003-0.007), respectively. The frequencies of the CYP2C9*1/*1, *1/*3, *1/*13 and *3/*3 genotypes were 0.904 (95% CI 0.890-0.918), 0.085 (95% CI 0.072-0.098), 0.009 (95% CI 0.005-0.013) and 0.001 (95% CI 0.000-0.002), respectively. In the pharmacokinetics studies, the AUC(0-∞) of losartan in CYP2C9*3/*3 subjects was 1.42-fold larger than that in CYP2C9*1/*1 subjects, and the AUC(0-∞) of E-3174, a more active metabolite of losartan, in CYP2C9*3/*3 subjects was only 12% of that in CYP2C9*1/*1 subjects. CONCLUSION: The results confirmed the frequencies of CYP2C9 genotypes in a large cohort of Koreans, and detected the CYP2C9*3/*3 genotype. CYP2C9*3/*3 subjects metabolized much less losartan into E-3174 than CYP2C9*1/*1 subjects.

Screening of genetic variations of SLC15A2, SLC22A1, SLC22A2 and SLC22A6 genes.

A growing list of membrane-spanning proteins involved in the transport of a large variety of drugs has been recognized and characterized to include peptide and organic anion/cation transporters. Given such an important role of transporter genes in drug disposition process, the role of single-nucleotide polymorphisms (SNPs) in such transporters as potential determinants of interindividual variability in drug disposition and pharmacological response has been investigated. To define the distribution of transporter gene SNPs across ethnic groups, we screened 450 DNAs in cohorts of 250 Korean, 50 Han Chinese, 50 Japanese, 50 African-American and 50 European-American ancestries for 64 SNPs in four transporter genes encoding proteins of the solute carrier family (SLC15A2, SLC22A1, SLC22A2 and SLC22A6). Of the 64 SNPs, 19 were core pharmacogenetic variants and 45 were HapMap tagging SNPs. Polymorphisms were genotyped using the golden gate genotyping assay. After genetic variability, haplotype structures and ethnic diversity were analyzed, we observed that the distributions of SNPs in a Korean population were similar to other Asian groups (Chinese and Japanese), and significantly different from African-American and European-American cohorts. Findings from this study would be valuable for further researches, including pharmacogenetic studies for drug responses.

Movements of individual BKCa channels in live cell membrane monitored by site-specific labeling using quantum dots.

The movements of BK(Ca) channels were investigated in live cells using quantum dots (QDs). The extracellular N-terminus was metabolically tagged with biotin, labeled with streptavidin-conjugated QDs and then monitored using real-time time-lapse imaging in COS-7 cells and cultured neurons. By tracking hundreds of channels, we were able to determine the characteristics of channel movements quantitatively. Channels in COS-7 cells exhibited a confined diffusion in an area of 1.915 µm(2), with an initial diffusion coefficient of 0.033 µm(2)/s. In neurons, the channel movements were more heterogeneous and highly dependent on subcellular location. While the channels in soma diffused slowly without clear confinement, axodendritic channels showed more rapid and pseudo-one-dimensional movements. Intriguingly, the channel movement in somata was drastically increased by the neuronal ß4 subunit, in contrast to the channels in the axodendritic area where the mobility were significantly decreased. Thus, our results demonstrate that the membrane mobility of BK(Ca) channels can be greatly influenced by the expression system used, subunit composition, and subcellular location. This QD-based, single-molecule tracking technique can be utilized to investigate the cellular mechanisms that determine the mobility as well as the localization of various membrane proteins in live cells.

Modulation of the conductance-voltage relationship of the BK(Ca) channel by shortening the cytosolic loop connecting two RCK domains.

Calcium-dependent gating of large-conductance calcium-activated potassium (BK(Ca)) channels is mediated by the intracellular carboxyl terminus, which contains two domains of regulator of K(+) conductance (RCK). In mammalian BK(Ca) channels, the two RCK domains are separated by a protein segment of 101 residues that is poorly conserved in evolution and predicted to have no regular secondary structures. We investigated the functional importance of this loop using a series of deletion mutations. We found that the length, rather than the specific sequence at the central region of the segment, is critical for the functionality of the channel. As the length of the loop is progressively shorted, the conductance-voltage relationship gradually shifts toward more positive voltages with a minimum length of 70 amino acids, in an apparent response to increased tension within the loop. Thus, the functional activity of the BK(Ca) channel can be modulated by altering the tension of this loop region.