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1.
Biochim Biophys Acta Mol Cell Res ; 1865(8): 1046-1059, 2018 08.
Article in English | MEDLINE | ID: mdl-29694914

ABSTRACT

GSK3ß interacting protein (GSKIP) is a naturally occurring negative regulator of GSK3ß and retains both the Protein Kinase A Regulatory subunit binding (PKA-RII) domain and GSK3ß interacting domain. Of these two domains, we found that PKA-RII is required for forming a working complex comprising PKA/GSKIP/GSK3ß/Drp1 to influence phosphorylation of Drp1 Ser637. In this study, bioinformatics and experimental explorations re-analyzing GSKIP's biofunctions suggest that the evolutionarily conserved Domain of Unknown Function (DUF727) is an ancestral prototype of GSKIP in prokaryotes, and acquired the C-terminal GSK3ß binding site (tail) in invertebrates except for Saccharomyces spp., after which the N-terminal PKA-RII binding region (head) evolved in vertebrates. These two regions mutually influence each other and modulate GSKIP binding to GSK3ß in yeast two-hybrid assays and co-immunoprecipitation. Molecular modeling showed that mammalian GSKIP could form a dimer through the L130 residue (GSK3ß binding site) rather than V41/L45 residues. In contrast, V41/L45P mutant facilitated a gain-of-function effect on GSKIP dimerization, further influencing binding behavior to GSK3ß compared to GSKIP wild-type (wt). The V41/L45 residues are not only responsible for PKA RII binding that controls GSK3ß activity, but also affect dimerization of GSKIP monomer, with net results of gain-of-function in GSKIP-GSK3ß interaction. In addition to its reported role in modulating Drp1, Ser637 phosphorylation caused mitochondrial elongation; we postulated that GSKIP might be involved in the Wnt signaling pathway as a scavenger to recruit GSK3ß away from the ß-catenin destruction complex and as a competitor to compete for GSK3ß binding, resulting in accumulation of S675 phosphorylated ß-catenin.


Subject(s)
Repressor Proteins/chemistry , Repressor Proteins/metabolism , Wnt Signaling Pathway , Binding Sites , Computational Biology , Cyclic AMP-Dependent Protein Kinases/metabolism , Dynamins , Evolution, Molecular , GTP Phosphohydrolases/chemistry , GTP Phosphohydrolases/metabolism , Glycogen Synthase Kinase 3 beta/metabolism , HEK293 Cells , Humans , Microtubule-Associated Proteins/chemistry , Microtubule-Associated Proteins/metabolism , Mitochondrial Proteins/chemistry , Mitochondrial Proteins/metabolism , Models, Molecular , Phosphorylation , Phylogeny , Protein Binding , Protein Domains , Protein Multimerization , Repressor Proteins/genetics , Serine/chemistry , Two-Hybrid System Techniques
2.
Biomed Rep ; 7(5): 416-422, 2017 Nov.
Article in English | MEDLINE | ID: mdl-29181154

ABSTRACT

Pluchea indica (L.) Less. is a perennial plant known for its versatile uses in traditional medicine. Previous findings have shown that the extracts of Pluchea indica possess significant anti-inflammatory, anti-ulcer and anti-tuberculosis activity. The aim of this study was to demonstrate the anticancer activity of the hexane fraction of P. indica root extract (H-PIRE) in human glioblastoma cells using flow cytometric and western blot analysis. The results shoewd that, H-PIRE suppressed the growth of glioblastoma cells in a dose-dependent manner. H-PIRE treatment markedly decreased the population of cells in S and G2/M phases. The significant upregulation of acidic vesicular organelle (AVO) was detected during H-PIRE treatment. The expression levels of microtubule-associated light chain 3-II (LC3-II) protein, phosphorylated JNK and phosphorylated p38 were significantly increased, confirming the occurrence of autophagy during the process. Finally, the combination treatment of H-PIRE and LY294002, a pan PI3K inhibitor, further decreased cell viability, suggesting an additive anticancer effect. Taken together, our results suggest that H-PIRE suppresses the proliferation of glioblastoma cells by inducing cell cycle arrest and autophagy.

3.
Cancer Biol Ther ; 17(10): 1070-1078, 2016 Oct 02.
Article in English | MEDLINE | ID: mdl-27636029

ABSTRACT

ROGDI is a protein that contains a leucine zipper domain and may be involved in cell proliferation. In addition, ROGDI is associated with genome stability by regulating the activity of a DNA damage marker, γ-H2AX. The role of ROGDI in tumor radiosensitization has not been investigated. Previous studies have indicated that radiosensitivity is associated with DNA repair and the cell cycle. In general, the G2/M DNA damage checkpoint is more sensitive to radiation, whereas the G1/S phase transition is more resistant to radiation. Inhibition of cyclin-dependent kinases (CDKs) can lead to a halt of cell cycle progression and a stay at different phases or checkpoints. Our data show that the downregulation of ROGDI led to a decreased expression of CDK 1, 2, cyclin A, B and resulted in a G2/M phase transition block. In addition, the downregulation of ROGDI increased cell accumulation at the G2 phase as detected using flow cytometry and decreased cell survival as revealed by clonogenic assay in HeLa and C33A cells following irradiation. These findings suggest that the downregulation of ROGDI can mediate radiosensitivity by blocking cells at G2/M, the most radiosensitive phase of the cell cycle, as well as exerting deleterious effects in the form of DNA damage, as shown by increased γ-H2AX activation.

4.
Molecules ; 20(6): 11508-23, 2015 Jun 22.
Article in English | MEDLINE | ID: mdl-26111179

ABSTRACT

Pluchea indica is used in traditional medicine for the treatment of lumbago, ulcer, tuberculosis and inflammation. The anti-cancer activities and the underlying molecular mechanisms of the ethanolic extracts of P. indica root (PIRE) were characterized in the present study. PIRE strongly inhibited the viability of the human nasopharyngeal carcinoma cells (NPC-TW 01 and NPC-TW 04) in a time- and dose-dependent manner. Migration of cancer cells was also suppressed by PIRE. In addition, PIRE significantly increased the occurrence of the cells in sub-G1 phase and the extent of DNA fragmentation in a dose-dependent manner, which indicates that PIRE significantly increased apoptosis in NPC cells. The apoptotic process triggered by PIRE involved up-regulation of pro-apoptotic Bax protein and down-regulation of anti-apoptotic Bcl-2 protein, consequently increasing the ratios of Bax/Bcl-2 protein levels. Moreover, the p53 protein was up-regulated by PIRE in a concentration-dependent manner. Therefore, PIRE could induce the apoptosis-signaling pathway in NPC cells by activation of p53 and by regulation of apoptosis-related proteins.


Subject(s)
Apoptosis/drug effects , Cell Proliferation/drug effects , Nasopharyngeal Neoplasms/drug therapy , Plant Extracts/administration & dosage , Asteraceae/chemistry , Carcinoma , Cell Line, Tumor , Humans , Nasopharyngeal Carcinoma , Nasopharyngeal Neoplasms/pathology , Plant Extracts/chemistry
5.
Biochim Biophys Acta ; 1853(8): 1796-807, 2015 Aug.
Article in English | MEDLINE | ID: mdl-25920809

ABSTRACT

GSK3ß binding of GSKIP affects neurite outgrowth, but the physiological significance of PKA binding to GSKIP remains to be determined. We hypothesized that GSKIP and GSK3ß mediate cAMP/PKA/Drp1 axis signaling and modulate mitochondrial morphology by forming a working complex comprising PKA/GSKIP/GSK3ß/Drp1. We demonstrated that GSKIP wild-type overexpression increased phosphorylation of Drp1 S637 by 7-8-fold compared to PKA kinase-inactive mutants (V41/L45) and a GSK3ß binding-defective mutant (L130) under H2O2 and forskolin challenge in HEK293 cells, indicating that not only V41/L45, but also L130 may be involved in Drp1-associated protection of GSKIP. Interestingly, silencing either GSKIP or GSK3ß but not GSK3α resulted in a dramatic decrease in Drp1 S637 phosphorylation, revealing that both GSKIP and GSK3ß are required in this novel PKA/GSKIP/GSK3ß/Drp1 complex. Moreover, overexpressed kinase-dead GSK3ß-K85R, which retains the capacity to bind GSKIP, but not K85M which shows total loss of GSKIP-binding, has a higher Drp1 S637 phosphorylation similar to the GSKIP wt overexpression group, indicating that GSK3ß recruits Drp1 by anchoring rather than in a kinase role. With further overexpression of either V41/L45P or the L130P GSKIP mutant, the elongated mitochondrial phenotype was lost; however, ectopically expressed Drp1 S637D, a phosphomimetic mutant, but not S637A, a non-phosphorylated mutant, restored the elongated mitochondrial morphology, indicating that Drp1 is a downstream effector of direct PKA signaling and possibly has an indirect GSKIP function involved in the cAMP/PKA/Drp1 signaling axis. Collectively, our data revealed that both GSKIP and GSK3ß function as anchoring proteins in the cAMP/PKA/Drp1 signaling axis modulating Drp1 phosphorylation.


Subject(s)
Cyclic AMP-Dependent Protein Kinases/metabolism , Cyclic AMP/metabolism , GTP Phosphohydrolases/metabolism , Glycogen Synthase Kinase 3/physiology , Microtubule-Associated Proteins/metabolism , Mitochondria/metabolism , Mitochondrial Proteins/metabolism , Repressor Proteins/physiology , Cells, Cultured , Dynamins , GTP Phosphohydrolases/genetics , Glycogen Synthase Kinase 3/metabolism , HEK293 Cells , HeLa Cells , Humans , Microtubule-Associated Proteins/genetics , Mitochondria/genetics , Mitochondrial Dynamics/physiology , Mitochondrial Proteins/genetics , Phosphorylation , Repressor Proteins/metabolism , Signal Transduction/genetics
6.
Molecules ; 19(11): 17464-77, 2014 Oct 29.
Article in English | MEDLINE | ID: mdl-25356567

ABSTRACT

Cancer stem cells (CSCs) are a subset of cancer cells in tumors or established cancer cell lines that can initiate and sustain the growth of tumors in vivo. Cancer stem cells can be enriched in serum-free, suspended cultures that allow the formation of tumorspheres over several days to weeks. Brefeldin A (BFA) is a mycotoxin that induces endoplasmic reticulum (ER) stress in eukaryotic cells. We found that BFA, at sub-microgram per milliliter concentrations, preferentially induced cell death in MDA-MB-231 suspension cultures (EC50: 0.016 µg/mL) compared to adhesion cultures. BFA also effectively inhibited clonogenic activity and the migration and matrix metalloproteinases-9 (MMP-9) activity of MDA-MB-231 cells. Western blotting analysis indicated that the effects of BFA may be mediated by the down-regulation of breast CSC marker CD44 and anti-apoptotic proteins Bcl-2 and Mcl-1, as well as the reversal of epithelial-mesenchymal transition. Furthermore, BFA also displayed selective cytotoxicity toward suspended MDA-MB-468 cells, and suppressed tumorsphere formation in T47D and MDA-MB-453 cells, suggesting that BFA may be effective against breast cancer cells of various phenotypes.


Subject(s)
Breast Neoplasms/pathology , Brefeldin A/pharmacology , Cell Movement/drug effects , Cell Survival/drug effects , Neoplastic Stem Cells/drug effects , Breast Neoplasms/metabolism , Cell Culture Techniques/methods , Cell Death/drug effects , Cell Line, Tumor , Down-Regulation/drug effects , Epithelial-Mesenchymal Transition/drug effects , Female , Humans , Hyaluronan Receptors/metabolism , Matrix Metalloproteinase 9/metabolism , Mycotoxins/pharmacology , Myeloid Cell Leukemia Sequence 1 Protein/metabolism , Neoplastic Stem Cells/metabolism , Neoplastic Stem Cells/pathology , Phenotype , Proto-Oncogene Proteins c-bcl-2/metabolism
7.
Kaohsiung J Med Sci ; 30(5): 219-23, 2014 May.
Article in English | MEDLINE | ID: mdl-24751383

ABSTRACT

Mammalian oligosaccharyltransferase complex subunit OSTC/DC2 protein has recently been shown to be a new subunit of the oligosaccharyltransferase; however, its physiological role is still unclear. Here, we report the expression pattern of OSTC/DC2 protein in the context of heat shock stress. Its upregulation was detected both in cells treated with heat shock in vitro and in an animal model of heat shock in vivo. Northern blot analysis indicated that OSTC/DC2 mRNA is ubiquitously expressed in various human tissues, with abundant expression in the placenta and liver. The temporal changes of OSTC/DC2 protein expression following acute heat shock in human malignant glioblastoma cell line U87MG and mice were analyzed by Western blot assay. In general, expression of OSTC/DC2 protein was elevated after heat shock; however, the time courses of the change of OSTC/DC2 protein expression varied in different tissues. In the cerebellum, heat shock induction of OSTC/DC2 protein and activation of AKT, a key regulator of stress response, followed a similar time course. These results suggest that the upregulation of OSTC/DC2, a novel component of the oligosaccharyltransferase complex, is part of the mammalian heat shock response.


Subject(s)
Heat-Shock Response , Hexosyltransferases/metabolism , Membrane Proteins/metabolism , Protein Subunits/metabolism , Animals , Cell Line, Tumor , Cerebellum/enzymology , Gene Expression Profiling , Heat-Shock Response/genetics , Hexosyltransferases/genetics , Humans , Membrane Proteins/genetics , Mice, Inbred BALB C , Phosphorylation , Protein Subunits/genetics , Proto-Oncogene Proteins c-akt/metabolism , RNA, Messenger/genetics , RNA, Messenger/metabolism
8.
Molecules ; 19(3): 3149-59, 2014 Mar 13.
Article in English | MEDLINE | ID: mdl-24633350

ABSTRACT

Cancer stem cells are a subset of cancer cells that initiate the growth of tumors. Low levels of cancer stem cells also exist in established cancer cell lines, and can be enriched in serum-free tumorsphere cultures. Since cancer stem cells have been reported to be resilient to common chemotherapeutic drugs in comparison to regular cancer cells, screening for compounds selectively targeting cancer stem cells may provide an effective therapeutic strategy. We found that 5-azacytidine (5-AzaC) selectively induced anoikis of MCF-7 in suspension cultures with an EC50 of 8.014 µM, and effectively inhibited tumorsphere formation, as well as the migration and matrix metalloproteinases-9 (MMP-9) activity of MCF-7 cells. Furthermore, 5-AzaC and radiation collaboratively inhibited MCF-7 tumorsphere formation at clinically relevant radiation doses. Investigating the underlying mechanism may provide insight into signaling pathways crucial for cancer stem cell survival and pave the way to novel potential therapeutic targets.


Subject(s)
Antimetabolites, Antineoplastic/pharmacology , Azacitidine/pharmacology , Breast Neoplasms/metabolism , Matrix Metalloproteinase 9/metabolism , Anoikis/drug effects , Cell Line, Tumor , Enzyme Activation/drug effects , Female , Humans , MCF-7 Cells , Spheroids, Cellular/drug effects , Tumor Cells, Cultured
9.
Anim Biotechnol ; 25(2): 119-27, 2014 Apr 03.
Article in English | MEDLINE | ID: mdl-24555797

ABSTRACT

Most species of penguins are sexual monomorphic and therefore it is difficult to visually identify their genders for monitoring population stability in terms of sex ratio analysis. In this study, we evaluated the suitability using melting curve analysis (MCA) for high-throughput gender identification of penguins. Preliminary test indicated that the Griffiths's P2/P8 primers were not suitable for MCA analysis. Based on sequence alignment of Chromo-Helicase-DNA binding protein (CHD)-W and CHD-Z genes from four species of penguins (Pygoscelis papua, Aptenodytes patagonicus, Spheniscus magellanicus, and Eudyptes chrysocome), we redesigned forward primers for the CHD-W/CHD-Z-common region (PGU-ZW2) and the CHD-W-specific region (PGU-W2) to be used in combination with the reverse Griffiths's P2 primer. When tested with P. papua samples, PCR using P2/PGU-ZW2 and P2/PGU-W2 primer sets generated two amplicons of 148- and 356-bp, respectively, which were easily resolved in 1.5% agarose gels. MCA analysis indicated the melting temperature (Tm) values for P2/PGU-ZW2 and P2/PGU-W2 amplicons of P. papua samples were 79.75°C-80.5°C and 81.0°C-81.5°C, respectively. Females displayed both ZW-common and W-specific Tm peaks, whereas male was positive only for ZW-common peak. Taken together, our redesigned primers coupled with MCA analysis allows precise high throughput gender identification for P. papua, and potentially for other penguin species such as A. patagonicus, S. magellanicus, and E. chrysocome as well.


Subject(s)
DNA Primers/genetics , DNA-Binding Proteins/genetics , Real-Time Polymerase Chain Reaction/methods , Sequence Analysis, DNA/methods , Sex Determination Analysis/methods , Spheniscidae/classification , Spheniscidae/genetics , Animals , Base Sequence , Female , Genetic Testing/methods , Male , Molecular Sequence Data , Reproducibility of Results , Sensitivity and Specificity , Sex Characteristics , Species Specificity , Transition Temperature
10.
Kaohsiung J Med Sci ; 29(9): 478-83, 2013 Sep.
Article in English | MEDLINE | ID: mdl-24018150

ABSTRACT

Although an increasing number of RNA adenosine-to-inosine (A-to-I) editing sites are being discovered, how the editing frequencies of these sites are modulated to fine-tune protein function in adaptive responses is not well understood. A previous study screening for heat tolerance in Drosophila mutants discovered a hypnos-2 mutant strain that was later found to be defective in dADAR, the Drosophila gene encoding the A-to-I editing enzyme. This supports the hypothesis that cells and organisms respond to stressful environments by ADAR (adenosine deaminase acting on RNA)-mediated RNA editing. Here, we investigated changes in the RNA A-to-I editing frequencies of 30 Drosophila nervous system targets in response to heat shock, a stress acclimatization that requires the dADAR function. To our surprise, most of these nervous system editing targets showed reduced editing. Our results suggest that a change in RNA editing pattern is a mechanism by which organisms acclimate to drastic environmental change. However, how RNA editing confers heat resistance is more complicated and requires further investigation.


Subject(s)
Adaptation, Physiological , Adenosine/genetics , Drosophila/genetics , Hot Temperature , Inosine/genetics , RNA Editing , Animals , Drosophila/physiology
11.
Molecules ; 18(9): 10242-53, 2013 Aug 22.
Article in English | MEDLINE | ID: mdl-23973996

ABSTRACT

Cancer stem cells (CSCs) are a small subset of cancer cells with indefinite potential for self-renewal and the capacity to drive tumorigenesis. Brefeldin A (BFA) is an antibiotic that is known to block protein transport and induce endoplasmic reticulum (ER) stress in eukaryotic cells, but its effects on colorectal CSCs are unknown. We investigated the inhibitory effect of BFA on human colorectal cancer Colo 205 cells. We found that BFA effectively reduced the survival of suspension Colo 205 cells (IC50 = ~15 ng/mL) by inducing apoptosis, and inhibited the clonogenic activity of Colo 205 CSCs in tumorsphere formation assay and soft agar colony formation assay in the same nanogram per milliliter range. We also discovered that at such low concentrations, BFA effectively induced endoplasmic reticulum (ER) stress response as indicated by the increased mRNA expression of ER stress-related genes, such as glucose-regulated protein 78 (GRP78), X-box binding protein 1 (XBP1), and C/EBP homologous protein (CHOP). Finally, we found that BFA reduced the activity of matrix metallopeptidase 9 (MMP-9). These findings suggest that BFA can effectively suppress the progression of colorectal cancer during the tumorigenesis and metastasis stages. These results may lead to the development of novel therapies for the treatment of colorectal cancer.


Subject(s)
Antineoplastic Agents/pharmacology , Brefeldin A/pharmacology , Matrix Metalloproteinase 9/metabolism , Neoplastic Stem Cells/drug effects , Cell Line, Tumor , Cell Survival/drug effects , Colorectal Neoplasms , Drug Screening Assays, Antitumor , Endoplasmic Reticulum Chaperone BiP , Endoplasmic Reticulum Stress/drug effects , Humans , Neoplastic Stem Cells/enzymology , Spheroids, Cellular/drug effects , Spheroids, Cellular/enzymology
12.
ScientificWorldJournal ; 2013: 804505, 2013.
Article in English | MEDLINE | ID: mdl-23737728

ABSTRACT

RNA editing is vital to provide the RNA and protein complexity to regulate the gene expression. Correct RNA editing maintains the cell function and organism development. Imbalance of the RNA editing machinery may lead to diseases and cancers. Recently, RNA editing has been recognized as a target for drug discovery although few studies targeting RNA editing for disease and cancer therapy were reported in the field of natural products. Therefore, RNA editing may be a potential target for therapeutic natural products. In this review, we provide a literature overview of the biological functions of RNA editing on gene expression, diseases, cancers, and drugs. The bioinformatics resources of RNA editing were also summarized.


Subject(s)
Gene Expression Regulation, Neoplastic/genetics , Genetic Therapy/methods , Models, Biological , Neoplasm Proteins/genetics , Neoplasms/drug therapy , Neoplasms/genetics , RNA Editing/genetics , Animals , Gene Expression Regulation, Neoplastic/drug effects , Humans
13.
Kaohsiung J Med Sci ; 29(4): 179-86, 2013 Apr.
Article in English | MEDLINE | ID: mdl-23541262

ABSTRACT

Dysregulations of RNA A-to-I editing are associated with developmental defects in mouse and human diseases. Although several methods of identifying RNA A-to-I editing sites are currently available, most of the critical editing targets responsible for the important biological functions of adenosine deaminases that act on RNA (ADARs) remain unknown. Here we report a modified I-specific cleavage method that improves the quality of the RNA product. Preliminary microarray comparison of RNAs subjected to I-specific cleavage or mock digestion reported 165 genes that showed more than 0.2-fold reductions due to the cleavage. Six of the 165 genes were randomly selected for further verification, and three were verified to be targets of I-specific cleavage. This method may provide an alternative method of identifying novel RNA A-to-I editing sites using a microarray and facilitate the inquiry into the roles of RNA A-to-I editing in various biological processes.


Subject(s)
Inosine/chemistry , RNA Cleavage , RNA Editing , Adenosine Deaminase/chemistry , Animals , Brain Chemistry , Mice , Mice, Inbred ICR , Microarray Analysis
14.
Phytochemistry ; 88: 67-73, 2013 Apr.
Article in English | MEDLINE | ID: mdl-23351982

ABSTRACT

Lawsonia inermis (Lythraceae) known as henna is one of the most popular and ancient plants used in cosmetics and hair dying. It is cultivated for its leaves but other parts such as seeds, flowers, stem bark and roots are also used in traditional medicine for millennia. Henna tattoo paste also proved to be beneficial for wound healing and in several skin diseases suggesting potent anti-inflammatory activity. To evaluate henna anti-inflammatory activity, 31 compounds, including three 1,5-diphenylpent-3-en-1-yne derivatives, lawsochylin A-C and three methyl naphthalene carboxylates, lawsonaphthoate A-C, were isolated from the stems and leaves of henna utilizing a bioassay-guided fractionation. The structures of the compounds were elucidated by spectroscopic data. Two compounds, lawsochylin A and lawsonaphthoate A showed potent anti-inflammatory activity by inhibition of superoxide anion generation (IC(50)=1.80 and 1.90 µg/ml) and elastase release (IC(50)=1.58 and 3.17 µg/ml) of human neutrophils in response to fMLP or cytochalasin B. Moreover, the known compounds, luteolin, apigenin, 4S-4-hydroxy-α-tetralone, and 2-butoxysuccinic acid, also showed potent inhibition of superoxide anion generation (IC(50)=0.75-1.78 µg/ml) and elastase release (IC(50)=1.62-3.61 µg/ml).


Subject(s)
Anti-Inflammatory Agents/chemistry , Anti-Inflammatory Agents/pharmacology , Carboxylic Acids/chemistry , Carboxylic Acids/pharmacology , Lawsonia Plant/chemistry , Neutrophils/drug effects , Carboxylic Acids/classification , Humans , Inhibitory Concentration 50 , Magnetic Resonance Spectroscopy , Molecular Structure , Naphthalenes/chemistry , Naphthalenes/classification , Naphthalenes/pharmacology , Plant Leaves/chemistry , Plant Stems/chemistry
15.
Mol Cell Probes ; 27(1): 38-45, 2013 Feb.
Article in English | MEDLINE | ID: mdl-22960667

ABSTRACT

RNA A-to-I editing is the most common single-base editing in the animal kingdom. Dysregulations of RNA A-to-I editing are associated with developmental defects in mouse and human diseases. Mouse knockout models deficient in ADAR activities show lethal phenotypes associated with defects in nervous system, failure of hematopoiesis and reduced tolerance to stress. While several methods of identifying RNA A-to-I editing sites are currently available, most of the critical editing targets responsible for the important biological functions of ADARs remain unknown. Here we report a method to systematically analyze RNA A-to-I editing targets by combining I-specific cleavage and exon array analysis. Our results show that I-specific cleavage on editing sites causes more than twofold signal reductions in edited exons of known targets such as Gria2, Htr2c, Gabra3 and Cyfip2 in mice. This method provides an experimental approach for genome-wide analysis of RNA A-to-I editing targets with exon-level resolution. We believe this method will help expedite inquiry into the roles of RNA A-to-I editing in various biological processes and diseases.


Subject(s)
Nerve Tissue Proteins/genetics , RNA Cleavage , RNA Editing , Receptors, AMPA/genetics , Receptors, GABA-A/genetics , Adaptor Proteins, Signal Transducing , Animals , Brain/cytology , Cell Line , Exons , Genome , HEK293 Cells , Humans , Male , Mice , Mice, Inbred ICR
16.
BMC Complement Altern Med ; 12: 265, 2012 Dec 26.
Article in English | MEDLINE | ID: mdl-23268709

ABSTRACT

BACKGROUND: Pluchea indica (L.) Less. (Asteraceae) is a perennial shrub plant with anti-inflammatory and antioxidant medicinal properties. However, the anti-cancer properties of its aqueous extracts have not been studied. The aim of this study was to investigate the anti-proliferation, anti-migration, and pro-apoptotic properties of crude aqueous extracts of P. indica leaf and root on human malignant glioma cancer cells and human cervical cancer cells, and the underlying molecular mechanism. METHODS: GBM8401 human glioma cells and HeLa cervical carcinoma cells were treated with various concentrations of crude aqueous extracts of P. indica leaf and root and cancer cell proliferation and viability were measured by cell growth curves, trypan blue exclusions, and the tetrazolium reduction assay. Effects of the crude aqueous extracts on focus formation, migration, and apoptosis of cancer cells were studied as well. The molecular mechanism that contributed to the anti-cancer activities of crude aqueous extracts of P. indica root was also examined using Western blotting analysis. RESULTS: Crude aqueous extracts of P. indica leaf and root suppressed proliferation, viability, and migration of GBM8401 and HeLa cells. Treatment with crude aqueous extracts of P. indica leaf and root for 48 hours resulted in a significant 75% and 70% inhibition on proliferation and viability of GBM8401 and HeLa cancer cells, respectively. Crude aqueous extracts of P. indica root inhibited focus formation and promoted apoptosis of HeLa cells. It was found that phosphorylated-p53 and p21 were induced in GBM8401 and HeLa cells treated with crude aqueous extracts of P. indica root. Expression of phosphorylated-AKT was decreased in HeLa cells treated with crude aqueous extracts of P. indica root. CONCLUSION: The in vitro anti-cancer effects of crude aqueous extracts of P. indica leaf and root indicate that it has sufficient potential to warrant further examination and development as a new anti-cancer agent.


Subject(s)
Apoptosis/drug effects , Asteraceae , Glioma/drug therapy , Phytotherapy , Plant Extracts/therapeutic use , Tumor Suppressor Protein p53/metabolism , Uterine Cervical Neoplasms/drug therapy , Antineoplastic Agents, Phytogenic/pharmacology , Antineoplastic Agents, Phytogenic/therapeutic use , Cell Movement/drug effects , Cell Proliferation/drug effects , Cyclin-Dependent Kinase Inhibitor p21/metabolism , Female , Glioma/metabolism , HeLa Cells , Humans , Phosphorylation , Plant Extracts/pharmacology , Uterine Cervical Neoplasms/metabolism
17.
BMC Complement Altern Med ; 12: 142, 2012 Aug 31.
Article in English | MEDLINE | ID: mdl-22937998

ABSTRACT

BACKGROUND: Methanolic extracts of Gracilaria tenuistipitata (MEGT) were obtained from the edible red algae. Previously, we found that water extract of G. tenuistipitata was able to modulate oxidative stress-induced DNA damage and its related cellular responses. METHODS: In this study, the methanol extraction product MEGT was used to evaluate the cell growth inhibition in oral cancer cells and its possible mechanism was investigated. RESULTS: The cell viability of MEGT treated Ca9-22 oral cancer cell line was significantly decreased in a dose-response manner (p < 0.05). The sub-G1 population and annexin V intensity of MEGT-treated Ca9-22 cancer cells were significantly increased in a dose-response manner (p < 0.0005 and p < 0.001, respectively). The γH2AX intensities of MEGT-treated Ca9-22 cancer cells were significantly increased in a dose-response manner (p < 0.05). The reactive oxygen species (ROS) and glutathione (GSH)-positive intensities of MEGT-treated Ca9-22 oral cancer cells were significantly increased and decreased, respectively, in a dose-response manner (p < 0.05). The DiOC2(3) intensity for mitochondrial membrane potential (MMP) of MEGT-treated Ca9-22 cancer cells was significantly decreased in a dose-response manner (p < 0.05). CONCLUSIONS: These results indicated that MEGT had apoptosis-based cytotoxicity against oral cancer cells through the DNA damage, ROS induction, and mitochondrial depolarization. Therefore, MEGT derived from the edible algae may have potential therapeutic effects against oral squamous cell carcinoma (OSCC).


Subject(s)
Apoptosis/drug effects , DNA Damage/drug effects , Gracilaria/chemistry , Growth Inhibitors/pharmacology , Mouth Neoplasms/physiopathology , Oxidative Stress/drug effects , Plant Extracts/pharmacology , Cell Line, Tumor , Cell Proliferation/drug effects , Humans , Mouth Neoplasms/drug therapy , Mouth Neoplasms/genetics , Mouth Neoplasms/metabolism
18.
Molecules ; 17(9): 10916-27, 2012 Sep 11.
Article in English | MEDLINE | ID: mdl-22968475

ABSTRACT

The water extract of Gracilaria tenuistipitata have been found to be protective against oxidative stress-induced cellular DNA damage, but the biological function of the ethanolic extracts of G. tenuistipitata (EEGT) is still unknown. In this study, the effect of EEGT on oral squamous cell cancer (OSCC) Ca9-22 cell line was examined in terms of the cell proliferation and oxidative stress responses. The cell viability of EEGT-treated OSCC cells was significantly reduced in a dose-response manner (p < 0.0001). The annexin V intensity and pan-caspase activity of EEGT-treated OSCC cells were significantly increased in a dose-response manner (p < 0.05 to 0.0001). EEGT significantly increased the reactive oxygen species (ROS) level (p < 0.0001) and decreased the glutathione (GSH) level (p < 0.01) in a dose-response manner. The mitochondrial membrane potential (MMP) of EEGT-treated OSCC cells was significantly decreased in a dose-response manner (p < 0.005). In conclusion, we have demonstrated that EEGT induced the growth inhibition and apoptosis of OSCC cells, which was accompanied by ROS increase, GSH depletion, caspase activation, and mitochondrial depolarization. Therefore, EEGT may have potent antitumor effect against oral cancer cells.


Subject(s)
Antineoplastic Agents, Phytogenic/pharmacology , Apoptosis/drug effects , Cell Proliferation/drug effects , Gracilaria , Membrane Potential, Mitochondrial/drug effects , Mouth Neoplasms/drug therapy , Plant Extracts/pharmacology , Caspases/metabolism , Cell Line, Tumor , Cell Survival/drug effects , Ethanol , Glutathione/analysis , Humans , Mitochondria/drug effects , Oxidative Stress/drug effects , Reactive Oxygen Species/metabolism
19.
Food Chem Toxicol ; 50(10): 3433-9, 2012 Oct.
Article in English | MEDLINE | ID: mdl-22847137

ABSTRACT

Areca nut (Areca catechu) is chewed regularly as a medical and psychoactive food by about 10% of the world population, in countries including India, Taiwan and parts of Southern Asia. Areca nut chewing during pregnancy has been associated with both lower birth weight and premature birth. Animals of low birth weights showed retardation of muscle development. Our previous study showed that arecoline, the major areca alkaloid, decreased the number of implanted embryos. Here we sought to determine the effects of arecoline in myogenic differentiation by in vitro assays using C2C12 myoblast cells. The results showed that arecoline higher than 0.4mM significantly increased apoptosis and decreased viability of C2C12 cells. Morphometric measurements of myotube formation and analyses of myogenic markers, myosin heavy chain and myogenin, revealed that myogenic differentiation was inhibited by 0.04-0.08 mM arecoline. Moreover, phosphorylated but not total STAT3 was significantly inhibited by arecoline during myotube formation. These results indicate that arecoline inhibits the myogenic differentiation of C2C12 cells by reducing the activation of STAT3, an upstream regulator of myogenesis. Improved understanding of the effects of arecoline during myogenic differentiation may help to establish public health policies and to develop potential treatments for such patients.


Subject(s)
Arecoline/pharmacology , Muscle Development/drug effects , Myoblasts/drug effects , STAT3 Transcription Factor/metabolism , Animals , Cell Differentiation , Gene Expression Regulation, Developmental/drug effects , Mice , Myoblasts/cytology , Myoblasts/physiology , Phosphorylation/drug effects , STAT3 Transcription Factor/genetics
20.
FEBS Lett ; 586(19): 3111-6, 2012 Sep 21.
Article in English | MEDLINE | ID: mdl-22884571

ABSTRACT

In the mammalian central nervous system transcripts of certain synaptic components are localized near the synapse, allowing for rapid regulation of protein levels. Here we test whether an mRNA localization mechanism also exists in the postsynaptic specialization induced by agrin in C2C12 myotubes. RT-PCR showed that Chrna1 was co-purified with nicotinic acetylcholine receptor (AChR) isolated by affinity column or by ultracentrifugation. In addition, Stau1 was found to interact with Chrna1 mRNA, and knocking down of Stau1 by RNAi resulted in defective AChR clustering. These results suggest that mRNA localization also participates in the formation of mammalian neuromuscular junction (NMJ).


Subject(s)
Agrin/metabolism , Muscle Fibers, Skeletal/metabolism , RNA, Messenger/genetics , RNA, Messenger/metabolism , Receptors, Nicotinic/genetics , Receptors, Nicotinic/metabolism , Animals , Cell Line , Gene Knockdown Techniques , Mice , RNA Interference , RNA-Binding Proteins/antagonists & inhibitors , RNA-Binding Proteins/genetics , RNA-Binding Proteins/metabolism , Synapses/metabolism
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