Inhibition Of Protein Phosphorylation Research Articles

FGF12 regulates cell cycle gene expression and promotes follicular granulosa cell proliferation through ERK phosphorylation in geese

The granulosa cells play an important role in the fate of follicular development or atresia in poultry. Fibroblast growth factor 12 (FGF12) is downregulated in atretic follicles and may be involved in regulating granulosa cell survival in previous studies, but its molecular mechanism remains unclear. In this study, FGF12 overexpression and knockdown models of goose granulosa cells were constructed to investigate its function. The downstream expression of the cell cycle pathway was analyzed by qPCR. Granulosa cell proliferative activity and apoptosis were detected by CCK8 and TUNEL. Protein phosphorylation levels of ERK and AKT were measured using Western blotting to analyze the key pathway of FGF12 regulation of granulosa cell proliferation. ERK protein phosphorylation inhibitor was added for further verification. After overexpression of FGF12, cell proliferation activity was increased, the expressions of cell cycle pathway genes CCND1, CCNA2, MAD2, and CHK1 were upregulated, the apoptosis of granulosa cell was decreased, and Caspase 3 gene and protein expression were downregulated. After the knockdown of FGF12, cell proliferation activity decreased, the expression of downstream genes in the cell cycle pathway was downregulated, the apoptosis of granulosa cells was increased, and the Bcl-2 gene and protein were downregulated. Overexpression of FGF12 promoted the synthesis of P4 and upregulates the expression of the STAR gene. Overexpression of FGF12 promoted ERK protein phosphorylation but did not affect AKT phosphorylation. The addition of ERK phosphorylation inhibitors resulted in the elimination of the increase in cell proliferative activity caused by FGF12 overexpression. In conclusion, FGF12 could promote proliferation and inhibit apoptosis of goose granulosa cells by increasing ERK phosphorylation.

Study on the Function and Mechanism of miR-585-3p Inhibiting the Progression of Ovarian Cancer Cells by Targeting FSCN1 to Block the MAPK Signaling Pathway

Ovarian cancer (OC) is the leading cause of death for women diagnosed with gynecological cancer. Studies have shown that dysregulated miRNA expression is related to various cancers, including OC. Here, we aimed to explore the biological function and mechanism of miR-585-3p in the occurrence and development of OC. The expression level of miR-585-3p was found to be low in OC tissues and cells. We analyzed the biological function of miR-585-3p in OC through in vitro cell experiments. The results indicated that overexpression of miR-585-3p inhibited the proliferation, invasion, and migration of SW626 cells, while low expression of miR-585-3p had the opposite effect in SKOV3 cells. We then screened the target genes of miR-585-3p through miRDB database and detected the expression of target genes in OC cells. FSCN1 was found to be most significantly upregulated in OC cells. Dual-luciferase reporter assays revealed FSCN1 as a potential target of miR-585-3p. Western blot analysis showed that miR-585-3p targeted FSCN1 to inhibit protein phosphorylation of ERK. In vivo animal experiments also confirmed that miR-585-3p targets FSCN1 to inhibit tumor growth and block the MAPK signaling pathway. In summary, miR-585-3p inhibits the proliferation, migration, and invasion of OC cells by targeting FSCN1, and its mechanism of action may be achieved by inhibiting the activation of the MAPK signaling pathway. miR-585-3p may serve as a potential biomarker and therapeutic target for OC.

PDIA4 promotes glioblastoma progression via the PI3K/AKT/m-TOR pathway

Protein disulfide isomerase A4 (PDIA4) is highly expressed in clear cell ovarian carcinoma and lung cancer. Through analysis of TCGA database and CGGA database, we noted that PDIA4 is a key promotor of glioblastoma (GBM). However, the detailed role and molecular mechanism of PDIA4 in GBM remain unclear. In this study, the expression pattern and biological role of PDIA4 in GBM was investigated. PDIA4 was overexpressed in GBM tumor samples and cell lines and positively correlated with pathological grades in glioma patients. In addition, downregulation of PDIA4 promoted apoptosis and inhibited proliferation of GBM. Meanwhile, there was a concurrent decrease in aerobic glycolysis metabolites. Mechanistically, PDIA4 downregulation promoted the apoptosis of GBM cells by increased the expression of apoptosis pathway proteins (caspase 3, caspase 9 and Bax). Downregulation of PDIA4 decreased energy demand and inhibited GBM growth in vitro and in vivo. Besides, such effect also inhibited the PI3K/AKT/m-TOR pathway by inhibiting protein phosphorylation levels of PI3K, AKT and m-TOR. After addition of PI3K/AKT/mTOR pathway activator 740Y–P, the effect of PDIA4 knockdown on GBM was reversed. Therefore, we believe that PDIA4 regulates the proliferation via activating the PI3K/AKT/m-TOR pathway and suppression of apoptosis in glioblastoma. It could be used as a potential target for the treatment of GBM.

Docking and dynamic simulation study of Crizotinib and Temozolomide drug with Glioblastoma and NSCLC target to identify better efficacy of the drug

BackgroundCrizotinib and Temozolomide are the two major chemotherapy drugs used for the treatment of cancers. Crizotinib is used as a target chemotherapy drug in many cancers. It mainly binds on the ATP binding regions of receptor tyrosine kinases (RTKs) targets and inhibits protein phosphorylation, which has already been reported. Temozolomide drug is known as the alkylating agent. Its mechanism of action is the methylation of DNA and thereby inhibiting DNA replication. However, the Temozolomide drug with protein level interaction of Glioblastoma Multiforme (GBM) and Non-small-cell lung carcinoma (NSCLC) of RTKs targets has not been reported so far. In the proposed work, we investigated the molecular level interaction of the Temozolomide drug in C-MET, C-ROS1, and ALK RTKs targets of GBM and NSCLC using an in silico study. We performed comparative analysis studies in both drugs' docked complexes based on their drug properties and complex energy (CE) to identify the better efficacy of the drug.ResultsFrom the docking studies, we could identify that the Temozolomide drug bounded protein complexes showed the least complex energy. The most stable complexes were identified from these docking studies by Molecular Dynamic simulation. In the proposed study, we found that the docked complex attained a stable conformation and least energy via solid hydrogen bond interactions between the amino acid residues and the drug at the binding sites of the proteins. The least energy and the hydrogen bond interaction of Temozolomide drug with the amino acid residues of the protein complexes of C-MET, C-ROS1 and ALK protein with their id name are: 2WGJ is − 11305.0830 (PRO1158, MET1160), 3ZBF is − 11,659.6814 (MET2029, GLU2027), and 2XP2 is − 11,734.7565 (ARG1275, ASP 1160, GLU1167).ConclusionOur studies revealed that the Temozolomide drug bounded protein complex showed the least energy when compared to Crizotinib. So it will give better interaction on the binding sites of proteins and thereby provide better inhibition in the treatment of target therapy of GBM and NSCLC.

Mutational mimics of allosteric effectors: a genome editing design to validate allosteric drug targets

Development of drugs that allosterically regulate enzyme functions to treat disease is a costly venture. Amino acid susbstitutions that mimic allosteric effectors in vitro will identify therapeutic regulatory targets enhancing the likelihood of developing a disease treatment at a reasonable cost. We demonstrate the potential of this approach utilizing human liver pyruvate kinase (hLPYK) as a model. Inhibition of hLPYK was the first desired outcome of this study. We identified individual point mutations that: 1) mimicked allosteric inhibition by alanine, 2) mimicked inhibition by protein phosphorylation, and 3) prevented binding of fructose-1,6-bisphosphate (Fru-1,6-BP). Our second desired outcome was activation of hLPYK. We identified individual point mutations that: 1) prevented hLPYK from binding alanine, the allosteric inhibitor, 2) prevented inhibitory protein phosphorylation, or 3) mimicked allosteric activation by Fru-1,6-BP. Combining the three activating point mutations produced a constitutively activated enzyme that was unresponsive to regulators. Expression of a mutant hLPYK transgene containing these three mutations in a mouse model was not lethal. Thus, mutational mimics of allosteric effectors will be useful to confirm whether allosteric activation of hLPYK will control glycolytic flux in the diabetic liver to reduce hepatic glucose production and, in turn, reduce or prevent hyperglycemia.

Human sperm motility, capacitation and acrosome reaction are impaired by 2-arachidonoylglycerol endocannabinoid.

The endocannabinoids are cannabinoids synthesized by mammalian tissues. These compounds are closely related to the regulation of the male reproductive system. However, little is known about the effects produced by 2-arachidonoylglycerol (2AG) on in vitro human sperm functions. This study was undertaken to determine the effects produced by 2AG on fresh human sperm and in the capacitation technique. Semen samples from healthy young men were exposed to different concentrations of 2AG before and during capacitation technique. In this work, we have demonstrated that 2AG induces the spontaneous acrosome reaction and reduces progressive motility in fresh human sperm. During the capacitation technique, sperm becomes more sensitive to low concentrations of 2AG, triggering the acrosome reaction and inhibiting protein phosphorylation. In summary, 2AG affects the in vitro functionality of human sperm by reducing motility, inhibiting capacitation and triggering the acrosome reaction.

Effect of protein phosphorylation inhibitor on production of parthenogenetic caprine embryos

The objective of the present study was to compare the effect of different concentrations of 6-Dimethylaminopurine (6-DMAP) treatment in chemical activated oocytes to compare the developmental potency of parthenogenetic caprine embryos. Morphologicaly matured oocytes were denuded and activated with 5 μm calcium ionophore (ionomycin) for 5 min. Activated oocytes were randomly divided into 5 groups: Group 1 oocytes were cultured in modified Charles Rosenkrans medium (mCR2aa), without DMAP treatment, group 2 cultured in mCR2aa with 2.5 mM DMAP, group 3 cultured in mCR2aa with 5 mM DMAP, group 4 cultured in mCR2aa with 10 mM DMAP, group 5 cultured in mCR2aa with 20 mM DMAP for 4 h. The presumptive zygotes of 5 groups were washed and cultured in the embryo development medium. Development of parthenotes was observed at every 48 h till day 12 post insemination. The percentage of cleavage, morula, blastocyst and hatched blastocyst production in groups 1, 2, 3, 4 and 5 were 61.76, 6.67, 0.00 and 0.00; 59.79, 27.43, 10.62 and 0.00; 72.43, 41.62, 10.66 and 1.52; 64.61, 50.44, 7.83 and 0.00; 63.19, 19.42, 3.88 and 0.00% respectively. Results indicated that 5 mM DMAP treatment for chemically activated oocyte is the best treatment for development of parthenogenetic caprine embryos. In conclusion, it can be stated that for the production of caprine parthenogenetic embryos in vitro, concentration of protein phosphorylation inhibitor plays an important role in the activation treatment. Optimized activation protocols could enhance the production of viable parthenogenetic embryos.

Dual ErbB1 and ErbB2 receptor tyrosine kinase inhibition exerts synergistic effect with conventional chemotherapy in pancreatic cancer

Patient survival in pancreatic cancer remains poor with gemcitabine (GEM)-based regimens. The target specific molecular agent lapatinib, a dual ErbB1 and ErbB2 receptor tyrosine kinase inhibitor, has shown significant activity against ErbB1 and ErbB2-expressing tumors. Since pancreatic tumors frequently overexpress these proteins, we investigated its effects, both alone and in conjunction with 5-FU or GEM. The pancreatic cancer cell lines PANC-1 and AsPC were treated with varying doses of lapatinib invitro. The effects on ErbB1/ErbB2 protein phosphorylation and on the cell survival protein survivin were determined by western blotting. Cytotoxicity was determined by MTT assay and apoptosis was measured using the caspase-3 colorimetric assay. Similar dose-response lapatinib experiments were conducted with varying concentrations of 5-FU or GEM and isobolograms were constructed to evaluate therapeutic synergy. Lapatinib inhibited protein phosphorylation in the range of 4-16µM, a clinically achievable concentration. The lapatinib-treated cells showed a dose-dependent inhibition of cell proliferation and induction of apoptosis at the same concentrations that blocked ErbB1/ErbB2 phosphorylation. The addition of 5-FU or GEM to these cells resulted in synergistic effects. The lapatinib-treated cells also demonstrated downregulation of survivin. Simultaneous dual ErbB1 and ErbB2 receptor tyrosine kinase inhibition with lapatinib results in significant reduction of pancreatic cancer cell growth and proliferation. These effects occur at clinically achievable concentrations and are synergistic with the effects of 5-FU or GEM. These findings support the potential role of lapatinib in the treatment of pancreatic cancer.

Growth factor regulation of intracellular pH homeostasis under hypoxic conditions in isolated equine articular chondrocytes

Hypoxia and acidosis are recognized features of inflammatory arthroses. This study describes the effects of IGF-1 and TGF-β(1) on pH regulatory mechanisms in articular cartilage under hypoxic conditions. Acid efflux, reactive oxygen species (ROS), and mitochondrial membrane potential were measured in equine articular chondrocytes isolated in the presence of serum (10% fetal calf serum), IGF-1 (1, 10, 50, 100 ng/ml) or TGF-β(1) (0.1, 1, 10 ng/ml) and then exposed to a short-term (3 h) hypoxic insult (1% O(2)). Serum and 100 ng/ml IGF-1 but not TGF-β(1) attenuated hypoxic regulation of pH homeostasis. IGF-1 appeared to act through mitochondrial membrane potential stabilization and maintenance of intracellular ROS levels in very low levels of oxygen. Using protein phosphorylation inhibitors PD98059 (25 µM) and wortmannin (200 nM) and Western blotting, ERK1/2 and PI-3 kinase pathways are important for the effect of IGF-1 downstream to ROS generation in normoxia but only PI-3 kinase is implicated in hypoxia. These results show that oxygen and growth factors interact to regulate pH recovery in articular chondrocytes by modulating intracellular oxygen metabolites.

Raf-1 Kinase Inhibitory Protein (RKIP) Mediates Ethanol-induced Sensitization of Secretagogue Signaling in Pancreatic Acinar Cells

Excessive alcohol consumption is associated with most cases of chronic pancreatitis, a progressive necrotizing inflammatory disease that can result in pancreatic insufficiency due to acinar atrophy and fibrosis and an increased risk of pancreatic cancer. At a cellular level acute alcohol exposure can sensitize pancreatic acinar cells to secretagogue stimulation, resulting in dysregulation of intracellular Ca(2+) homeostasis and premature digestive enzyme activation; however, the molecular mechanisms by which ethanol exerts these toxic effects have remained undefined. In this study we identify Raf-1 kinase inhibitory protein as an essential mediator of ethanol-induced sensitization of cholecystokinin- and carbachol-regulated Ca(2+) signaling in pancreatic acinar cells. We show that exposure of rodent acinar cells to ethanol induces protein kinase C-dependent Raf-1 kinase inhibitory protein phosphorylation, sensitization of cholecystokinin-stimulated Ca(2+) signaling, and potentiation of both basal and cholecystokinin-stimulated extracellular signal-regulated kinase activation. Furthermore, we show that either suppression of Raf-1 kinase inhibitory protein expression using short hairpin RNA or gene ablation prevented the sensitizing effects of ethanol on cholecystokinin- and carbachol-stimulated Ca(2+) signaling and intracellular chymotrypsin activation in pancreatic acinar cells, suggesting that the modulation of Raf-1 inhibitory protein expression may have future therapeutic utility in the prevention or treatment of alcohol-associated pancreatitis.

Protein phosphorylation on tyrosine restores expression and glycosylation of cyclooxygenase-2 by 2-deoxy-D-glucose-caused endoplasmic reticulum stress in rabbit articular chondrocyte

2-deoxy-D-glucose(2DG)-caused endoplasmic reticulum (ER) stress inhibits protein phosphorylation at tyrosine residues. However, the accurate regulatory mechanisms, which determine the inflammatory response of chondrocytes to ER stress via protein tyrosine phosphorylation, have not been systematically evaluated. Thus, in this study, we examined whether protein phosphorylation at tyrosine residues can modulate the expression and glycosylation of COX-2, which is reduced by 2DG-induced ER stress. We observed that protein tyrosine phosphatase (PTP) inhibitors, sodium orthovanadate (SOV), and phenylarsine oxide (PAO) significantly decreased expression of ER stress inducible proteins, glucose-regulated protein 94 (GRP94), and CCAAT/enhancer-binding-protein- related gene (GADD153), which was induced by 2DG. In addition, we demonstrated that SOV and PAO noticeably restored the expression and glycosylation of COX-2 after treatment with 2DG. These results suggest that protein phosphorylation of tyrosine residues plays an important role in the regulation of expression and glycosylation during 2DG-induced ER stress in rabbit articular chondrocytes.

Dual functional octreotide-modified liposomal irinotecan leads to high therapeutic efficacy for medullary thyroid carcinoma xenografts

Medullary thyroid carcinoma is a rare endocrine tumor, which shows overexpression of somatostatin receptor subtype 2. There is no systemic therapy for medullary thyroid carcinoma. Previously we reported that octreotide-PEG liposomes loaded with irinotecan, which target somatostatin receptor subtype 2, showed high therapeutic efficacy for medullary thyroid carcinoma xenografts compared with free irinotecan or non-targeted non-PEGylated liposomal irinotecan. In this study, we evaluated octreotide-PEG liposomes loaded with irinotecan in terms of the biodistribution of irinotecan and its active metabolite, and its therapeutic efficacy, compared with PEGylated liposomes. Furthermore, to elucidate the effect of octreotide ligand after cellular association, we assessed the cytotoxicity in tumor cells and the inhibition of protein phosphorylation in the tumor cells and xenografts using empty octreotide-PEG liposomes, which were loaded with no drug. In a therapeutic study, octreotide-PEG liposomes loaded with irinotecan significantly improved median survival compared with PEGylated liposomes. In tumor tissue at 6 h after injection, octreotide-PEG liposome-treated mice showed significantly higher concentrations of irinotecan and 7-ethyl-10-hydrocamptothecin compared with PEGylated liposome-treated mice, indicating that octreotide-PEG liposomes accumulated rapidly and to a high level in the tumor. Furthermore, empty octreotide-PEG liposome inhibited the phosphorylation of p70S6K in vitro and in vivo. These findings indicated that octreotide-PEG liposomal irinotecan has dual functions with targeted tumor delivery and assistance of cellular cytotoxicity, which led to higher therapeutic efficacy than PEGylated liposomes for medullary thyroid carcinoma xenografts.

Rb1 Gene Inactivation Expands Satellite Cell and Postnatal Myoblast Pools

Satellite cells are well known as a postnatal skeletal muscle stem cell reservoir that under injury conditions participate in repair. However, mechanisms controlling satellite cell quiescence and activation are the topic of ongoing inquiry by many laboratories. In this study, we investigated whether loss of the cell cycle regulatory factor, pRb, is associated with the re-entry of quiescent satellite cells into replication and subsequent stem cell expansion. By ablation of Rb1 using a Pax7CreER,Rb1 conditional mouse line, satellite cell number was increased 5-fold over 6 months. Furthermore, myoblasts originating from satellite cells lacking Rb1 were also increased 3-fold over 6 months, while terminal differentiation was greatly diminished. Similarly, Pax7CreER,Rb1 mice exhibited muscle fiber hypotrophy in vivo under steady state conditions as well as a delay of muscle regeneration following cardiotoxin-mediated injury. These results suggest that cell cycle re-entry of quiescent satellite cells is accelerated by lack of Rb1, resulting in the expansion of both satellite cells and their progeny in adolescent muscle. Conversely, that sustained Rb1 loss in the satellite cell lineage causes a deficit of muscle fiber formation. However, we also show that pharmacological inhibition of protein phosphatase 1 activity, which will result in pRb inactivation accelerates satellite cell activation and/or expansion in a transient manner. Together, our results raise the possibility that reversible pRb inactivation in satellite cells and inhibition of protein phosphorylation may provide a new therapeutic tool for muscle atrophy by short term expansion of the muscle stem cells and myoblast pool.

Effect of Phytohormones and Chemical Inhibitors on Pathogenesis-related Genes Identified by Differential Hybridization in Rice Suspension Culture Cells

In order to study disease resistance mechanisms in rice against the rice blast fungus Magnaporthe grisea, we screened fungal elicitor-responsive genes from rice suspension-cultured cells treated with fungal elicitors employing differential hybridization (DH). By DH screening, 31 distinct rice clones were isolated and a majority of them were full-length cDNAs encoding pathogenesisrelated (PR) genes. Sixteen of the 31 genes were upregulated at 4, 8, and 12 h following fungal elicitor treatment. To elucidate the effect of signal molecules and biotic elicitors on the regulation of rice defense genes, we further characterized the transcriptional expression patterns of representative isolated PR genes; OsGlu1, OsGlu2, OsTLP, OsRLK, and OsPR-10, following treatment with fungal elicitor, phytohormones, cycloheximide, and inhibitors of protein phosphorylation. Jasmonic acid (JA) induced transcriptional expression of OsGlu1, OsTLP, and OsRLK, but not of OsGlu2 and OsPR-10 at any of the tested time points. Salicylic acid (SA) and abscisic acid weakly induced the expression of OsTLP and OsRLK. SA showed an antagonistic effect with fungal elicitor and JA. Cycloheximide suppressed all these genes upon elicitor treatment, except for OsGlu2. Staurosporine only induced the expression of OsRLK. Application of calyculin A strongly induced OsRLK expression, but suppressed the expression of OsGlu2. Our study yielded a number of PR genes that play a role in defense mechanisms against the rice blast fungus, as well as contribute towards the elucidation of crosstalk between phytohormones and other modifications during defense signaling.

S100 proteins in health and disease

One class of proteins that has been increasingly emerging as a potentially important group of both molecular key players and biomarkers in the etiology, progression, manifestation and therapy of various inflammatory, neurodegenerative, metabolic and neoplastic disorders is the S100 family. S100 proteins are small, acidic, calcium-binding proteins, characterized by the presence of two calcium-binding EF-hand motifs, and found exclusively in vertebrates. The first member was identified in the bovine nervous system by Moore (1965). The name S100 was derived from the observation of the protein fraction remaining soluble after precipitation with 100% saturated ammonium sulfate at neutral pH. Subsequent studies demonstrated that this fraction contained predominantly two proteins, S100A1 and S100B. Since the first isolation and characterization of these 2 S100 proteins, at least 23 additional proteins have been assigned as members of the S100 family in humans (Marenholz et al. 2004, 2006). Twenty-one of them (S100A1–S100A18, and the multidomain proteins trichohyalin, filaggrin and repetin) are coded by genes clustered at chromosome locus 1q21, known as the epidermal differentiation complex, while the other genes belonging to the subfamilies of S100B, S100P, S100Z and S100G are, respectively, located at chromosome loci 21q22, 4p16, 5q14 and Xp22 (Santamaria-Kisiel et al. 2006). S100 proteins form homodimer, heterodimer and even oligomeric molecular assemblies and are expressed in a tissueand cell-specific manner, suggesting that each S100 protein may perform different functions (Donato 1999; Fritz and Heizmann 2006). Functional complexity and diversification of S100 proteins have been further achieved by differences in localization, e.g., intracellularly in cytoplasm and/or nucleus, extracellularly in various body fluid compartments, by action as autocrine or paracrine effectors, and by exhibition of different affinities toward calcium ions resulting in various degrees of conformational change and modes of interaction with a whole host of specific target proteins (Donato 1999; Leclerc et al. 2009). Moreover, several S100 proteins are known to bind to other divalent metal ions, such as magnesium, zinc and the transition metal copper with high affinity, perhaps implicating them additionally in the homeostasis of toxic metals and related pathophysiological conditions (Moroz et al. 2009). All of these result in a tremendous spectrum of pleiotropic intraand extracellular functions. In this regard, through, e.g., inhibition of protein phosphorylation, regulation of transcriptional factors, modulation of enzyme activity and cytoskeletal dynamics, S100 proteins have been linked to vital cellular processes, including cell cycle regulation, cell growth and differentiation, transcription, cell motility and invasion, extracellular signal transduction and intercellular adhesion (Santamaria-Kisiel et al. 2006, Leclerc et al. 2009). Among natural targets of extracellular S100 proteins, the multiligand or pattern recognition receptor for advanced glycation end products (RAGE) has gained significant importance (Hofmann et al. 1999; Leclerc et al. 2009). RAGE is a signal transduction receptor of the immunoglobulin superfamily and was first described as a receptor for end products of non-enzymatic glycation and glycoxidation of proteins (Schmidt et al. 1992). Importantly, RAGE also transduces signals stimulated by non-glycated proteins that J. Pietzsch (&) Department of Radiopharmaceutical Biology, Institute of Radiopharmacy, Research Center Dresden-Rossendorf, POB 510119, 01314 Dresden, Germany e-mail: j.pietzsch@fzd.de

Inhibition of Protein Phosphorylation in MIA Pancreatic Cancer Cells: Confluence of Metabolic and Signaling Pathways

Oxythiamine (OT), a transketolase inhibitor, is known to inhibit pancreatic cancer cell proliferation. In this study, we investigated the effect of inhibition of the transketolase pathway on signaling pathways in MIA PaCa cancer cells using in-house proteomic techniques. We hypothesized that OT alter protein phosphorylation thus affecting cell cycle arrest and cell proliferation. MIA PaCa-2 cells were cultured in media containing an algal (15)N amino acid mixture at 50% enrichment, with and without OT, to determine protein expression and synthesis. Analysis of cell lysates using two-dimensional gel electrophoresis matrix assisted laser desorption and ionization time-of-flight and time-of-flight mass spectrometry (2-DE-MALDI-TOF/TOF MS) identified 12 phosphor proteins that were significantly suppressed by OT treatment. Many of these proteins are involved in regulation of cycle activities and apoptosis. Among the proteins identified, expression of the phosphor heat shock protein 27 (Hsp27) was dramatically inhibited by OT treatment while the level of its total protein remained unchanged. Hsp27 expression and phosphorylation is known to be associated with drug resistance and cancer cell survival. The changes in phosphorylation of key proteins of cancer proliferation and survival suggest that protein phosphorylation is the confluence of the effects of OT on metabolic and signaling pathways.

Inhibitory effect of baicalein on IL‐6‐mediated signaling cascades in human myeloma cells

Interleukin-6 (IL-6) is an important growth factor for myeloma cells. IL-6 promotes the survival and proliferation of multiple myeloma (MM) cells through the phosphorylated proteins, including STAT3, MAPK, and Akt. Chemical components that suppress the signaling proteins' phosphorylation have a potential role for MM therapy. We recently identified that baicalein, a component of Scutellaria radix, suppressed proliferation and induced apoptosis of myeloma cells by blocking IkappaB-alpha degradation followed by down-regulating IL-6 and XIAP gene expression. In the present study of four myeloma cell lines, namely U266, NOP2, AMO1, and ILKM2, we demonstrated that baicalein not only inhibited IL-6-mediated phosphorylation of signaling proteins, such as Jak, STAT3, MAPK, and Akt, but also inhibited the expression of their target genes, such as bcl-xl. Finally, baicalein facilitated myeloma cell proliferation inhibited by dexamethasone. In contrast, baicalin, another major flavonoid derived from Scutellaria radix, had no significant effect on IL-6-mediated protein phosphorylation. Baicalein had no effect on Akt phosphorylation induced by the insulin-like growth factor-1 (IGF-1) in NOP2 cells. Compared with PD98059, an MAPK inhibitor, baicalein exhibited a stronger inhibitory effect on Erk(1/2) phosphorylation. Our results demonstrate that baicalein is a potent inhibitor of protein phosphorylation induced by IL-6, and thus may be a useful agent for the treatment of MM.

Regulation of Dauer Formation by O-GlcNAcylation in Caenorhabditis elegans

Modification of proteins at serine or threonine residues with N-acetylglucosamine, termed O-GlcNAcylation, plays an important role in most eukaryotic cells. To understand the molecular mechanism by which O-GlcNAcylation regulates the entry of Caenorhabditis elegans into the non-aging dauer state, we performed proteomic studies using two mutant strains: the O-GlcNAc transferase-deficient ogt-1(ok430) strain and the O-GlcNAcase-defective oga-1(ok1207) strain. In the presence of the dauer pheromone daumone, ogt-1 showed suppression of dauer formation, whereas oga-1 exhibited enhancement of dauer formation. Consistent with these findings, treatment of wild-type N2 worms with low concentrations of daumone and the O-GlcNAcase inhibitor O-(2-acetamido-2-deoxy-d-glucopyranosylidene)amino-N-phenylcarbamate (PUGNAc) enhanced dauer formation, which was dependent on intact O-GlcNAcylation metabolism. We also found that the treatment of daumone enhanced O-GlcNAcylation in vivo. Seven proteins, identified by coupled two-dimensional electrophoresis/liquid chromatography-mass spectroscopy (LC-MS) analysis, were differentially expressed in oga-1(ok1207) worms compared with wild-type N2 worms. The identities of these proteins suggest that O- GlcNAcylation influences stress resistance, protein folding, and mitochondrial function. Using O-GlcNAc labeling with fluorescent dye combined with two-dimensional electrophoresis/LC-MS analysis, we also identified five proteins that were differentially O-GlcNAcylated during dauer formation. Analysis of these candidate O-GlcNAcylated proteins suggests that O-GlcNAcylation may regulate cytoskeleton modifications and protein turnover during dauer formation.

Zinc inhibits protein phosphorylation in isolated sperm head membranes in Spisula solidissima

Zinc, when added to the media in concentrations as low as one micromolar, decreased protein phosphorylation in the isolated sperm head membranes in Spisula solidissima, 70-75%. Other divalent cations barium, strontium, cobalt, and copper, in similar concentrations exerted no effect. Sodium fluoride, a phosphatase inhibitor, had no effect on the zinc induced inhibition on protein phosphorylation and, when zinc was added to the media after the phosphorylation reaction of proteins, the inhibitory effect of zinc was lost. The autoradiography of -32P labeled proteins on SDS gel confirmed the inhibitory effect of zinc on protein phosphorylation. The inhibitory role of zinc on protein phosphorylation in the sperm membranes suggests a physiological role of this cation on membrane activity in the process of fertilization.

Bioactive sulfated sesterterpene alkaloids from the marine sponge Fasciospongia sp

Recent studies on signal transduction pathways have indicated various promising molecular targets for cancer treatment. Along these lines, a hyphae formation inhibition (HFI) assay [1] in Streptomyces 85E was adopted to rapidly screen and identify protein phosphorylation inhibitors. The organic extract of the marine sponge Fasciospongia sp. was found to significantly inhibit activity in the HFI assay. Bioassay-guided fractionation of this organic extract yielded two new unusual sesterterpene alkaloids (1 and 2), and a new sesterterpene sulfate (3), along with four known compounds. The structures were elucidated on the basis of spectroscopic analysis. Compounds 1-7 exhibited significant inhibitory activities in the HFI assay. These compounds were further evaluated in cytotoxicity assays with various cancer cell lines as described previously [2]. The structure elucidation and biological activities of these compounds is presented.