Differential regulation of CYP3A4 promoter activity by a new class of natural product derivatives binding to pregnane X receptor.

The pregnane X receptor (PXR) regulates drug metabolism by regulating the expression of drug-metabolizing enzymes such as cytochrome P450 3A4 (CYP3A4), which is involved in the metabolism of >50% of clinically prescribed drugs. The activity of PXR can be controlled by the binding of small molecule agonists or antagonists. Because of its unique ligand binding pocket, PXR binds promiscuously to structurally diverse chemicals. To study the structure-activity relationship, novel modulators for PXR are needed. Here we report the virtual screening of ∼25,000 natural product derivatives from the ZINC database using the Molecular Operating Environment docking software tool against the PXR-rifampicin complex X-ray crystal structure. Our screening resulted in identification of compounds based on the lowest S score, which measures Gibbs free energy. Interestingly, we found that the compounds that bind directly to PXR, as revealed in an intrinsic tryptophan fluorescence assay, modulate CYP3A4 promoter activity differentially in HepG2 cells. Mutational analysis and docking studies showed that these compounds bind broadly in the ligand binding pocket but interact with different amino acid residues. We further investigated the mechanism of binding by analyzing the functional groups that are important for distinguishing agonists from antagonists. The approach we used to identify novel modulators that bind to PXR can be useful for finding novel modulators of PXR.

The carbonylation and covalent dimerization of human superoxide dismutase 1 caused by its bicarbonate-dependent peroxidase activity is inhibited by the radical scavenger tempol.

Tempol (4-hydroxy-2,2,6,6-tetramethyl piperidine-1-oxyl) reduces tissue injury in animal models of various diseases via mechanisms that are not completely understood. Recently, we reported that high doses of tempol moderately increased survival in a rat model of ALS (amyotrophic lateral sclerosis) while decreasing the levels of oxidized hSOD1 (human Cu,Zn-superoxide dismutase) in spinal cord tissues. To better understand such a protective effect in vivo, we studied the effects of tempol on hSOD1 oxidation in vitro. The chosen oxidizing system was the bicarbonate-dependent peroxidase activity of hSOD1 that consumes H2O2 to produce carbonate radical, which oxidizes the enzyme. Most of the experiments were performed with 30 µM hSOD1, 25 mM bicarbonate, 1 mM H2O2, 0.1 mM DTPA (diethylenetriaminepenta-acetic acid) and 50 mM phosphate buffer at a final pH of 7.4. The results showed that tempol (5-75 µM) does not inhibit hSOD1 turnover, but decreases its resulting oxidation to carbonylated and covalently dimerized forms. Tempol acted by scavenging the carbonate radical produced and by recombining with hSOD1-derived radicals. As a result, tempol was consumed nearly stoichiometrically with hSOD1 monomers. MS analyses of turned-over hSOD1 and of a related peptide oxidized by the carbonate radical indicated the formation of a relatively unstable adduct between tempol and hSOD1-Trp32•. Tempol consumption by the bicarbonate-dependent peroxidase activity of hSOD1 may be one of the reasons why high doses of tempol were required to afford protection in an ALS rat model. Overall, the results of the present study confirm that tempol can protect against protein oxidation and the ensuing consequences.

Biological and structural evaluation of 10R- and 10S-methylthio-DDACTHF reveals a new role for sulfur in inhibition of glycinamide ribonucleotide transformylase.

Glycinamide ribonucleotide transformylase (GAR Tfase) is a folate-dependent enzyme in the de novo purine biosynthesis pathway, which has long been considered a potential target for development of anti-neoplastic therapeutics. Here we report the biological and X-ray crystallographic evaluations of both independent C10 diastereomers, 10S- and 10R-methylthio-DDACTHF, bound to human GAR Tfase, including the highest-resolution apo GAR Tfase structure to date (1.52 Å). Both diastereomers are potent inhibitors (Ki = 210 nM for 10R, and Ki = 180 nM for 10S) of GAR Tfase and exhibit effective inhibition of human leukemia cell growth (IC50 = 80 and 50 nM, respectively). Their inhibitory activity was surprisingly high, and these lipophilic C10-substituted analogues show distinct advantages over their hydrophilic counterparts, most strikingly in retaining potency in mutant human leukemia cell lines that lack reduced folate carrier protein activity (IC50 = 70 and 60 nM, respectively). Structural characterization reveals a new binding mode for these diastereoisomers, in which the lipophilic thiomethyl groups penetrate deeper into a hydrophobic pocket within the folate-binding site. In silico docking simulations of three other sulfur-containing folate analogues also indicate that this hydrophobic cleft represents a favorable region for binding lipophilic substituents. Overall, these results suggest sulfur and its substitutions play an important role in not only the binding of anti-folates to GAR Tfase but also the selectivity and cellular activity (growth inhibition), thereby presenting new possibilities for the future design of potent and selective anti-folate drugs that target GAR Tfase.

MicroRNA-503 suppresses proliferation and cell-cycle progression of endometrioid endometrial cancer by negatively regulating cyclin D1.

MicroRNAs (miRNAs) are post-transcriptional inhibitor regulators of gene expression that act by directly binding complementary mRNA and are key determinants of cancer initiation and progression. In this study, we revealed a role for the tumor-suppressor miRNA miR-503 in endometrioid endometrial cancer (EEC) cells. The miR-503 expression level gradually decreases across normal endometrial tissues, endometrial tissues with complex atypical hyperplasia, and EEC tissues. A relatively high level of miR-503 in EEC tissues indicates a longer survival time in EEC patients. The expression of a cell cycle-associated oncogene encoding cyclin D1 (CCND1) was inversely correlated with miR-503 expression in EEC tissues and cell lines. CCND1 has a binding sequence of miR-503 within its 3' untranslated region, and was confirmed to be a direct target of miR-503 by the fluorescent reporter assays. Increasing the miR-503 level in EEC cells suppressed cell viability, colon formation activity and cell-cycle progression, and the inhibited oncogenic phenotypes induced by miR-503 were alleviated by ectopic expression of CCND1 without the untranslated region sequence. Furthermore, in vivo studies also suggested a suppressive effect of miR-503 on EEC cell-derived xenografts. miR-503 increased in cell cycle-arrested EEC cells, and was restored to a normal level in EEC cells after cell cycle re-entry, while CCND1 displayed the opposite expression pattern. Collectively, this study suggested that miR-503 plays a tumor-suppressor role by targeting CCND1. Abnormal suppression of miR-503 leads to an increase in the CCND1 level, which may promote carcinogenesis and progression of EEC.

A general anaesthetic propofol inhibits aquaporin-4 in the presence of Zn²âº.

AQP4 (aquaporin-4), a water channel protein that is predominantly expressed in astrocyte end-feet, plays an important role in the brain oedema formation, and is thereby considered to be a potential therapeutic target. Using a stopped-flow analysis, we showed that propofol (2,6-diisopropylphenol), a general anaesthetic drug, profoundly inhibited the osmotic water permeability of AQP4 proteoliposomes in the presence of Zn²âº. This propofol inhibition was not observed in AQP1, suggesting the specificity for AQP4. In addition, the inhibitory effects of propofol could be reversed by the removal of Zn²âº. Other lipid membrane fluidizers also similarly inhibited AQP4, suggesting that the modulation of protein-lipid interactions plays an essential role in the propofol-induced inhibition of AQP4. Accordingly, we used Blue native PAGE and showed that the profound inhibition caused by propofol in the presence of Zn²âº is coupled with the reversible clustering of AQP4 tetramers. Site-directed mutagenesis identified that Cys²5³, located at the membrane interface connecting to the C-terminal tail, is responsible for Zn²âº-mediated propofol inhibition. Overall, we discovered that propofol specifically and reversibly inhibits AQP4 through the interaction between Zn²âº and Cys²5³. The findings provide new insight into the functional regulation of AQP4 and may facilitate the identification of novel AQP4-specific inhibitors.

ROS inhibitor N-acetyl-L-cysteine antagonizes the activity of proteasome inhibitors.

NAC (N-acetyl-L-cysteine) is commonly used to identify and test ROS (reactive oxygen species) inducers, and to inhibit ROS. In the present study, we identified inhibition of proteasome inhibitors as a novel activity of NAC. Both NAC and catalase, another known scavenger of ROS, similarly inhibited ROS levels and apoptosis associated with H2O2. However, only NAC, and not catalase or another ROS scavenger Trolox, was able to prevent effects linked to proteasome inhibition, such as protein stabilization, apoptosis and accumulation of ubiquitin conjugates. These observations suggest that NAC has a dual activity as an inhibitor of ROS and proteasome inhibitors. Recently, NAC was used as a ROS inhibitor to functionally characterize a novel anticancer compound, piperlongumine, leading to its description as a ROS inducer. In contrast, our own experiments showed that this compound depicts features of proteasome inhibitors including suppression of FOXM1 (Forkhead box protein M1), stabilization of cellular proteins, induction of ROS-independent apoptosis and enhanced accumulation of ubiquitin conjugates. In addition, NAC, but not catalase or Trolox, interfered with the activity of piperlongumine, further supporting that piperlongumine is a proteasome inhibitor. Most importantly, we showed that NAC, but not other ROS scavengers, directly binds to proteasome inhibitors. To our knowledge, NAC is the first known compound that directly interacts with and antagonizes the activity of proteasome inhibitors. Taken together, the findings of the present study suggest that, as a result of the dual nature of NAC, data interpretation might not be straightforward when NAC is utilized as an antioxidant to demonstrate ROS involvement in drug-induced apoptosis.

FDA-approved drugs and other compounds tested as inhibitors of human glutathione transferase P1-1.

OBJECTIVE: Glutathione transferase P1-1 (GST P1-1) is often overexpressed in tumor cells and is regarded as a contributor to their drug resistance. Inhibitors of GST P1-1 are expected to counteract drug resistance and may therefore serve as adjuvants in the chemotherapy of cancer by increasing the efficacy of cytostatic drugs. Finding useful inhibitors among compounds used for other indications would be a shortcut to clinical applications and a search for GST P1-1 inhibitors among approved drugs and other compounds was therefore conducted. METHODS: We tested 1040 FDA-approved compounds as inhibitors of the catalytic activity of purified human GST P1-1 in vitro. RESULTS: We identified chlorophyllide, merbromine, hexachlorophene, and ethacrynic acid as the most effective GST P1-1 inhibitors with IC50 values in the low micromolar range. For comparison, these compounds were even more potent in the inhibition of human GST A3-3, an enzyme implicated in steroid hormone biosynthesis. In distinction from the other inhibitors, which showed conventional inhibition patterns, the competitive inhibitor ethacrynic acid elicited strong kinetic cooperativity in the glutathione saturation of GST P1-1. Apparently, ethacrynic acid serves as an allosteric inhibitor of the enzyme. CONCLUSION AND PRACTICAL IMPLICATIONS: In their own right, the compounds investigated are less potent than desired for adjuvants in cancer chemotherapy, but the structures of the most potent inhibitors could serve as leads for the synthesis of more efficient adjuvants.

Diacylglycerol kinase θ couples farnesoid X receptor-dependent bile acid signalling to Akt activation and glucose homoeostasis in hepatocytes.

DGKs (diacylglycerol kinases) catalyse the conversion of diacylglycerol into PA (phosphatidic acid), a positive modulator of mTOR (mammalian target of rapamycin). We have found that chenodeoxycholic acid and the synthetic FXR (farnesoid X receptor) ligand GW4064 induce the mRNA and protein expression of DGKθ in the HepG2 cell line and in primary human hepatocytes. Reporter gene studies using 1.5 kB of the DGKθ promoter fused to the luciferase gene revealed that bile acids increase DGKθ transcriptional activity. Mutation of putative FXR-binding sites attenuated the ability of GW4046 to increase DGKθ luciferase activity. Consistent with this finding, ChIP (chromatin immunoprecipitation) assays demonstrated that bile acid signalling increased the recruitment of FXR to the DGKθ promoter. Furthermore, GW4064 evoked a time-dependent increase in the cellular concentration of PA. We also found that GW4064 and PA promote the phosphorylation of mTOR, Akt and FoxO1 (forkhead box O1), and that silencing DGKθ expression significantly abrogated the ability of GW4046 to promote the phosphorylation of these PA-regulated targets. DGKθ was also required for bile-acid-dependent decreased glucose production. Taken together, our results establish DGKθ as a key mediator of bile-acid-stimulated modulation of mTORC2 (mTOR complex 2), the Akt pathway and glucose homoeostasis.

MiR-133a modulates osteogenic differentiation of vascular smooth muscle cells.

Arterial calcification is a key pathologic component of vascular diseases such as atherosclerosis, coronary artery disease, and peripheral vascular disease. A hallmark of this pathological process is the phenotypic transition of vascular smooth muscle cells (VSMCs) to osteoblast-like cells. Several studies have demonstrated that microRNAs (miRNAs) regulate osteoblast differentiation, but it is unclear whether miRNAs also regulate VSMC-mediated arterial calcification. In the present study, we sought to characterize the role of miR-133a in regulating VSMC-mediated arterial calcification. Northern blotting analysis of VSMCs treated with ß-glycerophosphate demonstrated that miR-133a was significantly decreased during osteogenic differentiation. Overexpression of miR-133a inhibited VSMC transdifferentiation into osteoblast-like cells as evidenced by a decrease in alkaline phosphatase activity, osteocalcin secretion, Runx2 expression, and mineralized nodule formation. Conversely, the knockdown of miR-133a using an miR-133a inhibitor promoted osteogenic differentiation of VSMCs by increasing alkaline phosphatase activity, osteocalcin secretion, and Runx2 expression. Runx2 was identified as a direct target of miR-133a by a cotransfection experiment in VSMCs with luciferase reporter plasmids containing wild-type or mutant 3'-untranslated region sequences of Runx2. Furthermore, the pro-osteogenic effects of miR-133a inhibitor were abrogated in Runx2-knockdown cells, and the inhibition of osteogenic differentiation by pre-miR-133a was reversed by overexpression of Runx2, providing functional evidence that the effects of miR-133a in osteogenic differentiation were mediated by targeting Runx2. These results demonstrate that miR-133a is a key negative regulator of the osteogenic differentiation of VSMCs.

microRNA-133a regulates the cell cycle and proliferation of breast cancer cells by targeting epidermal growth factor receptor through the EGFR/Akt signaling pathway.

microRNAs are small, highly conserved, non-coding RNAs that regulate gene expression of target mRNAs through cleavage or translational inhibition, and are widely involved in carcinogenesis and cancer development. In this study, the expression profile of microRNA-133a (miR-133a) was examined in breast cancer cells and breast cancer tissues. The results showed that expression of miR-133a in both breast cancer cells and breast cancer tissues was significantly down-regulated. Over-expression of miR-133a in tumor cells arrested the cell cycle by drastically decreasing the G2 /S phase and retarded the newly synthesized DNA, suggesting a regulatory role for miR-133a in proliferation of breast cancer cells. Bioinformatics prediction showed that epidermal growth factor receptor (EGFR) is a potential target for miR-133a. A dual luciferase reporter gene assay showed that miR-133a bound to the 3' UTR of EGFR but not a mutated 3' UTR, thereby down-regulating the protein expression level. Accordingly, we found that expression of EGFR protein decreased with increased expression of miR-133a in MCF-7 and MDA-MB-231 cells. Over-expression of miR-133a in breast cancer cells resulted in suppression of the level of phosphorylated Akt protein (p-Akt) and inhibition of p-Akt nuclear translocation. These results demonstrate that miR-133a, which may act as a tumor suppressor in breast cancer, regulates the cell cycle and proliferation in tumorigenesis by targeting EGFR through the downstream signal molecule Akt. Overall, these results show that miR-133a may be used as biomarker and/or therapeutic target for diagnosis and therapy of breast cancer.

S100A11 is involved in the regulation of the stability of cell cycle regulator p21(CIP1/WAF1) in human keratinocyte HaCaT cells.

The cyclin-dependent kinase inhibitor p21(CIP1/WAF1) is a regulatory factor of the cell cycle. Its transcriptional activation and protein stability are tightly controlled by several distinct mechanisms. S100A11 is a member of the S100 family of Ca²âº-binding proteins involved in several biological processes, including cell cycle progression and signal transduction. In the present study, we show that down-regulation of S100A11 results in the reduction of p21 protein in human HaCaT keratinocytes. It appears that a ubiquitin-independent proteasomal degradation process is involved in p21 degradation in S100A11 down-regulated cells. The application of a proteasome inhibitor stabilized p21 protein in these cells. Analysis of distinct signal transduction pathways revealed a disturbed phosphatidylinositol-3-kinase/Akt pathway after S100A11 knockdown. We determined that the glycogen synthase kinase-3, which is negatively regulated by phosphatidylinositol 3-kinase/Akt, was activated in cells possessing knocked-down S100A11 and appears to be involved in p21 protein destabilization. The application of a specific inhibitor of glycogen synthase kinase 3 resulted in an increase of the p21 protein level in S100A11 down-regulated HaCaT cells. Glycogen synthase kinase 3 is able to phosphorylate p21 at T57, which induces p21 proteasomal turnover. Mutation of the glycogen synthase kinase 3 site threonine 57 into alanine (T57A) stabilizes p21 in HaCaT cells lacking S100A11. Beside decreased p21 protein, down-regulation of S100A11 triggered the induction of apoptosis in HaCaT cells. These observations suggest that S100A11 is involved in the maintenance of p21 protein stability and appears to function as an inhibitor of apoptosis in human HaCaT keratinocyte cells. Thus, the data shed light on a novel pathway regulating p21 protein stability.

PD173074, a selective FGFR inhibitor, reverses ABCB1-mediated drug resistance in cancer cells.

PURPOSE: Specific tyrosine kinase inhibitors were recently reported to modulate the activity of ABC transporters, leading to an increase in the intracellular concentration of their substrate drugs. In this study, we determine whether PD173074, a specific fibroblast growth factor receptor (FGFR) inhibitor, could reverse ABC transporter-mediated multidrug resistance. METHODS: 3-(4,5-Dimethylthiazol-yl)-2,5-diphenyllapatinibrazolium bromide assay was used to determine the effect of PD173074 on reversal of ABC transporter-mediated multidrug resistance (MDR). In addition, [³H]-paclitaxel accumulation/efflux assay, western blotting analysis, ATPase, and photoaffinity labeling assays were done to study the interaction of PD173074 on ABC transporters. RESULTS: PD173074 significantly sensitized both ABCB1-transfected and drug-selected cell lines overexpressing this transporter to substrate anticancer drugs colchicine, paclitaxel, and vincristine. This effect of PD173074 is specific to ABCB1, as no significant interaction was detected with other ABC transporters such as ABCC1 and ABCG2. The observed reversal effect seems to be primarily due to the decreased active efflux of [³H]-paclitaxel in ABCB1 overexpressing cells observed in efflux assay. In addition, no significant change in the ABCB1 expression was observed when ABCB1 overexpressing cells were exposed to 5 µM PD173074 for up to 3 days, thereby further suggesting its role in modulating the function of the transporter. In addition, PD173074 stimulated the ATPase activity of ABCB1 in a concentration-dependent manner, indicating a direct interaction with the transporter. Interestingly, PD173074 did not inhibit photolabeling of ABCB1 with [¹²5I]-iodoarylazidoprazosin (IAAP), showing that it binds at a site different from that of IAAP in the drug-binding pocket. CONCLUSIONS: Here, we report for the first time, PD173074, an inhibitor of the FGFR, to selectively reverse ABCB1 transporter-mediated MDR by directly blocking the efflux function of the transporter.

Role of cysteine residues in thermal inactivation of fungal Cel6A cellobiohydrolases.

Numerous protein engineering studies have focused on increasing the thermostability of fungal cellulases to improve production of fuels and chemicals from lignocellulosic feedstocks. However, the engineered enzymes still undergo thermal inactivation at temperatures well below the inactivation temperatures of hyperthermophilic cellulases. In this report, we investigated the role of free cysteines in the thermal inactivation of wild-type and engineered fungal family 6 cellobiohydrolases (Cel6A). The mechanism of thermal inactivation of Cel6A is consistent with disulfide bond degradation and thiol-disulfide exchange. Circular dichroism spectroscopy revealed that a thermostable variant lacking free cysteines refolds to a native-like structure and retains activity after heat treatment over the pH range 5-9. Whereas conserved disulfide bonds are essential for retaining activity after heat treatment, free cysteines contribute to irreversible thermal inactivation in engineered thermostable Cel6A as well as Cel6A from Hypocrea jecorina and Humicola insolens.

Expression and functional pathway analysis of nuclear receptor NR2F2 in ovarian cancer.

CONTEXT: Recent evidence implicates the orphan nuclear receptor, nuclear receptor subfamily 2, group F, member 2 (NR2F2; chicken ovalbumin upstream promoter-transcription factor II) as both a master regulator of angiogenesis and an oncogene in prostate and other human cancers. OBJECTIVE: The objective of the study was to determine whether NR2F2 plays a role in ovarian cancer and dissect its potential mechanisms of action. DESIGN, SETTING, AND PATIENTS: We examined NR2F2 expression in healthy ovary and ovarian cancers using quantitative PCR and immunohistochemistry. NR2F2 expression was targeted in established ovarian cancer cell lines to assess the impact of dysregulated NR2F2 expression in the epithelial compartment of ovarian cancers. RESULTS: Our results indicate that NR2F2 is robustly expressed in the stroma of healthy ovary with little or no expression in epithelia lining the ovarian surface, clefts, or crypts. This pattern of NR2F2 expression was markedly disrupted in ovarian cancers, in which decreased levels of stromal expression and ectopic epithelial expression were frequently observed. Ovarian cancers with the most disrupted patterns of NR2F2 were associated with significantly shorter disease-free interval by Kaplan-Meier analysis. Targeting NR2F2 expression in established ovarian cancer cell lines enhanced apoptosis and increased proliferation. In addition, we found that NR2F2 regulates the expression of NEK2, RAI14, and multiple other genes involved in the cell cycle, suggesting potential pathways by which dysregulated expression of NR2F2 impacts ovarian cancer. CONCLUSIONS: These results uncover novel roles for NR2F2 in ovarian cancer and point to a unique scenario in which a single nuclear receptor plays potentially distinct roles in the stromal and epithelial compartments of the same tissue.

Identification, expression profiling of a grass carp TLR8 and its inhibition leading to the resistance to reovirus in CIK cells.

TLR8 (toll-like receptor 8), a homolog of TLR3, TLR7 and TLR9 as prototypical intracellular members of TLR family, is generally associated with sensing single stranded RNA and plays a pivotal role in antiviral immune response. In this study, a TLR8 gene from grass carp Ctenopharyngodon idella (designated as CiTLR8) was obtained and characterized. The full-length cDNA of CiTLR8 was of 3766 bp. The open reading frame was of 3072 bp and encoded a polypeptide of 1023 amino acids, including seventeen LRR (leucine-rich repeat) motifs, one transmembrane domain and one TIR (toll/interleukin-1 receptor) domain. A single intron with the size of 839 bp was found on the neck of start codon (ATG). CiTLR8 mRNA was ubiquitously expressed in the 15 tested tissues and the expression level in gas bladder, spleen, brain, hindgut and trunk kidney tissues was high. Besides, the CiTLR8 expression in spleen and head kidney was significantly up-regulated and reached peak at 24 h post-injection of grass carp reovirus (GCRV). CiTLR8 transcription reached peak at 8 h and then declined below the normal level post-GCRV infection in the C. idella kidney (CIK) cell line; and it was rapidly and significantly down-regulated by the stimulation of the synthetic double-stranded RNA polyriboinosinic-polyribocytidylic acid sodium salt (poly I:C) in CIK cells in a dose and time-dependent manner. The inhibitor expression vectors were constructed and transfected into CIK cell line to obtain stably expressing shRNA targeting TLR8. In CiTLR8-knockdown cells, CiTLR7 transcript weakly increased, CiIFN-I mRNA was significantly down-regulated, and the expression of CiMyD88, CiIRF7 and CiMx1 scarcely changed. Post poly I:C stimulation, CiTLR8, CiTLR7 and CiMyD88 transcripts significantly increased, CiIRF7 was down-regulated after an initial phase of increase, and CiIFN-I and CiMx1 transcripts were up-regulated. After GCRV infection, the transcripts of CiTLR8, CiTLR7, CiMyD88 and CiIRF7 were up-regulated, but CiIFN-I and CiMx1 transcripts were inhibited. Nevertheless, cells transfected with pshTLR8 vectors had strong resistance against GCRV injection, suggesting CiTLR8 might play a negative role in antiviral immune response. These results collectively suggested that CiTLR8 was a novel member of TLR gene family, engaging in antiviral innate immune defense in C. idella, which laid a foundation for the further mechanism research of TLR8 in fishes.

Discovery of novel bromophenol 3,4-dibromo-5-(2-bromo-3,4-dihydroxy-6-(isobutoxymethyl)benzyl)benzene-1,2-diol as protein tyrosine phosphatase 1B inhibitor and its anti-diabetic properties in C57BL/KsJ-db/db mice.

In an effort to develop novel small molecule PTP1B inhibitors, a series of bromophenol derivatives were designed, synthesized and evaluated in vitro and in vivo. All of the synthesized compounds displayed weak to potent PTP1B inhibitory activities (5.62-96.25%) at 20 µg/mL. Among these compounds, 3,4-dibromo-5-(2-bromo-3,4-dihydroxy-6-(isobutoxymethyl)benzyl)benzene-1,2-diol (9) exhibited enhanced PTP1B inhibitory activity (IC50 = 1.50 µM) than the lead compound BDDPM (IC50 = 2.42 µM) and high selectivity against other PTPs (TCPTP, LAR, SHP-1 and SHP-2). Results of anti-diabetic assay using C57BL/KsJ-db/db mouse model demonstrated that compound 9 was effective at lowering blood glucose, total cholesterol and HbA1c (P < 0.01).

miR-181b modulates glioma cell sensitivity to temozolomide by targeting MEK1.

PURPOSE: Recent studies have reported that miR-181b contributes to chemoresistance in several cancer types and functions as a tumor suppressor in glioma. This study aimed to explore whether miR-181b could enhance the chemotherapeutic effect of temozolomide in glioma cells and sought to identify the candidate target genes which mediated the effect. METHODS: Using 48 frozen samples from patients with glioma who had received in vitro chemosensitivity assay, we measured MGMT promoter methylation status by methylation-specific PCR and miR-181b expression by qRT-PCR. Then, miR-181b expression level was correlated with temozolomide IC50 and MGMT promoter methylation status. To investigate the mechanism of miR-181b-induced chemosensitivity, assays were performed using stable miR-181b-expressing transfectants of glioma cell lines created by a lentiviral system. RESULTS: Glioma cells rich in miR-181b were more sensitive to temozolomide. miR-181b expression was not correlated with MGMT promoter methylation status. miR-181b combined with temozolomide enhanced glioma cell sensitivity and apoptosis. The effects were through posttranscriptional repression of MEK1. We demonstrated that miR-181b bound directly to the 3' untranslated regions of MEK1, thus reducing both the mRNA and protein levels of MEK1. Additionally, knockdown of MEK1 using small interfering RNA resulted in effects similar to ectopic miR-181b expression, whereas enforced expression of MEK1 lacking the 3' untranslated regions abrogated the effects. Finally, inverse correlation between miR-181b and MEK1 was established in glioma specimens. CONCLUSION: miR-181b independently predicted chemoresponse to temozolomide and enhanced temozolomide sensitivity via MEK1 downregulation. A combination of miR-181b and temozolomide may be an effective therapeutic strategy for gliomas.

Manipulation of PBF/PTTG1IP phosphorylation status; a potential new therapeutic strategy for improving radioiodine uptake in thyroid and other tumors.

CONTEXT: The clinical effectiveness of ablative radioiodine treatment of thyroid tumors is limited by the availability of the sodium iodide symporter (NIS) at the plasma membrane (PM) for uptake of ¹³¹I. A significant proportion of well-differentiated thyroid tumors are unable to concentrate sufficient radioiodine for effective therapy, and in other tumor models such as breast tumors, where radioiodine uptake would be an attractive therapeutic option, uptake is insufficient. OBJECTIVE: Pituitary tumor-transforming gene-binding factor (PBF; PTTG1IP) is overexpressed in multiple cancers and significantly decreases NIS expression at the PM. The goal of this study was to identify a method by which PBF repression of NIS may be overcome in human tumors. RESULTS: Here, we identify PBF as a tyrosine phosphoprotein that specifically binds the proto-oncogene tyrosine protein kinase Src in mass spectrometry, glutathione S-transferase pulldown and coimmunoprecipitation assays. Src induction leads to phosphorylation at PBF residue Y174. Abrogation of this residue results in PM retention and a markedly reduced ability to bind NIS. The Src inhibitor PP1 inhibits PBF phosphorylation in multiple cell lines in vitro, including human primary thyroid cells. Of direct clinical importance to the treatment of thyroid cancer, PP1 stimulates iodide uptake by transfected NIS in TPC1 thyroid carcinoma cells and entirely overcomes PBF repression of iodide uptake in human primary thyroid cells. CONCLUSIONS: We propose that targeting PBF phosphorylation at residue Y174 via tyrosine kinase inhibitors may be a novel therapeutic strategy to enhance the efficacy of ablative radioiodine treatment in thyroid and other endocrine and endocrine-related tumors.

Critical role of histone acetylation by p300 in human placental 11ß-HSD2 expression.

CONTEXT: Fetal overexposure to glucocorticoids leads to growth restriction. Optimal fetal glucocorticoid level is ensured by the expression of cortisol-inactivating enzyme 11ß-hydroxysteroid dehydrogenase type 2 (11ß-HSD2) in placental syncytiotrophoblasts. The transcription factor selective promoter factor 1 (Sp1) is known to up-regulate 11ß-HSD2 expression in the presence of enhanced histone acetylation in syncytiotrophoblasts, but the mechanisms underlying histone acetylation remain unknown. OBJECTIVES: The role of p300 in histone acetylation associated with 11ß-HSD2 expression in syncytiotrophoblasts was investigated. DESIGN: Distribution of p300 in human placenta was studied with immunohistochemistry. The role of p300 in histone-3 (H3) acetylation in association with 11ß-HSD2 expression was investigated in cultured primary human placental trophoblasts in the presence of small interfering RNA (siRNA)-mediated knockdown of p300, p300 inhibitor C646, or p300 overexpression. The interaction of Sp1 and p300 was studied with chromatin immunoprecipitation and coimmunoprecipitation. RESULTS: Intense staining of p300 was found in the nuclei of trophoblasts. Levels of p300 and acetyl H3K9 and H3K27 associated with 11ß-HSD2 promoter were increased in the course of syncytialization and by cAMP pathway activation. Chromatin immunoprecipitation and coimmunoprecipitation revealed p300 and Sp1 on 11ß-HSD2 promoter and in the same protein complex in the syncytiotrophoblasts. Overexpression of p300 enhanced 11ß-HSD2 expression, which was attenuated by Sp1 knockdown, whereas p300 knockdown and C646 reduced both basal and cAMP-stimulated acetylation of H3K9 and H3K27 associated with 11ß-HSD2 expression. CONCLUSIONS: Interaction of p300 with Sp1 plays a crucial role in histone acetylation associated with 11ß-HSD2 expression in syncytiotrophoblasts, which may have important implications in the establishment of the placental glucocorticoid barrier in gestation.

The DPP-4 inhibitor linagliptin restores ß-cell function and survival in human isolated islets through GLP-1 stabilization.

CONTEXT: Inhibition of dipeptidyl peptidase-4 (DPP-4) is a potent strategy to increase glucose-dependent insulinotropic polypeptide and glucagon like peptide 1 (GLP-1) induced insulin secretion in diabetes. It is important to know whether new drugs approved for the treatment of type 2 diabetes have direct effects on the ß-cell. OBJECTIVE: Herein we investigated the effect of linagliptin, a novel DPP-4 inhibitor, on ß-cell function and survival. DESIGN: Human islets were exposed to a diabetic milieu (11.1-33.3 mM glucose, 0.5 mM palmitate, the mixture of 2 ng/mL IL-1ß+1000 U/mL interferon-γ, or 50 µM H2O2) with or without 500 ng/mL IL-1 receptor antagonist (IL-1Ra) or 30-50 nM linagliptin. RESULTS: Linagliptin restored ß-cell function and turnover, which was impaired when islets were exposed to elevated glucose, palmitate, cytokines, or H2O2. Pretreatment with IL-1Ra was similarly effective, except against H2O2 treatment. Nitrotyrosine concentrations in islet lysates, an indicator of oxidative stress, were highly elevated under diabetic conditions but not in islets treated with linagliptin or IL-1Ra. Linagliptin also reduced cytokine secretion and stabilized GLP-1 in islet supernatants. CONCLUSIONS: We show that the novel DPP-4 inhibitor linagliptin protected from gluco-, lipo-, and cytokine-toxicity and stabilized active GLP-1 secreted from human islets. This provides a direct GLP-1 mediated protective effect of linagliptin on ß-cell function and survival.