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Oppositional defiant disorder and conduct disorder, collectively referred to as disruptive behavior disorders (DBDs), are prevalent psychiatric disorders in children. Early diagnosis of DBDs is crucial because they can increase the risks of other mental health and substance use disorders without appropriate psychosocial interventions and treatment. However, diagnosing DBDs is challenging as they are often comorbid with other disorders, such as attention-deficit/hyperactivity disorder, anxiety, and depression. In this study, a multimodal ensemble three-dimensional convolutional neural network (3D CNN) deep learning model was used to classify children with DBDs and typically developing children. The study participants included 419 females and 681 males, aged 108–131 months who were enrolled in the Adolescent Brain Cognitive Development Study. Children were grouped based on the presence of DBDs (n = 550) and typically developing (n = 550); assessments were based on the scores from the Child Behavior Checklist and on the Schedule for Affective Disorders and Schizophrenia for School-age Children-Present and Lifetime version for DSM-5. The diffusion, structural, and resting-state functional magnetic resonance imaging (rs-fMRI) data were used as input data to the 3D CNN. The model achieved 72% accuracy in classifying children with DBDs with 70% sensitivity, 72% specificity, and an F1-score of 70. In addition, the discriminative power of the classifier was investigated by identifying the cortical and subcortical regions primarily involved in the prediction of DBDs using a gradient-weighted class activation mapping method. The classification results were compared with those obtained using the three neuroimaging modalities individually, and a connectome-based graph CNN and a multi-scale recurrent neural network using only the rs-fMRI data.

Abstract Functional magnetic resonance imaging has revealed correlated activities in brain regions even in the absence of a task. Initial studies assumed this resting-state functional connectivity (FC) to be stationary in nature, but recent studies have modeled these activities as a dynamic network. Dynamic spatiotemporal models better model the brain activities, but are computationally more involved. A comparison of static and dynamic FCs was made to quantitatively study their efficacies in identifying intrinsic individual connectivity patterns using data from the Human Connectome Project. Results show that the intrinsic individual brain connectivity pattern can be used as a ‘fingerprint’ to distinguish among and identify subjects and is more accurately captured with partial correlation and assuming static FC. It was also seen that the intrinsic individual brain connectivity patterns were invariant over a few months. Additionally, biological sex identification was successfully performed using the intrinsic individual connectivity patterns, and group averages of male and female FC matrices. Edge consistency, edge variability and differential power measures were used to identify the major resting-state networks involved in identifying subjects and their sex.

Brain complexity estimated using sample entropy and multiscale entropy (MSE) has recently gained much attention to compare brain function between diseased or neurologically impaired groups and healthy control groups. Using resting-state functional magnetic resonance imaging (rfMRI) blood oxygen-level dependent (BOLD) signals in a large cohort (n = 967) of healthy young adults, the present study maps neuronal and functional complexities estimated by using MSE of BOLD signals and BOLD phase coherence connectivity, respectively, at various levels of the brain’s organization. The functional complexity explores patterns in a higher dimension than neuronal complexity and may better discern changes in brain functioning. The leave-one-subject-out cross-validation method is used to predict fluid intelligence using neuronal and functional complexity MSE values as features. While a wide range of scales was selected with neuronal complexity, only the first three scales were selected with functional complexity. Fewer scales are advantageous as they preclude the need for long BOLD signals to calculate good estimates of MSE. The presented results corroborate with previous findings and provide a baseline for other studies exploring the use of MSE to examine changes in brain function related to aging, diseases, and clinical disorders.

Cofactor F420 plays crucial roles in bacterial and archaeal metabolism, but its biosynthetic pathway is not fully understood. Here, the authors present the structure of one of the enzymes and provide experimental evidence for a substantial revision of the pathway, including the identification of a new intermediate.

Connexin43 mimetic peptide (Cx43MP) has been intensively investigated for its therapeutic effect in the management of inflammatory eye conditions, spinal cord injury, wound healing and ischemia-induced brain damage. Here, we report on a validated stabilityâindicating reversed-phase high performance liquid chromatography (RP-HPLC) method for the quantification of Cx43MP under stress conditions. These included exposure to acid/base, light, oxidation and high temperature. In addition, the degradation kinetics of the peptide were evaluated in bovine vitreous and drug-free human plasma at 37 °C. Detection of Cx43MP was carried out at 214 nm with a retention time of 7.5 min. The method showed excellent linearity over the concentration range of 0.9â250 µg/mL (R2 ⥠0.998), and the limits of detection (LOD) and quantification (LOQ) were found to be 0.90 and 2.98 μg/mL, respectively. The accuracy of the method determined by the mean percentage recovery at 7.8, 62.5 and 250 µg/mL was 96.79%, 98.25% and 99.06% with a RSD of < 2.2%. Accelerated stability studies revealed that Cx43MP was more sensitive to basic conditions and completely degraded within 24 h at 37 °C (0% recovery) and within 12 h at 80 °C (0.34% recovery). Cx43MP was found to be more stable in bovine vitreous (t1/2slow= 171.8 min) compared to human plasma (t1/2slow = 39.3 min) at 37 °C according to the two phase degradation kinetic model. These findings are important for further pre-clinical development of Cx43MP. Keywords: Connexin43 mimetic peptide, RP-HPLC, Stress degradation, Peptide stability, Bovine vitreous, Human plasma

The rapidly developing miniaturization in numerous fields require low-demanding but robust methods of nanomaterial production. Colloidal synthesis provides great flexibility in product material, size, and shape. Gold nanoparticle synthesis has been thoroughly studied, however, recent reports on mechanistic insights of crystal formation have been hindered by the numerous procedures and parameter optimization works. With every new study, scientists fill another blank space on the map of understanding anisotropic growth and find out the critical parameters. In the current work, we highlight the choice importance for surfactant supplier in achieving the gold nanotriangle formation. We systematically study the variation in the shape yield when utilizing five batches of cetyltrimethylammonium chloride (CTAC) from varied suppliers. Using analytical techniques, we search for deviations causing such variation, e.g. different impurity content. We found only a marginal effect of iodine contamination on the studied system, excluding this factor as decisive in contrast to what was proposed earlier in the literature, and leaving the high dependency of the yield to originate from yet unknown reagent characteristics. A deeper understanding of these factors would provide highly effective protocols lowering the reagent consumption and increasing the accessibility of nanomaterials manufactured in a sustainable manner., (© 2024. The Author(s).)

Tumor cells subvert immune surveillance by harnessing signals from immune checkpoints to acquire immune resistance. The protein PD-L1 is an important component in this process, and inhibition of PD-L1 elicits durable anti-tumor responses in a broad spectrum of cancers. However, immune checkpoint inhibition that target known pathways is not universally effective. A better understanding of the genetic repertoire underlying these processes is necessary to expand our knowledge in tumor immunity and to facilitate identification of alternative targets. Here, we present a CRISPR/Cas9 screen in human cancer cells to identify genes that confer tumors with the ability to evade the cytotoxic effects of the immune system. We show that the transcriptional regulator MLLT6 (AF17) is required for efficient PD-L1 protein expression and cell surface presentation in cancer cells. MLLT6 depletion alleviates suppression of CD8 + cytotoxic T cell-mediated cytolysis. Furthermore, cancer cells lacking MLLT6 exhibit impaired STAT1 signaling and are insensitive to interferon-γ-induced stimulation of IDO1, GBP5, CD74, and MHC class II genes. Collectively, our findings establish MLLT6 as a regulator of oncogenic and interferon-γ-associated immune resistance., (© 2020 The Authors. Published under the terms of the CC BY NC ND 4.0 license.)

Background: Mutations in CTNS -a gene encoding the cystine transporter cystinosin-cause the rare, autosomal, recessive, lysosomal-storage disease cystinosis. Research has also implicated cystinosin in modulating the mTORC1 pathway, which serves as a core regulator of cellular metabolism, proliferation, survival, and autophagy. In its severest form, cystinosis is characterized by cystine accumulation, renal proximal tubule dysfunction, and kidney failure. Because treatment with the cystine-depleting drug cysteamine only slows disease progression, there is an urgent need for better treatments., Methods: To address a lack of good human-based cell culture models for studying cystinosis, we generated the first human induced pluripotent stem cell (iPSC) and kidney organoid models of the disorder. We used a variety of techniques to examine hallmarks of cystinosis-including cystine accumulation, lysosome size, the autophagy pathway, and apoptosis-and performed RNA sequencing on isogenic lines to identify differentially expressed genes in the cystinosis models compared with controls., Results: Compared with controls, these cystinosis models exhibit elevated cystine levels, increased apoptosis, and defective basal autophagy. Cysteamine treatment ameliorates this phenotype, except for abnormalities in apoptosis and basal autophagy. We found that treatment with everolimus, an inhibitor of the mTOR pathway, reduces the number of large lysosomes, decreases apoptosis, and activates autophagy, but it does not rescue the defect in cystine loading. However, dual treatment of cystinotic iPSCs or kidney organoids with cysteamine and everolimus corrects all of the observed phenotypic abnormalities., Conclusions: These observations suggest that combination therapy with a cystine-depleting drug such as cysteamine and an mTOR pathway inhibitor such as everolimus has potential to improve treatment of cystinosis., (Copyright © 2020 by the American Society of Nephrology.)

Glutathione (GSH) grafted onto nanoliposomes (GSH-liposomes) have the potential to enhance drug delivery into the brain. GSH is known to be an unstable tripeptide, however, despite widespread use to promote active transport its stability has been largely ignored to date. Therefore this study focuses on the optimisation of GSH conjugation with liposomes, supported with a validated HPLC assay for GSH. An isocratic stability-indicating HPLC assay of GSH was developed after derivatisation of GSH with 5,5'-dithio-bis-2-nitrobenzoic acid and applied for efficient conjugation of GSH to DSPE-PEG2000-maleimide lipid, either in solution or in preformed liposomes (4% molar ratio) at pH 7.4. The conjugation rate was monitored by the HPLC assay to optimise the conjugation conditions, including GSH concentration, GSH:lipid ratio, reaction time, temperature and medium. The physiochemical properties of the resulting GSH-liposomes and their GSH densities were characterised. The HPLC method was linear in the range of 0.05-50 µg/ml, highly sensitive (limit of quantification 50 ng/ml), and accurate (98-102% recoveries) with less than 4% intra-day and inter-day variability. Interestingly, enhanced GSH stability was observed at higher GSH concentrations ≥2 mg/ml and mass spectroscopy confirmed that GSH degradation occurred predominantly by oxidation. Both the proton nuclear magnetic resonance ( 1 H NMR) spectra and HPLC analysis of GSH concentrations confirmed the formation of GSH-PEG-DSPE conjugate. Under the optimal conditions, complete conjugation was attained either by post-insertion or direct conjugation methods with the resulting GSH-liposomes attaining a GSH density of 4% with similar size (120 nm) and zeta potential (-26.7 ± 0.9 mV or -29.8 ± 1.5 mV). The study provides useful information on GSH stability for the optimisation of its conjugation in liposomal formulation., (Copyright © 2019 Elsevier B.V. All rights reserved.)

Cofactor F 420 plays critical roles in primary and secondary metabolism in a range of bacteria and archaea as a low-potential hydride transfer agent. It mediates a variety of important redox transformations involved in bacterial persistence, antibiotic biosynthesis, pro-drug activation and methanogenesis. However, the biosynthetic pathway for F 420 has not been fully elucidated: neither the enzyme that generates the putative intermediate 2-phospho-L-lactate, nor the function of the FMN-binding C-terminal domain of the γ-glutamyl ligase (FbiB) in bacteria are known. Here we present the structure of the guanylyltransferase FbiD and show that, along with its archaeal homolog CofC, it accepts phosphoenolpyruvate, rather than 2-phospho-L-lactate, as the substrate, leading to the formation of the previously uncharacterized intermediate dehydro-F 420 -0. The C-terminal domain of FbiB then utilizes FMNH 2 to reduce dehydro-F 420 -0, which produces mature F 420 species when combined with the γ-glutamyl ligase activity of the N-terminal domain. These new insights have allowed the heterologous production of F 420 from a recombinant F 420 biosynthetic pathway in Escherichia coli.

Scope: Blueberry consumption is believed to confer a cardiovascular health advantage, but the active compounds and effects require characterization. This study aims to identify the polyphenol metabolites in plasma after blueberry juice intake and determine their bioactivity on endothelial cells., Methods and Results: Three healthy individuals are recruited to obtain profiles of bioavailable plasma polyphenol metabolites following intake of blueberry juice. Of 33 phenolic compounds screened, 12 aglycone phenolic acids are detected and their maximum plasma concentrations and circulation time determined. Using this information, the effect of three physiologically relevant mixtures of blueberry-derived phenolic acids is investigated for their ability to induce nuclear factor erythroid 2-related factor 2 (Nrf2)-nuclear translocation and downstream gene expression in human endothelial cells. Pretreatment with the phenolic acids for 18 h results in a significant upregulation of the Nrf2-regulated antioxidant response proteins heme oxygenase 1 (HO-1) and glutamate-cysteine ligase modifier subunit (GCLM), following 6 h exposure to 2.5 μm H 2 O 2 ., Conclusion: Physiologically relevant concentrations of blueberry-derived aglycone phenolic acids can induce Nrf2-regulated antioxidant response proteins in vascular endothelial cells in response to low μm concentrations of H 2 O 2 . Our results represent an advance over previous studies that have used single compounds or high concentrations in cell-based investigations., (© 2017 WILEY-VCH Verlag GmbH & Co. KGaA, Weinheim.)

Connexin43 mimetic peptide (Cx43MP) has been intensively investigated for its therapeutic effect in the management of inflammatory eye conditions, spinal cord injury, wound healing and ischemia-induced brain damage. Here, we report on a validated stability-indicating reversed-phase high performance liquid chromatography (RP-HPLC) method for the quantification of Cx43MP under stress conditions. These included exposure to acid/base, light, oxidation and high temperature. In addition, the degradation kinetics of the peptide were evaluated in bovine vitreous and drug-free human plasma at 37 °C. Detection of Cx43MP was carried out at 214 nm with a retention time of 7.5 min. The method showed excellent linearity over the concentration range of 0.9-250 µg/mL ( R 2 ≥ 0.998), and the limits of detection (LOD) and quantification (LOQ) were found to be 0.90 and 2.98 μg/mL, respectively. The accuracy of the method determined by the mean percentage recovery at 7.8, 62.5 and 250 µg/mL was 96.79%, 98.25% and 99.06% with a RSD of < 2.2%. Accelerated stability studies revealed that Cx43MP was more sensitive to basic conditions and completely degraded within 24 h at 37 °C (0% recovery) and within 12 h at 80 °C (0.34% recovery). Cx43MP was found to be more stable in bovine vitreous (t 1/2slow = 171.8 min) compared to human plasma (t 1/2slow = 39.3 min) at 37 °C according to the two phase degradation kinetic model. These findings are important for further pre-clinical development of Cx43MP.

Noise-induced hearing loss (NIHL) is a global health problem affecting over 5% of the population worldwide. We have shown previously that acute noise-induced cochlear injury can be ameliorated by administration of drugs acting on adenosine receptors in the inner ear, and a selective A 1 adenosine receptor agonist adenosine amine congener (ADAC) has emerged as a potentially effective treatment for cochlear injury and resulting hearing loss. This study investigated pharmacokinetic properties of ADAC in rat perilymph after systemic (intravenous) administration using a newly developed liquid chromatography-tandem mass spectrometry detection method. The method was developed and validated in accordance with the USA FDA guidelines including accuracy, precision, specificity, and linearity. Perilymph was sampled from the apical turn of the cochlea to prevent contamination with the cerebrospinal fluid. ADAC was detected in cochlear perilymph within two minutes following intravenous administration and remained in perilymph above its minimal effective concentration for at least two hours. The pharmacokinetic pattern of ADAC was significantly altered by exposure to noise, suggesting transient changes in permeability of the blood-labyrinth barrier and/or cochlear blood flow. This study supports ADAC development as a potential clinical otological treatment for acute sensorineural hearing loss caused by exposure to traumatic noise., Competing Interests: The authors declare that they have no competing interests.

Cofactor F420is an electron carrier with a major role in the oxidoreductive reactions ofMycobacterium tuberculosis, the causative agent of tuberculosis. A γ-glutamyl ligase catalyzes the final steps of the F420biosynthesis pathway by successive additions ofl-glutamate residues to F420-0, producing a poly-γ-glutamate tail. The enzyme responsible for this reaction in archaea (CofE) comprises a single domain and produces F420-2 as the major species. The homologousM. tuberculosisenzyme, FbiB, is a two-domain protein and produces F420with predominantly 5-7l-glutamate residues in the poly-γ-glutamate tail. The N-terminal domain of FbiB is homologous to CofE with an annotated γ-glutamyl ligase activity, whereas the C-terminal domain has sequence similarity to an FMN-dependent family of nitroreductase enzymes. Here we demonstrate that full-length FbiB adds multiplel-glutamate residues to F420-0in vitroto produce F420-5 after 24 h; communication between the two domains is critical for full γ-glutamyl ligase activity. We also present crystal structures of the C-terminal domain of FbiB in apo-, F420-0-, and FMN-bound states, displaying distinct sites for F420-0 and FMN ligands that partially overlap. Finally, we discuss the features of a full-length structural model produced by small angle x-ray scattering and its implications for the role of N- and C-terminal domains in catalysis., (© 2016 by The American Society for Biochemistry and Molecular Biology, Inc.)

Hypoxia is a prevalent feature of many tumors contributing to disease progression and treatment resistance, and therefore constitutes an attractive therapeutic target. Several hypoxia-activated prodrugs (HAP) have been developed, including the phase III candidate TH-302 (evofosfamide) and the preclinical agent SN30000, which is an optimized analogue of the well-studied HAP tirapazamine. Experience with this therapeutic class highlights an urgent need to identify biomarkers of HAP sensitivity, including enzymes responsible for prodrug activation during hypoxia. Using genome-scale shRNA screens and a high-representation library enriched for oxidoreductases, we identified the flavoprotein P450 (cytochrome) oxidoreductase (POR) as the predominant determinant of sensitivity to SN30000 in three different genetic backgrounds. No other genes consistently modified SN30000 sensitivity, even within a POR-negative background. Knockdown or genetic knockout of POR reduced SN30000 reductive metabolism and clonogenic cell death and similarly reduced sensitivity to TH-302 under hypoxia. A retrospective evaluation of head and neck squamous cell carcinomas showed heterogeneous POR expression and suggested a possible relationship between human papillomavirus status and HAP sensitivity. Taken together, our study identifies POR as a potential predictive biomarker of HAP sensitivity that should be explored during the clinical development of SN30000, TH-302, and other hypoxia-directed agents., (©2015 American Association for Cancer Research.)

The antidiabetic drug metformin exhibits both chemopreventive and chemotherapeutic activity for multiple cancers including pancreatic cancer; however, the underlying mechanism of action of metformin is unclear. A recent study showed that metformin down-regulated specificity protein (Sp) transcription factors (TFs) Sp1, Sp3, and Sp4 in pancreatic cancer cells and tumors, and this was accompanied by down-regulation of several pro-oncogenic Sp-regulated genes. Treatment with metformin or down-regulation of Sp TFs by RNAi also inhibits two major pro-oncogenic pathways in pancreatic cancer cells, namely mammalian target of rapamycin (mTOR) signaling and epidermal growth factor (EGFR)-dependent activation of Ras. Metformin and Sp knockdown by RNAi decreased expression of the insulin-like growth factor-1 receptor (IGF-1R), resulting in inhibition of mTOR signaling. Ras activity was also decreased by metformin and Sp knockdown of EGFR, another Sp-regulated gene. Thus, the antineoplastic activities of metformin in pancreatic cancer are due, in part, to down-regulation of Sp TFs and Sp-regulated IGF-1R and EGFR, which in turn results in inhibition of mTOR and Ras signaling, respectively., (© 2014 by The American Society for Biochemistry and Molecular Biology, Inc.)

Introduction: Specificity protein (Sp) transcription factors (TFs) are members of the Sp/Kruppel-like factor family, and Sp proteins play an important role in embryonic and early postnatal development. Sp1 has been the most extensively investigated member of this family, and expression of this protein decreases with age, whereas Sp1 and other family members (Sp3 and Sp4) are highly expressed in tumors and cancer cell lines., Area Covered: The prognostic significance of Sp1 in cancer patients and the functional pro-oncogenic activities of Sp1, Sp3 and Sp4 in cancer cell lines are summarized. Several different approaches have been used to target downregulation of Sp TFs and Sp-regulated genes, and this includes identification of different structural classes of antineoplastic agents including NSAIDs, natural products and their synthetic analogs and several well-characterized drugs including arsenic trioxide, aspirin and metformin. The multiple pathways involved in drug-induced Sp downregulation are also discussed., Expert Opinion: The recognition by the scientific and clinical community that experimental and clinically used antineoplastic agents downregulate Sp1, Sp3 and Sp4, and pro-oncogenic Sp-regulated genes will facilitate future clinical applications for individual drug and drug combination therapies that take advantage of their unusual effects.

Unlabelled: NR4A1 (Nur77, TR3) is an orphan nuclear receptor that is overexpressed in pancreatic cancer and exhibits pro-oncogenic activity. RNA interference of NR4A1 expression in Panc-1 cells induced apoptosis and subsequent proteomic analysis revealed the induction of several markers of endoplasmic reticulum stress, including glucose-related protein 78 (GRP78), CCAAT/enhancer-binding protein-homologous protein (CHOP), and activating transcription factor-4 (ATF-4). Treatment of pancreatic cancer cells with the NR4A1 antagonist 1,1-bis(3'-indolyl)-1-(p-hydroxyphenyl)methane (DIM-C-pPhOH) gave similar results. Moreover, both NR4A1 knockdown and DIM-C-pPhOH induced reactive oxygen species (ROS), and induction of ROS and endoplasmic reticulum stress by these agents was attenuated after cotreatment with antioxidants. Manipulation of NR4A1 expression coupled with gene expression profiling identified a number of ROS metabolism transcripts regulated by NR4A1. Knockdown of one of these transcripts, thioredoxin domain containing 5 (TXNDC5), recapitulated the elevated ROS and endoplasmic reticulum stress; thus, demonstrating that NR4A1 regulates levels of endoplasmic reticulum stress and ROS in pancreatic cancer cells to facilitate cell proliferation and survival. Finally, inactivation of NR4A1 by knockdown or DIM-C-pPhOH decreased TXNDC5, resulting in activation of the ROS/endoplasmic reticulum stress and proapoptotic pathways., Implications: The NR4A1 receptor is pro-oncogenic, regulates the ROS/endoplasmic reticulum stress pathways, and inactivation of the receptor represents a novel pathway for inducing cell death in pancreatic cancer., (Mol Cancer Res; 12(4); 527-38. ©2014 AACR.)

Unlabelled: We have previously shown that adenosine amine congener (ADAC), a selective A1 adenosine receptor agonist, can ameliorate noise- and cisplatin-induced cochlear injury. Here we demonstrate the dose-dependent rescue effects of ADAC on noise-induced cochlear injury in a rat model and establish the time window for treatment., Methods: ADAC (25-300 μg/kg) was administered intraperitoneally to Wistar rats (8-10 weeks old) at intervals (6-72 hours) after exposure to traumatic noise (8-16 kHz, 110 dB sound pressure level, 2 hours). Hearing sensitivity was assessed using auditory brainstem responses (ABR) before and 12 days after noise exposure. Pharmacokinetic studies investigated ADAC concentrations in plasma after systemic (intravenous) administration., Results: ADAC was most effective in the first 24 hours after noise exposure at doses >50 μg/kg, providing up to 21 dB protection (averaged across 8-28 kHz). Pharmacokinetic studies demonstrated a short (5 min) half-life of ADAC in plasma after intravenous administration without detection of degradation products., Conclusion: Our data show that ADAC mitigates noise-induced hearing loss in a dose- and time-dependent manner, but further studies are required to establish its translation as a clinical otological treatment.

Several agents used for treatment of colon and other cancers induce reactive oxygen species (ROS) and this plays an important role in their anticancer activities. In addition to the well-known proapoptotic effects of ROS inducers, these compounds also decrease expression of specificity protein (Sp) transcription factors Sp1, Sp3 and Sp4 and several pro-oncogenic Spregulated genes important for cancer cell proliferation, survival and metastasis. The mechanism of these responses involve ROS-dependent downregulation of microRNA-27a (miR-27a) or miR-20a (and paralogs) and induction of two Sp-repressors, ZBTB10 and ZBTB4 respectively. This pathway significantly contributes to the anticancer activity of ROS inducers and should be considered in development of drug combinations for cancer chemotherapy.

2,3-Dihydro-5-methyl-3-([morpholinyl]methyl)pyrollo(1,2,3-de)-1,4-benzoxazinyl]-[1-naphthaleny]methanone [WIN 55,212-2, (WIN)] is a synthetic cannabinoid that inhibits RKO, HT-29, and SW480 cell growth, induced apoptosis, and downregulated expression of survivin, cyclin D1, EGF receptor (EGFR), VEGF, and its receptor (VEGFR1). WIN also decreased expression of specificity protein (Sp) transcription factors Sp1, Sp3, and Sp4, and this is consistent with the observed downregulation of the aforementioned Sp-regulated genes. In addition, we also observed by RNA interference (RNAi) that the oncogenic cap protein eIF4E was an Sp-regulated gene also downregulated by WIN in colon cancer cells. WIN-mediated repression of Sp proteins was not affected by cannabinoid receptor antagonists or by knockdown of the receptor but was attenuated by the phosphatase inhibitor sodium orthovanadate or by knockdown of protein phosphatase 2A (PP2A). WIN-mediated repression of Sp1, Sp3, and Sp4 was due to PP2A-dependent downregulation of microRNA-27a (miR-27a) and induction of miR-27a-regulated ZBTB10, which has previously been characterized as an "Sp repressor." The results show that the anticancer activity of WIN is due, in part, to PP2A-dependent disruption of miR-27a:ZBTB10 and ZBTB10-mediated repression of Sp transcription factors and Sp-regulated genes, including eIF4E., (©2013 AACR.)

Specificity protein (Sp) transcription factors Sp1, Sp3 and Sp4 are highly expressed in rhabdomyosarcoma (RMS) cells. In tissue arrays of RMS tumor cores from 71 patients, 80% of RMS patients expressed high levels of Sp1 protein, whereas low expression of Sp1 was detected in normal muscle tissue. The non-steroidal anti-inflammatory drug (NSAID) tolfenamic acid (TA) inhibited growth and migration of RD and RH30 RMS cell lines and also inhibited tumor growth in vivo using a mouse xenograft (RH30 cells) model. The effects of TA were accompanied by downregulation of Sp1, Sp3, Sp4 and Sp-regulated genes in RMS cells and tumors, and the role of Sp protein downregulation in mediating inhibition of RD and RH30 cell growth and migration was confirmed by individual and combined knockdown of Sp1, Sp3 and Sp4 proteins by RNA interference. TA treatment and Sp knockdown in RD and RH30 cells also showed that four genes that are emerging as individual drug targets for treating RMS, namely c-MET, insulin-like growth factor receptor (IGFR), PDGFRα and CXCR4, are also Sp-regulated genes. These results suggest that NSAIDs such as TA may have potential clinical efficacy in drug combinations for treating RMS patients., (Copyright © 2012 UICC.)

Aim: We hypothesized that the anticancer activity of cannabinoids was linked to induction of phosphatases., Materials and Methods: The effects of cannabidiol (CBD) and the synthetic cannabinoid WIN-55,212 (WIN) on LNCaP (prostate) and SW480 (colon) cancer cell proliferation were determined by cell counting; apoptosis was determined by cleavage of poly(ADP)ribose polymerase (PARP) and caspase-3 (Western blots); and phosphatase mRNAs were determined by real-time PCR. The role of phosphatases and cannabinoid receptors in mediating CBD- and WIN-induced apoptosis was determined by inhibition and receptor knockdown., Results: CBD and WIN inhibited LNCaP and SW480 cell growth and induced mRNA expression of several phosphatases, and the phosphatase inhibitor sodium orthovanadate significantly inhibited cannabinoid-induced PARP cleavage in both cell lines, whereas only CBD-induced apoptosis was CB1 and CB2 receptor-dependent., Conclusion: Cannabinoid receptor agonists induce phosphatases and phosphatase-dependent apoptosis in cancer cell lines; however, the role of the CB receptor in mediating this response is ligand-dependent.

Ethyl 2-((2,3-bis(nitrooxy)propyl)disulfanyl)benzoate (GT-094) is a novel nitric oxide (NO) chimera containing an nonsteroidal anti-inflammatory drug (NSAID) and NO moieties and also a disulfide pharmacophore that in itself exhibits cancer chemopreventive activity. In this study, the effects and mechanism of action of GT-094 were investigated in RKO and SW480 colon cancer cells. GT-094 inhibited cell proliferation and induced apoptosis in both cell lines and this was accompanied by decreased mitochondrial membrane potential (MMP) and induction of reactive oxygen species (ROS), and these responses were reversed after cotreatment with the antioxidant glutathione. GT-094 also downregulated genes associated with cell growth [cyclin D1, hepatocyte growth factor receptor (c-Met), epidermal growth factor receptor (EGFR)], survival (bcl-2, survivin), and angiogenesis [VEGF and its receptors (VEGFR1 and VEGFR2)]. Results of previous RNA interference studies in this laboratory has shown that these genes are regulated, in part, by specificity protein (Sp) transcription factors Sp1, Sp3, and Sp4 that are overexpressed in colon and other cancer cell lines and not surprisingly, GT-094 also decreased Sp1, Sp3, and Sp4 in colon cancer cells. GT-094-mediated repression of Sp and Sp-regulated gene products was due to downregulation of microRNA-27a (miR-27a) and induction of ZBTB10, an Sp repressor that is regulated by miR-27a in colon cancer cells. Moreover, the effects of GT-094 on Sp1, Sp3, Sp4, miR-27a, and ZBTB10 were also inhibited by glutathione suggesting that the anticancer activity of GT-094 in colon cancer cells is due, in part, to activation of an ROS-miR-27a:ZBTB10-Sp transcription factor pathway.

Ascorbic acid (vitamin C) inhibits cancer cell growth, and there is a controversy regarding the cancer chemoprotective effects of pharmacologic doses of this compound that exhibits prooxidant activity. We hypothesized that the anticancer activity of pharmacologic doses of ascorbic acid (<5 mM) is due, in part, to reactive oxygen species-dependent downregulation of specificity protein (Sp) transcription factors Sp1, Sp3, and Sp4 and Sp-regulated genes. In this study, ascorbic acid (1-3 mM) decreased RKO and SW480 colon cancer cell proliferation and induced apoptosis and necrosis, and this was accompanied by downregulation of Sp1, Sp3, and Sp4 proteins. In addition, ascorbic acid decreased expression of several Sp-regulated genes that are involved in cancer cell proliferation [hepatocyte growth factor receptor (c-Met), epidermal growth factor receptor and cyclin D1], survival (survivin and bcl-2), and angiogenesis [vascular endothelial growth factor (VEGF) and its receptors (VEGFR1 and VEGFR2)]. Other prooxidants such as hydrogen peroxide exhibited similar activities in colon cancer cells, and cotreatment with glutathione inhibited these responses. This study demonstrates for the first time that the anticancer activities of ascorbic acid are due, in part, to ROS-dependent repression of Sp transcription factors.

Arsenic trioxide exhibits antiproliferative, antiangiogenic and proapoptotic activity in cancer cells, and many genes associated with these responses are regulated by specificity protein (Sp) transcription factors. Treatment of cancer cells derived from urologic (bladder and prostate) and gastrointestinal (pancreas and colon) tumors with arsenic trioxide demonstrated that these cells exhibited differential responsiveness to the antiproliferative effects of this agent and this paralleled their differential repression of Sp1, Sp3 and Sp4 proteins in the same cell lines. Using arsenic trioxide-responsive KU7 and non-responsive 253JB-V bladder cancer cells as models, we show that in KU7 cells, < or =5 microM arsenic trioxide decreased Sp1, Sp3 and Sp4 and several Sp-dependent genes and responses including cyclin D1, epidermal growth factor receptor, bcl-2, survivin and vascular endothelial growth factor, whereas at concentrations up to 15 microM, minimal effects were observed in 253JB-V cells. Arsenic trioxide also inhibited tumor growth in athymic mice bearing KU7 cells as xenografts, and expression of Sp1, Sp3 and Sp4 was significantly decreased. Inhibitors of oxidative stress such as glutathione or dithiothreitol protected KU7 cells from arsenic trioxide-induced antiproliferative activity and Sp repression, whereas glutathione depletion sensitized 253JB-V cells to arsenic trioxide. Mechanistic studies suggested that arsenic trioxide-dependent downregulation of Sp and Sp-dependent genes was due to decreased mitochondrial membrane potential and induction of reactive oxygen species, and the role of peroxides in mediating these responses was confirmed using hydrogen peroxide., (Copyright 2010 Elsevier Inc. All rights reserved.)

1,1-Bis(3'-indolyl)-1-(p-bromophenyl)methane (DIM-C-pPhBr) and the 2,2'-dimethyl analog (2,2'-diMeDIM-C-pPhBr) inhibit proliferation and induce apoptosis in SW480 colon and Panc28 pancreatic cancer cells. In this study, treatment with 10-20 microM concentrations of these compounds for 24 h induced cleaved PARP and decreased survivin protein and mRNA expression in both cell lines. However, results of time course studies show that DIM-C-pPhBr and 2,2'-diMeDIM-C-pPhBr decrease survivin protein within 2 h after treatment, whereas survivin mRNA levels were decreased only at later time-points indicating activation of transcription-independent and -dependent pathways for downregulation of survivin. In addition, we also observed that gamma-radiation inhibited pancreatic and colon cancer cell growth and this was associated with enhanced expression of survivin after 24 (SW480) or 24 and 48 h (Panc28) and correlated with previous studies on the role of survivin in radiation-resistance. However, in cells co-treated with gamma-radiation plus DIM-C-pPhBr or 2,2'-diMeDIM-C-pPhBr, induction of survivin by gamma-radiation was inhibited after co-treatment with both compounds, suggesting applications for these drugs in combination cancer chemotherapy with gamma-radiation.