Kinetically Selective Inhibitors of Histone Deacetylase 2 (HDAC2) as Cognition Enhancers.

Aiming towards the development of novel nootropic therapeutics to address the cognitive impairment common to a range of brain disorders, we set out to develop highly selective small molecule inhibitors of HDAC2, a chromatin modifying histone deacetylase implicated in memory formation and synaptic plasticity. Novel ortho-aminoanilide inhibitors were designed and evaluated for their ability to selectively inhibit HDAC2 versus the other Class I HDACs. Kinetic and thermodynamic binding properties were essential elements of our design strategy and two novel classes of ortho-aminoanilides, that exhibit kinetic selectivity (biased residence time) for HDAC2 versus the highly homologous isoform HDAC1, were identified. These kinetically selective HDAC2 inhibitors (BRD6688 and BRD4884) increased H4K12 and H3K9 histone acetylation in primary mouse neuronal cell culture assays, in the hippocampus of CK-p25 mice, a model of neurodegenerative disease, and rescued the associated memory deficits of these mice in a cognition behavioural model. These studies demonstrate for the first time that selective pharmacological inhibition of HDAC2 is feasible and that inhibition of the catalytic activity of this enzyme may serve as a therapeutic approach towards enhancing the learning and memory processes that are affected in many neurological and psychiatric disorders.

In vitro evaluation and in vivo demonstration of a biomimetic, hemocompatible, microfluidic artificial lung.

Despite the promising potential of microfluidic artificial lungs, current designs suffer from short functional lifetimes due to surface chemistry and blood flow patterns that act to reduce hemocompatibility. Here, we present the first microfluidic artificial lung featuring a hemocompatible surface coating and a biomimetic blood path. The polyethylene-glycol (PEG) coated microfluidic lung exhibited a significantly improved in vitro lifetime compared to uncoated controls as well as consistent and significantly improved gas exchange over the entire testing period. Enabled by our hemocompatible PEG coating, we additionally describe the first extended (3 h) in vivo demonstration of a microfluidic artificial lung.

Phosphodiesterase 10A: a novel target for selective inhibition of colon tumor cell growth and ß-catenin-dependent TCF transcriptional activity.

The cyclic nucleotide phosphodiesterase 10A (PDE10) has been mostly studied as a therapeutic target for certain psychiatric and neurological conditions, although a potential role in tumorigenesis has not been reported. Here we show that PDE10 is elevated in human colon tumor cell lines compared with normal colonocytes, as well as in colon tumors from human clinical specimens and intestinal tumors from Apc(Min/+) mice compared with normal intestinal mucosa, respectively. An isozyme and tumor-selective role of PDE10 were evident by the ability of small-molecule inhibitors and small interfering RNA knockdown to suppress colon tumor cell growth with reduced sensitivity of normal colonocytes. Stable knockdown of PDE10 by short hairpin RNA also inhibits colony formation and increases doubling time of colon tumor cells. PDE10 inhibition selectively activates cGMP/cGMP-dependent protein kinase signaling to suppress ß-catenin levels and T-cell factor (TCF) transcriptional activity in colon tumor cells. Conversely, ectopic expression of PDE10 in normal and precancerous colonocytes increases proliferation and activates TCF transcriptional activity. These observations suggest a novel role of PDE10 in colon tumorigenesis and that inhibitors may be useful for the treatment or prevention of colorectal cancer.

Choline kinase inhibition induces exacerbated endoplasmic reticulum stress and triggers apoptosis via CHOP in cancer cells.

Endoplasmic reticulum (ER) is a central organelle in eukaryotic cells that regulates protein synthesis and maturation. Perturbation of ER functions leads to ER stress, which has been previously associated with a broad variety of diseases. ER stress is generally regarded as compensatory, but prolonged ER stress has been involved in apoptosis induced by several cytotoxic agents. Choline kinase α (ChoKα), the first enzyme in the Kennedy pathway, is responsible for the generation of phosphorylcholine (PCho) that ultimately renders phosphatidylcholine. ChoKα overexpression and high PCho levels have been detected in several cancer types. Inhibition of ChoKα has demonstrated antiproliferative and antitumor properties; however, the mechanisms underlying these activities remain poorly understood. Here, we demonstrate that ChoKα inhibitors (ChoKIs), MN58b and RSM932A, induce cell death in cancer cells (T47D, MCF7, MDA-MB231, SW620 and H460), through the prolonged activation of ER stress response. Evidence of ChoKIs-induced ER stress includes enhanced production of glucose-regulated protein, 78 kDa (GRP78), protein disulfide isomerase, IRE1α, CHOP, CCAAT/enhancer-binding protein beta (C/EBPß) and TRB3. Although partial reduction of ChoKα levels by small interfering RNA was not sufficient to increase the production of ER stress proteins, silencing of ChoKα levels also show a decrease in CHOP overproduction induced by ChoKIs, which suggests that ER stress induction is due to a change in ChoKα protein folding after binding to ChoKIs. Silencing of CHOP expression leads to a reduction in C/EBPß, ATF3 and GRP78 protein levels and abrogates apoptosis in tumor cells after treatment with ChoKIs, suggesting that CHOP maintains ER stress responses and triggers the pro-apoptotic signal. Consistent with the differential effect of ChoKIs in cancer and primary cells previously described, ChoKIs only promoted a transient and moderated ER stress response in the non-tumorogenic cells MCF10A. In conclusion, pharmacological inhibition of ChoKα induces cancer cell death through a mechanism that involves the activation of exaggerated and persistent ER stress supported by CHOP overproduction.

Activation of ERK signaling and induction of colon cancer cell death by piperlongumine.

Piperlongumine (PPLGM) is a bioactive compound isolated from long peppers that shows selective toxicity towards a variety of cancer cell types including colon cancer. The signaling pathways that lead to cancer cell death in response to PPLGM exposure have not been previously identified. Our objective was to identify the intracellular signaling mechanisms by which PPLGM leads to enhanced colon cancer cell death. We found that PPLGM inhibited the growth of colon cancer cells in time- and concentration-dependent manners, but was not toxic toward normal colon mucosal cells at concentrations below 10 µM. Acute (0-60 min) and prolonged (24h) exposure of HT-29 cells to PPLGM resulted in phosphorylation of ERK. To investigate whether ERK signaling was involved in PPLGM-mediated cell death, we treated HT-29 cells with the MEK inhibitor U0126, prior to treating with PPLGM. We found that U0126 attenuated PPLGM-induced activation of ERK and partially protected against PPLGM-induced cell death. These results suggest that PPLGM works, at least in part, through the MEK/ERK pathway to result in colon cancer cell death. A more thorough understanding of the molecular mechanisms by which PPLGM induces colon cancer cell death will be useful in developing therapeutic strategies to treat colon cancer.

Targeting genetic alterations in protein methyltransferases for personalized cancer therapeutics.

The human protein methyltransferases (PMTs) constitute a large enzyme class composed of two families, the protein lysine methyltransferases (PKMTs) and the protein arginine methyltransferases (PRMTs). Examples have been reported of both PKMTs and PRMTs that are genetically altered in specific human cancers, and in several cases these alterations have been demonstrated to confer a unique dependence of the cancer cells on PMT enzymatic activity for the tumorigenic phenotype. Examples of such driver alterations in PMTs will be presented together with a review of current efforts towards the discovery and development of small-molecule inhibitors of these enzymes as personalized cancer therapeutics.

Selective cyclooxygenase-2 silencing mediated by engineered E. coli and RNA interference induces anti-tumour effects in human colon cancer cells.

BACKGROUND: Cyclooxygenase-2 (COX-2) overexpression is strongly associated with colorectal tumourigenesis. It has been demonstrated that the chronic use of non-steroidal anti-inflammatory drugs (COX inhibitors) partially protects patients from colorectal cancer (CRC) development and progression but induces severe cardiovascular side effects. New strategies for selective COX-2 blockade are required. METHODS: We developed an improved technique, based on RNA interference (RNAi), to gain a selective COX-2 silencing in CRC cells by a tumour-dependent expression of anti-COX-2 short-hairpin RNA (shCOX-2). Anti-COX-2 shRNA-expressing vectors were delivered in CRC cells (in vitro) and in colon tissues (ex vivo) using engineered Escherichia coli strains, capable of invading tumour cells (InvColi). RESULTS: A highly tumour-dependent shCOX-2 expression and a significant COX-2 silencing were observed in CRC cells following InvColi strain infection. Cyclooxygenase-2 silencing was associated with a strong reduction in both proliferative and invasive behaviour of tumour cells. We also demonstrated a pivotal role of COX-2 overexpression for the survival of CRC cells after bacterial infection. Moreover, COX-2 silencing was achieved ex vivo by infecting colon tissue samples with InvColi strains, leading to anti-inflammatory and anti-tumour effects. CONCLUSION: Our RNAi/InvColi-mediated approach offers a promising tool for a highly selective COX-2 blockade in vitro and in vivo.

Development and testing of a CD-ROM program for improving adolescent knowledge of inflammatory bowel disease.

OBJECTIVE: The aim of this study was to design a theory-based educational program for adolescents with inflammatory bowel disease (IBD) using an interactive multimedia CD-ROM and to test its effectiveness in improving knowledge in IBD. MATERIALS AND METHODS: Curriculum-based instruction using educational theory and principles was designed for adolescents on an interactive multimedia CD-ROM. Twenty subjects completed summative evaluation of the CD-ROM measuring gain in knowledge about IBD immediately and 9 months after instruction. RESULTS: Subjects found the CD-ROM to be informative, appealing, and easy to use. The mean baseline score of the adolescents on the Crohn's and Colitis Knowledge questionnaire was 12.2 (standard deviation 5.14, range 3-24). After an average of 30 minutes of self-directed learning, adolescent subjects increased their posttest score to a mean of 19.8, a gain of 7.6 points over baseline (95% confidence interval 5.2-10.1, P < 0.0001). Knowledge of medications, disease complications, and gastrointestinal structure and function was gained and retained upon retesting at 9 months with a mean Crohn's and Colitis Knowledge questionnaire score of 17.5 (standard deviation 3.9, range 12-26), which was still an improvement over the mean pretest knowledge score of 12.2 (P < 0.001). CONCLUSIONS: Adolescents with IBD have low baseline knowledge about their disease. A rigorously developed interactive educational tool is now available for instructing adolescent patients about their IBD.

A randomized controlled trial of growth hormone in active pediatric Crohn disease.

OBJECTIVES: Growth hormone (GH) may reduce symptoms and improve growth in Crohn disease (CD). The effect on mucosal inflammation is not known. We hypothesized that GH would improve both clinical and mucosal disease activity and stimulate linear growth in pediatric CD. PATIENTS AND METHODS: Twenty patients ages 7 to 18 receiving corticosteroids (CTX) for active CD were randomized to begin GH, 0.075 mg x kg(-1) x day(-1) (group A), or continue CTX alone (group B). Clinical and endoscopic disease activities were assessed after 12 weeks. Group B began GH at 12 weeks, and clinical disease activity was assessed at 24 weeks. Subjects who experienced a clinical response after 12 weeks of GH therapy continued treatment for an additional 52 weeks, and linear growth was assessed. RESULTS: Sixty-five percent of patients receiving GH achieved clinical remission, compared with 20% treated with CTX alone (P = 0.03). Although endoscopic disease activity trended toward an improvement at week 12 in group A, this did not differ between the groups. Sixty-one percent of week 12 GH responders maintained their clinical response through week 64. Mean (95th confidence interval) height z score on GH increased from -1.1 (-1.6, -0.6) to -0.4 (-1, 0.2), P = 0.004 during this 52-week extension phase. GH was well tolerated with no unexpected safety signals. CONCLUSIONS: The addition of GH to CTX therapy did not induce a reduction in mucosal inflammation, relative to CTX alone. However, GH was safe and effective as an adjunct to CTX for treatment of clinical disease activity and growth failure in pediatric CD.

Increased proteasome subunit protein expression and proteasome activity in colon cancer relate to an enhanced activation of nuclear factor E2-related factor 2 (Nrf2).

An elevated proteasome activity contributes to tumorigenesis, particularly by providing cancer cells with antiapoptotic protection and efficient clearance from irregular proteins. Still, the underlying mechanisms are poorly known. In this study, we report that in colon cancer patients, higher proteasome activity was detected in tumoral tissue compared with surrounding normal tissue, and also that increased levels of proteasomal subunit proteins, such as S5a/PSMD4 and alpha-5/PSMA5, could be detected. Colon tumors showed higher nuclear levels of nuclear factor E2-related factor 2 (Nrf2), a transcription factor supposed to be involved in the control of proteasomal subunit protein expression. The induction or overexpression of Nrf2 led to stronger S5a and alpha-5 expression in the human colon cancer cell lines, Colo320 and Lovo, as well as in NCM460 colonocytes along with higher proteasome activity. The small interfering RNA (siRNA)-mediated Nrf2 knockdown decreased S5a and alpha-5 expression and reduced proteasome activity. Additionally, Nrf2-dependent S5a and alpha-5 expression conferred protection from tumor necrosis factor-related apoptosis-inducing ligand (TRAIL)-induced apoptosis, an effect preceded by an increased nuclear factor (NF)-kappaB activation and higher expression of antiapoptotic NF-kappaB target genes. These findings point to an important role of Nrf2 in the gain of proteasome activity, thereby contributing to colorectal carcinogenesis. Nrf2 may therefore serve as a potential target in anticancer therapy.

Deficits of mesolimbic dopamine neurotransmission in rat dietary obesity.

Increased caloric intake in dietary obesity could be driven by central mechanisms that regulate reward-seeking behavior. The mesolimbic dopamine system, and the nucleus accumbens in particular, underlies both food and drug reward. We investigated whether rat dietary obesity is linked to changes in dopaminergic neurotransmission in that region. Sprague-Dawley rats were placed on a cafeteria-style diet to induce obesity or a laboratory chow diet to maintain normal weight gain. Extracellular dopamine levels were measured by in vivo microdialysis. Electrically evoked dopamine release was measured ex vivo in coronal slices of the nucleus accumbens and the dorsal striatum using real-time carbon fiber amperometry. Over 15 weeks, cafeteria-diet fed rats became obese (>20% increase in body weight) and exhibited lower extracellular accumbens dopamine levels than normal weight rats (0.007+/-0.001 vs. 0.023+/-0.002 pmol/sample; P<0.05). Dopamine release in the nucleus accumbens of obese rats was stimulated by a cafeteria-diet challenge, but it remained unresponsive to a laboratory chow meal. Administration of d-amphetamine (1.5 mg/kg i.p.) also revealed an attenuated dopamine response in obese rats. Experiments measuring electrically evoked dopamine signal ex vivo in nucleus accumbens slices showed a much weaker response in obese animals (12 vs. 25x10(6) dopamine molecules per stimulation, P<0.05). The results demonstrate that deficits in mesolimbic dopamine neurotransmission are linked to dietary obesity. Depressed dopamine release may lead obese animals to compensate by eating palatable "comfort" food, a stimulus that released dopamine when laboratory chow failed.

Growth abnormalities persist in newly diagnosed children with crohn disease despite current treatment paradigms.

OBJECTIVES: We analyzed growth outcomes in children newly diagnosed with Crohn disease and determined whether growth abnormalities persist despite current therapies. PATIENTS AND METHODS: Clinical and growth data were prospectively obtained on an inception cohort younger than 16 years old at diagnosis and Tanner I to III during the study. RESULTS: In all, 176 children (mean age 10.1 years; 65% male) with mild (33%) or moderate/severe (67%) disease at diagnosis were studied. Disease activity at 1 year was inactive/mild (89%) or moderate/severe (11%). First-year treatments included immunomodulators (60%), corticosteroids (77%), 5-aminosalicylates (61%), infliximab (15%), and enteral nutrition (10%). By 2 years, 86% had received immunomodulators and 36% infliximab. Mean height z scores at diagnosis, 1 year, and 2 years were -0.49 +/- 1.2 standard deviations (SDs), -0.50 +/- 1.2, and -0.46 +/- 1.1, respectively. Of the subjects, 10%, 8%, and 6.5% had height z scores less than -2 SD at diagnosis, 1 year, and 2 years. A height velocity z score less than -1SD was seen in 45% of subjects at 1 year and 38% at 2 years. The mean height velocity z score, however, increased between 1 and 2 years from -0.71 to 0.26 (P < 0.03). Corticosteroid use greater than 6 months in the first year was associated with abnormal height velocity at 1 year (adjusted odds ratio = 4.5; 95% confidence interval [CI] = 2.2-9.6). No statistically significant effect on height velocity z scores was noted when comparing those receiving or not receiving infliximab. CONCLUSIONS: Growth delay persists in many children with CD following diagnosis, despite improved disease activity and the frequent use of immunomodulators and biologics. Additional strategies to improve growth outcomes require development.

Health values in adolescents with or without inflammatory bowel disease.

OBJECTIVE: To examine for differences in and predictors of health value/utility scores in adolescents with or without inflammatory bowel disease (IBD). STUDY DESIGN: Adolescents with IBD and healthy control subjects were interviewed in an academic health center. We collected sociodemographic data and measured health status, personal, family, and social characteristics, and spiritual well-being. We assessed time tradeoff (TTO) and standard gamble (SG) utility scores for current health. We performed bivariate and multivariable analyses with utility scores used as outcomes. RESULTS: Sixty-seven patients with IBD and 88 healthy control subjects 11 to 19 years of age participated. Among subjects with IBD, mean (SD) TTO scores were 0.92 (0.17), and mean (SD) SG scores were 0.97 (0.07). Among healthy control subjects, mean (SD) TTO scores were 0.99 (0.03) and mean (SD) SG scores were 0.98 (0.03). TTO scores were significantly lower (P= .001), and SG scores trended lower (P= .065) in patients with IBD when compared with healthy control subjects. In multivariable analyses controlling for IBD status, poorer emotional functioning and spiritual well-being were associated with lower TTO (R(2)=0.17) and lower SG (R(2)=0.22) scores. CONCLUSION: Direct utility assessment in adolescents with or without IBD is feasible and may be used to assess outcomes. Adolescents with IBD value their health state highly, although less so than healthy control subjects. Emotional functioning and spiritual well-being appear to influence utility scores most strongly.

Course and treatment of perianal disease in children newly diagnosed with Crohn's disease.

BACKGROUND: We sought to characterize perianal disease and its treatment in pediatric patients newly diagnosed with Crohn's disease. METHODS: Data were obtained from the Pediatric Inflammatory Bowel Disease (IBD) Collaborative Group Registry, a prospective, multicenter observational registry recording clinical and laboratory outcomes in children under 16 years of age newly diagnosed with IBD. Patients with Crohn's disease were selected who had data on perianal disease and at least 24 months of follow-up. The records of patients with a Pediatric Crohn's Disease Activity Index perianal subscore greater than 0 were reviewed, and patients with abscesses or fistulas were selected. The therapies used and the course of their perianal disease were then assessed. RESULTS: Of the 276 patients identified, 41 had perianal lesions within 30 days of diagnosis. Thirteen of these had skin tags and fissures only, whereas 28 had fistulas and/or abscesses. The latter lesions resolved by 1 year in 20 patients, and 8 had chronic/recurrent perianal disease persisting for more than 1 year following diagnosis. Patients with fistulizing disease were much more likely to be treated and were treated earlier with antibiotics, infliximab, and immunomodulators than were nonfistulizing patients. Patients who developed chronic perianal disease were more likely to have low body mass indices and required more perianal surgery than did patients whose perianal disease resolved. CONCLUSIONS: Approximately 10% of newly diagnosed pediatric patients with Crohn's disease will have perianal fistulas and/or abscesses at the time of diagnosis. Most of these will resolve within a year with medical therapy alone.

Effect of early immunomodulator use in moderate to severe pediatric Crohn disease.

BACKGROUND: The immunomodulators (IMs) 6-mercaptopurine and azathioprine decrease corticosteroid dependence and maintain remission in Crohn's disease (CD). We describe IM use in newly diagnosed pediatric CD, comparing outcomes of "early" versus "late" initiation of therapy. METHODS: Data were obtained from pediatric CD patients enrolled in a prospective, multicenter observational study. Moderate/severe disease patients treated with IM were compared for outcomes of remission, corticosteroid use, infliximab therapy, hospitalizations, and CD-related surgery based on timing of initiation of IM therapy. RESULTS: In all, 247 children met the criteria (60% male, mean age 11.9 years); 199 were treated with IM within 1 year of diagnosis; 150 between 0-3 months (early), 49 between 3-12 months (late). Both groups showed a decrease in corticosteroid use by 12 months, at which time proportionately fewer early group patients had received corticosteroids in the preceding quarter (22%) than late groups patients (41%)(P = 0.013). The number of hospitalizations per patient was also noted to be significantly lower in the early group over the 2-year follow-up (P = 0.03). No difference was noted in the rates of remission, infliximab use over time, or surgery. CONCLUSIONS: 80% of children with newly diagnosed moderate to severe CD are treated with IM within 1 year. Early IM use is associated with reduced corticosteroid exposure and possibly fewer hospitalizations per patient.

Development of normal human colon cell cultures to identify priority unregulated disinfection by-products with a carcinogenic potential.

Research was initiated to develop an in vitro system to identify disinfection by-products with a potential to transform normal human colonocytes into malignant cells. Tribromomethane and bromochloroacetic acid, rodent colon carcinogens, dibromonitromethane and tribromonitromethane, recently identified in drinking water, and azoxymethane, a classic colon carcinogen, were tested for the ability to transform NCM460 cells. The chronic toxicity was determined for the series of trihalomethanes, haloacetic acids and halonitromethanes as well as NCM460 cell enzymatic capabilities. The order of cytotoxicity was halonitromethanes > haloacetic acids > trihalomethanes. Cytotoxicity within a series increased with the degree of bromination and decreased with the molecular weight. The genotoxicity profile was similar to that for cytotoxicity. Enzymatic analysis demonstrated that NCM460 cells possess glutathione-S transerase-1-1 and CYP450 activity similar to that measured in the large intestine. NCM460 cells were exposed to 10(-6) M of the test chemicals for three days. While NCM460 cells from all treatments had the ability to grow in soft agar to some extent, only cells exposed to azoxymethane or tribromomethane were able to grow in media lacking serum and growth factors. When sub cultured, NCM460 cells exposed to 10(-9) M azoxymethane for three weeks formed colonies with morphology distinct from untreated cells.