Bidirectional reporter assay using HAL promoter and TOPFLASH improves specificity in high-throughput screening of Wnt inhibitors.

Constitutive activation of Wnt signaling plays an important role in colorectal and liver tumorigenesis. Cell-based assays using synthetic TCF/LEF (T-cell factor/lymphoid enhancer factor) reporters, as readouts of ß-catenin/TCF-dependent transcriptional activity, have contributed greatly to the discovery of small molecules that modulate Wnt signaling. In the present study, we report a novel screening method, called a bidirectional dual reporter assay. Integrated transcriptome analysis identified a histidine ammonia-lyase gene (HAL) that was negatively regulated by ß-catenin/TCF-dependent transcriptional activity. We leveraged a promoter region of the HAL gene as another transcriptional readout of Wnt signaling. Cells stably expressing both an optimized HAL reporter and the TCF/LEF reporter enabled bidirectional reporter activities in response to Wnt signaling. Increased HAL reporter activity and decreased TCF/LEF reporter activity were observed simultaneously in the cells when ß-catenin/TCF7L2 was inhibited. Notably, this method could decrease the number of false positives observed when screening an inhibitor library compared with the conventional TCF/LEF assay. We found that Brefeldin A, a disruptor of the Golgi apparatus, inhibited the Wnt/ß-catenin signaling pathway. The utility of our system could be expanded to examine other disease-associated pathways beyond the Wnt/ß-catenin signaling pathway.

Identification of therapeutic small-molecule leads in cultured cells using multiplexed pathway reporter readouts.

The rapid expansion of molecular screening libraries in size and complexity in the last decade has outpaced the discovery rate of cost-effective strategies to single out reagents with sought-after cellular activities. In addition to representing high-priority therapeutic targets, intensely studied cell signaling systems encapsulate robust reference points for mapping novel chemical activities given our deep understanding of the molecular mechanisms that support their activity. In this chapter, we describe strategies for using transcriptional reporters of several well-interrogated signal transduction pathways coupled with high-throughput biochemical assays to fingerprint novel compounds for drug target identification agendas.

Effects of dehydroepiandrosterone supplementation on cognitive function and activities of daily living in older women with mild to moderate cognitive impairment.

AIM: There is little evidence that dehydroepiandrosterone (DHEA) has beneficial effects on physical and psychological functions in older women. We investigated the effect of DHEA supplementation on cognitive function and ADL in older women with cognitive impairment. METHODS: A total of 27 women aged 65-90 years (mean ± standard deviation, 83 ± 6) with mild to moderate cognitive impairment (Mini-Mental State Examination, MMSE; 10-28/30 points), receiving long-term care at a facility in Japan were enrolled. Twelve women were assigned to receive DHEA 25 mg/day p.o. for 6 months. The control group (n = 15) matched for age and cognitive function was followed without hormone replacement. Cognitive function was assessed by MMSE and Hasegawa Dementia Scale-Revised (HDS-R), and basic activities of daily living (ADL) by Barthel Index at baseline, 3 and 6 months. Plasma hormone levels including testosterone, DHEA, DHEA-sulfate and estradiol were also followed up. RESULTS: After 6 months, DHEA treatment significantly increased plasma testosterone, DHEA and DHEA-sulfate levels by 2-3-fold but not estradiol level compared to baseline. DHEA administration increased cognitive scores and maintained basic ADL score, while cognition and basic ADL deteriorated in the control group (6-month change in DHEA group vs control group; MMSE, +0.6 ± 3.2 vs -2.1 ± 2.2, P < 0.05; HDS-R, +2.8 ± 2.8 vs -0.3 ± 4.1, P < 0.05; Barthel Index, +3.7 ± 7.1 vs -2.7 ± 4.6, P = 0.05). Among the cognitive domains, DHEA treatment improved verbal fluency (P < 0.05). CONCLUSION: DHEA supplementation in older women with cognitive impairment may have beneficial effects on cognitive function and ADL.

A randomized cross-over study of a traditional Japanese medicine (kampo), yokukansan, in the treatment of the behavioural and psychological symptoms of dementia.

The effectiveness and safety of yokukansan (TJ-54), a traditional Japanese medicine (kampo) for the treatment of the behavioural and psychological symptoms of dementia (BPSD), were evaluated in 106 patients diagnosed as having Alzheimer's disease (AD) (including mixed-type dementia) or dementia with Lewy bodies. Patients were randomly assigned to group A (TJ-54 treatment in period I and no treatment in period II; each period lasting 4 wk) or group B (no treatment in period I and TJ-54 treatment in period II). BPSD and cognitive functions were evaluated using the Neuropsychiatric Inventory (NPI) and the Mini-Mental State Examination (MMSE), respectively. Activities of daily living (ADL) were evaluated using Instrumental Activities of Daily Living (IADL) in outpatients and the Barthel Index in in-patients. For the safety evaluation, adverse events were investigated. Significant improvements in mean total NPI score associated with TJ-54 treatment were observed in both periods (Wilcoxon test, p=0.040 in period I and p=0.048 in period II). The mean NPI scores significantly improved during TJ-54 treatment in groups A and B (p=0.002 and p=0.007, respectively) but not during periods of no treatment. Among the NPI subscales, significant improvements were observed in delusions, hallucinations, agitation/aggression, depression, anxiety, and irritability/lability. The effects of TJ-54 persisted for 1 month without any psychological withdrawal symptoms in group A. TJ-54 did not show any effect on either cognitive function or ADL. No serious adverse reactions were observed. The present study suggests that TJ-54 is an effective and well-tolerated treatment for patients with BPSD.

A green tea component suppresses posttranslational expression of basic fibroblast growth factor in colorectal cancer.

BACKGROUND & AIMS: Green tea catechins are known to have anticarcinogenic effects. Epigallocatechin-3-gallate (EGCG) accounts for almost 50% of the total catechin content in green tea extract and has very potent antioxidant effects. EGCG also inhibits angiogenesis, possibly through the inhibition of proangiogenic factors including vascular endothelial growth factor (VEGF) and basic fibroblast growth factor (bFGF), which in turn, inhibits tumor growth and metastasis. However, the exact molecular mechanism by which EGCG suppresses bFGF expression is not known. Our objective was to elucidate the molecular mechanisms by which EGCG inhibits bFGF expression in colorectal cancer. METHODS: We examined posttranslational regulation of bFGF by EGCG in human colorectal cancer cells. We also examined bFGF in intestinal tumor formation of APC(Min/+) mice with and without catechin treatment. RESULTS: The bFGF protein was quickly degraded in the presence of EGCG, but a proteasome inhibitor suppressed this degradation. EGCG was also found to increase ubiquitination of bFGF and trypsin-like activity of the 20S proteasome, thereby resulting in the degradation of bFGF protein. Furthermore, EGCG suppressed tumor formation in APC(Min/+) mice, compared with vehicle-treated mice, in association with reduced bFGF expression. CONCLUSIONS: The ubiquitin-proteasome degradation pathway contributes significantly to down-regulation of bFGF expression by EGCG. Catechin compounds have fewer adverse effects than chemotherapeutic agents and hence can be used as proof-of-concept in cancer therapeutics to suppress growth and metastasis by targeting proteins such as bFGF.

Growth inhibition and apoptosis by (-)-epicatechin gallate are mediated by cyclin D1 suppression in head and neck squamous carcinoma cells.

In recent studies, green tea components have been shown to induce cell growth arrest and apoptosis in head and neck squamous cell carcinoma (HNSCC) cells. In this report, we have investigated the effects of epicatechin gallate (ECG), one of the catechins in green tea, on anti-cancer activity in vitro. We found that cyclin D1 was highly expressed in HNSCC cells, and ECG suppressed 90% of cyclin D1 expression in SCC7 cells. We have also evaluated the effect of ECG on cell growth and apoptosis, showing that ECG (50 microM) exhibited a significant inhibition (50%) on the growth of SCC7 cells via G1 cell cycle arrest. ECG suppressed cyclin D1 in SCC7 cells in a dose- and time-dependent manner, and the suppression of the beta-catenin pathway by ECG is one of the mechanism to facilitate ECG-induced cell growth arrest. These results suggest that ECG has a potential usage as a chemopreventive agent in HNSCC.

Anti-proliferative effect of horehound leaf and wild cherry bark extracts on human colorectal cancer cells.

Marubium vulgare (horehound) and Prunus serotina (wild cherry) have been traditionally used for the treatment of inflammatory-related symptoms such as cold, fever, and sore throat. In this report, we show that extracts of anti-inflammatory horehound leaves and wild cherry bark exhibit anti-proliferative activity in human colorectal cancer cells. Both horehound and wild cherry extracts cause suppression of cell growth as well as induction of apoptosis. We found that horehound extract up-regulates pro-apoptotic non-steroidal anti-inflammatory drug-activated gene (NAG-1) through transactivation of the NAG-1 promoter. In contrast, wild cherry extract decreased cyclin D1 expression and increased NAG-1 expression in HCT-116 and SW480 cell lines. Treatment with wild cherry extract resulted in the suppression of beta-catenin/T cell factor transcription, as assessed by TOP/FOP reporter constructs, suggesting that suppressed beta-catenin signaling by wild cherry extract leads to the reduction of cyclin D1 expression. Our data suggest the mechanisms by which these extracts suppress cell growth and induce apoptosis involve enhanced NAG-1 expression and/or down-regulation of beta-catenin signaling, followed by reduced cyclin D1 expression in human colorectal cancer cells. These findings may provide mechanisms for traditional anti-inflammatory products as cancer chemopreventive agents.