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Blockage of p53-MDM2 protein-protein interaction has long been a promising strategy of drug development for cancers with wild type p53. In this study, we report a new p53-MDM2 interaction inhibitor, CYZ2017, which could induce p53 nuclear translocation and possess p53-dependent anti-proliferation activity in a range of cancer cells. CYZ2017 treatment led to increase of p53 levels and induced the transactivation of its target genes p21. In addition, CYZ2017 induced G0/G1 cell cycle arrest and apoptosis in HCT116 cells. Besides, CYZ2017 suppressed tumor growth in a HCT116 xenograft model without visible toxicity. These results support that CYZ2017 might be a promising p53-MDM2 interaction inhibitor with good anti-tumor activity. Our finding provides some cues for further investigation of developing anti-tumor drugs based on the blockage of p53-MDM2 interaction.

Agonists of M1 muscarinic acetylcholine receptors are promising therapeutic agents for the treatment of Alzheimer's disease (AD). An example of one of these agents is xanomeline, which has been a leading candidate, however induces various unwanted adverse effects. 3‑[3‑(3‑florophenyl‑2‑propyn‑1‑ylthio)‑1,2,5‑thiadiazol-4-yl]-1,2,5,6-tetrahydro‑1‑methylpyridine oxalate (EUK1001), a fluorinated derivative of xanomeline, has been demonstrated to attenuate AD‑like neurodegenerative pathology in presenilin‑deficient mice, which has no β‑amyloid (Aβ) pathology. The present study assessed the effect of EUK1001 on the behavioral performance of the 3xTg‑AD model of AD. EUK1001 treatment decreased cognitive deficits in male and female AD mice in the Morris water maze test and novel object recognition tasks. EUK1001 also decreased Aβ42, however not Aβ40 in the cortex and hippocampus of AD mice. EUK1001 may also alter amyloid precursor protein processing to a nonamyloidgenic pathway in vitro. These results demonstrate that EUK1001 may ameliorate the cognitive dysfunction of AD mice, possibly by reducing Aβ production. Therefore, EUK1001 may be an effective treatment for AD.

Retinoic acid can cause many types of cells, including mouse neuroblastoma Neuro-2A cells, to differentiate into neurons. However, it is still unknown whether microRNAs (miRNAs) play a role in this neuronal differentiation. To address this issue, real-time polymerase chain reaction assays were used to detect the expression of several differentiation-related miRNAs during the differentiation of retinoic acid-treated Neuro-2A cells. The results revealed that miR-124 and miR-9 were upregulated, while miR-125b was downregulated in retinoic acid-treated Neuro-2A cells. To identify the miRNA that may play a key role, miR-124 expression was regulated by transfection of miRNA mimics or inhibitors. Morphological analysis results showed that inhibition of miR-124 expression reversed the effects of retinoic acid on neurite outgrowth. Moreover, miR-124 overexpression alone caused Neuro-2A cells to differentiate into neurons, and its inhibitor could block this effect. These results suggest that miR-124 plays an important role in retinoic acid-induced differentiation of Neuro-2A cells.

The leading cause of death among cancer patients is tumor metastasis. Tumor-derived exosomes are emerging as mediators of metastasis. In the present study, we demonstrated that exosomes play a pivotal role in the metastatic progression of colorectal cancer. First, a nude mouse model of colorectal cancer liver metastasis was established and characterized. Then, we demonstrated that exosomes from a highly liver metastatic colorectal cancer cell line (HT-29) could significantly increase the metastatic tumor burden and distribution in the mouse liver of Caco-2 colorectal cancer cells, which ordinarily exhibit poor liver metastatic potential. We further investigated the mechanisms by which HT-29-derived-exosomes influence the liver metastasis of colorectal cancer and found that mice treated with HT-29-derived exosomes had a relatively higher level of CXCR4 in the metastatic microenvironment, indicating that exosomes may promote colorectal cancer metastasis by recruiting CXCR4-expressing stromal cells to develop a permissive metastatic microenvironment. Finally, the migration of Caco-2 cells was significantly increased following treatment with HT-29-derived exosomes in vitro, further supporting a role for exosomes in modulating colorectal tumor-derived liver metastasis. The data from the present study may facilitate further translational medicine research into the prevention and treatment of colorectal cancer liver metastasis.

GPR12, a member of the orphan G-protein-coupled receptor family, constitutively activates the Gs protein and increases intracellular cyclic AMP concentrations. GPR12 can be activated by its known ligand-sphingosylphosphorylcholine, which regulates cellular physiological activities, including proliferation, neurite extension, cell clustering, and maintenance of meiotic arrest. However, signaling pathways involved in the GPR12-mediated physiological and biochemical changes are still not clearly illustrated. In the present study, heterologous GPR12 expression was demonstrated to promote proliferation and survival in human embryonic kidney 293 cells. Immunochemical analysis showed that Ki67, a prototypic cell cycle-related nuclear protein, might participate in the regulation of GPR12-mediated cell proliferation. Activation of extracellular signal-regulated protein kinase signaling and increased total Erk1/2 and B-cell lymphoma/leukemia-2 expression were also observed in HEK293 cells overexpressing human GPR12. In addition, we found that GPR12 promoted cell survival under serum deprivation, indicating that GPR12 may play a role in cell proliferation and survival.

Glutamate N-methyl-D-aspartate (NMDA) receptors play a pivotal role in different forms of memory. The dysfunction of NMDA receptors contributes to the pathology of central nervous system (CNS) disorders. To further investigate the role of the NMDA receptors in brain processes, we analyzed and compared the gene expression profiles in the hippocampus of NR2B overexpression-induced memory-enhanced mice (Tg mice) with those of their wild-type littermates. Results reveal that 249 genes, which are mainly involved in neurotransmission, signal transduction, cytoskeletal structure, hormone activity, and transcription, were significantly affected in Tg mice. Interestingly, the intracellular calcium channel proteins ryanodine receptor (RyR) 1 and 3, as well as functionally related proteins such as the histidine-rich calcium-binding protein and triadin 2, were upregulated. The Homer-1c protein was also increased in Tg mice and formed a complex with the RyR protein in the mouse brain, suggesting that Homer-1c is an important modulator in both intracellular calcium signaling and overall neuronal signaling by simultaneously interacting with the NMDA receptors and RyR. Western blot and real-time PCR results show that the expression of phospho-CREB, c-fos, and the immediate-early genes Egr2 and Egr4 were also upregulated in Tg mice. The current study demonstrates that a chronic increase in the activation of NMDA receptors affected the expression of a large number of genes and may provide important clues for a better understanding of the molecular mechanism of NMDA receptor-modulated learning and memory, as well as of CNS disorders.

Presenilin (PS) 1 and 2 conditional double knockout (cDKO) mice show progressive memory dysfunction and forebrain degeneration. Gene expression profiling results revealed a strong activation of immunity and inflammation responses in the brains of 10-month-old cDKO mice. As environmental enrichment (EE) has been shown to be able to improve memory and induce neurogenesis of the brain, we assessed the effects of EE on the memory performance and the neurodegeneration in cDKO mice. We found that EE effectively enhanced memory and partially rescued the forebrain atrophy of the cDKO mice. Our results suggest that immunity and inflammation could play important roles in the neurodegeneration of cDKO mice. Furthermore, the beneficial effects of EE may be associated with the inhibition of the expression of immunity and inflammation-related genes in the brain.

Protein tyrosine phosphatase 1B (PTP1B) has been proposed to be an effective target for the treatment of both type II diabetes and obesity. However, no PTP1B inhibitor has come into clinic application. Herein, we report mixed 3,3′-bisindoles as novel PTP1B inhibitors with low micromole-ranged inhibitory activity. The best active compound 9f inhibited PTP1B activity with an IC50 of 2.79 µM. Meanwhile, it had low cytotoxicity and enhanced glucose uptake in vitro. Further studies demonstrated that some of these active compounds had a specific selectivity over other PTPs. Computational analysis further showed the binding mode of compound 9f with the active pocket of PTP1B. Our studies provide a novel scaffold for further development of more promising PTP1B inhibitors and potential drugs for type II diabetes and obesity.

In addition to senile plaques and cerebral amyloid angiopathy, the hyperphosphorylation of tau protein and formation of intraneuronal neurofibrillary tangles (NFTs) represents another neuropathological hallmark in AD brain. Tau is a microtubule-associated protein and localizes predominantly in the axons of neurons with the primary function in maintaining microtubules stability. When the balance between tau phosphorylation and dephosphorylation is changed in favor of the former, tau is hyperphosphorylated and the level of the free tau fractions elevated. The hyperphosphorylation of tau protein and formation of NFTs represent a characteristic neuropathological feature in AD brain. We have discussed the role of Aβ in AD in our previous review, this review focused on the recent advances in tau-mediated AD pathology, mainly including tau hyperphosphorylation, propagation of tau pathology and the relationship between tau and Aβ.

Butyrate has been recently identified as a natural ligand of the G-protein-coupled receptor 41 (GPR41). In addition, it is an inhibitor of histone deacetylase (HDAC). Butyrate treatment results in the hyperacetylation of histones, with resultant multiple biological effects including inhibition of proliferation, induction of cell cycle arrest, and apoptosis, in a variety of cultured mammalian cells. However, it is not clear whether GPR41 is actively involved in the above-mentioned processes. In this study, we generated a stable cell line expressing the hGPR41 receptor in order to investigate the involvement of GPR41 on butyrate-induced biochemical and physiologic processes. We found that GPR41 activation may be a compensatory mechanism to counter the increase in histone H3 acetylation levels induced by butyrate treatment. Moreover, GPR41 had an inhibitory effect on the anti-proliferative, pro-apoptotic effects of butyrate. GPR41 expression induced cell cycle arrest at the G1-stage, while its activation by butyrate can cause more cells to pass the G1 checkpoint. These results indicated that GPR41 was associated with histone acetylation and might be involved in the acetylation-related regulation of cell processes including proliferation, apoptosis, and the cell cycle.

BACKGROUND: Curcumin has been demonstrated to have many neuroprotective properties, including improvement of cognition in humans and neurogenesis in animals, yet the mechanism of such effects remains unclear. METHODOLOGY: We assessed behavioural performance and hippocampal cell proliferation in aged rats after 6- and 12-week curcumin-fortified diets. Curcumin enhanced non-spatial and spatial memory, as well as dentate gyrate cell proliferation as compared to control diet rats. We also investigated underlying mechanistic pathways that might link curcumin treatment to increased cognition and neurogenesis via exon array analysis of cortical and hippocampal mRNA transcription. The results revealed a transcriptional network interaction of genes involved in neurotransmission, neuronal development, signal transduction, and metabolism in response to the curcumin treatment. CONCLUSIONS: The results suggest a neurogenesis- and cognition-enhancing potential of prolonged curcumin treatment in aged rats, which may be due to its diverse effects on genes related to growth and plasticity.

GPR12, an orphan G protein-coupled receptor, constitutively activates the Gs signaling pathway and further increases intracellular cyclic AMP. GPR12 overexpression has been reported to promote neurite extension in neurons or transform neuro2a neuroblastoma cells into neuron-like cells. However, the possible effects and mechanisms of GPR12 in the differentiation of PC12 cells are still unknown. The present study shows that GPR12 overexpression induced PC12 cells differentiation into neuron-like cells with enlarged cell sizes and neuritogenesis possibly via activation of Erk1/2 signaling and significantly increased the expression of several neurite outgrowth-related genes, including Bcl-xL, Bcl-2 and synaptophysin. These findings indicate that GPR12 may play a role in neurite outgrowth during PC12 cell differentiation.

A transgenic mouse model expressing Simian virus 40 T-antigen (SV40Tag) under the control of a tetracycline system was generated. In this model, a cerebellar tumor was developed after doxycycline hydrochloride treatment. Real time-polymerase chain reaction and immunohistochemistry results indicated that the SV40Tag gene was expressed in the tumor. Pathological analysis showed that the tumor belonged to medulloblastoma. Further molecular characterization of the tumor demonstrated that the insulin-like growth factor (IGF) signaling pathway was activated. We also found that the SV40Tag could bind and translocate insulin receptor substrate 1 into the nucleus in primary cultured tumor cells. The interaction between the IGF pathway and SV40Tag may contribute to the process of malignant transformation in medulloblastoma. This transgenic animal model provides an important tool for studies on the signal pathways involved in the preneoplastic process in medulloblastoma and could help to identify therapeutic targets for brain tumors.

Conditional double knockout of presenilin-1 and presenilin-2 (cDKO) in forebrain of mice led to brain atrophy, tau hyperphosphorylation, synaptic dysfunction and cognitive deficit. These brain changes recapitulated most of the neurodegenerative phenotypes of Alzheimer's disease (AD). In this report, we have investigated the effects of 4-month calorie restriction (CR) regimen on different phenotypes in cDKO mice. We found that CR improved novel object recognition and contextual fear conditioning memory in the cDKO mice. Histological and biochemical analysis showed that CR attenuated ventricle enlargement, caspase-3 activation and astrogliosis. In addition, the induction of tau hyperphosphorylation in the cDKO mice was reduced by CR, possibly through reduction of p25 accumulation and aberrant CDK5 activation. Finally, DNA microarray analysis demonstrated that CR could increase the expression of neurogenesis related genes and decrease the expression of inflammation related genes in the hippocampus of cDKO mice. The possible molecular mechanisms of the CR effects on alleviating AD pathogenesis have been discussed.

Neuromedin U 2 receptor (NMU2R) plays important roles for the regulation of food intake and body weight. However, the molecular mechanism underlying the action of NMU2R has not been clearly defined. We have taken chemical genetic approach to examine the involvement of peptides in the regulation of NMU2R effects. A cell-based reporter gene assay has been developed and used for the screening of human NMU2R agonist. Three natural product compounds, EUK2010, EUK2011 and EUK2012, were identified that could activate the reporter gene expression in the cell-based functional assay. Although these compounds showed high EC50 at hundreds micro-molar range, in vitro pharmacological analysis suggested that they were specific agonists for the human NMU2R. The natural compounds could decrease food intake and lead to the reduction of body weight in different animal models. To understand the molecular basis of the NMU2R regulation of food intake and body weight, we examined the expression of a number of key genes in hypothalamus and adipose tissues after oral administration of EUK2010 in mice. Our results demonstrated that the expression levels of a number of neuropeptide genes were altered after the treatment of EUK2010. Interestingly, EUK2010 increased the expression of Leptin in white fat. These results suggested that these peptides may participate in the regulation of NMU2R effects in mice.

NMDA receptors have been well shown to be involved in neuronal plasticity. In order to understand the role of NR2B subtype NMDA receptors in auditory function development, the present study investigated the effect of early auditory deprivation on the expression of NR2B mRNA in rat auditory cortex (AC) during postnatal development. For normal rats, the NR2B mRNA expression was highest at birth (postnatal day 1 [P1]) and declined rapidly to low level during adulthood. However, during the critical period of rat auditory development (two to three weeks after birth), there was a transient NR2B expression peak on postnatal day 21 (P21). For the auditory-deprived rats, the general declining trend of NR2B mRNA expression from birth to adult was similar to that observed in the normal group, whereas the expression level from P15 to P27 was significantly lower than normal and the transient peak on P21 disappeared. In both groups, the distribution pattern of NR2B mRNA-positive neurons was also examined in various layers and dorsal, medial and ventral subdistricts of AC. There is no significant effect on the spatial expression of the NR2B mRNA in the AC between normal and deprived group. Our results indicated that the early auditory deprivation decreased the expression levels of NR2B mRNA in AC during the critical period of rat auditory development, suggesting that NR2B plays an important role in the developmental plasticity of auditory function in rats.

AIM: To generate a SV40Tag transgenic tumor animal model and to study the mechanism underlying tumorigenesis. METHODS: A mammary gland expression vector containing SV40Tag DNA was generated. Transgene fragments were microinjeted into fertilized eggs of FVB mice. The genetically manipulated embryos were transferred into the oviducts of pseudo-pregnant female mice. PCR and Northern blot analysis were used for genotype analysis of F1 and F2 mice. Transgene expression was detected by RT-PCR and immunohistochemistry. RESULTS: SV40Tag gene was detected in two lines of transgenic mice. One of them delivered the transgene to F1 and a tumor was found in the pancreas of these mice. RT-PCR and immunohistochemistry showed that SV40Tag gene was expressed in the tumor. Pathological characterization of the transgenic mice demonstrated that the tumor belonged to pancreatic cystic neoplasm. CONCLUSION: SV40Tag transgenic mouse model can be successfully established. The transgenic mice develop a pancreatic tumor, which can be used for investigation of the molecular mechanism of tumorigenesis in vivo.