Baicalein is the flavonoids with multiple pharmacological activities. The aim of our study was to investigate the effects of baicalein on colorectal cancer (CRC) and to recognize the targets of baicalein treatment. To better understand baicalein's target, proteomic approaches were used to purify and identify the protein substrates using 2D difference gel electrophoresis (2D SDS-PAGE) to elucidate proteins differential display. Results from this study investigate that baicalein treatment of CRC cells results in reduced cell proliferation. As a result, differential protein displays between baicalein-treated and untreated CRC were determined and validated. There were 11 differentially expressed proteins between baicalein-treated and untreated CRC. Furthermore, we demonstrate that baicalein inhibits cancer cell proliferation and reduced reactive oxygen species (ROS) by up-regulating the levels of peroxiredoxin-6 (PRDX6). Knockdown of PRDX6 in baicalein-treated CRC cells by specific small interfering RNA resulted in ROS production and proliferation, opposite of the baicalein treatment scenario as indicated by cell cycle distribution. These results illustrate that baicalein up-regulates the expression of PRDX6, which attenuates the generation of ROS and inhibits the growth of CRC cells, whereas baicalein treatment have no effect on normal epithelial cells.

E-selectin expression by endothelial cells (ECs) is crucial for leukocyte recruitment during the inflammatory response. Macrophage accumulation and serum E-selectin elevation are features of type 2 diabetes mellitus. However, the interactions between macrophages and ECs in regulating vascular endothelial function are not clearly understood. We investigated the mechanisms underlying the modulation of EC E-selectin expression by high glucose (HG)-treated macrophages. Macrophage-conditioned media (MCM) were prepared from HG-treated macrophages. EC stimulation with HG-MCM induced increases the expression and secretion of E-selectin. By using specific inhibitors and small interfering RNAs, we demonstrate that the activation of the JNK and p38 MAPK pathways are critical for HG-MCM-induced E-selectin expression. Transcription factor ELISA and chromatin immunoprecipitation assays further showed that HG-MCM increases the NF-κB- and AP-1 DNA-binding activities in ECs. The inhibition of NF-κB and AP-1 activation by specific siRNAs blocks the HG-MCM-induced E-selectin promoter activity and expression. Protein arrays and blocking assays using neutralizing antibodies demonstrated that macrophage inflammatory protein 1α and 1β in HG-MCM are major mediators for the induction of EC E-selectin expression. These data support the hypothesis that E-selectin up-regulation stimulated by macrophages may play an active role in atherogenesis in the HG condition and suggest a new mechanism by which arterial disease is accelerated in diabetes.

Learnings from previous Roche p38-selective inhibitors were applied to a new fragment hit, which was optimized to a potent, exquisitely selective preclinical lead with a good pharmacokinetic profile.

The development of a new series of p38α inhibitors resulted in the identification of two clinical candidates, one of which was advanced into a phase 2 clinical study for rheumatoid arthritis. The original lead, an lck inhibitor that also potently inhibited p38α, was a screening hit from our kinase inhibitor library. This manuscript describes the optimization of the lead to p38-selective examples with good pharmacokinetic properties.

The c-Jun N-terminal kinase (JNK) pathway potentially links together the three major pathological hallmarks of Alzheimer's disease (AD): development of amyloid plaques, neurofibrillary tangles, and brain atrophy. As activation of the JNK pathway has been observed in amyloid models of AD in association with peri-plaque regions and neuritic dystrophy, as we confirm here for Tg2576/PS(M146L) transgenic mice, we directly tested whether JNK inhibition could provide neuroprotection in a novel brain slice model for amyloid precursor protein (APP)-induced neurodegeneration. We found that APP/amyloid beta (Abeta)-induced neurodegeneration is blocked by both small molecule and peptide inhibitors of JNK, and provide evidence that this neuroprotection occurs downstream of APP/Abeta production and processing. Our findings demonstrate that Abeta can induce neurodegeneration, at least in part, through the JNK pathway and suggest that inhibition of JNK may be of therapeutic utility in the treatment of AD.

Type I collagen constitutes a major portion of the extracellular matrix (ECM) in arterial wall and it is the major substrate for cell growth and differentiation. The goal of this study was to evaluate the differentiation and proliferation of placenta-derived multipotent cells (PDMCs) on polymerized type I collagen fibrils and monomer collagen. PDMCs grown on both polymerized collagen and monomer collagen with transforming growth factor (TGF)-beta treatment increases the expression of smooth muscle cell (SMC)-specific markers, including calponin, alpha-smooth muscle actin (alpha-SMA) and smooth muscle-myosin heavy chain (SM-MHC). Polymerized collagen increased the expressions of p21(CIP1) and p27(KIP1); decreased cyclin A, cyclin D1, cyclin-dependent protein kinase 2 (Cdk2); and led to G(0)/G(1) arrest in PDMCs. Furthermore, PDMC-differentiated SMCs exhibited significant collagen contractility in the presence or absence of endothelin-1 (ET-1) stimulation. By using specific inhibitors and small interfering RNA (siRNA), we demonstrated that p38 MAPK pathway and serum response factor (SRF)-DNA binding activity is critical for the polymerized collagen-induced PDMC differentiation into SMCs. Thus, polymerized collagen exhibits the great potential in inducing PDMCs differentiation into SMCs, and exerts anti-proliferative effect on PDMC-differentiated SMCs.

Background: Parkinson's disease (PD) is a progressive neurodegenerative condition characterized by an increasing loss of dopaminergic neurons resulting in motor dysfunction. However, cognitive impairments in PD patients are a common clinical feature that has gained increased attention. Objective: The purpose of the current study was to evaluate the effects of an MPTP-induced dopaminergic lesion in mice on social odor recognition (SOR) memory. Methods: Mice were acutely treated with MPTP and evaluated for memory impairments in the SOR assay and characterized using biochemical and immunohistochemical methods approximately 2 weeks later. Results: Here we demonstrate that SOR memory is sensitive to MPTP treatment and that it correlates with multiple measures of nigrostriatal integrity. MPTP treatment of C57BL/6N mice produced a profound decrease in dopamine levels, dopamine transporter binding and tyrosine hydroxylase immunoreactivity in the striatum. These impairments in stratial dopaminergic function were blocked by pretreatment with the MAO-B inhibitor deprenyl. Changes in the dopaminergic system parallel those observed in SOR with MPTP treatment impairing recognition memory in the absence of a deficit in odor discrimination during learning. Deprenyl pretreatment blocked the MPTP-induced impairment of SOR memory. Conclusion: The use of the SOR memory model may provide a preclinical method for evaluating cognitive therapies for PD.

Rationale: Hyperhomocysteinemia contributes to vascular dysfunction and risks of cardiovascular diseases. Stromal cell-derived factor (SDF)-1, a chemokine expressed by endothelial cells (ECs), is highly expressed in advanced atherosclerotic lesions. The interplays among homocysteine, chemokines, and shear stress in regulating vascular endothelial function are not clearly understood. Objective: To investigate the mechanisms for modulations of EC SDF-1 expression by homocysteine and shear stress. Methods and Results: Homocysteine stimulation induced dose- and time-dependent SDF-1 expression and phosphorylation of mitogen-activated protein kinases extracellular signal-regulated kinase, c-Jun N-terminal kinase (JNK), and p38. By using specific inhibitors, small interfering (si)RNA, and dominant negative mutants, we demonstrated that activation of JNK pathway is critical for the homocysteine-induced SDF-1 expression. Transcription factor ELISA and chromatin immunoprecipitation assays showed that homocysteine increased Sp1- and AP-1-DNA binding activities in ECs. Inhibition of Sp1 and AP-1 activations by specific siRNA blocked the homocysteine-induced SDF-1 promoter activity and expression. Preshearing of ECs for 1 to 4 hours at 20 dyn/cm(2) inhibited the homocysteine-induced JNK phosphorylation, Sp1 and AP-1 activation, and SDF-1 expression. The homocysteine-induced SDF-1 expression was suppressed by NO donor. Inhibitor or siRNA for endothelial NO synthase abolished the shear inhibition of SDF-1 expression. Conclusions: Our findings serve to elucidate the molecular mechanisms underlying the homocysteine induction of SDF-1 expression in ECs and the shear stress protection against this induction.

The systemic administration of 1-methyl-4-phenyl-1,2,3,6-tetrahydropyridine (MPTP) to mice produces a reliable and selective degeneration of the nigrostriatal pathway, a hallmark feature of Parkinson's disease (PD). Determining the brain concentrations of 1-methyl-4-phenyl pyridium (MPP(+)), the neurotoxic metabolite of MPTP, is critical for evaluating drugs designed to potentially treat PD. We have developed sensitive and specific quantitative methods for the determination of MPP(+) in mouse striatal tissue by liquid chromatography/tandem mass spectrometry. The separations were carried out based on reversed phase chromatography or cation exchange chromatography with volatile elution buffer. Neutralizing the brain sample with 0.2M phosphate buffer successfully solved a high-performance liquid chromatography (HPLC) peak tailing of MPP(+) in brain extracts with 0.4M perchloric acid (HClO(4)) under the reversed phase HPLC conditions, which significantly improved the sensitivity of the method. The HPLC peak shape of MPP(+) using cation exchange chromatography was not affected by the pH of the samples. Optimization of electrospray ionization (ESI) conditions for the quaternary ammonium compound MPP(+) established the limits of detection (LOD)(S/N=3) at 0.34pg/mg tissue and 0.007pg/mg tissue (5mul of injection) using the reversed phase liquid chromatography/tandem mass spectrometry (LC/MS/MS) and the cation exchange LC/MS/MS, respectively. Both methods were selective, precise (%R.S.D.<6%), and sensitive over a range of 0.001-1ng/mg tissue. The cation exchange method showed greater sensitivity and tolerance to low pH samples than the reversed phase method. The developed methods were applied to monitoring changes in MPP(+) concentrations in vivo. Two reference agents, R-(-) Deprenyl and MK-801, known to alter the concentration of MPP(+) in MPTP treated mice were evaluated.

The diuretic amiloride has recently proven neuroprotective in models of cerebral ischemia, a property attributable to the drug's inhibition of central acid-sensing ion channels (ASICs). Given that Parkinson's disease (PD), like ischemia, is associated with cerebral lactic acidosis, we tested amiloride in the MPTP-treated mouse, a model of PD also manifesting lactic acidosis. Amiloride was found to protect substantia nigra (SNc) neurons from MPTP-induced degeneration, as determined by attenuated reductions in striatal tyrosine hydroxylase (TH) and dopamine transporter (DAT) immunohistochemistry, as well as smaller declines in striatal DAT radioligand binding and dopamine levels. More significantly, amiloride also preserved dopaminergic cell bodies in the SNc. Administration of psalmotoxin venom (PcTX), an ASIC1a blocker, resulted in a much more modest effect, attenuating only the deficits in striatal DAT binding and dopamine. These findings represent the first experimental evidence of a potential role for ASICs in the pathogenesis of Parkinson's disease.