Emodin Suppresses Migration and Invasion through the Modulation of CXCR4 Expression in an Orthotopic Model of Human Hepatocellular Carcinoma

Accumulating evidence(s) indicate that CXCL12-CXCR4 signaling cascade plays an important role in the process of invasion and metastasis that accounts for more than 80% of deaths in hepatocellular carcinoma (HCC) patients. Thus, identification of novel agents that can downregulate CXCR4 expression and its associated functions have a great potential in the treatment of metastatic HCC. In the present report, we investigated an anthraquinone derivative, emodin for its ability to affect CXCR4 expression as well as function in HCC cells. We observed that emodin downregulated the expression of CXCR4 in a dose-and time-dependent manner in HCC cells. Treatment with pharmacological proteasome and lysosomal inhibitors did not have substantial effect on emodin-induced decrease in CXCR4 expression. When investigated for the molecular mechanism(s), it was observed that the suppression of CXCR4 expression was due to downregulation of mRNA expression, inhibition of NF-κB activation, and abrogation of chromatin immunoprecipitation activity. Inhibition of CXCR4 expression by emodin further correlated with the suppression of CXCL12-induced migration and invasion in HCC cell lines. In addition, emodin treatment significantly suppressed metastasis to the lungs in an orthotopic HCC mice model and CXCR4 expression in tumor tissues. Overall, our results show that emodin exerts its anti-metastatic effect through the downregulation of CXCR4 expression and thus has the potential for the treatment of HCC.     

Analysis of Human Blood Plasma Proteome from Ten Healthy Volunteers from Indian Population

Analysis of any mammalian plasma proteome is a challenge, particularly by mass spectrometry, due to the presence of albumin and other abundant proteins which can mask the detection of low abundant proteins. As detection of human plasma proteins is valuable in diagnostics, exploring various workflows with minimal fractionation prior to mass spectral analysis, is required in order to study population diversity involving analysis in a large cohort of samples. Here, we used ‘reference plasma sample’, a pool of plasma from 10 healthy individuals from Indian population in the age group of 25–60 yrs including 5 males and 5 females. The 14 abundant proteins were immunodepleted from plasma and then evaluated by three different workflows for proteome analysis using a nanoflow reverse phase liquid chromatography system coupled to a LTQ Orbitrap Velos mass spectrometer. The analysis of reference plasma sample a) without prefractionation, b) after prefractionation at peptide level by strong cation exchange chromatography and c) after prefractionation at protein level by sodium dodecyl sulfate polyacrylamide gel electrophoresis, led to the identification of 194, 251 and 342 proteins respectively. Together, a comprehensive dataset of 517 unique proteins was achieved from all the three workflows, including 271 proteins with high confidence identified by≥2 unique peptides in any of the workflows or identified by single peptide in any of the two workflows. A total of 70 proteins were common in all the three workflows. Some of the proteins were unique to our study and could be specific to Indian population. The high-confidence dataset obtained from our study may be useful for studying the population diversity, in discovery and validation process for biomarker identification.     

Molecular Dynamics in Protein—Single Stranded DNA Complexes. Two Distinct Nucleoside Mobilities in Poly(deoxythymidylic acid)—poly-L-lysine Complexes

Abstract

Stoichiometric amounts of poly-L-lysine were added to site-specifically spin labeled single stranded nucleic acids and the resulting complexes analyzed by electron spin resonance spectroscopy (ESR). The nucleic acids were spin labeled to different extents and with labels of varying tether length. The ESR data are used to determine nucleoside dynamics and some structural features in these complexes. It is concluded that two distinct base mobilities exist in the complexes; one set is characterized by a mean correlation time τR = 2 ns, and the other one by a τR ≤ 50 ns. A model is proposed which suggests that a poly-L-lys single stranded nucleic acid complex consists of low mobility segments flanked by more mobile bases. An interesting feature of the proposed model is its applicability to explain ESR data of single strand binding protein-spin labeled nucleic acid complexes, which can also be interpreted in terms of two distinct nucleoside mobility states. It is hypothesized that this phenomenon could be of biological significance for the release of protein ligands from a protein-nucleic acid complex.     

Effects of 12‐Hour Rotating Shifts on Menstrual Cycles of Photoelectronic Workers in Taiwan

12 h rotating shifts are common in high‐tech industries in Taiwan. The aim of this longitudinal study was to evaluate the effect of the disruption of circadian rhythms by the shift schedule on menstrual cycle length (MCL) and regularity of female workers at an optoelectronic company in Taiwan. We recruited females who worked rotating shifts in a clean room environment as the shift‐work group and female office workers who worked normal business hours as the comparison group. Every participant recorded their MCL for each menstruation cycle up to eight consecutive months prospectively and provided demographic characteristics, reproductive history, and menstrual characteristics. We collected data on 1,135 and 117 menstruation cycles in the shift‐work (n=280) and comparison groups (n=49). Whereas the two groups had similar group means for MCL and number of menstrual bleeding days, the prevalence of menstrual cycle irregularity (cycles<25 or>35 days) was higher in the shift‐work group (p=0.04). Univariate and multivariate logistic regression analyses demonstrated that rotating shift work was an independent predictor of menstrual cycle irregularity (odds ratio=1.71, 95% confidence interval: 1.03–2.88) after adjusting for shift‐work history, employment duration, coffee consumption, and pre‐employment menstrual cycle irregularity. Although further study is required to confirm our findings plus to explore prevention and control measures, our data indicate rotating shift work can increase the risk of MCL irregularity.     

Emergence of gp120 V3 Variants Confers Neutralization Resistance in an R5 Simian-Human Immunodeficiency Virus-Infected Macaque Elite Neutralizer That Targets the N332 Glycan of the Human Immunodeficiency Virus Type 1 Envelope Glycoprotein

Neutralization-resistant simian-human immunodeficiency virus AD8 (SHIV_AD8) variants that emerged in an infected macaque elite neutralizer targeting the human immunodeficiency virus type 1 (HIV-1) gp120 N332 glycan acquired substitutions of critical amino acids in the V3 region rather than losing the N332 glycosylation site. One of these resistant variants, carrying the full complement of gp120 V3 changes, was also resistant to the potent anti-HIV-1 monoclonal neutralizing antibodies PGT121 and 10-1074, both of which are also dependent on the presence of the gp120 N332 glycan.     

The Human Antimicrobial Peptide LL-37, but Not the Mouse Ortholog,mCRAMP, Can Stimulate Signaling by Poly(I:C) through a FPRL1-dependent Pathway

LL-37 is an antimicrobial peptide produced by human cells that can down-regulate the lipopolysaccharide-induced innate immune responses and up-regulate double-stranded (ds) RNA-induced innate responses through Toll-like receptor 3 (TLR3). The murine LL-37 ortholog, mCRAMP, also inhibited lipopolysaccharide-induced responses, but unlike LL-37, it inhibited viral-induced responses in mouse cells. A fluorescence polarization assay showed that LL-37 was able to bind dsRNA better than mCRAMP. In the human lung epithelial cell line BEAS-2B, LL-37, but not mCRAMP, colocalized with TLR3, and the colocalization was increased in the presence of dsRNA. The presence of poly(I:C) increased the accumulation of LL-37 in Rab5 endosomes. Signaling by cells induced with both LL-37 and poly(I:C) was sensitive to inhibitors that affect clathrin-independent trafficking, whereas signaling by poly(I:C) alone was not, suggesting that the LL-37-poly(I:C) complex trafficked to signaling endosomes by a different mechanism than poly(I:C) alone. siRNA knockdown of known LL-37 receptors identified that FPRL1 was responsible for TLR3 signaling induced by LL-37-poly(I:C). These results show that LL-37 and mCRAMP have different activities in TLR3 signaling and that LL-37 can redirect trafficking of poly(I:C) to effect signaling by TLR3 in early endosomes in a mechanism that involves FPRL1.     

Ablation of Promyelocytic Leukemia Protein (PML) Re-patterns Energy Balance and Protects Mice from Obesity Induced by a Western Diet

The promyelocytic leukemia protein is a well known tumor suppressor, but its role in metabolism is largely unknown. Mice with a deletion in the gene for PML (KO mice) exhibit altered gene expression in liver, adipose tissue, and skeletal muscle, an accelerated rate of fatty acid metabolism, abnormal glucose metabolism, constitutive AMP-activating kinase (AMPK) activation, and insulin resistance in skeletal muscle. Last, an increased rate of energy expenditure protects PML KO mice from the effects of obesity induced by a Western diet. Collectively, our study uncovers a previously unappreciated role of PML in the regulation of metabolism and energy balance in mice.     

The Peptidyl-prolyl Isomerase Pin1 Up-regulation and Proapoptotic Function in Dopaminergic Neurons: RELEVANCE TO THE PATHOGENESIS OF PARKINSON DISEASE*

Parkinson disease (PD) is a chronic neurodegenerative disease characterized by a slow and progressive degeneration of dopaminergic neurons in substantia nigra. The pathophysiological mechanisms underlying PD remain unclear. Pin1, a major peptidyl-prolyl isomerase, has recently been associated with certain diseases. Notably, Ryo et al. (Ryo, A., Togo, T., Nakai, T., Hirai, A., Nishi, M., Yamaguchi, A., Suzuki, K., Hirayasu, Y., Kobayashi, H., Perrem, K., Liou, Y. C., and Aoki, I. (2006) J. Biol. Chem. 281, 4117–4125) implicated Pin1 in PD pathology. Therefore, we sought to systematically characterize the role of Pin1 in PD using cell culture and animal models. To our surprise we observed a dramatic up-regulation of Pin1 mRNA and protein levels in dopaminergic MN9D neuronal cells treated with the parkinsonian toxicant 1-methyl-4-phenylpyridinium (MPP⁺) as well as in the substantia nigra of the 1-methyl-4-phenyl-1,2,3,6-tetrahydropyridine (MPTP)-induced PD mouse model. Notably, a marked expression of Pin1 was also observed in the substantia nigra of human PD brains along with a high co-localization of Pin1 within dopaminergic neurons. In functional studies, siRNA-mediated knockdown of Pin1 almost completely prevented MPP⁺-induced caspase-3 activation and DNA fragmentation, indicating that Pin1 plays a proapoptotic role. Interestingly, multiple pharmacological Pin1 inhibitors, including juglone, attenuated MPP⁺-induced Pin1 up-regulation, α-synuclein aggregation, caspase-3 activation, and cell death. Furthermore, juglone treatment in the MPTP mouse model of PD suppressed Pin1 levels and improved locomotor deficits, dopamine depletion, and nigral dopaminergic neuronal loss. Collectively, our findings demonstrate for the first time that Pin1 is up-regulated in PD and has a pathophysiological role in the nigrostriatal dopaminergic system and suggest that modulation of Pin1 levels may be a useful translational therapeutic strategy in PD.     

Purification of clinical-grade disulfide stabilized antibody fragment variable—Pseudomonas exotoxin conjugate (dsFv-PE38) expressed in Escherichia coli

Immunotoxins are rationally designed cancer targeting and killing agents. Disulfide stabilized antibody Fv portion—toxin conjugates (dsFv-toxin) are third generation immunotoxins containing only the antibody fragment variable portions and a toxin fused to the V_H or V_L. Pseudomonas exotoxin fragment (PE-38) is a commonly used toxin in immunotoxin clinical trials. dsFv-toxin purification was previously published, but the recovery was not satisfactory. This report describes the development of a cGMP production process of the dsFv-toxin that incorporated a novel purification method. The method has been successfully applied to the clinical manufacturing of two dsFv-PE38 immunotoxins, MR1-1 targeting EGFRvIII and HA22 targeting CD22. The two subunits, V_L and V_H PE-38 were expressed separately in Escherichia coli using recombinant technology. Following cell lysis, inclusion bodies were isolated from the biomass harvested from fermentation in animal source component-free media. The dsFv-toxin was formed after denaturation and refolding, and subsequently purified to homogeneity through ammonium sulfate precipitation, hydrophobic interaction and ion-exchange chromatography steps. It was shown, in a direct comparison experiment using MR1-1 as model protein, that the recovery from the new purification method was improved three times over that from previously published method. The improved recovery was also demonstrated during the clinical production of two dsFv-PE38 immunotoxins—MR1-1 and HA22.