Xiao-Lin Wang

Adenanthin targets peroxiredoxin I and II to induce differentiation of leukemic cells

Peroxiredoxins (Prxs) are potential therapeutic targets for major diseases such as cancers. However, isotype-specific inhibitors remain to be developed. We report that adenanthin, a diterpenoid isolated from the leaves of Rabdosia adenantha, induces differentiation of acute promyelocytic leukemia (APL) cells. We show that adenanthin directly targets the conserved resolving cysteines of Prx I and Prx II and inhibits their peroxidase activities. Consequently, cellular H(2)O(2) is elevated, leading to the activation of extracellular signal-regulated kinases and increased transcription of CCAAT/enhancer-binding protein β, which contributes to adenanthin-induced differentiation. Adenanthin induces APL-like cell differentiation, represses tumor growth in vivo and prolongs the survival of mouse APL models that are sensitive and resistant to retinoic acid. Thus, adenanthin can serve as what is to our knowledge the first lead natural compound for the development of Prx I- and Prx II-targeted therapeutic agents, which may represent a promising approach to inducing differentiation of APL cells.

Lipoprotein-Based Nanoparticles Rescue the Memory Loss of Mice with Alzheimer’s Disease by Accelerating the Clearance of Amyloid-Beta

Amyloid-beta (Aβ) accumulation in the brain is believed to play a central role in Alzheimers disease (AD) pathogenesis, and the common late-onset form of AD is characterized by an overall impairment in Aβ clearance. Therefore, development of nanomedicine that can facilitate Aβ clearance represents a promising strategy for AD intervention. However, previous work of this kind was concentrated at the molecular level, and the disease-modifying effectiveness of such nanomedicine has not been investigated in clinically relevant biological systems. Here, we hypothesized that a biologically inspired nanostructure, apolipoprotein E3-reconstituted high density lipoprotein (ApoE3-rHDL), which presents high binding affinity to Aβ, might serve as a novel nanomedicine for disease modification in AD by accelerating Aβ clearance. Surface plasmon resonance, transmission electron microscopy, and co-immunoprecipitation analysis showed that ApoE3-rHDL demonstrated high binding affinity to both Aβ monomer and oligomer. It also accelerated the microglial, astroglial, and liver cell degradation of Aβ by facilitating the lysosomal transport. One hour after intravenous administration, about 0.4% ID/g of ApoE3-rHDL gained access to the brain. Four-week daily treatment with ApoE3-rHDL decreased Aβ deposition, attenuated microgliosis, ameliorated neurologic changes, and rescued memory deficits in an AD animal model. The findings here provided the direct evidence of a biomimetic nanostructure crossing the blood-brain barrier, capturing Aβ and facilitating its degradation by glial cells, indicating that ApoE3-rHDL might serve as a novel nanomedicine for disease modification in AD by accelerating Aβ clearance, which also justified the concept that nanostructures with Aβ-binding affinity might provide a novel nanoplatform for AD therapy.

Application of high-performance liquid chromatography–mass spectrometry to detection of diuretics in human urine

A rapid, sensitive and reliable high-performance liquid chromatographic-mass spectrometric method for the detection of 25 diuretics in human urine has been developed. Atmosphere pressure chemical ionization (APCI) and electrospray ionization (ESI) modes were evaluated. A 2-ml volume of urine was extracted under basic conditions and separated on an Agilent Zorbax SB-C(18) column (150 x 2.1 mm, 5 microm). The mobile phase consisted of formic ammonium-formic acid buffer (pH 3.5) and acetonitrile. The effects of capillary temperature, sheath gas pressure and compositions of mobile phase on the sensitivity were studied. The recoveries of most of the diuretics were 75-95%. In the full scan mode, the limits of detection of the 25 diuretics were 0.25-25 ng/ml for APCI and 0.6-250 ng/ml for ESI. Under the optimal conditions, 14 diuretics from authentic urine samples were detected successfully by LC-APCI-MS. To obtain more fragmentation information on the chemical structure for positive confirmation, tandem mass analysis was also investigated.

Cellular internalization pathway and transcellular transport of pegylated polyester nanoparticles in Caco-2 cells

Biodegradable polyester nanoparticles have now attracted growing interest as promising drug delivery system. However, a fundamental understanding about its cellular transport as well as the influence by the polymeric architecture is still lack, which remains a significant obstacle to optimal nanocarrier design. In this work, using Caco-2 cell model, we characterized the cellular transport pathway of pegylated polyester nanoparticles and determined the effect of polymer architecture including PEG chain length and core material on its cellular interaction and transcellular transport. The nanoparticles were found to undergo an energy-dependent, lipid raft-mediated, but caveolae-independent endocytosis. PEG chain length (from 2000 to 5000 Da) and core material (PLA/PLGA) hardly affected the cellular interaction and the intracellular itinerary of the nanoparticles. However, in the case of transcellular transport, the maximal transcellular transport efficiency for its payload was achieved by the PEG5000-PLA40000 nanoparticles which present higher drug loading capacity and slower drug release. The findings here revealed the cellular interaction mechanism of pegylated polyester nanoparticles and provided evidence for the role of polymer architectures in modulating the transcellular permeability of the agents loaded by the nanoparticles, and would be helpful in improving carrier design to enhance drug delivery.

GM1-Modified Lipoprotein-like Nanoparticle: Multifunctional Nanoplatform for the Combination Therapy of Alzheimer's Disease.

Alzheimers disease (AD) exerts a heavy health burden for modern society and has a complicated pathological background. The accumulation of extracellular β-amyloid (Aβ) is crucial in AD pathogenesis, and Aβ-initiated secondary pathological processes could independently lead to neuronal degeneration and pathogenesis in AD. Thus, the development of combination therapeutics that can not only accelerate Aβ clearance but also simultaneously protect neurons or inhibit other subsequent pathological cascade represents a promising strategy for AD intervention. Here, we designed a nanostructure, monosialotetrahexosylganglioside (GM1)-modified reconstituted high density lipoprotein (GM1-rHDL), that possesses antibody-like high binding affinity to Aβ, facilitates Aβ degradation by microglia, and Aβ efflux across the blood-brain barrier (BBB), displays high brain biodistribution efficiency following intranasal administration, and simultaneously allows the efficient loading of a neuroprotective peptide, NAP, as a nanoparticulate drug delivery system for the combination therapy of AD. The resulting multifunctional nanostructure, αNAP-GM1-rHDL, was found to be able to protect neurons from Aβ(1-42) oligomer/glutamic acid-induced cell toxicity better than GM1-rHDL in vitro and reduced Aβ deposition, ameliorated neurologic changes, and rescued memory loss more efficiently than both αNAP solution and GM1-rHDL in AD model mice following intranasal administration with no observable cytotoxicity noted. Taken together, this work presents direct experimental evidence of the rational design of a biomimetic nanostructure to serve as a safe and efficient multifunctional nanoplatform for the combination therapy of AD.

Determination of glycine in biofluid by hydrophilic interaction chromatography coupled with tandem mass spectrometry and its application to the quantification of glycine released by embryonal carcinoma stem cells.

Because glycine plays a prominent role in living creatures, an accurate and precise quantitative analysis method for the compound is needed. Herein, a new approach to analyze glycine by hydrophilic interaction chromatography (HILIC) coupled with electrospray ionization tandem mass spectrometry (ESI-MS/MS) was developed. This method avoids the use of derivatization and/or ion-pairing reagents. N-methyl-D-aspartate (NMDA) is used as the internal standard (IS). The mobile phase for the isocratic elution consisted of 10 mM ammonium formate in acetonitrile-water (70:30, v/v, adjusted to pH 2.8 with formic acid), and a flow rate of 250 μL/min was used. Two microliters of sample was injected for analysis. The signal was monitored in the positive multiple reaction monitoring (MRM) mode. The total run time was 5 min. The dynamic range was 40-2000 ng/mL for glycine in the biological matrix. The LLOQ (lower limit of quantification) of this method was 40 ng/mL (80 pg on column). The validated method was applied to determine the dynamic release of glycine from P19 embryonal carcinoma stem cells (ECSCs). Glycine spontaneously released from the ECSCs into the intercellular space gradually increased from 331.02±60.36 ng/mL at 2 min in the beginning to 963.52±283.80 ng/mL at 60 min and 948.27±235.09 ng/mL at 120 min, finally reaching a plateau, indicating that ECSCs consecutively release glycine until achieving equilibration between the release and the reuptake of the compound; on the contrary, the negative control NIH/3T3 embryonic fibroblast cells did not release glycine. This finding will help to improve our understanding of the novel effects of neurotransmitters, including glycine, on non-neural systems.

Nanoformulated alpha-mangostin ameliorates Alzheimer's disease neuropathology by elevating LDLR expression and accelerating amyloid-beta clearance.

Alzheimers disease (AD), the most common form of dementia, is now representing one of the largest global healthcare challenges. However, an effective therapy is still lacking. Accumulation of amyloid-beta (Aβ) in the brain is supposed to trigger pathogenic cascades that eventually lead to AD. Therefore, Aβ clearance strategy is being actively pursued as a promising disease modifying therapy. Here, we found that α-mangostin (α-M), a polyphenolic xanthone derivative from mangosteen, up-regulated low density lipoprotein receptor (LDLR) expression in microglia and liver cells, and efficiently facilitated Aβ clearance. However, the in vivo application of α-M is limited due to its hydrophobic nature, poor aqueous solubility and stability, and thus low bioavailability and accumulation in the target organs. To overcome this limitation, α-M was encapsulated into the core of poly(ethylene glycol)-poly(l-lactide) (PEG-PLA) nanoparticles [NP(α-M)]. Such nanoencapsulation improved the biodistribution of α-M in both the brain and liver, enhanced the brain clearance of (125)I-radiolabeled Aβ1-42 in an LDLR-dependent manner, reduced Aβ deposition, attenuated neuroinflammatory responses, ameliorated neurologic changes and reversed behavioral deficits in AD model mice. These findings justified the concept that polyphenol-mediated modulation of LDLR expression might serve as a safe and efficient disease-modifying therapy for AD by accelerating Aβ clearance. It also demonstrated the powerful capacity of nanotechnology in modulating the biodistribution behavior of drug to improve its therapeutic efficacy in AD.

Determination of Raddeanin A in rat plasma by liquid chromatography-tandem mass spectrometry: Application to a pharmacokinetic study.

A simple, rapid and sensitive LC-MS/MS analysis method was developed and validated for the determination of Raddeanin A (RA) in rat plasma. Protein precipitation with three volumes of methanol as the precipitation reagent was used as the sample preparation method. The analysis process was performed on a Thermo Syncronis C18 column with the mobile phase of methanol-water (containing 5mM ammonium formate, pH 2.2) (85:15, v/v). RA and glycyrrhetinic acid (internal standard) were monitored under negative electrospray ionization in multiple reaction monitoring (MRM) mode. Retention time of RA and IS were 2.1 min and 3.5 min, respectively. The limit of detection was 5 ng/mL and the linear range was 50-50,000 ng/mL. The intra-day and inter-day precision was 1.87-2.94% and 3.25-5.36%, and the intra-day and inter-day accuracy ranged from 5.9% to 10.5% and 5.6% to 11.1%, respectively. The absolute recovery was above 90.3%. The method has been successfully translated to the pharmacokinetic study of RA in rats after intravenous and intraperitoneal administration (0.75 mg/kg).

Carbocysteine restores steroid sensitivity by targeting histone deacetylase 2 in a thiol/GSH-dependent manner.

Steroid insensitivity is commonly observed in patients with chronic obstructive pulmonary disease. Here, we report the effects and mechanisms of carbocysteine (S-CMC), a mucolytic agent, in cellular and animal models of oxidative stress-mediated steroid insensitivity. The following results were obtained: oxidative stress induced higher levels of interleukin-8 (IL-8) and tumor necrosis factor alpha (TNF-α), which are insensitive to dexamethasone (DEX). The failure of DEX was improved by the addition of S-CMC by increasing histone deacetylase 2 (HDAC2) expression/activity. S-CMC also counteracted the oxidative stress-induced increase in reactive oxygen species (ROS) levels and decreases in glutathione (GSH) levels and superoxide dismutase (SOD) activity. Moreover, oxidative stress-induced events were decreased by the thiol-reducing agent dithiothreitol (DTT), enhanced by the thiol-oxidizing agent diamide, and the ability of DEX was strengthened by DTT. In addition, the oxidative stress-induced decrease in HDAC2 activity was counteracted by S-CMC by increasing thiol/GSH levels, which exhibited a direct interaction with HDAC2. S-CMC treatment increased HDAC2 recruitment and suppressed H4 acetylation of the IL-8 promoter, and this effect was further ablated by addition of buthionine sulfoximine, a specific inhibitor of GSH synthesis. Our results indicate that S-CMC restored steroid sensitivity by increasing HDAC2 expression/activity in a thiol/GSH-dependent manner and suggest that S-CMC may be useful in a combination therapy with glucocorticoids for treatment of steroid-insensitive pulmonary diseases.

Development and validation of a quantitative liquid chromatography tandem mass spectrometry assay for pristimerin in rat plasma.

A sensitive, rapid and simple LC-MS/MS analysis method was developed and validated for the determination of pristimerin (PR) in rat plasma. Protein precipitation with four volumes of acetonitrile as the precipitation reagent was used as the sample preparation method. The analysis process was performed on a Merck ZIC-HILIC column with the mobile phase of acetonitrile-water (containing 5mM ammonium formate, pH 2.8) (85:15, v/v). PR (m/z 465.3-201.1) and glycyrrhetinic acid (internal standard, m/z 471.5-177.1) were monitored under positive electrospray ionization in multiple reaction monitoring (MRM) mode. Retention time of PR and IS was 2.45min and 2.4min, respectively. The limit of detection was 0.5ng/mL and the linear range was 1-500ng/mL. The intra-day and inter-day precision were 2.89-6.27% and 4.91-8.98%, and the intra-day and inter-day accuracy ranged from -5.81% to 8.64% and -7.37% to 9.57%, respectively. The matrix effects and absolute recovery ranged from 89.3% to 92.4% and 88.7% to 92.8%, respectively. The method has been successfully applied to the determination of PR concentration in rat plasma after intravenous administration (0.5mg/kg).