Combination of withaferin A and X-ray irradiation enhances apoptosis in U937 cells

Combined treatment with radiation has improved the outcome in various cancers and many radiosensitizers are used to enhance the therapeutic efficiency of radiotherapy. Withaferin A (Wit A), a natural compound derived from the medicinal plant Withania somnifera, has been reported for its anti-inflammatory and anti-tumor effects. In this study, we show that Wit A enhances the ionizing radiation (IR)-induced apoptosis in human lymphoma U937 cells. Wit A-enhanced IR-induced apoptosis is associated with the PARP cleavage, caspase-3 activation, as well as specifically down-regulation of anti-apoptotic protein Bcl-2, suggesting that Wit A may be useful as a potential radiosensitizer. In addition, pretreatment of U937 cells with SP600125 (JNK inhibitor) or SB203589 (p38 MAPK inhibitor) dose-dependently inhibited the proteolytic cleavage of PARP. We provide the evidence here that generation of reactive oxygen species (ROS), Bcl-2 down-regulation and activation of MAPKs pathway are critically involved in the apoptosis induced by Wit A and radiation.     

Discovery and applications of disulfide-rich cyclic peptides

Cyclic peptides typically have much higher stability and improved biopharmaceutical properties over their linear counterparts. Our work focuses on the discovery of naturally occurring disulfide-rich cyclic peptides and their applications in drug design. These peptides provide a design basis for re-engineering natural acyclic peptides to improve their biopharmaceutical properties by chemically linking their termini. Here we describe examples of the discovery of the cyclotide family of peptides, their chemical re-engineering to introduce desired pharmaceutical activities, studies of their biopharmaceutical properties and applications of cyclization technologies to naturally occurring toxins, including conotoxins and scorpion toxins. In the case of the conotoxin Vc1.1, we produced an orally active peptide with potential for the treatment of neuropathic pain by cyclising the native peptide. In the case of the scorpion toxin chlorotoxin, a cyclised derivative had improved biopharmaceutical properties as a tumour imaging agent over the naturally occurring linear chlorotoxin. Ongoing chemical and structural studies of these classes of disulfide-rich peptides promise to increase their value for use in dissecting biological processes in plants and mammals while also providing leads to new classes of biopharmaceuticals.     

Amphotericin B/Sterol Co-loaded PEG-Phospholipid Micelles: Effects of Sterols on Aggregation State and Hemolytic Activity of Amphotericin B

Purpose

To elucidate the effect of sterols on the aggregation of amphotericin B (AmB) in PEG-phospholipid micelles and its consequences on the hemolytic activity of AmB.

Methods

AmB-incorporated PEG-phospholipid micelles co-loaded with ergosterol, cholesterol, or 7-dehydrocholesterol were prepared at 4:1:1 and 20:5:1 ratios of polymer-to-sterol-to-AmB. The aggregation state of AmB was elucidated by UV?Cvis spectroscopy. AmB/sterol co-loaded PEG-phospholipid micelles were incubated with red blood cells and the hemolytic activity of AmB assessed by measurement of free hemoglobin.

Results

AmB in PEG-phospholipid micelles stayed mostly in a deaggregated state in the absence of sterol or with cholesterol, but aggregated in the presence of ergosterol or 7-dehydrocholesterol. The fraction of aggregated AmB in PEG-phospholipid micelles was lower at the 20:5:1 ratio. In an aggregated state or in the absence of sterol, AmB caused rapid and complete hemolysis. In contrast, deaggregated AmB co-loaded with cholesterol caused slower and incomplete hemolysis, especially at a 20:5:1 ratio.

Conclusions

The aggregation state of AmB in PEG-phospholipid micelles was sterol dependant. AmB/cholesterol co-loaded PEG-phospholipid micelles caused low in vitro hemolysis due to deaggregation of AmB and micellar stability, presumably owing to cholesterol/phospholipid versus cholesterol/AmB interactions in the interior core region.     

Ursolic acid from Oldenlandia diffusa induces apoptosis via activation of caspases and phosphorylation of glycogen synthase kinase 3 beta in SK-OV-3 ovarian cancer cells

Although ursolic acid isolated from Oldenlandia diffusa (Rubiaceae) was known to have anticancer activities in prostate, breast and liver cancers, the underlying mechanism of ursolic acid in ovarian cancer cells was not investigated so far. In the present study, the apoptotic mechanism of ursolic acid was elucidated in SK-OV-3 ovarian cancer cells by 2,3-bis(2-methoxy-4-nitro-5-sulphophenyl)-2H-tetrazolium-5-carboxanilide (XTT) assay, cell cycle analysis and Western blotting. Ursolic acid exerted cytotoxicity against SK-OV-3 and A2780 ovarian cancer cells with IC?? of ca. 50 and 65?μM, respectively. Apoptotic bodies were observed in ursolic acid treated SK-OV-3 cells. Also, ursolic acid significantly increased ethidium homodimer stained cells and sub-G1 apoptotic portion in SK-OV-3 cells. Consistently, Western blotting revealed that ursolic acid effectively cleaved poly(ADP-ribose) polymerase (PARP), caspase-9 and -3, suppressed the expression of survival genes such as c-Myc, Bcl-x(L) and astrocyte elevated gene (AEG)-1, and upregulated phosphorylation of extracellular signal-regulated kinase (ERK) in SK-OV-3 cells. Interestingly, ursolic acid suppressed β-catenin degradation as well as enhanced phosphorylation of glycogen synthase kinase 3 beta (GSK 3β). Furthermore, GSK 3β inhibitor SB216763 blocked the cleavages of caspase-3 and PARP induced by ursolic acid and proteosomal inhibitor MG132 disturbed down-regulation of β-catenin, activation of caspase-3 and decreased mitochondrial membrane potential (MMP) induced by ursolic acid in SK-OV-3 cells. Overall, our findings suggest that ursolic acid induces apoptosis via activation of caspase and phosphorylation of GSK 3β in SK-OV-3 cancer cells as a potent anti-cancer agent for ovarian cancer therapy.     

Acupuncture for Bell’s palsy: A systematic review and meta-analysis

<正>Objective:To assess the clinical evidence for and against acupuncture as a treatment for Bell's palsy.Methods:We conducted a literature search of 15 databases from their inception to December 2010 without language restrictions.We included all randomized clinical trials(RCTs) regardless of their controls. Methodological quality was evaluated using the Cochrane risk of bias assessment tool.Results:Of the 3474 articles,only eight RCTs met our inclusion criteria.Four RCTs tested the effects of acupuncture against drug therapy on disease response rate.The meta-analysis of these data showed significant improvements in the acupuncture group[n=463,risk ratio(RR)=1.07,95%CI:1.02 to 1.13;P=0.006,l~2=0%].Six RCTs tested the effects of acupuncture plus drug therapy versus drug therapy alone.The meta-analysis of this set of RCTs also showed the favorable effects of acupuncture on disease response rate(n=512,RR=1.11,95%CI:1.05 to 1.17; P=0.001,l~2=13%).Conclusions:The evidence supporting the effectiveness of acupuncture for treating Bell's palsy is limited.The number and quality of trials are too low to form firm conclusions.Further rigorous RCTs are warranted but need to overcome the many limitations of the current evidence.     

Multi-core vesicle nanoparticles based on vesicle fusion for delivery of chemotherapic drugs

The Pluronic nanoparticles (NPs) composed of Pluronic (F-68) and liquid polyethylene glycol (PEG, molecular wt: 400) containing docetaxel (DTX) were stabilized with the vesicle fusion. When DTX-loaded Pluronic NPs were mixed with vesicles in the aqueous medium, DTX-loaded Pluronic NPs were incorporated into vesicles to form multi-core vesicle NPs. The morphology and size distribution of multi-core vesicle NPs were observed using FE-SEM, cryo-TEM and a particle size analyzer. To apply multi-core vesicle NPs as a delivery system for DTX, a model anti-cancer drug, the release pattern of DTX was observed and the tumor growth was monitored by injecting the DTX-loaded multi-core vesicle NPs into the tail veins of tumor-bearing mice. We also evaluated the time-dependent excretion profile, in?vivo biodistribution, circulation time, and tumor targeting capability of multi-core vesicle NPs using a non-invasive live animal imaging technology.     

Tumor-homing photosensitizer-conjugated glycol chitosan nanoparticles for synchronous photodynamic imaging and therapy based on cellular on/off system

Herein, we developed the photosensitizer, protoporphyrin IX (PpIX), conjugated glycol chitosan (GC) nanoparticles (PpIX-GC-NPs) as tumor-homing drug carriers with cellular on/off system for photodynamic imaging and therapy, simultaneously. In order to prepare PpIX-GC-NPs, hydrophobic PpIXs were chemically conjugated to GC polymer and the amphiphilic PpIX-GC conjugates formed a stable nanoparticle structure in aqueous condition, wherein conjugated PpIX molecules formed hydrophobic inner-cores and they were covered by the hydrophilic GC polymer shell. Based on the nanoparticle structure, PpIX-GC-NPs showed the self-quenching effect that is 'off' state with no fluorescence signal and phototoxicity with light exposure. It is due to the compact crystallized PpIX molecules in the nanoparticles as confirmed by dynamic light scattering and X-ray diffraction methods. However, after cellular uptake, compact nanoparticle structure gradually decreased to generate strong fluorescence signal and singlet oxygen generation when irradiated. Importantly, PpIX-GC-NPs-treated mice presented prolonged blood circulation, enhanced tumor targeting ability, and improved in vivo therapeutic efficiency in tumor-bearing mice, compared to that of free PpIX-treated mice. These results proved that this tumor-homing cellular 'on/off' nanoparticle system of PpIX-GC-NPs has a great potential for synchronous photodynamic imaging and therapy in cancer treatment.     

The rapid production of high-titer porcine endogenous retrovirus(PERV)-B env pseudotype and construction of an EGFP-expressing replication competent PERV-A vector

Porcine endogenous retroviruses (PERVs) present a unique concern associated with xenotransplantation because they have been shown to infect certain human cells in vitro and it is also difficult to generate herds of pigs free of PERVs. A simple system for the production of high-titer MoMLV-PERV pseudotypes is reported; an EGFP-expressing replication-competent molecular clone that allows direct measurement of titer was also constructed. To improve the MLV-based retroviral vector system, a 2.1-kb PERV-B env product was amplified from PK-15 genomic DNA and cloned into the pCL-Eco retroviral vector. The titer of lacZ (PERV-B) from the 293 cells was about 1.0 × 10⁴ CFU/ml. In contrast, the titer of lacZ (PERV-B) from a conventional murine retroviral vector (split genome) was found to be 1.2 × 10² CFU/ml when the PERV-B env expression vector was transfected into TELCeB6 cells, which harbor MFGnlslacZ and the gag-pol-expressing vector. In addition, an infectious PERV-A clone containing enhanced GFP (EGFP) by using a PCR-based method was developed. This EGFP-expressing PERV-A-IRES-EGFP molecular clone was found to be stable genetically on transfection in 293 cells.     

Molecular identification and expression analysis of a novel tumor necrosis factor receptor from the black rockfish, Sebastes schlegelii

Members of the tumor necrosis factor (TNF) and TNF receptor (TNFR) superfamilies play crucial roles in both innate and adaptive immunity. In the present study, we isolated the full-length cDNA for black rockfish (Sebastes schlegelii) TNFR (BrTNFR). This cDNA is 2405 bp in length and contains a 939-bp open reading frame, a 27-bp 5′ untranslated region, and a 1439-bp 3′ untranslated region including a polyadenylation signal (AATAAA) and polyadenylation site. The 313-amino-acid predicted BrTNFR sequence is homologous to other TNFR sequences, contains four cysteine-rich domains and a death-effector domain (DED), and lacks a transmembrane region. Expression of BrTNFR mRNA was detected in eight different tissues from healthy black rockfish and was highest in peripheral blood lymphocytes and gills. In analyses of mitogen-stimulated BrTNFR expression in peripheral blood lymphocytes, expression of BrTNFR mRNA was observed between 1 and 24 h after stimulation with lipopolysaccharide, concanavalin A/phorbol myristate acetate, or poly I:C. Although the data suggest that BrTNFR represents an ancestral member of the TNFR superfamily, the orthology of TNFR in teleost fish is difficult to establish because few TNFRs have been identified in these species.     

Extracellular proteinase formation in carbon starving Aspergillus nidulans cultures--physiological function and regulation

Extracellular proteinase formation in carbon depleted cultures of the model filamentous fungus Aspergillus nidulans was studied to elucidate its regulation and possible physiological function. As demonstrated by gene deletion, culture optimization, microbial physiological and enzymological experiments, the PrtA and PepJ proteinases of A. nidulans did not appear to play a decisive role in the autolytic decomposition of fungal cells under the conditions we tested. However, carbon starvation induced formation of the proteinases observable in autolytic cultures. Similar to other degradative enzymes, production of proteinase was regulated by FluG-BrlA asexual developmental signaling and modulated by PacC-dependent pH-responsive signaling. Under the same carbon starved culture conditions, alterations of CreA, MeaB or heterotrimeric G protein mediated signaling pathways caused less significant changes in the formation of extracellular proteinases. Taken together, these results indicate that while the accumulation of PrtA and PepJ is tightly coupled to the initiation of autolysis, they are not essential for autolytic cell wall degradation in A. nidulans. Thus, as Aspergillus genomes contain a large group of genes encoding proteinases with versatile physiological functions, selective control of proteinase production in fungal cells is needed for the improved industrial use of fungi.