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- Antiproliferative and immunostimulatory activity of a protein from Pleurotus eryngii. [JOURNAL ARTICLE]
- J Sci Food Agric 2014 Mar 20.
Currently, the use of mushrooms as functional foods, nutraceuticals or phytopharmaceuticals source has risen. In contrast, the possible cellular cytotoxicity and immunostimulatory activity of Pleurotus eryngii (DC. ex Fr.) Quel protein (PEQP) is unknown. Here we report extraction, anti-tumorigenic and immunostimulatory activity of PEQP in vitro.PEQP was extracted from the dried fruiting bodies of P. eryngii, purified and characterised. Its in vitro antiproliferative activity was then evaluated in human non-small cell lung cancer A549 (NSCLC), stomach adenocarcinoma BGC-823, hepatocellular carcinoma HepG2 and gastric carcinoma HGC-27 cell lines using conventional cancer drugs (paclitaxel, doxorubicin and mitomycin C) as positive controls. The protein fractions (PEQP 1, 2, 3 and 4) obtained inhibited tumour cell proliferation dose-dependently with fraction PEQP 2 having significant (P < 0.05) toxicity in all tumour cells. PEQP had no significant toxicity on normal liver Chang cells but their proliferation was significantly inhibited by mitomycin C. Moreover, PEQP stimulated the proliferation, lysosomal enzyme activity, pinocytosis, nitric oxide and hydrogen peroxide production of RAW 264.7 cell lines dose-dependently.Based on these results, P. eryngii protein has a potential application in functional foods as a natural anti-tumour agent with immunostimulatory activity. © 2014 Society of Chemical Industry.
- Immunopotentiation of Pleurotus eryngii (DC. ex Fr.) Quel. [JOURNAL ARTICLE]
- J Ethnopharmacol 2014 Mar 18.
Pleurotus eryngii (DC. ex Fr.) Quel has been collected from the wild, cultivated and used in traditional medicines to treat various disorders and diseases since antiquity. In traditional Chinese medicine, the powdered fruiting bodies of Pleurotus eryngii were used for immunostimulation, skin-care, wound-healing, cancer and lumbago treatment. In the current study, we investigated the antiproliferative activity of Pleurotus eryngii powder on A549, BGC-823, HepG2 and HGC-27 cancer cells and its immunomodulating activity on macrophage, RAW 264.7 cells based on its active compound.A novel bioactive protein (PEP) was extracted from Pleurotus eryngii fruiting bodies powder and purified on DEAE-52, CM-52 and Superdex 75 column chromatographies using an ÄKTA purifier. Its cytotoxicity on A549, BGC-823, HepG2, HGC-27 and RAW 267.4 cell lines was then evaluated using MTT, alamar blue (AB), trypan blue (TB), neutral red (NR), lactate dehydrogenase (LDH), Annexin V FITC/PI and morphological change assays. Moreover, lysosomal enzyme activity, pinocytosis, nitric oxide (NO) and hydrogen peroxide (H2O2) production assays were used to examine immunomostimulatory activity of PEP on RAW 267.4 cells.Based on high performance gel permeation chromatography (HPGPC), Fourier transform infrared (FT-IR) and nuclear magnetic resonance (NMR) analyses, the isolated protein (PEP) had a molecular weight of 63kDa, a secondary (α-helical) structure and was mainly composed of arginine, serine and glycine. PEP significantly (P<0.05) inhibited A549, BGC-823, HepG2 and HGC-27 tumor cells proliferation dose-dependently with an IC50 range of 36.5±0.84 to 229.0±1.24µg/ml. Contrarily, PEP stimulated the proliferation of macrophages.Pleurotus eryngii fruiting bodies powder has a potential application as a natural antitumor agent with immunomodulatory activity, proposedly, by targeting the lysosomes of cancerous cells and stimulating macrophage-mediated immune responses.
- Size-dependent internalisation of folate-decorated nanoparticles via the pathways of clathrin and caveolae-mediated endocytosis in ARPE-19 cells. [Journal Article]
- J Pharm Pharmacol 2014 Apr; 66(4):564-73.
We aim to quantify the effect of size and degree of folate loading of folate-decorated polymeric nanoparticles (NPs) on the kinetics of cellular uptake and the selection of endocytic pathways in retinal pigment epithelium (RPE) cells.In this study, methoxy-poly(ethylene glycol)-b-polycaprolactone (mPEG-b-PCL) and folate-functionalized PEG-b-PCL were synthesized for assembling into nanoparticles with sizes ranging from 50 nm to 250 nm. These nanoparticles were internalized into ARPE-19 (human RPE cell line) via receptor-mediated endocytosis. A two-step endocytosis process mathematical model was adopted to quantify binding affinity and uptake kinetics of nanoparticles in RPE cells in uptake and inhibition studies.Nanoparticles with 100% folate loading have highest binding affinity and uptake rate in RPE cells. Maximum uptake rate (Vmax) of nanoparticles increased as the size of particles decreased from 250 nm to 50 nm. Endocytic pathway study was studied by using chlorpromazine and methyl-β-cyclodextran (MβCD), which are clathrin- and caveolae-mediated endocytosis inhibitors, respectively. Both chlorpromazine and MβCD inhibited the uptake of folate-decorated nanoparticles. Inhibition constant (Ki) and maximum uptake rate (Vmax) revealed that 50 nm and 120 nm folate-decorated nanoparticles were found to be internalized via both clathrin- and caveolae-mediated endocytosis. The 250 nm folate-decorated nanoparticles, however, were only internalized via caveolae-mediated pathway.Increased uptake rate of folate-decorated NPs into RPE cells is observed with increasing degree of folate modification. These NPs utilize both clathrin- and caveolae-mediated receptor-mediated endocytosis pathways to enter RPE cells upon size variation. The 50 nm NPs are internalized the fastest, with clathrin-mediated endocytosis as the preferred route. Uptake of 250 nm particles is the slowest and is dominated by caveolae-mediated endocytosis.
- Lysosomal enzymes may cross the blood-brain-barrier by pinocytosis: implications for enzyme replacement therapy. [Journal Article, Research Support, Non-U.S. Gov't]
- Med Hypotheses 2014 Apr; 82(4):478-80.
Here we hypothesized that the water-soluble lysosomal enzymes may cross the blood-brain-barrier and reach the brain using the mechanism of unspecific fluid-phase endocytosis. We also highlight studies that show that, at higher serum concentrations, a fraction of these proteins can reach the brain after intravenous injection, and we suggest some experiments to study this hypothesis. Finally we discuss the implications of this for treatments such as enzyme replacement of lysosomal storage disorders.
- PIP₃-dependent macropinocytosis is incompatible with chemotaxis. [Journal Article, Research Support, Non-U.S. Gov't]
- J Cell Biol 2014 Feb 17; 204(4):497-505.
In eukaryotic chemotaxis, the mechanisms connecting external signals to the motile apparatus remain unclear. The role of the lipid phosphatidylinositol 3,4,5-trisphosphate (PIP₃) has been particularly controversial. PIP₃ has many cellular roles, notably in growth control and macropinocytosis as well as cell motility. Here we show that PIP₃ is not only unnecessary for Dictyostelium discoideum to migrate toward folate, but actively inhibits chemotaxis. We find that macropinosomes, but not pseudopods, in growing cells are dependent on PIP₃. PIP₃ patches in these cells show no directional bias, and overall only PIP₃-free pseudopods orient up-gradient. The pseudopod driver suppressor of cAR mutations (SCAR)/WASP and verprolin homologue (WAVE) is not recruited to the center of PIP₃ patches, just the edges, where it causes macropinosome formation. Wild-type cells, unlike the widely used axenic mutants, show little macropinocytosis and few large PIP₃ patches, but migrate more efficiently toward folate. Tellingly, folate chemotaxis in axenic cells is rescued by knocking out phosphatidylinositide 3-kinases (PI 3-kinases). Thus PIP₃ promotes macropinocytosis and interferes with pseudopod orientation during chemotaxis of growing cells.
- Role of caveolin-1 in the biology of the blood-brain barrier. [Journal Article]
- Rev Neurosci 2014; 25(2):247-54.
Abstract Caveolin-1 is the principal marker of caveolae in endothelial cells. It plays an important role in physiological and pathological conditions of the blood-brain barrier and serves as a mediator in drug delivery through the blood-brain barrier. Caveolin-1 is related to the diminished expression of tight junction-associated proteins and metabolic pinocytosis vesicles when the blood-brain barrier is destroyed by outside invaders or malignant stimulus. The permeability of the blood-brain barrier, regulated by types of drugs or physical irradiation, is connected with drug transportation with the participation of caveolin-1. Caveolin-1, which serves as a platform or medium for signal transduction, cooperates with several signal molecules by forming a complex. Silencing of caveolin-1 and disruption of caveolae can attenuate or remove pathological damage and even engender the opposite effects in the blood-brain barrier. This review considers the role of caveolin-1 in the blood-brain barrier that may have profound implications for central nervous system disease and drug delivery through the blood-brain barrier.
- The effect of the neonatal Fc receptor on human IgG biodistribution in mice. [Journal Article]
- MAbs 2014 Mar-Apr; 6(2):502-8.
The neonatal Fc receptor (FcRn) plays a pivotal role in IgG homeostasis, i.e., it salvages IgG antibodies from lysosomal degradation following fluid-phase pinocytosis, thus preventing rapid systemic elimination of IgG. Recombinant therapeutic antibodies are typically composed of human or humanized sequences, and their biodistribution, or tissue distribution, is often studied in murine models, although, the effect of FcRn on tissue distribution of human IgG in rodents has not been investigated. In this report, an (125)I-labeled human IgG1 antibody was studied in both wild type C57BL/6 (WT) and FcRn knockout (KO) mice. Total radioactivity in both plasma and tissues (0-96hr post-dose) was measured by gamma-counting. Plasma exposure of human IgG1 were significantly lower in FcRn KO mice, which is consistent with the primary function of FcRn. Differences in biodistribution of human IgG to selected tissues were also observed. Among the tissue examined, the fat, skin and muscle showed a decrease in tissue-to-blood (T/B) exposure ratio of human IgG1 in FcRn KO mice comparing to the WT mice, while the liver, spleen, kidney, and lung showed an increase in the T/B exposure ratio in FcRn KO mice. A time-dependent change in the T/B ratios of human IgG1 was also observed for many tissues in FcRn KO mice. These results suggest that, in addition to its role in IgG elimination, FcRn may also play a role in antibody biodistribution.
- Cell-penetrating peptide-conjugated lipid nanoparticles for siRNA delivery. [Journal Article]
- Biochem Biophys Res Commun 2014 Feb 21; 444(4):599-604.
Lipid nanoparticles (LNP) modified with cell-penetrating peptides (CPP) were prepared for the delivery of small interfering RNA (siRNA) into cells. Lipid derivatives of CPP derived from protamine were newly synthesized and used to prepare CPP-decorated LNP (CPP-LNP). Encapsulation of siRNA into CPP-LNP improved the stability of the siRNA in serum. Fluorescence-labeled siRNA formulated in CPP-LNP was efficiently internalized into B16F10 murine melanoma cells in a time-dependent manner, although that in LNP without CPP was hardly internalized into these cells. In cells transfected with siRNA in CPP-LNP, most of the siRNA was distributed in the cytoplasm of these cells and did not localize in the lysosomes. Analysis of the endocytotic pathway indicated that CPP-LNP were mainly internalized via macropinocytosis and heparan sulfate-mediated endocytosis. CPP-LNP encapsulating siRNA effectively induced RNA interference-mediated silencing of reporter genes in B16F10 cells expressing luciferase and in HT1080 human fibrosarcoma cells expressing enhanced green fluorescent protein. These data suggest that modification of LNP with the protamine-derived CPP was effective to facilitate internalization of siRNA in the cytoplasm and thereby to enhance gene silencing.
- Low dose naltrexone (LDN) enhances maturation of bone marrow dendritic cells (BMDCs). [Journal Article, Research Support, Non-U.S. Gov't]
- Int Immunopharmacol 2013 Dec; 17(4):1084-9.
It has been demonstrated previously that immune cell activation and proliferation were sensitive to the effects of naltrexone, a non-peptidic δ-opioid receptor selective antagonist and opioid receptors on BMDCs have been detected . However, there is little prior data published on naltrexone and DCs. Therefore, we hypothesized that LDN could exert modulating effect on BMDCs. In present study, we studied influence of LDN on both phenotypic and functional maturation of BMDCs. Changes of BMDC post-treatment with LDN were evaluated using conventional light microscope and transmission electron microscopy (TEM); flow cytometry(FCM); cytochemistry; acid phosphatase activity(ACP) test; FITC-dextran bio-assay; mixed lymphocytes and enzyme-linked immunosorbent assay (ELISA). We have found that LDN enhances maturation of BMDCs as evidenced by 1) up-regulating the expression of MHC II, CD40, CD83, CD80 and CD86 molecules on BMDCs; 2) down-regulating the rates of pinocytosis and phagocytosis accompanied by the results of decreased ACP, and FITC-dextran bio-assay; 3) mounting potential of BMDCs to drive T cell; and 4) inducing secretion of higher levels of IL-12 and TNF-α. It is therefore concluded that LDN can efficiently promote the maturation of BMDCs via precise modulation inside and outside BMDCs. Our study has provided meaningful mode of action on the role of LDN in immunoregulation, and rationale on future application of LDN for enhancing host immunity in cancer therapy and potent use in the design of DC-based vaccines for a number of diseases.
- Gd loading by hypotonic swelling: an efficient and safe route for cellular labeling. [Journal Article, Research Support, Non-U.S. Gov't]
- Contrast Media Mol Imaging 2013 Nov-Dec; 8(6):475-86.
Cells incubated in hypo-osmotic media swell and their membranes become leaky. The flow of water that enters the cells results in the net transport of molecules present in the incubation medium directly into the cell cytoplasm. This phenomenon has been exploited to label cells with MRI Gd-containing contrast agents. It has been found that, in the presence of 100 mM Gd-HPDO3A in an incubation medium characterized by an overall osmolarity of 160 mOsm l⁻¹, each cell is loaded with amounts of paramagnetic complex ranging from 2 × 10⁹ to 2 × 10¹⁰ depending on the cell type. To obtain more insight into the determinants of cellular labeling by the 'hypo-osmotic shock' methodology, a study on cell viability, proliferation rate and cell morphology was carried out on J774A.1 and K562 cells as representative of cells grown in adhesion and suspended ones, respectively. Moreover a comparison of the efficiency of the proposed method with established cell labeling procedures such as pinocytosis and electroporation was carried out. Finally, the effects of the residual electric charge, the size and some structural features of the metal complex were investigated. In summary, the 'hypotonic shock' methodology appears to be an efficient and promising tool to pursue cellular labeling with paramagnetic complexes. Its implementation is straightforward and one may foresee that it will be largely applied in in vitro cellular labeling of many cell types.