Download the Free Unbound MEDLINE PubMed App to your smartphone or tablet.
Available for iPhone, iPad, iPod touch, and Android.
- [Ultrastructural changes in endothelial cells of blood capillaries of the Walker 256 carcinosarcoma in the application of melatonin]. [English Abstract, Journal Article]
- Vopr Onkol 2014; 60(2):80-3.
In Wistar rats with transplanted Walker 256 carcinosarcoma a use of melatonin as monoagent causes changes in intracellular organization of endothelial cells: reduced volume density of mitochondria, granular cytoplasmic network, micropinocytic vesicles, reduced the number density of free and attached ribosomes, which leads to increased apoptosis of tumor cells of Walker 256 carcinosarcoma.
- Lapatinib-incorporated lipoprotein-like nanoparticles: preparation and a proposed breast cancer-targeting mechanism. [Journal Article]
- Acta Pharmacol Sin 2014 Jun; 35(6):846-52.
Aim:Lapatinib is a dual inhibitor of EGFR and human epidermal growth factor receptor 2 (HER2), and used to treat advanced breast cancer. To overcome its poor water solubility, we constructed lapatinib-incorporated lipoprotein-like nanoparticles (LTNPs), and evaluated the particle characteristics and possible anti-breast cancer mechanisms.
Methods:LTNPs (lapatinib bound to albumin as a core, and egg yolk lecithin forming a lipid corona) were prepared. The particle characteristics were investigated using transmission electron microscopy (TEM) and atomic force microscopy (AFM). The uptake and subcellular localization of LTNPs, as well as the effects of LTNPs on cell cycle were examined in BT-474 human breast cancer cells in vitro. Mice bearing BT-474 subcutaneous xenograft were intravenously injected with coumarin-6 loaded LTNPs (30 mg/kg) to study the targeting mechanisms in vivo.
Results:The LTNPs particles were generally spherical but flexible under TEM and AFM, and approximately 62.1 nm in size with a zeta potential of 22.80 mV. In BT-474 cells, uptake of LTNPs was mediated by endosomes through energy-dependent endocytosis involving clathrin-dependent pinocytosis and macropinocytosis, and they could effectively escape from endosomes to the cytoplasm. Treatment of BT-474 cells with LTNPs (20 μg/mL) induced a significant cell arrest at G0/G1 phase compared with the same concentration of lapatinib suspension. In mice bearing BT-474 xenograft, intravenously injected LTNPs was found to target and accumulate in tumors, and colocalized with HER2 and SPRAC (secreted protein, acidic and rich in cysteine).
Conclusion:LTNPs can be taken up into breast cancer cells through specific pathways in vitro, and targeted to breast cancer xenograft in vivo via enhanced permeability and retention effect and SPARC.
- Minimally oxidized LDL inhibits macrophage selective cholesteryl ester uptake and native LDL-induced foam cell formation. [JOURNAL ARTICLE]
- J Lipid Res 2014 Jun 2.
Scavenger receptor-mediated uptake of oxidized LDL (oxLDL) is thought to be the major mechanism of foam cell generation in atherosclerotic lesions. Recent data has indicated that native LDL is also capable of contributing to foam cell formation via low-affinity, receptor-independent LDL particle pinocytosis and selective cholesteryl ester (CE) uptake. In the current investigation, Cu++-induced LDL oxidation was found to inhibit macrophage selective CE uptake. Impairment of selective CE uptake was significant with LDL oxidized for as little as 30 minutes and correlated with oxidative fragmentation of apolipoprotein B. In contrast, LDL aggregation, LDL CE oxidation and the enhancement of scavenger receptor-mediated LDL particle uptake required at least 3 hours of oxidation. Selective CE uptake did not require expression of the LDL receptor (LDL-R) and was inhibited similarly by LDL oxidation in LDL-R-/- versus WT macrophages. Inhibition of selective uptake was also observed when cells were pre-treated or co-treated with minimally oxidized LDL, indicating a direct inhibitory effect of this oxLDL on macrophages. Consistent with the effect on LDL CE uptake, minimal LDL oxidation almost completely prevented LDL-induced foam cell formation. These data demonstrate a novel inhibitory effect of mildly oxidized LDL that may reduce foam cell formation in atherosclerosis.
- Biochemical and biological properties of cortexillin III, a component of Dictyostelium DGAP1-cortexillin complexes. [JOURNAL ARTICLE]
- Mol Biol Cell 2014 May 7.
Cortexillins I, II and III are members of the α-actinin/spectrin subfamily of Dictyostelium calponin homology proteins. Unlike recombinant cortexillins I and II, which form homodimers as well as heterodimers in vitro, we find that recombinant cortexillin III is an unstable monomer but forms more stable heterodimers when coexpressed in E. coli with cortexillin I or II. Expressed cortexillin III also forms heterodimers with both cortexillin I and II in vivo and the heterodimers complex in vivo with DGAP1, a Dictyostelium GAP protein. Binding of cortexillin III to DGAP1 requires the presence of either cortexillin I or II, i.e. cortexillin III binds to DGAP1 only as a heterodimer, and the heterodimers form in vivo in the absence of DGAP1. Expressed cortexillin III colocalizes with cortexillins I and II in the cortex of vegetative amoebae, the leading edge of motile cells and the cleavage furrow of dividing cells. Co-localization of ctxIII and F-actin may require the heterodimer/DGAP1 complex. Functionally, cortexillin III may be a negative regulator of cell growth, cytokinesis, pinocytosis and phagocytosis as all are enhanced in cortexillin III-null cells.
- Simultaneous pH Measurement in Endocytic and Cytosolic Compartments in Living Cells using Confocal Microscopy. [Journal Article]
- J Vis Exp 2014; (86)
Intracellular pH is tightly regulated and differences in pH between the cytoplasm and organelles have been reported(1). Regulation of cellular pH is crucial for homeostatic control of physiological processes that include: protein, DNA and RNA synthesis, vesicular trafficking, cell growth and cell division. Alterations in cellular pH homeostasis can lead to detrimental functional changes and promote progression of various diseases(2). Various methods are available for measuring intracellular pH but very few of these allow simultaneous measurement of pH in the cytoplasm and in organelles. Here, we describe in detail a rapid and accurate method for the simultaneous measurement of cytoplasmic and organellar pH by using confocal microscopy on living cells(3). This goal is achieved with the use of two pH-sensing ratiometric dyes that possess selective cellular compartment partitioning. For instance, SNARF-1 is compartmentalized inside the cytoplasm whereas HPTS is compartmentalized inside endosomal/lysosomal organelles. Although HPTS is commonly used as a cytoplasmic pH indicator, this dye can specifically label vesicles along the endosomal-lysosomal pathway after being taken up by pinocytosis(3,4). Using these pH-sensing probes, it is possible to simultaneously measure pH within the endocytic and cytoplasmic compartments. The optimal excitation wavelength of HPTS varies depending on the pH while for SNARF-1, it is the optimal emission wavelength that varies. Following loading with SNARF-1 and HPTS, cells are cultured in different pH-calibrated solutions to construct a pH standard curve for each probe. Cell imaging by confocal microscopy allows elimination of artifacts and background noise. Because of the spectral properties of HPTS, this probe is better suited for measurement of the mildly acidic endosomal compartment or to demonstrate alkalinization of the endosomal/lysosomal organelles. This method simplifies data analysis, improves accuracy of pH measurements and can be used to address fundamental questions related to pH modulation during cell responses to external challenges.
- Synthesis of dye conjugates to visualize the cancer cells using fluorescence microscopy. [Journal Article]
- Appl Opt 2014 Apr 10; 53(11):2345-51.
The clinical diagnosis of most cancers is based on evaluation of histology microscopic slides to view the size and shape of cellular nuclei and morphological structure of tissue. To achieve this goal for in vivo and in-deep tissues, near infrared dyes-bovine serum albumin and immunoglobulin G conjugates were synthesized. The spectral study shows that the absorption and fluorescence of the dye conjugates are in the "tissue optical window" spectral ranges between 650 and 900 nm. The internalization and pinocytosis of the synthesized compounds were investigated at cell level using fluorescence microscopy to obtain the optimal concentration and staining time.
- Identification and quantification of host proteins in the vesicular fluid of porcine Taenia solium cysticerci. [JOURNAL ARTICLE]
- Exp Parasitol 2014 Apr 24.
The host-parasite relationship in cestode infections is complex. One feature of this bidirectional molecular communication is the uptake of host proteins by the parasite. Here we describe the presence of several host proteins in the vesicular fluid of Taenia solium cysticerci dissected from the central nervous system and the skeletal muscle of naturally infected pigs. Using two-dimensional electrophoresis we compared the protein patterns of vesicular fluids of cysticerci vs. the sera of cysticercotic pigs. We found that the vesicular fluids of both groups of cysts showed 17 protein spots matching with the pig's sera spots. After mass spectrometry sequencing of these spots, five host proteins were identified: hemoglobin, albumin, serpin A3-8, haptoglobin, rho GTPase-activating protein 36-like. Three of the 17 spots corresponded to host protein fragments: hemoglobin, albumin and serpin A3-8. IgG heavy and light chains were also identified by Western blot using a specific antibody. Quantitative estimations indicated that the host proteins represented 11-13% of the protein content in the vesicular fluids. We also calculated the relative abundance of these host proteins in the vesicular fluids; all were represented in similar relative abundances as in host sera. This suggests that uptake of host proteins by cysticerci proceeds through an unspecific mechanism such as non-specific fluid pinocytosis.
- Human Metapneumovirus SH and G Glycoproteins Inhibit Macropinocytosis-Mediated Entry into Human Dendritic Cells and Reduce CD4+ T Cell Activation. [JOURNAL ARTICLE]
- J Virol 2014 Mar 26.
Human metapneumovirus (HMPV) is a major etiologic agent of respiratory disease worldwide. HMPV reinfections are common in healthy adults and children, suggesting that the protective immune response to HMPV is incomplete and short-lived. We used gene-deletion viruses to evaluate the role of the attachment G and small hydrophobic SH glycoproteins on virus uptake by primary human monocyte-derived dendritic cells (MDDC) in vitro, and on subsequent MDDC maturation and activation of autologous T cells. HMPV with deletion of G and SH (ΔSHG) exhibited increased infectivity but had little effect on MDDC maturation. However, MDDC stimulated with ΔSHG induced increased proliferation of autologous Th1-polarized CD4+ T cells. This effect was independent of virus replication. Increased T cell proliferation was strictly dependent on contact between virus-stimulated MDDC and CD4+ T cells. Confocal microscopy revealed that deletion of SH and G was associated with an increased number of immunological synapses between memory CD4+ T cells and virus-stimulated MDDC. Uptake of HMPV by MDDC was found to be primarily by macropinocytosis. Uptake of wild-type (WT) virus was reduced compared to ΔSHG, indicative of inhibition by the SH and G glycoproteins. In addition, DC-SIGN-mediated endocytosis provided a minor alternative pathway that depended on SH and/or G and thus operated only for WT. Altogether our results show that SH and G glycoproteins reduce the ability of HMPV to be internalized by MDDC, resulting in a reduced ability of the HMPV-stimulated MDDC to activate CD4+ T cells. This study describes a previously unknown mechanism of virus immune evasion.Human metapneumovirus (HMPV) is a major etiologic agent of respiratory disease worldwide. HMPV reinfections are common in healthy adults and children, suggesting that the protective immune response to HMPV is incomplete and short-lived. We found that HMPV attachment G and small hydrophobic SH glycoproteins reduce the ability of HMPV to be internalized by macropinocytosis into human dendritic cells (DC). This results in a reduced ability of the HMPV-stimulated DC to activate Th1-polarized CD4+ T cells. These results contribute to a better understanding of the nature of incomplete protection against this important human respiratory virus, provide new information on the entry of HMPV into human cells, and describe a new mechanism of virus immune evasion.
- Vulnerability of glioblastoma cells to catastrophic vacuolization and death induced by a small molecule. [Journal Article]
- Cell 2014 Apr 10; 157(2):313-28.
Glioblastoma multiforme (GBM) is the most aggressive form of brain cancer with marginal life expectancy. Based on the assumption that GBM cells gain functions not necessarily involved in the cancerous process, patient-derived glioblastoma cells (GCs) were screened to identify cellular processes amenable for development of targeted treatments. The quinine-derivative NSC13316 reliably and selectively compromised viability. Synthetic chemical expansion reveals delicate structure-activity relationship and analogs with increased potency, termed Vacquinols. Vacquinols stimulate death by membrane ruffling, cell rounding, massive macropinocytic vacuole accumulation, ATP depletion, and cytoplasmic membrane rupture of GCs. The MAP kinase MKK4, identified by a shRNA screen, represents a critical signaling node. Vacquinol-1 displays excellent in vivo pharmacokinetics and brain exposure, attenuates disease progression, and prolongs survival in a GBM animal model. These results identify a vulnerability to massive vacuolization that can be targeted by small molecules and point to the possible exploitation of this process in the design of anticancer therapies.
- 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.