Several fixation and permeabilization techniques that enable the flow cytometric analysis of the cell contents have been introduced in recent years. These methods allow sensitive detection of intracellular antigens that facilitates the diagnosis of certain diseases. We have undertaken in this study to evaluate a simple method of fixation and permeabilization using 2% paraformaldehyde and Tween 20. Intracellular antigens in three different leukaemia cases were analysed. We found that the method was reliable and easy. Intracellular kappa light chains were found in abundance in a case of plasma cell leukaemia. CD3 and CD22 were found in greater amount intracellularly than on the surface in pre-T-ALL and pre-pre B-ALL respectively.
The role of membrane permeabilisation and disruption in the mechanism of action of some polymyxin analogues against Gram-negative organisms is contentious. The effects of polymyxin B (PMB) and its analogue polymyxin B nonapeptide (PMBN) on Escherichia coli envelopes should correlate, but previous work by other workers suggests that PMBN has a different mode of action. This study has reassessed the biochemical techniques used previously and has shown that, in contrast to previous studies, PMBN (a well-characterised antibacterial synergist) readily releases periplasmic proteins and lipopolysaccharide from treated E. coli at subinhibitory concentrations in normal physiological buffer conditions. We conclude that, when tested with appropriate methodology, PMBN closely correlates with the early effects of PMB on the cell envelope of E. coli and this study shows that it is now consistent with the accepted interactions of membrane-active agents against Gram-negative cells.
The aim of the present study is to formulate and investigate the potential of nanoemulsion formulation for topical delivery of ketoprofen. In this study, Palm Oil Esters (POEs) a newly introduced oil by Universiti Putra Malaysia researchers was chosen for the oil phase of the nanoemulsion, because the oil was reported to be a good vehicle for pharmaceutical use. Oil-in-water nanoemulsion was prepared by spontaneous emulsification method. The droplets size was studied by laser scattering spectroscopy (Nanophox) and Transmission Electron Microscopy (TEM). Franz diffusion cells were used, to determine the drug release and drug transferred through methyl acetate cellulose membrane (artificial membrane). The results of droplets size analysis shows the droplets are in the range of nanoemulsion which is below than 500 nm. The in vitro release profile shows a sufficient percentage of drugs released through the methyl acetate cellulose membrane. This initial study showed that the nanoemulsion formulated using POEs has great potential for topical delivery of ketoprofen.
The objective of the present study was to evaluate the effects of ultrasound on the growth of probiotics and bioconversion of isoflavones in prebiotic-soymilk. Previous studies have shown that ultrasound elevated microbial enzymatic activity and growth by altering cellular membranes. The growth of probiotics was significantly decreased (P < 0.05) immediately after ultrasound treatment, attributed to membrane permeabilization, cell lysis, and membrane lipid peroxidation upon ultrasound treatment. The ultrasound treatment also caused alteration at the acyl chain, polar head, and interface region of the probiotic membrane phospholipid bilayers. The cells treated with ultrasound showed recovery from injury with subsequent increase in growth upon fermentation in soymilk (P < 0.05). Ultrasound treatment at 100 W for 2 and 3 min also enhanced (P < 0.05) the intracellular and extracellular β-glucosidase activity of probiotics, leading to increased (P < 0.05) bioconversion of glucosides to aglycones in the prebiotic-soymilk. Our present study illustrated that ultrasound treatment could produce bioactive synbiotic-soymilk with increased concentrations of bioactive aglycones.
The objective of the present study was to evaluate the effect of electroporation on the membrane properties of lactobacilli and their ability to remove cholesterol in vitro. The growth of lactobacilli cells treated at 7.5 kV/cm for 4 ms was increased by 0.89 to 1.96 log(10) cfu/mL upon fermentation at 37 °C for 20 h, the increase being attributed to the reversible and transient formation of pores and defragmentation of clumped cells. In addition, an increase of cholesterol assimilation as high as 127.2% was observed for most cells electroporated at a field strength of 7.5 kV/cm for 3.5 ms compared with a lower field strength of 2.5 kV/cm. Electroporation also increased the incorporation of cholesterol into the cellular membrane, as shown by an increased cholesterol:phospholipids ratio (50.0-59.6%) upon treatment at 7.5 kV/cm compared with treatment at 2.5 kV/cm. Saturation of cholesterol was observed in different regions of the membrane bilayer such as upper phospholipids, apolar tail, and polar heads, as indicated by fluorescence anisotropy using 3 fluorescent probes. Electroporation could be a useful technique to increase the ability of lactobacilli to remove cholesterol for possible use as cholesterol-lowering adjuncts in the future.
The plant Artocarpus obtusus is a tropical plant that belongs to the family Moraceae. In the present study a xanthone compound Pyranocycloartobiloxanthone A (PA) was isolated from this plant and the apoptosis mechanism was investigated. PA induced cytotoxicity was observed using MTT assay. High content screening (HCS) was used to observe the nuclear condensation, cell permeability, mitochondrial membrane potential (MMP) and cytochrome c release. Reactive oxygen species formation was investigated on treated cells by using fluorescent analysis. Human apoptosis proteome profiler assays were performed to investigate the mechanism of cell death. In addition mRNA levels of Bax and Bcl2 were also checked using RT-PCR. Caspase 3/7, 8 and 9 were measured for their induction while treatment. The involvement of NF-κB was analyzed using HCS assay. The results showed that PA possesses the characteristics of selectively inducing cell death of tumor cells as no inhibition was observed in non-tumorigenic cells even at 30 μg/ml. Treatment of MCF7 cells with PA induced apoptosis with cell death-transducing signals, that regulate the MMP by down-regulation of Bcl2 and up-regulation of Bax, triggering the cytochrome c release from mitochondria to cytosol. The release of cytochrome c triggered the activation of caspases-9, then activates downstream executioner caspase-3/7 and consequently cleaved specific substrates leading to apoptotic changes. This form of apoptosis was found closely associated with the extrinsic pathway caspase (caspase-8) and inhibition of translocation of NF-κB from cytoplasm to nucleus. The results demonstrated that PA induced apoptosis of MCF7 cells through NF-κB and Bcl2/Bax signaling pathways with the involvement of caspases.
This study was set to investigate antiproliferative potential of dentatin (a natural coumarin isolated from Clausena excavata Burm. F) against prostate cancer and to delineate the underlying mechanism of action. Treatment with dentatin dose-dependently inhibited cell growth of PC-3 and LNCaP prostate cancer cell lines, whereas it showed less cytotoxic effects on normal prostate epithelial cell line (RWPE-1). The inhibitory effect of dentatin on prostate cancer cell growth was due to induction of apoptosis as evidenced by Annexin V staining and cell shrinkage. We found that dentatin-mediated accumulation of reactive oxygen species (ROS) and downregulated expression levels of antiapoptotic molecules (Bcl-2, Bcl-xL, and Survivin), leading to disruption of mitochondrial membrane potential (MMP), cell membrane permeability, and release of cytochrome c from the mitochondria into the cytosol. These effects were associated with induction of caspase-9, -3/7 activities, and subsequent DNA fragmentation. In addition, we found that dentatin inhibited TNF-α-induced nuclear translocation of p65, suggesting dentatin as a potential NF-κB inhibitor. Thus, we suggest that dentatin may have therapeutic value in prostate cancer treatment worthy of further development.
The aim of this study was to evaluate the effect of electroporation (2.5-7.5 kV cm⁻¹ for 3.0-4.0 ms) on the growth of lactobacilli and bifidobacteria, membrane properties and bioconversion of isoflavones in mannitol-soymilk.
The compound with R=CH2CH3 in Bi(S2CNR2)3 (1) is highly cytotoxic against a range of human carcinoma, whereas that with R=CH2CH2OH (2) is considerably less so. Both 1 and 2 induce apoptosis in HepG2 cells with some evidence for necrosis induced by 2. Based on DNA fragmentation, caspase activities and human apoptosis PCR-array analysis, both the extrinsic and intrinsic pathways of apoptosis have been shown to occur. While both compounds activate mitochondrial and FAS apoptotic pathways, compound 1 was also found to induce another death receptor-dependent pathway by induction of CD40, CD40L and TNF-R1 (p55). Further, 1 highly expressed DAPK1, a tumour suppressor, with concomitant down-regulation of XIAP and NF-κB. Cell cycle arrest at the S and G2/M phases correlates with the inhibition of the growth of HepG2 cells. The cell invasion rate of 2 is 10-fold higher than that of 1, a finding correlated with the down-regulation of survivin and XIAP expression by 1. Compounds 1 and 2 interact with DNA through different binding motifs with 1 interacting with AT- or TA-specific sites followed by inhibition of restriction enzyme digestion; 2 did not interfere with any of the studied restriction enzymes.
Literature data relevant to the decision to allow a waiver of in vivo bioequivalence (BE) testing for the approval of immediate-release (IR) solid oral dosage forms containing bisoprolol as the sole active pharmaceutical ingredient (API) are reviewed. Bisoprolol is classified as a Class I API according to the current Biopharmaceutics Classification System (BCS). In addition to the BCS class, its therapeutic index, pharmacokinetic properties, data related to the possibility of excipient interactions, and reported BE/bioavailability problems are taken into consideration. Qualitative compositions of IR tablet dosage forms of bisoprolol with a marketing authorization (MA) in ICH (International Conference on Harmonisation) countries are tabulated. It was inferred that these tablets had been demonstrated to be bioequivalent to the innovator product. No reports of failure to meet BE standards have been made in the open literature. On the basis of all these pieces of evidence, a biowaiver can currently be recommended for bisoprolol fumarate IR dosage forms if (1) the test product contains only excipients that are well known, and used in normal amounts, for example, those tabulated for products with MA in ICH countries and (2) both the test and comparator dosage form are very rapidly dissolving, or, rapidly dissolving with similarity of the dissolution profiles demonstrated at pH 1.2, 4.5, and 6.8.
The purification of human adipose-derived stem cells (hADSCs) from human adipose tissue cells (stromal vascular fraction) was investigated using membrane filtration through poly(lactide-co-glycolic acid)/silk screen hybrid membranes. Membrane filtration methods are attractive in regenerative medicine because they reduce the time required to purify hADSCs (i.e., less than 30 min) compared with conventional culture methods, which require 5-12 days. hADSCs expressing the mesenchymal stem cell markers CD44, CD73, and CD90 were concentrated in the permeation solution from the hybrid membranes. Expression of the surface markers CD44, CD73, and CD99 on the cells in the permeation solution from the hybrid membranes, which were obtained using 18 mL of feed solution containing 50 × 10⁴ cells, was statistically significantly higher than that of the primary adipose tissue cells, indicating that the hADSCs can be purified in the permeation solution by the membrane filtration method. Cells expressing the stem cell-associated marker CD34 could be successfully isolated in the permeation solution, whereas CD34⁺ cells could not be purified by the conventional culture method. The hADSCs in the permeation solution demonstrated a superior capacity for osteogenic differentiation based on their alkali phosphatase activity, their osterix gene expression, and the results of mineralization analysis by Alizarin Red S and von Kossa staining compared with the cells from the suspension of human adipose tissue. These results suggest that the hADSCs capable of osteogenic differentiation preferentially permeate through the hybrid membranes.
The aim of this study was to investigate the mode of action of the lavender essential oil (LV) on antimicrobial activity against multi-drug-resistant Escherichia coli J53 R1 when used singly and in combination with piperacillin.
Tanacetum polycephalum (L.) Schultz-Bip (Mokhaleseh) has been traditionally used in the treatment of headaches, migraines, hyperlipidemia and diabetes. The present study aimed to evaluate its anticancer properties and possible mechanism of action using MCF7 as an in vitro model. T. polycephalum leaves were extracted using hexane, chloroform and methanol solvents and the cytotoxicity was evaluated using the MTT assay. Detection of the early apoptotic cells was investigated using acridine orange/propidium iodide staining. An Annexin-V-FITC assay was carried out to observe the phosphatidylserine externalization as a marker for apoptotic cells. High content screening was applied to analyze the cell membrane permeability, nuclear condensation, mitochondrial membrane potential (MMP) and cytochrome c release. Apoptosis was confirmed by using caspase-8, caspase-9 and DNA laddering assays. In addition, Bax/Bcl-2 expressions and cell cycle arrest also have been investigated. MTT assay revealed significant cytotoxicity of T. Polycephalum hexane extract (TPHE) on MCF7 cells with the IC50 value of 6.42±0.35 µg/mL. Significant increase in chromatin condensation was also observed via fluorescence analysis. Treatment of MCF7 cells with TPHE encouraged apoptosis through reduction of MMP by down-regulation of Bcl-2 and up-regulation of Bax, triggering the cytochrome c leakage from mitochondria to the cytosol. The treated MCF7 cells significantly arrested at G1 phase. The chromatographic analysis elicited that the major active compound in this extract is 8β-hydroxy-4β,15-dihydrozaluzanin C. Taken together, the results presented in this study demonstrated that the hexane extract of T. Polycephalum inhibits the proliferation of MCF7 cells, resulting in the cell cycle arrest and apoptosis, which was explained to be through the mitochondrial pathway.
Alteration in the endothelium leading to increased vascular permeability contributes to plasma leakage seen in dengue hemorrhagic fever (DHF) and dengue shock syndrome (DSS). An earlier study showed that senescent endothelial cells (ECs) altered the ECs permeability. Here we investigated the susceptibility of senescing human umbilical vein endothelial cells (HUVECs) to dengue virus infection and determined if dengue virus infection induces HUVECs senescence. Our results suggest that DENV type-2 (DENV-2) foci forming unit (FFU) and extracellular virus RNA copy number were reduced by at least 35% and 85% in infection of the intermediate young and early senescent HUVECs, respectively, in comparison to infection of young HUVECs. No to low infectivity was recovered from infection of late senescent HUVECs. DENV infection also increases the percentage of HUVECs expressing senescence-associated (SA)-β-gal, cells arrested at the G2/M phase or 4N DNA content stage and cells with enlarged morphology, indicative of senescing cells. Alteration of HUVECs morphology was recorded using impedance-based real-time cell analysis system following DENV-2 infection. These results suggest that senescing HUVECs do not support DENV infection and DENV infection induces HUVECs senescence. The finding highlights the possible role of induction of senescence in DENV infection of the endothelial cells.
Tumor necrosis factor-related apoptosis-inducing ligand (TRAIL) is an attractive target for cancer therapy due to its ability to selectively induce apoptosis in cancer cells, without causing significant toxicity in normal tissues. We previously reported that galactoxyloglucan (PST001) possesses significant antitumor and immunomodulatory properties. However, the exact mechanism in mediating this anticancer effect is unknown. This study, for the first time, indicated that PST001 sensitizes non-small cell lung cancer (A549) and nasopharyngeal (KB) cells to TRAIL-mediated apoptosis. In vitro studies suggested that PST001 induced apoptosis primarily via death receptors and predominantly activated caspases belonging to the extrinsic apoptotic cascade. Microarray profiling of PST001 treated A549 and KB cells showed the suppression of survivin (BIRC5) and anti-apoptotic Bcl-2, as well as increased cytochrome C. TaqMan low density array analysis of A549 cells also confirmed that the induction of apoptosis by the polysaccharide occurred through the TRAIL-DR4/DR5 pathways. This was finally confirmed by in silico analysis, which revealed that PST001 binds to TRAIL-DR4/DR5 complexes more strongly than TNF and Fas ligand-receptor complexes. In summary, our results suggest the potential of PST001 to be developed as an anticancer agent that not only preserves innate biological activity of TRAIL, but also sensitizes cancer cells to TRAIL-mediated apoptosis.
Interferon-gamma (IFN-γ) is known to potentiate the progression of inflammatory diseases, such as inflammatory bowel disease and atherosclerosis. IFN-γ has been found to disrupt the barrier integrity of epithelial and endothelial cell both in vivo and in vitro. However, the mechanisms of IFN-γ underlying increased endothelial cell permeability have not been extensively elucidated. We reported that IFN-γ exhibits a biphasic nature in increasing endothelial permeability. The changes observed in the first phase (4-8 h) involve cell retraction and rounding in addition to condensed peripheral F-actin without a significant change in the F-/G-actin ratio. However, cell elongation, stress fiber formation, and an increased F-/G-actin ratio were noticed in the second phase (16-24 h). Consistent with our finding from the permeability assay, IFN-γ induced the formation of intercellular gaps in both phases. A delayed phase of increased permeability was observed at 12 h, which paralleled the onset of cell elongation, stress fiber formation, and increased F-/G-actin ratio. In addition, IFN-γ stimulated p38 mitogen-activated protein (MAP) kinase phosphorylation over a 24 h period. Inhibition of p38 MAP kinase by SB203580 prevented increases in paracellular permeability, actin rearrangement, and increases in the F-/G-actin ratio caused by IFN-γ. Our results suggest that p38 MAP kinase is activated in response to IFN-γ and causes actin rearrangement and altered cell morphology, which in turn mediates endothelial cell hyperpermeability. The F-/G-actin ratio might be involved in the regulation of actin distribution and cell morphology rather than the increased permeability induced by IFN-γ.
The aim of this study was to determine the in vitro effect of glutamine and insulin on apoptosis, mitochondrial membrane potential, cell permeability, and inflammatory cytokines in hyperglycemic umbilical vein endothelial cells.
The increase in endothelial permeability often promotes edema formation in various pathological conditions. Tumor necrosis factor-alpha (TNF-α), a pro-atherogenic cytokine, impairs endothelial barrier function and causes endothelial dysfunction in early stage of atherosclerosis. Asiaticoside, one of the triterpenoids derived from Centella asiatica, is known to possess antiinflammatory activity. In order to examine the role of asiaticoside in preserving the endothelial barrier, we assessed its effects on endothelial hyperpermeability and disruption of actin filaments evoked by TNF-α in human aortic endothelial cells (HAEC). TNF-α caused an increase in endothelial permeability to fluorescein isothiocyanate (FITC)-dextran. Asiaticoside pretreatment significantly suppressed TNF-α-induced increased permeability. Asiaticoside also prevented TNF-α-induced actin redistribution by suppressing stress fiber formation. However, the increased F to G actin ratio stimulated by TNF-α was not changed by asiaticoside. Cytochalasin D, an actin depolymerizing agent, was used to correlate the anti-hyperpermeability effect of asiaticoside with actin cytoskeleton. Surprisingly, asiaticoside failed to prevent cytochalasin D-induced increased permeability. These results suggest that asiaticoside protects against the disruption of endothelial barrier and actin rearrangement triggered by TNF-α without a significant change in total actin pool. However, asiaticoside seems to work by other mechanisms to maintain the integrity of endothelial barrier rather than stabilizing the F-actin organization.
The cytotoxic potential of ammonium-based deep eutectic solvents (DESs) with four hydrogen bond donors, namely glycerine (Gl), ethylene glycol (EG), triethylene glycol (TEG) and urea (U) were investigated. The toxicity of DESs was examined using In Vitro cell lines and In Vivo animal model. IC50 and selectivity index were determined for the DESs, their individual components and their combinations as aqueous solutions for comparison purposes. The cytotoxicity effect of DESs varied depending on cell lines. The IC50 for the GlDES, EGDES, UDES and TEGDES followed the sequence of TEGDES< GlDES< EGDES< UDES for OKF6, MCF-7, A375, HT29 and H413, respectively. GlDES was selective against MCF-7 and A375, EGDES was selective against MCF-7, PC3, HepG2 and HT29, UDES was selective against MCF-7, PC3, HepG2 and HT29, and TEGDES was selective against MCF-7 and A375. However, acute toxicity studies using ICR mice showed that these DESs were relatively toxic in comparison to their individual components. DES did not cause DNA damage, but it could enhance ROS production and induce apoptosis in treated cancer cells as evidenced by marked LDH release. Furthermore, the examined DESs showed less cytotoxicity compared with ionic liquids. To the best of our knowledge, this is the first time that combined In Vitro and In Vivo toxicity profiles of DESs were being demonstrated, raising the toxicity issue of these neoteric mixtures and their potential applicability to be used for therapeutic purposes.