Rhodamine 123 Research Articles

Reversal of Multidrug Resistance by the Synergistic Effect of Reversan and Hyperthermia to Potentiate the Chemotherapeutic Response of Doxorubicin in Glioblastoma and Glioblastoma Stem Cells.

The glioblastoma stem cell (GSC) population in glioblastoma multiforme (GBM) poses major complication in clinical oncology owing to increased resistance to chemotherapeutic drugs, thereby limiting treatment in patients with recurring glioblastoma. To completely eradicate glioblastoma, a single therapy module is not enough; therefore, there is a need to develop a multimodal approach to eliminate bulk tumors along with the CSC population. With an aim to target transporters associated with multidrug resistance (MDR), such as P-glycoprotein (P-gp), a small-molecule inhibitor, reversan (RV) was used along with multifunctional magnetic nanoparticles (MNPs) for hyperthermia (HT) therapy and targeted drug delivery. Higher efflux of free doxorubicin (Dox) from the cells was stabilized by encapsulation in PPS-MnFe nanoparticles, whose physicochemical properties were determined by dynamic light scattering (DLS) and transmission electron microscopy (TEM). Treatment with RV also enhanced the cellular uptake of PPS-MnFe-Dox, whereas RV and magnetic hyperthermia (MHT) together showed prolonged retention of fluorescence dye, Rhodamine123 (R123), in glioblastoma cells compared with individual treatment. Overall, in this work, we demonstrated the synergistic action of RV and HT to combat MDR in GBM and GSCs, and chemo-hyperthermia therapy enhanced the cytotoxic effect of the chemotherapeutic drug Dox (with lower effective concentration) and induced a higher degree of apoptosis compared to single-drug dosage.

Biliary organoids uncover delayed epithelial development and barrier function in biliary atresia.

Biliary atresia is a severe inflammatory and fibrosing cholangiopathy of neonates of unknown etiology. The onset of cholestasis at birth implies a prenatal onset of liver dysfunction. Our aim was to investigate the mechanisms linked to abnormal cholangiocyte development. We generated biliary organoids from liver biopsies of infants with biliary atresia and normal and diseased controls. Organoids emerged from biliary atresia livers and controls and grew as lumen-containing spheres with an epithelial lining of cytokeratin-19pos albuminneg SOX17neg cholangiocyte-like cells. Spheres had similar gross morphology in all three groups and expressed cholangiocyte-enriched genes. In biliary atresia, cholangiocyte-like cells lacked a basal positioning of the nucleus, expressed fewer developmental and functional markers, and displayed misorientation of cilia. They aberrantly expressed F-actin, β-catenin, and Ezrin, had low signals for the tight junction protein zonula occludens-1 (ZO-1), and displayed increased permeability as evidenced by a higher Rhodamine-123 (R123) signal inside organoids after verapamil treatment. Biliary atresia organoids had decreased expression of genes related to EGF signaling and FGF2 signaling. When treated with EGF+FGF2, biliary atresia organoids expressed differentiation (cytokeratin 7 and hepatocyte nuclear factor 1 homeobox B) and functional (somatostatin receptor 2, cystic fibrosis transmembrane conductance regulator [CFTR], aquaporin 1) markers, restored polarity with improved localization of F-actin, β-catenin and ZO-1, increased CFTR function, and decreased uptake of R123. Organoids from biliary atresia are viable and have evidence of halted epithelial development. The induction of developmental markers, improved cell-cell junction, and decreased epithelial permeability by EGF and FGF2 identifies potential strategies to promote epithelial maturation and function.

Pharmacokinetic interactions: The effects of selected herbal extracts on permeation of P-glycoprotein substrate drugs across excised pig intestinal tissue

Introduction: There is growing concern that co-administered herbal medicines may alter the pharmacokinetics and, therefore, the efficacy and toxicity of Western drugs. The aim of this study was to investigate the potential membrane permeation modulating effects of four herbal extracts, i.e., Harpagophytum procumbens, Hoodia gordonii, Leonotis leonurus, and Vitis vinifera on a model compound, Rhodamine 123 (RH-123). Methods: An in vitro permeation model, i.e., excised pig intestinal tissue, mounted to test chambers in a Sweetana-Grass diffusion apparatus, was used to measure the bi-directional transport of RH-123 in the presence and in the absence of four herbal extracts. The concentration of transported RH-123 in each sample was determined by means of fluorescence spectroscopic analysis. The integrity of the mounted jejunum tissue during experimentation was confirmed by measuring the permeation of Lucifer Yellow through these membranes. Trans-epithelial electrical resistance (TEER) of the mounted membranes was also measured at the onset and termination of each experiment to monitor whether tight junction modulation occurred. Results: H. procumbens extract increased the secretory transport of RH-123, indicative of the induction of P-glycoprotein (P-gp) mediated efflux. H. gordonii extract also increased RH-123’s absorptive transport, coupled with a subsequent decrease in its secretory transport, indicating the P-gp related efflux inhibition. Contrary, L. leonurus extract reduced RH-123’s absorptive transport, accompanied by an increase in its secretory transport. V. vinifera seed extract, however, increased both the absorptive and secretory transport of RH-123. A reduction in TEER was observed in the presence of V. vinifera extract, indicating the modulation of tight junction integrity. Conclusion: The ex vivo pharmacokinetics interactions recorded in the current study suggest that the co-administration of herbal medicines could alter the extent of membrane permeation of Western drugs.

Inhibition of autophagy enhances apoptosis induced by Ce6-photodynamic therapy in human colon cancer cells

ObjectiveTo evaluate the therapeutic effect of Chlorin e6 photodynamic therapy (Ce6-PDT) in human colorectal cancer cells and investigate the role of autophagy in Ce6-PDT. MethodsSW480 cells underwent Ce6-PDT with and without pretreatment with the autophagy inhibitor 3-methyladenine (3MA). Cell viability was assessed using 3-(4,5-dimethylthiazol-2-yl)-2, 5-diphenyltetrazolium bromide (MTT) assay. Apoptosis was evaluated using an Annexin V assay, using a rhodamine 123 (RH123) assay to evaluate mitochondrial membrane potential (MMP), and by measuring Caspase-3 and Bcl-2 protein expression using western blotting. Autophagy was evaluated by directly visualizing acridine orange-stained acidic vesicular organelles (AVOs) using fluorescent microscopy and by measuring LC3Ⅰ/Ⅱand Atg5 expression using western blotting. ResultsCe6-PDT decreased SW480 viability in a dose-dependent manner. Ce6-PDT induced apoptosis in SW480 cells via the mitochondrial apoptosis pathway as indicated by decreased mitochondrial membrane potential, increased Annexin V staining, and increased Caspase-3 expression. Ce6-PDT was also shown to induce autophagy as demonstrated by increased acridine-orange stained AVOs as well as increased expression of the autophagy-associated proteins Atg5. Inhibition of autophagy with 3MA potentiated SW480 cell response to Ce6-PDT and increased the rate of apoptosis in the treated cells. ConclusionsCe6-PDT induces autophagy and apoptosis of SW480 cells in a dose-dependent manner. Inhibition of autophagy increases the apoptosis induced by Ce6-PDT. Modulation of autophagy may be a potential therapeutic target for colon cancer cells treated with Ce6-PDT.

Possible Regulation of P-glycoprotein Function by Adrenergic Agonists in a Vascular-luminal Perfused Preparation of Small Intestine

Although the functions of small intestine are largely regulated by enteric nervous system (ENS), an independent intrinsic innervation, as well as central nervous system (CNS), the neural regulation of drug absorption from the small intestine still remains to be clarified. To obtain some information on it, the effect of adrenergic agonists on P-glycoprotein (P-gp) function was investigated by utilizing a vascular-luminal perfused rat small intestine. Adrenaline significantly decreased the secretion of rhodamine-123 (R-123) into the intestinal lumen, but dibutyryl cAMP (DBcAMP) significantly enhanced R-123 secretion. The inhibition study with quinidine clearly indicated that the decrease in secretory clearance of R-123 by adrenaline or the increase by DBcAMP would be attributed to the decrease or increase in P-gp activity, respectively. Expression levels of P-gp in whole mucosal homogenates were not changed at all by any chemicals examined, but those on brush border membrane (BBM) of intestinal epithelial cells were significantly decreased or increased by adrenaline or DBcAMP, respectively. Furthermore, changes in P-gp activity caused by adrenergic agonists and DBcAMP were significantly correlated with changes in expression level of P-gp in BBM, suggesting that the trafficking of P-gp from cytosolic pool to BBM would be regulated by adrenergic agonists and DBcAMP.

Mitochondrial ATP-Sensitive K+ Channel Opening Increased the Airway Smooth Muscle Cell Proliferation by Activating the PI3K/AKT Signaling Pathway in a Rat Model of Asthma.

Abnormal proliferation of airway smooth muscle cells (ASMCs) leads to airway remodeling and the development of asthma. This study aimed to assess whether mitochondrial ATP-sensitive K+ (mitoKATP) channels regulated the proliferation of ASMCs by regulating the phosphoinositide 3-kinase/protein kinase B (PI3K/AKT) pathway in asthmatic rats. Forty-eight Sprague Dawley rats were immunized with ovalbumin-containing alum to establish the asthma models. The ASMCs were isolated and identified by phase-contrast microscopic images and immunohistochemical staining for α-smooth muscle actin. The ASMCs were treated with a potent activator of mitoKATP, diazoxide, or an inhibitor of mitoKATP, 5-hydroxydecanoate (5-HD). Rhodamine-123 (R-123) was used for detecting the mitochondrial membrane potential (Δψm). The proliferation of ASMCs was examined by the MTT (3-[4,5-dimethylthiazol-2-yl]-2,5 diphenyl tetrazolium bromide) assay. The protein and mRNA expressions of AKT and p-AKT were detected using western blotting and quantitative real-time PCR. The results showed that diazoxide enhanced the mitoKATP channel opening in ASMCs in the rat model of asthma, while 5-HD impeded it. Diazoxide also increased ASMC proliferation in the rat model of asthma, whereas 5-HD alleviated it. However, LY294002, a PI3K/AKT pathway inhibitor, reversed the functional roles of diazoxide in the proliferation ability of ASMCs in the rat model of asthma. Furthermore, treatment with diazoxide induced the phosphorylation of AKT, and treatment with 5-HD decreased the phosphorylation of AKT in ASMCs in the rat model of asthma. In conclusion, the mitoKATP channel opening increased the proliferation of ASMCs by activating the PI3K/AKT signaling pathway in a rat model of asthma.

Improvement of light stability of DHR123 radio fluorogenic nano clay gel dosimeter by incorporating a new dispersant

A tissue-equivalent three-dimensional dosimeter is being developed to verify dose distributions for radiotherapy. Recently, we have reported a nanocomposite fluorescent gel (NC-RFG) dosimeter that enables readout by the fluorometry, which is more sensitive than absorption method in principle. However, irradiating by an excitation light for a dose information readout also produces fluorescent dyes and is not negligible in the low-dose irradiation range under 1 Gy. In this study, we investigate new additives to improve the NC-RFG’s light stability. We successfully suppressed the light-sensitive property of the NC-RFG to at least 10% of normal conditions by adding either N-vinylpyrrolidone or pyridine as a new dispersant that is presumed to break up aggregation of rhodamine 123 (RD123) and dihydrorhodamine 123 (DHR123) to stop photoexcited RD123 from oxidizing the adjacent DHR123. We also provide a detailed study of sensitivity characteristics such as dose rate dependence for fluorescent gel dosimeters.

Transfer of rhodamine-123\xa0into the brain and cerebrospinal fluid of fetal, neonatal and adult rats

BackgroundAdenosine triphosphate binding cassette transporters such as P-glycoprotein (PGP) play an important role in drug pharmacokinetics by actively effluxing their substrates at barrier interfaces, including the blood-brain, blood-cerebrospinal fluid (CSF) and placental barriers. For a molecule to access the brain during fetal stages it must bypass efflux transporters at both the placental barrier and brain barriers themselves. Following birth, placental protection is no longer present and brain barriers remain the major line of defense. Understanding developmental differences that exist in the transfer of PGP substrates into the brain is important for ensuring that medication regimes are safe and appropriate for all patients.MethodsIn the present study PGP substrate rhodamine-123 (R123) was injected intraperitoneally into E19 dams, postnatal (P4, P14) and adult rats. Naturally fluorescent properties of R123 were utilized to measure its concentration in blood-plasma, CSF and brain by spectrofluorimetry (Clariostar). Statistical differences in R123 transfer (concentration ratios between tissue and plasma ratios) were determined using Kruskal-Wallis tests with Dunn’s corrections.ResultsFollowing maternal injection the transfer of R123 across the E19 placenta from maternal blood to fetal blood was around 20 %. Of the R123 that reached fetal circulation 43 % transferred into brain and 38 % into CSF. The transfer of R123 from blood to brain and CSF was lower in postnatal pups and decreased with age (brain: 43 % at P4, 22 % at P14 and 9 % in adults; CSF: 8 % at P4, 8 % at P14 and 1 % in adults). Transfer from maternal blood across placental and brain barriers into fetal brain was approximately 9 %, similar to the transfer across adult blood-brain barriers (also 9 %). Following birth when placental protection was no longer present, transfer of R123 from blood into the newborn brain was significantly higher than into adult brain (3 fold, p < 0.05).ConclusionsAdministration of a PGP substrate to infant rats resulted in a higher transfer into the brain than equivalent doses at later stages of life or equivalent maternal doses during gestation. Toxicological testing of PGP substrate drugs should consider the possibility of these patient specific differences in safety analysis.

Influences of Hydrogen Bonding-Based Stabilization of Bolaamphiphile Layers on Molecular Diffusion within Organic Nanotubes Having Inner Carboxyl Groups.

This paper reports molecular diffusion behavior in two bolaamphiphile-based organic nanotubes having inner carboxyl groups with different inner dimeters (10 and 20 nm) and wall structures, COOH-ONT10nm and COOH-ONT20nm, using imaging fluorescence correlation spectroscopy (imaging FCS). The results were compared to those previously obtained in a similar nanotube with inner amine groups (NH2-ONT10nm). COOH-ONT10nm, as with NH2-ONT10nm, were formed from a rolled bolaamphiphile layer incorporating triglycine moieties, whereas COOH-ONT20nm consisted of four stacks of triglycine-free bolaamphiphile layers. Imaging FCS measurements were carried out for anionic sulforhodamine B (SRB), zwitterionic/cationic rhodamine B (RB), and cationic rhodamine-123 (R123) diffusing within ONTs (1-9 μm long) at different pH (3.4-8.4) and ionic strengths (1.6-500 mM). Diffusion coefficients (D) of these dyes in the ONTs were very small (0.01-0.1 μm2/s), reflecting the significant contributions of molecule-nanotube interactions to diffusion. The D of SRB was larger at higher pH and ionic strength, indicating the essential role of electrostatic repulsion that was enhanced by the deprotonation of the inner carboxyl groups. Importantly, the D of SRB was virtually independent of nanotube inner diameter and wall structure, indicating the diffusion of the hydrophilic molecule was controlled by short time scale adsorption/desorption processes onto the inner surface. In contrast, pH effects on D were less clear for relatively hydrophobic R123 and RB, suggesting the significant contributions of non-Coulombic interactions. Interestingly, the diffusion of these molecules in COOH-ONT20nm was slower than in COOH-ONT10nm. Slower diffusion in COOH-ONT20nm was attributable to relatively efficient partitioning of the hydrophobic dyes into the bolaamphiphile layers, which was reduced in COOH-ONT10nm due to the stabilization of its layer by polyglycine-II-type hydrogen bonding networks. These results show that, by tuning the bolaamphiphile structures and their intermolecular interactions, unique environments can be created within the nanospaces for enhanced molecular separations and reactions.

Redox-Sensitive and Hyaluronic Acid-Functionalized Nanoparticles for Improving Breast Cancer Treatment by Cytoplasmic 17α-Methyltestosterone Delivery

Novel reduction-responsive hyaluronic acid–chitosan–lipoic acid nanoparticles (HACSLA-NPs) were designed and synthesized for effective treatment of breast cancer by targeting Cluster of Differentiation 44 (CD44)-overexpressing cells and reduction-triggered 17α-Methyltestosterone (MT) release for systemic delivery. The effectiveness of these nanoparticles was investigated by different assays, including release rate, 3-(4,5-Dimethylthiazol-2-Yl)-2,5-Diphenyltetrazolium Bromide (MTT), lactate dehydrogenase (LDH), caspase-3 activity, Rhodamine 123 (RH-123), and Terminal deoxynucleotidyl transferase dUTP nick end labeling (TUNEL). In vitro experiments revealed that Methyltestosterone/Hyaluronic acid–chitosan–lipoic acid nanoparticles (MT/HACSLA-NPs) illustrated a sustained drug release in the absence of glutathione (GSH), while the presence of GSH led to fast MT release. HACSLA-NPs also showed high cellular internalization via CD44 receptors, quick drug release inside the cells, and amended cytotoxicity against positive CD44 BT-20 breast cancer cell line as opposed to negative CD44, Michigan Cancer Foundation-7 (MCF-7) cell line. These findings supported that these novel reduction-responsive NPs can be promising candidates for efficient targeted delivery of therapeutics in cancer therapy.

Anti-HIV and Anti-Hepatitis C Virus Drugs Inhibit P-Glycoprotein Efflux Activity in Caco-2 Cells and Precision-Cut Rat and Human Intestinal Slices.

P-glycoprotein (ABCB1), an ATP-binding-cassette efflux transporter, limits intestinal absorption of its substrates and is a common site of drug-drug interactions (DDIs). ABCB1 has been suggested to interact with many antivirals used to treat HIV and/or chronic hepatitis C virus (HCV) infections. Using bidirectional transport experiments in Caco-2 cells and a recently established ex vivo model of accumulation in precision-cut intestinal slices (PCIS) prepared from rat ileum or human jejunum, we evaluated the potential of anti-HIV and anti-HCV antivirals to inhibit intestinal ABCB1. Lopinavir, ritonavir, saquinavir, atazanavir, maraviroc, ledipasvir, and daclatasvir inhibited the efflux of a model ABCB1 substrate, rhodamine 123 (RHD123), in Caco-2 cells and rat-derived PCIS. Lopinavir, ritonavir, saquinavir, and atazanavir also significantly inhibited RHD123 efflux in human-derived PCIS, while possible interindividual variability was observed in the inhibition of intestinal ABCB1 by maraviroc, ledipasvir, and daclatasvir. Abacavir, zidovudine, tenofovir disoproxil fumarate, etravirine, and rilpivirine did not inhibit intestinal ABCB1. In conclusion, using recently established ex vivo methods for measuring drug accumulation in rat- and human-derived PCIS, we have demonstrated that some antivirals have a high potential for DDIs on intestinal ABCB1. Our data help clarify the molecular mechanisms responsible for reported increases in the bioavailability of ABCB1 substrates, including antivirals and drugs prescribed to treat comorbidity. These results could help guide the selection of combination pharmacotherapies and/or suitable dosing schemes for patients infected with HIV and/or HCV.

Preventive Effect of Opuntiol, Isolated from Opuntia ficus indica (L. Mill), Extract Against Ultraviolet A Radiation-Induced Oxidative Damages in NIH/3T3 Cells

Aim: We investigated the role of opuntiol, isolated from Opuntia ficus indica, against ultraviolet A waveband-mediated oxidative damages in the mouse embryonic fibroblast cell lines (NIH‐3T3). Materials and Methods: The antioxidant potential of opuntiol was carried out by hydroxyl radical, superoxide anion and DPPH radical scavenging assays. The preventive effect of opuntiol against UV-mediated cytotoxicity was revealed by MTT assay. Further, the oxidative end points during UV-exposure, in the presence and absence of opuntiol, was analyzed by DCFH-DA staining, rhodamine-123 staining and alkaline comet assay. Results: Opuntiol significantly neutralizes hydroxyl (OH•), superoxide anion (O2•–), hydrogen peroxide (H2O2), and 2,2-diphenyl-2-picrylhydrazyl (DPPH•) radicals in a concentration-dependent manner. In this study, the NIH-3T3 cells were treated with UVA-waveband in the presence and absence of opuntiol and oxidative damage markers were analyzed. We observed that opuntiol pretreatment (5 μM-20 μM) prevented 10 mJ/cm2UVA radiation-induced cytotoxicity in NIH-3T3 cells. Further, single UVA-radiation induces reactive oxygen species (ROS) through intracellular photosensitizers. Conversely, opuntiol pretreatment prevented UVA-mediated ROS generation and subsequent lipid peroxidation and loss of enzymatic antioxidants (superoxide dismutase [SOD], catalase, and glutathione peroxidase) in the NIH-3T3 cells. It has also been observed that the UVA-mediated ROS subsequently induces DNA damage and alters mitochondrial transmembrane potential (MMP). We noticed that opuntiol prevents UVA-radiation-mediated DNA single-strand breaks. Further, it prevents loss of MMPs and apoptotic morphological changes in the NIH-3T3 cells. Conclusion: Thus, these findings illustrate that opuntiol prevents UVA-radiation-mediated oxidative stress-related biochemical changes in the cellular system.

Enhancement of Berberine Hypoglycemic Activity by Oligomeric Proanthocyanidins.

This study investigated the possible enhancement of berberine’s (BB) hypoglycemic activity by oligomeric proanthocyanidins (OPCs) and its underlying mechanism. The hypoglycemic activity of the studied compounds was evaluated in diabetic db/db mice. The cellular uptake and efflux of BB with or without OPCs were investigated using Caco-2 intestinal cells. A pharmacokinetic study of BB and OPCs was performed in Sprague Dawley (SD) mice by oral administration of the study compounds. Liquid chromatography–tandem mass spectrometry (LC–MS/MS) was employed to determine the cellular efflux, retention, and the serum concentrations of the compounds. The results revealed that OPCs considerably potentiated the hypoglycemic efficacy of BB in diabetic db/db mice. In the in vitro experiments, OPCs significantly inhibited the efflux and increased the uptake of the P-glycoprotein (P-gp) substrate rhodamine-123 (R123) and BB in Caco-2 intestinal cells. Moreover, OPCs substantially reduced the expression of P-gp in Caco-2 cells. The inhibition of BB efflux by OPCs was translated into the improved pharmacokinetics in vivo. When co-administered, OPCs obviously increased the average maximum concentration of BB in mice. In summary, this study demonstrated that combination of BB with OPCs could significantly improve the pharmacokinetics and hypoglycemic efficacy of BB, which is valuable for future exploration of the combination of BB and OPCs as oral hypoglycemic agents.

P-gp modulating effect of Azadirachta indica extract in multidrug-resistant cancer cell lines.

The extract of Azadirachta indica, commonly known as neem, has found extensive use in traditional medicine for treating various human diseases. In this study, the effect of the 50% ethanol extract of A. indica (AI01) on P-glycoprotein (P-gp)-mediated multidrug resistance (MDR) was examined using MDR cell lines, specifically paclitaxel-resistant HepG2 (PR-HepG2) and doxorubicin (DOX)-resistant (DR) colon-26 cells. 96-h treatment of the two cell lines with AI01 (30 μg/mL) showed no effect on the expression of P-gp mRNA (human MDR1 and mouse mdr1b) and protein, while AI01 increased the accumulation of rhodamine 123, a P-gp substrate, in both PR-HepG2 and DR-colon-26 cells. The cytotoxic effects of 48-h treatment with AI01 on the viability of PR-HepG2 and DR-colon-26 cells were not observed. Therefore, 30 μg/mL AI01 may have no cytotoxic and P-gp-inducing effects. Finally, AI01 potentiated the sensitivity of PR-HepG2 and DR-colon-26 cell lines to DOX by 8.6- and 15.3-fold, respectively. These findings suggest that A. indica may be a promising source for a new class of P-gp modulators without cytotoxic/P-gp induction effects.

Synthesis of nanocomposites of iron oxide/gold (Fe3O4/Au) loaded on activated carbon and their application in water treatment by using sonochemistry: Optimization study

This paper focuses on the finding best operational conditions using response surface methodology (RSM) for Rhodamine123 (R123) and Disulfine blue (DSB) dyes removal by ultrasound assisted adsorption onto Au-Fe3O4 nanoparticles loaded on activated carbon (Au-Fe3O4 NPs-AC). The influences of variables such as initial R123 (X1) and DSB concentration (X2), pH (X3), adsorbent mass (X4) and sonication time (X5) on their removal were investigated by small central composite design (CCD) under response surface methodology. The significant variables and the possible interactions among variables were investigated and estimated accordingly. The best conditions were set as: 4min, 4.0, 0.025g, 13.5 and 26.5mgL−1 for sonication time, pH, adsorbent weight, initial R123 and DSB concentration, respectively. At above conditions, the adsorption equilibrium and kinetic follow the Langmuir isotherm and pseudo-second-order kinetic model, respectively. The maximum monolayer capacity (Qmax) of 71.46 and 76.38mgg−1 for R123 and DSB show sufficiency of model for well presentation of experimental data.

Effect of Curcuma comosa extracts on the functions of peptide transporter and P-glycoprotein in intestinal epithelial cells.

Curcuma comosa has been widely used as a herbal medicine in Thailand; however, it remains unclear whether C. comosa influences the absorption of drugs that are substrates for the transporters in the small intestine. In this study, we investigated the effect of C. comosa extracts on the functioning of peptide transporter 1 (PEPT1), an influx transporter, and P-glycoprotein (P-gp), an efflux transporter, in Caco-2 cells and rat intestine. In Caco-2 cells, the ethanolic extract of C. comosa (CCE) lowered the uptake of glycylsarcosine (Gly-Sar), a PEPT1 substrate, while it enhanced the uptake of rhodamine 123 (Rho123), a P-gp substrate, in a concentrationdependent manner. In addition, CCE inhibited apical-to-basal transport of Gly-Sar and basal-to-apical transport of Rho123. Furthermore, the absorption of cephalexin, another PEPT1 substrate, and the exsorption of Rho123 across the rat intestine were inhibited by CCE. Conversely, CCW, the hot water extract of C. comosa, suppresses the function of PEPT1 but not of P-gp in Caco-2 cells. These results suggest that C. comosa used as a herbal medicine in Thailand may affect the intestinal absorption of certain drugs.

In Vitro and In Vivo Activity of Fomitopsis Pinicola (Sw. Ex Fr.) Karst Chloroform (Fpkc) Extract Against S180 Tumor Cells

Background/Aims: Non-toxic fomitopsis is has been traditionally used in folk medicine in many countries for its anti-inflammatory and anti-vascular disease activities. The present study investigates the antitumor effect of Fomitopsis pinicola (Sw. Ex Fr.) Karst chloroform extract (FPKc) on S180 tumor cells in vitro and in vivo and we determined the underlying mechanisms. Methods: HPLC was employed to analyze the constituents of FPKc. In-vitro 3-(4,5-Dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide (MTT) assay was performed to quantify the growth inhibition of FPKc; Propidium iodide (PI) exclusion assay and scanning electron microscopy (SEM) were used to observe the damage on the cell membrane and the changes of the cell morphology; Staining with Hoechst 33342/propidium iodide (HO/PI) and the application of the Annexin V-FITC/PI analysis permitted to observe the cell death triggered by FPKc; DNA damage and cell cycle arrest were detected by flow cytometry; Rhodamine 123 (RH123) and Cytochrome C were used as dyes to investigate the alterations of the mitochondria. In-vivo tumor inhibition and mice survival time were determined. Results: The results of the HPLC assay indicated that FPKc might contain DA (dehydroeburiconic acid), PA (pachymic acid), and ES (ergosterol), at percentages of 0.25%, 17.8%, and 10.5%, respectively. Concerning the study of the biological function, the results showed that FPKc exhibited preferential and significant suppression of proliferation on specific cell lines including S180, HL-60, U937, K562, SMMC-7721, and Eca-109 cells, which induced plasma membrane and cell morphology damages, triggering S180 tumor-cells late apoptosis and leading to DNA damage and S phase arrest. Mitochondria were suspected to play a vital role in these changes. In vivo data indicated that FPKc inhibited the solid tumor growth and prolonged the survival time of tumor-bearing mice. Moreover, FPKc provoked only little damage on normal cells in vitro and also on normal tissues in vivo. Conclusion: FPKc inhibited S180 tumor cells growth and prolonged the lifespan of mice. In vitro, we found that FPKc induced S180 tumor cells apoptosis and cell cycle arrest, possibly via the mitochondrial pathway.

Effect of ketamine on mitochondrial function of rat neurons subjected to anoxia

Objective To investigate the effect of ketamine on the mitochondrial function of rat neurons subjected to anoxia. Methods Primarily cultured rat hippocampal neurons were seeded in culture dishes(35 mm in diameter)at the density of 5×105-1×106 cells/ml, and divided into 3 groups(n=11 each)using a random number table: control group, anoxia group and ketamine group.The neurons were exposed to 90% N2 plus 10% CO2 50 ml/min for 5 min in anoxia group.In ketamine group, ketamine was added to the culture medium with the final concentration of 20 μmol/L at 1 h before anoxia, and then the neurons were exposed to 90% N2 plus 10% CO2 50 ml/min for 5 min.After the end of treatment in each group, the dead neurons were detected using trypan blue staining, the ATP content was determined by ATP bioluminescence assay, and mitochondrial membrane potential was measured by rhodamine 123 staining. Results Compared with control group, the mortality rate of hippocampal neurons was significantly increased, and the ATP content and mitochondrial membrane potential were significantly decreased in anoxia group and ketamine group(P<0.05). Compared with anoxia group, the mortality rate of hippocampal neurons was significantly decreased, and the ATP content and mitochondrial membrane potential were significantly increased in ketamine group(P<0.05). Conclusion The mechanism by which ketamine ameliorates anoxia-induced damage to rat neurons is related to improved mitochondrial function. Key words: Ketamine; Hypoxia, brain; Neurons; Mitochondria

The application of P-gp inhibiting phospholipids as novel oral bioavailability enhancers — An in vitro and in vivo comparison

The efflux transporter P-glycoprotein (P-gp) significantly modulates drug transport across the intestinal mucosa, strongly reducing the systemic absorption of various active pharmaceutical ingredients. P-gp inhibitors could serve as helpful tools to enhance the oral bioavailability of those substances. As a membrane-associated protein P-gp is surrounded and influenced by phospholipids. Some synthetic phospholipids have been found to strongly reduce P-gp's activity. In this study two representative phospholipids, 1,2-dioctanoyl-sn-glycero-3-phosphocholine (8:0 PC) and 1,2-didecanoyl-sn-glycero-3-phosphocholine (10:0 PC), were compared with Tween® 80 and Cremophor® EL, both commonly used surfactants with P-gp inhibitory properties. Their influence on the cellular transport of the P-gp substrate rhodamine 123 (RH123) was examined using Caco-2 cell layers. In addition, fluorescence anisotropy measurements were performed in order to investigate their effect on membrane fluidity. Finally, we compared the phospholipids with Tween® 80 and the competitive P-gp inhibitor verapamil in an in vivo study, testing their effects on the oral bioavailability of the P-gp substrate drug ritonavir. Both phospholipids not only led to the strongest absorption of RH123, but a permeability enhancing effect was detected in addition to the P-gp inhibition. Their effects on membrane fluidity were not consistent with their P-gp inhibiting effects, and therefore suggested a more complex mode of action. Both phospholipids significantly increased the area under the ritonavir plasma level curve (AUC) within 150min by more than tenfold, but were inferior to Tween® 80, which showed superior solubilizing effects. Finally, these phospholipids represent a novel substance class showing a high permeabilization potential for P-gp substrates. Because of their physiological structure and intestinal degradability, good tolerability without systemic absorption is expected. Formulating P-gp substrates with an originally low oral bioavailability is a difficult task, requiring concerted interplay of all excipients. P-gp inhibiting phospholipids offer a new tool to help cope with these challenges.

Celastrol inhibits growth and induces apoptosis of human gallbladder cancer NOZ cells

Objective To investigate the effects of celastrol on the cell growth and apoptosis of human gallbladder cancer NOZ cells, and explore its potential molecular mechanism. Methods NOZ cell were cultured in vitro. And CCK-8 assay, Annexin V-FITC/PI staining method, cell cycle analysis were conducted to investigate the effects of celastrol on the growth and apoptosis of NOZ cells after being treated with drugs. The mitochondrial membrane potential and Bax and Bcl-2 protein expression level were determined by Rhodamine 123 and Western blot, respectively. Results Celastrol could inhibit NOZ cell growth, and the IC50 value was 5.3 μmol/L. Annexin-V/PI staining showed that cell apoptosis of NOZ cells were induced as the celastrol concentration increased, and the apoptosis ratio of control group was 4.4%, while the apoptosis rates of the test groups (2, 5, 10 μmol/L) were 7.4%, 27.1% and 43.4%, respectively. In addition, cell cycle analysis revealed that celastrol could induce G1-phase arrest. The G1-phase rate of control group was 25.6%, while the G1-phase rates of the test groups (2, 5, 10 μmol/L) were 36.5%, 45.7% and 92.5%, respectively. The mitochondrial membrane potential was measured after treatment with celastrol and the results indicated that the mitochondrial membrane potential was significantly decreased. Western Blot showed that the protein expression of Bax increased and Bcl-2 decreased in a time-dependent manner after treatment with celastrol. Conclusions Celastrol may inhibit cell proliferation of human gallbladder cancer NOZ cells and induce cell apoptosis partly by inducing the loss of mitochondrial membrane potential. Key words: Celastrol; Gallbladder cancer; Cell proliferation; Cell apoptosis