Improvement of Transmembrane Transport Mechanism Study of Imperatorin on P-Glycoprotein-Mediated Drug Transport.

P-glycoprotein (P-gp) affects the transport of many drugs; including puerarin and vincristine. Our previous study demonstrated that imperatorin increased the intestinal absorption of puerarin and vincristine by inhibiting P-gp-mediated drug efflux. However; the underlying mechanism was not known. The present study investigated the mechanism by which imperatorin promotes P-gp-mediated drug transport. We used molecular docking to predict the binding force between imperatorin and P-gp and the effect of imperatorin on P-gp activity. P-gp efflux activity and P-gp ATPase activity were measured using a rhodamine 123 (Rh-123) accumulation assay and a Pgp-Glo™ assay; respectively. The fluorescent probe 1,6-diphenyl-1,3,5-hexatriene (DPH) was used to assess cellular membrane fluidity in MDCK-MDR1 cells. Western blotting was used to analyze the effect of imperatorin on P-gp expression; and P-gp mRNA levels were assessed by qRT-PCR. Molecular docking results demonstrated that the binding force between imperatorin and P-gp was much weaker than the force between P-gp and verapamil (a P-gp substrate). Imperatorin activated P-gp ATPase activity; which had a role in the inhibition of P-gp activity. Imperatorin promoted Rh-123 accumulation in MDCK-MDR1 cells and decreased cellular membrane fluidity. Western blotting demonstrated that imperatorin inhibited P-gp expression; and qRT-PCR revealed that imperatorin down-regulated P-gp (MDR1) gene expression. Imperatorin decreased P-gp-mediated drug efflux by inhibiting P-gp activity and the expression of P-gp mRNA and protein. Our results suggest that imperatorin could down-regulate P-gp expression to overcome multidrug resistance in tumors.

Effects of surfactin on membrane models displaying lipid phase separation.

Surfactin, a bacterial amphiphilic lipopeptide is attracting more and more attention in view of its bioactive properties which are in relation with its ability to interact with lipids of biological membranes. In this work, we investigated the effect of surfactin on membrane structure using model of membranes, vesicles as well as supported bilayers, presenting coexistence of fluid-disordered (DOPC) and gel (DPPC) phases. A range of complementary methods was used including AFM, ellipsometry, dynamic light scattering, fluorescence measurements of Laurdan, DPH, calcein release, and octadecylrhodamine B dequenching. Our findings demonstrated that surfactin concentration is critical for its effect on the membrane. The results suggest that the presence of rigid domains can play an essential role in the first step of surfactin insertion and that surfactin interacts both with the membrane polar heads and the acyl chain region. A mechanism for the surfactin lipid membrane interaction, consisting of three sequential structural and morphological changes, is proposed. At concentrations below the CMC, surfactin inserted at the boundary between gel and fluid lipid domains, inhibited phase separation and stiffened the bilayer without global morphological change of liposomes. At concentrations close to CMC, surfactin solubilized the fluid phospholipid phase and increased order in the remainder of the lipid bilayer. At higher surfactin concentrations, both the fluid and the rigid bilayer structures were dissolved into mixed micelles and other structures presenting a wide size distribution.

Antifungal activity of lariciresinol derived from Sambucus williamsii and their membrane-active mechanisms in Candida albicans.

Lariciresinol is an enterolignan precursor isolated from the herb Sambucus williamsii, a folk medicinal plant used for its therapeutic properties. In this study, the antifungal properties and mode of action of lariciresinol were investigated. Lariciresinol displays potent antifungal properties against several human pathogenic fungal strains without hemolytic effects on human erythrocytes. To understand the antifungal mechanism of action of lariciresinol, the membrane interactions of lariciresinol were examined. Fluorescence analysis using the membrane probe 3,3'-diethylthio-dicarbocyanine iodide (DiSC(3)-5) and 1,6-diphenyl-1,3,5-hexatriene (DPH), as well as a flow cytometric analysis with propidium iodide (PI), a membrane-impermeable dye, indicated that lariciresinol was associated with lipid bilayers and induced membrane permeabilization. Therefore, the present study suggests that lariciresinol possesses fungicidal activities by disrupting the fungal plasma membrane and therapeutic potential as a novel antifungal agent for the treatment of fungal infectious diseases in humans.

Cholesterol-dependent induction of dendrite formation by ginsenoside Rh2 in cultured melanoma cells.

Herbal remedies containing root extracts of Panax ginseng are commonly used for complementary or alternative therapies. Ginsenosides, the major components of root extracts, are responsible for ginseng's pharmacological and biological effects; however, their mechanisms of action are unclear. We examined whether membrane cholesterol was involved in the mechanism of action of ginsenoside Rh2 in cultured cells. In B16 melanoma cells, Rh2 (18.5 µM) induced dendrite formation within 2 h. Depletion of cholesterol by pretreatment with 10 mM methyl-ß-cyclodextrin suppressed this effect of Rh2. Rh2 did not change the cellular cholesterol content and the immunofluorescence staining pattern of the lipid-raft-associated molecules, ganglioside GM3, Caveolin-1, Flotillin-1, and Flotillin-2, for up to 3 or 6 h. However, within 2 min of addition, Rh2 changed the fluorescence polarization of 1,6-diphenyl-1,3,5-hexatriene (DPH) but not of 1-[4-(trimethylammonio)phenyl]-6-phenyl-1,3,5-hexatriene (TMA-DPH). DPH is more sensitive than TMA-DPH to changes in the physical properties of membrane lipid bilayers regulated by cholesterol. These results suggest that Rh2 affects the physical properties of cholesterol-regulated membrane lipid bilayers and could lead to changes in cellular functions.

Membrane fluidity and surface changes during initiation of 1,2 dimethylhydrazine-induced colon carcinogenesis: protection by zinc.

The present study evaluated the modulatory effects of zinc on colonic membrane fluidity and surface abnormalities following 1,2 dimethylhydrazine (DMH)-induced colon carcinogenesis. Rats were segregated into four groups: normal control, DMH treated, zinc treated, DMH + zinc treated. Colon carcinogenesis was initiated through weekly subcutaneous injections of DMH (30 mg/kg body weight) for 8 weeks. Zinc (in the form of zinc sulphate) was supplemented to rats at a dose level of 227 mg/L in drinking water, ad libitum, for the entire duration of the study. Brush border membranes (BBM) were isolated from the colon of rats and the fluidity parameters were assessed by steady-state fluorescence polarization technique using the membrane extrinsic fluorophore 1,6-diphenyl-1,3,5-hexatriene (DPH). The translational diffusion was measured by using the excimer formation of pyrene incorporated in the membrane. The results demonstrated a significant increase in the polarization and anisotropy, accompanied by an increase in order parameter in the membrane preparations from the colon of DMH-injected rats. Further, studies with pyrene fluorophore indicated a marked decrease in membrane microviscosity following DMH treatment. However, the alterations in membrane fluorescence polarization and the fluidity parameters were completely restored following zinc treatment. Drastic alterations in colon surface were noticed after 8 weeks of DMH treatment. However, zinc treatment to DMH-treated rats greatly restored normalcy in the colonic surface. The study concludes that zinc has a strong membrane stabilizing effect and thus has a positive beneficial effect against chemically induced colonic preneoplastic progression in rats.

Isocryptomerin, a novel membrane-active antifungal compound from Selaginella tamariscina.

Isocryptomerin is a biflavonoid isolated from Selaginella tamariscina used in traditional medicine. In this study, we investigated novel antifungal properties of isocryptomerin. The results indicated that isocryptomerin exerted antifungal activity in an energy-independent manner without remarkable hemolytic effects. To understand mode of action(s) of isocryptomerin, we conducted experiments on Candida albicans, a noted human pathogenic fungal strain. Flow cytometric analysis with bis-(1,3-dibutylbarbituric acid) trimethine oxonol [DiBAC(4)(3)], a translational membrane potential dye, regeneration test with fungal protoplasts, and fluorescence analysis with 1,6-diphenyl-1,3,5-hexatriene (DPH), a probe for membrane studies by depolarization, indicated that isocryptomerin could depolarize fungal plasma membrane. In conclusion, the results suggested that the antifungal activities of isocryptomerin might be due to its membrane-disruption mechanism(s).

The alphaPROX assay: fluorescence screening of the inhibitory effects of hydrophilic antioxidants on protein oxidation.

We report on a new and convenient high-throughput fluorescence technique for determining antioxidant capacities of hydrophilic food samples. The new method is called alphaPROX (anti protein oxidation) and is based on an equimolar complex of diphenylhexatriene propionic acid (DPHPA) and bovine serum albumin (BSA) in aqueous buffer at pH 7.4. DPHPA is a reporter fluorophore that becomes nonfluorescent upon free radical-induced oxidation. In a typical assay, the DPHPA/BSA complex is challenged with peroxyl radicals and shows almost the same susceptibility to oxidation as unlabeled BSA. The progress of protein oxidation and its inhibition by antioxidants at physiological pH is determined from the time-dependent decrease in DPHPA fluorescence intensity. The alphaPROX method was compared to other techniques frequently used to measure antioxidant capacities. In this article, representative results are provided for the inhibitory effects of pure food components, fruit juices, wines, and various polar plant extracts on protein oxidation.

High-throughput fluorescence screening of antioxidative capacity in human serum.

Diphenylhexatriene-labeled phosphatidylcholine and propionic acid have been established as selective fluorescence markers for the continuous determination of oxidation processes in the lipid and aqueous phases of unfractionated human serum. Oxidation of the respective fluorophores leads to a decrease in fluorescence intensity from which the time-dependent degradation of the marker molecule can be determined. The lag times preceding the propagation of oxidation are representative for the antioxidative capacity of the system, which may be influenced by exogenous factors, e.g., the antioxidants from the diet. Supplementation of human serum by quercetin, rutin, vitamin E, vitamin C, or total apple phenolics in vitro led to a decrease in oxidizability depending on the oxidation marker and the hydrophobicity of the antioxidant. Quercetin and vitamin E showed a higher in vitro capacity of protecting lipoproteins against oxidation. In contrast, rutin and vitamin C were more efficient as inhibitors in the aqueous phase. The same effect on serum was found after dietary consumption of apples. This result is in line with the known observation that intake of plant polyphenols leads to an increase in serum levels of hydrophilic antioxidants.

Lysophosphatidylcholine modulates catalytically important motions of the Ca-ATPase phosphorylation domain.

Catalytically important motions of the Ca-ATPase, modulated by the physical properties of surrounding membrane phospholipids, have been suggested to be rate-limiting under physiological conditions. To identify the nature of the structural coupling between the Ca-ATPase and membrane phospholipids, we have investigated the functional and structural effects resulting from the incorporation of the lysophospholipid 1-myristoyl-2-hydroxy-sn-glycerol-3-phosphocholine (LPC) into native sarcoplasmic reticulum (SR) membranes. Nonsolubilizing concentrations of LPC abolish changes in fluorescence signals associated with either intrinsic or extrinsic chromophores that monitor normal conformational transitions accompanying calcium activation of the Ca-ATPase. There are corresponding decreases in the rates of calcium transport coupled to ATP hydrolysis, suggesting that LPC may increase conformational barriers associated with catalytic function. Fluorescence anisotropy measurements of the lipid analogue 1-(4-trimethylammoniumphenyl)-6-phenyl-1,3,5-hexatriene (TMA-DPH) partitioned into SR membranes indicate that LPC does not significantly modify lipid acyl chain rotational dynamics, suggesting differences in headgroup conformation between LPC and diacylglycerol phosphatidylcholines. Complementary measurements using phosphorescence anisotropy of erythrosin isothiocyanate at Lys464 on the Ca-ATPase provide a measure of the dynamic structure of the phosphorylation domain, and indicate that LPC restricts the amplitude of rotational motion. These results suggest a structural linkage between the cytosolic phosphorylation domain and the conformation of membrane phospholipid headgroups. Thus, changes in membrane phospholipid composition can modulate membrane surface properties and affect catalytically important motions of the Ca-ATPase in a manner that suggests a role for LPC generated during signal transduction.

Plasmatic and membrane lipid alterations in erythrocytes from diabetic rats fed either n-6 or n-3 fatty acids.

We examined the effect of dietary n-6 and n-3 fatty acids on the lipid composition and physical properties of erythrocyte membranes together with cholesterol and triglyceride plasmatic levels in normal and experimental diabetic rats. Plasmatic total cholesterol and triglyceride did not change in normal rats under the dietary regime, but both parameters decreased significantly in the diabetic animals after the consumption of either n-6 or n-3 fatty acids. Lipid analyses of erythrocyte membranes revealed a significant decrease in the total cholesterol together with an increase in the phospholipid amount in the diabetics compared to the normal rats. As a consequence, cholesterol/phospholipid ratio decreased in these groups of animals and markedly in those fed n-3 fatty acids. These changes would be responsible for the lower fluorescent polarization of DPH observed in the latter group. We conclude that equivalent and adequate amounts of dietary either n-6 or n-3 fatty acids produce plasmatic and red cell membrane lipid changes in diabetic rats that may improve the evolution of the disease.

Effects of dietary fish oil on plasma high density lipoprotein. Electron spin resonance and fluorescence polarization studies of lipid ordering and dynamics.

Dietary fish oils are implicated in reducing the incidence of coronary heart disease, perhaps by altering the properties of plasma lipoproteins. The hypothesis that omega-3 polyunsaturated fatty acids (PUFA) in fish oils produce changes in lipid ordering and dynamics within high density lipoprotein (HDL), thereby potentially modifying cholesterol transport, is investigated here. Rabbits were fed a diet supplemented with either 10% (by weight) menhaden oil (MO), a fish oil rich in omega-3 PUFAs, or hydrogenated cottonseed oil for a period of 12 weeks. HDL was isolated by sequential flotation ultracentrifugation from plasma drawn every 2 weeks. Gas chromatography confirmed that the predominant omega-3 PUFAs of fish oils, eicosapentaenoic 20:5 and docosahexaenoic 22:6 acids, were only incorporated into the triglyceride, phospholipid, and cholesteryl ester constituents of lipoproteins from rabbits on the MO diet. ESR of 5- and 16-doxyl stearic acids demonstrates that molecular order and dynamics within the outer monolayer of HDL is virtually unaffected. In contrast, ESR of cholesteryl 12-doxyl stearate indicates order is less within the inner apolar core of the lipoprotein for the MO diet than for the hydrogenated cottonseed oil diet. Fluorescence polarization of 1,6-diphenyl-1,3,5-hexatriene supports this finding. The greater disorder detected within HDL from rabbits fed fish oil may result in an enhancement of cholesterol exchange between lipoproteins and between lipoproteins and cells, which may have anti-atherogenic ramifications.