Internalization of FITC-albumin in Human Adipose-derived Stem Cells: Involvement of Clathrin and Caveolin.

Adipose tissue-derived stem cells (AdSCs) are one of the most promising cell types for cell-based therapies. In addition, AdSCs systematically injected into the body have been reported to localize to damaged tissues and certain types of tumor. As an important part of establishing a potent drug delivery system with AdSCs, the mechanism and efficiency of uptake into AdSCs has drawn much research attention. However, this remains to be fully clarified. The aim of this study was to examine the characteristics of endocytosis-mediated uptake in human AdSCs. We used fluorescein isothiocyanate-labeled albumin (FITC-albumin) as a potent marker of endocytosis. FITC-albumin uptake was time- and temperature-dependent. Confocal microscopy showed punctate localization of fluorescence in the cytoplasm. FITC-albumin uptake was inhibited by human serum albumin in a concentration-dependent manner. FITC-albumin uptake was inhibited by a metabolic inhibitor (2,4-dinitrophenol), a microtubule polymerization inhibitor (colchicine), an actin polymerization inhibitor (cytochalasin D), endosomal acidification inhibitors (chloroquine and bafilomycin A1), clathrin-dependent endocytosis inhibitors (chloropromazine, phenylarsine oxide, and Pitstop2), and caveolin-dependent endocytosis inhibitors (nystatin and methyl-ß-cyclodextrin). Furthermore, the knockdown of the clathrin heavy chain and caveolin-1 significantly reduced FITC-albumin uptake. These findings suggest that AdSCs take up albumin via endocytic pathways in which clathrin and caveolin are involved.

Fatty acids bound to albumin induce prostaglandin E2 production in human renal proximal tubular epithelial cell line HK-2.

Fatty acids bound to albumin have been reported to be involved in various responses in renal proximal tubular cells following albumin overload, leading to progression of tubulointerstitial damage in the kidneys. In addition, it has been reported that prostaglandin E2 (PGE2) plays an important role in nephrotoxicity. The aim of this study was to examine whether albumin-bound fatty acids induce PGE2 production in human renal proximal tubular epithelial cell line HK-2. Fatty acid-bearing human serum albumin increased PGE2 release in the culture medium in concentration-dependent and time-dependent manners, but fatty acid-depleted albumin had no effect on PGE2 production. Next, we investigated the effect of arachidonic acid, a precursor of eicosanoids, on PGE2 production. Arachidonic acid with fatty acid-free albumin significantly enhanced the release of PGE2 into the medium in a concentration-dependent manner. Furthermore, we examined the effect of arachidonic acid on mRNA expression of hypoxia inducible factor-1α (HIF-1α). Arachidonic acid increased HIF-1α mRNA expression in a concentration-dependent manner. These findings suggest that fatty acids, at least in part arachidonic acid, bound to albumin increase PGE2 production and expression of HIF-1α mRNA and protein, possibly resulting in various cell responses induced by albumin overload.

Pioglitazone induces hypoxia-inducible factor 1 activation in human renal proximal tubular epithelial cell line HK-2.

We previously reported that fatty acid-bearing albumin but not fatty acid-depleted albumin induces hypoxia-inducible factor-1 (HIF-1) activation in human renal proximal tubular epithelial cell line HK-2. Then, an increase in mRNA expression of peroxisome proliferator-activated receptor gamma (PPARγ) was observed on treatment with fatty acid-bearing albumin but not fatty acid-depleted albumin. The aim of this study was to determine whether a PPARγ agonist, pioglitazone, induces HIF-1 activation or not. Treatment with pioglitazone induced HIF-1α mRNA as well as PPARγ mRNA expression in a concentration dependent manner. In addition, pioglitazone increased HIF-1 target genes such as the mRNAs of glucose transporter 1 (GLUT1) and breast cancer resistance protein (BCRP/ABCG2), in a concentration-dependent manner. Consistent with the increases in GLUT1 and ABCG2 mRNAs, protein expression of GLUT1 and BCRP was increased by pioglitazone. In addition, GLUT inhibitor phloretin-sensitive D-[3H]glucose uptake activity and BCRP inhibitor Ko143-sensitive accumulation of Hoecsht33342, a BCRP substrate, were significantly enhanced by treatment with pioglitazone. These findings suggest that PPARγ activation by pioglitazone leads to HIF-1 protein expression induction followed by changes in HIF-1 target gene expression and protein product activity.

Cobalt Chloride Induces Expression and Function of Breast Cancer Resistance Protein (BCRP/ABCG2) in Human Renal Proximal Tubular Epithelial Cell Line HK-2.

The human breast cancer resistance protein (BCRP/ABCG2), a member of the ATP-binding cassette transporter family, is a drug transporter restricting absorption and enhancing excretion of many compounds including anticancer drugs. The cis-regulatory elements in the BCRP promoter include a hypoxia response element, i.e., the DNA binding site for hypoxia-inducible factor-1 (HIF-1). In this study, we investigated the effect of cobalt chloride, a chemical inducer of HIF-1α, on the expression and function of BCRP in human renal proximal tubular cell line HK-2. Cobalt chloride treatment significantly increased the mRNA expression of not only glucose transporter 1 (GLUT1), a typical HIF-1 target gene mRNA, but also ABCG2 mRNA in HK-2 cells. The BCRP inhibitor Ko143-sensitive accumulation of BCRP substrates such as Hoechst33342 and mitoxantrone was significantly enhanced by cobalt chloride treatment. In addition, treatment with cobalt chloride significantly increased the Ko143-sensitive accumulation of fluorescein isothiocyanate-labeled methotrexate in HK-2 cells. Furthermore, cobalt chloride treatment attenuated the cytotoxicity induced by mitoxantrone and methotrexate, which might be, at least in part, due to the increase in BCRP-mediated transport activity via HIF-1 activation. These findings indicate that HIF-1 activation protects renal proximal tubular cells against BCRP substrate-induced cytotoxicity by enhancing the expression and function of BCRP in renal proximal tubular cells.

Fatty acid-bearing albumin but not fatty acid-depleted albumin induces HIF-1 activation in human renal proximal tubular epithelial cell line HK-2.

Recently, we found that albumin overload induces expression of the transcription factor hypoxia-inducible factor-1α (HIF-1α) protein and several HIF-1 target genes in human renal proximal tubular epithelial cell line HK-2. In this study, the role of albumin-bound fatty acids in the albumin-induced HIF-1 activation was studied. The enhancing effect of fatty acid-bearing human serum albumin [FA(+)HSA] treatment on HIF-1α protein expression was much greater than that of fatty acid-depleted human serum albumin [FA(-)HSA] treatment. The FA(+)HSA treatment induced HIF-1 target gene mRNAs such as those of glucose transporter 1 (GLUT1), glyceraldehyde 3-phosphate dehydrogenase (GAPDH), and breast cancer resistance protein (BCRP) in concentration-dependent manners, while FA(-)HSA caused no significant increases in these mRNAs. Consistent with increased GLUT1 mRNA, GLUT1 protein expression and GLUT inhibitor cytochalasin B-sensitive d-[(3)H]glucose uptake activity were significantly enhanced by treatment with FA(+)HSA, but not with FA(-)HSA. These findings indicate that fatty acids bound to albumin play a crucial role in albumin-induced HIF-1 activation followed by changes in HIF-1 target gene expression and protein product activity.

Albumin overload induces expression of hypoxia-inducible factor 1α and its target genes in HK-2 human renal proximal tubular cell line.

The aim of this study was to investigate the effect of human serum albumin (HSA) overload on the expression of the transcription factor hypoxia-inducible factor-1α (HIF-1α) in human renal proximal tubular cell line HK-2. First, the cell viability and cytotoxic activity were examined to assess the cellular conditions in HK-2 cells with HSA treatment employed in this study. HSA treatment for 48h decreased the cell viability and increased the leakage of lactate dehydrogenase (LDH) into the medium in a concentration-dependent manner, but the toxicity was relatively mild. Western Blot analysis revealed that HSA treatment induced the expression of HIF-1α protein in a concentration-dependent manner without a change in ß-actin protein expression. Confocal microscopy analysis revealed that HIF-1α protein was predominantly localized in the nucleus but was also observed in the cytoplasm. The HIF-1 target gene mRNAs, glucose transporter 1 and glyceraldehyde 3-phosphate dehydrogenase, were up-regulated by HSA treatment, leading to the increases in the protein expression levels. In addition, the mRNA of HIF-1α was increased by HSA treatment. In conclusion, albumin loading induces HIF-1α in HK-2 cells, resulting in the increases in the expression of proteins of its target genes.

Characterization of protamine uptake by opossum kidney epithelial cells.

Protamine, a mixture of polypeptides that is rich in arginine, has been used clinically as an antidote to heparin overdoses and a complexing agent in a long-acting insulin preparation. When protamine is administered intravenously, its abundant accumulation in the kidneys has been reported. However, the renal uptake mechanism for protamine is not clear. In this study, we examined the transport mechanism for protamine in opossum kidney (OK) cells, a suitable in vitro model for renal proximal tubular epithelial cells. Flow cytometric analysis revealed that the association of fluorescein isothiocyanate (FITC)-labeled protamine from salmon (FITC-protamine) by OK cells was inhibited by unlabeled protamine in a concentration-dependent manner. The association of FITC-protamine was temperature- and energy-dependent. Confocal microscopy analysis showed that the fluorescence was localized in the cytoplasm and nucleus of OK cells. In addition, FITC-protamine association was inhibited by cationic drugs such as polycationic gentamicin and polymixin B, but it was increased by a basic amino acid, arginine. Inhibitors for clathrin- and caveolin-dependent endocytosis showed inhibitory effects on FITC-protamine association. Pretreatment with heparinase III partially but significantly decreased the association of FITC-protamine. These results suggest that protamine may be taken up by OK cells via receptor-mediated endocytosis, which may result in its localization in the cytoplasm and nucleus of the cells.

Effect of cigarette smoke extract on insulin transport in alveolar epithelial cell line A549.

BACKGROUND: The main purpose of this study was to evaluate the effect of cigarette smoke extract (CSE) on insulin transport in alveolar epithelial cells. METHODS: We first examined the effect of CSE pretreatment on cell viability, mRNA expression, and lamellar body structures in A549 cells. Then the effect of CSE pretreatment on FITC-insulin transport was examined. RESULTS: When A549 cells were treated with 30 µg/ml of CSE for 48 h, the expression of some mRNAs abundantly expressed in type II alveolar epithelial cells such as surfactant protein B was significantly increased. Lamellar bodylike structures became more evident with CSE treatment. FITC-insulin uptake from the apical side and subsequent efflux to the basal side was enhanced by CSE treatment in A549 cells. The enhancing effect of CSE on FITC-insulin uptake was concentration-dependent and reversible. A concentration-dependent enhancing effect of CSE on FITC-insulin uptake was also observed in normal, primary cultured alveolar type II epithelial cells isolated from rats. CONCLUSIONS: Treatment of A549 cells by CSE may direct the cells to a more type II-like phenotype. In accordance with this observation, FITC-insulin uptake was enhanced by CSE treatment. These results may partly explain the higher insulin absorption from the lung in smokers than in nonsmokers.

Nanoparticulation of probucol, a poorly water-soluble drug, using a novel wet-milling process to improve in vitro dissolution and in vivo oral absorption.

To improve the dissolution and oral absorption properties of probucol, a novel wet-milling process using the ULTRA APEX MILL was investigated. The particle size of bulk probucol powder was 17.1 µm. However, after wet-milling with dispersing agents such as Gelucire 44/14, Gelucire 50/13, vitamin E-TPGS, and Pluronic F-108, the probucol particle sizes decreased to about 77-176 nm. Scanning electron microscopy (SEM) analysis also suggested that the probucol particles were successfully milled into the nanometer range. An in vitro dissolution study showed that the dissolution rates of all nanopowders were several folds higher than those of the corresponding mixed powders. When orally administered to rats, the AUC values of probucol nanopowders treated with Gelucire 44/14 and 50/13, and vitamin E-TPGS were about 3.06-3.54-folds greater than that of the bulk powder. Therefore, through this study, we have developed a new pharmaceutical technique to improve the dissolution rate and oral absorption of probucol using the ULTRA APEX MILL by wet-milling with various dispersing agents.

Function and expression of ATP-binding cassette transporters in cultured human Y79 retinoblastoma cells.

The aim of this study was to reveal the expression and function of P-glycoprotein and multidrug resistance-associated proteins (MRP), members of the ATP-binding cassette (ABC) superfamily of drug transporters, in cultured human Y79 retinoblastoma cells. ABC transporter mRNA expression was evaluated by conventional reverse transcription-polymerase chain reaction (RT-PCR) and real-time PCR analyses. Cellular accumulation of rhodamine 123 (P-glycoprotein substrate), calcein (MRP substrate), and doxorubicin (P-glycoprotein/MRP substrate) was analyzed by fluorometry. Conventional RT-PCR analysis showed the expression of multidrug resistance 1 (MDR1), MRP1, MRP2 and lung resistance-related protein (LRP) mRNAs. Real-time RT-PCR analysis revealed that the expression levels of the MDR1 and MRP2 genes in Y79 cells were much lower than those in human intestinal cell line Caco-2, while the expression level of MRP1 was higher than that in Caco-2 cells. The accumulation of rhodamine 123 was not enhanced by verapamil or reversin 205, inhibitors of P-glycoprotein, indicating no function of P-glycoprotein in Y79 cells. The accumulation of calcein was significantly increased by various MRP inhibitors including probenecid, indicating that MRP functions in Y79 cells. The accumulation of doxorubicin was increased in the presence of metabolic inhibitors (10 mM 2-deoxyglucose and 5 mM sodium azide). However, most MRP inhibitors such as probenecid and indomethacin did not affect doxorubicin accumulation, while cyclosporin A and taclorimus significantly increased doxorubicin accumulation. These results suggest that MRP, but not P-glycoprotein, functions in Y79 cells, and that the efflux of doxorubicin from Y79 cells may be due to an ATP-dependent transporter, which has not been identified yet.

Pharmacokinetic-pharmacodynamic modelling of the hypoglycaemic effect of pulsatile administration of human insulin in rats.

The relationship between the plasma insulin (INS) concentration-time course and plasma glucose concentration-time course during and after pulsatile INS administration to rats was characterized using a pharmacokinetic-pharmacodynamic (PK-PD) model. A total INS dose of 0.5 IU/kg was intravenously injected in 2 to 20 pulses over a 2-h period. Compared with the single bolus administration, the area under the effect-time curve (AUE) increased depending on the number of pulses, and the AUEs for more than four pulses plateaued at a significantly larger value, which was similar to that after the infusion of a total of 0.5 IU/kg of INS over 2 h. No increase in plasma INS concentration occurred after pulsatile administration. Two indirect response models primarily reflecting the receptor-binding process (IR model) or glucose transporter 4 (GLUT4) translocation (GT model) were applied to describe the PK-PD relationship after single intravenous bolus administration of INS. These models could not explain the observed data after pulsatile administration. However, the IR-GT model, which was a combination of the IR and GT models, successfully explained the effects of pulsatile administration and intravenous infusion. These results indicate that the receptor-binding process and GLUT4 translocation are responsible for the change in AUE after pulsatile administration.

Effect of riboflavin deficiency on development of the cerebral cortex in Slc52a3 knockout mice.

Riboflavin transporter 3 (RFVT3), encoded by the SLC52A3 gene, is important for riboflavin homeostasis in the small intestine, kidney, and placenta. Our previous study demonstrated that Slc52a3 knockout (Slc52a3-/-) mice exhibited neonatal lethality and metabolic disorder due to riboflavin deficiency. Here, we investigated the influence of Slc52a3 gene disruption on brain development using Slc52a3-/- embryos. Slc52a3-/- mice at postnatal day 0 showed hypoplasia of the brain and reduced thickness of cortical layers. At embryonic day 13.5, the formation of Tuj1+ neurons and Tbr2+ intermediate neural progenitors was significantly decreased; no significant difference was observed in the total number and proliferative rate of Pax6+ radial glia. Importantly, the hypoplastic phenotype was rescued upon riboflavin supplementation. Thus, it can be concluded that RFVT3 contributes to riboflavin homeostasis in embryos and that riboflavin itself is required during embryonic development of the cerebral cortex in mice.

Effect of PEGylation of N-WASP181-200 on the inhibitory potency for renal aminoglycoside accumulation.

We previously showed that a 20-residue basic peptide, N-WASP181-200 (NISHTKEKKKGKAKKKRLTK), inhibits renal accumulation of aminoglycoside antibiotics such as gentamicin and arbekacin. The aim of this study is to determine whether PEGylation of N-WASP181-200 enhances its inhibitory potency for renal accumulation of aminoglycosides. N-terminally PEGylated peptide (PEG1k-N-W) was synthesized by conjugating N-WASP181-200 with PEG of approximately 1 kDa using the Fmoc protection/deprotection method. PEG1k-N-W decreased gentamicin binding to isolated rat renal brush-border membrane in a concentration-dependent manner, but the in vitro inhibitory potency of PEG1k-N-W was weaker than that of N-WAP181-200. On the other hand, under in vivo conditions, PEG1k-N-W decreased the renal accumulation of arbekacin more potently than N-WASP181-200. When injected intravenously, PEG1k-N-W showed a 1.7-fold longer plasma half-life relative to N-WASP181-200. In addition, the stability of N-WASP181-200 in renal brush-borer membrane suspension was found to be increased by PEGylation. Our findings suggest that PEGylation of N-WASP181-200 is a useful strategy for reducing dosage of the concomitant with which to decrease renal accumulation in the kidney, leading to prevention of aminoglycoside-induced nephrotoxicity.

Mechanism of insulin uptake in rat alveolar type II and type I-like epithelial cells.

The mechanism underlying the handling of protein and peptide drugs such as insulin in alveolar epithelial cells is still unclear. We therefore examined fluorescein isothiocyanate-labeled (FITC)-insulin uptake in rat primary cultured alveolar type II epithelial cells and in transdifferentiated type I-like cells. FITC-insulin uptake in these cells was much higher than those of FITC-immunoglobulin (IgG), transferrin, and dextran. FITC-insulin uptake was time- and concentration-dependent, and was almost completely inhibited by metabolic inhibitors in both cells, while bafilomycin A(1) inhibited the uptake only in type II cells. Inhibitors of clathrin- and caveolae-mediated endocytosis did not affect FITC-insulin uptake in either type of cell. Dynasore, a dynamin GTPase inhibitor, potently inhibited FITC-insulin uptake in type II cells. These results suggest that the characteristics of insulin uptake in type II and type I cells are different, and dynamin-dependent endocytosis that utilizes neither clathrin nor caveolae is involved in type II cells, while a dynamin-independent pathway is mainly involved in type I cells.

Nanoparticulation of poorly water soluble drugs using a wet-mill process and physicochemical properties of the nanopowders.

In order to improve the dissolution and oral absorption properties of poorly water soluble drugs such as omeprazole, albendazole and danazol, various dispersing agents were added to prepare nanopowder formulations using an ULTRA APEX MILL, which is a wet-mill instrument, and their physicochemical properties were evaluated. Using Pluronic F-108 or F-68 as dispersing agents, slurries containing drug particles having nanometer size were obtained for all model drugs tested. Omeprazole, a heat labile drug, was not degraded by wet-milling and the omeprazole nanoparticles in a milled slurry did not aggregate for 24 h after wet-milling. After lyophilization of these milled slurries containing drug nanoparticles, fine solid white nanopowders were obtained. Scanning electron microscopy (SEM) suggested that the model drugs were milled into nanometer size. X-ray powder diffraction (XRPD) patterns and Differential Scanning Calorimetry (DSC) curves confirmed that all milled drug nanopowders were crystalline, although milling of albendazole nanopowder transformed it to another crystal form. Wet-milling using an ULTRA APEX MILL offers a highly effective approach to produce stable drug nanopowders and is a very useful tool for bioavailability enhancement of poorly water soluble and heat labile drugs.

Novel polymyxin derivatives carrying only three positive charges are effective antibacterial agents.

The lack of novel antibiotics against gram-negative bacteria has reinstated polymyxins as the drugs of last resort to treat serious infections caused by extremely multiresistant gram-negative organisms. However, polymyxins are nephrotoxic, and this feature may complicate therapy or even require its discontinuation. Like that of aminoglycosides, the nephrotoxicity of polymyxins might be related to the highly cationic nature of the molecule. Colistin and polymyxin B carry five positive charges. Here we show that novel polymyxin derivatives carrying only three positive charges are effective antibacterial agents. NAB739 has a cyclic peptide portion identical to that of polymyxin B, but in the linear portion of the peptide, it carries the threonyl-D-serinyl residue (no cationic charges) instead of the diaminobutyryl-threonyl-diaminobutyryl residue (two cationic charges). The MICs of NAB739 for 17 strains of Escherichia coli were identical, or very close, to those of polymyxin B. Furthermore, NAB739 was effective against other polymyxin-susceptible strains of Enterobacteriaceae and against Acinetobacter baumannii. At subinhibitory concentrations, it dramatically sensitized A. baumannii to low concentrations of antibiotics such as rifampin, clarithromycin, vancomycin, fusidic acid, and meropenem. NAB739 methanesulfonate was a prodrug analogous to colistin methanesulfonate. NAB740 was the most active derivative against Pseudomonas aeruginosa. NAB7061 (linear portion of the peptide, threonyl-aminobutyryl) lacked direct antibacterial activity but sensitized the targets to hydrophobic antibiotics by factors up to 2,000. The affinities of the NAB compounds for isolated rat kidney brush border membrane were significantly lower than that of polymyxin B.

Influences of dosage regimen and co-administration of low-molecular weight proteins and basic peptides on renal accumulation of arbekacin in mice.

OBJECTIVES: The objectives of this study were to characterize renal accumulation of arbekacin, an aminoglycoside antibiotic for treatment of infections with methicillin-resistant Staphylococcus aureus, and to modulate renal uptake of arbekacin, leading to prevention of arbekacin-induced nephrotoxicity. METHODS: In vivo renal uptake studies were performed using mice. Renal concentrations of arbekacin after a bolus intravenous administration at various doses were analysed by HPLC. In addition, renal concentrations were investigated 24 h after an injection of arbekacin alone or in combination with low-molecular weight proteins and basic peptides. RESULTS: When administered by bolus injection at various doses, renal accumulation of arbekacin showed saturation kinetics with increasing dose. Renal concentration of arbekacin after a bolus administration remained constant from 4 to 24 h and subsequently decreased by a first-order process with a half-life of 42.7 h. The influences of three dosage regimens (a single injection of 4 mg/kg, two injections of 2 mg/kg and three injections of 1.33 mg/kg) were investigated. A single injection resulted in lower renal level of arbekacin than the multiple administrations. Co-administration of cytochrome c, lysozyme and N-WASP181-200 decreased renal accumulation of arbekacin in a dose-dependent manner. N-W(N1n), N-W(N1n,I2i,S3s) and N-W(N1n,K20k), in which the N- and/or C-terminal regions of N-WASP181-200 were substituted by one to three D-isomers, more potently decreased renal arbekacin accumulation than N-WASP181-200. CONCLUSIONS: These data may be useful for prevention of arbekacin-induced nephrotoxicity owing to reduction of renal accumulation of the aminoglycoside.

Modulation of function of multidrug resistance associated-proteins by Kaempferia parviflora extracts and their components.

In this study, the effects of extracts and flavone derivatives from the rhizome of Kaempferia parviflora on multidrug resistance associated-proteins (MRP)-mediated transport in A549 cells were examined. The cells employed express MRP1 and MRP2, but not P-glycoprotein. The cellular accumulation of calcein, an MRP substrate, was significantly increased by various MRP inhibitors without being affected by verapamil, a typical P-glycoprotein inhibitor. Ethanol and aqueous extracts from K. parviflora rhizome increased the accumulation of calcein and doxorubicin in A549 cells in a concentration-dependent manner. The inhibitory potency of the ethanol extract for MRP function was greater than that of the aqueous extract. Among six flavone derivatives isolated from K. parviflora rhizome, 5,7-dimethoxyflavone exhibited a maximal stimulatory effect on the accumulation of doxorubicin in A549 cells. The accumulation of doxorubicin was increased by four flavone derivatives without 5-hydroxy group, but not by the other two flavone derivatives with 5-hydroxy group. In addition, 5,7-dimethoxyflavone and 3,5,7,3',4'-pentamethoxyflavone decreased resistance to doxorubicin in A549 cells. These findings indicate that extracts and flavone derivatives from the rhizome of K. parviflora suppress MRP function, and therefore may be useful as modulators of multidrug resistance in cancer cells.

Effects of Kaempferia parviflora extracts and their flavone constituents on P-glycoprotein function.

The purpose of this study was to examine the effects of extracts and flavone derivatives from the rhizome of Kaempferia parviflora on P-glycoprotein (P-gp)-mediated transport in LLC-GA5-COL150, a transfectant cell line of a porcine kidney epithelial cell line LLC-PK1 with human MDR1 cDNA. Ethanol extract obtained from Kaempferia parviflora rhizome significantly increased the accumulation of rhodamine 123 and daunorubicin, P-gp substrates, in LLC-GA5-COL150 cells, but not in LLC-PK1 cells. The aqueous extract also increased the accumulation in LLC-GA5-COL150 cells with lower potency than the ethanol extract. The effects of flavone derivatives isolated from the rhizome of Kaempferia parviflora on P-gp function were examined. Among six flavones tested, 3,5,7,3',4'-pentamethoxyflavone most potently increased the accumulation of rhodamine 123 and daunorubicin in LLC-GA5-COL150 cells in a concentration-dependent manner. In addition, 5,7-dimethoxyflavone to lesser degree increased rhodamine 123 accumulation in LLC-GA5-COL150 cells. In contrast, the other four flavone derivatives had no significant effect on the accumulation of rhodamine 123 in LLC-GA5-COL150 cells in a concentration range tested. These results indicate that extracts and flavone derivatives from the rhizome of Kaempferia parviflora can inhibit P-gp function, which may be useful for overcoming P-gp-mediated multidrug resistance and improving the oral bioavailability of anticancer agents.

Role of intestinal efflux transporters in the intestinal absorption of methotrexate in rats.

The role of intestinal efflux transporters such as P-glycoprotein (P-gp), breast cancer resistance protein (BCRP) and multidrug resistance-associated proteins (MRPs) in intestinal absorption of methotrexate was examined in rats. In everted intestine, the mucosal efflux of methotrexate after application to serosal side was higher in jejunum than ileum, and the efflux in jejunum was suppressed by pantoprazole, a BCRP inhibitor, and probenecid, an MRP inhibitor, but not by verapamil, a P-gp inhibitor. The mucosal methotrexate efflux in ileum was suppressed by pantoprazole, but not by other inhibitors. On the other hand, the serosal efflux of methotrexate after application to mucosal side was greater in ileum than jejunum, and was suppressed by probenecid. In in-vivo rat studies, the intestinal absorption of methotrexate was significantly higher when methotrexate was administered to ileum than jejunum. Pantoprazole increased methotrexate absorption from jejunum and ileum. Probenecid increased the absorption of methotrexate from jejunum but decreased the absorption from ileum, as evaluated by peak plasma methotrexate levels. In conclusion, BCRP and MRPs are involved in the regional difference in absorption of methotrexate along the intestine, depending on their expression sites.