Effect of Carvacrol and Thymol on NorA efflux pump inhibition in multidrug-resistant (MDR) Staphylococcus aureus strains.

Undue exposure to antimicrobials has led to the acquisition and development of sophisticated bacterial resistance mechanisms, such as efflux pumps, which are able to expel or reduce the intracellular concentration of various antibiotics, making them ineffective. Therefore, inhibiting this mechanism is a promising way to minimize the phenomenon of resistance in bacteria. In this sense, the present study sought to evaluate the activity of the Carvacrol (CAR) and Thymol (THY) terpenes as possible Efflux Pump Inhibitors (EPIs), by determining the Minimum Inhibitory Concentration (MIC) and the association of these compounds in subinhibitory concentrations with the antibiotic Norfloxacin and with Ethidium Bromide (EtBr) against strains SA-1199 (wild-type) and SA-1199B (overexpresses NorA) of Staphylococcus aureus. In order to verify the interaction of the terpenes with the NorA efflux protein, an in silico molecular modeling study was carried out. The assays used to obtain the MIC of CAR and THY were performed by broth microdilution, while the Efflux Pump inhibitory test was performed by the MIC modification method of the antibiotic Norfloxacin and EtBr. docking was performed using the Molegro Virtual Docker (MVD) program. The results of the study revealed that CAR and THY have moderate bacterial activity and are capable of reducing the MIC of Norfloxacin antibiotic and EtBr in strains of S. aureus carrying the NorA efflux pump. The docking results showed that these terpenes act as possible competitive NorA inhibitors and can be investigated as adjuvants in combined therapies aimed at reducing antibiotic resistance.

The role of emodin on cisplatin resistance reversal of lung adenocarcinoma A549/DDP cell.

Exploring drugs that reverse drug resistance and increase the sensitivity of chemotherapy drugs could significantly improve treatment effect of cancer. Our study explored the reversal effect and possible molecular mechanisms of emodin on cisplatin resistance in A549/DDP cells. The IC50 and resistance index of cells were determined by Cell Counting Kit-8 assay. The ability of cell proliferation was evaluated by wound healing assay. Transwell assay was used to detect cell invasion and migration. Apoptosis induction rate was determined by flow cytometry assay and 4',6- diamidino- 2-phenylindole staining. Intracellular concentration was determined by HPLC. Western blot analysis was applied to determine expressions of nuclear factor kappa beta (NF-κB) and its downstream proteins. In this study, we found that the growth inhibitory effect of cisplatin was significantly enhanced by emodin in A549/DDP cells. The combined use of emodin with DDP can effectively promote lung cancer cells apoptosis and inhibit cell migration and invasion. Further investigation indicated that reinforcement effect of emodin and DDP may be associated with inhibition of NF-κB pathway and drug efflux-related proteins such as P-glycoprotein (P-gp), multidrug resistance-associated protein (MRP) and Glutathione S-transferase (GST). The key role of NF-κB was further confirmed by the application of NF-κB inhibitor Ammonium pyrrolidinedithiocarbamate. The intervention of both can significantly increase A549/DDP cell apoptosis and inhibit DDP-induced upregulation of P-gp, MRP and GST. Emodin reverses the cisplatin resistance of tumor cells by down-regulating expression of P-gp, MRP and GST, increasing the intracellular accumulation in A549/DDP cells, and the effect may be associated with the NF-κB pathways.

Antimicrobial activity and inhibition of the NorA efflux pump of Staphylococcus aureus by extract and isolated compounds from Arrabidaea brachypoda.

Arrabidaea brachypoda is a native shrub of the Brazilian Cerrado widely used in the folk medicine for treatment of renal diseases and articular pains. This study aimed to, first, evaluate the antimicrobial activity of both extracts and isolated molecules Brachydins BR-A and BR-B obtained from the flowers of A. brachypoda against Staphylococcus aureus, Escherchia coli and Candida albicans species. A second objective was to investigate if these natural products were able to potentiate the Norfloxacin activity against the strain Staphylococcus aureus SA1199-B that overexpress the norA gene encoding the NorA efflux pump. Extracts and isolated compounds were analyzed by HPLC-PDA and LC-ESI-MS respectively. Minimal inhibitory concentrations of Norfloxacin or Ethidium Bromide (EtBr) were determined in the presence or absence of ethanolic extract, dichloromethane fraction, as well as BR-A or BR-B by microdilution method. Only BR-B showed activity against Candida albicans. Addition of ethanolic extract, dichloromethane fraction or BR-B to the growth media at sub-inhibitory concentrations enhanced the activity of both Norfloxacin and EtBr against S. aureus SA1199-B, indicating that these natural products and its isolated compound BR-B were able to modulate the fluoroquinolone-resistance possibly by inhibition of NorA. Moreover, BR-B inhibited the EtBr efflux in the SA1199-B strain confirming that it is a NorA inhibitor. Isolated BR-B was able to inhibit an important mechanism of multidrug-resistance very prevalent in S. aureus strains, thus its use in combination with Norfloxacin could be considered as an alternative for the treatment of infections caused by S. aureus strains overexpressing norA.

Inhibition of Cyclic GMP Export by Multidrug Resistance Protein 4: A New Strategy to Treat Erectile Dysfunction?

BACKGROUND: Intracellular cyclic guanosine monophosphate (cGMP) concentrations are regulated by degradation enzymes (phosphodiesterases) and by active transport across the plasma membrane by multidrug resistance proteins (MRPs) 4 and 5. AIM: To evaluate the functional effect of MRP-4 inhibition and the role of MRP-4-mediated cGMP export in mouse corpora cavernosa. METHODS: Isometric tension of mouse corpora cavernosa was measured after cumulative addition of MK-571, an inhibitor of MRP-4, or sildenafil, a phosphodiesterase type 5 inhibitor. In addition, the effect of MRP-4 inhibition on cGMP-independent and cGMP-dependent relaxations was studied. In vivo intracavernosal pressure and mean arterial pressure measurements were performed after intracavernosal injection of MK-571. The effect of MRP-4 inhibition on cGMP content was determined using an enzyme immunoassay kit. OUTCOMES: Measurement of the effect of MK-571 on cGMP content, relaxant responses of mouse corpora cavernosa to cGMP-independent and cGMP-dependent vasodilating substances, and determination of the ratio of intracavernosal pressure to mean arterial pressure after intracavernosal injection of MK-571. RESULTS: MK-571 and sildenafil relaxed the corpora cavernosa concentration dependently, with sildenafil being the more potent relaxing compound. Furthermore, MK-571 enhanced relaxing responses to cGMP-dependent substances, such as sodium nitroprusside, sildenafil, acetylcholine, and electrical field stimulation, with the latter even under in vitro diabetic conditions. In contrast, cGMP-independent relaxations were not altered by MRP-4 inhibition. Intracavernosal administration of MK-571 significantly increased intracavernosal pressure, with minimal effect on mean arterial pressure. The cGMP analysis showed that MRP-4 inhibition was accompanied by increased cGMP levels. CLINICAL TRANSLATION: MRP-4, at least when targeted locally in the penis or when combined with a phosphodiesterase type 5 inhibitor, might be a valuable alternative strategy for the treatment of (diabetic) erectile dysfunction. STRENGTHS AND LIMITATIONS: This study is the first to demonstrate an in vitro direct relaxant and an in vivo pro-erectile effect of the MRP-4 inhibitor, MK-571, on mouse corpora cavernosa. However, the functional effect of MRP-5-mediated export in mouse corpora cavernosa was not explored, which has been suggested to play the predominant role in cGMP export. CONCLUSION: Inhibition of MRP-4 increases basal and stimulated levels of cGMP, leading to corpora cavernosa relaxation and penile erection. Therefore, in addition to degradation of cGMP, export of cGMP by MRP-4 could contribute substantially to regulating cGMP levels in mouse corpora cavernosa. Boydens C, Pauwels B, Vanden Daele L, Van de Voorde J. Inhibition of Cyclic GMP Export by Multidrug Resistance Protein 4: A New Strategy to Treat Erectile Dysfunction? J Sex Med 2017;14:502-509.

Acetazolamide Suppresses Multi-Drug Resistance-Related Protein 1 and P-Glycoprotein Expression by Inhibiting Aquaporins Expression in a Mesial Temporal Epilepsy Rat Model.

BACKGROUND Mesial temporal epilepsy (MTLE) is the most common type of focal epilepsy in adults, and is often drug-resistant. This study investigated the effects of aquaporins (AQP) inhibitor on multi-drug-resistant protein expression in an MTLE rat model. MATERIAL AND METHODS The MTLE rat model was established by injecting pilocarpine into rats. The MTLE rats were divided into an MTLE-6 h group, an MTLE-12 h group, and an MTLE-24 h group, together with a normal saline group (NS), to examine the AQP4 expression by using Western blot assay and immunohistochemistry assay. The other 18 MTLE model rats were used to observe the effects of the AQP4 inhibitor, acetazolamide, on the multi-drug-resistant protein 1 (MRP1) and P-glycoprotein (Pgp) by using Western blot and immunohistochemistry assays, respectively. RESULTS AQP4 expression was enhanced in hippocampal tissues of MTLE model rats compared to NS rats (P<0.05). More positively stained AQP4 was discovered in hippocampal tissues of MTLE model rats. AQP4 inhibitor significantly decreased multi-drug-resistant protein MRP1 and Pgp expression in the AQP4 inhibitor Interfere group and the AQP4 inhibitor Therapy group compared to the TMLE model group (P<0.05). CONCLUSIONS The present findings confirm that the AQP4 inhibitor, acetazolamide, effectively inhibits the multi-drug-resistant protein, MRP1, and Pgp, in the MTLE rat model.

Modulated function of multidrug resistance-associated proteins in cisplatin-induced acute renal failure rats.

The effect of cisplatin-induced acute renal failure (ARF) on the function and expression of multidrug resistanceassociated proteins (MRPs) was evaluated in rats. Rats received an intraperitoneal injection of cisplatin (9 mg/kg), and the induction of ARF state with high plasma concentrations of indoxyl sulfate and creatinine was observed 72 h after cisplatin treatment. The function of MRPs in the liver, kidney and brain was evaluated by measuring the tissue accumulation and biliary excretion of 2,4-dintrophenyl-S-glutathione (DNP-SG), a substrate for MRPs, after administration of 1-chloro-2,4-dintrobenzene (CDNB), a precursor of DNP-SG, in rats. The levels of MRP1-4 expression were evaluated by Western blot analysis. Effect of ARF plasma components on MRP function was also examined by using calcein acetoxymethyl ester (calcein-AM) in HepG2 cells. In ARF rats (72 h after cisplatin treatment), the accumulation of DNP-SG in the liver, kidney and brain was significantly higher than those in control and cisplatin-treated rats (1 h after treatment). In ARF rats, intrinsic biliary excretion clearance of DNP-SG, estimated by dividing the biliary excretion rate of DNP-SG with the liver concentration, was also significantly reduced, though the expression levels of MRP1-4 in the liver remained unchanged. ARF rat plasma (5%) significantly increased the accumulation of calcein, a MRP substrate, in HepG2 cells after application of calcein-AM. In conclusion, MRP function was found to be suppressed not only in the kidney but also in the liver and brain in cisplatin-induced ARF rats, possibly due to the accumulation of some MRP substrates/inhibitors in plasma.

Suppression of c-Myc is involved in multi-walled carbon nanotubes' down-regulation of ATP-binding cassette transporters in human colon adenocarcinoma cells.

Over-expression of ATP-binding cassette (ABC) transporters, a large family of integral membrane proteins that decrease cellular drug uptake and accumulation by active extrusion, is one of the major causes of cancer multi-drug resistance (MDR) that frequently leads to failure of chemotherapy. Carbon nanotubes (CNTs)-based drug delivery devices hold great promise in enhancing the efficacy of cancer chemotherapy. However, CNTs' effects on the ABC transporters remain under-investigated. In this study, we found that multiwalled carbon nanotubes (MWCNTs) reduced transport activity and expression of ABC transporters including ABCB1/Pgp and ABCC4/MRP4 in human colon adenocarcinoma Caco-2 cells. Proto-oncogene c-Myc, which directly regulates ABC gene expression, was concurrently decreased in MWCNT-treated cells and forced over-expression of c-Myc reversed MWCNTs' inhibitory effects on ABCB1 and ABCC4 expression. MWCNT-cell membrane interaction and cell membrane oxidative damage were observed. However, antioxidants such as vitamin C, ß-mecaptoethanol and dimethylthiourea failed to antagonize MWCNTs' down-regulation of ABC transporters. These data suggest that MWCNTs may act on c-Myc, but not through oxidative stress, to down-regulate ABC transporter expression. Our findings thus shed light on CNTs' novel cellular effects that may be utilized to develop CNTs-based drug delivery devices to overcome ABC transporter-mediated cancer chemoresistance.

Taurolithocholate-induced MRP2 retrieval involves MARCKS phosphorylation by protein kinase Cϵ in HUH-NTCP Cells.

UNLABELLED: Taurolithocholate (TLC) acutely inhibits the biliary excretion of multidrug-resistant associated protein 2 (Mrp2) substrates by inducing Mrp2 retrieval from the canalicular membrane, whereas cyclic adenosine monophosphate (cAMP) increases plasma membrane (PM)-MRP2. The effect of TLC may be mediated via protein kinase Cϵ (PKCϵ). Myristoylated alanine-rich C kinase substrate (MARCKS) is a membrane-bound F-actin crosslinking protein and is phosphorylated by PKCs. MARCKS phosphorylation has been implicated in endocytosis, and the underlying mechanism appears to be the detachment of phosphorylated myristoylated alanine-rich C kinase substrate (pMARCKS) from the membrane. The aim of the present study was to test the hypothesis that TLC-induced MRP2 retrieval involves PKCϵ-mediated MARCKS phosphorylation. Studies were conducted in HuH7 cells stably transfected with sodium taurocholate cotransporting polypeptide (HuH-NTCP cells) and in rat hepatocytes. TLC increased PM-PKCϵ and decreased PM-MRP2 in both HuH-NTCP cells and hepatocytes. cAMP did not affect PM-PKCϵ and increased PM-MRP2 in these cells. In HuH-NTCP cells, dominant-negative (DN) PKCϵ reversed TLC-induced decreases in PM-MRP2 without affecting cAMP-induced increases in PM-MRP2. TLC, but not cAMP, increased MARCKS phosphorylation in HuH-NTCP cells and hepatocytes. TLC and phorbol myristate acetate increased cytosolic pMARCKS and decreased PM-MARCKS in HuH-NTCP cells. TLC failed to increase MARCKS phosphorylation in HuH-NTCP cells transfected with DN-PKCϵ, and this suggested PKCϵ-mediated phosphorylation of MARCKS by TLC. In HuH-NTCP cells transfected with phosphorylation-deficient MARCKS, TLC failed to increase MARCKS phosphorylation or decrease PM-MRP2. CONCLUSION: Taken together, these results support the hypothesis that TLC-induced MRP2 retrieval involves TLC-mediated activation of PKCϵ followed by MARCKS phosphorylation and consequent detachment of MARCKS from the membrane.

Effect of various antibiotics on modulation of intestinal microbiota and bile acid profile in mice.

Antibiotic treatments have been used to modulate intestinal bacteria and investigate the role of intestinal bacteria on bile acid (BA) homeostasis. However, knowledge on which intestinal bacteria and bile acids are modified by antibiotics is limited. In the present study, mice were administered various antibiotics, 47 of the most abundant bacterial species in intestine, as well as individual BAs in plasma, liver, and intestine were quantified. Compared to the two antibiotic combinations (vancomycin+imipenem and cephalothin+neomycin), the three single antibiotics (metronidazole, ciprofloxacin and aztreonam) have less effect on intestinal bacterial profiles, and thus on host BA profiles and mRNA expression of genes that are important for BA homeostasis. The two antibiotic combinations decreased the ratio of Firmicutes to Bacteroidetes in intestine, as well as most secondary BAs in serum, liver and intestine. Additionally, the two antibiotic combinations significantly increased mRNA of the hepatic BA uptake transporters (Ntcp and Oatp1b2) and canalicular BA efflux transporters (Bsep and Mrp2), but decreased mRNA of the hepatic BA synthetic enzyme Cyp8b1, suggesting an elevated enterohepatic circulation of BAs. Interestingly, the two antibiotic combinations tended to have opposite effect on the mRNAs of most intestinal genes, which tended to be inhibited by vancomycin+imipenem but stimulated by cephalothin+neomycin. To conclude, the present study clearly shows that various antibiotics have distinct effects on modulating intestinal bacteria and host BA metabolism.

Induction of hepatic multidrug resistance-associated protein 3 by ethynylestradiol is independent of cholestasis and mediated by estrogen receptor.

Multidrug resistance-associated protein 3 (Mrp3; Abcc3) expression and activity are up-regulated in rat liver after in vivo repeated administration of ethynylestradiol (EE), a cholestatic synthetic estrogen, whereas multidrug resistance-associated protein 2 (Mrp2) is down-regulated. This study was undertaken to determine whether Mrp3 induction results from a direct effect of EE, independent of accumulation of any endogenous common Mrp2/Mrp3 substrates resulting from cholestasis and the potential mediation of estrogen receptor (ER). In in vivo studies, male rats were given a single, noncholestatic dose of EE (5 mg/kg s.c.), and basal bile flow and the biliary excretion rate of bile salts and glutathione were measured 5 hours later. This treatment increased Mrp3 mRNA by 4-fold, detected by real-time polymerase chain reaction, despite the absence of cholestasis. Primary culture of rat hepatocytes incubated with EE (1-10 µM) for 5 hours exhibited a 3-fold increase in Mrp3 mRNA (10 µM), consistent with in vivo findings. The increase in Mrp3 mRNA by EE was prevented by actinomycin D, indicating transcriptional regulation. When hepatocytes were incubated with an ER antagonist [7α,17ß-[9-[(4,4,5,5,5-Pentafluoropentyl)sulfinyl]nonyl]estra-1,3,5(10)-triene-3,17-diol (ICI182/780), 1 µM], in addition to EE, induction of Mrp3 mRNA was abolished, implicating ER as a key mediator. EE induced an increase in ER-α phosphorylation at 30 minutes and expression of c-Jun, a well-known ER target gene, at 60 minutes, as detected by Western blotting of nuclear extracts. These increases were prevented by ICI182/780. In summary, EE increased the expression of hepatic Mrp3 transcriptionally and independently of any cholestatic manifestation and required participation of an ER, most likely ER-α, through its phosphorylation.

Drug metabolism and transport during pregnancy: how does drug disposition change during pregnancy and what are the mechanisms that cause such changes?

There is increasing evidence that pregnancy alters the function of drug-metabolizing enzymes and drug transporters in a gestational-stage and tissue-specific manner. In vivo probe studies have shown that the activity of several hepatic cytochrome P450 enzymes, such as CYP2D6 and CYP3A4, is increased during pregnancy, whereas the activity of others, such as CYP1A2, is decreased. The activity of some renal transporters, including organic cation transporter and P-glycoprotein, also appears to be increased during pregnancy. Although much has been learned, significant gaps still exist in our understanding of the spectrum of drug metabolism and transport genes affected, gestational age-dependent changes in the activity of encoded drug metabolizing and transporting processes, and the mechanisms of pregnancy-induced alterations. In this issue of Drug Metabolism and Disposition, a series of articles is presented that address the predictability, mechanisms, and magnitude of changes in drug metabolism and transport processes during pregnancy. The articles highlight state-of-the-art approaches to studying mechanisms of changes in drug disposition during pregnancy, and illustrate the use and integration of data from in vitro models, animal studies, and human clinical studies. The findings presented show the complex inter-relationships between multiple regulators of drug metabolism and transport genes, such as estrogens, progesterone, and growth hormone, and their effects on enzyme and transporter expression in different tissues. The studies provide the impetus for a mechanism- and evidence-based approach to optimally managing drug therapies during pregnancy and improving treatment outcomes.

Let-7c sensitizes acquired cisplatin-resistant A549 cells by targeting ABCC2 and Bcl-XL.

Cancer cells that develop resistance to cisplatin (DDP) are a major clinical obstacle to the successful treatment of cancer, including non-small cell lung cancer (NSCLC). Recent studies have implicated dysregulation of microRNAs (miRNAs) function in chemoresistance. Here, we explored the role of let-7c in the acquisition of DDP-resistant phenotype in A549 cells. Let-7c was downregulated in A549/DDP cell compared with A549 cells. Modulation of let-7c altered the sensitivity of A549/DDP cells to DDP through regulating DDP-induced apopotis. Furthermore, ABCC2 and Bcl-XL were identified as targets of let-7c. ABCC2 and Bcl-XL knockdown increased DDP sensitivity and DDP-induced apoptosis in A549/DDP cells. In conclusion, our findings suggested for the first time that let-7c modulate DDP response in A549/DDP cells, and one of the mechanisms was through targeting ABCC2 and Bcl-XL. Thus, let-7c could be considered for potential therapeutic application for modulating DDP-based therapy.

Effect of overexpression of small non-coding DsrA RNA on multidrug efflux in Escherichia coli.

OBJECTIVES: Several putative and proven drug efflux pumps are present in Escherichia coli. Because many such efflux pumps have overlapping substrate spectra, it is intriguing that bacteria, with their economically organized genomes, harbour such large sets of multidrug efflux genes. To understand how bacteria utilize these multiple efflux pumps, it is important to elucidate the process of pump expression regulation. The aim of this study was to determine a regulator of the multidrug efflux pump in this organism. METHODS: We screened a genomic library of E. coli for genes that decreased drug susceptibility in this organism. The library was developed from the chromosomal DNA of the MG1655 strain, and then the recombinant plasmids were transformed into an acrB-deleted strain. Transformants were screened for resistance to various antibiotics including oxacillin. RESULTS: We found that the multidrug susceptibilities of the acrB-deleted strain were decreased by the overexpression of small non-coding DsrA RNA as well as by the overexpression of known regulators of multidrug efflux pumps. Plasmids carrying the dsrA gene conferred resistance to oxacillin, cloxacillin, erythromycin, rhodamine 6G and novobiocin. DsrA decreased the accumulation of ethidium bromide in E. coli cells. Furthermore, expression of mdtE was significantly increased by dsrA overexpression, and the decreased multidrug susceptibilities modulated by DsrA were dependent on the MdtEF efflux pump. CONCLUSIONS: These results indicate that DsrA modulates multidrug efflux through activation of genes encoding the MdtEF pump in E. coli.

Orange juice and its component, hesperidin, decrease the expression of multidrug resistance-associated protein 2 in rat small intestine and liver.

We investigated the effects of orange juice (OJ) or hesperidin, a component of OJ, on the pharmacokinetics of pravastatin (PRV) and the expression of both protein and mRNA of multidrug resistance-associated protein 2 (Mrp2) in the rat small intestine and liver. Eight-week-old male Sprague-Dawley rats were used in this study. OJ or a 0.079% hesperidin suspension was administered orally for 2 days. Tap water was given as a control. A single dose of PRV at 100 mg/kg p.o. was administered after 2 days of OJ, hesperidin, or tap water ingestion. The AUC, C(max), and t(1/2) values of PRV were significantly increased in OJ group. Mrp2 protein and mRNA levels in the small intestine and liver, respectively, were significantly decreased after the ingestion of OJ. The same results were obtained with hesperidin. These results suggest that the changes in PRV pharmacokinetic parameters and the decrease in Mrp2 expression caused by OJ are due to hesperidin in the juice.

Synergy-directed fractionation of botanical medicines: a case study with goldenseal (Hydrastis canadensis).

It is often argued that the efficacy of herbal medicines is a result of the combined action of multiple constituents that work synergistically or additively. Determining the bioactive constituents in these mixtures poses a significant challenge. We have developed an approach to address this challenge, synergy-directed fractionation, which combines comprehensive mass spectrometry profiling with synergy assays and natural products isolation. The applicability of synergy-directed fractionation was demonstrated using the botanical medicine goldenseal (Hydrastis canadensis) as a case study. Three synergists from goldenseal were identified, sideroxylin, 8-desmethyl-sideroxylin, and 6-desmethyl-sideroxylin. These flavonoids synergistically enhance the antimicrobial activity of the alkaloid berberine (also a constituent of H. canadensis) against Staphylococcus aureus by inhibition of the NorA multidrug resistance pump. The flavonoids possess no inherent antimicrobial activity against S. aureus; therefore, they could have been missed using traditional bioactivity-directed fractionation. The flavonoid synergists are present at higher concentration in extracts from H. canadensis leaves, while the antimicrobial alkaloid berberine is present at higher levels in H. canadensis roots. Thus, it may be possible to produce an extract with optimal activity against S. aureus using a combination of goldenseal roots and leaves.

Study on mechanism of low bioavailability of black tea theaflavins by using Caco-2 cell monolayer.

This study aimed to clarify the bioavailability mechanism of theaflavins by using the Caco-2 monolayer in vitro model. Prior to the transport of theaflavin (TF), theaflavin-3-gallate (TF3G), theaflavin-3'-gallate (TF3'G), and theaflavin-3, 3'-digallate (TFDG), we found the cytotoxicity of theaflavins was in the order of TF3'G > TFDG > TF3G > TF, suggesting the galloyl moiety enhances the cytotoxicity of theaflavins. Meantime, the galloyl moiety made theaflavins unstable, with the stability in the order of TF > TFDG > TF3G/TF3'G. Four theaflavins showed poor bioavailability with the Papp values ranging from 0.44 × 10-7 to 3.64 × 10-7 cm/s in the absorptive transport. All the theaflavins showed an efflux ratio of over 1.24. And it is further confirmed that P-glycoprotein (P-gp), multidrug resistance associated proteins (MRPs) and breast cancer resistance protein (BCRP) were all shown to contribute to the efflux transport of four theaflavins, with P-gp playing the most important role, followed by MRPs and BCRP. Moreover, theaflavins increased the expression of P-gp, MRP1, MPR3, and BCRP while decreased the expression of MRP2 at the transcription and translation levels. Additionally, the gallated theaflavins were degraded into simple theaflavins and gallic acids when transported through Caco-2 monolayers. Overall, the structural instability, efflux transporters, and cell metabolism were all responsible for the low bioavailability of four theaflavins in Caco-2 monolayers.

Synergism of hydroxyapatite nanoparticles and recombinant mutant human tumour necrosis factor-alpha in chemotherapy of multidrug-resistant hepatocellular carcinoma.

BACKGROUND/AIMS: Locoregional chemotherapy continues to be the mainstay for the treatment of unresectable hepatocellular carcinoma (HCC). One of the principal obstacles implicated in its unsuccessful therapy is multidrug resistance (MDR). Former studies have identified the multidrug-resistant nature and possible mechanisms of hepatoma cells both in vitro and in vivo. This work aimed to develop an effective strategy for the treatment of HCC with MDR. METHODS: The treatment was exploited to inhibit the MDR cells by co-administration of the recombinant mutant human tumour necrosis factor-alpha (rmhTNF-alpha), a sublethal dose of chemicals [adriamycin (ADM), mitomycin and 5-FU] and hydroxyapatite nanoparticles (nHAPs). Real-time quantitative reverse transcriptase-polymerase chain reaction and electrochemiluminescence Western blot were used to detect the expression of several related genes. RESULTS: The chemicals acted synergistically with rmhTNF-alpha and nHAP in suppressing the growth of hepatoma cells and inducing apoptosis of the cells, with the MDR phenotype reversed, as measured by intracellular ADM retention. Analysis of mRNA and protein revealed that rmhTNF-alpha inhibited the gene expression of XIAP, survivin, Ki67, PCNA, MDR1 and BCRP to some extent. Moreover, the inhibitory effects mentioned above could be as good or better than when nHAP is incorporated into the regimens. CONCLUSIONS: rmhTNF-alpha was not only able to restore the chemotherapeutic sensitivity to HepG2/ADM, its xenograft model and clinical samples but also further inhibited the growth of these tumours by a combination of nHAP. These results strongly suggested that chemicals in combination with rmhTNF-alpha and nHAP may be beneficial for the local treatment of advanced HCC.

Norlignans, acylphloroglucinols, and a dimeric xanthone from Hypericum chinense.

Two new norlignans, hyperiones A (1) and B (2), three new acylphloroglucinols, aspidinol C (3) and hyperaspidinols A (5) and B (6), the known compound aspidinol D (4), and the symmetrical dimeric xanthone hyperidixanthone (7) were isolated from Hypericum chinense. Their structures were established by spectroscopic analysis. In an antibacterial assay using a panel of multidrug-resistant (MDR) strains, compounds 3 and 4 exhibited promising activity against the NorA efflux protein overexpressing MDR Staphylococcus aureus strain SA-1199B with a minimum inhibitory concentration (MIC) of 2 µg/mL (8.4 µM) and 4 µg/mL (16.8 µM), respectively. The positive control antibiotic norfloxacin showed activity at MIC 32 µg/mL (100 µM).

Using a qPCR device to screen for modulators of ABC transporter activity: A step-by-step protocol.

INTRODUCTION: Adenosine triphosphate (ATP)-binding cassette (ABC) transporters are transmembrane proteins which actively transport a large variety of substrates across biological membranes. ABC transporter overexpression can be the underlying cause of multidrug resistance in oncology. Moreover, it has been revealed that increased ABCC1 transporter activity can ameliorate behavioural changes and Aß pathology in a rodent model of Alzheimer's disease and it is currently tested in AD patients. METHODS: Finding substances that modulate ABC transporter activity (inhibitors and activators) is of high relevance and thus, different methods have been developed to screen for potential modulators. For this purpose, we have developed a cell-based assay to measure the kinetics of ABCC1-mediated efflux of a fluorescent dye using a common qPCR device (Agilent AriaMx). RESULTS: We validated the specificity of our method with vanadate and benzbromarone controls. Furthermore, we provide a step-by-step protocol including statistical analysis of the resulting data and suggestions how to modify the protocol specifically to screen for activators of ABCC1. DISCUSSION: Our approach is biologically more relevant than cell-free assays. The continuous detection of kinetics allows for a more precise quantification compared with assays with single end-point measurements.

Sensitizing multi drug resistant Staphylococcus aureus isolated from surgical site infections to antimicrobials by efflux pump inhibitors.

BACKGROUND: Staphylococcus aureus is a common hospital acquired infections pathogen. Multidrug-resistant Methicillin-resistant Staphylococcus aureus represents a major problem in Egyptian hospitals. The over-expression of efflux pumps is a main cause of multidrug resistance. The discovery of efflux pump inhibitors may help fight multidrug resistance by sensitizing bacteria to antibiotics. This study aimed to investigate the role of efflux pumps in multidrug resistance. METHODS: Twenty multidrug resistant S. aureus isolates were selected. Efflux pumps were screened by ethidium bromide agar cartwheel method and polymerase chain reaction. The efflux pump inhibition by seven agents was tested by ethidium bromide agar cartwheel method and the effect on sensitivity to selected antimicrobials was investigated by broth microdilution method. RESULTS: Seventy percent of isolates showed strong efflux activity, while 30% showed intermediate activity. The efflux genes mdeA, norB, norC, norA and sepA were found to play the major role in efflux, while genes mepA, smr and qacA/B had a minor role. Verapamil and metformin showed significant efflux inhibition and increased the sensitivity to tested antimicrobials, while vildagliptin, atorvastatin, domperidone, mebeverine and nifuroxazide showed no effect. CONCLUSION: Efflux pumps are involved in multidrug resistance in Staphylococcus aureus. Efflux pump inhibitors could increase the sensitivity to antimicrobials.