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Thymoquinone (TQ) is the main constituent of the oil extracted from Nigella sativa seeds, which is known to be the active constituent responsible for many of the seed antioxidant and anti-inflammatory effects. The present study was designed to investigate whether TQ can protect against Alzheimer's amyloid-β peptide (Aβ) induced neurotoxicity in rat primary neurons. Cultured hippocampal and cortical neurons were treated with Aβ1-42 and TQ simultaneously for 72 h. Treatment with TQ efficiently attenuated Aβ1-42-induced neurotoxicity, as evidenced by improved cell viability. TQ also inhibited the mitochondrial membrane potential depolarization and reactive oxygen species generation caused by Aβ1-42. In addition, TQ restored synaptic vesicle recycling inhibition, partially reversed the loss of spontaneous firing activity, and inhibited Aβ1-42 aggregation in vitro. These beneficial effects may contribute to the protection against Aβ-induced neurotoxicity. In conclusion, our results suggested that TQ has neuroprotection potential against Aβ1-42 in rat hippocampal and cortical neurons and thus may be a promising candidate for Alzheimer disease treatment., (Copyright © 2013 Elsevier Inc. All rights reserved.)

We have investigated the involvement of intracellular pH (pH(i)) in the regulation of P-glycoprotein (P-gp) in K562/DOX cells. The selective Na(+)/H(+) exchanger1 (NHE1) inhibitor cariporide and the "high K(+)" buffer were used to induce the sustained intracellular acidification of the K562/DOX cells that exhibited more alkaline pH(i) than the K562 cells. The acidification resulted in the decreased P-gp activity with increased Rhodamine 123 (Rh123) accumulation in K562/DOX cells, which could be blocked by the P-gp inhibitor verapamil. Moreover, the acidification decreased MDR1 mRNA and P-gp expression, and promoted the accumulation and distribution of doxorubicin into the cell nucleus. Interestingly, these processes were all pH(i) and time-dependent. Furthermore, the change of the P-gp expression was reversible with the pH(i) recovery. These data indicate that the tumor multidrug resistance (MDR) mediated by P-gp could be reversed by sustained intracellular acidification through down-regulating the P-gp expression and activity, and there is a regulative link between the pH(i) and P-gp in K562/DOX cells.

Multidrug resistance (MDR), which is a significant impediment to the success of cancer chemotherapy, is attributable to the overexpression of membrane transport proteins, such as P-glycoprotein (P-gp), resulting in an increased drug efflux. In this study, we show that the histone deacetylase (HDAC) inhibitor apicidin leads to resistance of HeLa cells to paclitaxel through the induction of P-gp expression. Furthermore, apicidin dramatically increases the release of a fluorescent P-gp substrate, rhodamine 123, from cells. In parallel, apicidin resistance to the apoptotic potential of paclitaxel is associated with induction of P-gp expression in HeLa cells, as evidenced by specific inhibition of P-gp function using either the pharmacological inhibitor verapamil or RNA silencing. We also demonstrate the contribution of apicidin-induced functional P-gp expression to drug resistance using KB cells. Failure of P-gp induction by apicidin does not reverse paclitaxel-induced cytotoxicity in the cells. Although HDAC inhibitors are widely appreciated as a new class of anti-tumor agent, our findings clearly demonstrate that apicidin treatment may lead to P-gp-mediated resistance to other anti-tumor agents, suggesting a need for careful design of clinical applications using HDAC inhibitors.

The ABC transporter P-glycoprotein (P-gp) exerts a critical role in the systemic disposition of and exposure to lipophilic and amphipathic drugs, carcinogens, toxins, and other xenobiotics. The ability of P-gp to transfer a wide variety of structurally unrelated compounds from the cell interior across the membrane bilayer remains intriguing. Since dietary chemicals in green tea (and several other foods) appear to exert anticarcinogenic effects by an unknown mechanism, the constituents are frequently studied for interactions with various biomacromolecules as well as cytotoxins or isolated cells. We characterized several green tea catechins for their interaction with P-gp and their specific effects on P-gp export activity of several marker substrates. Some of these compounds inhibit the active efflux of the fluorescent markers LDS-751 (LDS) and rhodamine 123 (Rho) with low potency. Remarkably, others of these catechins facilitate the P-gp-mediated transport of LDS without affecting daunorubicin (DNR) transport or Rho. Moreover, (-)epicatechin, though an inhibitor of Rho transport, can significantly enhance the active net transport of another P-gp marker substrate, LDS. This result indicates that (-)epicatechin may bind to and activate an allosteric site that enhances P-gp overall function or efficiency. Such a mechanism of heterotropic allosteric enhancement of P-gp could serve as chemoprotective to many cells and contribute to the purported anticarcinogenic effect of green tea consumption.

Multidrug resistance (MDR) cells can be sensitized to anticancer drugs when treated concomitantly with chemosensitizers. In this study, chemosensitizing effects of 5,6,7,3',4'-pentamethoxyflavone (sinensetin) and its analogs were investigated with respect to in vitro efficacy and structure-activity relationship. Sinensetin reversed the resistance of P-glycoprotein (Pgp)-overexpressing AML-2/D100 to vincristine in a concentration-dependent manner. Chemosensitizing effect of sinensetin was 10- and 18-fold higher than those of 5,7,3',4'-tetramethoxyflavone and 3,7-dihydroxy-3',4'-dimethoxyflavone, respectively. Sinensetin cytotoxicity in AML-2/D100 was not changed by the complete inhibition of Pgp, suggesting that it is not a substrate for Pgp. Flow cytometry showed that sinensetin increased drug accumulation in the AML-2/D100 in a concentration-dependent manner. Unlike verapamil and cyclosporin A, the maximum non-cytotoxic concentrations of sinensetin were found to decrease the Pgp levels. Azidopine-binding assay showed that cyclosporin A or verapamil inhibited azidopine binding on Pgp partially but sinensetin did not. Taken together, these results suggest that sinensetin has a chemosensitizing effect in reversing Pgp-mediated MDR by increasing the intracellular accumulation of drugs without competition in a binding site of azidopine. Thus, sinensetin is anticipated as a novel and highly potent second-generation flavonoid chemosensitizer, since sinensetin has significant advantages of having a high therapeutic index, of being a non-transportable inhibitor, and of effecting no induction of Pgp.

Because MDR1 (P-glycoprotein) plays an important role in pharmacokinetics such as absorption and excretion of xenobiotics and multidrug resistance, an understanding of the factors regulating its function and expression is important. Here, the effects of digoxin on cell sensitivity to an anticancer drug, MDR1 function, and expression were examined by assessing the growth inhibition by paclitaxel, the transport characteristics of the MDR1 substrate Rhodamine123, and the level of MDR1 mRNA, respectively, using human colon carcinoma Caco-2 cells, which are widely used as a model of intestinal epithelial cells. The sensitivity to paclitaxel, an MDR1 substrate, in Caco-2 cells pretreated with digoxin was lower than that in non-treated cells. The accumulation of Rhodamine123 was reduced by pretreatment with digoxin and its efflux was enhanced. The level of MDR1 mRNA in Caco-2 cells was increased in a digoxin concentration-dependent manner. These results taken together suggested that digoxin up-regulates MDR1 in Caco-2 cells., ((C)2002 Elsevier Science (USA).)

We have examined the effects of verapamil and PSC 833 on cellular uptake and release of rhodamine 123 (R123) in two human cancer cell lines. Both verapamil and PSC 833 were able to increase R123 accumulation in the multidrug resistant (MDR) MV522/Q6 and KB-8-5 lines in the release study. However, the effects of these drugs on R123 accumulation during accumulation study were quite different. Incubation with PSC 833 increased R123 accumulation in both MDR lines. By contrast, incubation with verapamil only increased R123 accumulation in the KB-8-5 line. The failure of verapamil to increase R123 accumulation in the MV522/Q6 cells can be attributed to the presence of a carrier system in the parent MV522 cells that recognizes both R123 and verapamil, but not PSC 833, as substrates. These results imply that performing R123 accumulation study without first ascertaining possible role of a carrier system for cellular uptake of R123 and putative P-gp modulators might inadvertently lead one to draw improper conclusions on P-gp activity., (Copyright 2000 Academic Press.)

The inherent complexities of cholesterol disposition and metabolism preclude a single transmembrane active transport avenue for this steroid-precursor, cell-membrane constituent. Yet the ABC (ATP binding cassette) transporters are inextricably linked to elements of cholesterol disposition. Recent observations have suggested that, under certain settings, the ABC transporter P-glycoprotein (P-gp) performs a direct role in cholesterol disposition. The gene product of MDR1 (multidrug resistance transporter), P-glycoprotein also confers protection against xenobiotics. Using a whole cell assay in which the retention of a marker substrate is evaluated and quantified, we studied the ability of cholesterol to inhibit directly the function of this transporter. In a NIH-G185 cell line presenting an overexpressed amount of the human transporter P-gp, cholesterol caused dramatic inhibition of daunorubicin transport with an IC(50) of about 8 microM yet had no effect on the parent cell line nor rhodamine 123 transport. Additionally, using the ATP-hydrolysis assay, we showed that cholesterol increases P-gp-mediated ATP hydrolysis by approximately 1.6-fold with a K(s) of 5 microM. Suggesting that cholesterol directly interacts with the substrate binding site of P-gp, these results are consistent with cholesterol being transported by MDR1 P-gp., (Copyright 2000 Academic Press.)

In the present study we show that neutral hexanoyl-(glyco)sphingolipids inhibit P-glycoprotein (Pgp) activity in human ovarian 2780AD cells. By contrast, hexanoylceramide and the gangliosides GM(3) and GM(2) had no effect on Pgp activity, whereas sphingosine had a stimulating effect. In the case of hexanoylglucosylceramide, inhibition of Pgp activity by was reflected by a regained doxorubicin sensitivity of cells, which were grown in medium supplemented with the lipid. Our results lead to the conclusion that a direct transmodulation of Pgp activity by glycolipids occurs, depending on lipid headgroup structure, which can result in reduced resistance to the chemotherapeutic agent doxorubicin., (Copyright 1999 Academic Press.)

Human MDR1 P-glycoprotein (P-gp) is a membrane efflux pump for cytotoxics, whereas MDR3 P-gp is a phosphatidylcholine transporter. We have examined a role for MDR1 P-gp in phosphatidylcholine transport in MDR3-negative epithelial cells that have been induced to express the MDR1 P-gp by exposure to cytotoxics. The accumulation and retention of the fluorescently labelled phosphatidylcholine analogue, C12-NBD-PC, was studied in resistant, KBV1 and MCFadr, and sensitive, KB3-1 and MCF7, cells. Lower accumulation and decreased retention of C12-NBD-PC was evident in resistant cells, e.g., KBV1 accumulated 56%, and MCFadr accumulated 60%, of C12-NBD-PC levels in KB3-1 and MCF7, respectively. Treatment with the MDR1 P-gp inhibitor, verapamil, altered the kinetics of C12-NBD-PC in the resistant cells to more closely follow the pattern of C12-NBD-PC handling by sensitive cells. Comparison of C12-NBD-PC to that of the model MDR1 P-gp substrate, rhodamine-123, indicated phosphatidylcholine turnover kinetics by MDR1 P-gp to be relatively low. The transport by MDR1 P-gp of phosphatidylcholine from inner to outer membrane leaflet may regulate P-gp function and fulfill a role in the MDR1 multidrug-resistant phenotype., (Copyright 1999 Academic Press.)