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The Plasmodium falciparum genome encodes 16 members of ABC proteins, with one member of the ABCG subfamily (PfABCG). Analysis of PfABCG amino acid sequence shows equal sequence identity to hsABCG1 and G2. Using N-terminal directed antibody against a recombinant fragment of PfABCG, we show that PfABCG migrates with an apparent molecular mass of 65KDa polypeptide on SDS-PAGE. PfABCG is expressed in all four stages of the parasite erythrocytic life cycle, with lower and higher expression in ring and late trophozoite stages, respectively. The protein localizes to the plasma membrane and a novel spherical structure beneath the cell membrane. Similar localization is also observed in gametocytes where PfABCG is highly expressed. Analysis of PfABCG genomic sequences for polymorphisms and changes in protein expression between different strains of P. falciparum revealed identical nucleotide sequence among the different strains, but variable protein expression. PfABCG expression is least in HB3 chloroquine sensitive strain, while higher expression levels are seen in other chloroquine-sensitive and -resistant strains, with highest levels of expression in 7G8. The differential expression of PfABCG in three chloroquine-sensitive strains (e.g., 3D7, HB3 and D10) predicts the sensitivity of the different strains to ketotifen, an anti-histaminic drug, whereby low expression is associated with decreased sensitivity to ketotifen. Taken together, the results in this report provide the first description of native PfABCG expression and subcellular localization in asexual stages of the parasite and its localization in gametocytes. It remains to be determined if PfABCG is functionally equivalent to mammalian ABCG1, ABCG2 or both. [ABSTRACT FROM AUTHOR]

Abstract: DPPE, a tamoxifen derivative with antihistamine activity, was previously shown to potentiate the toxicity of chemotherapeutic drugs. Recently, a Phase III clinical study using doxorubicin with DPPE demonstrated significant increase in the overall survival of breast cancer patients. In this study we examined the effects of DPPE alone on the growth of drug sensitive and P-gp positive CHO cell line. Our results demonstrate DPPE is selectively toxic to P-gp positive cells and the sensitivity to DPPE alone correlated with the levels of P-gp expression. Moreover, in MDR cells, DPPE-induced apoptosis was significantly reduced with Bcl2 overexpression and in the presence of P-gp ATPase inhibitor, PSC833. Furthermore, knockdown of P-gp expression in MDR cells with P-gp-siRNA reversed DPPE sensitivity and increased their sensitivity to doxorubicin and taxol but not to cisplatin. The addition of DPPE to membrane fractions led to dose-dependent increase in P-gp ATPase that was inhibited with PSC833. Moreover, incubation of P-gp positive cells with DPPE led to a significant increase in superoxide levels and a drop in cellular ATP and GSH pools that were reversible with inhibitors of P-gp ATPase. The combined presence of DPPE and the mitochondria electron transport complex III inhibitor, antimycin A, synergized in their effects on the growth of MDR cells but had no effect on the growth of parental drug sensitive cells. Collectively, the results of this study provide a possible mechanism that may be relevant to the clinical results of DPPE in breast cancer trial and demonstrates DPPE as P-gp collateral sensitivity drug. [Copyright &y& Elsevier]

Abstract The malaria parasite, Plasmodium spp., produces hemozoin (Hz) crystals as a by-product of hemoglobin digestion. Purification methods used to remove host or parasite products adsorbed on Hz surface lead to variable and undetermined residues. This compositional variation likely accounts for the assortment of contradictory results in studies of Hz's biomineralization, immunomodulating properties, and the mechanism of action of some antimalarials. In this work, we study the surface of Hz cleaned with two methods, both reported in the literature, one stricter than the other. We find that biomolecules are adsorbed on Hz treated with either method, they bind through carboxylate groups, and may be present within Hz structure. Their composition and amount depend on the washing protocol, which also introduces contaminants. This finding led us to question the concept of "pure" Hz, and to propose x-ray photoelectron spectroscopy (XPS) and matrix-assisted laser desorption/ionization time of flight (MALDI-TOF) as characterization tools to assess surface contamination prior to further work on Hz crystals. Graphical abstract Organic contaminants are strongly adsorbed onto hemozoin surface despite extensive purification. The contaminants adsorb on hemozoin mainly through surface carboxylate groups, and could be present within its crystalline structure. These results explain discrepancies related to the immunomodulatory properties of hemozoin and will help designing new antimalarials and understanding hemozoin formation. Unlabelled Image Highlights • Organic contaminants remain adsorbed onto hemozoin even after purification. • The purification protocol introduces contamination to hemozoin surface. • The residual biomolecules could also be part of the crystalline structure of hemozoin. • Surface carboxylate groups in hemozoin are the main adsorption sites for biomolecules. [ABSTRACT FROM AUTHOR]

Malaria is a major disease in the tropics where chemotherapy remains the main mode of treatment and as such the rise and spread of drug-resistant malaria can lead to human tragedy. Two membrane transport proteins, PfMDR1 (Plasmodium falciparum multidrug resistance protein 1) and PfCRT (P. falciparum chloroquine resistance transporter), have been shown to cause resistance to several antimalarials. Both PfMDR1 and PfCRT are localized to the digestive vacuolar membrane and appear to regulate the transport of drugs and physiological metabolites. In this study we have used MK571, a 2-amino quinoline, to explore its interaction with PfMDR1 and PfCRT in chloroquine-sensitive and -resistant strains of P. falciparum. Our results show that chloroquine-resistant strains (e.g., K1, Dd2, and 7G8) are consistently more sensitive to MK571 than chloroquine-sensitive strains (e.g., 3D7, 106/1 and D10). This association, however, was not maintained with the chloroquine-resistant strain FCB which IC50 value was similar to chloroquine-sensitive strains. Moreover, the susceptibility of chloroquine-sensitive and -resistant strains to MK571 does not correlate with mutated PfCRT, nor is it reversible with verapamil; but correlates with mutations in PfMDR1. Furthermore, MK571 appears to target the parasite's digestive vacuole (DV), as demonstrated by the ability of MK571 to: (1) block the accumulation of the fluorescent dye Fluo-4 AM, a PfMDR1 substrate, into the digestive vacuole; (2) reduce the transvacuolar pH gradient; and (3) inhibit the formation of β-hematin in vitro. Moreover, the presence of non-toxic concentrations of MK571 sensitized both chloroquine-sensitive and -resistant parasites to mefloquine and halofantrine, likely by competing against PfMDR1-mediated sequestering of the drugs into the DV compartment and away from the drugs' cytosolic targets. Our data, nevertheless, found only a minimal decrease in MK571 IC50 value in FCB parasite which second pfmdr1 copy was inactivated via gene disruption. Taken together, the findings of this study suggest that MK571 interacts with native and mutant PfMDR1 and modulates the import of drugs or solutes into the parasite's DV and, as such, MK571 may be a useful tool in the characterization of PfMDR1 drug interactions and substrate specificity. [ABSTRACT FROM AUTHOR]

Abstract: The over-expression of ABCC1 transmembrane protein has been shown to cause multidrug resistance in tumor cell lines. ABCC1 is a member of the ABC transmembrane proteins that function as efflux pumps with diverse substrate specificity. Several endogenous cell metabolites, including the leukotriene C4 (LTC4) and glutathione (GSH) are substrates for ABCC1 protein. ABCC1 expression in certain tumor cells was demonstrated to confer hypersensitivity to glutathione modulating agents. In this report we have investigated the mechanism of collateral sensitivity seen in tumor cells over-expressing ABCC1 protein. The results of this study show that ABCC1 expression in tumor cells correlates with their hypersensitivity to various glutathione modulating agents, as demonstrated in H69AR-drug selected and HeLa/ABCC1-transfectant cells. This effect was triggered either through inhibition of GSH synthesis with BSO or by increasing ABCC1-mediated GSH transport with verapamil or apigenin. In addition, our results show that the hypersensitivity of ABCC1-expressing cells to BSO, verapamil or apigenin was preceded by an increase in reactive oxygen species (or ROS). A decrease in GSH level is also observed prior the increase in ROS. In addition, we show that hypersensitivity to the BSO, verapamil or apigenin leads to tumor cell death by apoptosis. Together, the results of this study demonstrate that ABCC1 potentiates oxidative stress in tumor cells through reductions in cellular GSH levels. [Copyright &y& Elsevier]

In malaria, Plasmodium parasites produce hemozoin (Hz) as a route to detoxify free heme released from the catabolism of hemoglobin. Hz isolated from the parasites is encapsulated in an organic layer constituted by parasite and host components. This organic coating may play a role in Hz formation and in the immunomodulatory properties attributed to Hz, and they may influence the mode of action of antimalarials that block Hz formation. In this work, we analyze the organic layer adhered to Hz, and find Na, Cl, Si, Ca and P present, in addition to organic material. Our results suggest that Na, Cl, and P adsorb during Hz release from the red blood cells, while Si and Ca derive from components present during Hz biomineralization within the digestive vacuole of the parasite. Overall, we show that inorganic elements associated with Hz surface provide insights into the biological functions of Plasmodium parasites. Hemozoin crystals collected from malaria parasites present inorganic elements of silicon, calcium, phosphorus, sodium, and chlorine, in addition to organic material. The speciation of the inorganic elements is discussed and points out to unexplored functions in Plasmodium 's biology and possibly other Apicomplexan parasites. Unlabelled Image • Inorganic ions are associated with the organic coating adsorbed on hemozoin surface. • Sodium, chlorine and phosphorus adsorb during hemozoin release from the red blood cells. • Silicon and calcium derive from components present within the parasite. • Silicon is present as silica and, likely, silicon bound to cellular components. • These ions may be related to yet unexplored biological functions of the parasite. [ABSTRACT FROM AUTHOR]