LmrA is an ATP binding cassette (ABC) multidrug transporter in Lactococcus lactis

The emergence of multidrug resistant cancer cells is a serious problem in the chemotherapeutic treatment of human tumors. In mammals, multidrug resistance based on the active extrusion of cytotoxic drugs from the cell is mediated by several members of the ATP-binding cassette (ABC) 1 superfamily. These include the multidrug resistance P-glycoprotein MDR1 (also termed ABCB1) and the protein MRP1 (multidrug resistance-associated protein 1, also termed ABCC1) (1). The breast cancer resistance protein (BCRP, also termed MXR, ABCP, or ABCG2) is one of the more recently discovered ABC multidrug transporters in human cancer cells. BCRP confers resistance on cells to (i) toxic ions such as rhodamine 123, (ii) anticancer agents including mitoxantrone and the anthracyclines daunomycin and doxorubicin, and (iii) the camptothecins topotecan and SN-38 (2-7). Overexpression of BCRP has been observed in several human cancer cell lines selected for drug resistance (2, 5, 8) as well as in tumor samples of cancer patients (9 -11). Recently, fumitremorgin C (FTC), a novel chemosensitizing agent, was identified and shown to reverse drug resistance in human BCRP-expressing cancer cells by inhibiting BCRP-mediated drug transport (12).BCRP is a 655-amino acid, 72.1-kDa protein and is the second member of the G subfamily of ABC transporters. Members of the G subfamily are all half-transporters and include among others (i) the Drosophila white, brown, and scarlet proteins, which are involved in the transport of eye pigment (13); (ii) ABCG1, which is thought to be involved in the transport of cholesterol and phospholipids (14); and (iii) heterodimeric ABCG5/ABCG8, which has been implicated in the transport of cholesterol and plant sterols (15). In contrast to P-glycoprotein MDR1 and MRP1, which are full size transporters, BCRP most likely functions as a homodimer (16).In normal tissue, high expression of the BCRP is found in stem cells (17), epithelial cells of small and large intestines, ducts and lobules of the breast, endothelial cells of veins and capillaries (18), and synchitiotrophoblastic cells of the placenta (19). The localization of BCRP suggests that it could have a potential role in protection against toxins. The recent observation in BCRP knock-out mice that BCRP protects against a chlorophyll-derived dietary phototoxin and protoporphyria is consistent with this notion (20).Previously, we have characterized the molecular basis of the drug specificity of LmrA, a half-transporter homologue of human P-glycoprotein MDR1, in the Gram-positive bacterium Lactococcus lactis (21,22). To allow a detailed comparison of BCRP and LmrA, human BCRP was functionally expressed in L. lactis using the nisin A-induced expression system that is used for the expression of LmrA. BCRP was active as an ATPdependent multidrug transporter in L. lactis and was able to interact with sterols. We conclude that the substrate specificity of BCRP partly overlaps with that proposed for ABCG1 and ABCG5/ABCG8. Our observations may suggest a physiological role f...

The concept that extracellular vesicles (EVs) from the diet can be absorbed by the intestinal tract of the consuming organism, be bioavailable in various organs, and in-turn exert phenotypic changes is highly debatable. Here, we isolate EVs from both raw and commercial bovine milk and characterize them by electron microscopy, nanoparticle tracking analysis, western blotting, quantitative proteomics and small RNA sequencing analysis. Orally administered bovine milk-derived EVs survive the harsh degrading conditions of the gut, in mice, and is subsequently detected in multiple organs. Milk-derived EVs orally administered to mice implanted with colorectal and breast cancer cells reduce the primary tumor burden. Intriguingly, despite the reduction in primary tumor growth, milk-derived EVs accelerate metastasis in breast and pancreatic cancer mouse models. Proteomic and biochemical analysis reveal the induction of senescence and epithelial-to-mesenchymal transition in cancer cells upon treatment with milk-derived EVs. Timing of EV administration is critical as oral administration after resection of the primary tumor reverses the pro-metastatic effects of milk-derived EVs in breast cancer models. Taken together, our study provides context-based and opposing roles of milk-derived EVs as metastasis inducers and suppressors.

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Summary Melaminophenyl arsenical drugs are a mainstay of chemotherapy against late‐stage African sleeping sickness, but drug resistance is increasingly prevalent. We describe here the characterization of two genes encoding putative metal–thiol conjugate transporters from Trypanosoma brucei. The two proteins, TbMRPA and TbMRPE, were each overexpressed in trypanosomes, with or without co‐expression of two key enzymes in trypanothione biosynthesis, ornithine decarboxylase and gamma‐glutamyl‐cysteine synthetase. Overexpression of gamma‐glutamyl‐cysteine synthetase resulted in a twofold increase in cellular trypanothione, whereas overexpression of ornithine decarboxylase had no effect on the trypanothione level. The overexpression of TbMRPA resulted in a 10‐fold increase in the IC50 of melarsoprol. The overexpression of the trypanothione biosynthetic enzymes alone gave two‐ to fourfold melarsoprol resistance, but did not enhance resistance caused by MRPA. Overexpression of TbMRPE had little effect on susceptibility to melarsoprol but did give two‐ to threefold resistance to suramin.

The human BCRP (breast cancer resistance protein, also known as ABCG2) is an ABC (ATP-binding cassette) transporter that extrudes various anticancer drugs from cells, causing multidrug resistance. To study the molecular determinants of drug specificity of BCRP in more detail, we have expressed wild-type BCRP (BCRP-R) and the drug-selected cancer cell line-associated R482G (Arg482-->Gly) mutant BCRP (BCRP-G) in Lactococcus lactis. Drug resistance and the rate of drug efflux in BCRP-expressing cells were proportional to the expression level of the protein and affected by the R482G mutation, pointing to a direct role of BCRP in drug transport in L. lactis. In agreement with observations in mammalian cells, the BCRP-R-mediated transport of the cationic substrates rhodamine 123 and tetramethylrosamine was significantly decreased compared with the activity of BCRP-G. In addition, BCRP-R showed an enhanced interaction with the anionic anticancer drug methotrexate when compared with BCRP-G, suggesting that structure/substrate specificity relationships in BCRP, as observed in eukaryotic expression systems, are maintained in prokaryotic L. lactis. Interestingly, BCRP-R exhibited a previously unestablished ability to transport antibiotics, unconjugated sterols and primary bile acids in L. lactis, for which the R482G mutation was not critical. Since Arg482 is predicted to be present in the intracellular domain of BCRP, close to transmembrane segment 3, our results point to a role of this residue in electrostatic interactions with charged substrates including rhodamine 123 and methotrexate. Since unconjugated sterols are neutral molecules and bile acids and many antibiotics are engaged in protonation/deprotonation equilibria at physiological pH, our observations may point either to a lack of interaction between Arg482 and neutral or neutralized moieties in these substrates during transport or to the interaction of these substrates with regions in BCRP not including Arg482.

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