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Papers by elias georges

The Journal of Biochemistry

The emergence of drug resistance remains an obstacle in the clinical treatment of cancer. Recent ... more The emergence of drug resistance remains an obstacle in the clinical treatment of cancer. Recent developments in the studies of drug resistance have identified compounds such as verapamil and tamoxifen that specifically target ABCB1-expressing multidrug-resistant (MDR) cells, through an ATP-dependent ROS-generating mechanism. In this report, we demonstrate that treatment of ABCB1-expressing MDR cells (CHORC5 or MDA-Doxo400) or individual clones of the latter with sub-lethal concentrations of tamoxifen or verapamil down-regulates ABCB1 protein and mRNA expression in surviving clones. Consequently, tamoxifen- and verapamil-treated cells show increased sensitivity to chemotherapeutic drugs (e.g., colchicine and doxorubicin) and decreased sensitivity to collateral sensitivity drugs (e.g., verapamil and tamoxifen). Importantly, we show for the first time that down-regulation of ABCB1 expression resulting from tamoxifen treatment and CRISPR-knockout of ABCB1 expression up-regulate α-enola...

Analytical Chemistry, 2019

Despite global efforts aimed at its elimination, malaria is still a significant health concern in... more Despite global efforts aimed at its elimination, malaria is still a significant health concern in many countries across the world. The disease is caused by blood-borne parasites, Plasmodium species, and is transmitted by female Anopheles mosquitoes and presents with generic febrile symptoms that are challenging to diagnose clinically. To adequately tackle this issue, an effective detection method is required for screening potential malaria patients for infection. To this day, the gold standard for malaria detection remains basic light microscopy of Giemsastained patient blood smears to first enable detection and manual counting to determine the parasite density by a microscopist. While effective at detecting parasites, this method requires both significant time and skilled personnel. As an alternate approach, we propose a new malaria detection method that we call third-harmonic generation image scanning cytometry (THGISC) based on the combination of third-harmonic generation imaging, high-speed motorized scanning, and automated software processing. Third-harmonic generation (THG) is a nonlinear optical process in which the frequency of incident photons is tripled within the sample material. We have previously demonstrated that hemozoin, a metabolic byproduct of the malaria parasite, presents a significant THG signal. We now present a practical approach that uses the selectivity of this contrast mechanism to perform label-free image scanning cytometry of patient blood smears for automated malaria detection. In this work, we applied this technique to lab-cultured parasites and parasites in whole blood obtained from malaria patients. We also compared its effectiveness to parasite counts obtained by classical methods. The ability to easily and rapidly determine parasitemia by THG offers potential not only for the easy confirmation of malaria diagnoses following symptoms, but also the tracking of treatment progress in existing patients, potentially allowing physicians to adjust medication and dosage for each individual.

Background P-glycoprotein (P-gp), a member of the ATP Binding Cassette B1 subfamily (ABCB1), conf... more Background P-glycoprotein (P-gp), a member of the ATP Binding Cassette B1 subfamily (ABCB1), confers resistance to clinically relevant anticancer drugs and targeted chemotherapeutics. However, paradoxically P-glycoprotein overexpressing drug resistant cells are “collaterally sensitive” to non-toxic drugs that stimulate its ATPase activity. MethodsCell viability assays were used to determine the effect of low concentrations of tamoxifen on the proliferation of multidrug resistant cells (CHORC5 and MDA-Doxo400), expressing P-gp, their parental cell lines (AuxB1 and MDA-MB-231) or P-gp-CRISPR knockout clones of AuxB1 and CHORC5 cells. Western blot analysis was used to estimate P-gp expression in different cell lines. Apoptosis of tamoxifen-induced cell death was estimated by flow cytometry using Annexin-V-FITC stained cells. Oxidative stress tamoxifen treated cells was determined by measuring levels of reactive oxygen specifies and reduced thiols using cell-permeant 2',7'-dichl...

Journal of Biological Chemistry, 1993

There is growing evidence for the direct role of P-glycoprotein mediating multidrug resistance in... more There is growing evidence for the direct role of P-glycoprotein mediating multidrug resistance in tumor cells. P-glycoprotein is thought to function as an energy-dependent drug efflux pump. The monoclonal antibody MRK-16 binds to an external domain of P-glycoprotein and partially inhibits drug efflux in multidrug-resistant cells. As an approach toward elucidating the mechanism by which MRK-16 affects drug transport, we undertook the definition of the precise binding site of this antibody. In this study we have mapped the epitope of MRK-16 monoclonal antibody to a resolution of a single amino acid using a series of overlapping synthetic peptides. We demonstrate that MRK-16 recognizes only the class I isoform (MDR1) of human P-glycoprotein and that its epitope encompasses at least two (first and fourth) of the six predicted extracellular peptide loops. These results suggest that the epitope of MRK-16 is discontinuous and that the sequences involved which are separated by about 625 amino acids in the linear sequence must be spatially situated in close proximity in the native protein. Based on these results, we present a model for transmembrane alpha-helical packing of P-glycoprotein in the lipid bilayer. This may have implications for understanding the function of P-glycoprotein in drug transport.

Biochemical and Biophysical Research Communications, 2021

The Plasmodium falciparum chloroquine resistance transporter (PfCRT) is a phospho-protein with th... more The Plasmodium falciparum chloroquine resistance transporter (PfCRT) is a phospho-protein with three identified phosphorylation sites (Ser33, Ser411 and Thr416) at its cytosolic N- and C-termini. In this study, we report on the characterization of PfCRT anti-serum and show the presence of three epitope-specific immunoglobulin (IgG) pools (i.e., IgG-P1, P2, and P3), each recognizing a different epitope in PfCRT cytoplasmic C-terminal. IgG-P2 bound the heptapeptide sequence (408NEDSEGE414), including Ser411. The effect of Ser411 phosphorylation on the binding specificity of IgG-P2 was confirmed using heptapeptides and full-length PfCRT with substitutions of Ser411 with aspartic acid (phospho-serine mimic) and alanine residues. Moreover, using purified IgG-P2, we show the presence of PfCRT homodimer that has un-phosphorylated Ser411 and migrates with an apparent molecular mass of 90 kDa on SDS-PAGE. In addition, parasite lysates showed PfCRT to be more phosphorylated at Ser411 in CQ-sensitive (3D7) than CQ-resistant (Dd2-H) strains of P. falciparum. Taken together, the findings of this study suggest a role for Ser411 phosphorylation in PfCRT structure-function.

La presente invention a trait a des procedes de detection de cellules neoplasiques ou endommagees... more La presente invention a trait a des procedes de detection de cellules neoplasiques ou endommagees et de detection de la multiresistance aux medicaments dans des cellules neoplasiques ou endommagees par la determination d'une croissance dans l'expression en surface cellulaire d'une proteine nucleophosmine (NPM) a la surface de telles cellules neoplasiques ou endommagees a multiresistance aux medicaments par rapport a l'expression de la proteine nucleophosmine a la surface d'une cellule normale.

International Journal of Biochemistry and Molecular Biology, Oct 12, 2010

Nucleophosmin (NPM/B23) is a multifunctional nucleolar protein to which both tumor-suppressor and... more Nucleophosmin (NPM/B23) is a multifunctional nucleolar protein to which both tumor-suppressor and oncogenic functions have been attributed. NPM/B23 has a variety of binding partners including ribosomes, nucleic acids, the centrosome and tumor suppressors such as p53 and p19ARF. These disparate functions are likely due to its ability to oligomerize and display molecular chaperone activity. In this report we identify a single amino acid residue, Cys(21), of nucleophosmin as important for the oligomerization and chaperone activity. Mutation of Cys(21) to aromatic hydrophobic residues (e.g., Phe or Try), but not to a conserved polar residue (e.g., Ser) inhibited the pentameric oligomerization of NPM/B23. However, only Phe substitution of Cys(21) drastically inhibited NPM/B23 chaperone activity. Interestingly, expression of Cys21Phe mutant in MCF7 cells demonstrated that this mutant protein does not co-polymerize with endogenous wild-type NPM/B23 and acts as negative dominant by destabil...

Corresponding Author: Elias Georges, Institute of Parasitology, Macdonald Campus, McGill 14 Unive... more Corresponding Author: Elias Georges, Institute of Parasitology, Macdonald Campus, McGill 14 University, Ste. Anne de Bellevue, Quebec, Canada, H9X-3V9; Tel: 514 398 8137; Fax: 514 398 15 7857; E-mail: Elias.Georges@McGill.CA 16 17 Running Title: 3-iodo Chloroquine Reverses PfCRT-mediated Drug Resistance 18

Journal of Inorganic Biochemistry, 2019

In malaria, Plasmodium parasites produce hemozoin (Hz) as a route to detoxify free heme released ... more 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.

International Journal of Antimicrobial Agents, 2017

Plasmodium falciparum infection of mature erythrocytes leads to heightened oxidative stress that ... more Plasmodium falciparum infection of mature erythrocytes leads to heightened oxidative stress that is tolerated in normal erythrocytes but not in erythrocytes from sickle cell, β-thalassaemia and glucose-6-phosphate dehydrogenase (G6PD) deficiency hosts. In this report, it was of interest to perturb the redox homeostasis of normal erythrocytes through drug-induced active efflux of glutathione via erythrocyte ABCC1, a member of the C-subfamily of the human ATP-binding cassette (ABC) transporters. To achieve this objective, we made use of apigenin (API), shown previously to activate ABCC1 glutathione efflux in mature erythrocytes. Our results show that API increased ABCC1-mediated efflux of calcein-AM from uninfected erythrocytes, which was reversed with MK571, an inhibitor of ABCC1 drug efflux. Similarly, addition of API to uninfected normal erythrocytes led to a dramatic increase in reactive oxygen species coupled with a significant decrease in intracellular glutathione. Moreover, using P. falciparum-infected normal erythrocytes, we demonstrate that increasing concentrations of API inhibited the proliferation of chloroquine-susceptible (3D7) and -resistant (Dd2) parasites in culture with similar 50% inhibitory concentration (IC) values (36.02 ± 2.4 µM and 34.45 ± 2.4 µM, respectively). Interestingly, the presence of API (25 µM) led to a three-fold decrease in the ICof artemisinin compared with artemisinin alone (2.8 ± 0.7 nM vs. 7.09 ± 1.5 nM, respectively). Taken together, the findings of this study demonstrate the feasibility of our proposed approach for the development of novel antimalarials by modulating host protein functions that leads to heightened oxidative stress in P. falciparum-infected erythrocytes and inhibits parasite proliferation.

Advances in Pharmacology, 1990

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Microbiology Research Journal International, 2021

The Plasmodium falciparum chloroquine resistance transporter (PfCRT) is localised on the parasite... more The Plasmodium falciparum chloroquine resistance transporter (PfCRT) is localised on the parasite digestive vacuole, an organelle that maintains an acidic lumen. Here we demonstrated the isolation of HEK-293F cells stably expressing wild type 3D7 and mutant Pfcrt alleles. Immuno-fluorescence staining of HEK-293F transfectants confirms the localization of Pfcrt alleles to the lysosomal vesicles. Moreover, cells expressing mut-PfCRTETSE showed greater lysosomal acidification as demonstrated by the dramatic increase in the accumulation of two weak bases, acridine orange and CytiPainter LysoOrange dyes. Furthermore, HEK-293 cells stably expressing mut-PfCRTETSE with a single substitution of proline 165 in transmembrane 4 completely inhibited the accumulation of weak bases. Taken together, our results demonstrate the role of Pro165 in PfCRT lysosomal acidification function in HEK-293F cells.

Biochemical and Biophysical Research Communications, 2016

The multidrug resistant associated protein 1 (or MRP1, ABCC1) encodes two cytoplasmic linker doma... more The multidrug resistant associated protein 1 (or MRP1, ABCC1) encodes two cytoplasmic linker domains (L0 and L1) composed of highly charged sequences with multiple protein kinase A/C phosphorylation sites. In this report we made use of the scanning peptide approach to identify MRP1 linker L1 (L1(MRP1)) interacting proteins. Scanning heptapeptides covering L1(MRP1) 126 amino acids (Ile(846)- Leu(972)) were synthesized and used in pull-down assays to isolate proteins from cell extracts (human multidrug resistant SCLC cell line; H69/AR). The results show four high affinity binding sequences in L1(MRP1) domain [(866)FLRTYAST(867); (906)SAGKQLQRQLSSS(912); (925)ISRHHNSTA(927) and (954)AQTGQVKLSVYW(959)] that bound ∼55 kDa and 110 kDa polypeptides. The latter polypeptides were identified by mass spectrometry as α- and β-tubulin monomers and dimers. Western blotting with monoclonal antibodies to α- and β-tubulin proteins confirmed the mass-spectrometry results. Moreover, using recombinant full-length GST-Linker 1 fusion polypeptide (GST-L1(MRP1)), we confirmed the peptide scanning approach demonstrating specific binding of tubulin to GST-L1(MRP1). Intriguingly, substitutions of serine residues in L1(MRP1) by aspartic acid, but not alanine, abolished its binding to tubulin, suggesting that phosphorylation of Ser(871, 915, 930, and 961) within L1(MRP1) may modulate its binding to tubulin. Taken together, the results of this study suggest possible interaction between MRP1 and tubulin that is modulated by phosphorylation of specific sequences in the L1(MRP1) domain.

The over-expression of α-enolase was demonstrated in several cancers, including lung, brain, brea... more The over-expression of α-enolase was demonstrated in several cancers, including lung, brain, breast, colon and prostate. In this report, we investigated the effects of α-enolase knockdown on the sensitivity of cancer cells to chemotherapeutic drugs. RNAi-mediated knockdown of α-enolase in A549 and H460 lung, MCF7 breast and CaOV3 ovarian cancer cells caused a significant increase in the sensitivity of these cells to antitubulin chemotherapeutics (e.g., vincristine and taxol), but not to doxorubicin, etoposide or cisplatinum. This is the first demonstration showing the effects of α-enolase expression on the sensitivity of tumor cells to clinically relevant chemotherapeutics.

Several studies have shown that the multidrug resistant protein MRP2 mediates the transport of ch... more Several studies have shown that the multidrug resistant protein MRP2 mediates the transport of chemotherapeutic drugs and normal cell metabolites, including Leukotriene C (LTC4); however direct binding of the LTC4 to MRP2 has not been demonstrated. In this study, a photoreactive analog of LTC4 (IAALTC4) was used to demonstrate its direct binding to MRP2. Our results show specific photoaffinity labeling of MRP2 with IAALTC4 in plasma membranes from MDCKII MRP2 cells. The photoaffinity labeling signal of MRP2 with IAALTC4 was much lower than that of MRP1, consistent with previous studies whereby the measured Km values of MRP1 and MRP2 for LTC4 were 1 µM and 0.1 µM LTC4, respectively. Competition of IAALTC4 photoaffinity labeling to MRP2 with MK571, a well characterized inhibitor of MRP2 function, showed ~75% reduction in binding in the presence of 50 µM excess MK571. Interestingly, unmodified LTC4 enhanced the photoaffinity labeling of IAALTC4 to MRP2, whereas excess GSH and Quercetin had no significant effect. Mild tryptic digestion of photoaffinity labeled MRP2 revealed several photoaffinity labeled peptides that localized the IAALTC4 binding to a 15 kDa amino acid sequence that contains transmembrane 16 and 17. Together these results provide the first demonstration of direct LTC4 binding to MRP2.