Analysis of DNA content in multidrug-resistant cells by image and flow cytometry

Abstract. Nuclear DNA content was assessed in multidrug-resistant (MDR) cells by image and flow cytometry. Two human MDR cell lines (K562-Dox and CEM-VLB) obtained by in vitro drug selection and overexpressing mdr1 gene were compared to their respective sensitive counterparts (K562 and CCRF-CEM) and to the MDR hamster LR73-R cell line obtained by transfection of mouse mdr1 cDNA. Both cell lines obtained by selection displayed a decreased DNA content, as measured by image cytometry after Feulgen staining, or by flow cytometry after staining with propidium iodide, ethidium bromide, or Hoechst 33342. This decrease was not accompanied by changes in cell cycle phase distribution of cells. Moreover, image cytometry of cells stained after various hydrolysis times in 5 M HCl indicated that MDR cells displayed the same hydrolysis kinetics and sensitivity as drug-sensitive cells with a well-preserved stoichiometry of the Feulgen reaction. LR73-R cells transfected with mdr1 cDNA exhibited only a very limited change in propidium iodide staining as compared with sensitive LR73 cells, suggesting that mdr1 gene overexpression alone could not account for the alterations in DNA content observed in the selected MDR cells.     

Identification of different isoforms of eEF1A in the nuclear fraction of human T-lymphoblastic cancer cell line specifically binding to aptameric cytotoxic GT oligomers.

GT oligomers, showing a dose-dependent cytotoxic effect on a variety of human cancer cell lines, but not on normal human lymphocytes, recognize and form complexes with nuclear proteins. By working with human T-lymphoblastic CCRF-CEM cells and by using MS and SouthWestern blotting, we identified eukaryotic elongation factor 1 alpha (eEF1A) as the main nuclear protein that specifically recognizes these oligonucleotides. Western blotting and supershift assays confirmed the nature of this protein and its involvement in forming a cytotoxicity-related complex (CRC). On the contrary, normal human lymphocytes did not show nuclear proteins able to produce CRC in a SouthWestern blot. Comparative bidimensional PAGE and Western-blotting analysis for eEF1A revealed the presence of a specific cluster of spots, focusing at more basic pH, in nuclear extracts of cancer cells but absent in those of normal lymphocytes. Moreover, a bidimensional PAGE SouthWestern blot demonstrated that cytotoxic GT oligomers selectively recognized the more basic eEF1A isoform expressed only in cancer cells. These results suggest the involvement of eEF1A, associated with the nuclear-enriched fraction, in the growth and maintenance of tumour cells, possibly modulated by post-translational processing of the polypeptide chain.     

Agents which reverse multidrug-resistance are inhibitors of [3H]vinblastine transport by isolated vesicles.

Resistance of human cancer cells to multiple cytotoxic hydrophobic agents (multidrug resistance) is due to overexpression of the MDR1 gene whose product is the ATP-dependent multidrug transporter, P-glycoprotein. We have previously reported that plasma membrane vesicles partially purified from multidrug-resistant human KB carcinoma cells, but not from drug-sensitive cells, accumulated [3H]vinblastine in an ATP-dependent manner (Horio, M., Gottesman, M.M. and Pastan, I. (1988) Proc. Natl. Acad. Sci. USA 85, 3580-3584). Certain calcium-channel blockers, quinidine, and phenothiazines are able to overcome multidrug resistance in cultured cells. In this work, the effect of these reversing agents on ATP-dependent vinblastine (VBL) transport by vesicles from drug-resistant KB cells has been characterized. Azidopine was the most potent inhibitor of ATP-dependent VBL uptake tested (ID50: concentration of inhibitor such that the transport of vinblastine is inhibited by 50%, less than 1 microM). Verapamil, quinidine, and the tiapamil analogue RO-11-2933 were potent but less effective inhibitors (ID50 less than 5 microM). Diltiazem, nifedipine and trifluoperazine were even less effective. These agents had no effect on Na(+)-dependent and Na(+)-independent L-leucine uptake by the vesicles, indicating that the inhibition of ATP dependent VBL transport by these agents is not a non-specific effect, as might result from leaks in the vesicle membrane. Verapamil, quinidine, azidopine and trifluoperazine increased the apparent Km value of vinblastine transport, suggesting that these agents may be competitive inhibitors of vinblastine transport.     

Transport properties of P-glycoprotein in plasma membrane vesicles from multidrug-resistant Chinese hamster ovary cells.

Multidrug resistant (MDR) cells overexpress a 170-180 kDa membrane glycoprotein, the P-glycoprotein, which is believed to export drugs in an ATP-dependent manner. Plasma membrane vesicles from the MDR CHRC5 cell line, but not the AuxB1 drug-sensitive parent, showed uptake of [3H]colchicine and [3H]vinblastine that was stimulated by the presence of ATP and an ATP-regenerating system. Steady-state uptake of drugs was achieved by 10 min and was stable for greater than 30 min. Non-hydrolysable ATP analogues were unable to support drug uptake, indicating that ATP hydrolysis is essential for transport. ATP-stimulated drug uptake appeared to result from drug transport into inside-out vesicles, since uptake was osmotically sensitive and could be prevented by detergent permeabilization. Steady-state uptake was half-maximal at 100 microM colchicine and 200 nM vinblastine and was inhibited by a 10-100-fold excess of MDR drugs and chemosensitizers, in the order vinblastine greater than verapamil greater than daunomycin greater than colchicine. In addition to being vanadate-sensitive, drug uptake was inhibited by 10-200 microM concentrations of several sulfhydryl-modifying reagents, suggesting that cysteine residues play an important role in drug transport. Vesicular colchicine was rapidly exchanged by an excess of unlabelled drug, demonstrating that drug association is the net result of opposing colchicine fluxes across the membrane.     

Increased mdr gene expression and decreased drug accumulation in a human colonic cancer cell line resistant to hydrophobic drug

An adriamycin-resistant human colonic cancer cell line was characterized. This clone exhibits the classical multidrug resistance (MDR) phenotype, being cross-resistant to hydrophobic drugs such as colchicine, and vinblastine. In contrast, this clone shows a normal response to DNA-damaging agents. The appearance of MDR in these cells was linked to a decreased accumulation of the drug [3H]colchicine as compared to the drug-sensitive cells. This MDR line expressed 80-100 fold increased levels of the specific 4.5-kb mdr mRNA, and a gene amplification. Our results indicate that MDR in human colonic cancer cells can result from increased expression of at least one member of the mdr gene family.     

Oligonucleotides blocking glucosylceramide synthase expression selectively reverse drug resistance in cancer cells

High glucosylceramide synthase (GCS) activity is one factor contributing to multidrug resistance (MDR) in breast cancer. Enforced GCS overexpression has been shown to disrupt ceramide-induced apoptosis and to confer resistance to doxorubicin. To examine whether GCS is a target for cancer therapy, we have designed and tested the effects of antisense oligodeoxyribonucleotides (ODNs) to GCS on gene expression and chemosensitivity in multidrug-resistant cancer cells. Here, we demonstrate that antisense GCS (asGCS) ODN-7 blocked cellular GCS expression and selectively increased the cytotoxicity of anticancer agents. Pretreatment with asGCS ODN-7 increased doxorubicin sensitivity by 17-fold in MCF-7-AdrR (doxorubicin-resistant) breast cancer cells and by 10-fold in A2780-AD (doxorubicin-resistant) ovarian cancer cells. In MCF-7 drug-sensitive breast cancer cells, asGCS ODN-7 only increased doxorubicin sensitivity by 3-fold, and it did not influence doxorubicin cytotoxicity in normal human mammary epithelial cells. asGCS ODN-7 was shown to be more efficient in reversing drug resistance than either the GCS chemical inhibitor d-threo-1-phenyl-2-decanoylamino-3-morpholino-1-propanol or the P-glycoprotein blocking agents verapamil and cyclosporin A. Experiments defining drug transport and lipid metabolism parameters showed that asGCS ODN-7 overcomes drug resistance mainly by enhancing drug uptake and ceramide-induced apoptosis. This study demonstrates that a 20-mer asGCS oligonucleotide effectively reverses MDR in human cancer cells.     

Alpha-anomeric configuration of GT oligodeoxynucleotide leads to loss of the specific aptameric and cytotoxic properties retained by the beta-anomeric analog

The development of antisense, antigene, or aptameric oligonucleotides to modulate in vivo cellular functions depends on using stable biologic molecules. Previous investigations showed that GT oligonucleotides could exert a specific, dose-dependent cytotoxic effect on human cancer cell lines. This is tightly related to the ability of these oligomers to specifically bind nuclear proteins, giving a complex of apparent molecular weight of 45 kDa. We demonstrated that with respect to the cytotoxic GT-beta-oligomer, alpha-anomeric GT analog did not alter the growth of the T lymphoblastic CCRF-CEM cell line, although the cells took it up efficiently. In agreement with this, GT-alpha-oligomer did not form the cytotoxicity-related 45-kDa complex with nuclear proteins. These findings likely could be related to the ability of GT-alpha to structure under nondenaturing conditions because of the high number of T in the sequence.     

Textural analysis of nuclear mitotic apparatus antigen (NuMA) spatial distribution in interphase nuclei from human drug-resistant CEM lymphoblasts.

In tumour cell lines, the resistance of cancer cells to a variety of structurally unrelated chemotherapeutic drugs is termed multidrug-resistance or MDR. We reported previously [6] that MDR leukemic cells displayed nuclear texture changes, as assessed by image cytometry. The nature of these changes remained uncertain but they could be associated with alterations of the nuclear matrix which could serve an important role in DNA organization and chromatin structure. Therefore, we have compared the textural features observed in G0/G1 nuclei from human leukemic CEM cells and their MDR variant CEM-VLB, after staining of either DNA by Feulgen method or nuclear matrix by immunodetection of NuMA antigen on DNase treated samples. Chromatin or NuMA distributions within the nucleus were evaluated by image cytometry. Changes in textural parameters indicate that modifications of NuMA distribution observed in MDR cells are parallel to those observed at the whole chromatin level (i.e., a more decondensed and coarse texture with increase of Energy and Long-run sections and decrease of Contrast and Short-run sections). Moreover, Optical Densities measurements indicate that MDR cells seem to contain less NuMA, a datum confirmed by immunoblotting of nuclear proteins. In conclusion, chromatin changes observed by image cytometry in drug-resistant human leukemic CEM cells appear associated with modifications of the nuclear matrix structure.     

Influence of IGF-I and cell density on MDR1 expression in the T-lymphoblastoid cell line CCRF-CEM

The debate about a direct or indirect effect of GH and IGF-I on the recurrence of malignancy, especially in the case of rhGH therapy in patients with leukemia, is still going on. Recent studies suggested that IGF-I plays a role in drug resistance during anticancer therapy. This resistance to diverse cytotoxic drugs, named multidrug-resistance (MDR), is mainly due to high levels of P-glycoprotein (P-gp). The gene encoding this membrane-associated transporter protein was named MDR1, and increased levels of P-gp are linked to enhanced MDR1 mRNA expression. Our aim was to investigate a possible effect of rhIGF-I on MDR1 gene expression in vitro. We cultured the T-lymphoblastoid cell line CCRF-CEM with different rhIGF-I concentrations (0, 5, 20 and 50 ng/ml) in serum-free medium for 3 days. CCRF-CEM cells are drug-sensitive and express MDR1 at low levels. MDR1 mRNA expression was measured by semiquantitative RT-PCR using a competitive assay with a heterologous DNA construct. In addition, GAPDH mRNA was amplified as an internal control for RNA integrity. P-gp activity was determined by a flow cytometric assay measuring rhodamine 123 accumulation. Furthermore, cell proliferation was monitored in all experiments. Our data do not support an effect of rhIGF-I on MDR1 mRNA expression, P-gp activity or cell proliferation in the CCRF-CEM cell line. MDR1 mRNA levels were inversely correlated to cell density with high significance (p < 0.0001). In conclusion, multidrug resistance linked to P-gp is not induced by IGF-I in CCRF-CEM cells. At high density, CCRF-CEM cells downregulate MDR1 gene expression. Our experimental model provides a very useful tool for monitoring the influence of growth factors on multidrug resistance in vitro.     

Suppression of multidrug resistance by treatment with TRAIL in human ovarian and breast cancer cells with high level of c-Myc

In this study, we investigated the role of c-Myc in overcoming multidrug resistance (MDR) in human ovarian and breast cancer cells by TRAIL. We showed that P-gp expressing MDR variants (Hey A8-MDR and MCF7-MDR cells) with high level of c-Myc were highly susceptible to TRAIL treatment when compared to their drug-sensitive parental human ovarian cancer Hey A8 and breast MCF-7 cells, respectively. Up-regulation of DR5 TRAIL receptor and down-regulation of c-FLIP and the promotion of caspase-dependent cell death, which contribute to TRAIL sensitization of MDR cells, were regulated by the over-expressed c-Myc in the MDR cells. After targeted inhibition of c-Myc with specific siRNA, these responses to TRAIL disappeared and TRAIL-induced apoptosis was also suppressed in MCF7-MDR cells. Treatment with TRAIL significantly reduced P-glycoprotein (P-gp)-mediated efflux of rhodamine123 in both Hey A8-MDR and MCF7-MDR cells. Furthermore, TRAIL significantly potentiated the cytotoxicity of vinblastine, vincristine, doxorubicin and VP-16 that are P-gp substrate anticancer drugs in both MDR cells, which resulted in the reversal effect of TRAIL on the MDR phenotype. The present study shows for the first time that elevated c-Myc expression in the MDR cells plays a critical role in overcoming MDR by TRAIL that can act as a specific sensitizer for P-gp substrate anticancer drug.     

Altered catalytic activity of and DNA cleavage by DNA topoisomerase II from human leukemic cells selected for resistance to VM-26

The simultaneous development of resistance to the cytotoxic effects of several classes of natural product anticancer drugs, after exposure to only one of these agents, is referred to as multiple drug resistance (MDR). At least two distinct mechanisms for MDR have been postulated: that associated with P-glycoprotein and that thought to be due to an alteration in DNA topoisomerase II activity (at-MDR). We describe studies with two sublines of human leukemic CCRF-CEM cells approximately 50-fold resistant (CEM/VM-1) and approximately 140-fold resistant (CEM/VM-1-5) to VM-26, a drug known to interfere with DNA topoisomerase II activity. Each of these lines is cross-resistant to other drugs known to affect topoisomerase II but not cross-resistant to vinblastine, an inhibitor of mitotic spindle formation. We found little difference in the amount of immunoreactive DNA topoisomerase II in 1.0 M NaCl nuclear extracts of the two resistant and parental cell lines. However, topoisomerase II in nuclear extracts of the resistant sublines is altered in both catalytic activity (unknotting) of and DNA cleavage by this enzyme. Also, the rate at which catenation occurs is 20-30-fold slower with the CEM/VM-1-5 preparations. The effect of VM-26 on both strand passing and DNA cleavage is inversely related to the degree of primary resistance of each cell line. Our data support the hypothesis that at-MDR is due to an alteration in topoisomerase II or in a factor modulating its activity.     

Carotenoids reverse multidrug resistance in cancer cells by interfering with ABC-transporters

Proteins of the ATP-binding cassette superfamily, mainly P-glycoprotein (P-gp; MDR1), play an important role in the development of multidrug resistance (MDR) in cancer cells and thus in the potential failure of chemotherapy. A selection of carotenoids (β-carotene, crocin, retinoic acid, canthaxanthin, and fucoxanthin) was investigated whether they are substrates of P-gp, and if they can reverse MDR in resistant Caco-2 and CEM/ADR5000 cells as compared to the sensitive parent cell line CCRF-CEM. The activity of ABC transporter was determined in resistant and sensitive cells by spectrofluorometry and flow cytometry using the substrates doxorubicin, rhodamine 123, and calcein as fluorescent probes. The carotenoids increased accumulation of these P-gp substrates in a dose-dependent manner indicating that they themselves also function as substrates. Fucoxanthin and canthaxanthin (50-100μM) produced a 3-5-fold higher retention of the fluorescent probes than the known competitive inhibitor verapamil. Carotenoids showed a low cytotoxicity in cells with MDR with IC(50) values between 100 and 200μM. The combination of carotenoids with eight structurally different cytotoxic agents synergistically enhanced their cytotoxicity in Caco-2 cells, probably by inhibiting the function of the ABC transporters. For example, fucoxanthin synergistically enhanced the cytotoxicity of 5-FU 53.37-fold, of vinblastine 51.01-fold, and of etoposide 12.47-fold. RT-PCR was applied to evaluate the mRNA levels of P-gp in Caco-2 cells after treatment with carotenoids. Fucoxanthin and canthaxanthin significantly decreased P-gp levels to 12% and 24%, respectively as compared to untreated control levels (p<0.001). This study implies that carotenoids may be utilised as chemosensitisers, especially as adjuvants in chemotherapy.     

Proteome analysis of vinca alkaloid response and resistance in acute lymphoblastic leukemia reveals novel cytoskeletal alterations

Vinca alkaloids are used widely in the treatment of both childhood and adult cancers. Their cellular target is the beta-tubulin subunit of alpha/beta-tubulin heterodimers, and they act to inhibit cell division by disrupting microtubule dynamics. Despite the effectiveness of these agents, drug resistance is a major clinical problem. To identify the underlying mechanisms behind vinca alkaloid resistance, we have performed high resolution differential proteome analysis. Treatment of drug-sensitive human leukemia cells (CCRF-CEM) with vincristine identified numerous proteins involved in the cellular response to vincristine. In addition, differential protein expression was analyzed in leukemia cell lines selected for resistance to vincristine (CEM/VCR R) and vinblastine (CEM/VLB100). This combined proteomic approach identified 10 proteins altered in both vinca alkaloid response and resistance: beta-tubulin, alpha-tubulin, actin, heat shock protein 90beta, 14-3-3tau, 14-3-3epsilon, L-plastin, lamin B1, heterogeneous nuclear ribonuclear protein-F, and heterogeneous nuclear ribonuclear protein-K. Several of these proteins have not previously been associated with drug resistance and are thus novel targets for elucidation of resistance mechanisms. In addition, seven of these proteins are associated with the tubulin and/or actin cytoskeletons. This study provides novel insights into the interrelationship between the microtubule and microfilament systems in vinca alkaloid resistance.     

Analysis of the steady-state and initial rate of doxorubicin efflux from a series of multidrug-resistant cells expressing different levels of P-glycoprotein.

Continuous monitoring of fluorescence (CMF) has been used to examine doxorubicin efflux from intact human myeloma cells. The time resolution of these measurements has enabled detailed comparison of the initial rates of efflux for the drug-sensitive myeloma line RPMI 8226 and a series of sequentially derived multidrug-resistant (MDR) lines expressing different amounts of human MDR protein (P-glycoprotein). Cells that are 3-, 10-, 60-, or 120-fold resistant to doxorubicin export approximately 10, 20, 30, or 33% more doxorubicin than the parental sensitive cells, respectively, when all are preloaded to the same level of total intracellular drug. Remarkably, however, when cells are loaded to the same level of exchangeable drug the initial rates of efflux are found to be virtually identical. This agreement between rates is apparently not dependent on the drug concentration. Approximately 50% of the increase in the steady-state level of doxorubicin efflux for the resistant cells is abolished upon glucose starvation. However, surprisingly, the apparent initial rates of efflux from the treated and untreated cells are found to be virtually the same. Pretreatment of the resistant cells with verapamil reduces the steady-state level of efflux but increases the apparent initial rate at some concentrations. Conversely, vincristine does not alter steady state but slows the initial rate of efflux from both sensitive and resistant cells by approximately the same extent. Finally, quite interestingly, a nearly linear relationship between pHi and relative steady state of efflux is found for the series of cell lines. These data are interpreted in terms of existing models for MDR.     

Ultrastructural effects of colchicine,vinblastine, and taxol in drug-sensitive and multidrug-resistant J 774.2 cells

Summary Cell lines derived from the murine macrophage-like cell J 774.2 are resistant to the cytotoxic effects of colchicine, vinblastine, and taxol. These multidrug-resistant (MDR) cells overproduce a family of 130–150 kDa P-glycoproteins (P-gp) associated with the plasma membrane region and display other typical features of the MDR phenotype. Ultrastructural analysis of drug-treated cells indicated that although hallmark structural effects engendered by each drug at efficacious doses were profound in the drug-sensitive J 774.2 cells, they were not evident in the similarly treated MDR cell lines. Thus, MDR phenotypic expression involved maintaining drug levels at subthreshold values so as to preclude the advent of these morphologic changes, and allowed vital tubulin-associated cellular processes, including replication, to occur. Using a polyclonal antibody specific for the P-gp, electron microscopic immunocytochemical evidence is presented for substantial association of P-gp with the plasma membrane/cell surface in the resistant cells which was not demonstrable in the drug-sensitive J 774.2 cells. This key cell surface localization of P-gp is germane to the postulated transport and related mechanisms whereby P-gp may play a pivotal role in endowing cells with multidrug resistance.     

Iron-transferrin-induced increase in protein kinase C activity in CCRF-CEM cells

Iron transferrin has been found to induce a mean 10-fold increase in the activity of protein kinase C in CCRF-CEM cells. This increase was not detectable up to 45 min after treatment of cells with iron transferrin, although after 60 min, a maximal increase in enzyme activity was observed. Similarly, iron transferrin at concentrations of 0.1-0.5 microgram/ml did not alter protein kinase C activity, while concentrations of iron transferrin of 1-100 micrograms/ml induced a maximal increase in enzyme activity. Apotransferrin and iron in the form of ferric citrate, as well as complexes of transferrin with copper, nickel, zinc, manganese, and cobalt did not increase protein kinase C activity. Additionally, CCRF-CEM cells pretreated with either actinomycin D or cycloheximide and then incubated with iron transferrin did not exhibit increased enzyme activity. Treatment with iron transferrin was found to have no effect on protein kinase C activity in normal human peripheral blood lymphocytes and in HL60, Daudi, and U937 cells. However, normal lymphocytes stimulated with phytohemagglutinin for 48 hr exhibited a 2-fold increase in protein kinase C activity following treatment with iron transferrin. These results indicate a specific effect of iron transferrin on protein kinase C activity in CCRF-CEM cells and in mitogen-stimulated human lymphocytes that may occur through increased synthesis of the enzyme.     

The multidrug transporter: rapid modulation of efflux activity monitored in single cells by the morphologic effects of vinblastine and daunomycin

Double-label fluorescence microscopy was used to demonstrate the efflux activity of the multidrug transporter in single cultured cells. NIH3T3 cells expressing a transfected MDR1 gene (NIH3T3-MDR) were treated with vinblastine or daunomycin. The accumulation of vinblastine was monitored by examining the morphology of tubulin in cells, using immunofluorescence. Overnight treatment of drug-sensitive cells caused disassembly of microtubules and formation of paracrystals; the absence of vinblastine effects was evident by the presence of intact microtubules. Daunomycin accumulation was detected in nuclei using the inherent fluorescence of the drug with rhodamine epifluorescence microscopy. Drug efflux in multidrug-resistant cells was inhibited with verapamil. When multidrug-resistant cells were treated overnight in vinblastine, an effect of 0.5 microM vinblastine on microtubules was seen only in the presence of verapamil. Similarly, when cells were treated with daunomycin, this drug accumulated in nuclei only when verapamil was present. When cells incubated with vinblastine and verapamil were washed free of drugs, they did not accumulate daunomycin in a subsequent incubation, indicating that the multidrug transporter was still active; this occurred even though the morphologic effects of vinblastine persisted. Cells incubated with vinblastine alone showed an immediate inhibition of efflux activity when verapamil was subsequently added with daunomycin. These results show that the efflux activity of the multidrug transporter can be rapidly manipulated by agents such as verapamil, despite a prior history of drug treatment, and that the effects of inhibition of the transporter are rapidly reversible.     

Quantitative study of the drug efflux kinetics from sensitive and MDR human breast cancer cells

Cancer multidrug resistance (MDR) is a major impediment to effective chemotherapy in human cancer, in which P-glycoprotein and Multidrug Resistance-Associated protein figure prominently. Design and exploitation of novel clinical MDR inhibitors is greatly hindered by a lack of understanding of drug efflux dynamics in drug-sensitive and resistant cells. The aim of our study was to provide a microelectrode method for measuring the multidrug transporter mediated efflux of doxorubicin as well as a corresponding data analysis method for quantifying the efflux kinetic parameters. We performed experiments using carbon fiber microelectrode to detect doxorubicin efflux from a monolayer of human breast cancer MCF-7 cells and derived MDR cells (MCF-7/ADR), established a material transport model and proposed a novel inverse method to quantitatively characterize the diffusion dynamics. The kinetic parameters of doxorubicin efflux from MCF-7 and MCF-7/ADR cells in the presence or absence of MDR inhibitors were estimated. Our investigations showed the average initial doxorubicin efflux rate of MCF-7/ADR that was 5.2 times faster than of MCF-7. After treatment by tetramethylpyrazine or verapamil, the drug efflux rate of the MCF-7/ADR cells was reduced by about half that of those without inhibitors. The novel methodology presented suggests new and expanded applications for computer-aided reconstruction of the drug efflux process, microelectrode design, and high-throughput drug screening.     

Multidrug resistant tumour cells shed more microvesicle-like EVs and less exosomes than their drug-sensitive counterpart cells

BackgroundMultidrug resistance (MDR) is a serious impediment to cancer treatment, with overexpression of drug efflux pumps such as P-glycoprotein (P-gp) playing a significant role. In spite of being a major clinical challenge, to date there is no simple, minimally invasive and clinically validated method for diagnosis of the MDR phenotype using non-tumour biological samples. Recently, P-gp has been found in extracellular vesicles (EVs) shed by MDR cancer cells. This study aimed to compare the EVs shed by MDR cells and their drug-sensitive cellular counterparts, in order to identify biomarkers of MDR.MethodsTwo pairs of MDR and drug-sensitive counterpart tumour cell lines were studied as models. EVs were characterized in terms of size and molecular markers and their protein content was investigated by proteomic analysis and Western blot.ResultsWe found that MDR cells produced more microvesicle-like EVs and less exosomes than their drug-sensitive counterpart. EVs from MDR cells contained P-gp and presented a different content of proteins known to be involved in the biogenesis of EVs, particularly in the biogenesis of exosomes.ConclusionsThe determination of the size and of this particular protein content of EVs shed by tumour cells may allow the development of a minimally-invasive simple method of detecting and predicting MDR.General significanceThis work describes for the first time that cancer multidrug resistant cells shed more microvesicle-like EVs and less exosomes than their drug-sensitive counterpart cells, carrying a specific content of proteins involved in EV biogenesis that could be further studied as biomarkers of MDR.