Multidrug resistance to chemotherapy is a major obstacle in the treatment of cancer patients. The best characterised mechanism responsible for multidrug resistance involves the expression of the MDR-1 gene product, P-glycoprotein. However, the resistance process is multifactorial. Studies of multidrug resistance mechanisms have relied on the analysis of cancer cell lines that have been selected and present cross-reactivity to a broad range of anticancer agents. This work characterises a multidrug resistant cell line, originally selected for resistance to the Vinca alkaloid vincristine and derived from the human erythroleukaemia cell K562. This cell line, named Lucena 1, overexpresses P-glycoprotein and have its resistance reversed by the chemosensitisers verapamil, trifluoperazine and cyclosporins A, D and G. Furthermore, we demonstrated that methylene blue was capable of partially reversing the resistance in this cell line. On the contrary, the use of 5-fluorouracil increased the resistance of Lucena 1. In addition to chemotherapics, Lucena 1 cells were resistant to ultraviolet A radiation and hydrogen peroxide and failed to mobilise intracellular calcium when thapsigargin was used. Changes in the cytoskeleton of this cell line were also observed.
Ouabain, an inhibitor of the Na+/K+-ATPase pump, was identified as an endogenous substance of human plasma. Ouabain has been studied for its ability to interfere with various regulatory mechanisms. Despite the studies portraying the ability of ouabain to modulate the immune response, little is known about the effect of this substance on the inflammatory process. The aim of this work was to study the effects triggered by ouabain on inflammation and nociceptive models. Ouabain produced a reduction in the mouse paw edema induced by carrageenan, compound 48/80 and zymosan. This anti-inflammatory potential might be related to the inhibition of prostaglandin E2, bradykinin, and mast-cell degranulation but not to histamine. Ouabain also modulated the inflammation induced by concanavalin A by inhibiting cell migration. Besides that, ouabain presented antinociceptive activity. Taken these data together, this work demonstrated, for the first time, that ouabain presented in vivo analgesic and anti-inflammatory effects.
The hallmark of CML (chronic myeloid leukaemia) is the BCR (breakpoint cluster region)–ABL fusion gene. CML evolves through three phases, based on both clinical and pathological features: a chronic phase, an accelerated phase and blast crisis. TKI (tyrosine kinase inhibitors) are the treatment modality for patients with chronic phase CML. The therapeutic potential of the TKI imatinib is affected by BCR–ABL dependent an independent mechanisms. Development of MDR (multidrug resistance) contributes to the overall clinical resistance. MDR involves overexpression of ABC -transporters (ATP-binding-cassette transporter) among other features. MDR studies include the analysis of cancer cell lines selected for resistance. CML blast crisis is accompanied by increased resistance to apoptosis. This work reviews the role played by the influx transporter OCT1 (organic cation transporter 1), by efflux ABC transporters, molecules involved in the modulation of apoptosis (p53, Bcl-2 family, CD95, IAPs (inhibitors of apoptosis protein)], Hh and Wnt/β-catenin pathways, cytoskeleton abnormalities and other features described in leukaemic cells of clinical samples and CML cell lines. An MDR cell line, Lucena-1, generated from K562 by stepwise exposure to vincristine, was used as our model and some potential anticancer drugs effective against the MDR cell line and patients’ samples are presented.
Ouabain, a potent inhibitor of the Na+, K+-ATPase, was identified as an endogenous substance. Recently, ouabain was shown to affect various immunological processes. We have previously demonstrated the ability of ouabain to modulate inflammation, but little is known about the mechanisms involved. Thus, the aim of the present work is to evaluate the immune modulatory role of ouabain on zymosan-induced peritonitis in mice. Our results show that ouabain decreased plasma exudation (33%). After induction of inflammation, OUA treatment led to a 46% reduction in the total number of cells, as a reflex of a decrease of polymorphonuclear leukocytes, which does not appear to be due to cell death. Furthermore, OUA decreased TNF-α (57%) and IL-1β (58%) levels, without interfering with IL-6 and IL-10. Also, in vitro experiments show that ouabain did not affect endocytic capacity. Moreover, electrophoretic mobility shift assay (EMSA) shows that zymosan treatment increased (85%) NF-κB binding activity and that ouabain reduced (30%) NF-κB binding activity induced by zymosan. Therefore, our data suggest that ouabain modulated acute inflammatory response, reducing the number of cells and cytokines levels in the peritoneal cavity, as well as NFκB activation, suggesting a new mode of action of this substance.
The multidrug-resistant (MDR) phenotype is multifactorial, and cell lines presenting multiple resistance mechanisms might be good models to understand the importance of the various pathways involved. The present work characterized a MDR chronic myeloid leukemia cell line, derived from K562 through a selective process using daunorubicin. This MDR cell line was shown to be resistant to vincristine, daunorubicin, and partially resistant to imatinib. It showed a slower duplication rate. Overexpression of ABCB1 and ABCC1 was observed at the protein and functional levels and the expression of CD95, a molecule related to cell death, was reduced in the MDR cell line. Conversely, no differences were observed related to the anti-apoptotic molecule Bcl-2 or p53 expression. The activation antigen CD69 was reduced in the MDR cell line and treatment with imatinib further decreased the expressed levels. Furthermore, secretion of IL-8 was diminished in the MDR cell line. When daunorubicin-selected cells were compared to another MDR cell line, Lucena 1, derived from the same parental line K562, and selected with vincristine, a different profile was observed in relation to most aspects studied. When both cell lines were silenced for ABCB1, differences in CD69 and CD95 were maintained, despite resistance reversal. These results reinforce the idea that cell lines selected in vitro may display multiple resistance strategies that may vary with the selective agent used as well as during different steps of the selection process.
The suggested involvement of ouabain in hypertension raised the need for a better understanding of its cellular action, but the mechanisms of ouabain toxicity are only now being uncovered. In the present study, we show that reduced glutathione (GSH) protected ouabain-sensitive (OS) cells from ouabain-induced toxicity and that the inhibition of GSH synthesis by D, L-buthionine-(S,R)-sulfoximine (BSO) sensitized ouabain-resistant (OR) cells. We could not observe formation of *OH or H2O2, but there was an increase in O2*-only in OS cells. Unexpectedly, an increased number of OR cells depolarized after treatment with ouabain, and BSO blocked this depolarization. Moreover, GSH increased ouabain-induced depolarization in OS cells. A sustained increase in tyrosine phosphorylation (P-Tyr) and Ras expression was observed after treatment of OS cells, and GSH prevented it. Conversely, BSO induced P-Tyr and Ras expression in ouabain-treated OR cells. The results obtained have three major implications: There is no direct correlation between membrane depolarization and ouabain-induced cell death; ouabain toxicity is not directly related to its classical action as a Na+, K+-ATPase inhibitor but seems to be associated to signal transduction, and GSH plays a major role in preventing ouabain-induced cell death.
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