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This study aimed to characterize the relationship between the COX2 and ALOX5 genes, as well as their link with the multidrug resistance (MDR) phenotype in sensitive (K562) and MDR (K562-Lucena and FEPS) erythroleukemia cells. For this, the inhibitors of 5-LOX (zileuton) and COX-2 (acetylsalicylic acid-ASA) and cells with the silenced ABCB1 gene were used. The treatment with ASA caused an increase in the gene expression of COX2 and ABCB1 in both MDR cell lines, and a decrease in the expression of ALOX5 in the FEPS cells. Silencing the ABCB1 gene induced a decrease in COX2 expression and an increase in the ALOX5 gene. Treatment with zileuton did not alter the expression of COX2 and ABCB1. Cytometry data showed that there was an increase in ABCB1 protein expression after exposure to ASA. In addition, the increased activity of ABCB1 in the K562-Lucena cell line indicates that ASA may be a substrate for this efflux pump, corroborating the molecular docking that showed that ASA can bind to ABCB1. Regardless of the genetic alteration in COX2 and ABCB1, the direct relationship between these genes and the inverse relationship with ALOX5 remained in the MDR cell lines. We assume that ABCB1 can play a regulatory role in COX2 and ALOX5 during the transformation of the parental cell line K562, explaining the increased gene expression of COX2 and decreased ALOX5 in the MDR cell lines., (Copyright © 2021 Elsevier Inc. All rights reserved.)

A new pterocarpanquinone (5a) was synthesized through a palladium catalyzed oxyarylation reaction and was transformed, through electrophilic substitution reaction, into derivatives 5b-d. These compounds showed to be active against human leukemic cell lines and human lung cancer cell lines. Even multidrug resistant cells were sensitive to 5a, which presented low toxicity toward peripheral blood mononuclear cells (PBMC) cells and decreased the production of TNF-alpha by these cells. In the laboratory these pterocarpanquinones were reduced by sodium dithionite in the presence of thiophenol at physiological pH, as NAD(P)H quinone oxidoredutase-1 (NQO1) catalyzed two-electron reduction, and the resulting hydroquinone undergo structural rearrangements, leading to the formation of Michael acceptors, which were intercepted as adducts of thiophenol. These results suggest that these compounds could be activated by bioreduction., (Copyright 2010 Elsevier Ltd. All rights reserved.)

Multidrug resistance related protein 1 (MRP1/ABCC1) is an ABC transporter protein related to the extrusion of reduced glutathione (GSH), oxidized glutathione (GSSG) and GSH-conjugates, as well as leukotriene C(4) and cyclopentane prostaglandins. Inhibition of ABCC1 activity impairs lymphocyte activation. The present work studied ABCC1 expression and activity on a murine macrophage cell line, RAW 267.4 and the effects of ABCC1 classical inhibitors, as well as GSH metabolism modulators, on LPS induced activation. Approximately, 75% of resting cells were positive for ABCC1 and the classical ABCC1 reversors (indomethacin, 0.1-2mM; probenecid, 0.1-10mM and MK571, 0.01-1mM) were able to enhance intracellular CFDA accumulation in a concentration-dependent manner, suggesting ABCC1 inhibition. After LPS (100ng/ml) activation 50% of the population was positive for ABCC1, and this protein was still active. In LPS-activated cells, ABCC1 activity was also impaired by BSO (1mM), an inhibitor of GSH synthesis. Conversely, GSH (5mM) reversed the BSO effect. ABCC1 inhibition by indomethacin, probenecid or MK571 decreased LPS induced nitrite production in a concentration-dependent manner, the same result was observed with BSO and again GSH reversed its effect. The ABCC1 reversors were also able to inhibit iNOS expression. In conclusion, LPS modulated the expression and activity of ABCC1 transporters in RAW macrophages and inhibitors of these transporters were capable of inhibiting nitrite production suggesting a role for ABCC1 transporters in the inflammatory process.

P-glycoprotein (Pgp/ABCB1) and multidrug resistance related protein 1 (MRP1/ABCC1) were first described in multidrug resistant tumor cells. It is presently known that both proteins are also expressed in a variety of normal cells, including lymphocytes. ABCB1 activity has already been detected in subpopulations of murine thymocytes, but there was little information on the expression or activity of ABCC1 in these cells. The present work studied in mice the expression of both proteins by RT-PCR and immunofluorescence. It was possible to identify the presence of ABCB1 and to detect the expression of ABCC1 in these cells. The functional activities of these proteins were also studied in vivo and in vitro measuring the extrusion of fluorescent dyes in association with MDR modulators. Cyclosporine A, verapamil and trifluoperazine inhibited the activity of thymic ABCB1. Indomethacin, probenecid and MK571 were effective in inhibiting ABCC1 activity by thymic cells. ABCB1 was only active in a small percentage of thymocytes being present in the immature double negative (not CD4 nor CD8) subpopulation and the mature single positive (CD4 or CD8) subpopulations. The functional activity of ABCC1, on the other hand, was more homogeneously distributed being found in all thymocyte subpopulations. Possible physiological roles for these transporters on thymocytes are discussed.

Ouabain (OUA) was capable of inhibiting peripheral blood lymphocyte (PBL) proliferation induced by phyothaemagglutinin (PHA) of phorbol ester (TPA), as measured by thymidine incorporation or cell cycle analysis. In this latter case it was possible to detect a block in the progression from G1 to S phase. This inhibition could not be reversed by interleukin (IL)-2 and was not due to an effect on CD 25 expression, as this molecule was only reduced in PHA cultures treated with OUA. Conversely, cultures activated by TPA and OUA showed an increased expression of CD25. The activation antigen CD69 was increased in both situation, suggesting that despite the absence of proliferative response the cells were being activated. The possibility that these cells were being deviated to the activation pathway leading to apoptosis is now under investigation. This study also suggested that CD25 induction may occur via different pathways, and that the selective effect of OUA for PHA-activated cells may become a useful tool for the understanding of the process.

We have recently reported that the diterpene jatrophone antagonizes the effects of phorbol ester in pharmacogical studies. In order to investigate further whether this action is associated with an inhibition of protein kinase C activity, we examined the effect of jatrophone on the stimulation of lymphocyte activities which are dependent on the protein kinase C pathway. Jatrophone (0.02-0.32 microM) caused concentration-dependent and equipotent inhibition of human lymphocyte proliferation induced by 5 micrograms/ml of phytohemagglutinin or by a combination of 100 ng/ml of 12-O-tetradecanoyl phorbol-13-acetate (TPA) plus 0.15 microM ionomicyn, with IC50 values (and their 95% confidence limits) of 53.4 (42.6-65.3) nM and 48.4 (39.4-59.8) nM, respectively. Jatrophone also blocked, in a concentration-dependent fashion, the murine lymphocyte proliferation stimulated by 5 micrograms/ml of concanavalin A, with an IC50 value of 63.5 (51.2-76.5) nM. The inhibition was not due to a toxic effect as the pre-incubation of lymphocytes for 48 h with 0.32 microM jatrophone did not impair the proliferation after removal of the diterpene from the culture medium. Human lymphocytes when pre-treated with 10 ng/ml TPA had a 3 times higher spontaneous natural killer activity against K562 cells and an increased expression of CD69. In addition, jatrophone inhibited both spontaneous and TPA-stimulated natural killer activity and the expression of CD69. Jatrophone concentrations that inhibited 75% of lymphocyte proliferation did not impair the intracellular increase in Ca2+ flux in lymphocytes stimulated by phytohemagglutinin. These results indicate that jatrophone is a potent inhibitor of activation of lymphocytes, probably through inhibition of the protein kinase C pathway.

A large amount of evidence points towards the potential role of lymphokine activated killer (LAK) cells as tools in the treatment of chronically stressed conditions, such as cancer. The modulation of this activity by biologically active endogenous compounds of the HPA (hypothalamic-pituitary-adrenal) axis, however, is not completely understood. Ouabain, a specific inhibitor of Na(+)-K(+)-ATPase, and now recognized as an endogenous component present in human plasma, was tested on IL-2 and TPA-activated killer cells. Ouabain was able to inhibit the generation of LAK activity, as well as to suppress either PHA or TPA-induced lymphocyte proliferation. Once the cells were triggered for cytotoxicity, however, ouabain was not able to interfere with their effector phase, as it did not show any effect when present only during the assay. TPA-induced "LAK-simile" cells displayed the same sensitivity towards ouabain as LAK cells did. Although the physiological relevance of endogenous ouabain secretion remains elusive, these effects of ouabain on LAK cytotoxicity should be considered in patients undergoing this kind of immunotherapy.

The capacity of CS-A to modify some inflammatory aspects of a cell mediated disease process, has been studied. In the present work, CS-A was shown to be effective at two levels during the development of experimental allergic encephalomyelitis (EAE) in rats. If CS-A was given at the time of the induction of the disease it inhibited lymphocyte proliferation in vivo, lymphocyte trapping into the draining lymph nodes, and delayed the subsequent infiltration of cells into the CNS and other inflammatory sites. When given around the time of disease manifestation, CS-A also reduced the rate of cell accumulation into the CNS and foot, but was without effect in modifying lymphocyte trapping in the draining nodes, despite the fact that this was still an ongoing process. Clinical signs were similarly delayed or reduced by both regimen of CS-A treatment. Treatment with CS-A did not lead, however, to long lasting unresponsiveness, since both treated groups suffered a relapse of disease at various times after treatment had been discontinued.