Márcia Alves Marques Capella

Multidrug resistance in tumour cells: characterization of the multidrug resistant cell line K562-Lucena 1

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.

A light in multidrug resistance: Photodynamic treatment of multidrug-resistant tumors

The major drawback of cancer chemotherapy is the development of multidrug-resistant (MDR) tumor cells, which are cross-resistant to a broad range of structurally and functionally unrelated agents, making it difficult to treat these tumors. In the last decade, a number of authors have studied the effects of photodynamic therapy (PDT), a combination of visible light with photosensitizing agents, on MDR cells. The results, although still inconclusive, have raised the possibility of treating MDR tumors by PDT. This review examines the growing literature concerning the responses of MDR cells to PDT, while stressing the need for the development of new photosensitizers that possess the necessary characteristics for the photodynamic treatment of this class of tumor.

Mechanisms of vanadate-induced cellular toxicity: role of cellular glutathione and NADPH

Besides its insulin-mimetic effects, vanadate is also known to have a variety of physiological and pharmacological properties, varying from induction of cell growth to cell death and is also a modulator of the multidrug resistance phenotype. However, the mechanisms underlying these effects are still not understood. The present report analyzes the mechanisms of vanadate toxicity in two cell lines previously found to have different susceptibilities to this compound. It was shown that catalase and GSH reversed the sensitivity of a vanadate-sensitive cell line and NADPH sensitized vanadate-resistant cells. NADPH also increased the residues of P-Tyr and the induction of Ras protein expression in vanadate-resistant cells, while GSH avoided these effects in vanadate-sensitive cells. Thus, it seems that the effects of vanadate in signal transduction are dependent on NADPH and are related to cell death. Based on the effects observed in the present study it was suggested that once inside the cell, vanadate is reduced to vanadyl in a process dependent on NADPH. Vanadyl then may react with H2O2 generating primarily peroxovanadium species (PV) rather than following the Fenton reaction. The PV compounds formed would be responsible for P-Tyr increase, Ras induction, and cell death. The results obtained also point to vanadate as a possible chemotherapic in the use of multidrug-resistant tumors.

Methylene blue reverts multidrug resistance: sensitivity of multidrug resistant cells to this dye and its photodynamic action

Photodynamic action has been advocated as an alternative treatment of tumors but the most common used dyes, hematoporphyrin derivatives, are substrate for P-glycoprotein. This study investigated the MDR-reverting properties of methylene blue (MB) and compared the sensitivity to its photodynamic action (PDA) in five cell lines that either express or do not express the MDR phenotype. MB was able to revert the MDR phenotype and there was no difference in sensitivity to MB-PDA between MDR and non-MDR cells, suggesting that MB has the advantage of being used simultaneously as a MDR reverser and a photodynamic agent.

Mechanisms of ouabain toxicity

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.

Differences in Sensitivity to UVC, UVB and UVA Radiation of a Multidrug‐Resistant Cell Line Overexpressing P‐Glycoprotein

Abstract— Multidrug resistance (MDR) is the phenomenon in which cultured tumor cells, selected for resistance to one chemotherapeutic agent, simultaneously acquire resistance to several apparently unrelated drugs. The MDR phenotype is multifactorial. The best‐studied mechanism involves the expression of a membrane protein that acts as an energy‐dependent efflux pump, known as P‐glycoprotein (Pgp), capable of extruding toxic materials from the cell. In this work, resistance to UVA radiation, but not to UVC nor UVB, was observed in an MDR leukemia cell line. This cell line overexpresses Pgp. To study the role of Pgp in the resistance to UVA radiation, two MDR modulators or reversing agents (verapamil and cyclosporin A) capable of blocking Pgp activity were used. Cell viability was assessed and the techniques of flow cytometry and fluorescence microscopy were employed to measure the extrusion of rhodamine 123 by the efflux pump. The results show that MDR modulators did not modify the resistance to UVA radiation. Furthermore, although cell viability was not significantly altered, Pgp function was impaired after UVA treatment, suggesting that this glycoprotein may be a physical target for oxidative damage, and that other factors may be responsible for the UVA resistance. In agreement with this, it was found that the resistant cell line presented a higher catalase activity than the parental (non‐MDR) cell line.

Vanadate Is Toxic to Adherent- Growing Multidrug-Resistant Cells

The development of multidrug resistance (MDR) is the primary cause of failure of cancer chemotherapy and circumventing this problem is a major challenge in oncology. Vanadate is known to inhibit the ATPase activity of the P-glycoprotein and multidrug-resistant associated protein. In the present study we show that adherent MDR cells are more sensitive to vanadate than adherent non-MDR ones, but the same is not true for suspension-growing cells. Vanadate induced stress fiber in the non-MDR adherent MDCK cell line, but destroyed the actin fibers of MDCK/60 and MA104 cells, two adherent MDR cell lines, suggesting that the sensitivity of these cells to vanadate is related to their actin cytoskeleton. The results suggest that vanadate may be used as an adjuvant in the chemotherapy of solid tumors, not only as an ATPase inhibitor but also because of its effect in the MDR cell cytoskeleton.

Sensitivity to microcystins: a comparative study in human cell lines with and without multidrug resistance phenotype.

Multidrug resistance (MDR) is an obstacle in cancer treatment. An understanding of how tumoral cells react to oxidants can help us elucidate the cellular mechanism involved in resistance. Microcystins are cyanobacteria hepatotoxins known to generate oxidative stress. The aim of this study was to compare the sensitivity to microcystins of human tumoral cell lines with (Lucena) and without (K562) MDR phenotype. Endpoints analyzed were effective microcystins concentration to 50% of exposed cells (EC50), antioxidant enzyme activity, lipid peroxidation, DNA damage, reactive oxygen species (ROS) concentration, and tubulin content. Lucena were more resistant and showed lower DNA damage than K562 cells (P < 0.05). Although microcystins did not alter catalase activity, a higher mean value was observed in Lucena than in K562 cells. Lucena cells also showed lower ROS concentration and higher tubulin content. The higher metabolism associated with the MDR phenotype should increase ROS concentration and make for an improved antioxidant defense against the toxic effects of microcystins.

Trypanosoma cruzi-cardiomyocytes: new contributions regarding a better understanding of this interaction

The present paper summarizes new approaches regarding the progress done to the understanding of the interaction of Trypanosoma cruzi-cardiomyocytes. Mannose receptors localized at the surface of heart muscle cell are involved in binding and uptake of the parasite. One of the most striking events in the parasite-heart muscle cells interaction is the disruption of the actin cytoskeleton. We have investigated the regulation of the actin mRNA during the cytopathology induced in myocardial cells by the parasite. T. cruzi invasion increases calcium resting levels in cardiomyocytes. We have previously shown that Ca2+ ATPase of the sarcoplasmic reticulum (SERCA) is involved in the invasion of T. cruzi in cardiomyocytes. Treating the cells with thapsigargin, a drug that binds to all SERCA ATPases and causes depletion of intracellular calcium stores, we found a 75% inhibition in the T. cruzi-cardiomyocytes invasion.

The role of KATP channels on propofol preconditioning in a cellular model of renal ischemia-reperfusion.

BACKGROUND: Propofol (2,6-diisopropylphenol) has been shown to protect several organs, including the kidneys, from ischemia-reperfusion (I-R)-induced injury. Although propofol affects adenosine triphosphate-sensitive potassium (KATP) channels in nonrenal tissues, it is still not clear by which mechanisms propofol protects renal cells from such damage. In this study, we investigated whether propofol induces renal preconditioning through renal KATP channels. METHODS: A reversible ATP depletion (antimycin A) followed by restoration of substrate supply in LLC-PK1 cells was used as an in vitro model of renal I-R. Cell viability was assessed by dimethylthiazol-diphenyltetrazol bromide and trypan blue dye exclusion test assays. Apoptosis was evaluated by annexin V–fluorescein isothiocyanate staining by flow cytometry and immunofluorescence. Propofol treatments were initiated at various time intervals: 1 or 24 h before ischemia, only during ischemia, or only during reperfusion. To evaluate the mechanisms of propofol protection, specific KATP channel inhibitors or activators were used in some experiments during propofol pretreatment. RESULTS: Propofol attenuated I-R injury on LLC-PK1 cells when present either 1 or 24 h before initiated I-R, and also during the recovery period, but not when added only during ischemia. Propofol pretreatment significantly protected LLC-PK1 from I-R-induced apoptosis. The protective effect of propofol was prevented by glibenclamide (a sarcolemmal ATP-dependent K+ channel blocker) and decreased by 5-hydroxidecanoic acid (a mitochondrial ATP-dependent K+ channel blocker), but it was not modified by diazoxide (a selective opener of ATP-sensitive K+ channel). CONCLUSION: Propofol protected cells against apoptosis induced by I-R. This protection was probably due to a preconditioning effect of propofol and was, at least in part, mediated by KATP channels.