Vivian M. Rumjanek

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.

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.

Ouabain Induces Apoptosis on PHA-Activated Lymphocytes

Apoptotic cell death plays a critical role in immune system homeostasis, and c-myc protooncogene deregulated expression is a component of this programmed genomic response. Pharmacological intervention and modulation of peripheral lymphocytes apoptosis would have important implications. The present results indicate that ouabain, a specific inhibitor of Na+K+-ATPase, promotes an increased expression of c-myc mRNA, and induces apoptosis in PHA-stimulated lymphocytes. Furthermore, this ouabain-induced apoptosis cannot be counteracted by the addition of exogenous IL-2.

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.

Anti-inflammatory and Antinociceptive Activity of Ouabain in Mice

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.

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.

New pterocarpanquinones: Synthesis, antineoplasic activity on cultured human malignant cell lines and TNF-α modulation in human PBMC cells

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.

On the molecular mechanisms for the highly procoagulant pattern of C6 glioma cells

Summary.  Background: That there is a correlation between cancer and procoagulant states is well‐known. C6 glioma cell line was originally induced in random‐bred Wistar–Furth rats and is morphologically similar to glioblastoma multiforme, the most common aggressive glioma resistant to therapeutic interventions. Objectives: In this study we analyzed the molecular mechanisms responsible for the highly procoagulant properties of C6 glioma cells. Methods: The presence of tissue factor (TF) and phosphatidylserine (PS) in C6 cells was investigated by flow‐cytometric and functional analyses. The assembly of extrinsic tenase, intrinsic tenase and prothrombinase complexes on these cells was studied using enzymatic assays employing plasma or purified proteins. Results: TF was identified by flow‐cytometric and functional [factor (F) Xa formation in the presence of cells and FVIIa] assays. Alternatively, conversion of FX into FXa was also observed in the presence of C6 cells, FIXa and FVIIIa. This effect was both cell‐ and FVIIIa‐dependent, being consistent with formation of the intrinsic tenase complex. C6 cells were also able to activate prothrombin in the presence of FXa and FVa, thus supporting formation of the prothrombinase complex. This ability was similar to positive controls performed with PS‐containing vesicles. Accordingly, exposure of PS on C6 cells was demonstrated by flow cytometry employing specific anti‐PS antibodies. In addition, annexin V, which blocks PS binding sites, inhibited FX and prothrombin conversion by their respective C6‐assembled activating complexes. Conclusion: C6 glioma cells support all procoagulant reactions leading to robust thrombin formation. This ability results from concomitant TF exposure and from the presence of the anionic lipid PS at the outer leaflet of cell membrane. Therefore, this animal cell line may be used to explore new aspects concerning the role of blood coagulation proteins in tumor biology, especially those affecting the central nervous system.

Simultaneous tissue factor expression and phosphatidylserine exposure account for the highly procoagulant pattern of melanoma cell lines.

A correlation between cancer and hypercoagulability has been described for more than a century. Patients with cancer are at increased risk for thrombotic complications, and the clotting initiator protein, tissue factor (TF), is possibly involved in this process. In addition to TF, the presence of negatively charged phospholipids, particularly phosphatidylserine (PS), is necessary to support some of the blood-clotting reactions. There are few reports describing PS exposure by tumor cells. In this study, we characterized the procoagulant properties of the murine B16F10 and the human WM-266-4 melanoma cell lines. Flow cytometry analyses showed constitutive TF expression by both cell lines, in contrast to negative staining observed for the nontumorigenic melanocyte lineage, melan-A. In addition, tumor cells accelerate plasma clotting in a number-dependent manner. For WM-266-4, this ability was partially reversed by an anti-TF antibody but not by aprotinin, a nonspecific serine-protease inhibitor. Furthermore, flow-cytometric analyses showed the presence of PS at the outer leaflet of both cell lines. This phenomenon was determinant for the assembly of the intrinsic tenase (FIXa/FVIIIa) and prothrombinase (FXa/FVa) complexes, resulting in the activation of FX to FXa and prothrombin to thrombin, respectively. As a result, incubation of WM-266-4 with human plasma produces robust thrombin generation. In conclusion, simultaneous TF expression and PS exposure are responsible for the highly procoagulant pattern of the aggressive melanoma cell lines B16F10 and WM-266-4. Therefore, these cell lines might be regarded as useful models for studying the role of blood coagulation proteins in tumor biology.

Pregnancy and the susceptibility of Lewis rats to experimental allergic encephalomyelitis.

Pregnant Lewis rats challenged with encephalitogen during the second or third week of gestation were afforded a high level of protection against experimental allergic encephalomyelitis, whilst females sensitised during the first week of pregnancy enjoyed only limited protection from clinical signs of disease. When rechallenged with encephalitogen, females which had been sensitised during pregnancy were marginally more susceptible to reinduction of clinical signs of disease than their virgin counterparts. Mothers inoculated during the first week of gestation were the only group to produce abnormal young.