Angel Ortega

Glutathione in Cancer Biology and Therapy

The glutathione (GSH) content of cancer cells is particularly relevant in regulating mutagenic mechanisms, DNA synthesis, growth, and multidrug and radiation resistance. In malignant tumors, as compared with normal tissues, that resistance associates in most cases with higher GSH levels within these cancer cells. Thus, approaches to cancer treatment based on modulation of GSH should control possible growth-associated changes in GSH content and synthesis in these cells. Despite the potential benefits for cancer therapy of a selective GSH-depleting strategy, such a methodology has remained elusive up to now. Metastatic spread, not primary tumor burden, is the leading cause of cancer death. For patient prognosis to improve, new systemic therapies capable of effectively inhibiting the outgrowth of seeded tumor cells are needed. Interaction of metastatic cells with the vascular endothelium activates local release of proinflammatory cytokines, which act as signals promoting cancer cell adhesion, extravasation, and proliferation. Recent work shows that a high percentage of metastatic cells with high GSH levels survive the combined nitrosative and oxidative stresses elicited by the vascular endothelium and possibly by macrophages and granulocytes. γ-Glutamyl transpeptidase overexpression and an inter-organ flow of GSH (where the liver plays a central role), by increasing cysteine availability for tumor GSH synthesis, function in combination as a metastatic-growth promoting mechanism. The present review focuses on an analysis of links among GSH, adaptive responses to stress, molecular mechanisms of invasive cancer cell survival and death, and sensitization of metastatic cells to therapy. Experimental evidence shows that acceleration of GSH efflux facilitates selective GSH depletion in metastatic cells.

Inhibition of cancer growth by resveratrol is related to its low bioavailability.

The relationship between resveratrol (RES) bioavalability and its effect on tumor growth was investigated. Tissue levels of RES were studied after i.v. and oral administration of trans-resveratrol (t-RES) to rabbits, rats, and mice. Half-life of RES in plasma, after i.v. administration of 20 mg t-RES/kg b.wt., was very short (e.g., 14.4 min in rabbits). The highest concentration of RES in plasma, either after i.v. or oral administration (e.g., 2.6 +/- 1.0 microM in mice 2.5 min after receiving 20 mg t-RES/kg orally), was reached within the first 5 min in all animals studied. Extravascular levels (brain, lung, liver, and kidney) of RES, which paralleled those in plasma, were always < 1 nmol/g fresh tissue. RES measured in plasma or tissues was in the trans form (at least 99%). Hepatocytes metabolized t-RES in a dose-dependent fashion (e.g., 43 nmol of t-RES/g x min in the presence of 20 microM tRES), which means that the liver can remove circulating RES very rapidly. In vitro B16 melanoma (B16M) cell proliferation and generation of reactive oxygen species (ROS) was inhibited by t-RES in a concentration-dependent fashion (100% inhibition of tumor growth was found in the presence of 5 microM t-RES). Addition of 10 microM H(2)O(2) to B16M cells, cultured in the presence of 5 microM t-RES, reactivated cell growth. Oral administration of t-RES (20 mg/kg twice per day; or included in the drinking water at 23 mg/l) did not inhibit growth of B16M inoculated into the footpad of mice (solid growth). However, oral administration of t-RES (as above) decreased hepatic metastatic invasion of B16M cells inoculated intrasplenically. The antimetastatic mechanism involves a t-RES (1 microM)-induced inhibition of vascular adhesion molecule 1 (VCAM-1) expression in the hepatic sinusoidal endothelium (HSE), which consequently decreased in vitro B16M cell adhesion to the endothelium via very late activation antigen 4 (VLA-4).

Oxidative stress in environmental-induced carcinogenesis

Reactive oxygen species (ROS) are the more abundant free radicals in nature and have been related with a number of tissue/organ injuries induced by xenobiotics, ischemia, activation of leucocytes, UV exposition, etc. Oxidative stress is caused by an imbalance between ROS production and a biological systems ability to readily detoxify these reactive intermediates or easily repair the resulting damage. Thus, oxidative stress is accepted as a critical pathophysiological mechanism in different frequent human pathologies, including cancer. In fact ROS can cause protein, lipid, and DNA damage, and malignant tumors often show increased levels of DNA base oxidation and mutations. Different lifestyle- and environmental-related factors (including, e.g., tobacco smoking, diet, alcohol, ionizing radiations, biocides, pesticides, viral infections) and other health-related factors (e.g. obesity or the aging process) may be procarcinogenic. In all these cases oxidative stress acts as a critical pathophysiological mechanism. Nevertheless it is important to remark that, in agreement with present knowledge, oxidative/nitrosative/metabolic stress, inflammation, senescence, and cancer are linked concepts that must be discussed in a coordinated manner.

Glutathione Is Recruited into the Nucleus in Early Phases of Cell Proliferation

We have studied the possible correlation between nuclear glutathione distribution and the progression of the cell cycle. The former was studied by confocal microscopy using 5-chloromethyl fluorescein diacetate and the latter by flow cytometry and protein expression of Id2 and p107. In proliferating cells, when 41% of them were in the S+G2/M phase of the cell cycle GSH was located mainly in the nucleus. When cells reached confluence (G0/G1) GSH was localized in the cytoplasm with a perinuclear distribution. The nucleus/cytoplasm fluorescence ratio for GSH reached a maximal mean value of 4.2 ± 0.8 at 6 h after cell plating. A ratio higher than 2 was maintained during exponential cell growth. In the G0/G1 phase of the cell cycle, the nucleus/cytoplasm GSH ratio decreased to values close to 1. We report here that cells concentrate GSH in the nucleus in the early phases of cell growth, when most of the cells are in an active division phase, and that GSH redistributes uniformly between the nucleus and the cytoplasm when cells reach confluence.

Glutathione in cancer cell death.

Glutathione (L-γ-glutamyl-L-cysteinyl-glycine; GSH) in cancer cells is particularly relevant in the regulation of carcinogenic mechanisms; sensitivity against cytotoxic drugs, ionizing radiations, and some cytokines; DNA synthesis; and cell proliferation and death. The intracellular thiol redox state (controlled by GSH) is one of the endogenous effectors involved in regulating the mitochondrial permeability transition pore complex and, in consequence, thiol oxidation can be a causal factor in the mitochondrion-based mechanism that leads to cell death. Nevertheless GSH depletion is a common feature not only of apoptosis but also of other types of cell death. Indeed rates of GSH synthesis and fluxes regulate its levels in cellular compartments, and potentially influence switches among different mechanisms of death. How changes in gene expression, post-translational modifications of proteins, and signaling cascades are implicated will be discussed. Furthermore, this review will finally analyze whether GSH depletion may facilitate cancer cell death under in vivo conditions, and how this can be applied to cancer therapy.

Complex I Dysfunction and Tolerance to Nitroglycerin: An Approach Based on Mitochondrial-Targeted Antioxidants

Nitroglycerin (GTN) tolerance was induced in vivo (rats) and in vitro (rat and human vessels). Electrochemical detection revealed that the incubation dose of GTN (5×10−6 mol/L) did not release NO or modify O2 consumption when administered acutely. However, development of tolerance produced a decrease in both mitochondrial O2 consumption and the Km for O2 in animal and human vessels and endothelial cells in a noncompetitive action. GTN tolerance has been associated with impairment of GTN biotransformation through inhibition of aldehyde dehydrogenase (ALDH)-2, and with uncoupling of mitochondrial respiration. Feeding rats with mitochondrial-targeted antioxidants (mitoquinone [MQ]) and in vitro coincubation with MQ (10−6 mol/L) or glutathione (GSH) ester (10−4 mol/L) prevented tolerance and the effects of GTN on mitochondrial respiration and ALDH-2 activity. Biotransformation of GTN requires functionally active mitochondria and induces reactive oxygen species production and oxidative stress within this organelle, as it is inhibited by mitochondrial-targeted antioxidants and is absent in HUVEC&rgr;0 cells. Experiments analyzing complex I–dependent respiration demonstrate that its inhibition by GTN is prevented by mitochondrial-targeted antioxidants. Furthermore, in presence of succinate (10×10−3 mol/L), a complex II electron donor added to bypass complex I–dependent respiration, GTN-treated cells exhibited O2 consumption rates similar to those of controls, thus suggesting that complex I was affected by GTN. We propose that, following prolonged treatment with GTN in addition to ALDH-2, complex I is a target for mitochondrially generated reactive oxygen species. Our data also suggest a role for mitochondrial-targeted antioxidants as therapeutic tools in the control of the tolerance that accompanies chronic nitrate use.

Acceleration of Glutathione Efflux and Inhibition of γ-Glutamyltranspeptidase Sensitize Metastatic B16 Melanoma Cells to Endothelium-induced Cytotoxicity

Highly metastatic B16 melanoma (B16M)-F10 cells, as compared with the low metastatic B16M-F1 line, have higher GSH content and preferentially overexpress BCL-2. In addition to its anti-apoptotic properties, BCL-2 inhibits efflux of GSH from B16M-F10 cells and thereby may facilitate metastatic cell resistance against endothelium-induced oxidative/nitrosative stress. Thus, we investigated in B16M-F10 cells which molecular mechanisms channel GSH release and whether their modulation may influence metastatic activity. GSH efflux was abolished in multidrug resistance protein 1 knock-out (MRP-/-1) B16M-F10 transfected with the Bcl-2 gene or in MRP-/-1 B16M-F10 cells incubated with l-methionine, which indicates that GSH release from B16M-F10 cells is channeled through MRP1 and a BCL-2-dependent system (likely related to an l-methionine-sensitive GSH carrier previously detected in hepatocytes). The BCL-2-dependent system was identified as the cystic fibrosis transmembrane conductance regulator, since monoclonal antibodies against this ion channel or H-89 (a protein kinase A-selective inhibitor)-induced inhibition of cystic fibrosis transmembrane conductance regulator gene expression completely blocked the BCL-2-sensitive GSH release. By using a perifusion system that mimics in vivo conditions, we found that GSH depletion in metastatic cells can be achieved by using Bcl-2 antisense oligodeoxynucleotide- and verapamil (an MRP1 activator)-induced acceleration of GSH efflux, in combination with acivicin-induced inhibition of γ-glutamyltranspeptidase (which limits GSH synthesis by preventing cysteine generation from extracellular GSH). When applied under in vivo conditions, this strategy increased tumor cytotoxicity (up to ∼90%) during B16M-F10 cell adhesion to the hepatic sinusoidal endothelium.

Natural polyphenols facilitate elimination of HT-29 colorectal cancer xenografts by chemoradiotherapy: a Bcl-2- and superoxide dismutase 2-dependent mechanism

Colorectal cancer is one of the most common malignancies worldwide. The treatment of advanced colorectal cancer with chemotherapy and radiation has two major problems: development of tumor resistance to therapy and nonspecific toxicity towards normal tissues. Different plant-derived polyphenols show anticancer properties and are pharmacologically safe. In vitro growth of human HT-29 colorectal cancer cells is inhibited (∼56%) by bioavailable concentrations of trans-pterostilbene (trans-3,5-dimethoxy-4′-hydroxystilbene; t-PTER) and quercetin (3,3′,4′,5,6-pentahydroxyflavone; QUER), two structurally related and naturally occurring small polyphenols. I.v. administration of t-PTER and QUER (20 mg/kg × day) inhibits growth of HT-29 xenografts (∼51%). Combined administration of t-PTER + QUER, FOLFOX6 (oxaliplatin, leucovorin, and 5-fluorouracil; a first-line chemotherapy regimen), and radiotherapy (X-rays) eliminates HT-29 cells growing in vivo leading to long-term survival (>120 days). Gene expression analysis of a Bcl-2 family of genes and antioxidant enzymes revealed that t-PTER + QUER treatment preferentially promotes, in HT-29 cells growing in vivo, (a) superoxide dismutase 2 overexpression (∼5.7-fold, via specificity protein 1-dependent transcription regulation) and (b) down-regulation of bcl-2 expression (∼3.3-fold, via inhibition of nuclear factor-κB activation). Antisense oligodeoxynucleotides to human superoxide dismutase 2 and/or ectopic bcl-2 overexpression avoided polyphenols and chemoradiotherapy-induced colorectal cancer elimination and showed that the mangano-type superoxide dismutase and Bcl-2 are key targets in the molecular mechanism activated by the combined application of t-PTER and QUER. [Mol Cancer Ther 2008;7(10):3330–42]

Natural polyphenols in cancer therapy.

Natural polyphenols are secondary metabolites of plants involved in defense against different types of stress. Extracts containing these compounds have been used for thousands of years in traditional eastern medicine. Polyphenols act on multiple targets in pathways and mechanisms related to carcinogenesis, tumor cell proliferation and death, inflammation, metastatic spread, angiogenesis, or drug and radiation resistance. Nevertheless, reported effects claimed for polyphenols are controversial, since correlations between in vitro effects and in vivo evidence are poorly established. The main discrepancy between health claims versus clinical observations is the frequent use of nonphysiologically relevant concentrations of these compounds and their metabolites in efficacy and mechanistic studies. The present review will discuss how in vivo administration correlates with polyphenol metabolism, toxicity, and bioavailability. Analysis of the general application of polyphenols in cancer therapy will be complemented by potential applications in the therapy of specific tumors, including melanoma, colorectal and lung cancers. Possible pharmaceutical formulations, structural modifications, combinations, and delivery systems aimed to increase bioavailability and/or biological effects will be discussed. Final remarks will include recommendations for future research and developments.

Endothelial nitric oxide synthase regulates N-Ras activation on the Golgi complex of antigen-stimulated T cells

Ras/ERK signaling plays an important role in T cell activation and development. We recently reported that endothelial nitric oxide synthase (eNOS)-derived NO regulates T cell receptor (TCR)-dependent ERK activation by a cGMP-independent mechanism. Here, we explore the mechanisms through which eNOS exerts this regulation. We have found that eNOS-derived NO positively regulates Ras/ERK activation in T cells stimulated with antigen on antigen-presenting cells (APCs). Intracellular activation of N-, H-, and K-Ras was monitored with fluorescent probes in T cells stably transfected with eNOS-GFP or its G2A point mutant, which is defective in activity and cellular localization. Using this system, we demonstrate that eNOS selectively activates N-Ras but not K-Ras on the Golgi complex of T cells engaged with APC, even though Ras isoforms are activated in response to NO from donors. We further show that activation of N-Ras involves eNOS-dependent S-nitrosylation on Cys118, suggesting that upon TCR engagement, eNOS-derived NO directly activates N-Ras on the Golgi. Moreover, wild-type but not C118S N-Ras increased TCR-dependent apoptosis, suggesting that S-nitrosylation of Cys118 contributes to activation-induced T cell death. Our data define a signaling mechanism for the regulation of the Ras/ERK pathway based on the eNOS-dependent differential activation of N-Ras and K-Ras at specific cell compartments.