Targeting glutathione S-transferase P and its interactome with selenium compounds in cancer therapy.

The glutathione (GSH) S-transferase family of detoxification and signalling proteins represents a major hub for the metabolism of Selenium-derived compounds. At the same time, these compounds can be used to modulate the expression and multiple activities of GSTs and other glutathione-dependent genes, that are important aspects in both the chemoprevention and therapy of drug-resistant cancers. In this context, the isoform GSTP-1 (GSTP) appears to play a fundamental role. Besides promoting GSH-dependent detoxification of cellular electrophiles, GSTP physically interacts with a number of small molecules and cellular proteins producing regulatory effects across the main signal transduction and transcription pathways (identified as the "regulatory interactome of GSTP"). An emerging molecular mechanism behind such regulatory function is the activity of GSTP as a redox chaperonine responsible for the selective glutathionylation of protein Cys residues in the different subcellular compartments. The redox-sensitive transcription factor Nrf2 was recently identified as one of the regulatory nodes of this interactome at the interface between inflammation, adaptive stress response, and cell death pathways. The influence of Nrf2 in the stress response to cellular electrophiles and its regulatory interaction with GSTP are discussed in this review suggesting the hypothesis that this interaction may represent the actual pharmacological target of Se compounds with thiol peroxidase activity. These points are critically evaluated with a view to further development of these compounds in cancer prevention and the chemotherapy of drug-resistant tumours.

CYP4F2 repression and a modified alpha-tocopherol (vitamin E) metabolism are two independent consequences of ethanol toxicity in human hepatocytes.

The expression of CYP4F2, a form of cytochrome P-450 with proposed role in α-tocopherol and long-chain fatty acid metabolism, was explored in HepG2 and HepaRG human hepatocytes during ethanol toxicity. Cytotoxicity, ROS production, and JNK and ERK1/2 kinase signaling increased in a dose and time-dependent manner during ethanol treatments; CYP4F2 gene expression decreased, while other CYP4F forms, namely 4F11 and 12, increased along with 3A4 and 2E1 isoforms. α-Tocopherol antagonized the cytotoxicity and CYP4F2 gene repression effect of ethanol in HepG2 cells. Ethanol stimulated the tocopherol-ω-hydroxylase activity and the other steps of vitamin E metabolism, which points to a minor role of CYP4F2 in this metabolism of human hepatocytes. PPAR-γ and SREBP-1c followed the same expression pattern of CYP4F2 in response to ethanol and α-tocopherol treatments. Moreover, the pharmacological inhibition of PPAR-γ synergized with ethanol in decreasing CYP4F2 protein expression, which suggests a role of this nuclear receptor in CYP4F2 transcriptional regulation. In conclusion, ethanol toxicity modifies the CYP expression pattern of human hepatic cells impairing CYP4F2 transcription and protein expression. These changes were associated with a lowered expression of the fatty acid biosynthesis regulators PPAR-γ and SREBP-1c, and with an increased enzymatic catabolism of vitamin E. CYP4F2 gene repression and a sustained vitamin E metabolism appear to be independent effects of ethanol toxicity in human hepatocytes.

Glutathione S-transferase pi expression regulates the Nrf2-dependent response to hormetic diselenides.

Glutathione S-transferase pi (GSTP), a phase II gene downstream of the nuclear factor erythroid 2-related factor 2 (Nrf2)-antioxidant-responsive element (ARE)/electrophile response element (EpRE) transcription pathway, plays a key role in both the signaling and detoxification response to Se-organic compounds with thiol peroxidase activity. We here investigated the role of GSTP on the Nrf2 activation response of cells challenged with a new class of diselenides derived from the basic structure of diphenyl diselenide [(PhSe)2]. These diselenides, and particularly 2,2'-diselenyl dibenzoic acid (DSBA), behave as mild thiol peroxidases leading to a moderate generation of H2O2 and NOx, and signaling of stress-activated and survival-promoting MAPKs, which ultimately control the mitochondrial pathway of apoptosis. Used in murine embryonic fibroblasts (MEFs) and HepG2 human hepatocarcinoma cells to produce submaximal conditions of stress, the diselenide compounds stimulated Nrf2 nuclear translocation and then the transcription of the same Nrf2 gene as well as of GSTP and other phase II genes. This resulted in a higher degree of protection against H2O2 cytotoxicity (hormetic effect). Diselenide toxicity increased in GSTP knockout MEFs by a higher generation of NOx and stress activated protein kinase (SAPK)/JNK activation. A lowered hormetic potential of these cells was observed in association with an abnormal expression and nuclear translocation of Nrf2 protein. Immunoprecipitation and affinity purification experiments revealed the existence of an Nrf2/GSTP complex in MEFs and HepG2 cells. Covalent oligomers of GSTP subunits were observed in DSBA-treated HepG2 cells. In conclusion, GSTP gene expression influences the Nrf2-dependent response to hormetic diselenides. Mechanistic interpretation for this GSTP-dependent effect may include a direct and redox-sensitive interaction of GSTP with Nrf2 protein.

Reaction kinetics and targeting to cellular glutathione S-transferase of the glutathione peroxidase mimetic PhSeZnCl and its D,L-polylactide microparticle formulation.

Catalytic properties and cellular effects of the glutathione peroxidase (GPx)-mimetic compound PhSeZnCl or its d,l-lactide polymer microencapsulation form (M-PhSeZnCl) were investigated and compared with the prototypical Se-organic compounds ebselen and diselenide (PhSe)2. PhSeZnCl was confirmed to catalyze the ping-pong reaction of GPx with higher Vmax than ebselen and (PhSe)2, but the catalytic efficiency calculated for the cosubstrates glutathione (GSH) and H2O2, and particularly the high reactivity against thiols (lowest KM for GSH in the series of test molecules), suggested poor biological applicability of PhSeZnCl as a GPx mimetic. Cytotoxicity of PhSeZnCl was demonstrated in various cancer cell lines via increased reactive oxygen species (ROS) generation, depletion of intracellular thiols, and induction of apoptosis. Experiments carried out in GSH S-transferase P (GSTP)-overexpressing K562 human erythroleukemia cells and in GSTP1-1-knockout murine embryonic fibroblasts (MEFs) demonstrated that this cytosolic enzyme represents a preferential target of the redox disturbances produced by this Se-compound with a key role in controlling H2O2 generation and the perturbation of stress/survival kinase signaling. Microencapsulation was adopted as a strategy to control the thiol reactivity and oxidative stress effects of PhSeZnCl, then assessing applications alternative to anticancer. The uptake of this "depowered" GPx-mimetic formulation, which occurred through an endocytosis-like mechanism, resulted in a marked reduction of cytotoxicity. In MCF-7 cells transfected with different allelic variants of GSTP, M-PhSeZnCl lowered the burst of cellular ROS induced by the exposure to extracellular H2O2, and the extent of this effect changed between the GSTP variants. Microencapsulation is a straightforward strategy to mitigate the toxicity of thiol-reactive Se-organic drugs that enhanced the antioxidant and cellular protective effects of PhSeZnCl. A mechanistic linkage of these effects with the expression pattern and signaling properties of GSTP . This has overcome the GPx-mimetic paradigm proposed for Se-organic drugs with a more pragmatic concept of GSTP signaling modulators.

Blood thiol status and erythrocyte glutathione-S-transferase in chronic kidney disease patients on treatment with frequent (daily) hemodialysis.

BACKGROUND: Chronic kidney disease (CKD) is a condition of impaired homeostasis of blood thiols characterized by a severe hyperhomocysteinemia (HH) and abnormal expression of the red blood cell glutathione (GSH)-consuming enzyme GSH S-transferase (eGST) (Galli et al., Clin Chem 1999). The correlation between plasma Hcy and eGST recently identified by our group (Dessì et al., Amino Acids 2012) suggests a role of this detoxifying enzyme in the impaired thiol status of CKD treated with hemodialysis therapy (HD). This retrospective study is aimed at investigating whether frequent HD can alleviate these biochemical symptoms of CKD. METHODS: Laboratory data of a population of 98 HD patients investigated for plasma Hcy and blood thiol status between 1999 and 2004 were examined. A frequent HD method carried out with a 2-h daily schedule (daily HD) (DHD) was compared with standard 4-h × 3/week protocol of HD (SHD) in either cross-sectional (n = 70 SHD vs. n = 28 DHD) and prospective A-B design (n = 18 SHD patients shifted to DHD). RESULTS: The results demonstrate that DHD produces a better correction than SHD of the uremic retention solute Hcy as well as of Cys and Cys-Gly measured in plasma. Such a correction effect of DHD on HH correlates with that on the detoxification enzyme eGST and on pGSH. CONCLUSIONS: These findings point to a role of frequent dialysis in the depuration of uremic retention solutes that may interfere with thiol metabolism and redox in HD patients. These solutes may include substrates of eGST that await further investigation for molecular identification and better removal by more efficient dialysis therapies.

Cannabidiol inhibits angiogenesis by multiple mechanisms.

BACKGROUND AND PURPOSE: Several studies have demonstrated anti-proliferative and pro-apoptotic actions of cannabinoids on various tumours, together with their anti-angiogenic properties. The non-psychoactive cannabinoid cannabidiol (CBD) effectively inhibits the growth of different types of tumours in vitro and in vivo and down-regulates some pro-angiogenic signals produced by glioma cells. As its anti-angiogenic properties have not been thoroughly investigated to date, and given its very favourable pharmacological and toxicological profile, here, we evaluated the ability of CBD to modulate tumour angiogenesis. EXPERIMENTAL APPROACH: Firstly, we evaluated the effect of CBD on human umbilical vein endothelial cell (HUVEC) proliferation and viability - through [3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide] assay and FACS analysis - and in vitro motility - both in a classical Boyden chamber test and in a wound-healing assay. We next investigated CBD effects on different angiogenesis-related proteins released by HUVECs, using an angiogenesis array kit and an ELISA directed at MMP2. Then we evaluated its effects on in vitro angiogenesis in treated HUVECs invading a Matrigel layer and in HUVEC spheroids embedded into collagen gels, and further characterized its effects in vivo using a Matrigel sponge model of angiogenesis in C57/BL6 mice. KEY RESULTS: CBD induced HUVEC cytostasis without inducing apoptosis, inhibited HUVEC migration, invasion and sprouting in vitro, and angiogenesis in vivo in Matrigel sponges. These effects were associated with the down-modulation of several angiogenesis-related molecules. CONCLUSIONS AND IMPLICATIONS: This study reveals that CBD inhibits angiogenesis by multiple mechanisms. Its dual effect on both tumour and endothelial cells supports the hypothesis that CBD has potential as an effective agent in cancer therapy.

Microsatellite instability and gastric non-invasive neoplasia in a high risk population in Cesena, Italy.

BACKGROUND/AIMS: In the natural history of gastric cancer, non-invasive neoplasia (NiN) precedes invasive carcinoma. A histological classification of gastric NiN has recently been proposed by a World Health Organisation international panel of experts. Genetic instability is known to be among the molecular pathways involved in gastric oncogenesis. In this retrospective cross sectional study, microsatellite instability (MSI) was analysed in a consecutive series of NiN and NiN related histological alterations from a northern Italian region at high risk for gastric cancer. PATIENTS/METHODS: Fifty five consecutive cases (indefinite for NiN, 29 cases; low grade NiN, 17 cases; high grade NiN, nine cases) were analysed by radioactive polymerase chain reaction and electrophoresis for microsatellite alterations at six loci (BAT25, BAT26, D2S123, D5S346, D17S250, and D3S1317). MSI was defined according to the Bethesda criteria distinguishing: (1) no instability in the analysed loci; (2) low frequency MSI (MSI-L); and (3) high frequency MSI (MSI-H). Immunohistochemical expression of MLH1 and MSH2 proteins was also analysed in all cases. RESULTS: Overall, MSI was found in 11 of 55 cases (indefinite for NiN, five of 29 (MSI-L, four; MSI-H, one); low grade NiN, three of 17 (MSI-L, one; MSI-H, two); high grade NiN, three of nine (MSI-L, one; MSI-H, two). CONCLUSIONS: In an Italian high risk area for gastric cancer, MSI is part of the spectrum of genetic alterations in gastric non-invasive neoplasia. In European populations at high risk of gastric cancer, DNA repair system alterations are thought to be among the early molecular events in gastric carcinogenesis.

Central and peripheral involvement of mu receptors in gastric secretory effects of opioids in the dog.

The effects of dermorphin and morphine on gastric acid secretion were studied in conscious dogs with both gastric fistulas (GF) and Heidenhain pouches (HP). Under basal conditions dermorphin and morphine, infused systemically at graded doses, produced a significant increase in acid secretion from both GF and HP. This increase was significantly inhibited by naloxone, naltrexone methylbromide and N-methyl-levallorphan methanesulphonate. Dermorphin did not modify the acid output stimulated by 2-deoxy-D-glucose from GF, while morphine significantly inhibited it; on the contrary acid secretion from HP was increased in this test by both dermorphin and morphine. Acid secretion from GF stimulated by pentagastrin was unaffected by morphine and significantly enhanced by dermorphin. Under these conditions a significant increase in acid secretion from HP was recorded with dermorphin and morphine. Naloxone and N-methyl-levallorphan methanesulphonate, given during pentagastrin-stimulated secretion, significantly inhibited acid output 'per se' from GF and HP and prevented the stimulatory effect of dermorphin and morphine. Bethanechol-induced secretion from GF and HP was significantly increased by both dermorphin and morphine. The present results demonstrate that opioids have simultaneous yet opposite effects on acid secretion in the dog and that mu receptors are involved in both the excitatory and inhibitory effects. Excitatory effects do not seem to be mediated via a vagal pathway (peripheral ?), in contrast to the inhibitory effects (central ?). The inhibitory effects of opiate antagonists on pentagastrin-stimulated secretion suggest a physiological role of peripheral opioid receptors in gastric acid secretion.