Synergism of small molecules targeting VDAC with sorafenib, regorafenib or lenvatinib on hepatocarcinoma cell proliferation and survival.

Voltage dependent anion channels (VDAC) in the outer mitochondrial membrane regulate the influx of metabolites that sustain mitochondrial metabolism and the efflux of ATP to the cytosol. Free tubulin and NADH close VDAC. The VDAC-binding small molecules X1 and SC18 modulate mitochondrial metabolism. X1 antagonizes the inhibitory effect of tubulin on VDAC. SC18 occupies an NADH-binding pocket in the inner wall of all VDAC isoforms. Here, we hypothesized that X1 and SC18 have a synergistic effect with sorafenib, regorafenib or lenvatinib to arrest proliferation and induce death in hepatocarcinoma cells. We used colony formation assays to determine cell proliferation, and a combination of calcein/propidium iodide, and trypan blue exclusion to assess cell death in the well differentiated Huh7 and the poorly differentiated SNU-449 cells. Synergism was assessed using the Chou-Talalay method. The inhibitory effect of X1, SC18, sorafenib, regorafenib and lenvatinib was concentration and time dependent. IC50s calculated from the inhibition of clonogenic capacity were lower than those determined from cell survival. At IC50s that inhibited cell proliferation, SC18 arrested cells in G0/G1. SC18 at 0.25-2 IC50s had a synergistic effect with sorafenib on clonogenic inhibition in Huh7 and SNU-449 cells, and with regorafenib or lenvatinib in SNU-449 cells. X1 or SC18 also had synergistic effects with sorafenib on promoting cell death at 0.5-2 IC50s for SC18 in Huh7 and SNU-449 cells. These results suggest that small molecules targeting VDAC represent a potential new class of drugs to treat liver cancer.

Retrograde signaling by a mtDNA-encoded non-coding RNA preserves mitochondrial bioenergetics.

Alveolar epithelial type II (AETII) cells are important for lung epithelium maintenance and function. We demonstrate that AETII cells from mouse lungs exposed to cigarette smoke (CS) increase the levels of the mitochondria-encoded non-coding RNA, mito-RNA-805, generated by the control region of the mitochondrial genome. The protective effects of mito-ncR-805 are associated with positive regulation of mitochondrial energy metabolism, and respiration. Levels of mito-ncR-805 do not relate to steady-state transcription or replication of the mitochondrial genome. Instead, CS-exposure causes the redistribution of mito-ncR-805 from mitochondria to the nucleus, which correlated with the increased expression of nuclear-encoded genes involved in mitochondrial function. These studies reveal an unrecognized mitochondria stress associated retrograde signaling, and put forward the idea that mito-ncRNA-805 represents a subtype of small non coding RNAs that are regulated in a tissue- or cell-type specific manner to protect cells under physiological stress.

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.

Protein Disulfide Isomerase Superfamily in Disease and the Regulation of Apoptosis.

Cellular homeostasis requires the balance of a multitude of signaling cascades that are contingent upon the essential proteins being properly synthesized, folded and delivered to appropriate subcellular locations. In eukaryotic cells the endoplasmic reticulum (ER) is a specialized organelle that is the central site of synthesis and folding of secretory, membrane and a number of organelletargeted proteins. The integrity of protein folding is enabled by the presence of ATP, Ca++, molecular chaperones, as well as an oxidizing redox environment. The imbalance between the load and capacity of protein folding results in a cellular condition known as ER stress. Failure of these pathways to restore ER homeostasis results in the activation of apoptotic pathways. Protein disulfide isomerases (PDI) compose a superfamily of oxidoreductases that have diverse sequences and are localized in the ER, nucleus, cytosol, mitochondria and cell membrane. The PDI superfamily has multiple functions including, acting as molecular chaperones, protein-binding partners, and hormone reservoirs. Recently, PDI family members have been implicated in the regulation of apoptotic signaling events. The complexities underlying the molecular mechanisms that define the switch from pro-survival to pro-death response are evidenced by recent studies that reveal the roles of specific chaperone proteins as integration points in signaling pathways that determine cell fate. The following review discusses the dual role of PDI in cell death and survival during ER stress.

Peroxiredoxin VI oxidation in cerebrospinal fluid correlates with traumatic brain injury outcome.

Traumatic brain injury (TBI) patients would benefit from the identification of reliable biomarkers to predict outcomes and treatment strategies. In our study, cerebrospinal fluid (CSF) from patients with severe TBI was evaluated for oxidant stress-mediated damage progression after hospital admission and subsequent ventriculostomy placement. Interestingly, substantial levels of peroxiredoxin VI (Prdx6), a major antioxidant enzyme normally found in astrocytes, were detected in CSF from control and TBI patients and were not associated with blood contamination. Functionally, Prdx6 and its associated binding partner glutathione S-transferase Pi (GSTP1-1, also detected in CSF) act in tandem to detoxify lipid peroxidation damage to membranes. We found Prdx6 was fully active in CSF of control patients but becomes significantly inactivated (oxidized) in TBI. Furthermore, significant and progressive oxidation of "buried" protein thiols in CSF of TBI patients (compared to those of nontrauma controls) was detected over a 24-h period after hospital admission, with increased oxidation correlating with severity of trauma. Conversely, recovery of Prdx6 activity after 24h indicated more favorable patient outcome. Not only is this the first report of an extracellular form of Prdx6 but also the first report of its detection at a substantial level in CSF. Taken together, our data suggest a meaningful correlation between TBI-initiated oxidation of Prdx6, its specific phospholipid hydroperoxide peroxidase activity, and severity of trauma outcome. Consequently, we propose that Prdx6 redox status detection has the potential to be a biomarker for TBI outcome and a future indicator of therapeutic efficacy.

Allelic variants of glutathione S-transferase P1-1 differentially mediate the peroxidase function of peroxiredoxin VI and alter membrane lipid peroxidation.

The dual-functioning antioxidant enzyme peroxiredoxin VI (Prdx6) detoxifies lipid peroxides particularly in biological membranes, and its peroxidase function is activated by glutathione S-transferase Pi (GSTP). The GSTP gene is polymorphic in humans, with the wild-type GSTP1-1A (Ile105, Ala114) and three variants: GSTP1-1B (Ile105Val, Ala114), GSTP1-1C (Ile105Val, Ala114Val), and GSTP1-1D (Ile105, Ala114Val). The focus of this study was to determine the influence of these polymorphisms on Prdx6 peroxidase function. Using extracellular generation of OH radicals and fluorescence (DPPP dye) detection, we found a fast (~300 s) onset of lipid peroxidation in membranes of MCF-7 cells transfected with a catalytically inactive Y7F mutant of GSTP1-1 and either GSTP1-1B or GSTP1-1D. However, this effect was not detected in cells expressing either GSTP1-1A or GSTP1-1C. Imaging of DPPP-labeled MCF-7 cells showed fluorescence localized in the plasma membrane, but intensity was substantially diminished in the GSTP1-1A- and GSTP1-1C-expressing cells. Moreover, in the Y7F mutant of GSTP1-1A-, GSTP1-1B-, and GST1-1D-expressing cells ()OH generation resulted (after 36 h) in plasma membrane-permeability-related cell death, whereas GSTP1-1A- and GSTP1-1C-expressing cells had significantly better survival. We used FRET analyses to measure in vitro binding of purified GSTP1-1 allelic variant proteins to purified recombinant Prdx6. The affinities for Prdx6 binding to GSH-loaded GSTP1-1's either mirrored their observed peroxidase activities (using phospholipid hydroperoxide as a substrate), GSTP1-1A>GSTP1-1C (K(D)=51.0 vs 57.0 nM), or corresponded to inactivation, GSTP1-1B (GSTP1-1D) (K(D)=101.0 (94.0) nM). In silico modeling of the GSTP1-1-Prdx6 heterodimer revealed that the sites of GSTP1-1 polymorphism (Ile105 and Ala114) are in close proximity to the binding interface. Thus, there is a hierarchy of effectiveness for polymorphic variants of GSTP1-1 to regulate Prdx6 peroxidase function, a feature that may influence human population susceptibilities to oxidant stress.

Preclinical pharmacokinetic analysis of NOV-002, a glutathione disulfide mimetic.

NOV-002 is a glutathione disulfide (GSSG) mimetic that is the subject of clinical investigation in oncology indications. GSSG is reduced by glutathione reductase (GR) to form glutathione (GSH), thereby maintaining redox homeostasis. The purpose of the study was to report the pharmacokinetic properties of NOV-002 and evaluate the effect that NOV-002 elicits in redox homeostasis. The pharmacokinetic analysis and tissue distribution of NOV-002 and GSH was evaluated in mice following a dose of 250 mg/kg, i.p. The redox potential and total protein thiol status was calculated. Here we show that NOV-002 is a substrate for GR and that GSH is a primary metabolite. Non-linear pharmacokinetic modeling predicted that the estimated absorption and elimination rate constants correspond to a half-life of approximately 13 min with an AUC of 1.18 µgh/mL, a C(max) of 2.16 µg/ml and a volume of distribution of 42.61 L/kg. In addition, measurement of the redox potential and total protein thiol status indicated the generation of a transient oxidative signal in the plasma compartment after administration of NOV-002. These results indicate that NOV-002 exerts kinetic and dynamic effects in mice consistent with the GSSG component as the active pharmacological constituent of the drug. A longer-lasting decrease in total plasma free thiol content was also seen, suggesting that the oxidative effect of the GSSG from NOV-002 was impacting redox homeostasis.

Protein cysteine sulfinic acid reductase (sulfiredoxin) as a regulator of cell proliferation and drug response.

Sulfiredoxin (Srx) is one of a family of low molecular weight sulfur containing proteins linked with maintenance of cellular redox balance. One function of Srx is the reduction of cysteine sulfinic acid to sulfenic acid in proteins subject to oxidative stress. Other redox active protein families have multiple functions in regulating redox and controlling proliferation/death pathways; increased Srx has been linked with oncogenic transformation. To explore the biological functions of Srx in tumors, we established cell lines that overexpress Srx. Enhanced levels of Srx promoted cell proliferation and enhanced cell death following cisplatin. Srx overexpression triggered an alteration in expression and phosphorylation of cell cycle regulators p21, p27 and p53; stabilized the phosphatase PTEN and, importantly, interacted directly with, and enhanced the activity of, phosphatase PTP1B. In turn, this promoted Src kinase activity by dephosphorylating its inhibitory tyrosine residue (Y530). Srx expression was stimulated by cell exposure to certain growth factors. These data support a role for Srx in controlling the phosphorylation status of key regulatory kinases through effects upon phosphatase activity with an ultimate effect on pathways that influence cell proliferation.

The ATP-binding cassette transporter ABCA2 as a mediator of intracellular trafficking.

ATP-binding cassette (ABC) transporters are a family of proteins that translocate molecules across cellular membranes. Substrates can include lipids, cholesterol and drugs. Mutations in ABC transporter genes can cause human pathologies and drug resistance phenotypes in cancer cells. ABCA2, the second member the A sub-family to be identified, was found at high levels in ovarian carcinoma cells resistant to the anti-cancer agent, estramustine (EM). In vitro models with elevated levels of ABCA2 are resistant to a variety of compounds, including estradiol, mitoxantrone and a free radical initiator, 2,2'-azobis-(2-amidinopropane) (AAPH). ABCA2 is most abundant in the central nervous system (CNS), ovary and macrophages. Enhanced expression of ABCA2 and related proteins, including ABCA1, ABCA4 and ABCA7, is found in human macrophages upon bolus cholesterol treatment. ABCA2 also plays a role in the trafficking of low-density lipoprotein (LDL)-derived free cholesterol and is coordinately expressed with genes involved in cholesterol homeostasis. Additionally, ABCA2 expression has been linked with gene cluster patterns consistent with pathologies including Alzheimer's disease (AD). A single-nucleotide polymorphism (SNP) in exon 14 of the ABCA2 gene was shown to be linked to early onset AD in humans, supporting the observation that ABCA2 expression influences levels of beta-amyloid peptide (Abeta), the primary component of senile plaques. ABCA2 may play a role in cholesterol transport and affect a cellular phenotype conducive to the pathogenesis of a variety of human diseases including AD, atherosclerosis and cancer.

Glutathione S-transferase polymorphisms: cancer incidence and therapy.

The super family of glutathione S-transferases (GSTs) is composed of multiple isozymes with significant evidence of functional polymorphic variation. Over the last three decades, data from cancer studies have linked aberrant expression of GST isozymes with the development and expression of resistance to a variety of chemicals, including cancer drugs. This review addresses how differences in the human GST isozyme expression patterns influence cancer susceptibility, prognosis and treatment. In addition to the well-characterized catalytic activity, recent evidence has shown that certain GST isozymes can regulate mitogen-activated protein kinases or can facilitate the addition of glutathione to cysteine residues in target proteins (S-glutathionylation). These multiple functionalities have contributed to the recent efforts to target GSTs with novel small molecule therapeutics. Presently, at least two drugs are in late-stage clinical testing. The evolving functions of GST and their divergent expression patterns in individuals make them an attractive target for drug discovery.

Alpha-tocopherol: roles in prevention and therapy of human disease.

Alpha-tocopherol, one of the eight isoforms of vitamin E, is the most potent fat-soluble antioxidant known in nature. For years, it was thought that alpha-tocopherol only functioned as a scavenger of lipid peroxyl radicals, specifically, oxidized low-density lipoprotein (oxLDL), thereby serving as a chief antioxidant for the prevention of atherosclerosis. In recent years, the many roles of alpha-tocopherol have been uncovered, and include not only antioxidant functions, but also pro-oxidant, cell signaling and gene regulatory functions. Decades of clinical and preclinical studies have broadened our understanding of the antioxidant vitamin E and its utility in a number of chronic, oxidative stress-induced pathologies. The results of these studies have shown promising, albeit mixed reviews on the efficacy of alpha-tocopherol in the prevention and treatment of heart disease, cancer and Alzheimer's disease. Future studies to uncover cellular and systemic mechanisms may help guide appropriate clinical treatment strategies using vitamin E across a diverse population of aging individuals.

The role of carotenoids in the prevention of human pathologies.

Reactive oxygen species (ROS) and oxidative damage to biomolecules have been postulated to be involved in the causation and progression of several chronic diseases, including cancer and cardiovascular diseases, the two major causes of morbidity and mortality in Western world. Consequently dietary antioxidants, which inactivate ROS and provide protection from oxidative damage are being considered as important preventive strategic molecules. Carotenoids have been implicated as important dietary nutrients having antioxidant potential, being involved in the scavenging of two of the ROS, singlet molecular oxygen (1O2) and peroxyl radicals generated in the process of lipid peroxidation. Carotenoids are lipophilic molecules which tend to accumulate in lipophilic compartments like membranes or lipoproteins. Chronic ethanol consumption significantly increases hydrogen peroxide and decreases mitochondrial glutathione (GSH) in cells overexpressing CYP2E1. The depletion of mitochondrial GSH and the rise of hydrogen peroxide are responsible for the ethanol-induced apoptosis. Increased intake of lycopene, a major carotenoid in tomatoes, consumed as the all-trans-isomer attenuates alcohol induced apoptosis in 2E1 cells and reduces risk of prostate, lung and digestive cancers. Cancer-preventive activities of carotenoids have been associated as well as with their antioxidant properties and the induction and stimulation of intercellular communication via gap junctions which play a role in the regulation of cell growth, differentiation and apoptosis. Gap junctional communication between cells which may be a basis for protection against cancer development is independent of the antioxidant property.

Trace elements in human physiology and pathology. Copper.

Copper is a trace element, important for the function of many cellular enzymes. Copper ions can adopt distinct redox states oxidized Cu(II) or reduced (I), allowing the metal to play a pivotal role in cell physiology as a catalytic cofactor in the redox chemistry of enzymes, mitochondrial respiration, iron absorption, free radical scavenging and elastin cross-linking. If present in excess, free copper ions can cause damage to cellular components and a delicate balance between the uptake and efflux of copper ions determines the amount of cellular copper. In biological systems, copper homeostasis has been characterized at the molecular level. It is coordinated by several proteins such as glutathione, metallothionein, Cu-transporting P-type ATPases, Menkes and Wilson proteins and by cytoplasmic transport proteins called copper chaperones to ensure that it is delivered to specific subcellular compartments and thereby to copper-requiring proteins.

Phytosterols in the prevention of human pathologies.

Coronary heart disease is a major health problem in developed countries. Many studies have shown that elevated serum concentrations of total or low-density-lipoprotein cholesterol (LDL cholesterol) are high risk factors, whereas high concentrations of high-density-lipoprotein cholesterol (HDL cholesterol) or a low LDL to HDL cholesterol ratio may protect against coronary heart disease. Plant sterols and stanols derived from vegetable oils or wood pulp have been shown to lower total and LDL cholesterol levels in humans by inhibiting cholesterol absorption from the intestine. These findings may lead to new therapeutic options to treat hypercholesterolemia. In addition, phytosterols may influence cell growth and apoptosis of tumor cells. However, they can interfere with the absorption of fat soluble vitamins and carotenoids.

The antioxidant role of selenium and seleno-compounds.

Selenium (Se) is an essential trace element for animals and humans that is obtained from dietary sources including cereals, grains and vegetables. The Se content of plants varies considerably according to its concentration in soil. Plants convert Se mainly into Se-methionine (Se-Met) and incorporate it into protein in place of methionine (Met). Selenocystine (Se-Cys), methyl-Se-Cys and gamma-glutamyl-Se-methyl-Cys are not significantly incorporated into plant protein and are at relatively low levels irrespective of soil Se content. Higher animals are unable to synthesize Se-Met and only Se-Cys was detected in rats supplemented with Se as selenite. Renal regulation is the mode by which whole body Se is controlled. Se is concentrated in hair and nail and it occurs almost exclusively in organic compounds. The potentiating effect of Se deficiency on lipid peroxidation is enhanced in some tissues by concurrent deficiency of copper or manganese. In the in vitro system, the chemical form of Se is an important factor in eliciting cellular responses. Although the cytotoxic mechanisms of selenite and other redoxing Se compounds are still unclear, it has been suggested that they derive from their ability to catalyze the oxidation of thiols and to produce superoxide simultaneously. Selenite-induced cytotoxicity and apoptosis in human carcinoma cells can be inhibited with copper (CuSO(4)) as an antioxidant. High doses of selenite result in induction of 8-hydroxydeoxyguanosine (8-OHdG) in mouse skin cell DNA and in primary human keratinocytes. It may cause DNA fragmentation and decreased DNA synthesis, cell growth inhibition, DNA synthesis, blockade of the cell cycle at the S/G(2)-M phase and cell death by necrosis. In contrast, in cells treated with methylselenocyanate or Se methylselenocysteine, the cell cycle progression was blocked at the G(1) phase and cell death was predominantly induced by apoptosis.

Gamma-glutamyl transpeptidase-deficient mice are resistant to the nephrotoxic effects of cisplatin.

We have proposed that the nephrotoxicity of cisplatin, a widely used chemotherapy drug, is the result of the binding of cisplatin to glutathione and the subsequent metabolism of the cisplatin-glutathione complex via a gamma-glutamyl transpeptidase (GGT)-dependent pathway in the proximal tubules. To test the hypothesis that GGT activity is essential for the nephrotoxicity of cisplatin, the effects of cisplatin were examined in wild-type and GGT-deficient mice. Mice were treated with 15 mg cisplatin/kg. Five days after treatment, renal histopathology, blood urea nitrogen levels, serum creatinine, platinum excretion, and platinum accumulation in the kidney were examined. Half the mice were supplemented with N-acetylcysteine, which has been shown to correct low levels of tissue glutathione in GGT-deficient mice. The data show that cisplatin was nephrotoxic in wild-type mice but not in GGT-deficient mice. The wild-type mice, with and without N-acetylcysteine supplementation, had significantly elevated levels of blood urea nitrogen, serum creatinine, and renal tubular necrosis. There was no evidence of nephrotoxicity in the GGT-deficient mice regardless of N-acetyl cysteine supplementation. No differences in platinum excretion were seen comparing wild-type and GGT-deficient mice, nor was there any significant difference in renal platinum accumulation. These data suggest that renal cisplatin toxicity is dependent on GGT activity, and is not correlated with uptake. The results support our hypothesis that the nephrotoxicity of cisplatin is the result of the metabolism of the drug through a GGT-dependent pathway.

Gamma-glutamyl transpeptidase accelerates tumor growth and increases the resistance of tumors to cisplatin in vivo.

We have shown previously that gamma-glutamyl transpeptidase (GGT) activity is essential for the nephrotoxicity of cisplatin. In this study we asked whether GGT activity was necessary for the antitumor activity of cisplatin. GGT was transfected into PC3 cells, a human prostate tumor cell line. Two independent GGT-positive cell lines were isolated and characterized. GGT cleaves extracellular glutathione providing the cells with access to additional cysteine. Expression of GGT had no effect on the growth rate of the cells in vitro where the culture medium contains high levels of cysteine. However, when the cells were injected into nude mice the GGT-positive tumors grew at more than twice the rate of the GGT-negative tumors. Weekly treatment with cisplatin was toxic to both GGT-positive and -negative tumors. The GGT-positive tumors were significantly more resistant to the toxicity of cisplatin than the GGT-negative tumors. Therefore, expression of GGT is required for the nephrotoxicity of cisplatin, but diminishes the tumor toxicity of the drug. These results indicate that the nephrotoxicity and the tumor toxicity of cisplatin are via two distinct pathways.

Recent developments in formulating model descriptors for subsurface transformation and sorption of trinitrotoluene.

Recent published data show that transformation and sorption are key processes involved in the subsurface transport of TNT. The state-of-the-art understanding of TNT soil transformation and sorption phenomena is summarized below: There is unequivocal evidence of reductive transformation of TNT in soils. However, soil properties affecting TNT transformation are only partially understood. Edaphic factors such as redox are probably important since highest reductive transformation rates occur under anaerobic conditions. TNT transformation products include 2A-DNT, 4A-DNT, 2,4-DANT, and 2,6-DANT. Azoxytoluenes have also been reported. Triaminotoluene may be an important TNT transformation product, but this product has not been routinely measured. TNT soil sorption is rapid and can be modeled as equilibrium controlled. Poor mass balances are difficult to interpret owing to lack of information on transformation products. Various irreversible disappearance mechanisms other than transformation to elutable transformation products are possible.