Cyclic strain modulates resistance to oxidant stress by increasing G6PDH expression in smooth muscle cells

Vascular smooth muscle cells (VSMC) may be subjected to mechanical forces, such as cyclic strain, that promote the formation of reactive oxygen species (ROS). We hypothesized that VSMC modulate this adverse milieu by increasing the expression of glucose-6-phosphate dehydrogenase (G6PDH) to maintain or restore intracellular glutathione (GSH) levels. Cyclic strain increased superoxide formation, which resulted in diminished GSH because of an increase in oxidized glutathione formation; there was also an increase in glutathione peroxidase and glutathione reductase activities. G6PDH activity and protein expression were enhanced concomitant with decreases in GSH levels and remained elevated until intracellular GSH levels were restored. To confirm the role of G6PDH in repleting GSH stores, we inhibited G6PDH activity with DHEA or inhibited enzyme expression with an antisense oligodeoxynucleotide. Diminished G6PDH activity or expression was associated with persistently depleted GSH levels and inhibition of the cyclic strain-mediated increase in glutathione reductase activity. These observations demonstrate that cyclic strain promotes oxidant stress in VSMC, which, in turn, induces G6PDH expression. When G6PDH is inhibited, GSH levels are not restored because of impaired glutathione reductase activity. These data suggest that G6PDH is a critical determinant of the response to oxidant stress in VSMC.     

Glutathione and p53 independently mediate responses against oxidative stress in ES cells.

We have investigated the roles of the antioxidant glutathione and p53 in the response of embryonic stem (ES) cells to oxidative stress. ES cells express gammaGCS, a critical enzyme in glutathione (GSH) biosynthesis. Treatment with the pro-oxidant menadione led to elevation of GSH, a strong apoptotic response and reduced clonogenic survival. Addition of BSO, a specific gammaGCS inhibitor depleted GSH pools and prevented the menadione-induced increase in GSH, sensitizing cells to oxidative insult. Although p53 status had no bearing on either the basal levels of GSH or the menadione-induced GSH response, the levels of menadione-induced apoptosis were reduced in the absence of p53. We conclude that the pathways involving p53 and GSH act independently to protect against the deleterious effects of oxidative damage. Furthermore, the presence of an intact p53 pathway confers a long-term growth advantage post oxidative stress. Thus, in the absence of p53 ES cells bearing genotoxic damage are less likely to be propagated, suggesting that p53-dependent apoptosis acts to limit the deleterious effects of oxidative stress during early development.     

H2O2 injury in beta thalassemic erythrocytes: protective role of catalase and the prooxidant effects of GSH

Redox-mediated injury is an important pathway in the destruction of beta thalassemic red blood cells (RBC). Because of the autoxidation of the unstable hemoglobin chains and subsequent release of globin free heme and iron, significant amounts of superoxide (O2-) and, more importantly, hydrogen peroxide (H2O2) are generated intracellularly. Hence, catabolism of H2O2 is crucial in preventing cellular injury. Removal of H2O2 is mediated via two primary pathways: GSH-dependent glutathione peroxidase or catalase. Importantly, both pathways are ultimately dependent on NADPH. In the absence of any exogenous oxidants, model thalassemic RBC demonstrated significantly decreased GSH levels (P < 0.001 at 20 h). Perhaps of greater pathophysiologic importance, however, was the finding that the model thalassemic RBC exhibited significantly (P < 0.001) decreased catalase activity. Following 20 h incubation at 37 degrees C only 61.5 +/- 2.9% of the initial catalase activity remained in the alpha-hemoglobin chain-loaded cells versus 104.6 +/- 4.5 and 108.2 +/- 3.2% in the control and control-resealed cells, respectively. The mechanism underlying the loss of both catalase activity and GSH appears to be the same in that both catabolic pathways require adequate NADPH levels. As shown in this study, model beta thalassemic cells are unable to maintain a normal ( approximately 1.0) NADPH/NADP(total) ratio and, after 20 h, the model beta thalassemic cells have a significantly (P < 0.001) lower ratio ( approximately 0.5) which is quite similar to a G6PD-deficient RBC. In support of these findings, direct inactivation of catalase gives rise to significantly increased oxidant damage. In contrast, GSH depletion is not closely associated with oxidant sensitivity. Indeed, the consumption of GSH noted in the thalassemic RBC may be via a prooxidant pathway as augmentation of cellular GSH levels actually enhances alpha-hemoglobin chain-mediated injury.     

Lipid hydroperoxide-induced apoptosis in human colonic CaCo-2 cells is associated with an early loss of cellular redox balance.

Apoptosis plays a critical role in maintaining homeostasis of the intestinal epithelium. Dietary oxidants like peroxidized lipids could perturb cellular redox status and disrupt mucosal turnover. The objective of this study was to delineate the role of lipid hydroperoxide (LOOH) -induced redox shifts in intestinal apoptosis using the human colonic CaCo-2 cell. We found that subtoxic concentrations of LOOH increased CaCo-2 cell apoptosis. This LOOH-induced apoptosis was associated with a significant decrease in the ratio of reduced glutathione-to-oxidized glutathione (GSH/GSSG), which preceded DNA fragmentation by 12 to 14 h, suggesting a temporal relationship between the two events. Oxidation of GSH with the thiol oxidant diamide caused significant decreases in cellular GSH and GSH/GSSG at 15 min that correlated with the activation of caspase 3 (60 min) and cleavage of PARP (120 min), confirming a temporal link between induction of cellular redox imbalance and initiation of apoptotic cell death. These kinetic studies further reveal that oxidant-mediated early redox change (within 1 h) was a primary inciting event of the apoptotic cascade. Once initiated, the recovery of redox balance did not prevent the progression of CaCo-2 cell apoptosis to its biological end point at 24 h. Collectively, the study shows that subtoxic levels of LOOH disrupt intestinal redox homeostasis, which contributes to apoptosis. These results provide insights into the mechanism of hydroperoxide-induced mucosal turnover that have important implications for understanding oxidant-mediated genesis of gut pathology.     

Chronic iron overload enhances inducible nitric oxide synthase expression in rat liver.

Iron is an essential micronutrient promoting oxidative stress in the liver of overloaded animals and human, which may trigger the expression of redox-sensitive genes. We have tested the hypothesis that chronic iron overload (CIO) enhances inducible nitric oxide synthase (iNOS) expression in rat liver by extracellular signal-regulated kinase (ERK1/2) and NF-kappaB activation. CIO (diet enriched with 3%(wt/wt) carbonyl-iron for 12 weeks) increased liver protein carbonylation and decreased reduced glutathione (GSH) content and the GSH/GSSG ratio after 6 weeks, parameters that are normalized after 8-12 weeks of treatment. These changes are paralleled by higher phosphorylated-ERK1/2 to non-phosphorylated-ERK1/2 ratios at 6 and 8 weeks, increased NF-kappaB DNA binding to the iNOS gene promoter at 8-12 weeks, and higher iNOS mRNA expression and activity at 8 and 12 weeks. It is concluded that CIO triggers liver oxidative stress at early times, with upregulation of iNOS expression involving the ERK/NF-kappaB pathway at later times, a finding that may represent a hepatoprotective mechanism against CIO toxicity in addition to the recovery of GSH homeostasis.     

Epidermal growth factor receptor is a common mediator of quinone-induced signaling leading to phosphorylation of connexin-43: role of glutathione and tyrosine phosphatases

Rat liver epithelial cells were exposed to three quinones with different properties: menadione (2-methyl-1,4-naphthoquinone, vitamin K3), an alkylating as well as redox-cycling quinone, the strongly alkylating p-benzoquinone (BQ), and the non-arylating redox-cycler, 2,3-dimethoxy-1,4-naphthoquinone (DMNQ). All three quinones induced the activation of extracellular signal-regulated kinase (ERK) 1 and ERK 2 via the activation of epidermal growth factor receptor (EGFR) and MAPK/ERK kinases (MEK) 1/2. ERK activation resulted in phosphorylation at Ser-279 and Ser-282 of the gap junctional protein, connexin-43, known to result in the loss of gap junctional intercellular communication. Another EGFR-dependent pathway was stimulated, leading to the activation of the antiapoptotic kinase Akt via phosphoinositide 3-kinase. The activation of EGFR-dependent signaling by these quinones was by different mechanisms: (i) menadione, but not BQ or DMNQ, inhibited a protein-tyrosine phosphatase regulating the EGFR, as concluded from an EGFR dephosphorylation assay; (ii) although menadione-induced activation of ERK was unimpaired by pretreatment of cells with N-acetyl cysteine, activation by BQ and DMNQ was prevented; (iii) cellular glutathione (GSH) levels were strongly depleted by BQ. The mere depletion of GSH by application of diethyl maleate EGFR-dependently activated ERK and Akt, thus mimicking BQ effects. GSH levels were only moderately decreased by menadione and not affected by DMNQ. In summary, EGFR-dependent signaling was mediated by protein-tyrosine phosphatase inactivation (menadione), GSH depletion (BQ), and redox-cycling (DMNQ), funneling into the same signaling pathway.     

Modulation of doxorubicin resistance by the glucose-6-phosphate dehydrogenase activity

How anti-neoplastic agents induce MDR (multidrug resistance) in cancer cells and the role of GSH (glutathione) in the activation of pumps such as the MRPs (MDR-associated proteins) are still open questions. In the present paper we illustrate that a doxorubicin-resistant human colon cancer cell line (HT29-DX), exhibiting decreased doxorubicin accumulation, increased intracellular GSH content, and increased MRP1 and MRP2 expression in comparison with doxorubicin-sensitive HT29 cells, shows increased activity of the PPP (pentose phosphate pathway) and of G6PD (glucose-6-phosphate dehydrogenase). We observed the onset of MDR in HT29 cells overexpressing G6PD which was accompanied by an increase in GSH. The G6PD inhibitors DHEA (dehydroepiandrosterone) and 6-AN (6-aminonicotinamide) reversed the increase of G6PD and GSH and inhibited MDR both in HT29-DX cells and in HT29 cells overexpressing G6PD. In our opinion, these results suggest that the activation of the PPP and an increased activity of G6PD are necessary to some MDR cells to keep the GSH content high, which is in turn necessary to extrude anticancer drugs out of the cell. We think that our data provide a new further mechanism for GSH increase and its effects on MDR acquisition.     

Adequate phenylalanine synthesis mediated by G protein is critical for protection from UV radiation damage in young etiolated Arabidopsis thaliana seedlings

Etiolated Arabidopsis thaliana seedlings, lacking a functional prephenate dehydratase1 gene (PD1), also lack the ability to synthesize phenylalanine (Phe) and, as a consequence, phenylpropanoid pigments. We find that low doses of ultraviolet (UV)-C (254 nm) are lethal and low doses of UV-B cause severe damage to etiolated pd1 mutants, but not to wild-type (wt) seedlings. Furthermore, exposure to UV-C is lethal to etiolated gcr1 (encoding a putative G protein-coupled receptor in Arabidopsis) mutants and gpa1 (encoding the sole G protein alpha subunit in Arabidopsis) mutants. Addition of Phe to growth media restores wt levels of UV resistance to pd1 mutants. The data indicate that the Arabidopsis G protein-signalling pathway is critical to providing protection from UV, and does so via the activation of PD1, resulting in the synthesis of Phe. Cotyledons of etiolated pd1 mutants have proplastids (compared with etioplasts in wt), less cuticular wax and fewer long-chain fatty acids. Phe-derived pigments do not collect in the epidermal cells of pd1 mutants when seedlings are treated with UV, particularly at the cotyledon tip. Addition of Phe to the growth media restores a wt phenotype to pd1 mutants.     

Mu- and kappa-opioids induce the differentiation of embryonic stem cells to neural progenitors

Growth factors, hormones, and neurotransmitters have been implicated in the regulation of stem cell fate. Since various neural precursors express functional neurotransmitter receptors, which include G protein-coupled receptors, it is anticipated that they are involved in cell fate decisions. We detected mu-opioid receptor (MOR-1) and kappa-opioid receptor (KOR-1) expression and immunoreactivity in embryonic stem (ES) cells and in retinoic acid-induced ES cell-derived, nestin-positive, neural progenitors. Moreover, these G protein-coupled receptors are functional, since [D-Ala(2),MePhe(4),Gly-ol(5)]enkephalin, a MOR-selective agonist, and U69,593, a KOR-selective agonist, induce a sustained activation of extracellular signal-regulated kinase (ERK) signaling throughout a 24-h treatment period in undifferentiated, self-renewing ES cells. Both opioids promote limited proliferation of undifferentiated ES cells via the ERK/MAP kinase signaling pathway. Importantly, biochemical and immunofluorescence data suggest that [D-Ala(2),MePhe(4),Gly-ol(5)]enkephalin and U69,593 divert ES cells from self-renewal and coax the cells to differentiate. In retinoic acid-differentiated ES cells, opioid-induced signaling features a biphasic ERK activation profile and an opioid-induced, ERK-independent inhibition of proliferation in these neural progenitors. Collectively, the data suggest that opioids may have opposite effects on ES cell self-renewal and ES cell differentiation and that ERK activation is only required by the latter. Finally, opioid modulation of ERK activity may play an important role in ES cell fate decisions by directing the cells to specific lineages.     

Schisandrin B elicits a glutathione antioxidant response and protects against apoptosis via the redox-sensitive ERK/Nrf2 pathway in H9c2 cells

This study investigated the signal transduction pathway involved in the cytoprotective action of (-)schisandrin B [(-)Sch B, a stereoisomer of Sch B]. Using H9c2 cells, the authors examined the effects of (-)Sch B on MAPK and Nrf2 activation, as well as the subsequent eliciting of glutathione response and protection against apoptosis. Pharmacological tools, such as cytochrome P-450 (CYP) inhibitor, antioxidant, MAPK inhibitor, and Nrf2 RNAi, were used to delineate the signaling pathway. (-)Sch B caused a time-dependent activation of MAPK in H9c2 cells, with the degree of ERK activation being much larger than that of p38 or JNK. The MAPK activation was followed by an increase in the level of nuclear Nrf2, an indirect measure of Nrf2 activation, and the eliciting of a glutathione antioxidant response. The activation of MAPK and Nrf2 seemed to involve oxidants generated from a CYP-catalyzed reaction with (-)Sch B. Both ERK inhibition by U0126 and Nrf2 suppression by Nrf2 RNAi transfection largely abolished the cytoprotection against hypoxia/reoxygenation-induced apoptosis in (-)Sch B-pretreated cells. (-)Sch B pretreatment potentiated the reoxygenation-induced ERK activation, whereas both p38 and JNK activations were suppressed. Under the condition of ERK inhibition, Sch B treatment did not protect against ischemia/reperfusion injury in an ex vivo rat heart model. The results indicate that (-)Sch B triggers a redox-sensitive ERK/Nrf2 signaling, which then elicits a cellular glutathione antioxidant response and protects against hypoxia/reoxygenation-induced apoptosis in H9c2 cells. The ERK-mediated signaling is also likely involved in the cardioprotection afforded by Sch B in vivo.     

Glutathione prevents inhibition of fibroblast-mediated collagen gel contraction by cigarette smoke

Cigarette smoke, the major risk factor for the development of emphysema, contains over 4,700 chemical compounds, including free radicals and other oxidants (10(14)/puff). An imbalance between oxidants and antioxidants has been proposed in the pathogenesis of chronic obstructive pulmonary disease. Inhibition of repair processes has been suggested to be one pathway contributing to the development of emphysema. We hypothesized that cigarette smoke inhibition of repair might result from a shift of the oxidant/antioxidant balance in favor of oxidants. To evaluate this hypothesis, N-acetyl-L-cysteine (NAC), which serves as a substrate for glutathione (GSH) production, and buthionine sulfoximine (BSO), which inhibits GSH production, were incubated in the presence and absence of cigarette smoke extract (CSE) with fibroblasts in three-dimensional collagen gels. Neither agent alone altered gel contraction. CSE inhibition of gel contraction, however, was mitigated by NAC and potentiated by BSO. Parallel effects were observed on cigarette smoke inhibition of fibronectin production and mRNA expression as well as by changes in intracellular GSH content. Pretreatment of fibroblasts with NAC or BSO resulted in similar effects, suggesting that neither agent was acting directly on smoke but, rather, was altering cellular response to smoke. In conclusion, smoke inhibition of fibroblast repair, as reflected by collagen gel contraction and fibronectin production, may be modulated by intracellular GSH levels.     

Lipid peroxide-induced redox imbalance differentially mediates CaCo-2 cell proliferation and growth arrest

Dietary oxidants like lipid hydroperoxides (LOOH) can perturb cellular glutathione/glutathione disulphide (GSH/GSSG) status and disrupt mucosal turnover. This study examines the effect of LOOH on GSH/GSSG balance and phase transitions in the human colon cancer CaCo-2 cell. LOOH at 1 or 5 micro m were noncytotoxic, but disrupted cellular GSH/GSSG and stimulated proliferative activity at 6 h that paralleled increases in ornithine decarboxylase activity, thymidine incorporation, expression of cyclin D1/cyclin-dependent kinase 4, phosphorylation of retinoblastoma protein, and cell progression from G0/G1 to S. At 24 h, LOOH-induced sustained GSH/GSSG imbalance mediated growth arrest at G0/G1 that correlated with suppression of proliferative activity and enhanced oxidative DNA damage. LOOH-induced cell transitions were effectively blocked by N-acetylcysteine. Collectively, the study shows that subtoxic LOOH levels induce CaCo-2 GSH/GSSG imbalance that elicits time-dependent cell proliferation followed by growth arrest. These results provide insights into the mechanism of hydroperoxide-induced disruption of mucosal turnover with implications for understanding oxidant-mediated genesis of gut pathology.     

Beneficial effect of (-)schisandrin B against 3-nitropropionic acid-induced cell death in PC12 cells

Huntington's disease (HD) is characterized by the dysfunction of mitochondrial energy metabolism, which is associated with the functional impairment of succinate dehydrogenase (mitochondrial complex II), and pyruvate dehydrogenase (PDH). Treatment with 3-nitropropionic acid (3-NP), a potent irreversible inhibitor of succinate dehydrogenase, replicates most of the pathophysiological features of HD. In the present study, we investigated the effect of (-)schisandrin B [(-)Sch B, a potent enantiomer of schisandrin B] on 3-NP-induced cell injury in rat differentiated neuronal PC12 cells. The 3-NP caused cell necrosis, as assessed by lactate dehydrogenase (LDH) leakage, and mitochondrion-dependent cell apoptosis, as assessed by caspase-3 and caspase-9 activation, in differentiated PC12 cells. The cytotoxicity induced by 3-NP was associated with a depletion of cellular reduced glutathione (GSH) as well as the activation of redox-sensitive c-Jun N-terminal kinase (JNK) pathway and the inhibition of PDH. (-)Sch B pretreatment (5 and 15 μM) significantly reduced the extent of necrotic and apoptotic cell death in 3-NP-challenged cells. The cytoprotection afforded by (-)Sch B pretreatment was associated with the attenuation of 3-NP-induced GSH depletion as well as JNK activation and PDH inhibition. (-)Sch B pretreatment enhanced cellular glutathione redox status and ameliorated the 3-NP-induced cellular energy crisis, presumably by suppressing the activated JNK-mediated PDH inhibition, thereby protecting against necrotic and apoptotic cell death in differentiated PC12 cells.     

Production of glutathione sulfonamide and dehydroglutathione from GSH by myeloperoxidase-derived oxidants and detection using a novel LC-MS/MS method

GSH is rapidly oxidized by HOCl (hypochlorous acid), which is produced physiologically by the neutrophil enzyme myeloperoxidase. It is converted into, mainly, oxidized glutathione. Glutathione sulfonamide is an additional product that is proposed to be covalently bonded between the cysteinyl thiol and amino group of the gamma-glutamyl residue of GSH. We have developed a sensitive liquid chromatography-tandem MS assay for the detection and quantification of glutathione sulfonamide as well as GSH and GSSG. The assay was used to determine whether glutathione sulfonamide is a major product of the reaction between GSH and HOCl, and whether it is formed by other two-electron oxidants. At sub-stoichiometric ratios of HOCl to GSH, glutathione sulfonamide accounted for up to 32% of the GSH that was oxidized. It was also formed when HOCl was generated by myeloperoxidase and its yield increased with the flux of oxidant. Of the other oxidants tested, only hypobromous acid and peroxynitrite produced substantial amounts of glutathione sulfonamide, but much less than with HOCl. Chloramines were able to generate detectable levels only when at a stoichiometric excess over GSH. We conclude that glutathione sulfonamide is sufficiently selective for HOCl to be useful as a biomarker for myeloperoxidase activity in biological systems. We have also identified a novel oxidation product of GSH with a molecular weight two mass units less than GSH, which we have consequently named dehydroglutathione. Dehydroglutathione represented a few percent of the total products and was formed with all of the oxidants except H2O2.     

Allicin inhibits cell growth and induces apoptosis through the mitochondrial pathway in HL60 and U937 cells

In this article, the effects of allicin, a biological active compound of garlic, on HL60 and U937 cell lines were examined. Allicin induced growth inhibition and elicited apoptotic events such as blebbing, mitochondrial membrane depolarization, cytochrome c release into the cytosol, activation of caspase 9 and caspase 3 and DNA fragmentation. Pretreatment of HL60 cells with cyclosporine A, an inhibitor of the mitochondrial permeability transition pore (mPTP), inhibited allicin-treated cell death. HL60 cell survival after 1 h pretreatment with cyclosporine A, followed by 16 h in presence of allicin (5 microM) was approximately 80% compared to allicin treatment alone (approximately 50%). Also N-acetyl cysteine, a reduced glutathione (GSH) precursor, prevented cell death. The effects of cyclosporine A and N-acetyl cysteine suggest the involvement of mPTP and intracellular GSH level in the cytotoxicity. Indeed, allicin depleted GSH in the cytosol and mitochondria, and buthionine sulfoximine, a specific inhibitor of GSH synthesis, significantly augmented allicin-induced apoptosis. In HL60 cells treated with allicin (5 microM, 30 min) the redox state for 2GSH/oxidized glutathione shifted from EGSH -240 to -170 mV. The same shift was observed in U937 cells treated with allicin at a higher concentration for a longer period of incubation (20 microM, 2 h). The apoptotic events induced by various concentrations of allicin correlate to intracellular GSH levels in the two cell types tested (HL60: 3.7 nmol/10(6) cells; U937: 7.7 nmol/10(6) cells). The emerging mechanistic basis for the antiproliferative function of allicin, therefore, involves the activation of the mitochondrial apoptotic pathway by GSH depletion and by changes in the intracellular redox status.     

Oxidant-mediated apoptosis in proximal tubular epithelial cells following ATP depletion and recovery

Oxidant-mediated apoptosis has been implicated in renal injury due to ischemia reperfusion (IR); however, the apoptotic signaling pathways following IR have been incompletely defined. The purpose of this study was to examine the role of oxidants on cell death in a model of in vitro simulated IR injury in renal proximal tubular epithelial cells by analyzing the effects of a cell-permeable superoxide dismutase mimetic, manganese (III) tetrakis (1-methyl-4-pyridyl) porphyrin pentachloride (MnTmPyP). Renal proximal tubular epithelial cells were ATP depleted for 2, 4, or 6 h, followed by 2 h of recovery. We found that MnTmPyP was effective in attenuating cytotoxicity (P<0.001) and decreasing steady-state oxidant levels (P<0.001) and apoptotic cell death (P<0.001) following ATP depletion-recovery. MnTmPyP treatment prevented the early cytosolic release of cytochrome c and increased Bcl-2 protein levels following short durations of ATP depletion-recovery. After longer periods of ATP depletion-recovery, we observed a significant increase in TNF-alpha protein levels (P<0.001) and caspase-8 activation (P<0.001), both of which were decreased (P<0.001) by treatment with MnTmPyP. Our results suggest that oxidant mediated apoptosis via the mitochondrial pathway during the early phase of ATP depletion and by activation of the receptor-mediated apoptotic pathway following longer durations of injury.     

MEK blockade overcomes the limited activity of palbociclib in head and neck cancer

Head and neck cancer (HNC) is characterized with multiple aberrations in cell cycle pathways, including amplification of cyclin D1. Palbociclib (PAL), a cyclin-dependent kinase 4/6 (CDK4/6) inhibitor, has been reported to regulate cell cycle progression in HNC. However, recent studies have revealed the acquired resistance of certain cells to PAL through activation of the mitogen-activated protein kinase kinase (MEK)/extracellular signal-regulated kinase (ERK) pathway. Therefore, we investigated whether the inhibition of MEK/ERK pathway by trametinib (TRA) may overcome the limited efficacy of PAL in HNC. We evaluated the effect of PAL alone and in combination with TRA on the viability of HNC cells, and found that the combination treatment synergistically inhibited the proliferation of HNC cells. The combination treatment induced G0/G1 cell cycle arrest and apoptotic cell death. In particular, apoptosis mediated by the combination treatment was accompanied with an increase in caspase-3 activity and the number of TUNEL-positive apoptotic cells. These results were consistent with the decrease in cell cycle progression and mitogen-activated protein kinase (MAPK) pathway activation. In a xenograft mouse model of HNC, PAL and TRA synergistically inhibited tumor growth and enhanced tumor cell apoptosis, consistent with the increase in the number of TUNEL-positive cells. The anti-proliferative effects were evident in tumor tissues subjected to the combination treatment as compared with those treated with single drug. Taken together, our study demonstrates that the combination of PAL and TRA exerts synergistic anticancer effects and inhibits cell cycle check points and MEK/ERK pathway in HNC, suggestive of their potential application for HNC treatment.     

Early redox imbalance mediates hydroperoxide-induced apoptosis in mitotic competent undifferentiated PC-12 cells

Our recent study has demonstrated that cellular redox imbalance can directly initiate apoptosis in a mitotic competent PC-12 cell line without the involvement of reactive oxygen species (ROS). However, whether cell apoptosis induced by ROS is, in fact, mediated by a loss of redox balance caused by the oxidant is unresolved. The linkage between oxidant-mediated apoptosis and the induction of cellular redox was examined in PC-12 cells using the oxidant, tert-butylhydroperoxide (TBH). TBH caused cell apoptosis in 24 h that was preceded by an early increase (30 min) in oxidized glutathione (GSSG). Pretreatment with N-acetyl cysteine prevented TBH-induced GSSG increases and cell apoptosis. Altered Bax/BcL-2 expression and release of mitochondrial cytochrome c occurred post-redox imbalance and was kinetically linked to caspase-3 activation and poly ADP-ribose polymerase cleavage. Moreover, cell apoptosis was attenuated by inhibition of caspase-9, but not caspase-8, and blockade of mitochondrial ROS generation and permeability transition pore attenuated caspase 3 activation and cell apoptosis. Collectively, these results show that TBH-induced GSSG elevation is associated with the disruption of mitochondrial integrity, activation of caspase-3 and cell apoptosis. This redox induction of the apoptotic cascade was dissociated from cellular GSH efflux.     

Actin polymerization in cellular oxidant injury

Microfilaments undergo an ATP-dependent disruption into shortened bundles following cellular exposure to oxidants. This phenomenon does not require a net change in the amount of polymerized actin. However, increased amounts of polymerized actin have been detected in oxidant-injured cells and it was the purpose of this study to determine the conditions under which the actin polymerization may occur. Utilizing the formation of oxidized glutathione (GSSG) as an indicator of cellular sulfhydryl oxidation, conditions were chosen to accentuate sulfhydryl oxidation within the target P388D1 cell line following exposure to the oxidants, H2O2 and diamide. Using the DNase I and flow cytometric assays of actin polymerization, significant polymerization of actin was detected only under conditions in which sulfhydryl oxidation occurred after exposure to the two oxidizing agents. Greater sulfhydryl oxidation early in the course of injury was associated with a greater rate and extent of actin polymerization in the injured cells. Experiments with cells depleted of glutathione (GSH) demonstrated that neither loss of GSH nor absolute levels of GSSG formed during oxidant exposure were responsible for the polymerization of actin. The data presented are consistent with the hypothesis that oxidizing conditions which induce significant sulfhydryl oxidation in target cells are correlated with assembly of polymerized actin and that this represents a process which is distinct and separate from the ATP-dependent gross disruption of microfilaments.