Accelerated metabolism and exclusion of 4-hydroxynonenal through induction of RLIP76 and hGST5.8 is an early adaptive response of cells to heat and oxidative stress

To explore the role of lipid peroxidation (LPO) products in the initial phase of stress mediated signaling, we studied the effect of mild, transient oxidative or heat stress on parameters that regulate the cellular concentration of 4-hydroxynonenal (4-HNE). When K562 cells were exposed to mild heat shock (42 degrees C, 30 min) or oxidative stress (50 microM H2O2, 20 min) and allowed to recover for 2 h, there was a severalfold induction of hGST5.8, which catalyzes the formation of glutathione-4-HNE conjugate (GS-HNE), and RLIP76, which mediates the transport of GS-HNE from cells (Awasthi, S., Cheng, J., Singhal, S. S., Saini, M. K., Pandya, U., Pikula, S., Bandorowicz-Pikula, J., Singh, S. V., Zimniak, P., and Awasthi, Y. C. (2000) Biochemistry 39, 9327-9334). Enhanced LPO was observed in stressed cells, but the major antioxidant enzymes and HSP70 remained unaffected. The stressed cells showed higher GS-HNE-conjugating activity and increased efflux of GS-HNE. Stress-pre-conditioned cells with induced hGST5.8 and RLIP76 acquired resistance to 4-HNE and H2O2-mediated apoptosis by suppressing a sustained activation of c-Jun N-terminal kinase and caspase 3. The protective effect of stress pre-conditioning against apoptosis was abrogated by coating the cells with anti-RLIP76 IgG, which inhibited the efflux of GS-HNE from cells, indicating that the cells acquired resistance to apoptosis by metabolizing and excluding 4-HNE at a higher rate. Induction of hGST5.8 and RLIP76 by mild, transient stress and the resulting resistance of stress-pre-conditioned cells to apoptosis appears to be a general phenomenon since it was not limited to K562 cells but was also evident in lung cancer cells, H-69, H-226, human leukemia cells, HL-60, and human retinal pigmented epithelial cells. These results strongly suggest a role of LPO products, particularly 4-HNE, in the initial phase of stress mediated signaling.     

Cells preconditioned with mild,transient UVA irradiation acquire resistance to oxidative stress and UVA-induced apoptosis: role of 4-hydroxynonenal in UVA-mediated signaling for apoptosis

Because 4-hydroxynonenal (4-HNE) has been suggested to be involved in oxidative stress-mediated apoptosis (Cheng, J. Z., Sharma, R., Yang, Y., Singhal, S. S., Sharma, A., Saini, M. K., Singh, S. V., Zimniak, P., Awasthi, S., and Awasthi, Y. C. (2001) J. Biol. Chem. 276, 41213-41223) and UVA irradiation also causes lipid peroxidation, we have examined the role of 4-HNE in UVA-mediated apoptosis. K562 cells irradiated with UVA (3.0 milliwatts/cm2) for 5, 15, and 30 min showed a time dependent increase in 4-HNE levels. As judged by the activation of caspases, apoptosis was observed only in cells irradiated for 30 min. Within 2 h of recovery in normal medium, 4-HNE levels in 5 and 15 min UVA, irradiated cells returned to the basal or even lower levels but in cells irradiated for 30 min, 4-HNE levels remained consistently higher. The cells irradiated with UVA for 5 min and allowed to recover for 2 h in normal medium (UVA-preconditioned cells) showed a remarkable induction of hGST5.8, which catalyzes conjugation of 4-HNE to glutathione (GSH), and RLIP76 (Ral BP-1), which mediates the transport of the conjugate, GS-HNE. In cells irradiated with UVA for 30 min the induction of RLIP76 or hGST5.8 was not observed. The preconditioned cells transported GS-HNE into the medium at a rate about 2-fold higher than the controls and the transport was inhibited (65%) by coating the cells with anti-RLIP76 IgG. Upon treatment with xanthine/xanthine oxidase (XA/XO), 4-HNE, or prolonged UVA exposure, the control cells showed a sustained activation of c-Jun N-terminal kinase (JNK) and apoptosis. However, in the UVA-preconditioned cells, apoptosis was not observed, and JNK activation was inhibited. This resistance of preconditioned cells to XA/XO-, 4-HNE-, or UVA-induced apoptosis could be abrogated when these cells were coated with anti-RLIP76 IgG to block the efflux of GS-HNE. These studies strongly suggest a role of 4-HNE in UVA-mediated apoptosis.     

Role of 4-hydroxynonenal in stress-mediated apoptosis signaling

In this mini review we summarize recent studies from our laboratory, which show the involvement of 4-hydroxynonenal (4-HNE) in cell cycle signaling. We demonstrate 4-HNE induced apoptosis in various cell lines is accompanied with c-Jun-N-terminal kinase and caspase-3 activation. Cells exposed to mild, transient, heat or oxidative stress acquire capacity to exclude intracellular 4-HNE at a faster rate by inducing hGST5.8 which conjugate 4-HNE to GSH, and RLIP76 which mediates the ATP-dependent transport of the GSH-conjugate of 4-HNE. The cells preconditioned with mild transient stress acquire resistance to H(2)O(2) and 4-HNE induced apoptosis by excluding intracellular 4-HNE at an accelerated pace. Furthermore, a decrease in intracellular concentration of 4-HNE achieved by transfecting cells with mGSTA4-4 or hGSTA4-4 results in a faster growth rate. These studies strongly suggest a role of 4-HNE in stress mediated signaling.     

Transport functions and physiological significance of 76 kDa Ral-binding GTPase activating protein (RLIP76)

We have recently demonstrated that a previously known Ral-binding GTPase activating protein, RLIP76, can also catalyze ATP-dependent transport of various structurally unrelated xeno- and endobiotics irrespective of their net charge (Awasthi et al., 2000, Biochemistry, 39: 9327). RLIP76 is a non-ATP binding cassette (ABC) protein but it has two ATP-binding sites and shows basal ATPase activity which is stimulated in the presence of its transport substrates (allocrites) such as doxorubicin (DOX) and S-(2,4-dinitrophenyl) glutathione (DNP-SG). Proteoliposomes reconstituted with purified RLIP76 catalyze ATP-dependent, saturable transport of DOX, as well as of glutathione-conjugates including leukotrienes (LTC4) and the GSH-conjugate of 4-hydroxynonenal (GS-HNE). In erythrocytes the majority of transport activity for DOX, GS-HNE, and LTC4 is accounted for by RLIP76. Cells exposed to mild oxidative stress show a rapid and transient induction of RLIP76 resulting in an increased efflux of GS-HNE and acquire resistance to oxidative stress mediated toxicity and apoptosis. Cells transfected with RLIP76 acquire resistance to DOX through increased efflux of the drug suggesting its possible role in the mechanisms of drug-resistance. In this article, we discuss the significance of transport functions of RLIP76 highlighting its role in the defense mechanisms against oxidative injury, and modulation of signaling mechanisms.     

Functional reconstitution of Ral-binding GTPase activating protein,RLIP76, in proteoliposomes catalyzing ATP-dependent transport of glutathione conjugate of 4-hydroxynonenal

Earlier studies from our laboratories have shown that RLIP76, a previously described Ral-binding GTPase activating protein (Jullien-Flores et al., 1995, J. Biol. Chem. 270: 22473), is identical with the xenobiotic transporter DNP-SG ATPase, and can catalyze ATP-dependent transport of glutathione-conjugates as well as doxorubin (Awasthi et al., 2000, Biochemistry, 39: 9327). We have now reconstituted purified bacterially expressed RLIP76 in proteoliposomes, and have studied ATP-dependent uptake of the glutathione conjugate of 4-hydroxynonenal (GS-HNE) by these vesicles. Results of these studies show that RLIP76 reconstituted in proteoliposomes catalyzes ATP-dependent transport of GS-HNE against a concentration gradient. The transport of GS-HNE is saturable with respect to ATP as well as GS-HNE with K(m) values of 1.4mM and 2.5 microM, respectively. These studies demonstrate that RLIP76 mediates active transport of GS-HNE, and are consistent with our previous work showing that RLIP76-mediated efflux of GS-HNE regulates the intracellular concentration of 4-HNE and thereby affects 4-HNE mediated signaling.     

Mitogenic responses of vascular smooth muscle cells to lipid peroxidation-derived aldehyde 4-hydroxy-trans-2-nonenal (HNE): role of aldose reductase-catalyzed reduction of the HNE-glutathione conjugates in regulating cell growth

Products of lipid peroxidation such as 4-hydroxy-trans-2-nonenal (HNE) trigger multiple signaling cascades that variably affect cell growth, differentiation, and apoptosis. Because glutathiolation is a significant metabolic fate of these aldehydes, we tested the possibility that the bioactivity of HNE depends upon its conjugation with glutathione. Addition of HNE or the cell-permeable esters of glutathionyl-4-hydroxynonenal (GS-HNE) or glutathionyl-1,4-dihydroxynonene (GS-DHN) to cultures of rat aortic smooth muscle cells stimulated protein kinase C, NF-kappaB, and AP-1, and increased cell growth. The mitogenic effects of HNE, but not GS-HNE or GS-DHN, were abolished by glutathione depletion. Pharmacological inhibition or antisense ablation of aldose reductase (which catalyzes the reduction of GS-HNE to GS-DHN) prevented protein kinase C, NF-kappaB, and AP-1 stimulation and the increase in cell growth caused by HNE and GS-HNE, but not GS-DHN. The growth stimulating effect of GS-DHN was enhanced in cells treated with antibodies directed against the glutathione conjugate transporters RLIP76 (Ral-binding protein) or the multidrug resistance protein-2. Overexpression of RLIP76 abolished the mitogenic effects of HNE and its glutathione conjugates, whereas ablation of RLIP76 using RNA interference promoted the mitogenic effects. Collectively, our findings suggest that the mitogenic effects of HNE are mediated by its glutathione conjugate, which has to be reduced by aldose reductase to stimulate cell growth. These results raise the possibility that the glutathione conjugates of lipid peroxidation products are novel mediators of cell signaling and growth.     

Effects of mGST A4 transfection on 4-hydroxynonenal-mediated apoptosis and differentiation of K562 human erythroleukemia cells

Cellular levels of downstream products of membrane lipid oxidation appear to regulate differentiation in K562 human erythroleukemia cells. 4-Hydroxynonenal (4-HNE) is a diffusible and relatively stable product of peroxidation of arachidonic and linoleic acids, cellular levels of which are regulated through metabolism to glutathione (GSH) conjugate by glutathione S-transferases (GSTs). A group of immunologically related alpha-class mammalian GSTs expressed in mice (mGST A4-4), rat (rGST A4-4), human (hGST A5.8), and other species, as well as the more distantly related human hGST A4-4, preferentially utilize 4-HNE as a substrate and are suggested to be major determinants of intracellular levels of 4-HNE. Present studies were designed to examine the effects of 4-HNE on K562 cells and to study the effect of transfection of mGSTA4-4 in these cells. Exposure of K562 cells to 20 microM 4-HNE for 2 h resulted in a rapid erythroid differentiation of K562 cells, as well as apoptosis evidenced by characteristic DNA laddering. Stable transfection of cells with mGST A4-4 resulted in a fivefold increase in GST-specific activity toward 4-HNE compared with wild-type or vector-only transfected cells. The mGST A4-4-transfected cells were resistant to the cytotoxic, apoptotic, and differentiating effects of 4-HNE. The mGST A4 transfection also conferred resistance to direct oxidative stress (IC(50) of H(2)O(2) 22, 23, and 35 microM for wild-type, vector-transfected, and mGST A4-transfected cells, respectively). mGST A4-4-transfected cells also showed a higher rate of proliferation compared with wild-type or vector-transfected K562 cells (doubling time 22.1 +/- 0.7, 31 +/- 1.2, and 29 +/- 0.6 h, respectively). Cellular 4-HNE levels determined by mass spectrometry were lower in mGST A4-4-transfected cells compared to cells transfected with vector alone (5.9 pmol/5 x 10(7) cells and 62.9 pmol/5 x 10(7) cells, respectively). Our studies show that 4-HNE can induce erythroid differentiation in K562 cells and that overexpression of mGST A4 suppresses 4-HNE levels and inhibits erythroid differentiation and apoptosis.     

Regulation of 4-hydroxynonenal-mediated signaling by glutathione S-transferases

4-Hydroxynonenal (HNE), one of the major end products of lipid peroxidation, has been shown to be involved in signal transduction and available evidence suggests that it can affect cell cycle events in a concentration-dependent manner. Glutathione S-transferases (GSTs) can modulate the intracellular concentrations of HNE by affecting its generation during lipid peroxidation by reducing hydroperoxides and also by converting it into a glutathione conjugate. We have recently demonstrated that overexpression of the Alpha class GSTs in cells leads to lower steady-state levels of HNE, and these cells acquire resistance to apoptosis induced by lipid peroxidation-causing agents such as H(2)O(2), UVA, superoxide anion, and pro-oxidant xenobiotics, suggesting that signaling for apoptosis by these agents is transduced through HNE. Cells with the capacity to exclude HNE from the intracellular environment at a faster rate are relatively more resistant to apoptosis caused by H(2)O(2), UVA, superoxide anion, and pro-oxidant xenobiotics as well as by HNE, suggesting that HNE may be a common denominator in mechanisms of apoptosis caused by oxidative stress. We have also shown that transfection of adherent cells with HNE-metabolizing GSTs leads to transformation of these cells due to depletion of HNE. These recent studies from our laboratories, which strongly suggest that HNE is a key signaling molecule and that GSTs, being determinants of its intracellular concentrations, can regulate stress-mediated signaling, are reviewed in this article.     

Resveratrol protects against 4-HNE induced oxidative stress and apoptosis in Swiss 3T3 fibroblasts

Here we report on the marked protective effect of resveratrol on 4-hydroxynonenal (4-HNE) induced oxidative stress and apoptotic death in Swiss 3T3 fibroblasts. 4-HNE, one of the major aldehydic products of the peroxidation of membrane w-6 polyunsaturated fatty acids, has been suggested to contribute to oxidant stress mediated cell injury. Indeed, in vitro treatment of 3T3 fibroblasts with 4-HNE induced a condition of oxidative stress as monitored by the oxidation of dichlorofluorescein diacetate; this reaction was prevented when cells were pretreated with resveratrol. Further, 4-HNE-treated fibroblasts eventually underwent apoptotic death as determined by differential staining and internucleosomal DNA fragmentation. Resveratrol pretreatment also prevented 4-HNE induced DNA fragmentation and apoptosis. These observations are consistent with a potential role of lipid peroxidation-derived products in programmed cell death and demonstrate that resveratrol can counteract this effect by quenching cell oxidative stress.     

Glutathione S-transferases as antioxidant enzymes: small cell lung cancer (H69) cells transfected with hGSTA1 resist doxorubicin-induced apoptosis

It has been suggested that the alpha-class glutathione S-transferases (GSTs) protect various cell types from oxidative stress and lipid peroxidation (LPO). In order to examine the protective role of alpha-class GST isozyme hGSTA1-1 against doxorubicin (DOX)-induced lipid peroxidation, cytotoxicity, and apoptosis, human small cell lung cancer (SCLC) H69 cells were stably transfected with hGSTA1. Immunological and biochemical characterization of hGSTA1-transfected cells revealed the expression of functionally active hGSTA1-1 localized near the cellular plasma membranes. hGSTA1-transfected cells acquired significantly increased resistance to the DOX-induced cytotoxicity by suppressing lipid peroxidation levels in these cells. Overexpression of hGSTA1-1 in cells inhibited DOX-mediated depletion of GSH and higher GSH levels were found in DOX-treated hGSTA1-transfected cells as compared with empty vector-transfected controls. hGSTA1-1 overexpression also provided protection to cells from DOX-induced apoptosis by inhibiting phosphorylation of c-Jun-N-terminal kinases (JNK), caspase-3 activation, and by preserving the levels of anti-apoptotic protein Bcl-2. These results are consistent with the idea that the alpha-class GSTs provide protection against oxidative stress by attenuating lipid peroxidation and these enzymes can modulate signaling for apoptosis.     

Role of 4-hydroxy-<Emphasis Type="Italic">trans</Emphasis>-2-nonenal in cell functions

The role of lipid peroxidation product 4-hydroxy-trans-2-nonenal (4-HNE) in functional activity of cells under normal and different pathological conditions is discussed. Different pathways of 4-HNE metabolism in tissues are analyzed, with particular focus on the role the glutathione system in this process. 4-HNE is implicated in regulation of cell growth, proliferation, differentiation, and apoptosis. 4-HNE and metabolic products of other antioxidants (carotenoids) resemble each other in chemical nature of the product and influence general pathways of signal transduction. Manifestation of 4-HNE toxicity under oxidative stress conditions is regarded as a link to many diseases whose pathogenesis is connected with modifications of proteins and nucleic acids.     

Molecular mechanisms of ALDH3A1-mediated cellular protection against 4-hydroxy-2-nonenal

Evidence suggests that aldehydic molecules generated during lipid peroxidation (LPO) are causally involved in most pathophysiological processes associated with oxidative stress. 4-Hydroxy-2-nonenal (4-HNE), the LPO-derived product, is believed to be responsible for much of the cytotoxicity. To counteract the adverse effects of this aldehyde, many tissues have evolved cellular defense mechanisms, which include the aldehyde dehydrogenases (ALDHs). Our laboratory has previously characterized the tissue distribution and metabolic functions of ALDHs, including ALDH3A1, and demonstrated that these enzymes may play a significant role in protecting cells against 4-HNE. To further characterize the role of ALDH3A1 in the oxidative stress response, a rabbit corneal keratocyte cell line (TRK43) was stably transfected to overexpress human ALDH3A1. These cells were studied after treatment with 4-HNE to determine their abilities to: (a) maintain cell viability, (b) metabolize 4-HNE and its glutathione conjugate, (c) prevent 4-HNE-protein adduct formation, (d) prevent apoptosis, (e) maintain glutathione homeostasis, and (f) preserve proteasome function. The results demonstrated a protective role for ALDH3A1 against 4-HNE. Cell viability assays, morphological evaluations, and Western blot analyses of 4-HNE-adducted proteins revealed that ALDH3A1 expression protected cells from the adverse effects of 4-HNE. Based on the present results, it is apparent that ALDH3A1 provides exceptional protection from the adverse effects of pathophysiological concentrations of 4-HNE such as may occur during periods of oxidative stress.     

Adaptive response induced by lipid peroxidation products in cell cultures

The adaptive response induced by the lipid peroxidation products, such as phosphatidylcholine hydroperoxide, lysophosphatidylcholine (LysoPC), 15-deoxy-Delta(12,14)-prostaglandin J(2), 4-hydroxynonenal (4-HNE), hydroxyoctadecadienoic acid, 7-hydroxycholesterol, and cholesterol 5beta,6beta-epoxide, was investigated in this study. Although these products have been implicated in oxidative stress-related diseases, pretreatment with such compounds at sublethal concentrations significantly protected PC12 cells against subsequent oxidative stress induced by 6-hydroxydopamine. Moreover, 4-HNE and LysoPC also exhibited adaptive protection in human arterial endothelial cells. These findings suggest a general hormetic effect of such compounds in cell cultures and may lead to a reappraisal of the eventual role of reactive oxygen species and lipid peroxidation in organisms.     

Role of glutathione S-transferases in oxidative stress-induced male germ cell apoptosis

Cellular apoptosis in a tissue may occur for the maintenance of proper ratio of cells or because of toxic effects of free radicals or other agents. Male germ cell apoptosis is pivotal in maintaining the proper functioning of the testis, but it is not clear how free radicals affect germ cells and what the defense mechanisms are that are used by these cells to combat the toxic effects of the products of oxidative stress. This study shows that male germ cells are susceptible to H(2)O(2)-induced stress and, upon exposure to H(2)O(2) in vitro, demonstrate a typical apoptotic phenotype that includes DNA fragmentation and formation of DNA ladders. Other changes include considerable accumulation of products of lipid peroxidation in the germ cells after exposure to H(2)O(2). Evidence is presented for the existence of multiple isoforms of glutathione S-transferases (GSTs) that possess both transferase and Se-independent peroxidase activity. Germ cell GST activity increases after H(2)O(2) exposure. If this increase in activity is inhibited with suitable inhibitors, the formation of products of lipid peroxidation is augmented, resulting in germ cell apoptosis. Also, when constitutive GST activity is inhibited, accumulation of products of lipid peroxidation occurs, resulting in increased cellular apoptosis. These data show that GSTs form a part of adaptive response of germ cells to oxidative stress and are important constituents in detoxifying the products of lipid peroxidation.     

Retinol dehydrogenase 12 detoxifies 4-hydroxynonenal in photoreceptor cells

Mutations of the photoreceptor retinol dehydrogenase 12 (RDH12) gene cause the early onset retinal dystrophy Leber congenital amaurosis (LCA) by mechanisms not completely resolved. Determining the physiological role of RDH12 in photoreceptors is the focus of this study. Previous studies showed that RDH12, and the closely related retinol dehydrogenase RDH11, can enzymatically reduce toxic lipid peroxidation products such as 4-hydroxynonenal (4-HNE), in vitro. To explore the significance of this activity, we investigated the ability of RDH11 and RDH12 to protect stably transfected HEK-293 cells against the toxicity of 4-HNE. Both enzymes protected against 4-HNE modification of proteins and 4-HNE-induced apoptosis in HEK-293 cells. In the retina, exposure to bright light induced lipid peroxidation, 4-HNE production, and 4-HNE modification of proteins in photoreceptor inner segments, where RDH11 and RDH12 are located. In mouse retina, RDH12—but not RDH11—protected against adduct formation, suggesting that 4-HNE is a physiological substrate of RDH12. RDH12—but not RDH11—also protected against light-induced apoptosis of photoreceptors. We conclude that in mouse retina RDH12 reduces 4-HNE to a nontoxic alcohol, protecting cellular macromolecules against oxidative modification and protecting photoreceptors from light-induced apoptosis. This activity is of particular significance to the understanding of the molecular mechanisms of RDH12-induced LCA.     

Ethanol-induced modulation of hepatocellular extracellular signal-regulated kinase-1/2 activity via 4-hydroxynonenal

Modulation of the extracellular signal-regulated kinases (ERK-1/2), a signaling pathway directly associated with cell proliferation, survival, and homeostasis, has been implicated in several pathologies, including alcoholic liver disease. However, the underlying mechanism of ethanol-induced ERK-1/2 modulation remains unknown. This investigation explored the effects of ethanol-associated oxidative stress on constitutive hepatic ERK-1/2 activity and assessed the contribution of the lipid peroxidation product 4-hydroxynonenal (4-HNE) to the observations made in vivo. Constitutive ERK-1/2 phosphorylation was suppressed in hepatocytes isolated from rats chronically consuming ethanol for 45 days. This observation was associated with an increase in 4-HNE-ERK monomer adduct concentration and a hepatic cellular and lobular redistribution of ERK-1/2 that correlated with 4-HNE-protein adduct accumulation. Chronic ethanol consumption was also associated with a decrease in hepatocyte nuclear ELK-1 phosphorylation, independent of changes in total nuclear ELK-1 protein. Primary hepatocytes treated with concentrations of 4-HNE consistent with those occurring during oxidative stress displayed a concentration-dependent decrease in constitutive ERK-1/2 phosphorylation, activity, and nuclear localization that negatively correlated with 4-HNE-ERK-1/2 monomer adduct accumulation. These data paralleled the decreased phosphorylation of the downstream kinase ELK-1. Molar ratios of purified ERK-2 to 4-HNE consistent with pathologic ratios found in vivo resulted in protein monomer-adduct formation across a range of concentrations. Collectively, these data demonstrate a novel association between ethanol-induced lipid peroxidation and the inhibition of constitutive ERK-1/2, and suggest an inhibitory mechanism mediated by the lipid peroxidation product 4-hydroxynonenal.     

Revealing mechanisms of selective,concentration-dependent potentials of 4-hydroxy-2-nonenal to induce apoptosis in cancer cells through inactivation of membrane-associated catalase

Tumor cells generate extracellular superoxide anions and are protected against superoxide anion-mediated intercellular apoptosis-inducing signaling by the expression of membrane-associated catalase. 4-Hydroxy-2-nonenal (4-HNE), a versatile second messenger generated during lipid peroxidation, has been shown to induce apoptosis selectively in malignant cells. The findings described in this paper reveal the strong, concentration-dependent potential of 4-HNE to specifically inactivate extracellular catalase of tumor cells both indirectly and directly and to consequently trigger apoptosis in malignant cells through superoxide anion-mediated intercellular apoptosis-inducing signaling. Namely, 4-HNE caused apoptosis selectively in NOX1-expressing tumor cells through inactivation of their membrane-associated catalase, thus reactivating subsequent intercellular signaling through the NO/peroxynitrite and HOCl pathways, followed by the mitochondrial pathway of apoptosis. Concentrations of 4-HNE of 1.2 µM and higher directly inactivated membrane-associated catalase of tumor cells, whereas at lower concentrations, 4-HNE triggered a complex amplificatory pathway based on initial singlet oxygen formation through H₂O₂ and peroxynitrite interaction. Singlet-oxygen-dependent activation of the FAS receptor and caspase-8 increased superoxide anion generation by NOX1 and amplification of singlet oxygen generation, which allowed singlet-oxygen-dependent inactivation of catalase. 4-HNE and singlet oxygen cooperate in complex autoamplificatory loops during this process. The finding of these novel anticancer pathways may be useful for understanding the role of 4-HNE in the control of malignant cells and for the optimization of ROS-dependent therapeutic approaches including antioxidant treatments.     

The lipid peroxidation end-product 4-HNE induces COX-2 expression through p38MAPK activation in 3T3-L1 adipose cell

Oxidative stress and low grade chronic inflammation are increased in accumulating fat. Our objective was to test whether 4-hydroxynonenal (4-HNE), an end-product of lipid peroxidation, affects cyclooxygenases in 3T3-L1 adipose cells. 4-HNE increased COX-2 mRNA and protein expression and p38MAP-kinase phosphorylation in a dose-dependent manner. Pretreatment of 3T3-L1 cells by a selective inhibitor of p38MAPK (PD 169316) abolished 4-HNE and glucose oxidase induced COX-2 expression. Our results show that oxidative stress induces COX-2 expression through the production of 4-HNE which activates p38MAPKinase, suggesting that 4-HNE links oxidative stress and chronic inflammation through the activation of cyclooxygenase.