Exposure of HL-60 human leukaemic cells to 4-hydroxynonenal promotes the formation of adduct(s) with alpha-enolase devoid of plasminogen binding activity.

HNE (4-hydroxynonenal), the major product of lipoperoxidation, easily reacts with proteins through adduct formation between its three main functional groups and lysyl, histidyl and cysteinyl residues of proteins. HNE is considered to be an ultimate mediator of toxic effects elicited by oxidative stress. It can be detected in several patho-physiological conditions, in which it affects cellular processes by addition to functional proteins. We demonstrated in the present study, by MS and confirmed by immunoblotting experiments, the formation of HNE-alpha-enolase adduct(s) in HL-60 human leukaemic cells. Alpha-enolase is a multifunctional protein that acts as a glycolytic enzyme, transcription factor [MBP-1 (c-myc binding protein-1)] and plasminogen receptor. HNE did not affect alpha-enolase enzymatic activity, expression or intracellular localization, and did not change the expression and localization of MBP-1 either. Confocal and electronic microscopy results confirmed the plasma membrane, cytosolic and nuclear localization of alpha-enolase in HL-60 cells and demonstrated that HNE was colocalized with alpha-enolase at the surface of cells early after its addition. HNE caused a dose- and time-dependent reduction of the binding of plasminogen to alpha-enolase. As a consequence, HNE reduced adhesion of HL-60 cells to HUVECs (human umbilical vein endothelial cells). These results could suggest a new role for HNE in the control of tumour growth and invasion.

4-Hydroxynonenal and PPARgamma ligands affect proliferation, differentiation, and apoptosis in colon cancer cells.

PPARgamma ligands inhibit growth and induce apoptosis of various cancer cells. 4-Hydroxynonenal (HNE), a product of lipid peroxidation, inhibits proliferation and induces differentiation or apoptosis in neoplastic cells. The aim of this work was to investigate the effects of PPARgamma ligands (rosiglitazone and 15-deoxy-prostaglandin J2 (15d-PGJ2)) and HNE, alone or in association, on proliferation, apoptosis, differentiation, and growth-related and apoptosis-related gene expression in colon cancer cells (CaCo-2 cells). PPARgamma ligands inhibited cell proliferation (IC50 was 37.47+/-6.6 microM, for 15d-PGJ2, and 170.34+/-20 microM for rosiglitazone). HNE (1 microM) inhibited cell growth by 70%. Apoptosis was induced by 15d-PGJ2 and HNE and, to a minor extent, rosiglitazone. Differentiation was induced by rosiglitazone and by 15d-PGJ2, but not by HNE. PPARgamma ligands inhibited c-myc expression. HNE induced a transitory increase in c-myc expression and a subsequent down-regulation. HNE induced p21 expression, whereas PPARgamma ligands did not. Expression of the bax gene was increased by HNE and 15d-PGJ2, but not by rosiglitazone. No synergism or antagonism was found between HNE and PPARgamma ligands. Both apoptosis and differentiation induction may be responsible for the inhibition of proliferation by PPARgamma ligands; apoptosis and c-myc and p21 expression seem to be involved in the inhibition of proliferation by HNE.

4-Hydroxynonenal inhibits telomerase activity and hTERT expression in human leukemic cell lines.

4-Hydroxynonenal (HNE), produced during oxidative stress, has an antiproliferative/differentiative effect in several tumor cells. Recently, it has been observed that oxidative stress accelerates telomere loss. The length of telomeres depends on the telomerase activity, and the catalytic subunit of telomerase (hTERT) is strongly up-regulated in most human cancers and inhibited by differentiating agents. In this paper the inhibitory effect of HNE on telomerase activity and hTERT expression in three human leukemic cell lines (HL-60, U937, ML-1) is reported. To investigate the molecular mechanism involved in hTERT down-regulation by HNE, the expression of several transcription factors was also studied: in all these cell lines, c-Myc was inhibited, Mad-1 was up-regulated, and Sp-1 was not affected. Moreover, in p53 wild-type ML-1 cells, HNE up-regulated p53 expression. In HL-60 cells, DNA binding activity of c-Myc and Mad-1 to the E-box sequence of the hTERT promoter was inhibited and up-regulated, respectively. In summary, HNE inhibits telomerase activity via decreased hTERT promoter activity, by modulating c-Myc/Mad-1 transcription factor expression.

4-Hydroxynonenal modulation of p53 family gene expression in the SK-N-BE neuroblastoma cell line.

4-Hydroxynonenal (HNE), a product of lipid peroxidation, inhibits proliferation of several tumor cells. The p53 tumor suppressor protein plays a critical role in cell cycle control, by inducing p21 expression, and in apoptosis, by inducing bax expression. Recently, two other proteins with many p53-like properties, TAp73 (p73) and TAp63 (p63), have been discovered. SK-N-BE human neuroblastoma cells express the three p53 family proteins and can be used for the study of their induction. We investigated HNE action in the control of proliferation, differentiation, and apoptosis in SK-N-BE cells and the HNE effect on the expression of p53, p63, p73, p21, bax, and G1 cyclins. Retinoic acid (RA) was used as a positive control. HNE inhibited cell proliferation without inducing differentiation; it decreased S-phase cells and increased the number of apoptotic cells. RA reduced the proportion of S-phase cells and did not induce apoptosis. HNE increased p53, p73, p63, p21, and bax expression at different time points. HNE reduced cyclin D2 expression and the phosphorylation of pRb protein. Our results demonstrated that HNE inhibits SK-N-BE cell proliferation by increasing the expression of p53 family proteins and p53 target proteins which modulate cell cycle progression and apoptosis.

4-hydroxynonenal and regulation of cell cycle: effects on the pRb/E2F pathway.

The hypothesis that 4-hydroxynonenal (HNE), a product of lipid peroxidation, might negatively affect cell proliferation, arose from the observation that lipid peroxidation is very low in tumors. In leukemic cells HNE inhibited cell growth and reduced c-myc and c-myb expression. HNE also induced differentiation in different leukemic cell lines. In HL-60 human leukemic cells, HNE induced the accumulation of cells in the G(0)/G(1) phase of the cell cycle accompanied by a decrease of cyclins D1, D2, and A. Moreover, HNE caused an increase in p21 expression. As cyclin D/CDK2 and cyclin A/CDK2 phosphorylate pRB, these findings suggested that pRb phosphorylation could be affected by HNE. Hypophosphorylated pRb binds and inactivates the E2F transcription factors. HNE induced the dephosphorylation of pRb and the increase in pRb/E2F1 complexes, whereas pRb/E2F4 complexes were reduced, because HNE downregulated E2F4 protein expression. The analysis of E2F binding to the P2 c-myc promoter revealed that HNE caused a decrease in "free" E2F, as well as an increase in pRb (and pRB family members) bound to E2F, with consequent repression of the transcription. In conclusion, HNE reduces E2F transcriptional activity by modifying a number of genes involved in regulation of the pRb/E2F pathway.

Synergistic effect of 4-hydroxynonenal and PPAR ligands in controlling human leukemic cell growth and differentiation.

Peroxisome proliferator-activated receptors play an important role in the differentiation of different cell lines. In this study we demonstrate that PPAR-alpha ligands (clofibrate and ciprofibrate) and PPAR-gamma ligands (troglitazone and 15d-prostaglandin J2) inhibit growth and induce monocytic differentiation in HL-60 cells, whereas only PPAR-gamma ligands inhibit growth of U937 cells. Differentiation was demonstrated by the analysis of surface antigen expression CD11b and CD14, and by the characteristic morphological changes. PPAR-gamma ligands are more effective than PPAR-alpha ligands in the inhibition of cell growth and in the induction of differentiation. The physiological product of lipid peroxidation, 4-hydroxynonenal (HNE), which alone induces granulocytic-like differentiation of HL-60 cells, potentiates the monocytic differentiation induced by ciprofibrate, troglitazone, and 15d-prostaglandin J2. The same HNE treatment significantly inhibits U937 cell growth and potentiates the inhibition of cell growth in PPAR-gamma ligand-treated cells. However, HNE does not induce a significant number of CD14-positive U937 cells. HNE causes a great increase of PPAR-gamma expression in both HL-60 and U937 cells, whereas it does not modify the PPAR-alpha expression. This observation may account for the high synergistic effect displayed by HNE and PPAR-gamma ligands in the inhibition of cell growth and differentiation induction. These results represent the first evidence of the involvement of a product of lipid peroxidation in the modulation of PPAR ligand activity and suggest a relationship between HNE and PPAR ligand pathways in leukemic cell growth and differentiation.

The "two-faced" effects of reactive oxygen species and the lipid peroxidation product 4-hydroxynonenal in the hallmarks of cancer.

Reacytive Oxygen Species (ROS) have long been considered to be involved in the initiation, progression and metastasis of cancer. However, accumulating evidence points to the benefical role of ROS. Moreover, ROS production, leading to apoptosis, is the mechanism by which many chemotherapeutic agents can act. Beside direct actions, ROS elicit lipid peroxidation, leading to the production of 4-hydroxynoneal (HNE). Interestingly, HNE also seems to have a dual behaviour with respect to cancer. In this review we present recent literature data which outline the "two-faced" character of oxidative stress and lipid peroxidation in carcinogenesis and in the hallmarks of cancer.

4-hydroxynonenal, a lipid peroxidation product of dietary polyunsaturated fatty acids, has anticarcinogenic properties in colon carcinoma cell lines through the inhibition of telomerase activity.

The effects of polyunsaturated fatty acids (PUFAs) obtained from the diet on colorectal cancer have been widely explored. However, controversial results have been obtained about the role played by the lipid peroxidation products of PUFAs, such as 4-hydroxy-nonenal (HNE), in the control of colon cancer growth. This aldehyde, indeed, showed both procarcinogenic and protective effects. In an attempt to verify the action of HNE, we studied the effects of a low dose of HNE (1 microM), similar to those "physiologically" found in normal cells and plasma, on telomerase activity, a key parameter of malignant transformation. Caco-2 cells were exposed to HNE and, paralleling cell growth inhibition, we observed the down-regulation of telomerase activity and hTERT expression. Similar effects have also been observed in HT-29 cells, in which HNE inhibited cell proliferation, telomerase activity and hTERT expression, suggesting that the inhibition of telomerase activity could be a general mechanism involved in the antiproliferative effect exerted by this aldehyde. Finally, we elucidated the mechanism of hTERT inhibition by HNE. A reduction of GSH content preceded the decrease of telomerase activity, but this only partially explained the telomerase activity inhibition. The major mechanism of HNE action seems to be the modulation of expression and activity of transcription factors belonging to the Myc/Mad/Max network. Since the presence of PUFAs in the diet exposes epithelial colon cells to HNE, this aldehyde could contribute to cell growth control through the inhibitory action on telomerase activity and hTERT expression, suggesting a protective effect on colon mucosa.

4-Hydroxynonenal affects pRb/E2F pathway in HL-60 human leukemic cells.

4-Hydroxynonenal (HNE), a highly reactive product of lipid peroxidation, has an antiproliferative effect in several tumor cell lines and provokes alteration of cell cycle progression in HL-60 cells. HNE down-regulates c-myc expression in K562, HL-60, and MEL cells. This prompted us to study the cascade of phenomena that, starting from the CKIs expression and the phosphorylation of pRb, arrives at the E2F binding to consensus sequence in the P2 promoter of the c-myc gene. Treatment of HL-60 cells with HNE (1 microM) causes a p53-independent increase of p21(WAF1/CIP1) expression, pRb dephosphorylation, a decrease of low molecular weight E2F complexes and an increase of high molecular weight E2F complexes bound to P2 c-myc promoter. E2F4 expression is reduced by HNE treatment as well as the amount of pRb/E2F4 complexes, whereas the amount of pRb/E2F1 complexes is increased. In conclusion, HNE can affect the pRb/E2F pathway by modifying the expression of several genes involved in the control of cell proliferation.