Cristina Toaldo

MicroRNA expression changes during human leukemic HL-60 cell differentiation induced by 4-hydroxynonenal, a product of lipid peroxidation

4-Hydroxynonenal (HNE) is one of several lipid oxidation products that may have an impact on human pathophysiology. It is an important second messenger involved in the regulation of various cellular processes and exhibits antiproliferative and differentiative properties in various tumor cell lines. The mechanisms by which HNE affects cell growth and differentiation are only partially clarified. Because microRNAs (miRNAs) have the ability to regulate several cellular processes, we hypothesized that HNE, in addition to other mechanisms, could affect miRNA expression. Here, we present the results of a genome-wide miRNA expression profiling of HNE-treated HL-60 leukemic cells. Among 470 human miRNAs, 10 were found to be differentially expressed between control and HNE-treated cells (at p<0.05). Six miRNAs were down-regulated (miR-181a*, miR-199b, miR-202, miR-378, miR-454-3p, miR-575) and 4 were up-regulated (miR-125a, miR-339, miR-663, miR-660). Three of these regulated miRNAs (miR-202, miR-339, miR-378) were further assayed and validated by quantitative real-time RT-PCR. Moreover, consistent with the down-regulation of miR-378, HNE also induced the expression of the SUFU protein, a tumor suppressor recently identified as a target of miR-378. The finding that HNE could regulate the expression of miRNAs and their targets opens new perspectives on the understanding of HNE-controlled pathways. A functional analysis of 191 putative gene targets of miRNAs modulated by HNE is discussed.

Induction of cell cycle arrest and DNA damage by the HDAC inhibitor panobinostat (LBH589) and the lipid peroxidation end product 4-hydroxynonenal in prostate cancer cells

Histone deacetylase inhibitors (HDACIs) are promising antineoplastic agents for the treatment of cancer. Here we report that the lipid peroxidation end product 4-hydroxynonenal (HNE) significantly potentiates the anti-tumor effects of the HDAC inhibitor panobinostat (LBH589) in the PC3 prostate cancer cell model. Panobinostat and HNE inhibited proliferation of PC3 cells and the combination of the two agents resulted in a significant combined effect. Cell cycle analysis revealed that both single agents and, to a greater extent, their combined treatment induced G2/M arrest, but cell death occurred in the combined treatment only. Furthermore, HNE and, to a greater extent, the combined treatment induced dephosphorylation of Cdc2 leading to progression into mitosis as confirmed by α-tubulin/DAPI staining and phospho-histone H3 (Ser10) analysis. To evaluate possible induction of DNA damage we utilized the marker phosphorylated histone H2A.X. Results showed that the combination of panobinostat and HNE induced significant DNA damage concomitant with the mitotic arrest. Then, by using androgen receptor (AR)-expressing PC3 cells we observed that the responsiveness to HNE and panobinostat was independent of the expression of functional AR. Taken together, our data suggest that HNE potentiates the antitumoral effect of the HDACI panobinostat in prostate cancer cells.

Nuclear factor erythroid 2-related factor-2 activity controls 4-hydroxynonenal metabolism and activity in prostate cancer cells

4-Hydroxynonenal (HNE) is an end product of lipoperoxidation with antiproliferative and proapoptotic properties in various tumors. Here we report a greater sensitivity to HNE in PC3 and LNCaP cells compared to DU145 cells. In contrast to PC3 and LNCaP cells, HNE-treated DU145 cells showed a smaller reduction in growth and did not undergo apoptosis. In DU145 cells, HNE did not induce ROS production and DNA damage and generated a lower amount of HNE-protein adducts. DU145 cells had a greater GSH and GST A4 content and GSH/GST-mediated HNE detoxification. Nuclear factor erythroid 2-related factor-2 (Nrf2) is a regulator of the antioxidant response. Nrf2 protein content and nuclear accumulation were higher in DU145 cells compared to PC3 and LNCaP cells, whereas the expression of KEAP1, the main negative regulator of Nrf2 activity, was lower. Inhibition of Nrf2 expression with specific siRNA resulted in a reduction in GST A4 expression and GS-HNE formation, indicating that Nrf2 controls HNE metabolism. In addition, Nrf2 knockdown sensitized DU145 cells to HNE-mediated antiproliferative and proapoptotic activity. In conclusion, we demonstrated that increased Nrf2 activity resulted in a reduction in HNE sensitivity in prostate cancer cells, suggesting a potential mechanism of resistance to pro-oxidant therapy.

The role of PPAR ligands in controlling growth-related gene expression and their interaction with lipoperoxidation products

Peroxisome proliferators-activated receptors (PPARs) are ligand-activated transcription factors that belong to the nuclear hormone receptor superfamily. The three PPAR isoforms (α, γ and β/δ) have been found to play a pleiotropic role in cell fat metabolism. Furthermore, in recent years, evidence has been found regarding the antiproliferative, proapoptotic, and differentiation-promoting activities displayed by PPAR ligands, particularly by PPARγ ligands. PPAR ligands affect the expression of different growth-related genes through both PPAR-dependent and PPAR-independent mechanisms. Moreover, an interaction between PPAR ligands and other molecules which strengthen the effects of PPAR ligands has been described. Here we review the action of PPAR on the control of gene expression with particular regard to the effect of PPAR ligands on the expression of genes involved in the regulation of cell-cycle, differentiation, and apoptosis. Moreover, the interaction between PPAR ligands and 4-hydroxynonenal (HNE), the major product of the lipid peroxidation, has been reviewed.

Increase of telomerase activity and hTERT expression in myelodysplastic syndromes

Background: National Cancer Institutes (NCI) designated cancer centers use one of three organizational structures. The hypothesis of this study is that there are differences in the amount of annual NCI funding per faculty member based on a cancer center’s organizational structure. The study also considers the impact of secondary factors (i.e. the existence of a clinical program, the region and the size of the city in which the cancer center is located) on funding and the number of Howard Hughes Medical Institute (HHMI) investigators at each cancer center. Methods: Online research and telephone interviews with each cancer center were used to gather information, including: organizational structure, the presence of a clinical program, the number of faculty members, and the number of Howard Hughes Medical Institute investigators. Statistical tests were used to assess the impact which organizational structure has on the amount of funding per faculty member and number of HHMI investigators. Results: Of the 63 cancer centers, 44 use a matrix structure, 16 have a freestanding structure, and 3 have a Department of Oncology structure. Kruskal-Wallis tests reveal no statistically significant differences in the amount of funding per faculty member or the number of HHMI investigators between cancers with a matrix, freestanding or Department of Oncology structure. Conclusion: While the results seem to suggest that the organizational structure of a given cancer center does not impact the amount of NCI funding or number of HHMI investigators which it attracts, the existence of this relationship is likely masked by the small sample size in this study. Further studies may be appropriate to examine the effect organizational structure has on other measurements which are relevant to cancer centers, such as quality and quantity of research produced.

Exposure of HL-60 human leukaemic cells to 4-hydroxynonenal promotes the formation of adduct(s) with α-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.

Rosiglitazone and AS601245 decrease cell adhesion and migration through modulation of specific gene expression in human colon cancer cells.

PPARs are nuclear receptors activated by ligands. Activation of PPARγ leads to a reduction of adhesion and motility in some cancer models. PPARγ transcriptional activity can be negatively regulated by JNK-mediated phosphorylation. We postulated that the use of agents able to inhibit JNK activity could increase the effectiveness of PPARγ ligands. We analysed the effects of rosiglitazone (PPARγ ligand) and AS601245 (a selective JNK inhibitor) alone or in association on adhesion and migration of CaCo-2, HT29, and SW480 human colon cancer cells and investigated, through microarray analysis, the genes involved in these processes. Cell adhesion and migration was strongly inhibited by rosiglitazone and AS601245. Combined treatment with the two compounds resulted in a greater reduction of the adhesion and migration capacity. Affymetrix analysis in CaCo-2 cells revealed that some genes which were highly modulated by the combined treatment could be involved in these biological responses. Rosiglitazone, AS601245 and combined treatment down-regulated the expression of fibrinogen chains in all three cell lines. Moreover, rosiglitazone, alone or in association with AS601245, caused a decrease in the fibrinogen release. ARHGEF7/β-PIX gene was highly down-regulated by combined treatment, and western blot analysis revealed that β-PIX protein is down-modulated in CaCo-2, HT29 and SW480 cells, also. Transfection of cells with β-PIX gene completely abrogated the inhibitory effect on cell migration, determined by rosiglitazone, AS601245 and combined treatment. Results demonstrated that β-PIX protein is involved in the inhibition of cell migration and sustaining the positive interaction between PPARγ ligands and anti-inflammatory agents in humans.

PPARγ ligands inhibit telomerase activity and hTERT expression through modulation of the Myc/Mad/Max network in colon cancer cells

In human cells the length of telomeres depends on telomerase activity. This activity and the expression of the catalytic subunit of human telomerase reverse transcriptase (hTERT) is strongly up‐regulated in most human cancers. hTERT expression is regulated by different transcription factors, such as c‐Myc, Mad1 and Sp1. In this study, we demonstrated that 15d‐PG J2 and rosiglitazone (an endogenous and synthetic peroxisome proliferators activated receptor γ (PPARγ) ligand, respectively) inhibited hTERT expression and telomerase activity in CaCo‐2 colon cancer cells. Moreover, both ligands inhibited c‐Myc protein expression and its E‐box DNA binding activity. Additionally, Mad1 protein expression and its E‐box DNA binding activity were strongly increased by 15d‐PG J2 and, to a lesser extent, by rosiglitazone. Sp1 transcription factor expression and its GC‐box DNA binding activity were not affected by both PPARγ ligands. Results obtained by transient transfection of CaCo‐2 cells with pmaxFP‐Green‐PRL plasmid constructs containing the functional hTERT core promoter (including one E‐box and five GC‐boxes) and its E‐box deleted sequences, cloned upstream of the green fluorescent protein reporter gene, demonstrated that 15d‐PG J2, and with minor effectiveness, rosiglitazone, strongly reduced hTERT core promoter activity. E‐boxes for Myc/Mad/Max binding showed a higher activity than GC‐boxes for Sp1. By using GW9662, an antagonist of PPARγ, we demonstrated that the effects of 15d‐PG J2 are completely PPARγ independent, whereas the effects of rosiglitazone on hTERT expression seem to be partially PPARγ independent. The regulation of hTERT expression by 15d‐PG J2 and rosiglitazone, through the modulation of the Myc/Max/Mad1 network, may represent a new mechanism of action of these substances in inhibiting cell proliferation.

PPARγ in coronary atherosclerosis: In vivo expression pattern and correlations with hyperlipidemic status and statin treatment

OBJECTIVE Peroxisome proliferator-activated receptor-γ (PPARγ) is involved in regulation of macrophage inflammation and in atherosclerosis. Herein we investigate the influence of statin treatment on PPARγ expression in coronary artery disease. METHOD PPARγ expression was investigated in coronary atherosclerotic atherectomies (N=48) and arteries (N=12) from patients with stable or unstable coronary syndromes or undergoing cardiac transplantation for end-stage ischemic cardiomyopathy, respectively, by immunohistochemistry. Plaque components and tissue factor immunoreactivity were also investigated. Atherectomies were obtained from de novo culprit lesions of hypercholesterolemic (16 statin-treated and 16 untreated) and normolipidemic (N=16) patients. Furthermore, PPARγ expression was evaluated in patients peripheral blood monocytes and in monocytic U937 cells after atorvastatin incubation, by Western blot analysis. RESULT PPARγ expression was higher in coronary plaques and peripheral blood monocytes of statin-treated patients, and it significantly increased in monocytes after 24h atorvastatin incubation (p<0.05). Intra-plaque macrophage content, atheroma, neoangiogenesis and hemorrhage, and circulating CRP levels were lower in statin-treated than untreated hypercholesterolemic patients and comparable with normolipidemic subjects. PPARγ immunoreactivity was localized to neointima and media, its distribution pattern being different from that of tissue factor. CONCLUSION PPARγ expression was enhanced in statin-treated patients with different distribution and behavior as compared to atheroma, macrophage content, tissue factor immunoreactivity and serum CRP. In vitro studies showed increased PPARγ expression in monocytes after atorvastatin incubation. These findings provide further evidence as to the protective role of statins in coronary artery disease and their influence on PPARγ expression in coronary plaques and on the inflammatory status of patients.

AS601245, an Anti-Inflammatory JNK Inhibitor, and Clofibrate Have a Synergistic Effect in Inducing Cell Responses and in Affecting the Gene Expression Profile in CaCo-2 Colon Cancer Cells

PPARαs are nuclear receptors highly expressed in colon cells. They can be activated by the fibrates (clofibrate, ciprofibrate etc.) used to treat hyperlipidemia. Since PPARα transcriptional activity can be negatively regulated by JNK, the inhibition of JNK activity could increase the effectiveness of PPARα ligands. We analysed the effects of AS601245 (a JNK inhibitor) and clofibrate alone or in association, on proliferation, apoptosis, differentiation and the gene expression profile of CaCo-2 human colon cancer cells. Proliferation was inhibited in a dose-dependent way by clofibrate and AS601245. Combined treatment synergistically reduced cell proliferation, cyclin D1 and PCNA expression and induced apoptosis and differentiation. Reduction of cell proliferation, accompanied by the modulation of p21 expression was observed in HepG2 cells, also. Gene expression analysis revealed that some genes were highly modulated by the combined treatment and 28 genes containing PPRE were up-regulated, while clofibrate alone was ineffective. Moreover, STAT3 signalling was strongly reduced by combined treatment. After combined treatment, the binding of PPARα to PPRE increased and paralleled with the expression of the PPAR coactivator MED1. Results demonstrate that combined treatment increases the effectiveness of both compounds and suggest a positive interaction between PPARα ligands and anti-inflammatory agents in humans.