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Biomedical subjects

R A Canuto

Publications and source records attributed to R A Canuto.

At least 19 recordsLinked to original sources
PubMed2006

HMG-CoA reductase and PPARalpha are involved in clofibrate-induced apoptosis in human keratinocytes.

Contrasting data have been reported on the effects of clofibrate, a PPARalpha agonist and hypolipidemic drug. The carcinogenic and anti-apoptotic effects have been demonstrated especially in rodents in both "in vivo" and "in vitro" experiments. In contrast, in rat and human hepatoma cell lines, several reports have shown its concentration-dependent pro-apoptotic effect. No epidemiological data exist about its carcinogenetic effect in man. This study shows that clofibrate also induced apoptosis in a human non-tumour cell line, NCTC 2544, which shares the characteristic of proliferation with tumour cells. Both HMG-CoA reductase and PPARalpha were found to be involved in the signal transduction pathway inducing apoptosis, the former being the principal target: HMG-CoA reductase decreased and PPARalpha increased. Changes in HMG-CoA reductase expression caused activation of parameters leading to apoptosis via the mitochondria pathway. Clofibrate must be considered a pro-apoptotic molecule at concentrations of 0.25 mM and above: the effect is exercised not only on tumour cells but also on normal human proliferating cells. Clofibrate should thus be regarded as a potential drug to reduce the number of proliferating cells in pathological conditions.

Apoptosis↗
PubMed2001

Increase in class 2 aldehyde dehydrogenase expression by arachidonic acid in rat hepatoma cells.

Aldehyde dehydrogenase (ALDH) is a family of several isoenzymes important in cell defence against both exogenous and endogenous aldehydes. Compared with normal hepatocytes, in rat hepatoma cells the following changes in the expression of ALDH occur: cytosolic class 3 ALDH expression appears and mitochondrial class 2 ALDH decreases. In parallel with these changes, a decrease in the polyunsaturated fatty acid content in membrane phospholipids occurs. In the present study we demonstrated that restoring the levels of arachidonic acid in 7777 and JM2 rat hepatoma cell lines to those seen in hepatocytes decreases hepatoma cell growth, and increases class 2 ALDH activity. This latter effect appears to be due to an increased gene transcription of class 2 ALDH. To account for this increase, we examined whether peroxisome-proliferator-activated receptors (PPARs) or lipid peroxidation were involved. We demonstrated a stimulation of PPAR expression, which is different in the two hepatoma cell lines: in the 7777 cell line, there was an increase in PPAR alpha expression, whereas PPAR gamma expression increased in JM2 cells. We also found increased lipid peroxidation, but this increase became evident at a later stage when class 2 ALDH expression had already increased. In conclusion, arachidonic acid added to the culture medium of hepatoma cell lines is able to partially restore the normal phenotype of class 2 ALDH, in addition to a decrease in cell growth.

Aldehyde Dehydrogenase↗
PubMed2001

The effect of a novel irreversible inhibitor of aldehyde dehydrogenases 1 and 3 on tumour cell growth and death.

Aldehyde dehydrogenases (ALDHs) are a family of several isoenzymes expressed in various tissues and in all subcellular fractions. In some tumours, there is an increase of ALDH activity, especially that of class 1 and 3. The increase in the activity of these isoenzymes is correlated with cell growth and drug resistance shown by these cells. It has been observed that hepatoma cells expressing low ALDH3 activity are more susceptible to growth inhibition by low concentration of lipid peroxidation products than hepatoma cells expressing high ALDH3 activity. The products of lipid peroxidation are good substrates for ALDH, but when their intracellular levels are increased in hepatoma cells treated repeatedly with prooxidants, they inhibit ALDH3 and bring about growth inhibition or cell death. As a follow up to the work previously reported on S-methyl 4-amino-4-methylpent-2-ynethioate, a synthetic suicide inhibitor of ALDH1, which induced bcl2 overexpressing cells into apoptosis and exhibited an ED50 of 400 microM, a novel broad spectrum inhibitor of ALDH1 and ALDH3 was synthesised. This new compound (ATEM) is a suicide inhibitor of ALDH1, an irreversible inhibitor of ALDH3 and exhibits an ED50 of 10-25 microM on rat cultured hepatoma cells. Four hours after treatment with 25 microM ATEM, ALDH activity using benzaldehyde or propionaldehyde in hepatoma cells was decreased by 40% and cell number by 15% compared with controls. As cell growth did not resume when the inhibitor was removed from the culture medium, it suggested strongly that ALDHs play a pivotal role in mediating cell death.

Aldehyde Dehydrogenase↗
PubMed2001

Inhibition of cytosolic class 3 aldehyde dehydrogenase by antisense oligonucleotides in rat hepatoma cells.

Aldehyde dehydrogenases (ALDHs) are a superfamily of several isoenzymes widely expressed in bacteria, yeast, plant and animals. Three major classes of ALDHs have been traditionally identified, classes 1, 2 and 3. Both exogenous and endogenous aldehydes, including aldehydes derived from lipid peroxidation, are oxidized by the ALDH superfamily. Several changes in ALDH isoenzyme expression take place in hepatoma cells, in particular cytosolic class 3 ALDH (ALDH3), not expressed in normal hepatocytes, appears and increases with the degree of deviation. It has been demonstrated that cytosolic ALDH3 is important in determining the resistance of tumor cells to antitumor drugs, such as cyclophosphamide. Moreover, hepatoma-associated ALDH3 seems to be important in metabolizing aldehydes derived from lipid peroxidation, and in particular the cytostatic aldehyde 4-hydroxynonenal (4-HNE). We demonstrated previously that restoring endogenous lipid peroxidation in hepatoma cells by enriching them with arachidonic acid causes a decrease of mRNA, protein and enzyme activity of ALDH3 and that this decrease reduces cell growth and/or causes cell death, depending on basal class 3 ALDH activity. To confirm the correlation between inhibition of class 3 ALDH and reduction of cell proliferation, we exposed hepatoma cells to antisense oligonucleotides (ODNs) against ALDH3. In JM2 hepatoma cell line, with high ALDH3 activity, the exposure to antisense ODNs significantly decreases mRNA and enzyme activity (90%). At the same time, cell growth was reduced by about 70%. The results confirm that in hepatoma cells ALDH3 expression is closely related with cell growth, and that its inhibition is important in reducing the proliferation of hepatoma cells overexpressing ALDH3.

Aldehyde Dehydrogenase↗
PubMed2000

Effect of bioactive aldehydes on cell proliferation and c-myc expression in HL-60 human leukemic cells.

Lipid peroxidation produces several toxic carbonyls, including biologically active aldehydes. In previous studies, we demonstrated that 4-hydroxynonenal (HNE), one of the major products of lipoperoxidation, inhibited growth and c-myc expression in K562 and HL-60 human leukemic cells. In this study, we compared the HNE effects with those of 4-hydroxyoctenal (HOE), 4-hydroxyundecenal (HUE; different lengths of the lipophilic tail), and the analogous aldehydes 2-trans-nonanal (lacking the OH group) and nonenal (lacking the OH group and the trans CC double bond), on HL-60 cell proliferation and c-myc expression. HUE and HOE inhibited growth and c-myc expression in a dose-dependent fashion, with an effectiveness comparable with that of HNE, whereas 2-nonenal and nonanal did not affect these parameters. Our results showed that different aldehydes produced from lipid peroxidation may contribute to growth inhibition by c-myc downregulation and that the molecular features involved seem to be the hydroxy group and the trans CC double bond.

Aldehydes↗
PubMed1999

Dose-dependent inhibition of cell proliferation induced by lipid peroxidation products in rat hepatoma cells after enrichment with arachidonic acid.

Polyunsaturated fatty acids (PUFA) are important constituents of membrane phospholipids, whose levels are decreased in some tumor cells. This deficiency may cause alterations in signal transduction and an interruption of normal cellular events. The enrichment of tumor cells with PUFA may stimulate or inhibit tumor growth, probably depending on the type of PUFA and the cellular concentration of aldehydes derived from restored lipid peroxidation. We examined the effect of several doses of prooxidant on the growth of hepatoma cells with different aldehyde dehydrogenase activities, enriched with arachidonic acid. Two doses of prooxidant were sufficient to reduce growth of hepatoma cells with low aldehyde dehydrogenase activity, whereas three doses were necessary for those with high enzyme activity. In both cases, lipid peroxidation products blocked the cells in the S phase.

Animals↗
PubMed1999

Inhibition of class-3 aldehyde dehydrogenase and cell growth by restored lipid peroxidation in hepatoma cell lines.

Hepatoma cells have a below-normal content of polyunsaturated fatty acids; this reduces lipid peroxidation and the production of cytotoxic and cytostatic aldehydes within the cells. In proportion to the degree of deviation, hepatoma cells also show an increase in the activity of Class-3 aldehyde dehydrogenase, an enzyme important in the metabolism of lipid peroxidation products and also in that of several drugs. When hepatoma cells with different degrees of deviation were enriched with arachidonic acid and stimulated to peroxidize by ascorbate/iron sulphate, their growth rate was reduced in proportion to the quantity of aldehydes produced and to the activity of aldehyde dehydrogenase. Therefore, 7777 cells, less deviated and with low Class-3 aldehyde dehydrogenase activity, were more susceptible to lipid peroxidation products than JM2 cells. It is noteworthy that repeated treatments with prooxidant also caused a decrease in mRNA and activity of Class-3 aldehyde dehydrogenase, contributing to the decreased growth and viability. Thus, Class-3 aldehyde dehydrogenase could be considered relevant for the growth of hepatoma cells, since it defends them against cell growth inhibiting aldehydes derived from lipid peroxidation.

Aldehyde Dehydrogenase↗
PubMed1999

Liver cancer is induced by a subnecrogenic dose of DENA when associated with fasting/refeeding: role of glutathione-transferase and lipid peroxidation.

In previous studies, we reported that fasting/refeeding has a role in sustaining the initiation of liver cancer by a subnecrogenic (noninitiating) dose of diethylnitrosamine (DENA). This research investigated whether the metabolic alterations imposed by fasting/refeeding provide an imbalance between the generation of carcinogenic molecules and the scavenger defense mechanisms in rat liver. Metabolism of DENA, levels of reduced glutathione (GSH) and GSH transferase (GST) activity, as well as basal and stimulated malondialdehyde (MDA) production, were examined. Rats fasted for 4 days showed a decrease in the liver levels of GSH, GST activity, monounsaturated fatty acids and % of labeled nuclei. After 1 day of refeeding, at which point DENA was administered, the levels of GSH recovered, GST activity remained below control values, basal and stimulated MDA production and content of total polyunsaturated fatty acids in liver phospholipids decreased. One day after DENA treatment, MDA production further decreased, although the % of labeled nuclei increased. No significant changes in the content of arachidonic acid, the main target of peroxidation, were observed at any time. The results indicated that the induction of the hepatocellular carcinoma was associated with a depression of GST activity and lipid peroxidation when rats were given 20 mg/kg of DENA after 1 day of refeeding after 4-day fasting.

Animals↗
PubMed1998

Peroxisome proliferators induce apoptosis in hepatoma cells.

In the AH-130 hepatoma, a poorly differentiated tumor, maintained by weekly transplantations in rats, a low percentage of cells spontaneously underwent apoptosis, mainly during the transition from logarithmic- to stationary-growth phase. It was possible to induce massive apoptosis of cells by treating them with clofibrate, a peroxisome proliferator and hypolipidemic drug. Similar results were obtained with HepG2 cells. With 1 mM clofibrate, apoptosis began to manifest itself after 1 h of treatment in vitro, and was assessed by morphological analysis, by DNA fragmentation carried out with agarose gel electrophoresis, and with flow cytometric determination of terminal deoxynucleotidyl transferase-mediated dUTP-biotin nick end-labeling. The mechanisms whereby clofibrate induces apoptosis are still unclear. Since the peroxisome proliferator-activated receptor was expressed at a very low level and was not stimulated by clofibrate in the AH-130 hepatoma cells, its involvement seems unlikely. Moreover, lipid peroxidation was not increased after clofibrate treatment. Phospholipids and cholesterol were significantly decreased. The decreased cholesterol content might suggest an inhibition of the mevalonate pathway and, therefore, of isoprenylation of proteins involved in cell proliferation.

Animals↗
PubMed1997

Rapid and extensive lethal action of clofibrate on hepatoma cells in vitro.

Clofibrate, for a long time in use as a hypolipidemic drug, is a well known peroxisomal proliferator (PP) and hepatocarcinogen in rodents. We show here that in vitro 1 mM clofibrate induces a rapid and massive death of rat AH-130 hepatoma cells. Cell death was prominent already after 4 h of treatment, with a characteristic ;apoptotic' pattern by conventional microscopy. This was further supported by the pronounced chromatin condensation detectable on 4',6-diamine-2'-phenylindole dihydrochloride (DAPI) staining, the clearcut internucleosomal DNA fragmentation on agarose-gel electrophoresis (ladder pattern), and the accumulation of markedly hypochromic cells observed in flow cytometric DNA histograms. Consistently with the apoptotic features of the process, some parameters commonly used to detect cell death, such as plasma membrane permeabilization to trypan blue or propidium iodide, lack of mitochondrial retention of rhodamine 123, or extracellular release of lactate dehydrogenase, were all virtually negative. However, these same parameters became markedly positive after 24 h of treatment, which was suggestive for the occurrence of ;secondary' necrosis among AH-130 cells. By a combination of flow cytometric parameters, after 4 h on 1 mM clofibrate only 41% of the AH-130 cells could still be categorized as viable (i.e., non-apoptotic and non-necrotic), while 46% of cells appeared apoptotic and 13% necrotic. At 24 h, 67% of cells were necrotic, 20% apoptotic and only 13% non-apoptotic and non-necrotic. Apoptosis was also extensive in AH-130 cells treated with another PP such as nafenopin at 1 mM concentration and in human hepatoma HepG2 cells treated with clofibrate. By contrast, clofibrate did not cause apoptosis on primary rat hepatocyte cultures. These observations indicate that: (i) apart from their well-known cell growth-promoting action, PPs such as clofibrate or nafenopin may exert a substantial cytotoxic action on targets such as the AH-130 or HepG2 hepatoma cells; (ii) this cell death evolves from an initial 'apoptotic' to an eventual ;necrotic' pattern; (iii) detection of cell death requires the adoption of a full panel of tests, adequate to cover the whole evolving death pattern, while such tests may even be substantially misleading whenever applied individually; (iv) the cytotoxicity of clofibrate and similar agents on normal and, particularly, tumoural cells may deserve careful reevaluation.

Journal Article↗
PubMed1997

Comparative evaluation of cytotoxicity and metabolism of four aldehydes in two hepatoma cell lines.

The metabolism of acetaldehyde (ACA), benzaldehyde (BA), propionaldehyde (PA) and valeraldehyde (VA) has been studied in two hepatoma cell lines, the rat HTC and mouse Hepa 1c1c7 cells. The cytotoxicity of the four aldehydes to these two cell lines has been compared. The end-points for evaluating cytotoxicity were 1) total macromolecular content (TMC) of confluent cultures, and 2) colony forming ability of dividing cells. These two assay systems had different sensitivities for the toxicity of aldehydes, probably due to different numbers of target cells. The activities of aldehyde dehydrogenases (NAD- and NADP-dependent, ALDH), alcohol dehydrogenase and aldehyde reductase were markedly greater in the HTC cell line compared to the Hepa 1c1c7 cell line, especially with BA as substrate. The cytotoxicities of aldehydes were generally stronger in the HTC cell line than in the Hepa 1c1c7 cell line; with the CF test. Particularly, BA was highly toxic to the HTC cells, which possessed the highest ALDH levels. Moreover, the treatment with (diethylamino)benzaldehyde, an ALDH inhibitor, completely abolished the toxicity of BA. Taken together, all these findings suggest that several cell lines expressing different aldehyde metabolizing activities could be used especially in the pre-screening phase to distinguish the metabolism-dependent cytotoxic effects from the metabolism independent effects.

Acetaldehyde↗
PubMed1996

Enzymatic pattern of aldehyde metabolism during HL-60 cell differentiation.

A number of metabolic changes, including modification of different enzyme activities, are linked to the acquisition of differentiated phenotype in HL-60 cells. Enzymes metabolizing aldehydes contribute to maintaining the intracellular steady-state concentration of aldehydes derived from lipid peroxidation. 4-Hydroxynonenal is one of the most important aldehydes produced by this process, and it is able to inhibit proliferation and induce differentiation of HL-60 human leukemic cells. We have now demonstrated that, after induction of HL-60 cell differentiation by 4-hydroxynonenal or DMSO, glutathione transferase activity increases in parallel to the degree of differentiation induction. Moreover, in 4-hydroxynonenal- or DMSO-treated cells, the concentration of reduced glutathione decreases five days after treatment. The rise of glutathione transferase activity, as well as the decrease of reduced glutathione, are possibly linked to the increase of detoxification capability of differentiated cells.

Alcohol Dehydrogenase↗
PubMed1995

Hepatocellular metabolism of 4-hydroxy-2,3-nonenal is impaired in conditions of chronic cholestasis.

4-Hydroxy-2,3-nonenal is a major aldehydic end-product of lipid peroxidation known to exert several biological and cytotoxic effects and to be produced during conditions of chronic cholestasis. Here we report that viable hepatocytes isolated from cholestatic livers of bile duct-ligated rats (BDL hepatocytes) show a significantly lower rate of HNE metabolism than control cells. This feature is likely to be the consequence of a significant inhibition in the activity of HNE-metabolizing cytosolic glutathione-S-transferase and alcohol dehydrogenase in BDL hepatocytes. Particulate NADP-dependent aldehyde dehydrogenase was also inhibited. No significant change was found for aldehyde reductase activity. A decreased hepatocellular metabolism of HNE can expose liver parenchymal and non-parenchymal cells to cytotoxic as well as pro-inflammatory and pro-fibrogenic effects of HNE, contributing to the development of chronic cholestatic liver damage.

Alcohol Dehydrogenase↗