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Aldehyde dehydrogenase 3 expression is decreased by clofibrate via PPAR gamma induction in JM2 rat hepatoma cell line
Chemico-Biological Interactions 143 /144 (2003) 29 /35 www.elsevier.com/locate/chembioint
Aldehyde dehydrogenase 3 expression is decreased by clofibrate via PPAR gamma induction in JM2 rat hepatoma cell line Rosa A. Canuto *, Marina Maggiora, Antonella Trombetta, Germana Martinasso, Giuliana Muzio Dipartimento di Medicina ed Oncologia Sperimentale, Universita` di Torino, Corso Raffaello 30, 10125 Torino, Italy
Abstract In normal liver aldehyde dehydrogenase 3 (ALDH3) is poorly expressed. In hepatoma cells, its expression increases in direct correlation with the degree of deviation and increased ALDH3 activity is one cause of resistance to the toxicity of drugs and lipid peroxidation aldehydes. Hepatoma cells with high ALDH3 content are more resistant to the cytotoxic effect of aldehydes than those with low ALDH3, and inhibition of the enzyme with aldehydes, specific inhibitors or antisense oligonucleotides (AS-ODN), decreases cell growth. It remains open how ALDH3 influences cell growth or cell phenotype. Recently, we have shown that enrichment of a highly deviated rat hepatoma cell line, JM2, with arachidonic acid, a natural ligand of peroxisome proliferator activated receptors (PPARs), inhibits growth, partially restores ALDH2 and ALDH3 to their normal levels and induces PPAR expression. In the present study we address the effect of clofibrate, a hypolipidemic drug and synthetic PPAR ligand on ALDH gene expression. We show that treatment of JM2 cells with clofibrate inhibits cell growth, induces PPARg and decreases ALDH3 expression. To determine the relationship between PPARg and ALDH3 expression, we exposed JM2 cells to AS-ODN against PPARg. AS-ODN reduced PPARg content and prevented the inhibitory effect of clofibrate on cell proliferation and ALDH3 expression. Since these results indicate that ALDH3 expression is under PPAR control, we examined the 5? flanking sequence of the ALDH3 gene, but were unable to find any sequence similar to any known peroxisome proliferator response element. We thus believe that the effect of PPARg on ALDH3 occurs via other transcription factors, whose identity remain to be determined. The results indicate that PPARg plays a key role in regulation of growth and differentiation of hepatoma cells, and that ALDH3 collaborates in modulating cell proliferation and in determining some aspects of the hepatoma phenotype, i.e. resistance to drugs and to lipid peroxidation products. # 2002 Elsevier Science Ireland Ltd. All rights reserved. Keywords: Lipid peroxidation; Proliferation; Peroxisome proliferator-activated receptors
* Corresponding author. Tel.: /39-011-6707781; fax: /39-011-6707753 E-mail address: [email protected] (R.A. Canuto). 0009-2797/02/$ - see front matter # 2002 Elsevier Science Ireland Ltd. All rights reserved. PII: S 0 0 0 9 - 2 7 9 7 ( 0 2 ) 0 0 1 6 9 - 2
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R.A. Canuto et al. / Chemico-Biological Interactions 143 /144 (2003) 29 /35
1. Introduction Various metabolic pathways are involved in the detoxification of aldehydes to less reactive forms: aldehyde dehydrogenases (ALDHs), alcohol dehydrogenases, aldo-keto reductases, glutathione-Stransferases. ALDHs oxidise aldehydes to the corresponding carboxylic acids, using NAD or NADP as coenzymes, and they have been discovered in practically all organisms and found to exist in multiple isoforms [1 /3]. More than 160 ALDH cDNAs or genes have been isolated and sequenced from various sources: bacteria, yeast, fungi, plant and animals [4 /6]. In eukaryotes, 86 distinct cDNAs/genes of ALDH have been determined, and are divided into 18 families [7]. With regard to function, ALDHs exhibit a rather broad substrate specificity and many of them can oxidize several aliphatic and aromatic aldehydes [5]. A cytosolic form, ALDH3 (ALDH3A1), is highly expressed in the stomach, lung and cornea, but poorly expressed, if at all, in normal liver. In rat liver cells, cytosolic ALDH3 is induced by polycyclic aromatic hydrocarbons (PAH) or chlorinated compounds such as TCDD [8]. These compounds are ligands for the aryl hydrocarbon (Ah) receptor, a transcription factor that binds a consensus sequence, Ah responsive element (AhRE), in the promoter of target genes [9]. PAH and chlorinated compounds are an important class of environmental pollutants that produce a wide range of toxic and carcinogenic effects. Whereas in the liver ALDH3 is poorly expressed if at all, in hepatoma cells its expression increases in direct correlation with the degree of tumour deviation [10]. The increase of ALDH3 activity is evident in human and rat hepatoma cell lines, in hepatomas induced in rats by administration of chemical carcinogens [10,11], and in human hepatocellular carcinoma tissues from approximately 50% of patients with this form of cancer. The increase of ALDH3 expression in hepatoma cells is one cause of their resistance to drug toxicity and the consequent poor response to certain antitumour drugs. In particular, ALDH3 is responsible for cell tumour resistance to the oxazaphosphorine antineoplastic agents (eg.
cyclophosphamide). Sreerama and Sladek [12] report that interindividual variation in the activity of ALDH3 and ALDH1 in breast cancer cells could contribute to the very varied clinical response to cyclophosphamide. ALDH3 is also important in the metabolism of aldehydes derived from lipid peroxidation. Lipid peroxidation is a consequence of oxidative stress on polyunsaturated fatty acids and results in the formation of reactive by-products such as lipid hydroperoxides and aldehydes. The a,b-unsaturated aldehyde 4-hydroxynonenal is the most reactive and cytotoxic of the aldehyde by-products of lipid peroxidation [13]; it has been shown to have a variety of effects in biological systems, most often resulting in cytotoxicity. Hepatoma cells with a high ALDH3 content are more resistant to the cytostatic and cytotoxic effect of lipid peroxidation aldehydes than those with a low content [14]. As consequence of the above, inhibiting cytosolic ALDH3 may be a useful approach to increasing susceptibility of hepatoma cells to drugs and lipid peroxidation products, thereby reducing cell proliferation and inducing the normal phenotype. Reducing ALDH3 activity though inhibition by aldehydes generated by lipid peroxidation, specific ALDH3 inhibitors, or by ALDH3-specific antisense oligonucleotides (AS-ODN), hepatoma cells show decreased growth but not decreased cell viability in tumour cells with high ALDH3 activity [14 /17]. It remains open how cytosolic ALDH3 influences cell growth or the phenotype of hepatoma cells. To address this problem, we chose to investigate the expression of ALDH3 after treating hepatoma cells with clofibrate, a hypolipidemic drug that belongs to the peroxisome proliferator activated receptors (PPAR) ligands, since clofibrate has an inhibitory effect on the proliferation of hepatoma cells and influences ALDH. Clofibrate is known to induce the microsomal ALDH3 mRNA level in Hepa-1c1c7 hepatoma cells [18], to repress expression of mitochondrial ALDH2 in normal liver [19], and to cause an increase in liver ALDH activity located in peroxisomes and microsomes, whereas it does not alter the enzyme activity in the cytoplasmic fraction of liver [20].
R.A. Canuto et al. / Chemico-Biological Interactions 143 /144 (2003) 29 /35
2. Materials and methods 2.1. Culture conditions and treatments JM2 rat hepatoma cells were seeded (20 000 cells/cm2) and maintained for 24 h in DMEM/F12 medium plus 2 mM glutamine, 1% antibiotic/ antimycotic solution and 10% newborn calf serum (medium A). Treatment with clofibrate: 24 h after cell seeding, clofibrate dissolved in DMSO was added at final concentrations of 0.25, 0.5 or 1 mM. Treatment with clofibrate and AS-ODN against PPARg: 24 h after cell seeding, medium A was removed and replaced with OPTI-MEM I medium supplemented with 5 nM AS-ODN prepared as follows. AS-ODN, phosphorothioated at their 5? and 3? terminus and complementary to the nucleotide sequences 1 /22 of rat PPARg (GenBank accession number AB011365), was used. ASODN (10 pmoles/100 ml sterile water) and lipofectin (10 ml) were diluted separately in 200 ml of OPTI-MEM I. Lipofectin was incubated for 45 min at room temperature, gently mixed with ASODN, maintained for 15 min at room temperature to allow formation of the AS-ODN/Lipofectin complex, and then diluted to 2 ml with OPTIMEM I. 24 h later, the medium supplemented with AS-ODN was replaced with fresh identical ASODN- supplemented medium, and 0.5 mM clofibrate was added to the cells. Control cells were grown in OPTI-MEM I medium supplemented with lipofectin and given solvent or AS-ODN alone. At the different experimental times (see figure legends) cells from each treatment were harvested and used for the following determinations. 2.2. Cell proliferation Cell proliferation was evaluated as the number of cells present in the monolayer and in the culture medium. 2.3. Northern blot and western blot analysis Northern blot and Western blot analysis was performed as described in Canuto et al. [14,16].
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Polyclonal anti-PPARa or anti-PPARg antibodies, and monoclonal anti-p-Erk1,2 or anti-c-myc antibody were used. The cDNA probe and the polyclonal antibody for ALDH3 were kind gifts from Dr R. Lindahl. 2.4. Aldehyde dehydrogenase activity ALDH3 activity was determined in cytosol obtained from JM2 hepatoma cells treated or not with 0.5 mM clofibrate. Preparation of the cytosol and determination of ALDH3 activity were as described in Canuto et al. [15] 2.5. Protein determination Protein content was determined by the biuret method [21]. 2.6. Statistical analysis All data were expressed as means9/S.D. The significance of differences between group means was assessed by variance analysis, followed by the Newman-Keuls test.
3. Results and discussion Our previous results showed that it is possible to reduce cell proliferation in hepatoma cells by increasing the products of lipid peroxidation, following enrichment of cells with arachidonic acid. We have also demonstrated that clofibrate will induce rapid and extensive apoptosis or inhibit proliferation of hepatoma cells ([14,22 /24], Canuto et al., unpublished). Both arachidonic acid and clofibrate are ligands for PPARs, which are nuclear receptors for several chemicals. PPARs are present in three different isotypes, PPARa, b/d, and g [25]. Emerging evidence indicates that PPAR ligands can suppress the growth of different types of cancer. In the case of arachidonic acid treatment, the reduction of cell proliferation parallels the reduction of cytosolic ALDH3 expression. The correlation between proliferation and cytosolic ALDH3 was confirmed by using specific inhibitors or AS-ODN against ALDH3 [15,16]. In the light
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of these results we decided to examine whether cytosolic ALDH3 activity was also lowered when cell proliferation was reduced by clofibrate. The mechanism underlying the possible inhibition of cytosolic ALDH3 activity was also investigated. The experiments were performed on JM2 hepatoma cell line, a highly deviated hepatoma as demonstrated by its high content of cytosolic ALDH3 and poor response to inducers of cytochrome P450 [10,26]. Clofibrate inhibited JM2 cell proliferation in a dose-dependent manner (Fig. 1). The growth inhibition was not due to cell death as demonstrated by our inability to detect cells detached from the monolayer at any concentration or at any time (data not shown). The decrease in cell proliferation was accompanied by a decrease in the activity of the MAPK pathway as evidenced by a significant decrease in the phosphorylated form of Erk1, 2 and by decreased c-myc levels, a consequence of reduced MAPK pathway activity [27] (Fig. 2). Cytosolic ALDH3 expression was evaluated to detect any correlation with clofibrateinduced changes in cell proliferation. ALDH3 mRNA content, as determined by Northern blot, decreased in the presence of clofibrate at 24 h of exposure (Fig. 3, lane 2), whereas ALDH3 enzyme activity was found to decrease at 48 h of clofibrate exposure (Table 1). The delayed decrease in ALDH3 enzyme activity relative to the mRNA
Fig. 1. Cell growth in JM2 hepatoma cells exposed to 0.25, 0.5 or 1 mM clofibrate. Values are means9/S.D. of four experiments. Means with different letters are statistically different (P B/0.001) from one another as determined by variance analysis followed by the Newman-Keuls test. C, control cells; Cl, cells treated with clofibrate.
Fig. 2. Protein content of PPARg, the phosphorylated form of Erk1,2, and c-myc in JM2 hepatoma cells exposed to clofibrate for 24 h. Lane 1, control cells; Lane 2, cells exposed to 0.25 mM clofibrate; Lane 3, cells exposed to 0.5 mM clofibrate.
Fig. 3. mRNA content of ALDH3 in JM2 hepatoma cells exposed to 0.5 mM clofibrate and AS-ODN against PPARg. Lanes 1, control cells; Lanes 2, cells exposed to 0.5 mM clofibrate; Lanes 3, cells exposed to AS-ODN against PPARg; Lanes 4, cells exposed to AS-ODN against PPARg and 0.5 mM clofibrate.
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Table 1 ALDH3 activity in cytosol isolated from JM2 hepatoma cells treated with clofibrate 24 h
48 h
C
Cl 0.5 mM
C
Cl 0.5 mM
458
446
357.8
134.7
Data are expressed as nmol of NADPH produced/min/mg of protein and are the mean of two experiments. 2.5 mM benzaldehyde was used as substrate.
decrease is explained by the approximately 96 h half-life of the protein. This result prompted us to consider whether changes in cytosolic ALDH3 were correlated with cell proliferation, but not its primary cause. It might perhaps be better considered as linked to the phenotype changes. To understand the mechanisms underlying the decrease of ALDH3 expression, PPARa and g expression were determined. PPARa was not detected in untreated JM2 cells and did not change after clofibrate treatment (data not shown), whereas PPARg, which is present in untreated JM2 cells, significantly increased with clofibrate (Fig. 2). PPARa is especially involved in peroxisome proliferation and lipid metabolism, whereas PPARg is more closely linked to induction of differentiation and to the inhibition of cell proliferation. Treatment of JM2 hepatoma cells with another PP, arachidonic acid, also induced PPARg, but not PPARa [24]. The possible correlation between PPARg and cytosolic ALDH3 activity was investigated by using AS-ODN against PPARg mRNA. ASODN abolished the antiproliferative effect of clofibrate (Fig. 4) and at the same time also prevented the decrease in ALDH3 mRNA levels (Fig. 3). PPARg decreased in the presence of its AS-ODN and was not increased in the presence of clofibrate plus AS-ODN, the content being similar to that of control cells (Fig. 5). On the contrary, inhibiting ALDH3 by AS-ODN against ALDH3 did not change PPARg expression (data not shown). Since these results suggest that cytosolic ALDH3 expression is under PPAR control, we examined 8 kb of 5? flanking sequence of the
Fig. 4. Cell growth in JM2 hepatoma cells exposed to AS-ODN against PPARg. Values reported are numbers of cells in the monolayer, and are means9/S.D. of three experiments. Means with different letters are statistically different (P B/0.05) from one another as determined by variance analysis followed by the Newman-Keuls test. C, control cells; Cl, cells treated with 0.5 mM clofibrate; ODN, cells treated with 5 nM AS-ODN against PPARg.
Fig. 5. Protein content of PPARg in JM2 hepatoma cells exposed to clofibrate and to AS-ODN against PPARg. Lane 1, control cells; Lane 2, cells exposed to 0.5 mM clofibrate; Lane 3, cells exposed to AS-ODN against PPARg; Lane 3, cells exposed to AS-ODN against PPARg and to 0.5 mM clofibrate.
ALDH3 gene, but without finding any sequence similar to known peroxisome proliferator response element, as reported in Ref. [28]. Thus we believe that the effect of PPARg on ALDH3 expression occurs via other transcription factors that remain to be determined. The results indicate that, as in many other different tumour types, PPARg plays a key role in regulation of growth and differentiation of hepatoma cells, and that ALDH3 collaborates in modulating cell proliferation and in determining some aspects of the hepatoma phenotype, i.e. resistance to drugs and to cytostatic lipid peroxidation products. Therefore, it is likely that the growth inhibitory effect of clofibrate on tumour cells is associated with induction of a more
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differentiated phenotype. The ability of PPARg to control expression of genes involved in differentiation and cell proliferation suggests that PPARg ligands might be used as therapeutic agents in the treatment of tumours, and that the evaluation of cytosolic ALDH3 expression is a marker of differentiation of tumour cells derived from the liver.
[12]
[13]
[14]
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Aldehyde dehydrogenase 3 expression is decreased by clofibrate via PPAR gamma induction in JM2 rat hepatoma cell line Rosa A. Canuto *, Marina Maggiora, Antonella Trombetta, Germana Martinasso, Giuliana Muzio Dipartimento di Medicina ed Oncologia Sperimentale, Universita` di Torino, Corso Raffaello 30, 10125 Torino, Italy
Abstract In normal liver aldehyde dehydrogenase 3 (ALDH3) is poorly expressed. In hepatoma cells, its expression increases in direct correlation with the degree of deviation and increased ALDH3 activity is one cause of resistance to the toxicity of drugs and lipid peroxidation aldehydes. Hepatoma cells with high ALDH3 content are more resistant to the cytotoxic effect of aldehydes than those with low ALDH3, and inhibition of the enzyme with aldehydes, specific inhibitors or antisense oligonucleotides (AS-ODN), decreases cell growth. It remains open how ALDH3 influences cell growth or cell phenotype. Recently, we have shown that enrichment of a highly deviated rat hepatoma cell line, JM2, with arachidonic acid, a natural ligand of peroxisome proliferator activated receptors (PPARs), inhibits growth, partially restores ALDH2 and ALDH3 to their normal levels and induces PPAR expression. In the present study we address the effect of clofibrate, a hypolipidemic drug and synthetic PPAR ligand on ALDH gene expression. We show that treatment of JM2 cells with clofibrate inhibits cell growth, induces PPARg and decreases ALDH3 expression. To determine the relationship between PPARg and ALDH3 expression, we exposed JM2 cells to AS-ODN against PPARg. AS-ODN reduced PPARg content and prevented the inhibitory effect of clofibrate on cell proliferation and ALDH3 expression. Since these results indicate that ALDH3 expression is under PPAR control, we examined the 5? flanking sequence of the ALDH3 gene, but were unable to find any sequence similar to any known peroxisome proliferator response element. We thus believe that the effect of PPARg on ALDH3 occurs via other transcription factors, whose identity remain to be determined. The results indicate that PPARg plays a key role in regulation of growth and differentiation of hepatoma cells, and that ALDH3 collaborates in modulating cell proliferation and in determining some aspects of the hepatoma phenotype, i.e. resistance to drugs and to lipid peroxidation products. # 2002 Elsevier Science Ireland Ltd. All rights reserved. Keywords: Lipid peroxidation; Proliferation; Peroxisome proliferator-activated receptors
* Corresponding author. Tel.: /39-011-6707781; fax: /39-011-6707753 E-mail address: [email protected] (R.A. Canuto). 0009-2797/02/$ - see front matter # 2002 Elsevier Science Ireland Ltd. All rights reserved. PII: S 0 0 0 9 - 2 7 9 7 ( 0 2 ) 0 0 1 6 9 - 2
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1. Introduction Various metabolic pathways are involved in the detoxification of aldehydes to less reactive forms: aldehyde dehydrogenases (ALDHs), alcohol dehydrogenases, aldo-keto reductases, glutathione-Stransferases. ALDHs oxidise aldehydes to the corresponding carboxylic acids, using NAD or NADP as coenzymes, and they have been discovered in practically all organisms and found to exist in multiple isoforms [1 /3]. More than 160 ALDH cDNAs or genes have been isolated and sequenced from various sources: bacteria, yeast, fungi, plant and animals [4 /6]. In eukaryotes, 86 distinct cDNAs/genes of ALDH have been determined, and are divided into 18 families [7]. With regard to function, ALDHs exhibit a rather broad substrate specificity and many of them can oxidize several aliphatic and aromatic aldehydes [5]. A cytosolic form, ALDH3 (ALDH3A1), is highly expressed in the stomach, lung and cornea, but poorly expressed, if at all, in normal liver. In rat liver cells, cytosolic ALDH3 is induced by polycyclic aromatic hydrocarbons (PAH) or chlorinated compounds such as TCDD [8]. These compounds are ligands for the aryl hydrocarbon (Ah) receptor, a transcription factor that binds a consensus sequence, Ah responsive element (AhRE), in the promoter of target genes [9]. PAH and chlorinated compounds are an important class of environmental pollutants that produce a wide range of toxic and carcinogenic effects. Whereas in the liver ALDH3 is poorly expressed if at all, in hepatoma cells its expression increases in direct correlation with the degree of tumour deviation [10]. The increase of ALDH3 activity is evident in human and rat hepatoma cell lines, in hepatomas induced in rats by administration of chemical carcinogens [10,11], and in human hepatocellular carcinoma tissues from approximately 50% of patients with this form of cancer. The increase of ALDH3 expression in hepatoma cells is one cause of their resistance to drug toxicity and the consequent poor response to certain antitumour drugs. In particular, ALDH3 is responsible for cell tumour resistance to the oxazaphosphorine antineoplastic agents (eg.
cyclophosphamide). Sreerama and Sladek [12] report that interindividual variation in the activity of ALDH3 and ALDH1 in breast cancer cells could contribute to the very varied clinical response to cyclophosphamide. ALDH3 is also important in the metabolism of aldehydes derived from lipid peroxidation. Lipid peroxidation is a consequence of oxidative stress on polyunsaturated fatty acids and results in the formation of reactive by-products such as lipid hydroperoxides and aldehydes. The a,b-unsaturated aldehyde 4-hydroxynonenal is the most reactive and cytotoxic of the aldehyde by-products of lipid peroxidation [13]; it has been shown to have a variety of effects in biological systems, most often resulting in cytotoxicity. Hepatoma cells with a high ALDH3 content are more resistant to the cytostatic and cytotoxic effect of lipid peroxidation aldehydes than those with a low content [14]. As consequence of the above, inhibiting cytosolic ALDH3 may be a useful approach to increasing susceptibility of hepatoma cells to drugs and lipid peroxidation products, thereby reducing cell proliferation and inducing the normal phenotype. Reducing ALDH3 activity though inhibition by aldehydes generated by lipid peroxidation, specific ALDH3 inhibitors, or by ALDH3-specific antisense oligonucleotides (AS-ODN), hepatoma cells show decreased growth but not decreased cell viability in tumour cells with high ALDH3 activity [14 /17]. It remains open how cytosolic ALDH3 influences cell growth or the phenotype of hepatoma cells. To address this problem, we chose to investigate the expression of ALDH3 after treating hepatoma cells with clofibrate, a hypolipidemic drug that belongs to the peroxisome proliferator activated receptors (PPAR) ligands, since clofibrate has an inhibitory effect on the proliferation of hepatoma cells and influences ALDH. Clofibrate is known to induce the microsomal ALDH3 mRNA level in Hepa-1c1c7 hepatoma cells [18], to repress expression of mitochondrial ALDH2 in normal liver [19], and to cause an increase in liver ALDH activity located in peroxisomes and microsomes, whereas it does not alter the enzyme activity in the cytoplasmic fraction of liver [20].
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2. Materials and methods 2.1. Culture conditions and treatments JM2 rat hepatoma cells were seeded (20 000 cells/cm2) and maintained for 24 h in DMEM/F12 medium plus 2 mM glutamine, 1% antibiotic/ antimycotic solution and 10% newborn calf serum (medium A). Treatment with clofibrate: 24 h after cell seeding, clofibrate dissolved in DMSO was added at final concentrations of 0.25, 0.5 or 1 mM. Treatment with clofibrate and AS-ODN against PPARg: 24 h after cell seeding, medium A was removed and replaced with OPTI-MEM I medium supplemented with 5 nM AS-ODN prepared as follows. AS-ODN, phosphorothioated at their 5? and 3? terminus and complementary to the nucleotide sequences 1 /22 of rat PPARg (GenBank accession number AB011365), was used. ASODN (10 pmoles/100 ml sterile water) and lipofectin (10 ml) were diluted separately in 200 ml of OPTI-MEM I. Lipofectin was incubated for 45 min at room temperature, gently mixed with ASODN, maintained for 15 min at room temperature to allow formation of the AS-ODN/Lipofectin complex, and then diluted to 2 ml with OPTIMEM I. 24 h later, the medium supplemented with AS-ODN was replaced with fresh identical ASODN- supplemented medium, and 0.5 mM clofibrate was added to the cells. Control cells were grown in OPTI-MEM I medium supplemented with lipofectin and given solvent or AS-ODN alone. At the different experimental times (see figure legends) cells from each treatment were harvested and used for the following determinations. 2.2. Cell proliferation Cell proliferation was evaluated as the number of cells present in the monolayer and in the culture medium. 2.3. Northern blot and western blot analysis Northern blot and Western blot analysis was performed as described in Canuto et al. [14,16].
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Polyclonal anti-PPARa or anti-PPARg antibodies, and monoclonal anti-p-Erk1,2 or anti-c-myc antibody were used. The cDNA probe and the polyclonal antibody for ALDH3 were kind gifts from Dr R. Lindahl. 2.4. Aldehyde dehydrogenase activity ALDH3 activity was determined in cytosol obtained from JM2 hepatoma cells treated or not with 0.5 mM clofibrate. Preparation of the cytosol and determination of ALDH3 activity were as described in Canuto et al. [15] 2.5. Protein determination Protein content was determined by the biuret method [21]. 2.6. Statistical analysis All data were expressed as means9/S.D. The significance of differences between group means was assessed by variance analysis, followed by the Newman-Keuls test.
3. Results and discussion Our previous results showed that it is possible to reduce cell proliferation in hepatoma cells by increasing the products of lipid peroxidation, following enrichment of cells with arachidonic acid. We have also demonstrated that clofibrate will induce rapid and extensive apoptosis or inhibit proliferation of hepatoma cells ([14,22 /24], Canuto et al., unpublished). Both arachidonic acid and clofibrate are ligands for PPARs, which are nuclear receptors for several chemicals. PPARs are present in three different isotypes, PPARa, b/d, and g [25]. Emerging evidence indicates that PPAR ligands can suppress the growth of different types of cancer. In the case of arachidonic acid treatment, the reduction of cell proliferation parallels the reduction of cytosolic ALDH3 expression. The correlation between proliferation and cytosolic ALDH3 was confirmed by using specific inhibitors or AS-ODN against ALDH3 [15,16]. In the light
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of these results we decided to examine whether cytosolic ALDH3 activity was also lowered when cell proliferation was reduced by clofibrate. The mechanism underlying the possible inhibition of cytosolic ALDH3 activity was also investigated. The experiments were performed on JM2 hepatoma cell line, a highly deviated hepatoma as demonstrated by its high content of cytosolic ALDH3 and poor response to inducers of cytochrome P450 [10,26]. Clofibrate inhibited JM2 cell proliferation in a dose-dependent manner (Fig. 1). The growth inhibition was not due to cell death as demonstrated by our inability to detect cells detached from the monolayer at any concentration or at any time (data not shown). The decrease in cell proliferation was accompanied by a decrease in the activity of the MAPK pathway as evidenced by a significant decrease in the phosphorylated form of Erk1, 2 and by decreased c-myc levels, a consequence of reduced MAPK pathway activity [27] (Fig. 2). Cytosolic ALDH3 expression was evaluated to detect any correlation with clofibrateinduced changes in cell proliferation. ALDH3 mRNA content, as determined by Northern blot, decreased in the presence of clofibrate at 24 h of exposure (Fig. 3, lane 2), whereas ALDH3 enzyme activity was found to decrease at 48 h of clofibrate exposure (Table 1). The delayed decrease in ALDH3 enzyme activity relative to the mRNA
Fig. 1. Cell growth in JM2 hepatoma cells exposed to 0.25, 0.5 or 1 mM clofibrate. Values are means9/S.D. of four experiments. Means with different letters are statistically different (P B/0.001) from one another as determined by variance analysis followed by the Newman-Keuls test. C, control cells; Cl, cells treated with clofibrate.
Fig. 2. Protein content of PPARg, the phosphorylated form of Erk1,2, and c-myc in JM2 hepatoma cells exposed to clofibrate for 24 h. Lane 1, control cells; Lane 2, cells exposed to 0.25 mM clofibrate; Lane 3, cells exposed to 0.5 mM clofibrate.
Fig. 3. mRNA content of ALDH3 in JM2 hepatoma cells exposed to 0.5 mM clofibrate and AS-ODN against PPARg. Lanes 1, control cells; Lanes 2, cells exposed to 0.5 mM clofibrate; Lanes 3, cells exposed to AS-ODN against PPARg; Lanes 4, cells exposed to AS-ODN against PPARg and 0.5 mM clofibrate.
R.A. Canuto et al. / Chemico-Biological Interactions 143 /144 (2003) 29 /35
33
Table 1 ALDH3 activity in cytosol isolated from JM2 hepatoma cells treated with clofibrate 24 h
48 h
C
Cl 0.5 mM
C
Cl 0.5 mM
458
446
357.8
134.7
Data are expressed as nmol of NADPH produced/min/mg of protein and are the mean of two experiments. 2.5 mM benzaldehyde was used as substrate.
decrease is explained by the approximately 96 h half-life of the protein. This result prompted us to consider whether changes in cytosolic ALDH3 were correlated with cell proliferation, but not its primary cause. It might perhaps be better considered as linked to the phenotype changes. To understand the mechanisms underlying the decrease of ALDH3 expression, PPARa and g expression were determined. PPARa was not detected in untreated JM2 cells and did not change after clofibrate treatment (data not shown), whereas PPARg, which is present in untreated JM2 cells, significantly increased with clofibrate (Fig. 2). PPARa is especially involved in peroxisome proliferation and lipid metabolism, whereas PPARg is more closely linked to induction of differentiation and to the inhibition of cell proliferation. Treatment of JM2 hepatoma cells with another PP, arachidonic acid, also induced PPARg, but not PPARa [24]. The possible correlation between PPARg and cytosolic ALDH3 activity was investigated by using AS-ODN against PPARg mRNA. ASODN abolished the antiproliferative effect of clofibrate (Fig. 4) and at the same time also prevented the decrease in ALDH3 mRNA levels (Fig. 3). PPARg decreased in the presence of its AS-ODN and was not increased in the presence of clofibrate plus AS-ODN, the content being similar to that of control cells (Fig. 5). On the contrary, inhibiting ALDH3 by AS-ODN against ALDH3 did not change PPARg expression (data not shown). Since these results suggest that cytosolic ALDH3 expression is under PPAR control, we examined 8 kb of 5? flanking sequence of the
Fig. 4. Cell growth in JM2 hepatoma cells exposed to AS-ODN against PPARg. Values reported are numbers of cells in the monolayer, and are means9/S.D. of three experiments. Means with different letters are statistically different (P B/0.05) from one another as determined by variance analysis followed by the Newman-Keuls test. C, control cells; Cl, cells treated with 0.5 mM clofibrate; ODN, cells treated with 5 nM AS-ODN against PPARg.
Fig. 5. Protein content of PPARg in JM2 hepatoma cells exposed to clofibrate and to AS-ODN against PPARg. Lane 1, control cells; Lane 2, cells exposed to 0.5 mM clofibrate; Lane 3, cells exposed to AS-ODN against PPARg; Lane 3, cells exposed to AS-ODN against PPARg and to 0.5 mM clofibrate.
ALDH3 gene, but without finding any sequence similar to known peroxisome proliferator response element, as reported in Ref. [28]. Thus we believe that the effect of PPARg on ALDH3 expression occurs via other transcription factors that remain to be determined. The results indicate that, as in many other different tumour types, PPARg plays a key role in regulation of growth and differentiation of hepatoma cells, and that ALDH3 collaborates in modulating cell proliferation and in determining some aspects of the hepatoma phenotype, i.e. resistance to drugs and to cytostatic lipid peroxidation products. Therefore, it is likely that the growth inhibitory effect of clofibrate on tumour cells is associated with induction of a more
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differentiated phenotype. The ability of PPARg to control expression of genes involved in differentiation and cell proliferation suggests that PPARg ligands might be used as therapeutic agents in the treatment of tumours, and that the evaluation of cytosolic ALDH3 expression is a marker of differentiation of tumour cells derived from the liver.
[12]
[13]
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