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The effect of a novel irreversible inhibitor of aldehyde dehydrogenases 1 and 3 on tumour cell growth and death
Chemico-Biological Interactions 130 – 132 (2001) 209 – 218
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The effect of a novel irreversible inhibitor of aldehyde dehydrogenases 1 and 3 on tumour cell growth and death Rosa Angela Canuto a,*, Giuliana Muzio a, Raffaella A. Salvo a, Marina Maggiora a, Antonella Trombetta a, Jaqueline Chantepie b, Guy Fournet c, Uwe Reichert d, Gerard Quash b a
Department of Experimental Medicine and Oncology, Uni6ersity of Turin, Corso Raffaello 30, 10125 Turin, Italy b Laboratoire d’Immunochimie, Faculte´ de Me´decine Lyon Sud, Chemin du Petit Re6oyet-B.P. 12, 69921 Oullins, Cedex, France c Laboratorie de Chimie Organique 1, UMR, Villeurbanne, France d Galderma Research and De6elopment SNC, Valbonne, France
Abstract 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-2ynethioate, a synthetic suicide inhibitor of ALDH1, which induced bcl2 overexpressing cells into apoptosis and exhibited an ED50 of 400 mM, 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 mM on rat * Corresponding author. Tel.: + 39-11-6707781; fax: +39-11-6707753. E-mail address: [email protected] (R.A. Canuto). 0009-2797/01/$ - see front matter © 2001 Elsevier Science Ireland Ltd. All rights reserved. PII: S 0 0 0 9 - 2 7 9 7 ( 0 0 ) 0 0 2 8 0 - 5
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cultured hepatoma cells. Four hours after treatment with 25 mM 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. © 2001 Elsevier Science Ireland Ltd. All rights reserved. Keywords: Hepatoma cells; Aldehyde dehydrogenases; Chemical inhibitors
1. Introduction Aldehyde dehydrogenases (ALDHs) are a family of several isoenzymes, involved in the metabolism of exogenous or endogenous aldehydes — benzaldehyde, acetaldehyde, aldehydes derived from the lipid peroxidation process (for example, 4-hydroxynonenal), methional derived from methionine metabolism, and aldehydes derived from drugs that are metabolised by cells into active compounds (for example, cyclophosphamide and derivatives) [1–3]. ALDHs are, thus, involved in the detoxification of compounds that are toxic to the cell; moreover, they are involved in the activation of compounds that exert a beneficial effect on the cell, for example, retinals [1].
Fig. 1. Molecular structure of ALDH inhibitors. The molecules differ from each other in the substituent group in C4. Table 1 Aldehyde dehydrogenase activity in cytosol isolated from hepatocytes and hepatoma cell linesa Cells
BA
PA
Hepatocytes 7777 JM2
2.27 90.51a 1.23 90.19b 597.63 9 93.21c
2.33 90.17a 4.35 90.46b 41.73 96.89c
a Data are expressed as nmoles of NAD(P)H produced/min/mg of protein and represent mean9 S.D. of three experiments. Abbreviations — BA, benzaldehyde; PA, propionaldehyde. For each substrate, means with different letters are statistically different (PB0.001) from one another as determined by ANOVA followed by the Newman–Keuls test.
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Fig. 2. Effect of ATE on growth of JM2 hepatoma cells. Control cells were given an equal quantity of ethanol. Control cells and cells treated with ATE were harvested at the times indicated in the figure. Data are mean 9 S.D. of four experiments and are expressed as percentage of inhibition determined in cells treated with the inhibitor versus the control value. The S.Ds. are less than 10% of the means. The means with different letters are statistically different (PB 0.05) from one another as determined by ANOVA followed by the Newman–Keuls test. Inhibition of control cells is thus 0% (a).
In hepatoma cells, there is an increase in class 1 and 3 ALDH activity. The increase is correlated to the degree of cell deviation [3]. Previous research has shown that hepatoma cells with a high content of ALDH (i.e. JM2) are more resistant to the antiproliferative effect of lipid peroxidation aldehydes than those cells with a lower ALDH content (i.e. 7777). Restoring lipid peroxidation by enriching hepatoma cells with arachidonic acid and by exposing them to pro-oxidants causes a decrease in cell growth or cell death in 7777, both of which occur earlier and to a greater extent than in JM2 [4,5]. Enrichment with arachidonic acid is necessary to induce lipid peroxidation, since in basal conditions, hepatoma cells have a reduced content of arachidonic acid in membrane phospholipids. JM2 cells become susceptible to the toxic effects of aldehydes when ALDH 3 activitity is decreased by the aldehydes produced by the JM2 cells themselves [4,5]. Based on these results, we decided to inhibit ALDH activity in situ using specific chemical inhibitors and assess the effects of reduced ALDH activity on cell growth. 4-Amino-4-methyl-2-pentyne-1-al (ampal) and its derivative thioampal (ATE), two irreversible inhibitors of ALDH [6], were synthesised and their effect on tumour cells tested. Ampal has been demonstrated to inhibit cell growth of several
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transformed cell lines, such as glioblastoma U251, HeLa, L1210, without affecting normal human embryonic fibroblasts (MRC-5); ATE has been shown to inhibit cell growth of HeLa cells at a lower concentration than that of MRC-5 [7]. This paper evaluates the effect of ALDH inhibitors on the growth of hepatoma cells with high ALDH content. ATE, which is more effective than ampal, and two other newly synthesised inhibitors, also derived from ampal, were used. 2. Materials and methods
2.1. Culture conditions JM2 hepatoma cells were seeded (15 000 cells per cm2) and maintained for 24 h in medium A (DMEM/F12 plus 2 mM glutamine, 1% antibiotic/antimycotic solution) supplemented with 10% newborn calf serum; 24 h later, medium A was removed and replaced with medium B [medium A plus 0.4% of albumin, 1% ITS (insulin transferrin, sodium selenite), 1% nonessential amino acids, 1% vitamin solution] supplemented or not with the different inhibitors. The inhibitors were dissolved in absolute ethanol (maximum final concentration 0.5%) and added to the cells at different concentrations.
Fig. 3. Effect of ATE on ALDH activity determined in the cytosolic fractions of JM2 hepatoma cells. Presentation and statistical analysis of data as in Fig. 2.
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Fig. 4. Effect of ATEB on growth of JM2 hepatoma cells. Control cells and cells treated with ATEB were harvested at the times indicated in the figure. Presentation and statistical analysis of data as in Fig. 2.
At the different experimental times, cells were harvested and analysed for cell growth and enzyme activity. The 7777 hepatoma cells were seeded (20 000 cells per cm2) and maintained for 72 h in the medium A. At 72 h, cells were harvested and analysed for enzyme activity in the cytosolic fraction with benzaldehyde or propionaldehyde as substrate. Rat hepatocytes were isolated and harvested as described in Canuto et al. 1999 [3]. The enzyme activity was determined in the cytosolic fraction with benzaldehyde or propionaldehyde as substrate.
2.2. Cell growth Cell growth was evaluated as the number of cells present in the monolayer and in the culture medium. The percentage inhibition produced in cells treated with the inhibitor was calculated versus the control value.
2.3. Enzyme acti6ity determination ALDH activity was determined as described in Canuto et al. (1996) using benzaldehyde and NADP as substrates for ALDH3 and propionaldehyde and NAD for ALDH1[4]. The activity was determined in cytosolic fractions obtained as described in Canuto et al. 1999 [3]. The percentage inhibition produced in cells treated with the inhibitor was calculated versus the control value.
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2.4. Protein determination Proteins were determined by the biuret method [8].
2.5. Statistical analysis All data are expressed as mean9 S.D. The significance of differences between group means was assessed by the analysis of variance (ANOVA), followed by the Newman – Keuls test. Student t-test was used when the means of only two groups were compared. 3. Results and discussion Hepatoma cells show changes in ALDH isoenzyme expression in comparison with normal cells — an increase of cytosolic class 3 and class 1 ALDH (Table 1) and a decrease of mitochondrial ALDH 2 are evident [9]. The increase of ALDH
Fig. 5. Effect of ATEB on ALDH activity determined in the cytosolic fractions of JM2 hepatoma cells. Presentation and statistical analysis of data as in Fig. 2.
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Fig. 6. Effect of ATEM on growth of JM2 hepatoma cells. The numbers are the concentrations of ATEM expressed in mM. Presentation and statistical analysis of data as in Fig. 2.
3 and 1 is correlated to the degree of deviation; in the more deviated JM2 hepatoma cells, the ALDH activity is higher than in the less deviated 7777 hepatoma cells, with all substrates used (Table 1). 7777 hepatoma cells show ALDH activity values similar to those of hepatocytes. Based on this correlation, we believe that ALDH activity also correlated with cell growth and that its inhibition might inhibit cell growth. For this reason, several inhibitors were synthesised in order to find the inhibitor that is the most efficient against ALDH 1 or 3, that reduces growth of tumour cells, and, at the same time, the least toxic towards normal cells. JM2 hepatoma cell lines were used as hepatoma cells, because they have high content of ALDH 3 and also contain more ALDH 1 than the less deviated 7777 cells. Fig. 1 shows the molecular structure of the three inhibitors used, all derived from ampal, which is the first inhibitor synthesised against ALDH [6]. The three inhibitors differ from ampal in the substituent group at C1 and differ from each other for the substituent at C4. The first inhibitor, ATE, caused a decrease in the cell number in JM2 cells after 48 h at 500 mM concentration; inhibition was about 50%. After this time, the cell number tended to increase, however, it did not return to control levels. ALDH activity, using benzaldehyde as substrate, was inhibited by about 60% after 24 h of treatment. The inhibition was the same at 48 h, but at 72 h was decreased to about
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30% (Fig. 2). Activity with propionaldehyde was not inhibited significantly until 48 h of treatment and remained inhibited about 30% through 72 h (Fig. 2). With the second inhibitor, ATEB, cell growth inhibition was evident at 24 h after administration and increased thereafter (Fig. 3). The concentration used was as high as the concentration of the first inhibitor used, but growth inhibition was higher, being about 40% of control value at 24 h and increasing thereafter. Enzyme activity was also inhibited with both the substrates used, benzaldehyde and propionaldehyde, and the inhibition was about 90% with benzaldehyde at 24 h and remained constant thereafter (Fig. 4). The second inhibitor was more efficient against activity and cell growth, but the effective concentration remained too high. Therefore, a third inhibitor was synthesised, by changing the group at C4, and we found that this third molecule, ATEM, was more efficient than the former two. In fact, cell growth inhibition of about 50, 80 and 90% was achieved, respectively, at 24, 48 and 72 h after exposure to 25 mM ATEM (Figs. 5 and 6). 60% inhibition was also achieved after 72 h of treatment with 10 mM ATEM. The ALDH activity in cells treated with 25 mM ATEM was determined at 4 h and, there was a significant decrease in enzyme activity at this time (Fig. 7). The percentage inhibition was about 50%, using benzaldehyde or propionaldehyde as substrate. Importantly, inhibition of enzyme activity precedes the inhibition of cell growth. Both inhibitors, ATEB and ATEM, have the same effect on other types of tumour cells (data not shown).
Fig. 7. Effect of ATEM on ALDH activity determined in the cytosolic fractions of JM2 hepatoma cells at 4 h. * ‘Student t-test’, PB0.001 (treated cells versus control cells).
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In conclusion, inhibition of ALDH activity causes inhibition of hepatoma cell growth, and inhibition of either ALDH 3 or 1 or both is correlated with the ability of hepatoma cells to grow. The next step will be to find the substrates of ALDH able to interfere with hepatoma cell growth. At this time, we can hypothesise that two ALDH substrates may effect cell growth — methional, and/or 4-hydroxynonenal. The first is an intermediate of methionine metabolic pathway and has been shown to induce apoptosis in murine BAF3 lymphoid cells [10]; the second is a product of lipid peroxidation, and has been shown to inhibit cell growth in many different tumour cell lines [3,11,12].
Acknowledgements This study was supported by grants from the Italian Ministry for University, Scientific and Technological Research (60% and Cofinanziamento 1997, 1999).
References [1] R. Lindahl, Aldehyde dehydrogenases and their role in carcinogenesis, CRC Crit. Rev. Biochem. Mol. Biol. 27 (1992) 283–335. [2] V. Vasiliou, C. Kozak, R. Lindahl, D.W. Nebert, Mouse microsomal class 3 aldehyde dehydrogenase: ALDH3 cDNA sequence, inducibility by dioxine and clofibrate, and geneting mapping, DNA Cell Biol. 15 (1996) 235–245. [3] R.A. Canuto, M. Ferro, G. Muzio, A.M. Bassi, G. Leonarduzzi, M. Maggiora, D. Adamo, G. Poli, R. Lindahl, Role of aldehyde metabolizing enzymes in mediating effects of aldehyde products of lipid peroxidation in liver cells, Carcinogenesis 15 (1994) 1359 – 1364. [4] R.A. Canuto, M. Ferro, M. Maggiora, R. Federa, O. Brossa, A.M. Bassi, R. Lindahl, G. Muzio, In hepatoma cell lines restored lipid peroxidation affects cell viability inversely to aldehyde metabolizing enzyme activity, Adv. Exp. Med. Biol. 414 (1996) 113 – 122. [5] R.A Canuto, G. Muzio, M. Ferro, M. Maggiora, R. Federa, A.M. Bassi, R. Lindahl, M.U. Dianzani, Inhibition of class-3 aldehyde dehydrogenase and cell growth by restored lipid peroxidation in hepatoma cell lines, Free Radic. Biol. Med. 26 (1999) 333 – 340. [6] V. Quemener, J.P. Moulinoux, C. Martin, F. Darsel, W. Guegan, J. Faivre, G. Quash, dehydrogenase activity in xenografted human brain tumor in nude mice. Preliminary results in human glioma biopsies, J. Neuro-Oncol. 9 (1990) 115 – 123. [7] G. Quash, G. Fournet, C. Raffin, J. Chantepie, Y. Michal, J. Gore, U. Reichert, A thioesther analogue of an aminoacetylenic aldehyde is a suicide inhibitor of aldehyde dehydrogenase and an inducer of apoptosis in mouse lymphoid cells overexpressing the bcl-2 gene, Adv. Exp. Med. Biol. 463 (1999) 97–106. [8] A.G. Gornall, C.J. Bardawill, M. David, Determination of serum proteins by means of the biuret reaction, J. Biol. Chem. 177 (1949) 751 – 766. [9] R.A. Canuto, M. Ferro, R.A. Salvo, A.M. Bassi, M. Terreno, M.U. Dianzani, R. Lindahl, G. Muzio, Effect of arachidonic acid alone or with prooxidant on aldehyde dehydrogenases in hepatoma cells, Adv. Exp. Med. Biol. 463 (1999) 133 – 142. [10] A.M. Roch, G. Panaye, Y. Michal, G. Quash, Methional, a cellular metabolite, induces apoptosis preferentially in G2/M-synchronized BAF3 murine lymphoid cells, Cytometry 31 (1998) 10 – 19.
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[11] G. Barrera, C. Di Mauro, R. Muraca, D. Ferrero, G. Cavalli, V.M. Fazio, L. Paradisi, M.U. Dianzani, Induction of differentiation in human HL-60 cells by 4-hydroxy-nonenal, a product of lipid peroxidation, Exp. Cell Res. 197 (1991) 148 – 152. [12] G. Barrera, R. Muraca, S. Pizzimenti, A. Serra, C. Rosso, G. Saglio, M.G. Farace, V. Fazio, M.U. Dianzani, Inhibition of c-myc expression induced by 4-hydroxy-nonenal, a product of lipid peroxidation, in the HL-60 human leukemic cell line, Biochem. Biophys. Res. Commun. 203 (1994) 553–561.
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The effect of a novel irreversible inhibitor of aldehyde dehydrogenases 1 and 3 on tumour cell growth and death Rosa Angela Canuto a,*, Giuliana Muzio a, Raffaella A. Salvo a, Marina Maggiora a, Antonella Trombetta a, Jaqueline Chantepie b, Guy Fournet c, Uwe Reichert d, Gerard Quash b a
Department of Experimental Medicine and Oncology, Uni6ersity of Turin, Corso Raffaello 30, 10125 Turin, Italy b Laboratoire d’Immunochimie, Faculte´ de Me´decine Lyon Sud, Chemin du Petit Re6oyet-B.P. 12, 69921 Oullins, Cedex, France c Laboratorie de Chimie Organique 1, UMR, Villeurbanne, France d Galderma Research and De6elopment SNC, Valbonne, France
Abstract 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-2ynethioate, a synthetic suicide inhibitor of ALDH1, which induced bcl2 overexpressing cells into apoptosis and exhibited an ED50 of 400 mM, 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 mM on rat * Corresponding author. Tel.: + 39-11-6707781; fax: +39-11-6707753. E-mail address: [email protected] (R.A. Canuto). 0009-2797/01/$ - see front matter © 2001 Elsevier Science Ireland Ltd. All rights reserved. PII: S 0 0 0 9 - 2 7 9 7 ( 0 0 ) 0 0 2 8 0 - 5
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cultured hepatoma cells. Four hours after treatment with 25 mM 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. © 2001 Elsevier Science Ireland Ltd. All rights reserved. Keywords: Hepatoma cells; Aldehyde dehydrogenases; Chemical inhibitors
1. Introduction Aldehyde dehydrogenases (ALDHs) are a family of several isoenzymes, involved in the metabolism of exogenous or endogenous aldehydes — benzaldehyde, acetaldehyde, aldehydes derived from the lipid peroxidation process (for example, 4-hydroxynonenal), methional derived from methionine metabolism, and aldehydes derived from drugs that are metabolised by cells into active compounds (for example, cyclophosphamide and derivatives) [1–3]. ALDHs are, thus, involved in the detoxification of compounds that are toxic to the cell; moreover, they are involved in the activation of compounds that exert a beneficial effect on the cell, for example, retinals [1].
Fig. 1. Molecular structure of ALDH inhibitors. The molecules differ from each other in the substituent group in C4. Table 1 Aldehyde dehydrogenase activity in cytosol isolated from hepatocytes and hepatoma cell linesa Cells
BA
PA
Hepatocytes 7777 JM2
2.27 90.51a 1.23 90.19b 597.63 9 93.21c
2.33 90.17a 4.35 90.46b 41.73 96.89c
a Data are expressed as nmoles of NAD(P)H produced/min/mg of protein and represent mean9 S.D. of three experiments. Abbreviations — BA, benzaldehyde; PA, propionaldehyde. For each substrate, means with different letters are statistically different (PB0.001) from one another as determined by ANOVA followed by the Newman–Keuls test.
R.A. Canuto et al. / Chemico-Biological Interactions 130–132 (2001) 209–218
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Fig. 2. Effect of ATE on growth of JM2 hepatoma cells. Control cells were given an equal quantity of ethanol. Control cells and cells treated with ATE were harvested at the times indicated in the figure. Data are mean 9 S.D. of four experiments and are expressed as percentage of inhibition determined in cells treated with the inhibitor versus the control value. The S.Ds. are less than 10% of the means. The means with different letters are statistically different (PB 0.05) from one another as determined by ANOVA followed by the Newman–Keuls test. Inhibition of control cells is thus 0% (a).
In hepatoma cells, there is an increase in class 1 and 3 ALDH activity. The increase is correlated to the degree of cell deviation [3]. Previous research has shown that hepatoma cells with a high content of ALDH (i.e. JM2) are more resistant to the antiproliferative effect of lipid peroxidation aldehydes than those cells with a lower ALDH content (i.e. 7777). Restoring lipid peroxidation by enriching hepatoma cells with arachidonic acid and by exposing them to pro-oxidants causes a decrease in cell growth or cell death in 7777, both of which occur earlier and to a greater extent than in JM2 [4,5]. Enrichment with arachidonic acid is necessary to induce lipid peroxidation, since in basal conditions, hepatoma cells have a reduced content of arachidonic acid in membrane phospholipids. JM2 cells become susceptible to the toxic effects of aldehydes when ALDH 3 activitity is decreased by the aldehydes produced by the JM2 cells themselves [4,5]. Based on these results, we decided to inhibit ALDH activity in situ using specific chemical inhibitors and assess the effects of reduced ALDH activity on cell growth. 4-Amino-4-methyl-2-pentyne-1-al (ampal) and its derivative thioampal (ATE), two irreversible inhibitors of ALDH [6], were synthesised and their effect on tumour cells tested. Ampal has been demonstrated to inhibit cell growth of several
212
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transformed cell lines, such as glioblastoma U251, HeLa, L1210, without affecting normal human embryonic fibroblasts (MRC-5); ATE has been shown to inhibit cell growth of HeLa cells at a lower concentration than that of MRC-5 [7]. This paper evaluates the effect of ALDH inhibitors on the growth of hepatoma cells with high ALDH content. ATE, which is more effective than ampal, and two other newly synthesised inhibitors, also derived from ampal, were used. 2. Materials and methods
2.1. Culture conditions JM2 hepatoma cells were seeded (15 000 cells per cm2) and maintained for 24 h in medium A (DMEM/F12 plus 2 mM glutamine, 1% antibiotic/antimycotic solution) supplemented with 10% newborn calf serum; 24 h later, medium A was removed and replaced with medium B [medium A plus 0.4% of albumin, 1% ITS (insulin transferrin, sodium selenite), 1% nonessential amino acids, 1% vitamin solution] supplemented or not with the different inhibitors. The inhibitors were dissolved in absolute ethanol (maximum final concentration 0.5%) and added to the cells at different concentrations.
Fig. 3. Effect of ATE on ALDH activity determined in the cytosolic fractions of JM2 hepatoma cells. Presentation and statistical analysis of data as in Fig. 2.
R.A. Canuto et al. / Chemico-Biological Interactions 130–132 (2001) 209–218
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Fig. 4. Effect of ATEB on growth of JM2 hepatoma cells. Control cells and cells treated with ATEB were harvested at the times indicated in the figure. Presentation and statistical analysis of data as in Fig. 2.
At the different experimental times, cells were harvested and analysed for cell growth and enzyme activity. The 7777 hepatoma cells were seeded (20 000 cells per cm2) and maintained for 72 h in the medium A. At 72 h, cells were harvested and analysed for enzyme activity in the cytosolic fraction with benzaldehyde or propionaldehyde as substrate. Rat hepatocytes were isolated and harvested as described in Canuto et al. 1999 [3]. The enzyme activity was determined in the cytosolic fraction with benzaldehyde or propionaldehyde as substrate.
2.2. Cell growth Cell growth was evaluated as the number of cells present in the monolayer and in the culture medium. The percentage inhibition produced in cells treated with the inhibitor was calculated versus the control value.
2.3. Enzyme acti6ity determination ALDH activity was determined as described in Canuto et al. (1996) using benzaldehyde and NADP as substrates for ALDH3 and propionaldehyde and NAD for ALDH1[4]. The activity was determined in cytosolic fractions obtained as described in Canuto et al. 1999 [3]. The percentage inhibition produced in cells treated with the inhibitor was calculated versus the control value.
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2.4. Protein determination Proteins were determined by the biuret method [8].
2.5. Statistical analysis All data are expressed as mean9 S.D. The significance of differences between group means was assessed by the analysis of variance (ANOVA), followed by the Newman – Keuls test. Student t-test was used when the means of only two groups were compared. 3. Results and discussion Hepatoma cells show changes in ALDH isoenzyme expression in comparison with normal cells — an increase of cytosolic class 3 and class 1 ALDH (Table 1) and a decrease of mitochondrial ALDH 2 are evident [9]. The increase of ALDH
Fig. 5. Effect of ATEB on ALDH activity determined in the cytosolic fractions of JM2 hepatoma cells. Presentation and statistical analysis of data as in Fig. 2.
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215
Fig. 6. Effect of ATEM on growth of JM2 hepatoma cells. The numbers are the concentrations of ATEM expressed in mM. Presentation and statistical analysis of data as in Fig. 2.
3 and 1 is correlated to the degree of deviation; in the more deviated JM2 hepatoma cells, the ALDH activity is higher than in the less deviated 7777 hepatoma cells, with all substrates used (Table 1). 7777 hepatoma cells show ALDH activity values similar to those of hepatocytes. Based on this correlation, we believe that ALDH activity also correlated with cell growth and that its inhibition might inhibit cell growth. For this reason, several inhibitors were synthesised in order to find the inhibitor that is the most efficient against ALDH 1 or 3, that reduces growth of tumour cells, and, at the same time, the least toxic towards normal cells. JM2 hepatoma cell lines were used as hepatoma cells, because they have high content of ALDH 3 and also contain more ALDH 1 than the less deviated 7777 cells. Fig. 1 shows the molecular structure of the three inhibitors used, all derived from ampal, which is the first inhibitor synthesised against ALDH [6]. The three inhibitors differ from ampal in the substituent group at C1 and differ from each other for the substituent at C4. The first inhibitor, ATE, caused a decrease in the cell number in JM2 cells after 48 h at 500 mM concentration; inhibition was about 50%. After this time, the cell number tended to increase, however, it did not return to control levels. ALDH activity, using benzaldehyde as substrate, was inhibited by about 60% after 24 h of treatment. The inhibition was the same at 48 h, but at 72 h was decreased to about
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30% (Fig. 2). Activity with propionaldehyde was not inhibited significantly until 48 h of treatment and remained inhibited about 30% through 72 h (Fig. 2). With the second inhibitor, ATEB, cell growth inhibition was evident at 24 h after administration and increased thereafter (Fig. 3). The concentration used was as high as the concentration of the first inhibitor used, but growth inhibition was higher, being about 40% of control value at 24 h and increasing thereafter. Enzyme activity was also inhibited with both the substrates used, benzaldehyde and propionaldehyde, and the inhibition was about 90% with benzaldehyde at 24 h and remained constant thereafter (Fig. 4). The second inhibitor was more efficient against activity and cell growth, but the effective concentration remained too high. Therefore, a third inhibitor was synthesised, by changing the group at C4, and we found that this third molecule, ATEM, was more efficient than the former two. In fact, cell growth inhibition of about 50, 80 and 90% was achieved, respectively, at 24, 48 and 72 h after exposure to 25 mM ATEM (Figs. 5 and 6). 60% inhibition was also achieved after 72 h of treatment with 10 mM ATEM. The ALDH activity in cells treated with 25 mM ATEM was determined at 4 h and, there was a significant decrease in enzyme activity at this time (Fig. 7). The percentage inhibition was about 50%, using benzaldehyde or propionaldehyde as substrate. Importantly, inhibition of enzyme activity precedes the inhibition of cell growth. Both inhibitors, ATEB and ATEM, have the same effect on other types of tumour cells (data not shown).
Fig. 7. Effect of ATEM on ALDH activity determined in the cytosolic fractions of JM2 hepatoma cells at 4 h. * ‘Student t-test’, PB0.001 (treated cells versus control cells).
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In conclusion, inhibition of ALDH activity causes inhibition of hepatoma cell growth, and inhibition of either ALDH 3 or 1 or both is correlated with the ability of hepatoma cells to grow. The next step will be to find the substrates of ALDH able to interfere with hepatoma cell growth. At this time, we can hypothesise that two ALDH substrates may effect cell growth — methional, and/or 4-hydroxynonenal. The first is an intermediate of methionine metabolic pathway and has been shown to induce apoptosis in murine BAF3 lymphoid cells [10]; the second is a product of lipid peroxidation, and has been shown to inhibit cell growth in many different tumour cell lines [3,11,12].
Acknowledgements This study was supported by grants from the Italian Ministry for University, Scientific and Technological Research (60% and Cofinanziamento 1997, 1999).
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