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Anti-tumor-promoting effects of glycoglycerolipid analogues on two-stage mouse skin carcinogenesis
Cancer Letters 161 (2000) 201±205
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Anti-tumor-promoting effects of glycoglycerolipid analogues on two-stage mouse skin carcinogenesis Diego Colombo a,*, Federica Compostella a, Fiamma Ronchetti a, Antonio Scala a, Lucio Toma b, Masashi Kuchide c, Harukuni Tokuda c, Hoyoku Nishino c a
Dipartimento di Chimica e Biochimica Medica, UniversitaÁ di Milano, Via Saldini 50, 20133 Milan, Italy b Dipartimento di Chimica Organica, UniversitaÁ di Pavia, Via Taramelli 10, 27100 Pavia, Italy c Department of Biochemistry, Kyoto Prefectural University of Medicine, Kamigyo-ku, Kyoto 602, Japan Received 28 July 2000; received in revised form 17 September 2000; accepted 18 September 2000
Abstract Four glycoglycerolipid analogues, 1-O-hexanoyl-2-O-b -d-glucopyranosyl-sn-glycerol (1), 1-O-hexanoyl-2-O-b -d-galactopyranosyl-sn-glycerol (2), 2-O-(6-O-hexanoyl-b -d-galactopyranosyl)-sn-glycerol (3) and 2-O-(6-O-hexanoyl-a -d-galacto pyranosyl)-sn-glycerol (4), potent in vitro inhibitors of 12-O-tetradecanoylphorbol-13-acetate (TPA) induced Epstein±Barr virus early antigen (EBV-EA) activation, were submitted to an in vivo two-stage mouse skin carcinogenesis test, using dimethylbenz[a]anthracene (DMBA) and TPA. The study was extended to two deacylated galactosylglycerol structures, 1-O-b -d-galactopyranosyl-sn-glycerol (5) and 3-O-b -d-galactopyranosyl-sn-glycerol (6). All the tested compounds exhibited remarkable anti-tumor-promoting effects on mouse skin tumor promotion, the 1-hexanoate 2 being the most active among the glycoglycerolipids until now studied. q 2000 Elsevier Science Ireland Ltd. All rights reserved. Keywords: Glycoglycerolipids analogues; Skin tumor, Anti-tumor-promoting effect
1. Introduction Cancer chemoprevention is an emerging ®eld for cancer control. In general, when new compounds are evaluated as chemopreventors, preliminary screenings in vitro are performed, which are followed, for the compounds able to prevent more strongly the tumor-promoting step, by in vivo assays. A large number of natural and synthetic glycoglycerolipids have been assayed in in vitro tests [1±5], showing remarkable inhibitory effects on the activation of the Epstein±Barr virus early antigen (EBV* Corresponding author. Tel.: 139-02-70645230; fax: 139-0270645221. E-mail address: [email protected] (D. Colombo).
EA) induced by the tumor promoter 12-O-tetradecanoylphorbol-13-acetate (TPA); conversely, to our knowledge only one of the natural compounds was examined in vivo [4]. In the last few years, in order to enhance the antitumor-promoting activity of this class of compounds, we prepared synthetic analogues structurally related to the natural products. They differed in several structural features, namely the sugar moiety, the position of the glycerol linkage to the sugar, the length and location of the acyl chain, and the anomeric con®guration of the sugar [6±9]. Primary in vitro studies [10±13] of the inhibitory effects on Epstein±Barr virus early antigen (EBV± EA) activation induced by TPA in Raji cells of
0304-3835/00/$ - see front matter q 2000 Elsevier Science Ireland Ltd. All rights reserved. PII: S 0304-383 5(00)00610-8
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D. Colombo et al. / Cancer Letters 161 (2000) 201±205
these synthetic analogues revealed that they all showed a signi®cant activity, which was modulated by the structural characteristics, in particular by the length of the acyl chain: the best activity being reached in the presence of a hexanoyl chain on one of the primary hydroxyl groups [11]. 1-O-Hexanoyl-2-O-b -d-glucopyranosyl-sn-glycerol (1), 1-O-hexanoyl-2-O-b -d-galactopyranosyl-snglycerol (2), 2-O-(6-O-hexanoyl-b -d-galactopyranosyl)-sn-glycerol (3) resulted the most active among compounds of b anomeric con®guration [11], and comparable activity showed 2-O-(6-O-hexanoyl-a d- g alacto p y r an o syl )-s n-gl ycerol ( 4 ) a mong compounds of a anomeric con®guration [8]. Moreover, among non acylated glycosylglycerols, 1-O-b d-galactopyranosyl-sn-glycerol (5) showed the highest activity [13]. These promising in vitro results prompted us to assay the inhibitory effect of the synthetic glycoglycerolipids analogues 1±4 on mouse skin tumor promotion in an in vivo two-stage mouse skin carcinogenesis test and in this paper we report the results. Moreover, as also compounds lacking the acyl chain resulted active in the in vitro tests, though to a lesser extent than the hexanoates, the study was extended to the deacylated compounds 1-O- and 3-O-b -d-galactopyranosyl-sn-glycerol (5) and (6), the latter showing the skeleton of the natural galactoglycerolipids.
2. Materials and methods 2.1. Test products Glycoglycerolipid analogues 1±3 were synthesized according to Colombo et al. [6,7]. Compound 4 (mp. 788C; [a ]D20 1 98 (c 1, methanol); 1H-NMR and mass spectrometry data consistent with its structure) was synthesized according to Colombo et al. [8]. 1-Oand 3-O-b -d-galactopyranosyl-sn-glycerol (5) and (6) were prepared as described in ref [9]. 2.2. Short term in vitro bioassay for anti-tumor promoters The inhibitions, determined using a short term in vitro test for Epstein±Barr virus activation in Raji cells induced by 12-O-tetradecanoylphorbol-13-acetate (TPA), are described in refs [10,11] for compounds 1±3, and in Ref. [13] for compounds 5 and 6. Compound 4 [8] completely inhibited EBV activation at a concentration of 1 £ 103 molar ratio toward TPA (32 pmol) and showed an inhibitory effect on EBV activation of 10:7 ^ 1:2 (percentage to positive control ^ standard error) at a concentration of 5 £ 10 2 mol ratio/TPA concentration, whereas no cytotoxicity was exhibited by the sample at the same concentrations. 2.3. In vivo two-stage mouse skin carcinogenesis test Female ICR mice were obtained at 5±6 weeks of age from SLC Co. Ltd. (Shizuoka, Japan). Groups of animals (15 animals per group) were housed in bunches of ®ve in polycarbonate cages and given food ad libitum. Mice were permitted free access to MP solid diet (Oriental yeast Co., Ltd. Chiba, Japan) and drinking water at all times during the study. The back of each mouse was shaved with surgical clippers before the ®rst day of initiation. The mice were initiated with dimethylbenz[a]anthracene (DMBA; 390 nmol) in acetone (0.1 ml). One week after initiation, they were promoted twice a week by application of TPA (1.7 nmol) in acetone (0.1 ml). The mice were treated with the test compounds (85 nmol, 50 mol ratio/TPA) in acetone (0.1 ml) 1 h before each TPA treatment. The incidence of papillomas was observed weekly for 20 weeks. The differences in mouse skin papillomas between control and experiments were
D. Colombo et al. / Cancer Letters 161 (2000) 201±205
203
Fig. 1. Inhibitory effects of compounds 1±6 (85 nmol) on DMBA-TPA mouse skin carcinogenesis. All mice were initiated with DMBA (390 nmol) and promoted with TPA (1.7 nmol) twice a week starting 1 week after initiation. (A and C) percentage of mice with papillomas; (B and D) averaged number of papillomas per mouse (X TPA alone; W TPA 1 1; A TPA 1 2; O TPA 1 3; B TPA 1 4; w TPA 1 5; V TPA 1 6). At 20 weeks of promotion the averaged number of papillomas per mouse was reduced, with respect to the control group, by 56, 71, 64, 49, 57 and 46% (P , 0:001 in all the cases), respectively for 1±6.
analyzed by means of the Student's t-test at 20 weeks of promotion, whereas the differences in tumor bearing mice were analyzed by means of the x 2-test. After 20 weeks of promotion, there was no statistically signi®cant difference in body weights between control and any treated group (control: 54 ^ 2:8 g, treated: 56 ^ 2:3 g at end of experiment). 3. Results and discussion Four synthetic equiactive glycoglycerolipid analogues 1±4, which showed a signi®cant inhibitory activ-
ity in previously performed in vitro assays of TPAinduced EBV-EA activation, were submitted to an in vivo two-stage carcinogenesis test of mouse skin papillomas using DMBA as an initiator and TPA as a promoter, in order to evaluate their inhibitory effects. The assays were performed also on deacylated 1-O- and 3-O-galactosylglycerols 5 and 6, the former being the most active among the studied glycosylglycerols, the latter showing the basic skeleton of natural galactoglycerolipids. The activities, evaluated by both the incidence (percentage of mice bearing papillomas) (Fig. 1A,C and Table 1) and the multiplicity (average numbers of
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papillomas per mouse) (Fig. 1B,D and Table 1), were compared with those of the control group. As shown in Fig. 1, in the control, 100% of the mice bore papillomas even at only 10 weeks of promotion, and about nine papillomas were formed per mouse after 20 weeks of promotion. On the other hand, when compounds 1±6 were applied before each TPA treatment, they all delayed the formation of papillomas and lowered the number of papillomas per mouse, being 2 the most potent compound. In fact while in each group treated with compounds 1, 3, 4 and 6, at 10 weeks of promotion mice bore papillomas in a percentage from 33 to 53%, these percentages were of about 12% in the case of compounds 2 and 5; at 20 weeks of promotion only compound 2 showed about 53% of papillomas incidence, which varied from 79 to 94% for the other compounds (Fig. 1A,C). Compound 2 was the most effective also in reducing the number of papillomas per mouse, which after 10 weeks of promotion were about 0.5 for compounds 2 and 5 and 1±2 for the other compounds, while after 20 weeks of promotion they were suppressed with respect to control by 71% for compound 2 and by 46±64% for the other compounds (Fig. 1B,D). The decrease of the number of papillomas per mouse was signi®cant in the treated cases with respect to the control; in fact, in all the cases P , 0:001 (Student's t-test). Also, the differences among the treatment groups were signi®cant: 2 with respect to 1 (P , 0:002), to 3 (P , 0:1), to 4 (P , 0:001), to 5 (P , 0:005) and to 6 (P , 0:001). Similarly, 5 resulted better than 6 (P , 0:02). Less signi®cant appear the differences in the incidence of papillomas analyzed by the x 2±test; however compound 2 resulted, after 20 weeks, signi®cantly Table 1 Final papilloma incidence and multiplicity for compounds 1±6
TPA alone TPA 1 1 TPA 1 2 TPA 1 3 TPA 1 4 TPA 1 5 TPA 1 6
Papilloma incidence (%)
Papilloma multiplicity (m ^ SD)
100 80 53 80 87 93 93
9.1 ^ 1.6 4.0 ^ 0.9 2.6 ^ 1.2 3.3 ^ 0.9 4.6 ^ 1.1 3.9 ^ 1.1 4.9 ^ 0.8
different from the control (P , 0:005) and superior to the other compounds. It is noteworthy that 1-O-hexanoyl-2-O-b -d-galactopyranosyl-sn-glycerol (2), which shows in vitro an inhibitory effect comparable to that of compounds 1, 3 and 4, displays a higher potency in the in vivo twostage carcinogenesis, whereas in the case of the deacylated compounds 5 and 6, the in vitro experiments are con®rmed, being 1-O-b -D-galactopyranosyl-sn-glycerol (5) more active than its 3-Odiastereoisomer. Until now, only one example of an in vivo study appeared in the literature [4], which describes the anti-tumor-promoting activity on mouse skin of a natural glycoglycerolipid (DLGG) (DLGG 1,2-di-O-a -linolenoyl-3-O-b -d-galactopyranosyl-snglycerol) from Citrus hystrix. Interestingly, the synthetic analogue 2 here reported shows higher activity than the potent natural compound DLGG, because it displays an inhibitory effect similar to DLGG when its concentration is about half [4]. At present, the mechanism of glycoglycerolipids inhibitory effect on tumor promoting activity is not known; the inhibitors probably interfere with the TPA-induced promotional events, interacting with the receptor of tumor promoters protein kinase C [14,15]. The study of the structure-activity relationship may provide some insight; in particular, from this in vivo study we can conclude that the presence of the hexanoyl moiety at the 1-position of the glycerol backbone and the axial con®guration of the hydroxyl in the 4-position of the sugar are important for a remarkable activity. Acknowledgements This study was supported in part by Ministero dell UniversitaÁ e della Ricerca Scienti®ca e Tecnologica Rome, Italy (PRIN99: Chemistry of Bioactive Organic Compounds) and University of Milan, Italy and in part by Grants-in-Aid from the Ministry of Education, Science and Culture, and Ministry of Health and Welfare Japan. References [1] A. Nagatsu, M. Watanabe, K. Ikemoto, M. Hashimoto, N.
D. Colombo et al. / Cancer Letters 161 (2000) 201±205
[2]
[3]
[4]
[5]
[6]
[7]
Murakami, J. Sakakibara, H. Tokuda, H. Nishino, A. Iwashima, K. Yazawa, Synthesis and structure-anti-tumorpromoting activity relationship of monogalactosyl diacylglycerols, Bioorg. Med. Chem. Lett. 4 (1994) 1619±1622. H. Shirahashi, N. Murakami, M. Watanabe, A. Nagatsu, J. Sakakibara, H. Tokuda, H. Nishino, A. Iwashima, Glycolipids. VIII. Isolation and identi®cation of antitumor-promoting principles from the fresh-water cyanobacterium Phormidium tenue, Chem. Pharm. Bull. 41 (1993) 1664±1666. H. Shirahashi, T. Morimoto, A. Nagatsu, N. Murakami, K. Tatta, J. Sakakibara, H. Tokuda, H. Nishino, Antitumorpromoting activities of various synthetic 1-O-acyl-3-O-(6 0 O-acyl-b-d-galactopyranosyl)-sn-glycerols related to natural product from freshwater cyanobacterium Anabaena ¯os-aquae f.¯os-aquae, Chem. Pharm. Bull. 44 (1996) 1404±1406. N. Murakami, Y. Nakamura, K. Koshimizu, H. Ohigashi, Glyceroglycolipids from Citrus hystrix, a traditional herb in Thailand, potently inhibit the tumor-promoting activity of 12O-tetradecanoylphorbol 13-acetate in mouse skin, J. Agric. Food Chem. 43 (1995) 2779±2783. T. Morimoto, A. Nagatsu, N. Murakami, J. Sakakibara, H. Tokuda, H. Nishino, A. Iwashima, Anti-tumour-promoting glyceroglycolipids from the green alga, Chlorella vulgaris, Phytochemistry 40 (1995) 1433±1437. D. Colombo, F. Ronchetti, A. Scala, I.M. Taino, L. Toma, A facile lipase catalyzed access to fatty acid monoesters of 2-Ob-d-glucosylglycerol, Tetrahedron: Asymmetry 7 (1996) 771±777. D. Colombo, F. Ronchetti, A. Scala, L. Toma, Bioactive glycoglycerolipid analogues: an expeditious enzymatic approach to mono and diesters of 2-O-b-d-galactosylglycerol, Tetrahedron: Asymmetry 9 (1998) 2113±2119.
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[8] D. Colombo, F. Compostella, F. Ronchetti, A. Scala, L. Toma, H. Tokuda, H. Nishino, Glycoglycerolipid analogues active as anti-tumor-promoters: the in¯uence of the anomeric con®guration, Eur. J. Med. Chem. (2000) in press. [9] D. Colombo, F. Ronchetti, A. Scala, I.M. Taino, F.M. Albini, L. Toma, Optically pure 1-O- and 3-O-b-d-glucosyl and galactosylglycerols through lipase-catalyzed transformations, Tetrahedron Lett. 36 (1995) 4865±4868. [10] D. Colombo, A. Scala, I.M. Taino, L. Toma, F. Ronchetti, H. Tokuda, H. Nishino, A. Nagatsu, J. Sakakibara, Inhibitory effects of fatty acid monoesters of 2-O-b-d-glucosylglycerol on Epstein±Barr virus activation, Cancer Lett. 123 (1998) 83± 86. [11] D. Colombo, F. Compostella, F. Ronchetti, A. Scala, L. Toma, T. Mukainaka, A. Nagatsu, T. Konoshima, H. Tokuda, H. Nishino, Inhibitory effects of monoacylated 2-O-b-galactosylglycerols on Epstein±Barr virus activation: the signi®cant role of the hexanoyl chain, Cancer Lett. 143 (1999) 1±4. [12] D. Colombo, F. Compostella, F. Ronchetti, A. Scala, L. Toma, H. Tokuda, H. Nishino, Chemoenzymatic synthesis and antitumor promoting activity of 6 0 - and 3-esters of 2-O-b-dglucosylglycerol, Bioorg. Med. Chem. 7 (1999) 1867±1871. [13] D. Colombo, A. Scala, I.M. Taino, L. Toma, F. Ronchetti, 1-O-, 2-O- and 3-O-b-Glycosyl-sn-glycerols: structure-antitumor-promoting activity relationship, Bioorg. Med. Chem. Lett. 6 (1996) 1187±1190. [14] Y. Nishizuka, Studies and perspectives of protein kinase C, Science 233 (1986) 305±312. [15] J.A. Bomser, K.W. Singletary, B. Meline, Inhibition of 12-Otetradecanoylphorbol-13-acetate (TPA)-induced mouse skin ornitine decarboxylase and protein kinase C by polyphenolics from grapes, Chem. Biol. Interact. 127 (2000) 45±59.
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Anti-tumor-promoting effects of glycoglycerolipid analogues on two-stage mouse skin carcinogenesis Diego Colombo a,*, Federica Compostella a, Fiamma Ronchetti a, Antonio Scala a, Lucio Toma b, Masashi Kuchide c, Harukuni Tokuda c, Hoyoku Nishino c a
Dipartimento di Chimica e Biochimica Medica, UniversitaÁ di Milano, Via Saldini 50, 20133 Milan, Italy b Dipartimento di Chimica Organica, UniversitaÁ di Pavia, Via Taramelli 10, 27100 Pavia, Italy c Department of Biochemistry, Kyoto Prefectural University of Medicine, Kamigyo-ku, Kyoto 602, Japan Received 28 July 2000; received in revised form 17 September 2000; accepted 18 September 2000
Abstract Four glycoglycerolipid analogues, 1-O-hexanoyl-2-O-b -d-glucopyranosyl-sn-glycerol (1), 1-O-hexanoyl-2-O-b -d-galactopyranosyl-sn-glycerol (2), 2-O-(6-O-hexanoyl-b -d-galactopyranosyl)-sn-glycerol (3) and 2-O-(6-O-hexanoyl-a -d-galacto pyranosyl)-sn-glycerol (4), potent in vitro inhibitors of 12-O-tetradecanoylphorbol-13-acetate (TPA) induced Epstein±Barr virus early antigen (EBV-EA) activation, were submitted to an in vivo two-stage mouse skin carcinogenesis test, using dimethylbenz[a]anthracene (DMBA) and TPA. The study was extended to two deacylated galactosylglycerol structures, 1-O-b -d-galactopyranosyl-sn-glycerol (5) and 3-O-b -d-galactopyranosyl-sn-glycerol (6). All the tested compounds exhibited remarkable anti-tumor-promoting effects on mouse skin tumor promotion, the 1-hexanoate 2 being the most active among the glycoglycerolipids until now studied. q 2000 Elsevier Science Ireland Ltd. All rights reserved. Keywords: Glycoglycerolipids analogues; Skin tumor, Anti-tumor-promoting effect
1. Introduction Cancer chemoprevention is an emerging ®eld for cancer control. In general, when new compounds are evaluated as chemopreventors, preliminary screenings in vitro are performed, which are followed, for the compounds able to prevent more strongly the tumor-promoting step, by in vivo assays. A large number of natural and synthetic glycoglycerolipids have been assayed in in vitro tests [1±5], showing remarkable inhibitory effects on the activation of the Epstein±Barr virus early antigen (EBV* Corresponding author. Tel.: 139-02-70645230; fax: 139-0270645221. E-mail address: [email protected] (D. Colombo).
EA) induced by the tumor promoter 12-O-tetradecanoylphorbol-13-acetate (TPA); conversely, to our knowledge only one of the natural compounds was examined in vivo [4]. In the last few years, in order to enhance the antitumor-promoting activity of this class of compounds, we prepared synthetic analogues structurally related to the natural products. They differed in several structural features, namely the sugar moiety, the position of the glycerol linkage to the sugar, the length and location of the acyl chain, and the anomeric con®guration of the sugar [6±9]. Primary in vitro studies [10±13] of the inhibitory effects on Epstein±Barr virus early antigen (EBV± EA) activation induced by TPA in Raji cells of
0304-3835/00/$ - see front matter q 2000 Elsevier Science Ireland Ltd. All rights reserved. PII: S 0304-383 5(00)00610-8
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D. Colombo et al. / Cancer Letters 161 (2000) 201±205
these synthetic analogues revealed that they all showed a signi®cant activity, which was modulated by the structural characteristics, in particular by the length of the acyl chain: the best activity being reached in the presence of a hexanoyl chain on one of the primary hydroxyl groups [11]. 1-O-Hexanoyl-2-O-b -d-glucopyranosyl-sn-glycerol (1), 1-O-hexanoyl-2-O-b -d-galactopyranosyl-snglycerol (2), 2-O-(6-O-hexanoyl-b -d-galactopyranosyl)-sn-glycerol (3) resulted the most active among compounds of b anomeric con®guration [11], and comparable activity showed 2-O-(6-O-hexanoyl-a d- g alacto p y r an o syl )-s n-gl ycerol ( 4 ) a mong compounds of a anomeric con®guration [8]. Moreover, among non acylated glycosylglycerols, 1-O-b d-galactopyranosyl-sn-glycerol (5) showed the highest activity [13]. These promising in vitro results prompted us to assay the inhibitory effect of the synthetic glycoglycerolipids analogues 1±4 on mouse skin tumor promotion in an in vivo two-stage mouse skin carcinogenesis test and in this paper we report the results. Moreover, as also compounds lacking the acyl chain resulted active in the in vitro tests, though to a lesser extent than the hexanoates, the study was extended to the deacylated compounds 1-O- and 3-O-b -d-galactopyranosyl-sn-glycerol (5) and (6), the latter showing the skeleton of the natural galactoglycerolipids.
2. Materials and methods 2.1. Test products Glycoglycerolipid analogues 1±3 were synthesized according to Colombo et al. [6,7]. Compound 4 (mp. 788C; [a ]D20 1 98 (c 1, methanol); 1H-NMR and mass spectrometry data consistent with its structure) was synthesized according to Colombo et al. [8]. 1-Oand 3-O-b -d-galactopyranosyl-sn-glycerol (5) and (6) were prepared as described in ref [9]. 2.2. Short term in vitro bioassay for anti-tumor promoters The inhibitions, determined using a short term in vitro test for Epstein±Barr virus activation in Raji cells induced by 12-O-tetradecanoylphorbol-13-acetate (TPA), are described in refs [10,11] for compounds 1±3, and in Ref. [13] for compounds 5 and 6. Compound 4 [8] completely inhibited EBV activation at a concentration of 1 £ 103 molar ratio toward TPA (32 pmol) and showed an inhibitory effect on EBV activation of 10:7 ^ 1:2 (percentage to positive control ^ standard error) at a concentration of 5 £ 10 2 mol ratio/TPA concentration, whereas no cytotoxicity was exhibited by the sample at the same concentrations. 2.3. In vivo two-stage mouse skin carcinogenesis test Female ICR mice were obtained at 5±6 weeks of age from SLC Co. Ltd. (Shizuoka, Japan). Groups of animals (15 animals per group) were housed in bunches of ®ve in polycarbonate cages and given food ad libitum. Mice were permitted free access to MP solid diet (Oriental yeast Co., Ltd. Chiba, Japan) and drinking water at all times during the study. The back of each mouse was shaved with surgical clippers before the ®rst day of initiation. The mice were initiated with dimethylbenz[a]anthracene (DMBA; 390 nmol) in acetone (0.1 ml). One week after initiation, they were promoted twice a week by application of TPA (1.7 nmol) in acetone (0.1 ml). The mice were treated with the test compounds (85 nmol, 50 mol ratio/TPA) in acetone (0.1 ml) 1 h before each TPA treatment. The incidence of papillomas was observed weekly for 20 weeks. The differences in mouse skin papillomas between control and experiments were
D. Colombo et al. / Cancer Letters 161 (2000) 201±205
203
Fig. 1. Inhibitory effects of compounds 1±6 (85 nmol) on DMBA-TPA mouse skin carcinogenesis. All mice were initiated with DMBA (390 nmol) and promoted with TPA (1.7 nmol) twice a week starting 1 week after initiation. (A and C) percentage of mice with papillomas; (B and D) averaged number of papillomas per mouse (X TPA alone; W TPA 1 1; A TPA 1 2; O TPA 1 3; B TPA 1 4; w TPA 1 5; V TPA 1 6). At 20 weeks of promotion the averaged number of papillomas per mouse was reduced, with respect to the control group, by 56, 71, 64, 49, 57 and 46% (P , 0:001 in all the cases), respectively for 1±6.
analyzed by means of the Student's t-test at 20 weeks of promotion, whereas the differences in tumor bearing mice were analyzed by means of the x 2-test. After 20 weeks of promotion, there was no statistically signi®cant difference in body weights between control and any treated group (control: 54 ^ 2:8 g, treated: 56 ^ 2:3 g at end of experiment). 3. Results and discussion Four synthetic equiactive glycoglycerolipid analogues 1±4, which showed a signi®cant inhibitory activ-
ity in previously performed in vitro assays of TPAinduced EBV-EA activation, were submitted to an in vivo two-stage carcinogenesis test of mouse skin papillomas using DMBA as an initiator and TPA as a promoter, in order to evaluate their inhibitory effects. The assays were performed also on deacylated 1-O- and 3-O-galactosylglycerols 5 and 6, the former being the most active among the studied glycosylglycerols, the latter showing the basic skeleton of natural galactoglycerolipids. The activities, evaluated by both the incidence (percentage of mice bearing papillomas) (Fig. 1A,C and Table 1) and the multiplicity (average numbers of
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D. Colombo et al. / Cancer Letters 161 (2000) 201±205
papillomas per mouse) (Fig. 1B,D and Table 1), were compared with those of the control group. As shown in Fig. 1, in the control, 100% of the mice bore papillomas even at only 10 weeks of promotion, and about nine papillomas were formed per mouse after 20 weeks of promotion. On the other hand, when compounds 1±6 were applied before each TPA treatment, they all delayed the formation of papillomas and lowered the number of papillomas per mouse, being 2 the most potent compound. In fact while in each group treated with compounds 1, 3, 4 and 6, at 10 weeks of promotion mice bore papillomas in a percentage from 33 to 53%, these percentages were of about 12% in the case of compounds 2 and 5; at 20 weeks of promotion only compound 2 showed about 53% of papillomas incidence, which varied from 79 to 94% for the other compounds (Fig. 1A,C). Compound 2 was the most effective also in reducing the number of papillomas per mouse, which after 10 weeks of promotion were about 0.5 for compounds 2 and 5 and 1±2 for the other compounds, while after 20 weeks of promotion they were suppressed with respect to control by 71% for compound 2 and by 46±64% for the other compounds (Fig. 1B,D). The decrease of the number of papillomas per mouse was signi®cant in the treated cases with respect to the control; in fact, in all the cases P , 0:001 (Student's t-test). Also, the differences among the treatment groups were signi®cant: 2 with respect to 1 (P , 0:002), to 3 (P , 0:1), to 4 (P , 0:001), to 5 (P , 0:005) and to 6 (P , 0:001). Similarly, 5 resulted better than 6 (P , 0:02). Less signi®cant appear the differences in the incidence of papillomas analyzed by the x 2±test; however compound 2 resulted, after 20 weeks, signi®cantly Table 1 Final papilloma incidence and multiplicity for compounds 1±6
TPA alone TPA 1 1 TPA 1 2 TPA 1 3 TPA 1 4 TPA 1 5 TPA 1 6
Papilloma incidence (%)
Papilloma multiplicity (m ^ SD)
100 80 53 80 87 93 93
9.1 ^ 1.6 4.0 ^ 0.9 2.6 ^ 1.2 3.3 ^ 0.9 4.6 ^ 1.1 3.9 ^ 1.1 4.9 ^ 0.8
different from the control (P , 0:005) and superior to the other compounds. It is noteworthy that 1-O-hexanoyl-2-O-b -d-galactopyranosyl-sn-glycerol (2), which shows in vitro an inhibitory effect comparable to that of compounds 1, 3 and 4, displays a higher potency in the in vivo twostage carcinogenesis, whereas in the case of the deacylated compounds 5 and 6, the in vitro experiments are con®rmed, being 1-O-b -D-galactopyranosyl-sn-glycerol (5) more active than its 3-Odiastereoisomer. Until now, only one example of an in vivo study appeared in the literature [4], which describes the anti-tumor-promoting activity on mouse skin of a natural glycoglycerolipid (DLGG) (DLGG 1,2-di-O-a -linolenoyl-3-O-b -d-galactopyranosyl-snglycerol) from Citrus hystrix. Interestingly, the synthetic analogue 2 here reported shows higher activity than the potent natural compound DLGG, because it displays an inhibitory effect similar to DLGG when its concentration is about half [4]. At present, the mechanism of glycoglycerolipids inhibitory effect on tumor promoting activity is not known; the inhibitors probably interfere with the TPA-induced promotional events, interacting with the receptor of tumor promoters protein kinase C [14,15]. The study of the structure-activity relationship may provide some insight; in particular, from this in vivo study we can conclude that the presence of the hexanoyl moiety at the 1-position of the glycerol backbone and the axial con®guration of the hydroxyl in the 4-position of the sugar are important for a remarkable activity. Acknowledgements This study was supported in part by Ministero dell UniversitaÁ e della Ricerca Scienti®ca e Tecnologica Rome, Italy (PRIN99: Chemistry of Bioactive Organic Compounds) and University of Milan, Italy and in part by Grants-in-Aid from the Ministry of Education, Science and Culture, and Ministry of Health and Welfare Japan. References [1] A. Nagatsu, M. Watanabe, K. Ikemoto, M. Hashimoto, N.
D. Colombo et al. / Cancer Letters 161 (2000) 201±205
[2]
[3]
[4]
[5]
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