Chapter 25
Antitumor-Promoting Effects of Gallotannins, Ellagitannins, and Flavonoids in Mouse Skin In Vivo 1
1
1
2
3
J. P. Perchellet , H. U. Gali , E. M . Perchellet , P. E. Laks , V. Bottari , R. W. Hemingway , and A. Scalbert 4
5
1
Anti-Cancer Drug Laboratory, Division of Biology, Kansas State University, Ackert Hall, Manhattan, KS 66506-4901 Institute of Wood Research, Michigan Technological University, Houghton, MI 49931-1295 Silva S.r.l., 12080 San Michele Mondovi (Cuneo), Italy Southern Forest Experiment Station, U.S. Department of Agriculture, Forest Service, Pineville, LA 71360-5500 Laboratoire de Chimie Biologique, Institut National de la Recherche Agronomique, Institut National Agronomique, Paris-Grignon, 78850 Thiverval-Grignon, France 2
3
4
5
Hydrolyzable (HTs) and condensed tannins (CTs) were tested topically for their ability to inhibit the biochemical and biological effects of 12-0-tetradecanoylphorbol-13-acetate (TPA) in mouse epidermis in vivo. Overall, commercial tannic acid (TA), ellagic acid (EA), and n-propyl gallate (PG) inhibit the promotion of skin papillomas and carcinomas by TPA in relation with their ability to inhibit TPA-induced epidermal ornithine decarboxylase (ODC) activity, hydroperoxide (HPx) production, and D N A synthesis. Pure pentagalloylglucose, castalagin, vescalagin, catechin dialkyl ketals, and epicatechin-4-alkylsulphides or heterogenous sumac leaf T A , Aleppo gall TA, tara pod TA, loblolly pine bark CT, guamuchil bark CT, and southern red oak bark CT also inhibit these biochemical markers of TPA promotion to various degrees. When applied to initiated skin 20 min before each promotion treatment, the different T A samples all remarkably inhibit complete tumor promotion by TPA. Sumac leaf T A is the most effective. The antitumor-promoting activity of a T A pretreatment can be further enhanced by the application of T A 24 h after each promotion treatment with TPA. Commercial T A and Aleppo gall T A inhibit the second stage of tumor promotion by mezerein but not the first stage of tumor promotion by TPA. Therefore, tannins in general might be valuable to prevent and/or inhibit tumor propagation, the only reversible stage of tumorigenesis. 0097-6156/94/0546-0303$07.25/0 © 1994 American Chemical Society
304
FOOD PHYTOCHEMICALS I: FRUITS AND VEGETABLES
Tannins are found in high concentrations throughout the plant kingdom and are the sources of reactive polyphenols with great structural diversity (1-3). The HTs are esters of gallic acid (GA) or hexahydroxydiphenic acid (HHDP) and the CTs derive from the condensation of flavan-3,4-diol (Figure 1). Tannin extracts are often collections of HTs or CTs with undefined degree of polymerization, molecular weight (M.W.), monomer unit composition, and type of linkage. "Hydrolyzable" Tannins OH
OH Gallic acid Gallotannins
OH Hexahydroxydiphenic acid Ellagitannins
" C o n d e n s e d " Tannins
OH Flavan-3,4-diol (Polyflavanoids) Proanthocyanidins
Figure 1. Classes and monomer units of tannins (adapted from ref. 3). Structures of HTs and CTs The HTs, or more correctly gallotannins and ellagitannins, are easily split into sugars and phenolic carboxylic acids. Upon acid hydrolysis, gallotannins and ellagitannins yield G A and EA, respectively. HTs, therefore, have a sugar core with pendant esterified G A or HHDP constituents and may possess a variable number of depsidically linked galloyl units in a polygalloyl chain (Figure 2). The CTs, or more correctly proanthocyanidins or polyflavonoids, have no sugars, do not readily break down, and almost invariably contain one of or both of the flavan-3-ols, (+)-catechin or (-)-epicatechin. CT monomer units are flavonoids consisting of 2 aromatic rings A and Β joined through a pyran ring C. Ten classes of CTs are distinguished on the basis of the hydroxylation patterns of the A - and fi rings. In oligomeric and polymeric CTs, a variable number of these skeletons are bound together by interflavonoid linkages occurring at one or more sites (Figure 2). Tannins and Multistage Carcinogenesis Experimental skin carcinogenesis has been divided into tumor initiation, promotion, and progression (4-6). This sequence is also called the multistage model of carcinogenesis. A subcarcinogenic dose of 7,12-dimethylbenz[a]anthra cene (DMBA) produces no tumors during the lifespan of the animal but, after metabolic activation, the electrophilic ultimate carcinogen interacts covalently with, and mutates, epidermal D N A to initiate skin carcinogenesis. Tumor initiation is generally regarded as a permanent alteration of the cell genotype with as yet no neoplastic phenotype. The correlation between tumor initiation and resistance of epidermal cells to signals for terminal differentiation suggests that the initiating event in skin carcinogenesis causes a genetic alteration in the program of terminal differentiation.
25. PERCHELLET ET AL.
Gallotannins, Ellagitannins, and Flavonoids 305
Repetitive applications of the most potent tumor promoter TPA are required to trigger molecular events leading the immediate progeny of the initiated epidermal cells to the formation of growing skin tumors and achieve complete tumor promotion. It is theorized that TPA stimulates the expression of the abnormal genetic information within the initiated cells which, because of their altered program of differentiation, acquire a neoplastic phenotype (conversion) and a proliferative advantage over their normal neighbors (propagation). Proanthocyanidins
Gallotannins
OH Galloyl unit
Figure 2. Examples of linkages between monomer units of HTs and CTs (adapted from ref. 3). One to four applications of TPA are sufficient to trigger the first, partially irreversible, stage of promotion called "conversion." Multiple applications of the ineffective promoter mezerein are then required to achieve the second stage of promotion called "propagation" and complete the promotion process. Neither treatment alone is sufficient in CF-1 mice. The long-lasting effects of TPA that are essential for stage 1 promotion persist for almost 2 months before declining whereas those of mezerein in stage 2 promotion are rapidly reversible and require a certain frequency of application in order to induce tumors. The molecular mechanism by which tumor promoters select the mutation-bearing initiated epidermal cells and induce their neoplastic transformation and clonal expansion into skin tumors is not fully understood. Finally, tumor progression requires a number of genetic alterations and is correlated to the level of D N A damage, chromosomal aberration, and aneuploidy.
306
FOOD PHYTOCHEMICALS I: FRUITS AND VEGETABLES
Benign neoplastic cells that have accumulated additional sublethal genetic alterations, besides those associated with tumor initiation, may be selected to develop malignant characteristics. E A and HTs, such as commercial TA, have already been shown to inhibit mutation, tumor initiation, and complete carcinogenesis, which are irreversible events (7-24). Therefore, it is crucial to determine if HTs can also inhibit the reversible phase of tumor promotion. When the tumor promoter TPA is applied topically to mouse skin at time 0, it triggers 3 major biochemical markers of tumor promotion: the induction of ODC activity at 5 h and the stimulation of HPx production and D N A synthesis at 16 h (25). Much of the biochemical significance of tannins may be linked to macromolecule complexation, mineral chelation, and antioxidation (1-3). Recently, we found that commercial TA, Ε A, and several G A derivatives applied topically to mouse skin can inhibit ODC, a marker of TPA promotion, by up to 85% (26). Objectives of Current Investigation New studies were designed to determine if the antioxidant activities of commercial HTs would enable them to inhibit TPA-stimulated HPx production and D N A synthesis in mouse epidermis in vivo, and the promotion of papillomas and carcinomas by TPA in initiated skin (27-29). Moreover, the antitumor-promoting effects of different T A extracts were compared. Vegetable tannins are common dietary components but CTs are far more abundant in food than are HTs. Therefore, the present study was also undertaken to determine if pure or heterogenous HTs and CTs prepared from various sources share the ability to inhibit the biochemical events linked to skin tumor promotion by TPA. Methods of Study Biochemical Markers of Skin Tumor Promotion. A l l tumor promoters were delivered to the shaved backs of female CF-1 mice in 0.2 ml acetone. HTs and CTs were also applied topically in 0.4 ml of the same solvent at various times before or after, and to the same area of skin as, each application of tumor promoter. Ε A and chestnut wood tannin were administered similarly in 0.4 ml methanol:acetone (55:45) and watenacetone (35:65), respectively. Doses of all T A samples and chestnut wood tannin were expressed in μιηοΐ based on average M.W.s of 1701 and 1100, respectively. At the appropriate times after TPA treatment, the mice were killed, their skins were excised, the epidermis was separated from the dermis, and homogenates were prepared by pooling the epidermises from 2 skins (26-29). Epidermal ODC activity was determined 5 h after a single TPA treatment by measuring the release of C 0 from L-[l- C]ornithine-HCl (30). The HPxproducing activity of the epidermis was assayed by a modification of the ferrithiocyanate method 16 h after the last of 2 applications of TPA at a 48 h interval (25,31). D N A synthesis was determined 16 h after a single TPA treatment. The mice were injected i.p. with 30 μα H-thymidine and the rate of incorporation of this radiolabeled precursor into epidermal D N A was measured by liquid scintillation counting after a 40 min period of pulse-labeling (25,32). 1 4
14
2
3
Tumor Induction Experiments. In the initiation-complete tumor promotion protocol (4-6), skin tumors were initiated in all female CF-1 mice by a single topical application of 100 nmol D M B A in 0.2 ml acetone. Two weeks later, all
25. PERCHELLET ET AL.
Gallotannins, Ellagitannins, and Flavonoids
mice were promoted twice a week (on days 1 and 4) with 8.5 nmol TPA for the rest of the experiments (20 or 40 weeks). In the initiation/two-stage promotion protocol (4-6), skin tumors were initiated in all female S E N C A R mice by a single application of 25 nmol D M B A . Two weeks later, all mice were promoted twice a week for 2 weeks with 4.25 nmol T P A (stage 1) and then twice a week for 18 weeks with 4.25 nmol mezerein (stage 2). Except where otherwise specified, HTs were applied 20 min before, and to the same area of skin as, each TPA treatment in complete and stage 1 tumor promotion or each mezerein treatment in stage 2 tumor promotion. Initially, there were 36 mice in each treatment group. The incidence and yield of skin tumors were recorded weekly and once every 2 weeks, respectively. The graphs of tumor data include 2 or 4 panels. (A) Average number of papillomas/ survivor. (B) Percentage of survivors with papillomas. (C) Average number of car cinomas/mouse and (D) percentage of mice with carcinomas based on the number of survivors in each group at the time of appearance of the first carcinoma in the experiment. Statistics for the differences between the means of papillomas/ mouse or carcinomas/mouse were performed using Student's Mest. Differences between papilloma and carcinoma incidences were compared using the χ statistic. The level of significance was set in both cases at ρ < 0.05. 2
Antitumor-Promoting Effects of Commercial HTs Commercial P G , E A , and T A (Figure 3) were from Sigma Chemical Co. Commercial EA, the dilactone form of HHDP, was purified from chestnut bark but the source of commercial T A was unknown. Commercial T A , usually Chinese gallotannin, is a mixture of molecules with a core of β-penta-O-galloyl-D-glucose to which other (approximately 2) galloyl residues are linked. Thus, T A samples are heterogenous because of the variation in the number of depsidically linked galloyl groups in the polygalloyl chain (Figure 3). OH
(n = 0,1,2) Figure 3. Structures of PG, E A and TA.
OH
307
308
FOOD PHYTOCHEMICALS I: FRUITS AND VEGETABLES
Inhibition of Complete Tumor Promotion. Commercial TA, EA, and P G — the treatments that most inhibit the ODC and HPx responses to TPA (26,29) — also inhibit remarkably the formation of papillomas and carcinomas when applied topically to DMBA-initiated skin 20 min before each promotion treatment with TPA (27,28). Antitumor-promoting Activity of Commercial TA. In control mice promoted with TPA only, the first papillomas appear at 7 weeks, the incidence and yield of papillomas reach a plateau at 16 weeks, and maximal tumor promotion is observed at 22 weeks with 97% of the mice bearing papillomas and an average of 12 papillomas/mouse (Figures 4 and 5). That commercial T A is an outstanding inhibitor of TPA promotion is illustrated by the fact that, at this stage of 22 weeks, 2.5, 5, 10, or 20 μπιοί of TA inhibit the tumor incidence by 50-100% and the tumor yield by 80-100% (Figure 4). Incredibly, 20 μπιοί of TA can prolong the latency period for papilloma development by 20 weeks so that the first tumors appear at 27 weeks instead of 7 weeks in the control group. After 22 weeks, some of the mice promoted in the presence of TA develop new papillomas while the control mice treated only with T P A have decreasing numbers of papillomas. But the T A treatments still inhibit the incidence of papillomas by 40-80% and the yield of papillomas by 70-95% at 40 weeks, suggesting that this HT decreases the tumorpromoting activity of TPA and does not simply delay or slow down the rate of tumor development. Less than 10% of the benign skin papillomas promoted by TPA progress to malignant skin carcinomas starting at week 26 and this event is also delayed by 2 11 weeks in the presence of increasing doses of TA (Figure 4). The treatments with 2.5, 5, and 10 μπιοί of TA inhibit both the incidence and yield of carcinomas at 40 weeks by 70-90%. Obviously, so few papillomas are promoted in the presence of 20 μπιοί of T A that none can progress to carcinomas before 40 weeks. Therefore, commercial T A clearly inhibits the promotion of both skin papillomas and carcinomas by TPA. These chronic topical applications of T A are not toxic because the body weights and rates of survival are identical in all groups up to 32 weeks, whether or not they receive TA. Moreover, the protective effect of TA is illustrated by the fact that, between weeks 32 and 40, the rate of survival drops from 100 to 50% in the control mice promoted only with TPA but it remains at 100% in all the groups promoted in the presence of TA. There are several explanations for the fluctuations and apparent declines in the number of papillomas after 22 weeks, especially in the control mice promoted with T P A only (Figure 4). The local and systemic toxicity of chronic T P A treatment and the high tumor burden decrease the rate of survival and the dead mice lost, especially those bearing carcinomas, are likely to have more papillomas than the group average. Furthermore, with increasing time there is coalescence of several small neighboring papillomas that combine together to form a single big papilloma and the continued conversion of papillomas to carcinomas results in tumors disappearing from Chart A to appear in Chart C (Figure 4). Antitumor-promoting Activities of EA and PG. From this tumor experiment, it appears that TA is the most effective anti-tumor promoter among the commercial HTs tested. Topical application of 5 μπιοί E A or 20 μπιοί P G 20 min before each TPA treatment also inhibits the incidence and yield of skin papillomas
25.
PERCHELLET ET AL.
Gallotannins, Ellagitannins, and Flavonoids 309
Weeks of T P A Treatment
Figure 4. Inhibition of TPA promotion ( · ) by 2.5 (•), 5 (•), 10 (A), and 20 (À) μπιοί TA. (Reproduced with permission from ref. 28. Copyright 1992 IRL Press.)
310
FOOD PHYTOCHEMICALS I: FRUITS AND VEGETABLES
Weeks of TPA Treatment
Figure 5. Inhibition of TPA promotion ( · ) by 5 μπιοί E A (O) or 20 μιηοΐ PG (A). (Adapted from ref. 28.)
25. PERCHELLET ET AL.
Gallotannins, Ellagitannins, and Flavonoids
and carcinomas promoted by this compound but E A and P G are somewhat less potent than equal doses of T A (Figure 5). Overall, the antitumor-promoting activities of commercial T A , E A , and P G in the initiation-complete tumor promotion protocol correlate well with their combined inhibitory effects on the 3 major biochemical markers of TPA promotion. Inhibition of the Biochemical Markers of Tumor Promotion. In Table I, the magnitudes of the inhibitory effects of commercial T A , E A , and P G on the biochemical and biological effects of TPA have been summarized. TPA-induced ODC activity and HPx production are 2 events that have been postulated to complement each other to trigger enough D N A synthesis and compensatory cell proliferation to achieve the prolonged hyperplastic response required for tumor propagation (25). On an equal dose basis, T A is as good an antioxidant as P G and less effective than ΕA (29) but it is a much more potent inhibitor of ODC induction than either of these two compounds (26). Consequently, T A also inhibits the stimulation of epidermal D N A synthesis and the promotion of skin tumors by TPA to a greater degree than similar doses of E A or PG. Indeed, the mean inhibitions of the ODC, HPx, and D N A responses to TPA by TA, EA, and P G match closely the means by which these HTs inhibit the incidence and yield of skin tumors promoted by TPA (Table I).
Table I. Comparison of the Abilities of T A , E A and P G to Inhibit the Biochemical and Biological Effects of T P A in Mouse Skin In Vivo Biochemical markers of tumor promotion
Skin tumor promotion at plateau
3
Treatment
TPA (8.5 nmol) + E A (5 μπιοί) + T A (5 μπιοί) + P G (20 μπιοί) + T A (20 μπιοί) a e
a
DNA ODC HPx induction production synthesis 100 90 41 48 14
b
e
100 0 27 1 1
b
100 69 50 82 41
d
Tumor incidence 100 63 48 62 25
e
e
Tumor yield 100 40 21 25 4
f
d
Percent of TPA effect. 1957% of control; 283% of control; 486% of control; 95.7% of mice with papillomas; 11.01 papillomas/mouse. f
Antitumor-Promoting Effects of Pure or Heterogenous HTs Several samples of pure or heterogenous HTs and CTs prepared from various sources were screened for their antitumor-promoting effects in mouse epidermis in vivo. Heterogenous T A Samples from Various Sources. Table II shows that topical applications of sumac leaf T A (from Rhus coriaria), Aleppo gall T A (from Quercus infectoria), and tara pod T A (from Caesalpinia spinosa) all mimic or even
311
312
FOOD PHYTOCHEMICALS I: FRUITS AND VEGETABLES
surpass the inhibitory effects of commercial T A on TPA-induced ODC activity and HPx production in mouse epidermis in vivo. But chestnut wood tannin (from Castanea sativa) inhibits only the HPx response to TPA, and to a lesser degree than the other T A samples tested (Table II). At a higher dose, sumac leaf TA, Aleppo gall TA, and tara pod T A also mimic more or less the inhibition of TPA-stimulated D N A synthesis caused by commercial T A (Table III). Aleppo gall TA, however, which inhibits ODC induction and HPx production the most (Table II), is not the best inhibitor of epidermal D N A synthesis (Table III) or skin tumor promotion (Figure 6) during T P A treatment, suggesting that one cannot always predict accurately the antitumor-promoting potential of HT extracts based solely on the magnitudes of their inhibitions of the ODC and HPx responses to TPA. Other inhibitory effects still unknown must play a role in the mechanism of anti-tumor promotion by TA. Inhibition of Complete Tumor Promotion. The effectiveness of Aleppo gall T A , tara pod T A , and sumac leaf T A as inhibitors of complete tumor promotion by T P A was compared at 10 μπιοί (Figure 6), a dose at which it is possible to determine if these T A extracts are more potent than commercial T A (Figure 4). In the control mice promoted only with TPA, skin tumor promotion is maximal as early as 12 weeks with about 84% of the mice bearing papillomas and 12 papillomas/mouse. At this stage, topical applications of Aleppo gall T A , commercial T A , tara pod T A , and sumac leaf T A 20 min before each T P A treatment inhibit the tumor incidence by 29-55% and the tumor yield by 80-88% (Figure 6). After 20 weeks of TPA promotion, the same T A samples are still able to inhibit significantly the tumor incidence by 16-46% and the tumor yield by 5378%. Interestingly, sumac leaf TA, which is the best inhibitor of TPA-stimulated D N A synthesis (Table III), is also the most effective against skin tumor promotion by TPA. At 20 weeks, the inhibitory effects of sumac leaf T A are significantly greater than those of Aleppo gall T A and commercial T A on the tumor incidence and those of Aleppo gall T A on the tumor yield. Body weights and rates of survival were identical in all treatment groups up to the end of the experiment. Thus, all T A extracts tested so far are anti-tumor promoters but their efficacy may vary considerably depending on their origin.
Effects of T A Post-Treatments. Since commercial T A can inhibit the HPx response to TPA even when it is administered 24 h after the tumor promoter (29), it is of interest to determine if such T A post-treatment can also inhibit complete tumor promotion by TPA. As shown in Figure 7, 10 μπιοί commercial T A applied 24 h after each promotion treatment with TPA do not inhibit skin tumor promotion, suggesting that the ability of T A to decrease the HPx-producing activity of the epidermis previously treated with T P A is not sufficient in itself to prevent the promotion of skin tumors. Even though T A may exert potent antioxidant effects in the epidermis at any time (29), it must be applied at a time when it can prevent TPA from triggering the other biochemical events linked to skin tumor promotion in order to successfully decrease the tumor-promoting activity of this agent. Indeed, T A applied 12-15 h after TPA fails to alter the peak stimulation of D N A synthesis observed 16 h after the tumor promoter (data not shown). T A post-treatment at +24 h, however, enhances significantly the ability of TA pretreatment at -20 min to
25. PERCHELLET ET AL.
Gallotannins, Ellagitannins, and Flavonoids 313
Table II. Inhibitory Effects of Heterogenous HT Samples from Various Sources on TPA-induced Ornithine Decarboxylase Activity (ODC) and Hydroperoxide (HPx) Production in Mouse Epidermis In Vivo b
3
Treatment (Dose/application) Control TPA (8.5 nmol) + commercial T A (5 μπιοί) + sumac leaf T A (5 μπιοί) + Aleppo pod T A (5 μπιοί) + tara pod T A (5 μπιοί) + chestnut wood tannin (5 μπιοί)
ODC activitv nmol C 0 / h / % o f %of mg protein control TPA 2
0
HPx production nmol H 0 / 4 h/ % o f %oi control TPA mg protein 2
2
100 2854 1129
100 37
10.8 ± 0 . 5 28.9 ± 1.1 15.0 ±0.7
100 268 100 139 23
917
30
20.2 ± 1.6
187
52
3.27 ±0.33** 798
25
10.8 ±2.8*** 100
0
6.65 ±0.53
1622
55
18.3 ± 4 . 0
11.68 ± 1.18
2849
100
0.41 ±0.05 11.70 ±0.99 4.63 ±0.35 3.76 ±0.44*
a
169
42
23.3 ±2.0**** 216
69
b
T A s applied 20 min before each TPA treatment; Five h after a single TPA treatment, mean ± S.D. (n=6); Sixteen h after 2 TPA treatments 48 h apart, mean ± S.D. (n=4). *p
