ANTITUMOR-PROMOTI!"iG ACTIVITIES OF TANNIC ACID, ELLAGIC ACID, AND SEVERAL GALLIC ACID DERIVATIVES IN MOUSE SKIN Jean-Pierre Perchellet, Hala U. Gali, Elisabeth M. Perchellet, Darren S. Klish, and Andrew D. Armbrust Anti-Cancer Drug Laboratory Kansas State University Manhattan, KS 66506-4901
ABSTRACT Naturally occurring plant phenols with antimutagenic and anticarcinogenic activities were tested for their abilities to inhibit the biochemical and biological effects of the potent tumor promoter 12- O-tetradecanoylphorbol-13-acetate (TPA) in mouse epidermis in vivo. When applied topically to mouse skin, tannic acid (TA), ellagic acid, and several gallic acid derivatives all inhibit TPA-induced ornithine decarboxylase activity, hydroperoxide production, and DNA synthesis, three biochemical markers of skin tumor promotion. Moreover, in the two-step initiationpromotion protocol, the same phenolic compounds also inhibit the incidence and yield of skin tumors promoted by TPA. TA is the most effective of these treatments. Since they are already known to inhibit tumor initiation, the plant phenols protecting against skin tumor promotion by TPA may be universal inhibitors of multistage carcinogenesis. TA and other polyphenols, therefore, might be valuable in cancer therapy and/or prevention.
INTRODUCTION Skin tumorigenesis is a multistage process. 1,2 The sequence of tumor initiation, stage 1 (conversion) and stage 2 (propagation) promotion, and progression is depicted in figure 1 where symbols represent large populations of epidermal stem Plant Polyphenols. Edited by R.W. Hemingway and P.E. Laks. Plenum Press. New York. 1992
783
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cells. Tumor initiation occurs when normal cells are subjected to sub carcinogenic events triggering DNA mutation. At first, the mutated genes are not expressed (proto-oncogenes), and there is no phenotypic transformation of the initiated cells. However, a few tumor-promoting events, which induce the expression of the mutated genes (oncogenes), convert the initiated cells into dormant tumor cells. The
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Figure 1. The multistage process of skin tumorigenesis. [Figure reproduced by with permission from lSI Atlas of Science: Pharmacol. 2:325 (1988)].2
Antitumor Activity
785
initiating event in skin carcinogenesis causes a genetic alteration in the program ofterminal differentiation. 3 In stage 1, therefore, a few 12-O-tetradecanoylphorbol13-acetate (TPA) treatments may accelerate the differentiation and elimination of the normal keratinocytes and favor indirectly the clonal expansion of the neoplastically transformed cells, which have become resistant to the TPA-induced signals of terminal differentiation. 4 Finally, in stage 2, repeated tumor-promoting events are required to provide some of the dormant tumor cells with a proliferative advantage leading to the formation of a visible skin tumor. Rapidly proliferating neoplastic cells that have only those genetic lesions specific for initiation form benign skin papillomas (Pa). But, at any stage of the process, the cells that acquire other types of DNA lesions and chromosomal aberrations that increase their degree of aneuploidy and malignant potential may progress to invasive skin carcinomas (Ca). Because tumor initiation is irreversible, it is fundamental to the prevention of neoplasia to identify the anti-cancer drugs that are the most effective against the reversible propagation phase of tumorigenesis. Tannic acid (TA), gallic acid (GA), and ellagic acid (EA) have substantial potential for decreasing the risk of tumorigenicity.5,6 Reactive O 2 species (ROS) generated directly or indirectly by chemical carcinogens and tumor promoters are implicated at all stages of skin tumorigenesis. 7 Interestingly, the naturally occurring plant phenols TA and EA are anti-oxidants known to inhibit skin tumor initiation and complete carcinogenesis by polycyclic aromatic hydrocarbons (PARs), but their antitumor-promoting activities have not been tested. 7 EA is a candidate cancer chemopreventive agent 8 because its pharmacology is known, it is inexpensive and well tolerated, it is an anti-oxidant as effective as, or better than, a-tocopherol or tert-butylhydroxyanisole, and it shows inhibitory activity against lipid peroxidation. 5 The inhibition of skin tumor initiation and carcinogenesis by polyphenolic compounds may result from decreased metabolic activation and increased conjugation reactions, as indicated by the findings that TA and EA inhibit epidermal aryl hydrocarbon hydroxylase activity, induce glutathione S-transferase activities, and decrease the formation of ultimate carcinogens and their covalent binding to epidermal DNA. 9 - 18 The ability of polyphenols to bind to DNA and scavenge the ultimate forms of the carcinogens may also playa role in the mechanism by which they inhibit mutagenesis and carcinogenesis.1 9 ,2o Since the abilities of TA and EA to alter the effects of the tumor promoters are unknown, the present study was undertaken to determine whether such plant phenols with antimutagenic and anticarcinogenic activities can also inhibit the biochemical and biological effects of the potent tumor promoter TPA in mouse epidermis in vivo.
BIOCHEMICAL STUDIES The mechanisms by which TPA induces the expression of altered genes and the clonal expansion of the transformed progenitor cells may be mediated, at least in part, through Ca2+ mobilization and protein kinase C (PKC) activation. 21 - 23 The induction of epidermal ornithine decarboxylase (ODC) activity by TPA is an excellent biochemical marker of stage 2 promotion. 1,24 Moreover, prolonged stimulations of hydroperoxide (RPx) production 25 ,26 and DNA synthesis27 in TPA-treated epidermis are required to achieve the propagation phase of skin tumorigenesis. But
786
Perchellet
the magnitude of tumor promoter-induced DNA synthesis may be linked to HPx production rather than ODC induction.28 Each of these three responses, therefore, appears to be essential but not sufficient for tumor promotion. The increased HPxproducing activity of the epidermis may be required in stage 2 to eliminate some populations of normal cells sensitive to free radical-induced DNA strand breaks, terminal differentiation, and cytotoxicity, and facilitate prolonged compensatory waves of DNA synthesis and mitosis in the partly synchronized populations of transformed stem cells that resist and/or survive the oxidative stress caused by the tumor promoters. The first goal was to determine whether TA, EA, and several GA derivatives could inhibit these biochemical events linked to skin tumor promotion by TPA. Female CF-1 mice, 9 weeks old, were used throughout, and their dorsal skins were shaved before experimentation. 29 Solutions of tumor promoters were prepared in acetone and delivered to the shaved backs of individual mice in a volume of 0.2 mL. Multiple tumor promoter treatments were administered at 72-hour intervals. TA and the various GA derivatives (all from Sigma Chemical Co., St. Louis, Missouri) were dissolved in acetone and applied topically in a volume of 0.4 mL at the appropriate times before or after, and to the same area of skin as, each application of tumor promoter. EA (from Sigma) was dissolved in a mixture ofmethanol:acetone (55:45) and administered topically to the skin in 0.4 mL. The 10 J.tmol treatments of EA were delivered in two consecutive applications of 0.4 ml containing 5 J.tmol. Controls were treated with acetone or the above mixture of vehicles only. In every experiment, all mice received the same volume of solvent. Inhibition of TPA-Induced ODe Activity Epidermal ODC activities were determined 5 hours after tumor promoter treatment by measuring the release of 14eo2 from L-[l- 14 C]ornithine-HCI (55 mCi/mmol) essentially as described previously.29 The results (fig. 2) represent the mean values ± SD of two different experiments, each performed in triplicate (with two epidermises/replicate). The Students t test was used to determine the statistical significance of the difference between two sample means. The dose of20 J.tmol ofTA inhibits maximally TPA-induced ODe activity when it is applied 20 min before TPA, and its effectiveness is lost at treatment times further from the time of application of TPA (fig. 2). But TA is able to inhibit the ODC response to TPA when administered over a long period oftime extending from 3 hours before to 1 hour after the application of the tumor promoter. The ability of TA to inhibit TPA-induced ODC activity when applied 20 min before the tumor promoter becomes apparent with 0.5 J.tmol and is clearly dose dependent (fig. 3). The most effective dose of TA, 20 J.tmol, inhibits the ODC response to TPA by at least 85 percent. As reported previously,24,29 epidermal ODC activity peaks 5 hours after TPA treatment (time 0) and returns to basal level at 12 hours (fig. 4). When applied 20 minutes before the tumor promoter, 20 J.tmol of TA inhibit remarkably TPAinduced ODC activity at all time points studied without altering the general time course observed previously.
Antitumor Activity
787
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Figure 2. Effect of the time of application of TA on TPA-induced ODe activity in mouse epidermis in vivo. TA (20 ILmol) was applied (.) at various times before or after treatment with 8.5 nmol of TPA (time 0). TPA-induced ODe activity at +5 hours in the absence of TA treatment was 8.87 ± 0.68 nmol CO 2 /1 hour/mg protein (100 ± 8 percent, ~). Basal ODe activity in control mice (0.45 ± 0.04 nmol CO 2 ) has been subtracted from the data. a No significant difference versus TPAj bp < 0.0005, significantly smaller versus TPAj cp < .0005, significantly smaller versus TA 40 min before TPAj dp < 0.005, significantly smaller versus TPA. [Figure reproduced with permission from Cancer Res. 51:2820 (1991).41]
So far, the studies of plant phenols as inhibitors of mutagenesis and carcinogenesis have focused on (EA).5,6 Therefore, the treatments with TA, EA, GA, GA lauryl ester (GALE), GA methyl ester (GAME), and n-propyl gallate (PG) were compared for their effectiveness as inhibitors of TPA-induced ODe activity (table 1). TA is by far the most effective treatment for inhibiting the action of TPA as compared with the inhibitory effects of the other compounds tested. EA cannot be tested at 20 J.lmol because of its poor solubility in topically applicable solvents. Doses of 5 J.lmol or more are required to demonstrate the inhibitory effect of EA, which is the least effective treatment against ODe induction by TPA. Although slightly more effective than EA, GA, and its derivatives all inhibit the ODe response to TPA to a much lesser degree than TA. Since the efficacies of ODe induction and skin tumor promotion are maximal when the time interval between repeated TPA treatments is 72 hours,3o it is important to compare the inhibitory effects of TA on ODe induction by single vs. multiple treatments with TPA (fig. 5). The specific activities of epidermal ODe are increased more and more by successive applications of TPA at 72-hour intervals and reach a plateau of maximal stimulation after the third treatment. TA pretreatments inhibit remarkably not only the induction of ODe activity produced by a single application of TPA but also the greater ODe responses elicited by each successive tumor promoter treatment.
788
Perchellet
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Figure 3. Dose-response curve showing the inhibitory effect of TA on TPAinduced ODe activity in mouse epidermis in vivo. The indicated doses of TA were applied (.) 20 minutes before 8.5 nmol of TPA. TPA-induced ODe activity at +5 hours in the absence of TA treatment was 7.47 ± 0.54 nmol e02/1 hour/mg protein (100 ± 7 percent, ~). Basal ODe activity in controls (0.40 ± 0.03 nmol CO 2) has been subtracted. a No significant difference versus TPA; bp < 0.01, significantly smaller versus TPA; cp < 0.005, significantly smaller versus TPA but no significance versus 0.5 IJmol of TA before TPA; dp < 0.005, significantly smaller versus 50 IJmol of TA before TPA. 41
Finally, TA was tested for its ability to inhibit the ODC responses to other phorbol ester and non-phorbol ester tumor promoters applied twice at a 72-hour interval (table 2). The ODC-inducing activities of several structurally different agents with various tumor-promoting activities have been reported. 24 ,26,28,29,31-38 In general, the phorbol-related diterpene esters and the indole alkaloids with complete and/or stage 2 skin tumor-promoting activities are more potent inducers of epidermal ODC activity than other classes of tumor promoters such as the diacylglycerols, peroxides, anthrones, Ca2+ ionophores, and n-alkanes, which must be applied at much higher doses in order to induce this enzyme activity. Whatever their magnitudes, all these tumor promoter effects are dramatically inhibited by TA. The induction of ODC activity, the first and rate-limiting enzyme in polyamine biosynthesis, is essential but not sufficient for tumor promotion. 1,2,24 All inhibitors of ODC induction are also effective against tumor promotion. 1,2,24,39 The significance of the inhibitory effects of TA, EA, and several GA derivatives on the ODC marker of tumor promotion has been discussed recently.4o,41 The mechanism of this inhibition is unknown. The mice show no stress or discomfort following repeated applications of 20 J.\mol of TA. Cytotoxicity is unlikely since the inhibitory effect
789
Antitumor Activity 9 c
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Figure 4. Time-response curves showing the inhibitory effects ofTA on TPAinduced ODe activity in mouse epidermis in vivo. Mice were treated with (.) or without (O) 20 pmol of TA 20 min before 8.5 nmol of TPA (time O). ODe activity was determined at the indicated times following TPA treatment. Basal ODe activity in controls (0.43 ± 0.05 nmol e0 2 /1 hour/mg protein) has been subtracted. tip < 0.0005, significantly greater versus control; b no significance versus TA + TPA at 7.5 hours but P < 0.025, significantly greater versus TA + TPA at 10 hours; cp < 0.05, significantly smaller versus TPA at 5 hours; dp < 0.005, significantly smaller versus TPA at 10 hours and greater versus TA + TPA at 12.5 hours; e no significance versus control or TA + TPA at 12.5 hours.41
of TA is rapidly reversible (fig. 2), and similar TA and EA treatments enhance the activities of other epidermal enzymes. 12 ,17 Macroscopic examination of the skins collected after single or multiple TA + TPA treatments shows that they have been very well protected from the hyperplastic and inflammatory activities of the tumor promoter as compared with the skins receiving TPA alone. Moreover, TA applied within 10 minutes of the time of sacrifice (5 hours after TPA treatment) alters neither the pH of the ODC-containing supernatant nor the activity of the TPAinduced enzyme, suggesting that the observed inhibitory effects are not simply due to traces of plant phenols remaining in the epidermal extracts. TPA induces ODC activity by a signal transduction mechanism involving Ca2 + mobilization, PKC induction, ODe mRNA expression, and de novo protein synthesis. 21 ,23,24,42 The fact that TA is more effective against ODC induction when applied before rather than after TPA treatment (fig. 2) suggests that this compound does not interact directly with the enzyme but interferes with the actions of the
790
Perchellet
Table 1. Comparison of the Inhibitory Effects of Various Plant Phenols on TPA-Induced ODC Activity in Mouse Epidermis In Vivo 41 Treatment G
Control TPA
ODC activity at 5 hours nmol CO 2 /1h/mg Percent of Percent of protein control TPA
(8.5 nmol) + EA (2 pmol) + TA (2 pmol) + EA (5 pmol) + TA (5 pmol) + EA (10 pmol) + GA (10 pmol) + GALE (10 pmol) + GAME (10 pmol) + PG (10 pmol) + TA (10 pmol)
0.43 6.77 7.01 4.19 6.12 3.26 5.29 3.75 3.65 4.72 4.28 2.17
± 0.03 ± 0.56 ± 0.75 b ± 0.31 ± 0.50 c ± 0.40 ± 0.38 ± 0.22d ± 0.20 ± 0.45 e ± 0.36 ± 0.271
100 1574 1630 974 1423 758 1230 872 849 1098 995 505
100 104 59 90 45 77 52 51 68 61 27
GIndicated compounds applied 20 minutes before TPA. bNo significant difference versus TPA cp < 0.05, significantly smaller versus TPA dNo significance versus TPA + GALE (10 /lmol); P < 0.01, significantly smaller versus TPA PG (10 /lmol). eNo significance versus TPA + PG (10 /lmol); P < 0.05, significantly smaller versus TPA + EA (10 /lmol). Ip < 0.0005, significantly smaller versus TPA + GALE (10 /lmol) and TPA + TA (5 /lmol).
+
tumor promoter and/or the molecular pathways regulating enzyme activities. (-)-Epigallocatechin gallate (EGCG) slightly inhibits the specific binding of 3H_ TPA to mouse skin, decreases the number of phorbol ester receptors, and prevents the activation of PKC by teleocidin. 43 On an equal dose basis, TA is the most effective of the polyphenols tested as an inhibitor of TPA-induced ODC activity. TA is also the most potent of the plant phenols at inhibiting epidermal monooxygenase activities and PAH metabolism, and covalent binding to epidermal DNA. 9 ,lO TA pretreatments inhibit the ODCinducing activity of TPA by about 85 percent. That TA inhibits the time course for ODe induction by TPA at each time point studied in figure 4 suggests that this compound does not simply delay the peak of TPA-induced ODe activity. EA is at least 50 percent less effective than TA at inhibiting the ODe response to TPA. Higher, perhaps more effective doses of this compound cannot be tested because they cannot be dissolved and spread in topically applicable solvents. The poor absorption of EA and the oxidative breakdown of its polyphenolic structure may limit the effectiveness of this treatment against tumorigenesis. 6 ,16
Antitumor Activity
791
4
5
Number of Applications of TPA Figure 5. Inhibitory effects of TA on the ODe responses to multiple applications of TPA in mouse epidermis in vivo. Mice were treated with (.) or without (0) 20 I'mol of TA 20 min before each application of 8.5 nmol of TPA repeated at 72-hour intervals. ODe activity was determined 5 hours after the indicated number of TPA treatments. Basal ODe activity in controls (0.38 ± 0.03 nmol e0 2 /1 hour/mg protein) has been subtracted. aNo significance versus third or fourth applications of acetone + TPA; bN 0 significance versus third or fourth applications of TA + TPAY
Because the magnitude of ODC induction almost doubles after the second TPA treatment (fig. 5), it is more accurate to compare the limited ODC-inducing activities of weak tumor promoters after two applications at a 72-hour interval (table 2). Under these conditions, the abilities of several structurally different tumor promoters to induce ODC activity reflect their tumor-promoting activities. The order of effectiveness of the phorbol esters is TPA > phorbol-12,13-didecanoate > phorbol-12,13-dibenzoate. 12-Deoxyphorbol-13-tetradecanoate is sixteenfold more irritant but induces ODC activity (table 2) and skin tumor promotion44 as much as TPA. Mezerein and 12-0-retinoylphorbol-13-acetate are very weak complete tumor promoters but induce ODC activity as much as TPA and are excellent in stage 2 of promotion. 1,2 However, the PKC activator and stage 2 tumor promoter, 1,2dioctanoyl-sn-glycerol,45 is a weak inducer of ODC activity (table 2).33 Although the Ca2+ ionophore A23187 is a significant ODC inducer (table 2) that stimulates HPx production and DNA synthesis almost as much as TPA and mezerein,26,28 its tumor-promoting activity has only been demonstrated in stage 1. 1 The synthetic analogs of the teleocidin A type,46 (-)-indolactam V and (-)-7-octylindolactam V, which are moderate and potent activators of PKC and bind to the same site on
792
Perchellet
Table 2. Inhibitory Effects of TA on the ODe Responses to Various Tumor Promoters in Mouse Epidermis In Vivo U Treatment"
ODC activity at 5 hours nmol C02 /lh/mg Percent of Percent of protein control tumor promoter
Control TPA (8.5 nmol) +TA 12-Deoxyphorboll3tetradecanoate (8.5 nmol) Mezerein (8.5 nmol)
+TA +TA
12- 0-Retinoylphorbol13-acetate (8.5 nmol) Phorbol-12,I3-didecanoate (8.5 nmol) Phorbol-12,I3-dibenzoate (8.5 nmol) A23187 (0.2 ttmol)
+TA +TA +TA +TA
1,2-Dioctanoyl-snglycerol (5 ttmol) (-)-Indolactam V (85 nmol) (- )-7-0ctylindolactam V (8.5 nmol) H 20 2 (100 ttmol)
+TA +TA +TA +TA
Benzoyl peroxide (100 ttmo1) Anthralin (250 nmol) Chrysarobin (250 nmol) n-Dodecane (25 mg)
+TA +TA +TA +TA
0.35 ± 0.04 9.21 ± 0.82 2.64 ± 0.16 9.77 ± 0.64b 2.26 ± 0.12 10.89 ± 1.02 c 2.39 ± 0.18 10.45 ± 1.18d 1.82 ± 0.30 5.28 ± 0.52 2.16 ± 0.36 1.82 ± 0.17 0.81 ± 0.10 1.58 ± 0.10 0.55 ± 0.03 1.20 ± 0.19 0.67 ± 0.14 4.49 ± 0.29 0.83 ± 0.07 7.55 ± 0.83 e 1.31 ± 0.22 1.44 ± 0.12 0.85 ± 0.13 0.89 ± 0.09 0.63 ± 0.031 1.95 ± 0.15 0.81 ± 0.05 0.91 ± 0.08 0.43 ± 0.039 2.82 ± 0.26 2.12 ± 0.36h
100 2630 754 2791 646 3111 683 2986 520 1509 617 519 231 451 157 343 191 1282 237 2157 374 411 243 254 180 556 231 260 123 806 606
100 26 100 20 100 19 100 15 100 37 100 31 100 16 100 38 100 12 100 13 100 46 100 52 100 29 100 15 100 72
"Treatment applied twice at a 72-hour interval (20 ttmol of TA. applied 20 minutes before each tumor promoter). bNo significance versus TPA. cp < 0.01, significantly greater versus TPA. dp < 0.05, significantly greater versus TPA. ep < 0.005, significantly smaller versus TPA. fp < 0.0005, significantly smaller versus benzoylperoxide. 9p < 0.005, significantly greater versus control. hp < 0.005, significantly smaller versus n-dodecane.
the enzyme at which the phorbol esters act, also induce remarkably ODC activity (table 2) in relation with their binding affinities for the TPA receptor. 38 The free radical generator benzoyl peroxide, which induces epidermal PKC and xanthine
Antitumor Activity
793
oxidase activities, hyperplasia, and morphological changes similar to those caused by TPA, is a good tumor progress or but a weak inducer of ODe activity in relation with its weak tumor-promoting activity.1,31,32,47-50 The alkane n-dodecane, a weak tumor promoter active primarily in stage 2, produces very little inflammation or ODC induction after a single 50 mg treatment, but its hyperplastic activity after four 50 mg treatments is comparable to that of TPA or mezerein. 37 ,51 But a dose of 25 mg of n-dodecane applied twice at a 72-hour interval is a rather good inducer of ODC activity (table 2). Since the magnitude and time course for the effects of anthralin and chrysarobin on epidermal ODC activity and polyamine and DNA synthesis are very different from those of TPA and mezerein, the mechanism by which the anthrones promote skin tumors may be somewhat different from that of the diterpenes. 34 - 36 In any case, the ability of TA to inhibit all the ODC responses elicited by such different tumor promoters suggests that 1) the inhibitory effects of the polyphenols on ODC induction are not specific for the TPA-altered enzyme, and 2) the plant phenols are likely to inhibit other biochemical events linked to skin tumor promotion by these various agents.
Inhibition of TPA-Induced HPx Production The evidence for the role of ROS in tumor promotion has been reviewed. 2,7,52,53 Briefly, 1) tumor promoters increase the generation and decrease the degradation of ROS, 2) organic peroxides and free radical-generating systems exhibit tumorpromoting activities and mimic or enhance some of the molecular events linked to tumor promotion, and 3) various antioxidants and free radical scavengers inhibit the biochemical and biological effects of the tumor promoters. The HPx response linked to skin tumor promotion has been characterized recently.25,26,28 Complete and stage 2 tumor promoters increase remarkably the HPx-producing activity of the epidermis while decreasing its natural antioxidant protection. Initiated and neoplastically transformed epidermal cells may become resistant to ROS-induced DNA strand breakage and terminal differentiation. 54 TPA-induced HPx production, therefore, might accelerate the differentiation and elimination of the normal stem cells that are sensitive to oxidative stress and trigger the prolonged compensatory stimulation of DNA synthesis and tumor cell proliferation required to achieve the propagation phase of skin tumorigenesis. The following study was initiated to determine if the antioxidant activities of EA and other plant phenols would enable them to inhibit the HPx marker of skin tumor promotion. The epidermal production of HPx was determined 16 hours after the last of two applications of TPA at a 48-hour interval (table 3), a time when maximal TPA-induced HPx formation is observed. 26 ,28 The HPx-producing activities of the epidermal homogenates were assayed at acid pH by a modification of the ferrithiocyanate method essentially as described previously.26,28,55 The absorption of the red ferrithiocyanate complex formed in the presence of peroxide was measured by double-beam spectrophotometry against a reagent blank at 480 nm, and the levels of HPx were quantitated with reference to calibration curves prepared under similar conditions with standards of H20 2 ranging from 10 to 200 j.tM. The results are the means ± SD of duplicate determinations of HPx levels from four groups of
794
Perchellet
Table 3. Comparison of the Inhibitory Effects of Various Doses of TA and EA on TPA-Induced HPx Production in Mouse Epidermis In Vivo Treatment ll
HPx formation at 16 hours nmol H2 0 2 /4h/ Percent of Percent of TPA mg protein control
Control
13.0
± 0.9
100
+ TA (1 #Lmol) + TA (2 #Lmol) + TA (5 #Lmol) + TA (10 #Lmol) + TA (20 #Lmol)
36.2 27.5 23.9 21.1 15.2 13.1
± 4.1 ± 3.4b ± 2.1 ± 1.9 ± 1.0c ± 0.5 d
278 211 184 162 117 101
100 62 47 35 9 0
+ EA (0.05 #Lmol) + EA (0.1 #Lmol) + EA (0.2 #Lmol) + EA (0.5 #Lmol) + EA (1 #Lmol)
31.7 28.6 21.9 14.4 13.0
± 3.3 e ± 1.5 b ± 1.5 ± 0.7' ± 0.3 d
244 220 168 111 100
81 67 38 6 0
TPA (8.5 nmol)
IITreated twice at a 48-hour interval (dose/application). bp < 0.025, significantly smaller versus TPA. cp < 0.025, significantly greater versus control. dNo significant difference versus control. eNo significant difference versus TPA. Jp < 0.05, significantly greater versus control.
mice in two different experiments; each group contained the combined epidermal homogenates prepared from two mice. The t test is used. When applied 20 minutes before each tumor promoter treatment, TA and EA inhibit totally the HPx response to TPA (table 3). These inhibitory effects are dose dependent. On an equal dose basis, EA is at least 10 times more potent than TA as an inhibitor of TPA-induced HPx production. Except PG, GA derivatives are all less effective than TA at inhibiting the HPx response to TPA (data not shown). EA and TA are antioxidants so potent that they can inhibit totally the HPx response to TPA even when they are applied 8 hours before or after the tumor promoter (data not shown), a period of time much longer than that observed for their effectiveness against ODe induction (fig. 2). Antioxidants generally inhibit the ODe-inducing activity of TPA. 7 ,31,32 ODe induction and HPx production are both essential for stage 2 promotion. However, there is no apparent correlation between oxidant generation and ODe induction since the HPx response to TPA does not require ODe induction and is not essential for ODe induction. 26,28 This hypothesis is substantiated by the finding that topical applications of doses of EA < 2 pmol, which are not sufficient to affect the ODe response to the tumor promoter (table
795
Antitumor Activity
1), inhibit totally TPA-induced HPx production in mouse epidermis in vivo (table 3). Therefore, there is no indication that the polyphenols inhibit the induction of ODC activity by TPA because of their antioxidant activities.
Inhibition of TPA-Induced DNA Synthesis The alteration of epidermal DNA synthesis by the complete and stage 2 tumor promoters, TPA and mezerein, in vivo, is characterized by an early 12-hour period of inhibition followed by two peaks of maximal stimulation at about 16 and 32 hours. 27 ,36,56 Moreover, the conversion phase of carcinogenesis elicited when TPA is used as a stage 1 promoter either shortly before of after tumor initiation (fig. 1) has been shown to require undisturbed DNA synthesis at 18 hours.57 Therefore, EA and TA were tested for their abilities to alter the integrity ofthe TPA-induced DNA synthesis process (table 4). The rate of incorporation of [methyl-3H]thymidine (59 Ci/mmol) into epidermal DNA was determined 16 hours after TPA treatment. The mice received 30 /lCi of [3H]thymidine i.p. and were killed after a 40-min period of pulse-labeling. The DNA contained in the epidermal homogenates prepared from two mice was extracted, and hydrolyzed from the acid-insoluble precipitate with 3 ml of 0.5N HCI0 4 for 15 min at 90 °C. 28 ,32,56 The radioactivity incorporated into each sample was determined by liquid scintillation counting in 0.2-mL aliquots of the above hydrolysates. The DNA content of each sample was determined by the diphenylamine procedure with calf thymus DNA as the standard. 58 Results are the means ± SD of two different experiments, each performed in triplicate (with
Table 4. Inhibitory Effects of EA and TA on TPA-Stimulated DNA Synthesis in Mouse Epidermis In Vivo Treatment B
[3H]Thymidine incorporation into epidermal DNA at 16 hours cpm//-tg DNA
Percent of TPA
48.6
± 6.8
100
(8.5 nmol)
233.2
± 27.1
480
100
+ EA (5 /-tmol)
173.4
± 15.9 b
357
68
+ TA (5 /-tmol)
138.7
± 17.5 c
285
49
Control TPA
Percent of control
BEA and TA applied 1 hour after TPA. bp < 0.005, significantly smaller versus TPA. cp < 0.005, significantly smaller venms TPA + EA.
796
Perchellet
two epidermises/replicate). The t test is used. As shown in table 4, EA and, to a greater degree, TA, both inhibit the peak stimulation of epidermal DNA synthesis observed 16 hours after TPA treatment.
TUMOR EXPERIMENT Finally, the TA, EA, and GA derivative treatments shown to inhibit the ODC, HPx, and DNA markers oftumor promotion were tested for their abilities to inhibit the tumor-promoting activity of TPA. The development of skin tumors requires repeated applications of a tumor promoter to initiated skin. 1 ,2 Because the biochemical, histological, and morphological effects triggered by TPA on mouse skin are maximal after three to five applications of this compound,26,28,29,32,49,59 the ability of TA to inhibit to about the same degree the greater ODC and HPx responses elicited by each successive application of TPA suggests that TA may also decrease the biological activity of chronic TPA treatment in a two-stage initiation-promotion protocol. 'Tumors were initiated in all mice by a single topical application of 0.1 "mol of 7,12-dimethylbenz[a]anthracene in 0.2 ml of acetone. Two weeks following initiation, all mice were promoted twice a week (on days 1 and 4) with 8.5 nmol ofTPA in 0.2 ml of acetone for the duration of the experiment. The various doses of TA, EA, and GA derivatives were all applied in 0.4 mL of solvent 20 minutes before each promotion treatment with TPA. Initially, there were 36 mice in each treatment group. The incidence of skin tumors was recorded weekly. Statistical analyses of the differences between the means of papillomas per mouse were performed using Student's t test, whereas, differences between papilloma incidences were compared using the X2 statistic. The level of significance was set in both cases at P ~ 0.05. After 22 weeks, all the plant phenols tested inhibit remarkably both the incidence and the yield of skin papillomas promoted by TPA (table 5). In all treatment groups, TA, EA, and the GA derivatives inhibit the number of tumors/mouse to a greater degree than the number of mice with tumors. Overall, TA is the best inhibitor, and in a dose-dependent manner, of the tumor-promoting activity of TPA. At 20 "mol, TA affords total protection against TPA promotion. On an equal dose basis, EA is less potent than TA at inhibiting the number of papillomas/mouse. With the exception of PG, GA and its derivatives are the least effective inhibitors of tumor promotion. That chronic applications of plant phenols do not induce toxic effects in vivo is suggested by the fact that, after 22 weeks of promotion, the weight and rate of survival of the animals treated twice a week with 2.5 to 20 /lmol of TA, EA, GA, GALE, GAME, or PG are identical with those of mice receiving TPA only. ODC induction in early stage 2 and the subsequent activation of polyamine biosynthesis have been suggested to complement the HPx-mediated events in order to trigger enough DNA synthesis and compensatory cell proliferation in late stage 2 to achieve tumor propagation. 28 This might explain why there is a correlation between the antitumor-promoting activities of the compounds tested and the mean values of their inhibitory effects on ODC induction plus HPx production. For instance, 5 /lmol ofTA inhibit ODC induction to a greater degree than a similar
TPA 8.5 nmol) + TA (2.5 I'mol) + TA (5 /-Imol) + TA (10 I'mol) + TA (20 I'mol) + EA (5 /-Imol) + GA (20 I'mol) + GALE (10 I'mol) + GAME (20 I'mol) + PG (20 I'mol)
Promotion Treatment a
31.5 30.7 30.8 31.6 30.7 31.6 32.4 32.9 32.6 31.1
(g)
Weight/ Mouse
aTwo treatments per week dose application. bp < 0.005, significantly smaller vs. group 1. cp < 0.05, significantly smaller vs. group 1.
1 2 3 4 5 6 7 8 9 10
Groups
100 97 97 100 97 100 100 100 97 100
97 51 51 30 0 50 83 b 78 79 58
12.1 ± 1.5 2.5 ± 1.1 1.7 ± 1.0 1.5 ± 1.2 0 4.2 ± 3.3 6.9 ± 2.2 4.3 ± 2.0 7.5 ± 3.7 c 2.9 ± 1.3
100 21 14 12 0 35 57 36 62 24
Observations at 22 Weeks Percent of Percent of Papillomas/Mouse Number Percent of Survival Mice with TPA Papillomas
Papillomas by TPA
Table 5. Inhibitory Effects of TA, EA, and GA Derivatives on the Promotion of Mouse Skin
~
---l
---l
ABSTRACT Naturally occurring plant phenols with antimutagenic and anticarcinogenic activities were tested for their abilities to inhibit the biochemical and biological effects of the potent tumor promoter 12- O-tetradecanoylphorbol-13-acetate (TPA) in mouse epidermis in vivo. When applied topically to mouse skin, tannic acid (TA), ellagic acid, and several gallic acid derivatives all inhibit TPA-induced ornithine decarboxylase activity, hydroperoxide production, and DNA synthesis, three biochemical markers of skin tumor promotion. Moreover, in the two-step initiationpromotion protocol, the same phenolic compounds also inhibit the incidence and yield of skin tumors promoted by TPA. TA is the most effective of these treatments. Since they are already known to inhibit tumor initiation, the plant phenols protecting against skin tumor promotion by TPA may be universal inhibitors of multistage carcinogenesis. TA and other polyphenols, therefore, might be valuable in cancer therapy and/or prevention.
INTRODUCTION Skin tumorigenesis is a multistage process. 1,2 The sequence of tumor initiation, stage 1 (conversion) and stage 2 (propagation) promotion, and progression is depicted in figure 1 where symbols represent large populations of epidermal stem Plant Polyphenols. Edited by R.W. Hemingway and P.E. Laks. Plenum Press. New York. 1992
783
784
Perchellet
cells. Tumor initiation occurs when normal cells are subjected to sub carcinogenic events triggering DNA mutation. At first, the mutated genes are not expressed (proto-oncogenes), and there is no phenotypic transformation of the initiated cells. However, a few tumor-promoting events, which induce the expression of the mutated genes (oncogenes), convert the initiated cells into dormant tumor cells. The
o~ -sUbcarciiiCiQiiniC=r===::-1
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Response Modification PromotabiHty.: Alterations in Programs of Grow1h and Oifferen- I tiatian, Reslsmce to Cytotoxic AQa'IIs and intercellular Ccmmunicotion. :
I
~~==~~~~~~~T'---I-----At AIrt TIne the initiated Cells May Carry or Acquire AdcItional DNA Lesions, Chromosomal Aberrations
No S1aQe I
:{ ?
Promoter
y·
Lonv-Iasting Events • Convert to PrornoIability· the Cells that Have Been or Be Initiated
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Some of the Initiated Cells Acquire the Critical Alterations (Clone Size? Reqlired for Promolability
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I
.!,
1J
!
Chromosomally t-------'-------------!-
Stage 2 Selective Clonal that Acquired the matability
Po
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v l.ntabIe KII-yotype,
of the initiated Cells Required for Pro-
Chromoaomally Altered Dormant TIIIIOr Cells
- - - - - - -- --
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~ Ca o Po ---;--:--+)
Po
Co
0
Chromasamally Normal Proliferating TIIIIOr Cells (No Malignant Potential)
IBENIGN
TUMORS
Co
'ilal'l0I0I1-.ai
I
Figure 1. The multistage process of skin tumorigenesis. [Figure reproduced by with permission from lSI Atlas of Science: Pharmacol. 2:325 (1988)].2
Antitumor Activity
785
initiating event in skin carcinogenesis causes a genetic alteration in the program ofterminal differentiation. 3 In stage 1, therefore, a few 12-O-tetradecanoylphorbol13-acetate (TPA) treatments may accelerate the differentiation and elimination of the normal keratinocytes and favor indirectly the clonal expansion of the neoplastically transformed cells, which have become resistant to the TPA-induced signals of terminal differentiation. 4 Finally, in stage 2, repeated tumor-promoting events are required to provide some of the dormant tumor cells with a proliferative advantage leading to the formation of a visible skin tumor. Rapidly proliferating neoplastic cells that have only those genetic lesions specific for initiation form benign skin papillomas (Pa). But, at any stage of the process, the cells that acquire other types of DNA lesions and chromosomal aberrations that increase their degree of aneuploidy and malignant potential may progress to invasive skin carcinomas (Ca). Because tumor initiation is irreversible, it is fundamental to the prevention of neoplasia to identify the anti-cancer drugs that are the most effective against the reversible propagation phase of tumorigenesis. Tannic acid (TA), gallic acid (GA), and ellagic acid (EA) have substantial potential for decreasing the risk of tumorigenicity.5,6 Reactive O 2 species (ROS) generated directly or indirectly by chemical carcinogens and tumor promoters are implicated at all stages of skin tumorigenesis. 7 Interestingly, the naturally occurring plant phenols TA and EA are anti-oxidants known to inhibit skin tumor initiation and complete carcinogenesis by polycyclic aromatic hydrocarbons (PARs), but their antitumor-promoting activities have not been tested. 7 EA is a candidate cancer chemopreventive agent 8 because its pharmacology is known, it is inexpensive and well tolerated, it is an anti-oxidant as effective as, or better than, a-tocopherol or tert-butylhydroxyanisole, and it shows inhibitory activity against lipid peroxidation. 5 The inhibition of skin tumor initiation and carcinogenesis by polyphenolic compounds may result from decreased metabolic activation and increased conjugation reactions, as indicated by the findings that TA and EA inhibit epidermal aryl hydrocarbon hydroxylase activity, induce glutathione S-transferase activities, and decrease the formation of ultimate carcinogens and their covalent binding to epidermal DNA. 9 - 18 The ability of polyphenols to bind to DNA and scavenge the ultimate forms of the carcinogens may also playa role in the mechanism by which they inhibit mutagenesis and carcinogenesis.1 9 ,2o Since the abilities of TA and EA to alter the effects of the tumor promoters are unknown, the present study was undertaken to determine whether such plant phenols with antimutagenic and anticarcinogenic activities can also inhibit the biochemical and biological effects of the potent tumor promoter TPA in mouse epidermis in vivo.
BIOCHEMICAL STUDIES The mechanisms by which TPA induces the expression of altered genes and the clonal expansion of the transformed progenitor cells may be mediated, at least in part, through Ca2+ mobilization and protein kinase C (PKC) activation. 21 - 23 The induction of epidermal ornithine decarboxylase (ODC) activity by TPA is an excellent biochemical marker of stage 2 promotion. 1,24 Moreover, prolonged stimulations of hydroperoxide (RPx) production 25 ,26 and DNA synthesis27 in TPA-treated epidermis are required to achieve the propagation phase of skin tumorigenesis. But
786
Perchellet
the magnitude of tumor promoter-induced DNA synthesis may be linked to HPx production rather than ODC induction.28 Each of these three responses, therefore, appears to be essential but not sufficient for tumor promotion. The increased HPxproducing activity of the epidermis may be required in stage 2 to eliminate some populations of normal cells sensitive to free radical-induced DNA strand breaks, terminal differentiation, and cytotoxicity, and facilitate prolonged compensatory waves of DNA synthesis and mitosis in the partly synchronized populations of transformed stem cells that resist and/or survive the oxidative stress caused by the tumor promoters. The first goal was to determine whether TA, EA, and several GA derivatives could inhibit these biochemical events linked to skin tumor promotion by TPA. Female CF-1 mice, 9 weeks old, were used throughout, and their dorsal skins were shaved before experimentation. 29 Solutions of tumor promoters were prepared in acetone and delivered to the shaved backs of individual mice in a volume of 0.2 mL. Multiple tumor promoter treatments were administered at 72-hour intervals. TA and the various GA derivatives (all from Sigma Chemical Co., St. Louis, Missouri) were dissolved in acetone and applied topically in a volume of 0.4 mL at the appropriate times before or after, and to the same area of skin as, each application of tumor promoter. EA (from Sigma) was dissolved in a mixture ofmethanol:acetone (55:45) and administered topically to the skin in 0.4 mL. The 10 J.tmol treatments of EA were delivered in two consecutive applications of 0.4 ml containing 5 J.tmol. Controls were treated with acetone or the above mixture of vehicles only. In every experiment, all mice received the same volume of solvent. Inhibition of TPA-Induced ODe Activity Epidermal ODC activities were determined 5 hours after tumor promoter treatment by measuring the release of 14eo2 from L-[l- 14 C]ornithine-HCI (55 mCi/mmol) essentially as described previously.29 The results (fig. 2) represent the mean values ± SD of two different experiments, each performed in triplicate (with two epidermises/replicate). The Students t test was used to determine the statistical significance of the difference between two sample means. The dose of20 J.tmol ofTA inhibits maximally TPA-induced ODe activity when it is applied 20 min before TPA, and its effectiveness is lost at treatment times further from the time of application of TPA (fig. 2). But TA is able to inhibit the ODC response to TPA when administered over a long period oftime extending from 3 hours before to 1 hour after the application of the tumor promoter. The ability of TA to inhibit TPA-induced ODC activity when applied 20 min before the tumor promoter becomes apparent with 0.5 J.tmol and is clearly dose dependent (fig. 3). The most effective dose of TA, 20 J.tmol, inhibits the ODC response to TPA by at least 85 percent. As reported previously,24,29 epidermal ODC activity peaks 5 hours after TPA treatment (time 0) and returns to basal level at 12 hours (fig. 4). When applied 20 minutes before the tumor promoter, 20 J.tmol of TA inhibit remarkably TPAinduced ODC activity at all time points studied without altering the general time course observed previously.
Antitumor Activity
787
~
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+1
,
I
I
+2
Time Before or After TPA (hours)
Figure 2. Effect of the time of application of TA on TPA-induced ODe activity in mouse epidermis in vivo. TA (20 ILmol) was applied (.) at various times before or after treatment with 8.5 nmol of TPA (time 0). TPA-induced ODe activity at +5 hours in the absence of TA treatment was 8.87 ± 0.68 nmol CO 2 /1 hour/mg protein (100 ± 8 percent, ~). Basal ODe activity in control mice (0.45 ± 0.04 nmol CO 2 ) has been subtracted from the data. a No significant difference versus TPAj bp < 0.0005, significantly smaller versus TPAj cp < .0005, significantly smaller versus TA 40 min before TPAj dp < 0.005, significantly smaller versus TPA. [Figure reproduced with permission from Cancer Res. 51:2820 (1991).41]
So far, the studies of plant phenols as inhibitors of mutagenesis and carcinogenesis have focused on (EA).5,6 Therefore, the treatments with TA, EA, GA, GA lauryl ester (GALE), GA methyl ester (GAME), and n-propyl gallate (PG) were compared for their effectiveness as inhibitors of TPA-induced ODe activity (table 1). TA is by far the most effective treatment for inhibiting the action of TPA as compared with the inhibitory effects of the other compounds tested. EA cannot be tested at 20 J.lmol because of its poor solubility in topically applicable solvents. Doses of 5 J.lmol or more are required to demonstrate the inhibitory effect of EA, which is the least effective treatment against ODe induction by TPA. Although slightly more effective than EA, GA, and its derivatives all inhibit the ODe response to TPA to a much lesser degree than TA. Since the efficacies of ODe induction and skin tumor promotion are maximal when the time interval between repeated TPA treatments is 72 hours,3o it is important to compare the inhibitory effects of TA on ODe induction by single vs. multiple treatments with TPA (fig. 5). The specific activities of epidermal ODe are increased more and more by successive applications of TPA at 72-hour intervals and reach a plateau of maximal stimulation after the third treatment. TA pretreatments inhibit remarkably not only the induction of ODe activity produced by a single application of TPA but also the greater ODe responses elicited by each successive tumor promoter treatment.
788
Perchellet
~ >-
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~
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U III
C
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0.5
2
,
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5102050
Tannic Acid (~mol)
Figure 3. Dose-response curve showing the inhibitory effect of TA on TPAinduced ODe activity in mouse epidermis in vivo. The indicated doses of TA were applied (.) 20 minutes before 8.5 nmol of TPA. TPA-induced ODe activity at +5 hours in the absence of TA treatment was 7.47 ± 0.54 nmol e02/1 hour/mg protein (100 ± 7 percent, ~). Basal ODe activity in controls (0.40 ± 0.03 nmol CO 2) has been subtracted. a No significant difference versus TPA; bp < 0.01, significantly smaller versus TPA; cp < 0.005, significantly smaller versus TPA but no significance versus 0.5 IJmol of TA before TPA; dp < 0.005, significantly smaller versus 50 IJmol of TA before TPA. 41
Finally, TA was tested for its ability to inhibit the ODC responses to other phorbol ester and non-phorbol ester tumor promoters applied twice at a 72-hour interval (table 2). The ODC-inducing activities of several structurally different agents with various tumor-promoting activities have been reported. 24 ,26,28,29,31-38 In general, the phorbol-related diterpene esters and the indole alkaloids with complete and/or stage 2 skin tumor-promoting activities are more potent inducers of epidermal ODC activity than other classes of tumor promoters such as the diacylglycerols, peroxides, anthrones, Ca2+ ionophores, and n-alkanes, which must be applied at much higher doses in order to induce this enzyme activity. Whatever their magnitudes, all these tumor promoter effects are dramatically inhibited by TA. The induction of ODC activity, the first and rate-limiting enzyme in polyamine biosynthesis, is essential but not sufficient for tumor promotion. 1,2,24 All inhibitors of ODC induction are also effective against tumor promotion. 1,2,24,39 The significance of the inhibitory effects of TA, EA, and several GA derivatives on the ODC marker of tumor promotion has been discussed recently.4o,41 The mechanism of this inhibition is unknown. The mice show no stress or discomfort following repeated applications of 20 J.\mol of TA. Cytotoxicity is unlikely since the inhibitory effect
789
Antitumor Activity 9 c
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Figure 4. Time-response curves showing the inhibitory effects ofTA on TPAinduced ODe activity in mouse epidermis in vivo. Mice were treated with (.) or without (O) 20 pmol of TA 20 min before 8.5 nmol of TPA (time O). ODe activity was determined at the indicated times following TPA treatment. Basal ODe activity in controls (0.43 ± 0.05 nmol e0 2 /1 hour/mg protein) has been subtracted. tip < 0.0005, significantly greater versus control; b no significance versus TA + TPA at 7.5 hours but P < 0.025, significantly greater versus TA + TPA at 10 hours; cp < 0.05, significantly smaller versus TPA at 5 hours; dp < 0.005, significantly smaller versus TPA at 10 hours and greater versus TA + TPA at 12.5 hours; e no significance versus control or TA + TPA at 12.5 hours.41
of TA is rapidly reversible (fig. 2), and similar TA and EA treatments enhance the activities of other epidermal enzymes. 12 ,17 Macroscopic examination of the skins collected after single or multiple TA + TPA treatments shows that they have been very well protected from the hyperplastic and inflammatory activities of the tumor promoter as compared with the skins receiving TPA alone. Moreover, TA applied within 10 minutes of the time of sacrifice (5 hours after TPA treatment) alters neither the pH of the ODC-containing supernatant nor the activity of the TPAinduced enzyme, suggesting that the observed inhibitory effects are not simply due to traces of plant phenols remaining in the epidermal extracts. TPA induces ODC activity by a signal transduction mechanism involving Ca2 + mobilization, PKC induction, ODe mRNA expression, and de novo protein synthesis. 21 ,23,24,42 The fact that TA is more effective against ODC induction when applied before rather than after TPA treatment (fig. 2) suggests that this compound does not interact directly with the enzyme but interferes with the actions of the
790
Perchellet
Table 1. Comparison of the Inhibitory Effects of Various Plant Phenols on TPA-Induced ODC Activity in Mouse Epidermis In Vivo 41 Treatment G
Control TPA
ODC activity at 5 hours nmol CO 2 /1h/mg Percent of Percent of protein control TPA
(8.5 nmol) + EA (2 pmol) + TA (2 pmol) + EA (5 pmol) + TA (5 pmol) + EA (10 pmol) + GA (10 pmol) + GALE (10 pmol) + GAME (10 pmol) + PG (10 pmol) + TA (10 pmol)
0.43 6.77 7.01 4.19 6.12 3.26 5.29 3.75 3.65 4.72 4.28 2.17
± 0.03 ± 0.56 ± 0.75 b ± 0.31 ± 0.50 c ± 0.40 ± 0.38 ± 0.22d ± 0.20 ± 0.45 e ± 0.36 ± 0.271
100 1574 1630 974 1423 758 1230 872 849 1098 995 505
100 104 59 90 45 77 52 51 68 61 27
GIndicated compounds applied 20 minutes before TPA. bNo significant difference versus TPA cp < 0.05, significantly smaller versus TPA dNo significance versus TPA + GALE (10 /lmol); P < 0.01, significantly smaller versus TPA PG (10 /lmol). eNo significance versus TPA + PG (10 /lmol); P < 0.05, significantly smaller versus TPA + EA (10 /lmol). Ip < 0.0005, significantly smaller versus TPA + GALE (10 /lmol) and TPA + TA (5 /lmol).
+
tumor promoter and/or the molecular pathways regulating enzyme activities. (-)-Epigallocatechin gallate (EGCG) slightly inhibits the specific binding of 3H_ TPA to mouse skin, decreases the number of phorbol ester receptors, and prevents the activation of PKC by teleocidin. 43 On an equal dose basis, TA is the most effective of the polyphenols tested as an inhibitor of TPA-induced ODC activity. TA is also the most potent of the plant phenols at inhibiting epidermal monooxygenase activities and PAH metabolism, and covalent binding to epidermal DNA. 9 ,lO TA pretreatments inhibit the ODCinducing activity of TPA by about 85 percent. That TA inhibits the time course for ODe induction by TPA at each time point studied in figure 4 suggests that this compound does not simply delay the peak of TPA-induced ODe activity. EA is at least 50 percent less effective than TA at inhibiting the ODe response to TPA. Higher, perhaps more effective doses of this compound cannot be tested because they cannot be dissolved and spread in topically applicable solvents. The poor absorption of EA and the oxidative breakdown of its polyphenolic structure may limit the effectiveness of this treatment against tumorigenesis. 6 ,16
Antitumor Activity
791
4
5
Number of Applications of TPA Figure 5. Inhibitory effects of TA on the ODe responses to multiple applications of TPA in mouse epidermis in vivo. Mice were treated with (.) or without (0) 20 I'mol of TA 20 min before each application of 8.5 nmol of TPA repeated at 72-hour intervals. ODe activity was determined 5 hours after the indicated number of TPA treatments. Basal ODe activity in controls (0.38 ± 0.03 nmol e0 2 /1 hour/mg protein) has been subtracted. aNo significance versus third or fourth applications of acetone + TPA; bN 0 significance versus third or fourth applications of TA + TPAY
Because the magnitude of ODC induction almost doubles after the second TPA treatment (fig. 5), it is more accurate to compare the limited ODC-inducing activities of weak tumor promoters after two applications at a 72-hour interval (table 2). Under these conditions, the abilities of several structurally different tumor promoters to induce ODC activity reflect their tumor-promoting activities. The order of effectiveness of the phorbol esters is TPA > phorbol-12,13-didecanoate > phorbol-12,13-dibenzoate. 12-Deoxyphorbol-13-tetradecanoate is sixteenfold more irritant but induces ODC activity (table 2) and skin tumor promotion44 as much as TPA. Mezerein and 12-0-retinoylphorbol-13-acetate are very weak complete tumor promoters but induce ODC activity as much as TPA and are excellent in stage 2 of promotion. 1,2 However, the PKC activator and stage 2 tumor promoter, 1,2dioctanoyl-sn-glycerol,45 is a weak inducer of ODC activity (table 2).33 Although the Ca2+ ionophore A23187 is a significant ODC inducer (table 2) that stimulates HPx production and DNA synthesis almost as much as TPA and mezerein,26,28 its tumor-promoting activity has only been demonstrated in stage 1. 1 The synthetic analogs of the teleocidin A type,46 (-)-indolactam V and (-)-7-octylindolactam V, which are moderate and potent activators of PKC and bind to the same site on
792
Perchellet
Table 2. Inhibitory Effects of TA on the ODe Responses to Various Tumor Promoters in Mouse Epidermis In Vivo U Treatment"
ODC activity at 5 hours nmol C02 /lh/mg Percent of Percent of protein control tumor promoter
Control TPA (8.5 nmol) +TA 12-Deoxyphorboll3tetradecanoate (8.5 nmol) Mezerein (8.5 nmol)
+TA +TA
12- 0-Retinoylphorbol13-acetate (8.5 nmol) Phorbol-12,I3-didecanoate (8.5 nmol) Phorbol-12,I3-dibenzoate (8.5 nmol) A23187 (0.2 ttmol)
+TA +TA +TA +TA
1,2-Dioctanoyl-snglycerol (5 ttmol) (-)-Indolactam V (85 nmol) (- )-7-0ctylindolactam V (8.5 nmol) H 20 2 (100 ttmol)
+TA +TA +TA +TA
Benzoyl peroxide (100 ttmo1) Anthralin (250 nmol) Chrysarobin (250 nmol) n-Dodecane (25 mg)
+TA +TA +TA +TA
0.35 ± 0.04 9.21 ± 0.82 2.64 ± 0.16 9.77 ± 0.64b 2.26 ± 0.12 10.89 ± 1.02 c 2.39 ± 0.18 10.45 ± 1.18d 1.82 ± 0.30 5.28 ± 0.52 2.16 ± 0.36 1.82 ± 0.17 0.81 ± 0.10 1.58 ± 0.10 0.55 ± 0.03 1.20 ± 0.19 0.67 ± 0.14 4.49 ± 0.29 0.83 ± 0.07 7.55 ± 0.83 e 1.31 ± 0.22 1.44 ± 0.12 0.85 ± 0.13 0.89 ± 0.09 0.63 ± 0.031 1.95 ± 0.15 0.81 ± 0.05 0.91 ± 0.08 0.43 ± 0.039 2.82 ± 0.26 2.12 ± 0.36h
100 2630 754 2791 646 3111 683 2986 520 1509 617 519 231 451 157 343 191 1282 237 2157 374 411 243 254 180 556 231 260 123 806 606
100 26 100 20 100 19 100 15 100 37 100 31 100 16 100 38 100 12 100 13 100 46 100 52 100 29 100 15 100 72
"Treatment applied twice at a 72-hour interval (20 ttmol of TA. applied 20 minutes before each tumor promoter). bNo significance versus TPA. cp < 0.01, significantly greater versus TPA. dp < 0.05, significantly greater versus TPA. ep < 0.005, significantly smaller versus TPA. fp < 0.0005, significantly smaller versus benzoylperoxide. 9p < 0.005, significantly greater versus control. hp < 0.005, significantly smaller versus n-dodecane.
the enzyme at which the phorbol esters act, also induce remarkably ODC activity (table 2) in relation with their binding affinities for the TPA receptor. 38 The free radical generator benzoyl peroxide, which induces epidermal PKC and xanthine
Antitumor Activity
793
oxidase activities, hyperplasia, and morphological changes similar to those caused by TPA, is a good tumor progress or but a weak inducer of ODe activity in relation with its weak tumor-promoting activity.1,31,32,47-50 The alkane n-dodecane, a weak tumor promoter active primarily in stage 2, produces very little inflammation or ODC induction after a single 50 mg treatment, but its hyperplastic activity after four 50 mg treatments is comparable to that of TPA or mezerein. 37 ,51 But a dose of 25 mg of n-dodecane applied twice at a 72-hour interval is a rather good inducer of ODC activity (table 2). Since the magnitude and time course for the effects of anthralin and chrysarobin on epidermal ODC activity and polyamine and DNA synthesis are very different from those of TPA and mezerein, the mechanism by which the anthrones promote skin tumors may be somewhat different from that of the diterpenes. 34 - 36 In any case, the ability of TA to inhibit all the ODC responses elicited by such different tumor promoters suggests that 1) the inhibitory effects of the polyphenols on ODC induction are not specific for the TPA-altered enzyme, and 2) the plant phenols are likely to inhibit other biochemical events linked to skin tumor promotion by these various agents.
Inhibition of TPA-Induced HPx Production The evidence for the role of ROS in tumor promotion has been reviewed. 2,7,52,53 Briefly, 1) tumor promoters increase the generation and decrease the degradation of ROS, 2) organic peroxides and free radical-generating systems exhibit tumorpromoting activities and mimic or enhance some of the molecular events linked to tumor promotion, and 3) various antioxidants and free radical scavengers inhibit the biochemical and biological effects of the tumor promoters. The HPx response linked to skin tumor promotion has been characterized recently.25,26,28 Complete and stage 2 tumor promoters increase remarkably the HPx-producing activity of the epidermis while decreasing its natural antioxidant protection. Initiated and neoplastically transformed epidermal cells may become resistant to ROS-induced DNA strand breakage and terminal differentiation. 54 TPA-induced HPx production, therefore, might accelerate the differentiation and elimination of the normal stem cells that are sensitive to oxidative stress and trigger the prolonged compensatory stimulation of DNA synthesis and tumor cell proliferation required to achieve the propagation phase of skin tumorigenesis. The following study was initiated to determine if the antioxidant activities of EA and other plant phenols would enable them to inhibit the HPx marker of skin tumor promotion. The epidermal production of HPx was determined 16 hours after the last of two applications of TPA at a 48-hour interval (table 3), a time when maximal TPA-induced HPx formation is observed. 26 ,28 The HPx-producing activities of the epidermal homogenates were assayed at acid pH by a modification of the ferrithiocyanate method essentially as described previously.26,28,55 The absorption of the red ferrithiocyanate complex formed in the presence of peroxide was measured by double-beam spectrophotometry against a reagent blank at 480 nm, and the levels of HPx were quantitated with reference to calibration curves prepared under similar conditions with standards of H20 2 ranging from 10 to 200 j.tM. The results are the means ± SD of duplicate determinations of HPx levels from four groups of
794
Perchellet
Table 3. Comparison of the Inhibitory Effects of Various Doses of TA and EA on TPA-Induced HPx Production in Mouse Epidermis In Vivo Treatment ll
HPx formation at 16 hours nmol H2 0 2 /4h/ Percent of Percent of TPA mg protein control
Control
13.0
± 0.9
100
+ TA (1 #Lmol) + TA (2 #Lmol) + TA (5 #Lmol) + TA (10 #Lmol) + TA (20 #Lmol)
36.2 27.5 23.9 21.1 15.2 13.1
± 4.1 ± 3.4b ± 2.1 ± 1.9 ± 1.0c ± 0.5 d
278 211 184 162 117 101
100 62 47 35 9 0
+ EA (0.05 #Lmol) + EA (0.1 #Lmol) + EA (0.2 #Lmol) + EA (0.5 #Lmol) + EA (1 #Lmol)
31.7 28.6 21.9 14.4 13.0
± 3.3 e ± 1.5 b ± 1.5 ± 0.7' ± 0.3 d
244 220 168 111 100
81 67 38 6 0
TPA (8.5 nmol)
IITreated twice at a 48-hour interval (dose/application). bp < 0.025, significantly smaller versus TPA. cp < 0.025, significantly greater versus control. dNo significant difference versus control. eNo significant difference versus TPA. Jp < 0.05, significantly greater versus control.
mice in two different experiments; each group contained the combined epidermal homogenates prepared from two mice. The t test is used. When applied 20 minutes before each tumor promoter treatment, TA and EA inhibit totally the HPx response to TPA (table 3). These inhibitory effects are dose dependent. On an equal dose basis, EA is at least 10 times more potent than TA as an inhibitor of TPA-induced HPx production. Except PG, GA derivatives are all less effective than TA at inhibiting the HPx response to TPA (data not shown). EA and TA are antioxidants so potent that they can inhibit totally the HPx response to TPA even when they are applied 8 hours before or after the tumor promoter (data not shown), a period of time much longer than that observed for their effectiveness against ODe induction (fig. 2). Antioxidants generally inhibit the ODe-inducing activity of TPA. 7 ,31,32 ODe induction and HPx production are both essential for stage 2 promotion. However, there is no apparent correlation between oxidant generation and ODe induction since the HPx response to TPA does not require ODe induction and is not essential for ODe induction. 26,28 This hypothesis is substantiated by the finding that topical applications of doses of EA < 2 pmol, which are not sufficient to affect the ODe response to the tumor promoter (table
795
Antitumor Activity
1), inhibit totally TPA-induced HPx production in mouse epidermis in vivo (table 3). Therefore, there is no indication that the polyphenols inhibit the induction of ODC activity by TPA because of their antioxidant activities.
Inhibition of TPA-Induced DNA Synthesis The alteration of epidermal DNA synthesis by the complete and stage 2 tumor promoters, TPA and mezerein, in vivo, is characterized by an early 12-hour period of inhibition followed by two peaks of maximal stimulation at about 16 and 32 hours. 27 ,36,56 Moreover, the conversion phase of carcinogenesis elicited when TPA is used as a stage 1 promoter either shortly before of after tumor initiation (fig. 1) has been shown to require undisturbed DNA synthesis at 18 hours.57 Therefore, EA and TA were tested for their abilities to alter the integrity ofthe TPA-induced DNA synthesis process (table 4). The rate of incorporation of [methyl-3H]thymidine (59 Ci/mmol) into epidermal DNA was determined 16 hours after TPA treatment. The mice received 30 /lCi of [3H]thymidine i.p. and were killed after a 40-min period of pulse-labeling. The DNA contained in the epidermal homogenates prepared from two mice was extracted, and hydrolyzed from the acid-insoluble precipitate with 3 ml of 0.5N HCI0 4 for 15 min at 90 °C. 28 ,32,56 The radioactivity incorporated into each sample was determined by liquid scintillation counting in 0.2-mL aliquots of the above hydrolysates. The DNA content of each sample was determined by the diphenylamine procedure with calf thymus DNA as the standard. 58 Results are the means ± SD of two different experiments, each performed in triplicate (with
Table 4. Inhibitory Effects of EA and TA on TPA-Stimulated DNA Synthesis in Mouse Epidermis In Vivo Treatment B
[3H]Thymidine incorporation into epidermal DNA at 16 hours cpm//-tg DNA
Percent of TPA
48.6
± 6.8
100
(8.5 nmol)
233.2
± 27.1
480
100
+ EA (5 /-tmol)
173.4
± 15.9 b
357
68
+ TA (5 /-tmol)
138.7
± 17.5 c
285
49
Control TPA
Percent of control
BEA and TA applied 1 hour after TPA. bp < 0.005, significantly smaller versus TPA. cp < 0.005, significantly smaller venms TPA + EA.
796
Perchellet
two epidermises/replicate). The t test is used. As shown in table 4, EA and, to a greater degree, TA, both inhibit the peak stimulation of epidermal DNA synthesis observed 16 hours after TPA treatment.
TUMOR EXPERIMENT Finally, the TA, EA, and GA derivative treatments shown to inhibit the ODC, HPx, and DNA markers oftumor promotion were tested for their abilities to inhibit the tumor-promoting activity of TPA. The development of skin tumors requires repeated applications of a tumor promoter to initiated skin. 1 ,2 Because the biochemical, histological, and morphological effects triggered by TPA on mouse skin are maximal after three to five applications of this compound,26,28,29,32,49,59 the ability of TA to inhibit to about the same degree the greater ODC and HPx responses elicited by each successive application of TPA suggests that TA may also decrease the biological activity of chronic TPA treatment in a two-stage initiation-promotion protocol. 'Tumors were initiated in all mice by a single topical application of 0.1 "mol of 7,12-dimethylbenz[a]anthracene in 0.2 ml of acetone. Two weeks following initiation, all mice were promoted twice a week (on days 1 and 4) with 8.5 nmol ofTPA in 0.2 ml of acetone for the duration of the experiment. The various doses of TA, EA, and GA derivatives were all applied in 0.4 mL of solvent 20 minutes before each promotion treatment with TPA. Initially, there were 36 mice in each treatment group. The incidence of skin tumors was recorded weekly. Statistical analyses of the differences between the means of papillomas per mouse were performed using Student's t test, whereas, differences between papilloma incidences were compared using the X2 statistic. The level of significance was set in both cases at P ~ 0.05. After 22 weeks, all the plant phenols tested inhibit remarkably both the incidence and the yield of skin papillomas promoted by TPA (table 5). In all treatment groups, TA, EA, and the GA derivatives inhibit the number of tumors/mouse to a greater degree than the number of mice with tumors. Overall, TA is the best inhibitor, and in a dose-dependent manner, of the tumor-promoting activity of TPA. At 20 "mol, TA affords total protection against TPA promotion. On an equal dose basis, EA is less potent than TA at inhibiting the number of papillomas/mouse. With the exception of PG, GA and its derivatives are the least effective inhibitors of tumor promotion. That chronic applications of plant phenols do not induce toxic effects in vivo is suggested by the fact that, after 22 weeks of promotion, the weight and rate of survival of the animals treated twice a week with 2.5 to 20 /lmol of TA, EA, GA, GALE, GAME, or PG are identical with those of mice receiving TPA only. ODC induction in early stage 2 and the subsequent activation of polyamine biosynthesis have been suggested to complement the HPx-mediated events in order to trigger enough DNA synthesis and compensatory cell proliferation in late stage 2 to achieve tumor propagation. 28 This might explain why there is a correlation between the antitumor-promoting activities of the compounds tested and the mean values of their inhibitory effects on ODC induction plus HPx production. For instance, 5 /lmol ofTA inhibit ODC induction to a greater degree than a similar
TPA 8.5 nmol) + TA (2.5 I'mol) + TA (5 /-Imol) + TA (10 I'mol) + TA (20 I'mol) + EA (5 /-Imol) + GA (20 I'mol) + GALE (10 I'mol) + GAME (20 I'mol) + PG (20 I'mol)
Promotion Treatment a
31.5 30.7 30.8 31.6 30.7 31.6 32.4 32.9 32.6 31.1
(g)
Weight/ Mouse
aTwo treatments per week dose application. bp < 0.005, significantly smaller vs. group 1. cp < 0.05, significantly smaller vs. group 1.
1 2 3 4 5 6 7 8 9 10
Groups
100 97 97 100 97 100 100 100 97 100
97 51 51 30 0 50 83 b 78 79 58
12.1 ± 1.5 2.5 ± 1.1 1.7 ± 1.0 1.5 ± 1.2 0 4.2 ± 3.3 6.9 ± 2.2 4.3 ± 2.0 7.5 ± 3.7 c 2.9 ± 1.3
100 21 14 12 0 35 57 36 62 24
Observations at 22 Weeks Percent of Percent of Papillomas/Mouse Number Percent of Survival Mice with TPA Papillomas
Papillomas by TPA
Table 5. Inhibitory Effects of TA, EA, and GA Derivatives on the Promotion of Mouse Skin
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