Enhancement by Methionine Enkephalin of Colon

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[CANCER RESEARCH 51. 785-788. February 1, 1991] Enhancement by Methionine Enkephalin of Colon Carcinogenesis Induced by Azoxymethane Hiroyasu lishi,1 Masaharu Tatsuta, Miyako Baba, Shigeru Okuda, and Haruo Taniguchi

Departments of Gastrointestinal Oncology [H. I., M. T., M. B.], Gastroenterology fS. O.], and Pathology [H. T.], The Center for Adult Diseases, Osaka 3-3, Nakamichi 1-chÃ´me,Higashinari-ku, Osaka 53 7, Japan

ABSTRACT Experimental Design. The animals were randomly divided into four groups of 25 rats each and were given ten weekly s.c. injections of 7.4 The effect of the opioid receptor agonist methionine enkephalin (Met- mg/kg of body weight of AOM (Sigma Chemical, St. Louis, MO) in enkephalin) and the opioid receptor antagonist naloxone on colonie 0.9% NaCl solution. From the start of administration of the carcinogen, carcinogenesis induced by azoxymethane was investigated in Wistar rats. the rats also received the following injections on alternate days until Rats received ten weekly injections of 7.4 mg/kg of body weight of the end of the experiment: Group 1, olive oil, 1 ml/kg of body weight; azoxymethane and injections of Met-enkephalin (50 Mg/kg of body Group 2, Met-enkephalin, 50 Mg/kgof body weight; Group 3, naloxone, weight), naloxone (2 mg/kg of body weight), or Met-enkephalin (50 MK/ 2 mg/kg of body weight; and Group 4, Met-enkephalin, 50 Mg/kg of kg of body weight) plus naloxone (2 mg/kg of body weight) once every 2 body weight plus naloxone, 2 mg/kg of body weight. days. In wk 40, the group treated with Met-enkephalin had a significantly Met-enkephalin (Sigma), naloxone (Sigma), and Met-enkephalin increased incidence of colonie tumors. A combination of Met-enkephalin plus naloxone were injected s.c. as suspensions in a volume of 1 ml/kg and naloxone attenuated the enhancing effect by Met-enkephalin on the of body weight of olive oil, between 2 and 3 p.m. each day, various sites development of colonie tumors. Administration of naloxone alone had no being chosen for injections. Group 1 received injections of equal influence on colonie tumorigenesis. During and after administration of amounts of the vehicle (olive oil) only. the carcinogen, the bromodeoxyuridine-labeling indices of the colon Histological Observations. Five rats in each group were sacrificed in mucosa and/or cancers were significantly increased in rats treated with experimental wk 8 for determination of the labeling index of the colonie Met-enkephalin. However, a combination of Met-enkephalin and nalox mucosa. Other animals were killed if they became moribund, and all one significantly decreased the labeling indices of the colon mucosa and/ survivors were sacrificed at the end of wk 40. All the animals sacrificed or cancers. These findings indicate that Met-enkephalin enhanced colon during the study were necropsied, and the internal organs were carefully carcinogenesis and that naloxone attenuated this enhancement. Because examined. The body weight and colon length were measured. The large naloxone is an opioid receptor antagonist, these findings also indicate intestine was opened, pinned flat on a cork mat, and fixed with buffered that the enhancing effect of Met-enkephalin on colon carcinogenesis may picric acid-formaldehyde solution (7). The fixed colon was cut into five be mediated through opioid receptors. segments of equal length, which are referred to hereafter as Part 1 (adjacent to the anal orifice) to Part 5 (adjacent to the cecum). Tumor- bearing areas and areas suspected of having lesions were excised and INTRODUCTION embedded in paraffin. Semiserial sections 5 Mmthick were cut to expose the central part of the tumor or the stalk, when present, and were Opioid peptides, enkephalins, and endorphins, originally iso stained with hematoxylin and eosin. In addition to tumors, flat mucosa lated from the brain, have been detected immunocytochemically from each segment of the fixed colon with no visible tumors was cut and radioimmunologically in the digestive system and shown into two strips 3 mm wide, which were embedded in paraffin. Thin to be localized in the nerve fibers of the myenteric plexus (1). sections were prepared and were inspected microscopically for tumor Although the physiological role of endogenous opiates is still foci. All sections were examined without knowledge of which group unclear, recent investigations demonstrate the involvement of they were from. endogenous peptides in regulation of tumor growth (2). The Classification of Colon Tumors. Colon tumors induced by AOM were classified histologically into adenomas, CIS, and adenocarcinomas (8). results were conflicting: opioid peptides were found to exert The adenocarcinomas were further classified as either well-differen either a stimulatory (3-5) or an inhibitory (6) effect on tumor tiated or mucinous carcinomas. growth, depending on the experimental conditions. However, Measurement of Labeling Indices of Colon Mucosa and Colon Can they suggested that Met-enkephalin2 might affect colon carci cers. The labeling indices of colon mucosa and/or colon cancers were nogenesis. To test this possibility, we examined the effect of measured in wk 8 and 40 with an immunohistochemical analysis kit Met-enkephalin on the development of colonie tumors induced for assaying the incorporation of bromodeoxyuridine (Becton-Dickin- by AOM in Wistar rats. son, Mountain View, CA) (9, 10), by the modified method described by Tada et al. (Il ). For this, the rats were fasted for 12 h and then received s.c. injections: Group 1, olive oil, 1 ml/kg of body weight; MATERIALS AND METHODS Group 2, Met-enkephalin, 50 Mg/kgof body weight; Group 3, naloxone, 2 mg/kg of body weight; and Group 4, Met-enkephalin, 50 Mg/kg of Animals. One hundred 7-wk-old male Wistar rats were purchased body weight plus naloxone, 2 mg/kg of body weight. One h later, the from SLC, Japan (Shizuoka, Japan). The animals were housed in animals received an i.p. injection of bromodeoxyuridine of 20 mg/kg suspended, wire-bottomed metal cages in animal quarters with con trolled temperature (21-22Â°C),humidity (30 to 50%), and light (12-h of body weight, and after another hour they were sacrificed with ether. The colon was removed and fixed in 70% ethanol for 4 h. Parts 2 cycle), and they had free access to tap water and regular chow pellets (distal portion) and 4 (proximal portion) of the colon were then excised (Oriental Yeast, Tokyo, Japan). and embedded in paraffin. Received 8/1/90; accepted 11/9/90. For analysis of the labeling indices of colon mucosa and colon The costs of publication of this article were defrayed in part by the payment cancers, the numbers of bromodeoxyuridine-labeled and unlabeled cells of page charges. This article must therefore be hereby marked advertisement in in the zone of proliferating cells were counted (12) without knowledge accordance with 18 U.S.C. Section 1734 solely to indicate this fact. 1To whom requests for reprints should be addressed. of which treatment group the samples were from. For analysis of the 2The abbreviations used are: Met-enkephalin, methionine enkephalin; AOM, labeling index of colon cancers, the numbers of bromodeoxyuridine- azoxymethane; CIS, carcinoma in situ; GABA, 7-amino-n-butyric acid. labeled and unlabeled cancer cells in the cancerous colon lesions were 785

Downloaded from cancerres.aacrjournals.org on October 1, 2021. © 1991 American Association for Cancer Research. MET-ENKEPHALIN ENHANCEMENT OF COLON CANCERS counted. On the basis of these measurements, we derived the labeling bined administration of Met-enkephalin and naloxone de index as the number of bromodeoxyuridine-labeled cells/the total num creased the incidence of well-differentiated adenocarcinomas, ber of cells within the zone of proliferation or colon cancer lesion. Statistical Analysis. Data were analyzed by the x2 test. Fisher's exact but the difference was not statistically significant. probability test, or by one-way analysis of variance with Dunn's multiple Colon Length and Labeling Indices of Colon Mucosa and Colon comparison (13-15). Data are given as the mean Â±SE. Differences Cancers. Table 3 summarizes data on the colon length and bromodeoxyuridine-labeling indices of the colon mucosa and were regarded as significant when the calculated P value was less than 0.05. colon cancers in wk 8 and 40. At both times examined, there was no significant difference in the colon lengths in the four groups. Table 3 also shows that administration of Met-enkeph RESULTS alin (Group 2) led to a significant increase in the labeling indices Incidence and Number of Colon Tumors. Animals that sur of both parts of the colon mucosa and/or of colon cancers at vived for more than 36 wk were included in the effective both times examined than in Group 1 (olive oil). However, the numbers because the first colon tumor was found in a rat in concomitant use of Met-enkephalin and naloxone (Group 4) Group 1 that died in wk 36. Three, one, one, and one rat in significantly reduced the labeling indices of colon mucosa and/ Groups 1 to 4, respectively, were killed before wk 36 because or colon cancers, as compared with those in Group 2 (Met- they became moribund. No tumors were found in any of these enkephalin). animals, which were excluded from the effective numbers. The incidence of colon tumors and their number per rat and DISCUSSION the body weight in each group are summarized in Table 1. At wk 40, the animals that had received either Met-enkephalin or The data presented in this paper show that the opioid peptide naloxone had slightly, but not significantly, lower body weights Met-enkephalin enhanced colon carcinogenesis induced by than did the olive oil-treated rats. AOM in Wistar rats. Treatment of rats with Met-enkephalin In Group 1 (olive oil only), colon tumors were found in 10 in depot form every other day from the start of the experiment (59%) of 17 rats examined. In Group 2 (Met-enkephalin), the led to a significant increase in the incidence of colon cancers in incidence of colon tumors was significantly higher than that wk40. in Group 1. However, concomitant administration of Met- The mechanisms involved in this effect of Met-enkephalin enkephalin and naloxone (Group 4) significantly reduced the are not fully understood, but five possible direct and indirect incidence of colon tumors as compared with that in Group 2. mechanisms may be considered. One possibility would involve Administration of naloxone alone (Group 3) had little or no an inhibitory effect on acetylcholine release in the central and influence on the incidence of colon tumors. In Group 1, the peripheral nervous systems (16, 17). Vizi et al. (18) found that average number of colon tumors per rat was 0.7 Â±0.2. Admin Met-enkephalin inhibited the release of [14C]acetylcholine from istration of Met-enkephalin (Group 2) slightly increased the the myenteric plexus in a dose-related fashion. Acetylcholine number of colon tumors per rat, but the increase was not has been demonstrated to be capable of influencing cell division statistically significant. (19). Tutton (20) showed that cholinoceptor stimulation, either HistolÃ³gica! Types of Colon Tumors. The distribution of dif by injection of carbachol or by inhibition of acetylcholinester- ferent histolÃ³gica! types of colon tumors is summarized in Table ase, resulted in an increase in the mitotic rate in the crypts of 2. There was no significant difference in the distributions of LieberkÃ¼hnin rat jejunum. Gurkalo and Volfson (21) suggested adenomas, CIS, and adenocarcinomas in the four groups. How that pharmacological compounds that enhanced the activity of ever, Table 2 shows that, in Group 1 (olive oil), 22% of the the sympathetic nerves stimulated carcinogenesis, whereas adenocarcinomas were of the well-differentiated type, whereas those that enhanced cholinergic functions inhibited carcinogen in Group 2 (Met-enkephalin), well-differentiated adenocarci esis. We recently found that the long-term administration of nomas were significantly more frequent than in Group 1. Corn- the parasympathomimetic compound neostigmine resulted in a significant decrease in the incidence and number per rat of Table1 IncidenceandnumberofcolontumorsinAOM-treatedrats colon tumors in wk 40 (22). Moreover, we also found that (g)Group Body wt of rats prolonged administration of parasympatholytic atropine re Iffective with colon of tu Treatment"1 wk381 no.tumors17 mors/rat0.7 sulted in a significant increase in the number of gastric cancers oil Â± Â± Â±0.2 (23). 2 Met-enkephalin 167 Â±3 377 Â±7 19 18(95)'' 1.3 Â±0.2 A second mechanism may involve an effect on the secretion 3 Naloxone 168 Â±2 376 Â±8 19 12(63) 0.8 Â±0.2 of anterior pituitary hormones. Endogenous opioid peptides 4OliveMet-enkephalin + 166 Â±2"340389 Â±76No. 1910(59)c10(53)'No. 0.8 Â±0.2 naloxoneInitial164 participated in control of anterior pituitary hormone function. "Treatment regimens: In Group 1 (olive oil), rats were given 10 weekly s.c. Endogenous opioid peptides (24, 25) can increase pituitary injections of 7.4 mg/kg of body weight of AOM and were also given s.c. the prolactin and growth hormone secretion and reduce gonadotro- vehicle (olive oil) every other day until the end of the experiment. In Group 2 pin and thyrotropin secretion. Dorchester and Haist (26) found (Met-enkephalin), rats were given 10 weekly s.c. injections of 7.4 mg/kg of body weight of AOM and were also given s.c. 50 fig/kg of body weight of Met- that the weight and secretin content of atrophie small intestine enkephalin in olive oil every other day until the end of the experiment. In Group of hypophysectomized rats were increased by multiple injec 3 (naloxone). rats were given 10 weekly s.c. injections of 7.4 mg/kg of body tions of growth hormone, corticotropin, or anterior pituitary weight of AOM and were also given s.c. 2 mg/kg of body weight of naloxone in olive oil every other day until the end of the experiment. In Group 4 (Met- extracts. These hormones may affect cancer growth. enkephalin + naloxone), rats were given 10 weekly s.c. injections of 7.4 mg/kg of A third mechanism is an effect on GABA. Brennan et al. (27) body weight of AOM and were also given s.c. 50 /ig/kg of body weight of Met- showed that Met-enkephalin provided a very potent stimulus enkephalin and 2 mg/kg of body weight of naloxone in olive oil every other day until the end of the experiment. for the release of GABA from nerve endings, and that the * Mean Â±SE. stimulation of release by Met-enkephalin was not prevented by c Numbers in parentheses, percentage. **Significantly different from the value for Group 1 at P < 0.05. naloxone. The dose of naloxone used in this experiment is the 'Significantly different from the value for Group 2 at P< 0.01. upper range for specific opiate antagonism, and it is likely that 786

Table 2 HistolÃ³gica! types and depth of involvement of colon tumors in AOM-treated rats ColontumorHistolÃ³gica! typeGroup typeWell involvementSubmucosalof differ layer Treatment"1 no.12 cinoma9(75) no.9 entiated2(22) layer2(22) or deeper7(78) Olive oil (25)' 2 Met-enkephalin 25 8(32) 1(4) 16(64) 167 12(75)c 4(25) 4(25) 12(75) 3 Naloxone 16 7(44) 2(12) 7(44) 4(57) 3(43) 2(29) 5(71) 4 Met-enkephalin + naloxoneTotal 15Adenoma38(53)CIS0(0)1(7)Adenocar-6(40)Total 6AdenocarcinomaHistolÃ³gica!2(33)Mucinous7(78)4(67)Depth 2(33)Muscle 4(67) " For an explanation of treatments, see Table 1. * Numbers in parentheses, percentage. ' Significantly different from the value for Group 1 at P < 0.02.

Table 3 Colon length and labeling indices of colon mucosa and colon tumors Bromodeoxyuridine-labeling index Experimental Colon length wk Group Treatment" (cm) Distal portion Proximal portion Cancer 840!2341234Olive oilMet-enkephalinNaloxoneMet-enkephalin Â±0.3*24.5 Â±0.010.43 Â±0.325.0 (b)f0.020.02 Â±0.02(a)0.33 Â±0.425.8 Â±0.010.31 naloxoneOlive + Â±0.326.8 (a)"0.010.01 Â±0.02(d)0.28

oilMet-enkephalinNaloxoneMet-enkephalin Â±0.326.4 Â±0.010.40 Â±0.020.43 Â±0.226.4 (b)0.010.21 Â±0.02(b)0.27 (a)0.33Â±0.03 Â±0.326.6 Â±0.010.28 Â±0.020.30 + naloxone25.5 Â±0.20.300.420.290.270.210.340.200.020.020.01 (d)0.34 Â±0.01 (d)0.31 Â±0.03 (c) " For an explanation of treatments, see Table 1. * Mean Â±SE. ' Significantly different from the value for Group 1: (a) P < 0.01; (b) P< 0.001. d Significantly different from the value for Group 2: (c) P < 0.01; (d) P < 0.001. at the dose used, naloxone has a number of nonopiate actions. (36) found that opioid peptides, including Met-enkephalin, Naloxone has been reported to activate the central GABAergic exert a strong growth-promoting effect on nervous tissue in system. Recently, however, we examined the effects of GABA, culture. The present study showed that administration of Met- the GABAA receptor agonist muscimol, and the GABAB recep enkephalin significantly increased the bromodeoxyuridine- tor agonist baclofen on the development of gastric cancers labeling indices of the colon mucosa and cancers during and induced by jV-methyl-./V'-nitro-./V-nitrosoguanidine, and found after treatment with the carcinogen AOM. that GABA and baclofen, but not muscimol, inhibit gastric In this work, we found that prolonged administration of Met- carcinogenesis (28). enkephalin enhanced development of colonie tumors, and that A fourth mechanism is an immunomodulation with Met- the opioid receptor antagonist naloxone inhibited the enhancing enkephalin. Opioid peptides modulate both cellular and hu effect of Met-enkephalin. This finding indicates that the effect moral immune function via an opiate receptor-mediated action. of Met-enkephalin may be mediated through opioid receptors. 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Hiroyasu Iishi, Masaharu Tatsuta, Miyako Baba, et al.

Cancer Res 1991;51:785-788.

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