[CANCER RESEARCH 49, 843-846, February 15, 1989] Promotion by Neurotensin of Gastric Carcinogenesis Induced by TV-Methyl-W-nitro-W-nitrosoguanidine in Wistar Rats Masaharu Tat sut a, ' Hiroyasu lishi, Miyako Baba, and Haruo Taniguchi

Departments of Gastrointestinal Oncology [M. T., H. l., M. B,] and Pathology [H. T.], The Center for Adult Diseases, Osaka, 3-3, Nakamichi Ì-chôme, Higashinari-ku, Osaka 537, Japan

ABSTRACT water containing MNNG (25 ^g/ml) for 25 wk. The MNNG (Aldrich Chemical Co., Milwaukee, WI) was dissolved in drinking water at a The effects of neurotensin on the incidence and histology of gastric cancers induced by Ar-methyl-Ar'-nitro-Ar-nitrosoguanidine were investi concentration of 1 mg/ml and kept in a cool dark place. The stock solution was replaced within 1 wk. Just before use, the stock solution gated in Wistar rats. Rats were given 100 or 200 MUper kg of body was diluted to 25 Mg/ml and given to the rats every other day from weight of neurotensin s.c. every other day in depot form after 25 wk of bottles covered with aluminum foil to prevent denaturation of MNNG P.O.treatment with the carcinogen. Prolonged alternate-day administra by light. The MNNG in the stock solution and in the diluted solution tion of neurotensin at 200 ¿tgperkg of body weight resulted in a significant was stable for at least 1 wk and 2 days, respectively (12). From Wk 26, increase in the incidence of gastric cancers of the glandular stomach by the MNNG-treated rats were given normal tap water and divided into Wk 52. However, it did not influence the histológica!appearance of the three groups. The three groups were treated as follows until the end of gastric cancers. Furthermore, it caused a significant increase in the the experiment. Group 1 (25 rats) received the vehicle, olive oil, only. labeling indices of the epithelial cells of the antrum and of gastric cancers. Groups 2 and 3 (25 rats each) received neurotensin in depot form at In contrast, the administration of neurotensin at 100 UKper kg of body dosages of 100 and 200 Mgper kg of body weight per day, respectively. weight had a slight, but not significant, influence on the development of From Wk 26, Group 4 (20 rats) received olive oil only, but was not gastric cancers. These findings indicate that neurotensin promotes gastric treated with MNNG nor neurotensin. Groups 5 and 6 (20 rats each) carcinogenesis, and that this effect may be related to its effect in increas received neurotensin only in depot form at dosages of 100 and 200 /ig ing proliferation of epithelial cells in the antral mucosa and in gastric per kg of weight per day, respectively, but were not treated with MNNG. cancers. Neurotensin (Peptide Institute, Inc., Osaka, Japan) was given as suspension in olive oil. Injections were given s.c. in various sites every INTRODUCTION other day in a volume of 1 ml per kg of body weight, between 2 and 3 Neurotensin is one of the gastrointestinal peptides present in p.m. each day. The rats in Groups 1 and 4 received 1 ml per kg of body brain and gut (1-3). The pharmacological and biochemical weight of plain olive oil, administered as for Groups 2 and 3. The experimental groups were kept in different cages in the same properties of neurotensin strongly suggest that the peptide acts room throughout the experiment and had free access to regular chow as a neurotransmitter or neuromodulator in the central nervous pellets (Oriental Yeast Co., Tokyo, Japan). system and as a hormone in the periphery (4, 5). Specific Animals that survived for more than 45 experimental wk were neurotensin receptors have been characterized not only in brain included in the effective numbers, because the first tumor of the glan and in intact cells of neural or nonneural origin, but also in dular stomach was found in a rat in Group 1 that died in Wk 46. gastrointestinal membrane preparations (6). Thus, neurotensin Animals were killed when they became moribund during the experi has important roles in the physiology and pathophysiology of ment, or at the end of Wk 52. All rats were autopsied, and the stomach the gastrointestinal system. and other organs were carefully examined. The stomach was opened along the greater curvature, pinned flat on a cork mat, and fixed with Recently, a number of reports have appeared regarding the Zamboni's solution (13) for histológica!examination. The fixed stom effect of gastrointestinal peptides on the growth of experimen ach was cut into longitudinal strips 3 mm wide. The specimens were tally induced tumors (7). We previously reported that prolonged administration of tetragastrin in depot form after MNNG2 embedded in paraffin, and serial sections 5 ¿imthickwere stained with hematoxylin and eosin. Sections were examined without knowledge of treatment resulted in a significant reduction in the incidence of which group they were from. gastric cancers in the glandular stomach of Wistar rats (8). Histologically, adenocarcinomas were defined as lesions in which Recently we found that this effect may be closely related to the neoplastic glands had penetrated the muscularis mucosae to involve the effect of gastrin in decreasing cell proliferation of the antral submucosa or deeper layers. As previously reported, the adenocarci mucosa (9). Feurle et al. (IO, 11) found that neurotensin can nomas were classified as highly well differentiated, well differentiated, act as a trophic factor on gastric antrum, leading to an increase or poorly differentiated (9). On the basis of their mucin-producing activity, well-differentiated adenocarcinomas were subdivided into a in the thickness of the gastric antrum. Moreover, Amar et al. (6) identified neurotensin receptors in the human colonie ade- common type and a mucinous type, and poorly differentiated cancers were subdivided into anaplastic and signet-ring cell carcinomas. nocarcinoma cell line. These findings suggest that neurotensin The labeling indices of the gastric mucosa and gastric cancers were affects the growth of gastric cancers. Therefore, in the present examined in Wk 30 and/or Wk 52. The labeling indices were measured work, we examined the effect on the development of gastric with an immunohistochemical analysis kit for assaying BrdUrd incor cancers of treating rats with neurotensin after MNNG treat poration (14,15) (Becton Dickinson Immunocytometry System, Moun ment. tain View, CA), by the modified method described by Tada et al. (16). Briefly, the rats were starved for 12 h and then received one of the MATERIALS AND METHODS following s.c. injections: 1 ml per kg of body weight olive oil (Groups 1 and 4); or 100 or 200 ^g per kg of body weight neurotensin (Groups A total of 135 young male Wistar rats about 5 wk old, initially weighing 90 to 100 g, was used. Seventy-five rats were given drinking 2 and 5 and Groups 3 and 6, respectively). Three h later, the rats received an i.p. injection of 20 mg per kg of body weight of BrdUrd, Received 4/29/88; revised 9/16/88; accepted 11/7/88. and they were killed l h later with ether. Before killing, blood was The costs of publication of this article were defrayed in part by the payment obtained by cardiac puncture for determination of gastrin. The stomach of page charges. This article must therefore be hereby marked advertisement in was fixed in 70% ethanol for 4 h. Sections 3 ^m thick were immersed accordance with 18 U.S.C. Section 1734 solely to indicate this fact. ' To whom requests for reprints should be addressed. in 2 N HC1 solution for 30 min at room temperature and then in 0. l M 2The abbreviations used are: MNNG, /V-methyN/V'-nitro-A'-nUrosoguanidine; Na2B4O7 to neutralize the acid. The sections were then stained with Brill rd, bromodeoxyuridine. anti-BrdUrd monoclonal antibody (diluted 1:100) for 2 h at room 843

Downloaded from cancerres.aacrjournals.org on September 28, 2021. © 1989 American Association for Cancer Research. PROMOTION BY NEUROTENSIN OF GASTRIC CANCERS temperature, washed, stained with biotin-conjugated horse anti-mouse Table 1 Incidence of gastric cancers in each group antibody (at a dilution of 1:200) for 30 min, and stained with avidin- (g)Group123456Treatment"MNNG Body wt of rats biotin-peroxidase complex for 30 min. The reaction product was located no. of with gastric with 3,3'-diaminobenzidine tetrahydrochloride. Cells containing rats181920101010No.cancer5 ±6'337 (28)'10(53)14 BrdUrd were identified by the presence of dark pigment over their oilMNNG+ olive ±6394 nuclei. For analysis of the labeling index of the gastric mucosa, the +neurotensin,lOOxg/kgMNNG±5334 ±9393 numbers of BrdUrd-labeled and unlabeled cells in the zone of prolifer +neurotensin,200 ±3352 ±7425 (70)''0(0)0(0)0(0) ating cells were counted without knowing which treatment group the Mg/kgOlive samples were from (17). The zone of proliferating cells in the fundic oilNeurotensin, ±7359 ±6400 mucosa was defined as a rectangle 250 pm wide between the highest 100Neurotensin, ±9355 ±7408 and lowest labeled cells in a well-oriented section. Ten such rectangular 200Mg/kgWk26335 ±2Wk52402±6Effective areas were selected in each rat. In the antral mucosa, all cells below the highest labeled cell in each pit-gland column were regarded as being " Treatment regimens: MNNG + olive oil, 1 ml/kg of olive oil given every within the zone of the proliferating cells. We selected 100 well-oriented other day after MNNG treatment for 25 wk; MNNG + neurotensin, 100 »ig/kg, column pits and glands in each rat. From these measurements we 100 »ig/kgof neurotensin in depot form given every other day after MNNG derived labeling index (number of BrdUrd-labeled cells/total number treatment for 25 wk; MNNG + neurotensin, 200 ng/kg, 200 Mg/kgof neurotensin in depot form given every other day after MNNG treatment for 25 wk; olive oil, of cells within the zone of proliferating cells). For analysis of labeling 1 ml/kg of olive oil given from Wk 26, but not treated with MNNG; neurotensin, index of the gastric cancers, the numbers of BrdUrd-labeled and unla 100 Mg/kg, 100 Mg/kg of neurotensin in depot form given every other day from beled cells were counted in 20 or more neoplastic glands at the periph Wk 26, but not treated with MNNG; neurotensin, 200 Mg/kg, 200 fig/kg of neurotensin in depot form given every other day from Wk 26, but not treated eral part of the tumors. The labeling index of the cancers is expressed with MNNG. as the number of BrdUrd-labeled cells/total number of cancer cells. * Mean ±SE. Gastric acid secretion and serum gastrin level were assessed in Wk ' Numbers in parentheses, percentage. ''Significance of difference from the value in Group l,P< 0.05. 30 and/or Wk 52. Gastric secretions were collected for 3 h by the method of Shay et al. (18). Briefly, the animals were starved for 24 h and then anesthetized with ether, and the stomach pylorus was ligated. dular stomach were identified by histology as adenocarcinomas. Then the rats received one of the following s.c. injections: 1 ml per kg Almost all were highly well differentiated, and no poorly differ of body weight of olive oil (Groups 1 and 4); or 100 or 200 fig per kg entiated cancers were found in any rat. There were no significant of body weight of neurotensin (Groups 2 and 5 and Groups 3 and 6, differences in the histológica!types of adenocarcinomas among respectively). Three h later, the rats were anesthetized with ether, the the three groups. Table 2 also shows that there were no signif stomach was rapidly removed, and the fluid in the gastric cavity was collected. The volume of fluid was measured, and its acid content was icant differences in depth of involvement of gastric cancers determined by titrating a 2-ml portion with 0.1 N NaOH to pH 7.0 among the three groups. using a glass electrode. Then the acid output was calculated. Labeling Index, Serum Gastrin Level, and Gastric Acid Secre Gastrin content of the serum was assayed with a radioimmunoassay tion. Table 3 summarizes the data on the labeling indices of the kit from Dainabot Radioisotope Laboratories, Ltd. (Tokyo, Japan) gastric mucosa and gastric cancers, the serum gastrin level, and (19). gastric acid secretion in Wk 30 and 52. At both times, Groups Results were analyzed by the x2 test (20) or one-way analysis of 3 (MNNG and neurotensin at 200 Mg/kg) and 6 (neurotensin variance with Dunn's multiple comparison (20-22). Data are given as the mean ±SE. "Significant" indicates a calculated P value of less than at 200 Mg/kg) had a significantly elevated labeling index in the antral mucosa, but not in the fundic mucosa, compared to the 0.05. control Groups 1 and 4, respectively. The gastric cancers in Group 3 also had a significantly elevated labeling index as RESULTS compared to Group 1. However, the labeling indices of antral Incidence of Gastric Cancer. Five rats each of MNNG-treated mucosa in Groups 2 (MNNG and neurotensin at 100 Mg/kg) groups and ten rats each of MNNG-untreated groups were and 5 (neurotensin at 100 Mg/kg) and gastric cancers in Group killed in Wk 30 for determination of the labeling index of the 2 were slightly, but not significantly, higher than in control gastric mucosa, serum gastrin level, or gastric acid secretion. groups. At both times, administration of neurotensin in MNNG- Two rats in Group 1 and one rat in Group 2 died before Wk treated and untreated rats resulted in a dose-dependent increase 40. No tumors were found in these animals, and they were excluded from the effective numbers. in the serum gastrin levels, but the differences were not statis In Wk 52, all rats that had received neurotensin had slightly, tically significant. At both times, administration of neurotensin also caused a dose-dependent decrease in the gastric acid secre but not significantly, lower body weights than the control tion in MNNG-untreated rats, but it caused a dose-dependent groups. increase in the gastric acid secretion in MNNG-treated rats The incidences of gastric cancers in each group are summa rized in Table 1. In Group 1 (MNNG and olive oil), gastric although the differences were not statistically significant. cancers were found in 5 (28%) of 18 rats examined. In Group 3 (MNNG and neurotensin at 200 Mg/kg), the incidence of DISCUSSION gastric cancers was significantly higher than in Group 1. In Group 2 (MNNG and neurotensin at 100 Mg/kg), it was slightly, In the present work, we found that long-term administration but not significantly, higher than in Group 1. No gastric cancers of neurotensin in depot form after MNNG treatment for 25 wk were found in Groups 4 (olive oil alone), 5 (neurotensin at 100 resulted in a significant increase in the incidence of gastric /ig/kg), and 6 (neurotensin at 200 Mg/kg). adenocarcinomas in the glandular stomach and a significant All cancers were found in the antral mucosa, and no métas increase in the labeling index of antral mucosa. The mechanism tases were seen in any rats. of this effect of neurotensin is unknown, although three possible Histológica!Types and Depth of Involvement of Gastric Can explanations may be considered. cers. Table 2 shows data on the incidence of different histolog- The first explanation is the effect of gastrin on alterations in ical types and the depth of involvement of the gastric cancers cell proliferation kinetics. The trophic effects of gastrin on the in MNNG-treated groups. All the tumors induced in the glan- fundic mucosa are well established (23). However, in rats, 844

Downloaded from cancerres.aacrjournals.org on September 28, 2021. © 1989 American Association for Cancer Research. PROMOTION BY NEUROTENSIN OF GASTRIC CANCERS Table 2 Histological types and depth of involvement of gastric cancers in MNNG-treated rats differentiatedGroup Well involvementSubmucosa4(80)of of gastric well propria or Treatment"1 cancers5 differentiated4 type1(20) type0(0) deeper1(20) MNNG + olive oil (80)' 2 MNNG + neurotensin,No. ISHighly 15(100)Common 0(0)Mucinous 0(0)Depth 13(87)Muscularis 2(13)

MNNG + neurotensin, 21 20 (95) 1(5) 0(0) 18(86) 3(14)

" For explanation of treatments, see Table 1. ' Numbers in parentheses, percentage.

Table 3 Labeling indices of gastric mucosa and gastric cancers, serum gastrin levels, and gastric acid secretion index'Autrui gas acid Experimental trin level secretion wk3052Group123456123456Treatment"MNNG mucosa0.20 Cancer0.13mucosa (pg/ml)253 (meq/h)0.070 oilMNNG+ olive ±o.or0.20 ±0.010.15 ±22262 +neurotensin.100Mg/kgMNNG ±0.020.21 ±0.020.22 ±26310

+neurotensin.200 ±0.020.20 e)'0.14±0.02 (a, ±37230 ^g/kgOlive oilNeurotensin, ±0.020.19 ±0.010.15 ±26279 ±0.0060.060 100>"g/kgNeurotensin, +0.020.20 ±0.020.26 ±36325 ±0.0070.049

200eg/kgMNNG ±0.020.20 ±0.02(g)0.12 ±57278 ±0.0070.033

oilMNNG+ olive ±0.010.20 ±0.200.15±0.01 1.31 ±41386 130.041±0.0 +neurotensin,100Mg/kgMNNG ±0.010.20 ±0.240.25±0.01 1.58 ±40406 ±0.0120.063

+neurotensin,200 ±0.020.20 00.1 ±0.01 (b, d) 3.71 ±0.62 (c, ±69241 ±0.0340.062 /ig/kgOlive oilNeurotensin, ±0.020.20 5±0.020.18 ±19267 ±0.0060.055 100*

200Mg/kgFundic ±0.02Labeling ±0.01 (h)Serum ±16Gastric ±0.003

" For explanation of treatments, see Table 1. '' Labeling index was expressed as the number of BrdUrd-labeled nuclei/total number of cells within the zone of proliferating cells for gastric mucosa, and the number of BrdUrd-labeled nuclei/total number of cancer cells examined for cancer. ' Mean ±SE. **Significance of difference from the value in Group 1: P < 0.01 (a); P < 0.001 (b); P < 0.05 (c). Significance of difference from the value in Group 2: P < 0.001 (d); P < 0.01 (e); P < 0.05 (0. Significance of difference from the values in Groups 4 and 5: both, P < 0.001 (g); both, P < 0.01 (h). pentagastrin inhibits normal cell proliferation in the antral These findings suggest that endogenous hypergastrinemia may mucosa (24). We previously found that prolonged administra not be related to the promotion by neurotensin of gastric tion of tetragastrin in depot form caused a significant decrease carcinogenesis, but it is still unclear why neurotensin increased in the incidence and number of gastric cancers induced by the gastric acid secretion in MNNG-treated rats. MNNG and a significant increase in the labeling index of the The second possibility is the effect of neurotensin on intra- fundic mucosa but a significant decrease in that of the antral cellular cyclic GMP. Receptors for neurotensin have been dem mucosa (9). The effect of hypergastrinemia on gastric carcino- onstrated in gastrointestinal membrane preparations (6). Amar genesis is still unclear. We recently found that administration et al. (29) found that neurotensin increases the intracellular of cysteamine caused a significant increase in the serum gastrin cyclic GMP concentration in neuroblastoma N1E-115 cells and level and gastric acid secretion and a significant decrease in the that the cyclic GMP response is mediated by the association of labeling index of the antral mucosa and the incidence of gastric neurotensin with its specific receptors (29). There is much cancer induced by MNNG (25). Deveny et al. (26) reported that evidence that cyclic GMP is involved in the control of growth the endogenous hypergastrinemic state exerts a protective effect and differentiation of various cell types (30, 31). Cyclic GMP against gastric cancer induced by MNNG in rats only when the has been reported to stimulate growth of both fibroblasts and gastric acid secretion is high, but hypergastrinemia associated lymphocytes in culture (32), and increases in cellular cyclic with hyposecretion of acid or achlorhydria has no inhibitory GMP content have been implicated in the initiation of prolif effect on the development of gastric cancers. In the present eration of these cells (32). DeRubertis et al. (33) found that the work, we found that administration of neurotensin resulted in cyclic GMP content of human colon adenocarcinomas was a dose-dependent increase in the serum gastrin level. Infusion greater than that of the surrounding mucosa. of neurotensin i.v. is reported to decrease pentagastrin- or meal- The third possibility is the effects of neurotensin on inositol stimulated gastric acid secretion (27, 28). In the present work, phospholipid metabolism and protein kinase C activation. Sev we found that neurotensin decreased the gastric acid secretion eral neurotransmitters and hormones are known to stimulate in MNNG-untreated rats, but increased acid secretion in the hydrolysis of inositol phospholipid through stimulation of MNNG-treated rats in a dose-dependent manner. However, a polyphosphoinositide-specific phospholipase C, leading to an neurotensin did not cause an increase in the labeling index of increased formation of inositol 1,4,5-triphosphate and inositol the fundic mucosa and decrease in that of the antral mucosa. 1,4-bisphosphate (34, 35). The hydrolysis of inositol phospho- 845

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Downloaded from cancerres.aacrjournals.org on September 28, 2021. © 1989 American Association for Cancer Research. Promotion by Neurotensin of Gastric Carcinogenesis Induced by N-Methyl-N′-nitro-N-nitrosoguanidine in Wistar Rats

Masaharu Tatsuta, Hiroyasu Iishi, Miyako Baba, et al.

Cancer Res 1989;49:843-846.

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