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Enhancement by peptide histidine isoleucine of experimental carcinogenesis in the colon of rats induced by azoxymethane
ELSEVIER
Cancer Letters 94 (1995) 49-54
CANCER LETTERS
Enhancement by peptide histidine isoleucine of experimental carcinogenesis in the colon of rats induced by azoxymethane Hiroyasu Iishi’ a,*,Masaharu Tatsutaa, Miyako Babaa, Kazushige Isekib, Hiroyuki Ueharaa, Akihiko Nakaizumia “Depurtment bDepartment
of Gastrointestinal
Oncology, The Centerfor Adult Diseases, Osaka, 3-3, Nakamirhi I-rhome, Higashinari-ku. Osaka 537, Japan The Centerjor Adult Diseases, Osaka, 3-3, Nakamichi 1-chome, Higashinari-ku, Osaka 537, Japan
qf Gastroenterology.
Received 3 April 1995; accepted 20 April 1995
Abstract
The effects of peptide histidine isoleucine (PHI) on the incidence and histology of colon tumors induced by azoxymethane (AOM), and on the labeling index of colon mucosa were investigated in Wistar rats. Rats received weekly S.C.injections of 7.4 mg/kg body weight of AOM for 10 weeks, and of 1.0 or 4.0 nmoykg body weight of PHI until the end of the experiment in week 35. Administration of PHI at the higher, but not the lower dosage, significantly increased the incidence of colon tumors. PHI had no influence on the histology of colon tumors or adenocarcinomas. It also caused significant increase in the labeling index of colon epithelial cells. These findings indicate that PHI enhances colon carcinogenesis, and that its effect may be related to increasing proliferation of colon epithelial cells. Keywords:
Peptide histidine isoleucine; Colon cancer; Azoxymethane;
1. Introduction
Peptide histidine isoleucine (PHI) is a 27-amino acid regulatory peptide and is presentat high concentration in neural elementsthroughout the mammalian gastrointestinal tract [1,2]. It is very similar to vasoactive intestinal peptide (VIP) in structure [3] and biological activities [4]. The vascular action of PHI has been reported to be mediated by its binding to a VIP-preferring receptor [5]. However, there is evidence for the possibleexistence of a specific receptor * Corresponding 9727749.
author,
Tel.:
+81
0304.3835/95/%09.50 Q 1995 Elsevier SSDI 0304-3835(95)03823-F
6 9721181;
Fax:
Science Ireland
+81
6
Rat
for PHI [4]. A biological activity of PHI differing from that of VIP is stimulation of luteinizing hormone secretion after its injection into the preoptic area of rat brain [6]. Moreover, pharmacological desensitization studies have demonstrated distinct receptors for PHI and VIP in the opossuminternal anal sphincter [7]. We previously found that long-term administration of VIP before and during azoxymethane (AOM)treatment significantly increased the incidence of colon tumors [8]. This finding and the above results suggestthat PHI may influence colon carcinogenesis. Therefore, in the present study, we investigated the effect of prolonged administration of PHI on the de-
Ltd. All rights reserved
velopment of colon tumors M’istar rat5. 2. Materials
induced by AOM
in
and methods
Ninety young Wlstar rats, initially weighing 170i 40 g! were used in this study. The animals were pul-chased from Japan SLC (Shizuoka. Japan). and housed In suspended, wire-bottomed metal cages in 0ur animal quarter at controlled temperature (20.--i‘7°C) and humidity (30-50%), with a 12 h/J2 h !igbtidark cvcle. Regular chow pellets (Oriental t’cast. Tnkvoi were availabls ad libitum. -\ -..
-1 i.
Cat-citwgen and treatmetlr The ratx were randomly divided into three groups ;)f 30 rars each, and were given weekly S.C. injections :I! 7.4 mg/kg body weight of AOM (Sigma, St. Louis. IZIO) In 0.9% NaCl solution for JO weeks. From the \tart of administration of the carcinogen, the rats re,,eived the following S.C. injections until the end of the rxpenment In week 35: Group 1. the control group. was given only the vehicle, plam olive oil; Groups 2 and 3 were given 1.O and 4.0 nmol/kg body weight of PHI, respectively. PHI (Bachem, Torrance, ‘,‘A) was prepared as a suspension in olive oil. InjecFIC)~~ were given S.C. in a volume ot 1 ml/kg body weight. between 1400 and IS00 h every other day. ‘~21itbuSsitrc of injection being chosen
The first tumor of the colon was found in a rat m t;r~up 1 killed in week 31. SKIanimal\ that survived ior more than 31 weeks were included in effective oumbers. Rats were killed when they became mori-, bund. and surviving ammals were killed at the end ot week 35. The internal organs of all animals killed during the experiment or m week 35 were carefull) fxantined. The colons were opened, pinned flat on a .:wk mat. and fixed with buffered picric acidformaldehyde solution [9]. The fixed colon was cut into five 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 bearing lesions were excised and embedded in paraffin. Semiserial, 5,um thick cections were cut to expose the central part of the
tumor or the stalk. when present, and were stained \lith hematoxylin and eosin. In addition to tumors, flat mucosa from each segment of the fixed colon with no visible tumors was cut into 3-mm wide strips, which were embedded in paraffin. Thin sections were prepared and examined microscopically for tumor foci. All sections were examined without knowledge pf their group of origin.
Adenomas were defined histologically as lesions m which neoplastic cells were confined to the mucosal layer, while adenocarcinomas were defined as lesions in which neoplastic cells had penetrated the muscularis mucosae to invade the submucosal layer or deeper layers. As reported previously [IO]. the adenocarcinomaa were further classified as either well-differentiated or mutinous carcinomas. In the former, tumor cells were found in acinal clusters simulating the glandular crypts of normal colon mucosa. In the latter, mucin secretion was active, resulting in tnucinous nodules containing large amounts of cxtracellular mucin. with only a few isolated groups of tumor cells, 2.5. Measurement of labeling index of colon mucosa The labeling index of colon epithelial cells was measuredin weeks 9 and 35 with an immunohistochemical analysis kit for bromodeoxyuridine (BrdUrd) incorporation (Becton-Dickinson, Mountain View. Cl\;) 111.121. For the assay, five rats In each group were starved for 12 h and then received S.C. I ml/kg body weight of olive oil (Group 1I, or 1.i) ntnol/kg body weight (Group 2) or 4.0 nmol/kg body weight (Group 3) of PHI in olive oil. One hour later. the animalsreceived an i.p. injection of BrdUrd (20 me/kg body weight) and were killed with ether 1 h later. The colon was removed and fixed in 70% ethanol for 4 h. Parts 2 (distal portion) and 3 (proximal portion) of the colon were then excised and embedded in paraffin. Thin sections of 3,um thickness were immersed in 2 N HCI solution for 30 min and then in 0. I M Na2BJ0,. Slides were also immersed in 0.3% H20z in methanol for 30 min to block endogenous peroxidase activity. and then treated with 10% horse serum. The specimenswere incubated with anti-BrdUrd monoclonal antibody (diluted 1:X) for 2 h, washed, stained with biotin-
H. Iishi et al. I Cancer
conjugated horse anti-mouse antibody (Vector Laboratories, Burlingame, CA; diluted 1:200) for 30 min, and stained with avidin-biotin-peroxidase complex (Vector Laboratories) for 30 min. The reaction product was detected with 3,3-diaminobenzidine tetrahydrochloride. Cells that contained BrdUrd were identified by the presence of a dark pigment over their nuclei. To analyze the labeling index of colon mucosa, we counted the numbers of BrdUrd-labeled and unlabeled cells in the zone of proliferating cells without knowledge of which treatment group the samples were from. The zone of proliferating cells was defined as that below the highest BrdUrd-labeled cells [13]. We selected 50-well oriented columns of pits and glands from each rat. On the basis of these measurements, we calculated the labeling index as the number of BrdUrd-labeled cells/total cells within the zone of proliferating cells. 2.6. Measurement of serum gastrin level The serum gastrin level in the basal state was examined in weeks 9 and 35. For this, five rats in each group were starved for 12 h and then anesthetized, and blood was obtained. Its gastrin content was assayed with a radioimmunoassay kit from Dainabot (Tokyo) [14]. 2.7. Statistical analysis Data were analyzed by theX2 test or Fisher’s exact probability test, or by one-way analysis of variance with Dunn’s multiple comparison [15-171. Data are means + SE. Differences were considered to be significant at a calculated P value of less than 0.05. Table
1 2 3
94 (1995)
49-54
3. Results 3.1. lncidences and numbers of colon tumors and body weights Ten rats in each group were killed in week 9 for determination of the labeling index of colon mucosa and measurement of the serum gastrin level. Two rats in Groups 1 and 2, and one rat in Group 3 were killed before week 3 1, because they became moribund. No tumors were found in any of these rats, which were excluded from the effective numbers. The incidence of colon tumors and their number per tumor-bearing rat, and the body weights in each group are summarized in Table 1. Administration of PHI at either dosage had no significant influence on the body weight in week 35. In Group 1 (olive oil only), colon tumors were found in 7 (39%) of 18 rats examined, and the average number of tumors per tumor-bearing rat was 1.6 + 0.3. Administration of PHI at 4.0 nmol/kg body weight (Group 3) significantly increased the incidence of colon tumors, but not their number per rat. In Group 2 treated with PHI at 1.0 nmol/kg body weight, the incidence of colon tumors and their number per rat were similar to those in control Group 1. 3.2. Histological types of colon tumors and adenocarcinomas Table 2 shows the distribution of different histological types of colon tumors, and histological types and depths of involvement of adenocarcinomas. In Group 1 (olive oil only), 64% of the tumors were identified histologically as adenocarcinomas, and 29% and 43% of the adenocarcinomas were well-
1
Incidences Group
Letters
no.
and numbers
of colon tumors,
Treatmenta
Olive oil PHI 1 .O nmol/kg PHI 4.0 nmobkg
and body weights
of AOM-treated
rats
Initial
35 weeks
Effective no. of rats
183+ 1 183 + 2 189+2
390+8 391 + 9 382+5
18 18 19
Body weight(g)
No. of rats with colon tumors (a)
No. of colon tumors per tumorbearing rat
7 (39) 10 (56) 15 (79)h
1.6 + 0.3 1.3 + 0.2 1.1 kO.1
a Treatment: rats were given 10 weekly SC. injections of 7.4 mg/kg body weight of AOM, and also S.C. injections (Group l), 1 .O nmoiikg body weight of PHI (Group 2). or 4 nmoYkg body weight of PHI (Group 3). b Significantly different from the value for Group 1 at P < 0.05.
of the vehicle,
olive oil
,\.
H. lixfzr et 01 i Gmcer
I aM:
types of colon tumors
c:t imp nil. Trtu1mentJ
and ndenocarcinomas tokln
olive oi! PHf I 0 nmol/kg PHi 4.0 nmolikg For explnnntion
of treatments,
m AOM-treated ---
49.. V
rats
tumor,s C%,i
Total no
-I
94 (199.5)
1
Histological _.~.
I
Letter3
___-I_ II 13 Ii
Adenomns
Adenocarcinomns Adenocnrcinomas
Histology
f %)
Depth of involvement
Well rliffcrentinted
Uucinous
Submucosa
Muscle layer or deeper
2 (29) 2 (25) 5 (38)
s (71) 6 (75) 8 (62)
4 (57) 4 (SO) 6 (46)
3 (43) 4 (SO) 7 (54)
(%)
111 4 (36) 5 US) 4 (24)
7 (64: 8032~ I.3 17f1)
see Table 1
differentiated cancers and more deeply invasive, respectively. Administration of PHI at 4.0 nmol/kg body weight slightly, but not significantly, increased the incidences of total adenocarcinomas, welldifferentiated cancers and more deeply invasive cani:c1‘s 3.3. Labeling indices qf colon mucosaand serum jpxsfrin 1evei.i Table 3 summarizesdata on the labeling indices of colon mucos;~and basalserumgastrin levels in weeks ij and 35. At both times, administration of -1.0nmol/kg body weight of PHI (Group 3) signifi,cantly increasedthe labeling index of epithelial cells of the proximal and distal colon mucosaand significantly decreased the serum gastrin level. PHI at i-0 nmol/kg body weight (Group 2) did not affect the
labeling index of colon mucosaor the serum gastrin level. 4. Discussion PHI hasmarked sequencehomology with VIP 131. Furthermore, PHI and VIP are co-synthesized from a common precursor [ 181and co-localized in the central and peripheral nervous systems[19]. It is uncertain whether PHI exerts its action via a mechanismor receptor distinct from that for VIP [5]. There is evidencefar the possibleexistence of a separatereceptor for PHI 171.However, the present study showed that prolonged administration of PHI, like that of VIP, enhanced AOM-induced colon carcinogenesis in Wistar rats, resulting in significant increase in the incidenceof colon tumors in week 35.
H. lishi et al. I Cancer
The mechanism(s) of this effect of PHI is not yet known, but three possibilities may be considered. One possible explanation involves stimulation of adenylate cyclase. Cauvin et al. [20] compared the relative potencies and efficacies of the two forms of rat PHI to stimulate adenylate cyclase in membranes from rat tissues and cells (liver, lung, pancreas, anterior pituitary, circulating lymphocytes, and early thymocytes of the 51E cell line) and found that PHI stimulated adenylate cyclase in these tissues and cell membranes. There is much evidence that cyclic AMP is involved in the control of growth and differentiation of various cell types [21,22]. Under certain experimental conditions, cyclic AMP is a positive rather than a negative signal for hepatocyte growth [23]. Moreover, adenylate cyclase activity has been reported to be higher in rapidly proliferating crypt cells of the intestinal mucosa than in slowly growing superficial cells [24]. A second possibility involves an effect on gastrin secretion. Wolfe et al. [l] examined whether PHI inhibited antral gastrin, and found that PHI might exert a physiological inhibitory effect on antral gastrin cells at several steps along the biosynthetic pathway. Furthermore, they [25] found that PHI inhibited basal expression of the gastrin gene at all levels examined, and also decreased carbachol-stimulated antral gastrin release. Several lines of investigations suggest that gastrin may have important growthstimulating roles in human colorectal cancer. This hormone has a trophic effect on the colonic epithelium in rodents [26] and also stimulates proliferation of normal and malignant colonic epithelial cells in vitro [27,28]. However, studies by Penman et al. [29] on the effect of hypergastrinemia induced by omeprazole on the development of colorectal tumors induced by AOM showed that chronic omeprazole treatment resulted in appreciable hypergastrinemia and significant decrease in the tumor incidence. Previously, we [30] also found that prolonged administration of tetragastrin in depot form during and after intrarectal instillation of N-methyl-N’-nitro-Nnitrosoguanidine into Wistar rats resulted in significant reduction in the incidence of colon tumors in experimental week 35. A third possibility involves increase in activity of the sympathetic nervous system by PHI. Nilsson et al. [3 I] studied the effects of PHI on the release of
Letters
94 (1995)
49-54
53
[3H]norepinephrine from the pig choroid plexus elicited by electrical field stimulation, and found that PHI enhanced norepinephrine release. There is evidence for neural control of cell proliferation in various cell systems, including rat jejunal and colonic crypts [32,33]. Beta-adrenergic agents were shown to inhibit cell division, and alpha-adrenergic agents to promote it [34]. Previously, we [35] examined the effect of chemical sympathectomy induced by 6hydroxydopamine on the incidence of colon tumors induced by AOM, and on the norepinephrine concentration in the colon wall and the labeling index of colon mucosa, and found that prolonged administration of 6-hydroxydopamine caused significant reductions in the incidence of colon tumors, the tissue norepinephrine concentration, and the labeling index of colon mucosa. These findings indicate that norepinephrine is closely related to colon carcinogenesis. The present results showed that prolonged treatment with PHI enhanced colon carcinogenesis. The exact mechanism of this effect is not known, but may be related to the effect of PHI in increasing cell proliferation of colon epithelial cells. These results suggest that PHI and VIP have the same enhancing effect on colon carcinogenesis. Acknowledgments This work was supported in part by a Grant-in-Aid from the Ministry of Health and Welfare for a Comprehensive lo-Year Strategy for Cancer Control, Japan. References VI
Wolfe, M.M.. Mailhard, M.E., Dunn, B.M., Green, E.P. and Karnik, P.S. (1989) Inhibition of antral gastrin cells by peptide histidine isoleucine. Am. J. Physiol., 257, G328G333. Y., Christofides, N.D., Blank, M.A., Yanaihara., [21 Yiangou, N., Tatemoto, K., Bishop, A.E., Polak, J.M. and Bloom, S.R. (1985) Molecular forms of peptide histidine isoleucine-like immunoreactivity in the gastrointestinal tract: nonequimolar levels of peptide histidine isoleucine and vasoactive intestinal peptide in the stomach explained by the presence of a big peptide histidine isoleucine-like molecule. Gastroenterology, 89, 516-524. [31 Tatemoto, K. and Mutt, V. (1981) Isolation and characterization of intestinal peptide porcine PHI (PHI-27). a new member of the glucagon-secretin family. Proc. Nat]. Acad. Sci. USA. 78,6603X5607. O.P. (1990) PHI preferentially [41 Huang, M. and Rorstad,
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azoxymethane-induced experimental in Wistar rats by h-hydroxydopamine ?()I
H
(1992) colon Int.
J.
Cancer Letters 94 (1995) 49-54
CANCER LETTERS
Enhancement by peptide histidine isoleucine of experimental carcinogenesis in the colon of rats induced by azoxymethane Hiroyasu Iishi’ a,*,Masaharu Tatsutaa, Miyako Babaa, Kazushige Isekib, Hiroyuki Ueharaa, Akihiko Nakaizumia “Depurtment bDepartment
of Gastrointestinal
Oncology, The Centerfor Adult Diseases, Osaka, 3-3, Nakamirhi I-rhome, Higashinari-ku. Osaka 537, Japan The Centerjor Adult Diseases, Osaka, 3-3, Nakamichi 1-chome, Higashinari-ku, Osaka 537, Japan
qf Gastroenterology.
Received 3 April 1995; accepted 20 April 1995
Abstract
The effects of peptide histidine isoleucine (PHI) on the incidence and histology of colon tumors induced by azoxymethane (AOM), and on the labeling index of colon mucosa were investigated in Wistar rats. Rats received weekly S.C.injections of 7.4 mg/kg body weight of AOM for 10 weeks, and of 1.0 or 4.0 nmoykg body weight of PHI until the end of the experiment in week 35. Administration of PHI at the higher, but not the lower dosage, significantly increased the incidence of colon tumors. PHI had no influence on the histology of colon tumors or adenocarcinomas. It also caused significant increase in the labeling index of colon epithelial cells. These findings indicate that PHI enhances colon carcinogenesis, and that its effect may be related to increasing proliferation of colon epithelial cells. Keywords:
Peptide histidine isoleucine; Colon cancer; Azoxymethane;
1. Introduction
Peptide histidine isoleucine (PHI) is a 27-amino acid regulatory peptide and is presentat high concentration in neural elementsthroughout the mammalian gastrointestinal tract [1,2]. It is very similar to vasoactive intestinal peptide (VIP) in structure [3] and biological activities [4]. The vascular action of PHI has been reported to be mediated by its binding to a VIP-preferring receptor [5]. However, there is evidence for the possibleexistence of a specific receptor * Corresponding 9727749.
author,
Tel.:
+81
0304.3835/95/%09.50 Q 1995 Elsevier SSDI 0304-3835(95)03823-F
6 9721181;
Fax:
Science Ireland
+81
6
Rat
for PHI [4]. A biological activity of PHI differing from that of VIP is stimulation of luteinizing hormone secretion after its injection into the preoptic area of rat brain [6]. Moreover, pharmacological desensitization studies have demonstrated distinct receptors for PHI and VIP in the opossuminternal anal sphincter [7]. We previously found that long-term administration of VIP before and during azoxymethane (AOM)treatment significantly increased the incidence of colon tumors [8]. This finding and the above results suggestthat PHI may influence colon carcinogenesis. Therefore, in the present study, we investigated the effect of prolonged administration of PHI on the de-
Ltd. All rights reserved
velopment of colon tumors M’istar rat5. 2. Materials
induced by AOM
in
and methods
Ninety young Wlstar rats, initially weighing 170i 40 g! were used in this study. The animals were pul-chased from Japan SLC (Shizuoka. Japan). and housed In suspended, wire-bottomed metal cages in 0ur animal quarter at controlled temperature (20.--i‘7°C) and humidity (30-50%), with a 12 h/J2 h !igbtidark cvcle. Regular chow pellets (Oriental t’cast. Tnkvoi were availabls ad libitum. -\ -..
-1 i.
Cat-citwgen and treatmetlr The ratx were randomly divided into three groups ;)f 30 rars each, and were given weekly S.C. injections :I! 7.4 mg/kg body weight of AOM (Sigma, St. Louis. IZIO) In 0.9% NaCl solution for JO weeks. From the \tart of administration of the carcinogen, the rats re,,eived the following S.C. injections until the end of the rxpenment In week 35: Group 1. the control group. was given only the vehicle, plam olive oil; Groups 2 and 3 were given 1.O and 4.0 nmol/kg body weight of PHI, respectively. PHI (Bachem, Torrance, ‘,‘A) was prepared as a suspension in olive oil. InjecFIC)~~ were given S.C. in a volume ot 1 ml/kg body weight. between 1400 and IS00 h every other day. ‘~21itbuSsitrc of injection being chosen
The first tumor of the colon was found in a rat m t;r~up 1 killed in week 31. SKIanimal\ that survived ior more than 31 weeks were included in effective oumbers. Rats were killed when they became mori-, bund. and surviving ammals were killed at the end ot week 35. The internal organs of all animals killed during the experiment or m week 35 were carefull) fxantined. The colons were opened, pinned flat on a .:wk mat. and fixed with buffered picric acidformaldehyde solution [9]. The fixed colon was cut into five 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 bearing lesions were excised and embedded in paraffin. Semiserial, 5,um thick cections were cut to expose the central part of the
tumor or the stalk. when present, and were stained \lith hematoxylin and eosin. In addition to tumors, flat mucosa from each segment of the fixed colon with no visible tumors was cut into 3-mm wide strips, which were embedded in paraffin. Thin sections were prepared and examined microscopically for tumor foci. All sections were examined without knowledge pf their group of origin.
Adenomas were defined histologically as lesions m which neoplastic cells were confined to the mucosal layer, while adenocarcinomas were defined as lesions in which neoplastic cells had penetrated the muscularis mucosae to invade the submucosal layer or deeper layers. As reported previously [IO]. the adenocarcinomaa were further classified as either well-differentiated or mutinous carcinomas. In the former, tumor cells were found in acinal clusters simulating the glandular crypts of normal colon mucosa. In the latter, mucin secretion was active, resulting in tnucinous nodules containing large amounts of cxtracellular mucin. with only a few isolated groups of tumor cells, 2.5. Measurement of labeling index of colon mucosa The labeling index of colon epithelial cells was measuredin weeks 9 and 35 with an immunohistochemical analysis kit for bromodeoxyuridine (BrdUrd) incorporation (Becton-Dickinson, Mountain View. Cl\;) 111.121. For the assay, five rats In each group were starved for 12 h and then received S.C. I ml/kg body weight of olive oil (Group 1I, or 1.i) ntnol/kg body weight (Group 2) or 4.0 nmol/kg body weight (Group 3) of PHI in olive oil. One hour later. the animalsreceived an i.p. injection of BrdUrd (20 me/kg body weight) and were killed with ether 1 h later. The colon was removed and fixed in 70% ethanol for 4 h. Parts 2 (distal portion) and 3 (proximal portion) of the colon were then excised and embedded in paraffin. Thin sections of 3,um thickness were immersed in 2 N HCI solution for 30 min and then in 0. I M Na2BJ0,. Slides were also immersed in 0.3% H20z in methanol for 30 min to block endogenous peroxidase activity. and then treated with 10% horse serum. The specimenswere incubated with anti-BrdUrd monoclonal antibody (diluted 1:X) for 2 h, washed, stained with biotin-
H. Iishi et al. I Cancer
conjugated horse anti-mouse antibody (Vector Laboratories, Burlingame, CA; diluted 1:200) for 30 min, and stained with avidin-biotin-peroxidase complex (Vector Laboratories) for 30 min. The reaction product was detected with 3,3-diaminobenzidine tetrahydrochloride. Cells that contained BrdUrd were identified by the presence of a dark pigment over their nuclei. To analyze the labeling index of colon mucosa, we counted the numbers of BrdUrd-labeled and unlabeled cells in the zone of proliferating cells without knowledge of which treatment group the samples were from. The zone of proliferating cells was defined as that below the highest BrdUrd-labeled cells [13]. We selected 50-well oriented columns of pits and glands from each rat. On the basis of these measurements, we calculated the labeling index as the number of BrdUrd-labeled cells/total cells within the zone of proliferating cells. 2.6. Measurement of serum gastrin level The serum gastrin level in the basal state was examined in weeks 9 and 35. For this, five rats in each group were starved for 12 h and then anesthetized, and blood was obtained. Its gastrin content was assayed with a radioimmunoassay kit from Dainabot (Tokyo) [14]. 2.7. Statistical analysis Data were analyzed by theX2 test or Fisher’s exact probability test, or by one-way analysis of variance with Dunn’s multiple comparison [15-171. Data are means + SE. Differences were considered to be significant at a calculated P value of less than 0.05. Table
1 2 3
94 (1995)
49-54
3. Results 3.1. lncidences and numbers of colon tumors and body weights Ten rats in each group were killed in week 9 for determination of the labeling index of colon mucosa and measurement of the serum gastrin level. Two rats in Groups 1 and 2, and one rat in Group 3 were killed before week 3 1, because they became moribund. No tumors were found in any of these rats, which were excluded from the effective numbers. The incidence of colon tumors and their number per tumor-bearing rat, and the body weights in each group are summarized in Table 1. Administration of PHI at either dosage had no significant influence on the body weight in week 35. In Group 1 (olive oil only), colon tumors were found in 7 (39%) of 18 rats examined, and the average number of tumors per tumor-bearing rat was 1.6 + 0.3. Administration of PHI at 4.0 nmol/kg body weight (Group 3) significantly increased the incidence of colon tumors, but not their number per rat. In Group 2 treated with PHI at 1.0 nmol/kg body weight, the incidence of colon tumors and their number per rat were similar to those in control Group 1. 3.2. Histological types of colon tumors and adenocarcinomas Table 2 shows the distribution of different histological types of colon tumors, and histological types and depths of involvement of adenocarcinomas. In Group 1 (olive oil only), 64% of the tumors were identified histologically as adenocarcinomas, and 29% and 43% of the adenocarcinomas were well-
1
Incidences Group
Letters
no.
and numbers
of colon tumors,
Treatmenta
Olive oil PHI 1 .O nmol/kg PHI 4.0 nmobkg
and body weights
of AOM-treated
rats
Initial
35 weeks
Effective no. of rats
183+ 1 183 + 2 189+2
390+8 391 + 9 382+5
18 18 19
Body weight(g)
No. of rats with colon tumors (a)
No. of colon tumors per tumorbearing rat
7 (39) 10 (56) 15 (79)h
1.6 + 0.3 1.3 + 0.2 1.1 kO.1
a Treatment: rats were given 10 weekly SC. injections of 7.4 mg/kg body weight of AOM, and also S.C. injections (Group l), 1 .O nmoiikg body weight of PHI (Group 2). or 4 nmoYkg body weight of PHI (Group 3). b Significantly different from the value for Group 1 at P < 0.05.
of the vehicle,
olive oil
,\.
H. lixfzr et 01 i Gmcer
I aM:
types of colon tumors
c:t imp nil. Trtu1mentJ
and ndenocarcinomas tokln
olive oi! PHf I 0 nmol/kg PHi 4.0 nmolikg For explnnntion
of treatments,
m AOM-treated ---
49.. V
rats
tumor,s C%,i
Total no
-I
94 (199.5)
1
Histological _.~.
I
Letter3
___-I_ II 13 Ii
Adenomns
Adenocarcinomns Adenocnrcinomas
Histology
f %)
Depth of involvement
Well rliffcrentinted
Uucinous
Submucosa
Muscle layer or deeper
2 (29) 2 (25) 5 (38)
s (71) 6 (75) 8 (62)
4 (57) 4 (SO) 6 (46)
3 (43) 4 (SO) 7 (54)
(%)
111 4 (36) 5 US) 4 (24)
7 (64: 8032~ I.3 17f1)
see Table 1
differentiated cancers and more deeply invasive, respectively. Administration of PHI at 4.0 nmol/kg body weight slightly, but not significantly, increased the incidences of total adenocarcinomas, welldifferentiated cancers and more deeply invasive cani:c1‘s 3.3. Labeling indices qf colon mucosaand serum jpxsfrin 1evei.i Table 3 summarizesdata on the labeling indices of colon mucos;~and basalserumgastrin levels in weeks ij and 35. At both times, administration of -1.0nmol/kg body weight of PHI (Group 3) signifi,cantly increasedthe labeling index of epithelial cells of the proximal and distal colon mucosaand significantly decreased the serum gastrin level. PHI at i-0 nmol/kg body weight (Group 2) did not affect the
labeling index of colon mucosaor the serum gastrin level. 4. Discussion PHI hasmarked sequencehomology with VIP 131. Furthermore, PHI and VIP are co-synthesized from a common precursor [ 181and co-localized in the central and peripheral nervous systems[19]. It is uncertain whether PHI exerts its action via a mechanismor receptor distinct from that for VIP [5]. There is evidencefar the possibleexistence of a separatereceptor for PHI 171.However, the present study showed that prolonged administration of PHI, like that of VIP, enhanced AOM-induced colon carcinogenesis in Wistar rats, resulting in significant increase in the incidenceof colon tumors in week 35.
H. lishi et al. I Cancer
The mechanism(s) of this effect of PHI is not yet known, but three possibilities may be considered. One possible explanation involves stimulation of adenylate cyclase. Cauvin et al. [20] compared the relative potencies and efficacies of the two forms of rat PHI to stimulate adenylate cyclase in membranes from rat tissues and cells (liver, lung, pancreas, anterior pituitary, circulating lymphocytes, and early thymocytes of the 51E cell line) and found that PHI stimulated adenylate cyclase in these tissues and cell membranes. There is much evidence that cyclic AMP is involved in the control of growth and differentiation of various cell types [21,22]. Under certain experimental conditions, cyclic AMP is a positive rather than a negative signal for hepatocyte growth [23]. Moreover, adenylate cyclase activity has been reported to be higher in rapidly proliferating crypt cells of the intestinal mucosa than in slowly growing superficial cells [24]. A second possibility involves an effect on gastrin secretion. Wolfe et al. [l] examined whether PHI inhibited antral gastrin, and found that PHI might exert a physiological inhibitory effect on antral gastrin cells at several steps along the biosynthetic pathway. Furthermore, they [25] found that PHI inhibited basal expression of the gastrin gene at all levels examined, and also decreased carbachol-stimulated antral gastrin release. Several lines of investigations suggest that gastrin may have important growthstimulating roles in human colorectal cancer. This hormone has a trophic effect on the colonic epithelium in rodents [26] and also stimulates proliferation of normal and malignant colonic epithelial cells in vitro [27,28]. However, studies by Penman et al. [29] on the effect of hypergastrinemia induced by omeprazole on the development of colorectal tumors induced by AOM showed that chronic omeprazole treatment resulted in appreciable hypergastrinemia and significant decrease in the tumor incidence. Previously, we [30] also found that prolonged administration of tetragastrin in depot form during and after intrarectal instillation of N-methyl-N’-nitro-Nnitrosoguanidine into Wistar rats resulted in significant reduction in the incidence of colon tumors in experimental week 35. A third possibility involves increase in activity of the sympathetic nervous system by PHI. Nilsson et al. [3 I] studied the effects of PHI on the release of
Letters
94 (1995)
49-54
53
[3H]norepinephrine from the pig choroid plexus elicited by electrical field stimulation, and found that PHI enhanced norepinephrine release. There is evidence for neural control of cell proliferation in various cell systems, including rat jejunal and colonic crypts [32,33]. Beta-adrenergic agents were shown to inhibit cell division, and alpha-adrenergic agents to promote it [34]. Previously, we [35] examined the effect of chemical sympathectomy induced by 6hydroxydopamine on the incidence of colon tumors induced by AOM, and on the norepinephrine concentration in the colon wall and the labeling index of colon mucosa, and found that prolonged administration of 6-hydroxydopamine caused significant reductions in the incidence of colon tumors, the tissue norepinephrine concentration, and the labeling index of colon mucosa. These findings indicate that norepinephrine is closely related to colon carcinogenesis. The present results showed that prolonged treatment with PHI enhanced colon carcinogenesis. The exact mechanism of this effect is not known, but may be related to the effect of PHI in increasing cell proliferation of colon epithelial cells. These results suggest that PHI and VIP have the same enhancing effect on colon carcinogenesis. Acknowledgments This work was supported in part by a Grant-in-Aid from the Ministry of Health and Welfare for a Comprehensive lo-Year Strategy for Cancer Control, Japan. References VI
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