B. Evers

Role of bombesin on gut mucosal growth

ObjectiveThe authors examined the effects of exogenous bombesin (BBS) on gut mucosal growth in chowfed rats and the mucosal regeneration after gut atrophy brought about by feeding an elemental diet and after intestinal injury produced by methotrexate (MTX). Summary Background DataBombesin is one of many gastrointestinal peptides implicated in the regulation of gut mucosal growth. Although BBS is known to stimulate growth of normal pancreatic tissue, the trophic effect of BBS on gut mucosa is less clear and its exact role in gut mucosal regeneration and repair is not known. MethodsRats were fed a regular chow diet (control) or an elemental diet plus either saline or BBS (10 μg/ kg). In another experiment, rats fed a chow diet and treated with saline or BBS were given MTX (20 μg/kg) or a single intraperitoneal injection. In all experiments, small and large bowel mucosa and pancreas were removed and analyzed for BBS-mediated proliferation. ResultsBombesin produced significant mucosal proliferation of the small bowel at day 14, but not at day 7, in rats fed regular chow. In contrast, BBS treatment for 7 days produced significant proliferation in both the atroophic and injured gut mucosa of rats given elemental diet or MTX. ConclusionsBombesin may be an important enterotrophic factor for normal mucosal proliferation and may be clinically beneficial as an agent to restore or maintain gut mucosa during periods of atrophy or injury.

Caloric restriction increases the expression of heat shock protein in the gut.

OBJECTIVE The authors determined whether caloric restriction (CR) either acutely or chronically, alters heat shock protein 70 (hsp70) gene expression in the gut. SUMMARY BACKGROUND DATA Caloric restriction prolongs the life span and delays age-related disease (e.g., cancer) in mammals; the mechanisms responsible for these effects are not known. Heat shock proteins are a group of stress-responsive genes of which the most prominent member is hsp70. METHODS In the first experiment, adult (4-month-old) rats (n = 3/group) were killed after a 48-hour fast or 6 and 24 hours after refeeding. In addition, three rats (controls) were killed without fasting or refeeding. The stomach was removed and RNA was extracted for hsp70 gene expression. In the second experiment, aged (22- to 26-month-old) rats were fed ad libitum (AL) or a CR diet (60% caloric intake of AL diet). Rats were killed, the stomach and duodenum were removed, and RNA was extracted for determination of hsp70 gene expression. RESULTS In the first experiment, hsp70 mRNA levels were increased approximately threefold in the stomach of rats fasted for 48 hours; levels decreased to control values by 6 and 24 hours after refeeding. In the second experiment, hsp70 mRNA levels were increased significantly in both the stomach and duodenum of aged CR rats compared with AL controls. CONCLUSIONS The authors have demonstrated that hsp70 mRNA levels are increased in the proximal gut of young and old rats, either acutely (with fasting) or with CR. Increased expression of the cytoprotective hsp70 gene in the gut may provide a possible cellular mechanism for the beneficial effects noted with CR.

Heat shock proteins are differentially expressed in human gastrointestinal cancers.

The heat shock proteins (Hsp) are stress-responsive genes present in all species; increases of Hsp can confer chemotherapeutic resistance to certain cancers. The purpose of this study was to determine Hsp expression in human gastric, pancreatic and colon cancers. Gastric (n = 3), pancreatic (n = 6) and colon (n = 8) cancers were extracted for RNA and protein, and Northern and Western blots performed. We found that hsp70 and hsp27 mRNA levels were differentially expressed in the gastrointestinal cancers; mRNA expression closely correlated with protein levels suggesting regulation at the level of transcription. In addition, Hsp90 and BiP proteins were constitutively expressed in the gastrointestinal cancers. We conclude that the Hsp are differentially expressed in human gastric, pancreatic and colon cancers; these increases in Hsp occur constitutively and are not the result of physiological or environmental stresses. Increases of Hsp expression in cancer cells may enhance resistance and account for the altered sensitivity of certain gastrointestinal cancers to chemotherapeutic agents.

Neurotensin stimulates growth of colon cancer.

Neurotensin (NT), a peptide from the distal gut that is released by fat ingestion, stimulates the growth of normal small bowel and colonic mucosa. The purpose of this study was to determine whether chronic administration of NT would affect the growth of a mouse colon cancer (MC-26) and a human colon cancer (LoVo) in vivo. In experiment 1, male Balb/c mice were inoculated with MC-26 cells (5 x 10(4)) and then randomized to four treatment groups receiving either saline (control) or NT (150, 300 or 600 micrograms kg-1) administered subcutaneously (s.c.) every 8 h for 21 days. In experiment 2, 60 mice with MC-26 tumours were randomized to receive saline (control) or NT (300 or 600 micrograms kg-1) for 28 days, and survival was then assessed. In experiment 3, 16 athymic nude mice with LoVo tumour xenografts were randomized to receive either saline (control) or NT (600 micrograms kg-1). We found that administration of NT (300 and 600 micrograms kg-1) significantly stimulated mean tumour area, weight and DNA, RNA and protein content of MC-26 tumours. In addition, the survival rate of mice bearing MC-26 tumours and treated with either dose of NT was significantly decreased compared with the control group given saline injections. Similarly, NT (600 micrograms kg-1) stimulated growth (tumour area, weight and nucleic acid contents) of the human colon cancer, LoVo. We conclude that NT acts as a tropic factor for the colon cancer cell lines MC-26 and LoVo in vivo. NT may play an important role in growth regulation of certain colon cancers.

Neurotensin expression and release in human colon cancers.

Neurotensin (NT), a distal gut peptide released by intraluminal fats, is trophic for normal small bowel and colonie mucosa. In addition, NT stimulates growth of certain colon cancers; the mechanism for this effect is not known. The purpose of this study was to determine whether human colon cancers (HCC) (1) express the mRNA for NT/neuromedin N (N), (2) produce NT peptide, and (3) express the mRNA for a functional NT receptor (NTR). RNA was extracted from four HCC cell lines in culture, nine HCC lines established in athymic nude mice, and from six HCC and adjacent normal mucosa from freshly resected operative specimens; the RNA was analyzed for NT/N mRNA by Northern hybridization with a complementary DNA probe. Neurotensin peptide content, NTR expression, and intracellular Ca++ ([Ca++]1) mobilization in response to NT were evaluated in three HCC cell lines (LoVo, HT29, HCT116). Neurotensin/N mRNA transcripts were identified in all four of the HCC cell lines and in one of nine HCC in nude mice. Neurotensin expression was found in two of six freshly resected HCC and in none of the six corresponding samples of normal mucosa. Neurotensin peptide was identified by RIA in LoVo, HT29, and HCT116. In addition, NTR mRNA was found in HT29 and HCT116. Neurotensin stimulated [Ca++]1 mobilization in HCT116 (without serum) and in LoVo (with 0.25% serum). These findings demonstrate the presence of NT/N mRNA and NT peptide and the presence of a functional NTR in certain HCC. Neurotensin, a potent trophic factor for normal gut mucosa, may function as an autocrine growth factor in certain human colon cancers.

Bombesin stimulates mucosal growth in jejunal and ileal Thiry-Vella fistulas

OBJECTIVE The authors determined whether the trophic effects of bombesin (BBS) on the small bowel mucosa are mediated by either nonluminal factors or endogenous luminal secretion. SUMMARY BACKGROUND DATA The gut hormone bombesin stimulates growth of small bowel mucosa. The mechanisms responsible for this trophic effect are not known. METHODS Rats underwent construction of a Thiry-Vella fistula (TVF) of either the jejunum or ileum. On postoperative day 10, the two groups were subdivided to receive either saline (control) or bombesin (10 micrograms/kg, subcutaneously, three times a day). After 14 days, rats were killed and the TVF was removed. The mucosa was scraped and weighed, and DNA and protein content was determined. RESULTS Bombesin significantly increased mucosal weight and DNA and protein content of both the jejunal and ileal TVF compared with the control rats. CONCLUSIONS Bombesin-mediated stimulation of small bowel mucosal growth is mediated by factors that are independent of luminal contents and pancreaticobiliary secretion. Bombesin may prove to be an important enterotrophic factor for gut mucosal proliferation.

Endogenous cholecystokinin regulates growth of human cholangiocarcinoma.

Exogenous administration of cholecystokinin (CCK) or caerulein inhibits growth of SLU-132, a human cholangiocarcinoma that we have shown to possess receptors for CCK. Chronic administration of cholestyramine, a resin that binds bile salts, increases release of CCK and growth of the pancreas in guinea pigs. Feeding the bile salt, taurocholate, inhibits meal-stimulated release of CCK. The purpose of this study was to determine whether endogenous CCK affects growth of the human cholangiocarcinoma, SLU-132. We implanted SLU-132 subcutaneously into athymic nude mice. The bile salt pool was depleted by feeding 4% cholestyramine for 40 days, either alone or enriched with 0.5% taurocholate for 32 days. When the mice were killed, tumors and pancreas were removed. Cholestyramine significantly inhibited the growth of SLU-132 and stimulated growth of the normal pancreas. Feeding of taurocholate acted to stimulate tumor growth. These results demonstrate that endogenous levels of CCK regulate growth of this human cholangiocarcinoma. Our findings suggest that manipulation of levels of endogenous gut hormones may, in the future, play a role in management of patients with certain gastrointestinal cancers.

Analysis of multiple molecular changes in human endocrine tumours

To define the molecular changes occurring in endocrine tumours, we have analysed three human endocrine tumours established in our laboratory: BON, a functioning carcinoid tumour from the pancreas; SIM, a nonfunctioning carcinoid of the ileum; and STAN, a pheochromocytoma. A homozygous point mutation of the N-ras gene was identified at codon 61 in BON cells in conjunction with overexpression of N-ras mRNA and protein. BON cells also exhibited increased expression of c-myc and cdc2 kinase mRNA and protein; TGF-beta 1, p53 and retinoblastoma (RB) mRNA and protein levels were decreased. In addition, increased expression of the mdm2 oncogene and both the truncated and the wild-type RB protein were noted in BON. SIM cells exhibited moderately increased N-ras and c-myc mRNA levels along with decreased levels of RB mRNA and protein. Similar to BON and SIM, analysis of STAN showed increased N-ras and c-myc levels. Our data show multiple molecular changes in the three human endocrine tumours with the BON cell line exhibiting the most dramatic changes. Furthermore, our data suggest the existence of different molecular pathways in the pathogenesis of endocrine tumours. These cell lines will provide unique in vitro models to further analyse the significance of these molecular alterations.

Comparative effects of neurotensin and neuromedin N on growth of human pancreatic cancer, MIA PaCa-2

Neurotensin (NT), an important regulatory hormone of the gut, stimulates growth of the human pancreatic cancer cell line MIA PaCa-2 in vitro. The purpose of our study was to compare the stimulatory effects of NT and neuromedin N (NMN), a structurally related hexapeptide, on the growth of MIA PaCa-2. In addition, the effects of NT on the growth of MIA PaCa-2 xenografts and normal GI tissues were assessed in athymic nude mice. MIA PaCa-2 cells, plated in serum-free media, were treated with either NT (10(-12)-10(-6) M) or NMN (10(-11)-10(-7) M) and cells were counted. For the in vivo study, MIA PaCa-2 cells were inoculated sc into 30 athymic nude mice and then randomized to two groups to receive either NT (600 micrograms kg-1, sc, tid) or vehicle. At sacrifice (day 35), the xenografted tumours, as well as normal host pancreas, jejunum and ileum were removed, weighed, and assayed for DNA, RNA and protein. Both NT and NMN stimulated the growth of MIA PaCa-2 cells in vitro with maximal (approximately 30%) increases occurring with dosages of 10(-9) M. In vivo, NT had a transient effect on xenografted MIA PaCa-2 tumour area with increases noted on days 21 and 25 of the study. Conversely, NT significantly stimulated the growth of jejunum and ileum, with a more pronounced effect noted in the jejunum. NT and NMN have similar growth-stimulatory effects on MIA PaCa-2 cells in vitro, which suggests an interaction through the same receptor.(ABSTRACT TRUNCATED AT 250 WORDS)

Amiloride inhibits the growth of human colon cancer cells in vitro

Cytoplasmic alkalinization induced by activation of the Na+/H+ antiport plays an essential role in the initiation of cell proliferation. In the present study we examined the effects of amiloride, a specific and reversible inhibitor of Na+/H+ antiporter, on the growth of human colon cancer cells (HT-29). Amiloride (50-800 microM) inhibited the growth of HT-29 cells in a dose-dependent fashion. Forty-three percent inhibition of growth was found at an amiloride concentration of 400 microM after 4 days of treatment. The inhibitory effect of amiloride on growth of HT-29 cells was reversible since removal of amiloride by a media change after 48 h treatment lead to rapid regrowth to control levels. The reversibility of growth inhibition suggests that amiloride is not a non-specific cytotoxin for HT-29 cells. We examined the possible mechanisms for the inhibitory effects of amiloride. Amiloride (400 microM) completely abolished serum-stimulated ODC activity and inhibited difluoromethylornithine (DMFO)-stimulated putrescine uptake by 56%. We conclude that amiloride inhibits the in vitro growth of human colon cancer cells; since ODC-activity and polyamine transport were both inhibited, the inhibitory effects may be mediated in part by polyamine-dependent processes. Amiloride may be a useful agent in the treatment of colon cancer.