Gastrointestinal hormones as potential adjuvant treatment of exocrine pancreatic adenocarcinoma

InternationalJournalof Pancreatology.vol.24, no. 3, 169-180,December1998 9 Copyright1998by HumanaPressInc. All rightsof any naturewhatsoeverreserved. 0169-4197/98/24:169-180/$11.00

Gastrointestinal Hormones as Potential Adjuvant Treatment of Exocrine Pancreatic Adenocarcinoma William E. Fisher,*,1 Peter Muscarella, 2 Laszlo G. Boros, z and William J. Schirmer z tDepartment of Surgery, Baylor College of Medicine, Houston, TX; and 2Department of Surgery, Ohio State University, Columbus, OH

Summary Conclusion. Gastrointestinal hormones and their antagonists can alter the growth of pancreatic adenocarcinoma in vitro and in vivo. The potential clinical benefit of this approach deserves further study. Background. Epithelial cell growth is normally under hormonal control. Hormones also affect the growth of many epithelial cancers, and this fact is used to modify tumor growth. Pancreatic epithelial cell growth is under the influence of gastrointestinal hormones. This article reviews experiments designed to determine the effect of gastrointestinal hormones on the growth of pancreatic adenocarcinoma. Methods. Eighty-eight articles were identified from a Medline search using the terms pancreatic adenocarcinoma and the individual names of gastrointestinal hormones. The experimental design and results of these studies are reviewed. Results. In general, somatostatin, vasoactive intestinal polypeptide, pancreatic polypeptide, and pancreastatin inhibit pancreatic adenocarcinoma growth. Cholecystokinin, secretin, bombesin, gastrin, EGF, TGF-o~, insulin, and IGF-1 have a growth-promoting effect. Key Words: Pancreatic adenocarcinoma; gastrointestinal hormones.

Introduction

was possible (1). Another surgeon, Huggins, eventually won the Nobel Prize in 1966 for demonstrating regression of cancers by endocrine manipulation. He demonstrated that antiandrogenic treatment consisting of orchiectomy or the administration of estrogens could produce long-term regression in patients with advanced disseminated prostatic carcinoma (2). Today, dramatic results are not uncommon after hormonal treatment of prostatic, breast, endometrial, and ovarian cancers, even in patients with advanced disease (3,4). A similar approach to pancreatic cancer, a malignancy that usually presents in an advanced stage, may hold great promise. The fact that many gastrointestinal hormones affect the growth of the normal exocrine pancreas

Hormonal manipulation can be used to control the growth of some cancers originating from organs normally under hormonal control. In 1895 the Scottish surgeon Beatson performed bilateral oophorectomy on a woman with advanced breast carcinoma. This resulted in dramatic regression of her disease and demonstrated that hormonal control of cancer Received April 17, 1998; Revised and Accepted August 10, 1998. *Author to whom all correspondence and reprint requests should be addressed: Baylor College of Medicine, Houston VAMC, Surgical Service (112). Houston, TX 77030.

169

170 suggests that these peptides could be used to slow the growth of pancreatic cancer analogous to the success with sex hormone manipulation in other tumors. Gastrointestinal hormones, such as somatostatin, that normally inhibit the division of the exocrine pancreatic cells may also slow the growth of exocrine pancreatic cancer and provide a relatively nontoxic therapeutic option. It has also been postulated that antagonists of growth-promoting hormones, such as cholecystokinin, may be beneficial in the adjuvant treatment of pancreatic cancer. Alternatively, growthpromoting hormones, by increasing tumor cell division, may increase the sensitivity of a pancreatic tumor to adjuvant cytotoxic treatment. To provide a more substantial rationale for extending the old concept of sex hormonal therapy to gastrointestinal hormonal therapy for pancreatic cancer, it must be proven that these hormones can indeed affect the growth of autologous malignant cells. In the laboratory, several experimental steps are commonly taken to demonstrate the potential of a hormone in manipulating the growth of pancreatic cancer. The growth effect of the hormone must be shown in vitro using various proliferation assays. The growth effect should also be demonstrated in vivo, commonly by studying the effect of the hormone on the growth of established tumor xenografts in athymic nude mice or by studying the effect of the hormone on carcinogenesis. Evidence that the tumors produce receptors for the hormone are important in clarifying the mechanism of altered growth. Finally, if receptors are important, neutralization of the hormone or the receptor with an antibody or specific antagonist should inhibit the growth effect. By summarizing the results of many such experiments, this review provides a substantial rationale for further study into the use of gastrointestinal hormones as adjuvant treatment of pancreatic cancer.

Gastrointestinal Hormones That May Inhibit Pancreatic Cancer Growth Somatostatin Somatostatin is a naturally occurring 14-amino acid peptide secreted by the D-cells in the pancreatic islets. It has been characterized as the "universal off switch" because it inhibits the release of growth hormone and essentially all gastrointestinal hormones. Somatostatin is known to inhibit not only pancreatic endocrine and exocrine secretion but also International Journal of Pancreatology

Fisher et al. DNA synthesis (5). However, a role for somatostatin and its analogs in pancreatic cancer therapy has yet to be established. Initial studies suggest that these agents could provide a useful and relatively nontoxic adjuvant therapy. The exact mechanism of the antiproliferative effect of somatostatin is not known. The inhibitory effect could be mediated through specific, highaffinity somatostatin receptors on the tumor cell surface. Susini et al. (6) demonstrated that the antiproliferative effect mediated by somatostatin receptors involves the activation ofa tyrosine phosphatase. Alternatively, the antitumor effect could be independent of somatostatin receptors. Somatostatin could inhibit tumor proliferation by inhibiting the secretion of other gastrointestinal hormones thought to be important in pancreatic cancer growth. In addition, somatostatin has been shown to selectively decrease splanchnic blood flow and inhibit angiogenesis, either of which might interfere with pancreatic tumor growth. Somatostatin and its analog RC-160 have been shown to inhibit p r e n e o p l a s t i c c h a n g e s and decrease the incidence of tumors in hamsters exposed to the pancreatic carcinogen BOP (7,8). Histologic evaluation showed an increase in the number of apoptotic tumor cells. RC-160 given after the induction of cancer has also been shown to decrease tumor weight and prolong survival of hamsters with nitrosamine-induced pancreatic cancer (9-11). Somatostatin in combination with 5FU has been shown to inhibit tumorous pancreas weight and preneoplastic changes by 76%, suggesting potentially quite good results when adjuvant treatment with gastrointestinal hormones is combined with standard chemotherapy (12). Fekete et al. (13) showed that receptors for somatostatin are present on the normal hamster pancreas and on nitrosamineinduced pancreatic cancer. In addition, the number of receptors increased on the tumor cells after treatment with the somatostatin analog RC-160. They were also able to demonstrate somatostatin receptors on normal human pancreas and three human pancreatic cancer specimens (13). In later studies, these results were confirmed and different analogs of somatostatin were found to have variable affinities for the receptors on tumor cells, suggesting that some analogs could be therapeutically superior to others (14). Volume 24, 1998

Gastrointestinal Hormones as Adjuvant Treatment Liebow et al. (15) showed that growth of the human pancreatic cancer cell line MIA PaCa-2 was inhibited in cell culture by somatostatin-14. Stimulated growth of these cells by epidermal growth factor (EGF) was also inhibited in cell culture by three long-acting analogs of somatostatin (16). The degree of stimulation of tyrosine phosphatase correlated with the effectiveness of each analog suggesting that somatostatin may act by dephosphorylating the EGF receptor (EGF-R). Takeda and Escribano (17) studied the effect of somatostatin on the growth of two human pancreatic cancer cell lines, BxPC-3 and SOJ-6, using a clonogenic assay in soft agar. Colony formation by the BxPC-3 cell line was decreased by somatostatin u'eatment but had no effect on SOJ-6 growth (17). Growth of the MIA PaCa-2 cells implanted subcutaneously in nude mice was dose-dependently inhibited by twice daily injections of the somatostatin analog octreotide (250 and 2500 lag/kg) (18). Radulovic et al. (19) also showed that microcapsules of RC-160, another somatostatin analog, releasing 1250 lag/kg/d also significantly inhibited growth of MIA PaCa-2 tumors in nude mice (19). They then treated nude mice bearing MIA PaCa-2 tumors for 4 wk with a somatostatin analog containing methotrexate and found tumor inhibition to be enhanced. Binding affinity of the somatostatin analog on the MIA PaCa-2 cells was decreased only slightly by the addition of the cytotoxic radical (20). By contrast, Gillespie et al. (21) were not able to demonstrate receptors or a growth effect with somatostatin-14 or its analog RC-160 on the MIA PaCa-2 cell line or on another human pancreatic cancer cell line, Panc-1. Poston et al. (22) studied the effect of SMS 201995, a somatostatin analog, on the growth of two human pancreatic cancers, SKI and PGER, in nude mice inoculated with tumor chunks. Somatostatin inhibited the growth of SKI but not PGER (22). Both tumor types were subsequently shown to have highaffinity cholecystokinin (CCK) receptors, but only the SKI cell line was shown to have somatostatin receptors (23). A somatostatin analog has been shown to inhibit the growth of SKI and CAV, two human pancreatic cancers maintained as nude mouse xenotransplants (24). However, recent clinical trials of somatostatin analogs in the adjuvant treatment of pancreatic cancer have failed to demonstrate a response. Klijn and coworkers (25) treated 14 patients with metastatic International Journal of Pancreatology

171 pancreatic cancer with three daily sc injections of 100-200 lag of the somatostatin analog Sandostatin for an average of 7 wk and observed no antitumor effect (25). Nineteen patients with advanced exocrine pancreatic carcinoma were given the somatostatin analog BIM 23014, using a range of doses from 250 gg/d to 1 mg/d for 2 mo. One patient had a partial response, 6 had stable disease, and 11 had progressive disease (26). Huguier and coworkers (27), in a randomized prospective study of 86 patients given a similar treatment regimen, demonstrated no significant increase in median survival according to life-table analysis. We have also shown that growth of the MIA PaCa-2 cell line is inhibited by somatostatin and octreotide in vitro and in vivo (28). However, the growth of four other human pancreatic cancer cell lines--Capan- 1, Capan-2, CAV, and Panc-l--was not inhibited by somatostatin in cell culture or as xenografts in nude mice. Continuous sc infusion of octreotide (500 lag/ kg/d) for 36 d by osmotic pumps did not alter plasma levels of CCK, EGF, insulin-like growth factor I (IGF-I), or insulin. Only the MIA PaCa-2 cells were shown to possess mRNA for the somatostatin receptor subtype II and high-affinity cell surface somatostatin receptors (29). We have concluded that expression of somatostatin receptors is Critical for human pancreatic cancer to respond to treatment with somatostatin and its analogs.

Vasoactive Intestinal Peptide Vasoactive intestinal peptide (VIP) is a 28-amino acid peptide involved in the regulation of the exocrine pancreas. VIP may alter the replication of epithelium by acting through cyclic adenosine monophosphate (cAMP). Functional receptors for VIP have been demonstrated on human pancreatic cancer cells, and stimulation of these cells with VIP caused a fivefold increase in cAMP production (30). Ruellan et al. (31) demonstrated VIP receptors on the human pancreatic cancer cell line Capan-1 by competitive binding techniques with 125I-VIP, but was unable to demonstrate a significant change in cell proliferation when the cells were cultured with VIP. Poston et al. (32) were able to demonstrate VIP receptors on H2T, a hamster pancreatic carcinoma cell line, but not on MIA PaCa-2, a human pancreatic cancer cell line. VIP inhibited the growth of the H2T tumors in nude mice but had no effect on the MIA PaCa-2 human pancreatic tumors (32). Virgolini et Volume 24, 1998

172 al. (33) recently reported a technique of gamma-camera scanning after iv injection of 123I-VIP for the localization of tumors. Tumor masses were visualized in 10 of 12 patients with primary or recurrent pancreatic adenocarcinoma, suggesting a high prevalence of VIP receptors on human pancreatic cancer (33). These studies suggest that there may be a role for VIP in the adjuvant treatment of human pancreatic cancer. In addition, the presence of VIP receptors may predict growth inhibition by exogenous VIP. We used reverse transcriptase polymerase chain reaction to examine 11 histologically proven human pancreatic adenocarcinomas from our patients undergoing laparotomy, and 6 were found to express VIP receptor mRNA. However, we also examined the human pancreatic cancer cell lines Capan-1, CAV, Panc-1, and MIA PaCa-2 using competitive binding techniques on whole-cell preparations, but we could not demonstrate cell surface VIP receptors or an antiproliferative effect by VIP in vitro. These unpublished preliminary data suggest that VIP receptors are not expressed in many pancreatic cancers.

Pancreatic Polypeptide Family Pancreatic polypeptide, first isolated by Kimmel and colleagues in 1968, is a 36-amino acid peptide secreted from the F-cells, which are most prominently found in the periphery of the islets in the head of the pancreas (34). Pancreatic polypeptide binds to specific receptors and inhibits exocrine pancreatic secretion of enzyme, bicarbonate, and water and decreases pancreatic blood flow (35). In 1980 Tatemoto and Mutt (36) isolated peptide YY (PYY), and neuropeptide Y (NPY), also 36-amino acid peptides sharing about 50% homology with pancreatic polypeptide and having similar actions. PYY and its synthetic analog BIM-43004-1 have been shown to cause significant reduction in growth of the human ductal pancreatic cancer cell line MIA PaCa-2 in vitro (37). This analog of PYY was subsequently shown to bind to receptors on these pancreatic cancer cells, decrease intracellular cAMP levels, and suppress tumor growth in vivo (38). By contrast, another study reported increased incorporation of 3H-thymidine in MIA PaCa-2 cells and two other cell lines, one human (Capan-2) and the other a hamster pancreatic adenocarcinoma (H2T), after exposure to NPY and PYY (39). We have examined the effect of pancreatic polypeptide on the growth of the Capan-2 and H2T International Journal of Pancreatology

Fisher et al. cell lines and examined the cells for pancreatic polypeptide receptors using competitivebinding assays with 125I-PP. Dose-dependent inhibition of tumor cell proliferation was observed when the H2T cells were cultured with increasing concentrations of pancreatic polypeptide from 10-1~ to 10-7M. However, no growth effect was detected with Capan-2. Neither cell line could be shown to have pancreatic polypeptide receptors by competitive binding studies. Pancreatic polypeptide, as well as other members of this family of related peptides, may exert an inhibitory effect on pancreatic ductal adenocarcinoma cells. Although we were unable to demonstrate receptors in our preliminary unpublished work, others have shown that specific receptors appear to be involved in the mechanism of growth inhibition. Further studies are warranted to determine the usefulness of these peptides and their analogs, either alone or in combination with chemotherapy and radiation, in the adjuvant treatment of pancreatic cancer.

Pancreastatin Pancreastatin is a recently discovered pancreatic islet peptide product that inhibits insulin, and possibly somatostatin release, and augments glucagon release (40,41). In addition to its effects on the endocrine pancreas, pancreastatin inhibits pancreatic exocrine secretion (42). Smith et al. (43) evaluated the effect of pancreastatin on the growth of the normal pancreas and two human pancreatic cancer cell lines, MIA PaCa-2 and SW- 1990, in vitro and in vivo (43). Pancreastatin in doses of 10-t 1-10-8M significantly inhibited DNA synthesis, as measured by incorporation of 3H-thymidine, in the normal pancreas and in both human pancreatic cancer cell lines. However, pancreastatin given subcutaneously (100 gg/kg) twice daily for 35 d, starting 3 wk after tumor cell inoculation, caused only a transient decrease in tumor volume but did not alter final tumor weight of MIA PaCa-2 xenografts to nude mice. We have examined a number of human pancreatic carcinoma cell lines (BxPC-3, Capan-1, Capan-2, CAV, HS766T, MIA PaCa-2, and Panc-1) and the hamster pancreatic adenocarcinoma cell line, H2T, for pancreastatin receptors by competitive binding assays on whole-cell preparations. None of these cell lines were found to have pancreastatin receptors. This unpublished preliminary data suggests that most pancreatic cancer cells do not express receptors for pancreastatin. Volume 24, 1998

Gastrointestinal Hormones as Adjuvant Treatment Gastrointes~uff Hormones That May Promote Pancreatic Cancer Growth Cholecystokinin CCK, a peptide hormone produced in the upper small intestine, is known to stimulate the secretion and growth of the normal exocrine pancreas (44). Many studies have been conducted to examine the role of CCK in the development of pancreatic adenocarcinoma and promotion of its growth once established. Ductal adenocarcinoma of the pancreas develops in hamsters after the administration of nitrosamines, such as N-nitrosobis(2-oxopropyl)amine (BOP). The effect of CCK on the pancreatic carcinogenicity of BOP has been studied by several groups of investigators in the hamster model. Pour et al. (45) showed that CCK inhibited pancreatic cancer induction when given prior to or simultaneously with BOP. However, CCK administered after BOP or simultaneously with BOP for 20 wk did not alter the incidence of pancreatic tumors (45). Johnson et al. (46) confirmed these results (46). By contrast, Howatson and Carter (47) found an enhancing effect of CCK on BOP carcinogenesis and AndrenSandberg et al. (48) observed no effects at all. These contradictory results may be owing to the sequence of the carcinogen and CCK administration. In the studies with an inhibitory effect, CCK was given before or simultaneously with BOP, whereas in the other studies CCK was given after BOP. The inhibitory effects of CCK on the carcinogenicity of BOP observed in these studies could be caused by CCK's ability to modify DNA alkylation (49). In the rat, azaserine has been used to induce acinar pancreatic cancers. Rats treated with CCK and the pancreatic carcinogen, azaserine, develop more adenocarcinomas than control animals treated with the carcinogen alone (50). Lorglumide, a CCK antagonist, inhibited the enhancing effect of CCK on pancreatic carcinogenesis in this model. Therefore, CCK has been shown to increase the incidence of pancreatic carcinoma in some experimental models, and this effect can be inhibited by CCK antagonists. Through its proliferative effect, CCK may make the stimulated exocrine pancreatic cells more sensitive to carcinogens. The contradictory findings in some studies may be related to the dose and timing of CCK administration. However, the majority of International Journal of Pancreatology

173 the experimental data suggests that CCK modulates the induction of experimental pancreatic cancer. The effect of CCK on the growth of established pancreatic cancer has also been studied by many investigators. Townsend et al. (51) showed that CCK in combination with secretin promotes the growth of established hamster pancreatic adenocarcinoma (H2T) cell implants. Final tumor weight in animals treated with ip injections of CCK (15 pg/kg/d) and secretin (75 lag&g/d) for 15-20 d was 230 mg, compared with 126 mg in the untreated controls (51). The effect of the CCK receptor antagonist L-364,718 on the growth of nitrosamine-induced pancreatic carcinoma transplants in the hamster has also been studied (52). The CCK antagonist significantly reduced tumor growth when given immediately after tumor transplantation and also in animals with established tumors. CCK has also been shown to inhibit the growth of established pancreatic acinar cell carcinoma tumors originally induced by azaserine and maintained by successive transplantations in rats. When the tumor transplants became palpable, rats were treated three times daily for 2 wk with CCK (4 l.tg/kg) or its antagonist CR 1409 (10 mg/ kg) twice daily, or both. Tumor weight was decreased after CCK treatment, and this effect was partially reversed by CR 1409, which by itself also reduced tumor growth (53). Bell et al. (54) has demonstrated overexpression of high-affinity CCK-8 receptors in premalignant and malignant azaserine-induced tumors in the rat. They suggest that novel expression of CCK receptors may be generated by gene mutation or amplification during carcinogenesis and that this may play an important role in promoting tumor growth. Smith et al. (55) showed that CCK stimulates the growth of six human pancreatic cancer cell lines-SW-1990, Panc-1, MIA PaCa-2, BxPC-3, RWP-2, and Capan-2--in tissue culture. CCK receptors were demonstrated on the Panc-1 cells by competitive binding studies using membrane preparations and 125I-CCK (56). However, Hudd et al. (57) were not able to demonstrate any effect of CCK on the growth of the Panc- 1 or MIA PaCa-2 cell lines xenografted in nude mice. Once tumors were established (5 mm in the longest axis), the mice received CCK (50 lag&g) by ip injection twice daily for 2 wk (57). By contrast, Upp and coworkers (58) concluded that the presence of CCK receptors on human pancreatic cancer cells Volume 24, 1998

1 74 predicts the response to CCK. They demonstrated that CCK stimulated the growth of SKI, a CCK receptor-positive human pancreatic cancer, when xenografted in nude mice. This effect was blocked by the CCK receptor antagonists proglumide and asperlicin (59). They found that CCK had no effect on the growth of CAV xenografts, a CCK receptornegative human pancreatic cancer. Growth of the human pancreatic carcinoma cell line KP-IN was stimulated by CCK (10-11-10-12M) in tissue culture, and this effect was blocked by the CCK receptor antagonist CR 1505. In addition, daily injections of CR 1505 (35 mg/kg) diminished the number of tumor colonies in the liver that were formed after an intrasplenic injection of tumor cells in nude mice (60). Smith et al. (61) showed that CCK ( 10-12_ 10-8M) increases proliferation of SW-1990 human pancreatic cancer cells in tissue culture. CCK (25 gg/kg) twice daily for 20 d also significantly increased weight, protein content, and DNA content of SW- 1990 pancreatic cancer xenografts in nude mice. The CCK receptor antagonist L364,718 has been shown to significantly decrease the volume, weight, protein content, and DNA content of SW-1990 xenografts in nude mice (62). The effect of cisplatin, a chemotherapeutic agent, and the CCK antagonist, MK-329 (same molecule as L364,718) alone and in combination on the growth of the human pancreatic cancer cell line MIA PaCa-2 was determined in a clonogenic assay. Both drugs exerted an antiproliferative effect when used alone. However, a synergistic cytotoxic effect was observed with combined therapy (63). Administration of MK-329 has been shown to reduce the growth of PGER, a CCK receptor-positive cell line in athymic mice (64). MK-329 has been used in a clinical trial involving 18 patients with advanced pancreatic cancer (65). Patients were given 5 mg orally twice a day for 4 wk as outpatients, and no concomitant antineoplastic therapy was allowed. After 4 wk the dose was doubled, for the second half of the study, in patients without toxicity who failed to achieve an objective antitumor response. As a whole, no significant tumor response was seen; however, the CCK receptor status of the tumors was unknown. Since CCK is trophic for the pancreas and appears to promote the growth of some pancreatic cancers, it International Journal of Pancreatology

Fisher et al. has been postulated that CCK antagonists may be beneficial in the adjuvant treatment of pancreatic cancer. However, it has also been postulated that CCK itself, by promoting tumor cell division, may increase the sensitivity of a pancreatic tumor to cytotoxic treatment. In a phase I study, 14 patients with pancreatic cancer were given conventional FAM chemotherapy (5-fluorouracil, adriamycin, and mitomycin C), along with CCK 25-33 (1 gg/h) and secretin (6 gg/h) intravenously over 144 h. There was no increase in side effects, including pancreatitis, and there was no evidence of increased tumor growth induced by the hormones and not compensated for by FAM (66). Further studies are warranted to determine the role of CCK and its antagonists in the adjuvant treatment of pancreatic cancer. Although there is a substantial body of literature supporting a role for CCK in the modulation of pancreatic cancer growth, our unpublished preliminary data do not support this hypothesis. We have examined five human pancreatic cancer cell lines--Capan-l, Capan-2, CAV, MIA PaCa-2, and Panc-l--for the presence of CCK receptors using 125I-CCK-8 and determined the response of these cell lines to CCK-8 in tissue culture. We were unable to demonstrate CCK receptors on any of these cell lines and observed no increase in the incorporation of 3H-thymidine when the cells were cultured in the presence of CCK-8 (10-11-10-7M).

Bombesin Bombesin is a gastrointestinal hormone originally isolated from the skin of a frog (Bombina bombina) and subsequently found to be homologous with human gastrin-releasing peptide (GRP). As its name implies, GRP stimulates the release of gastrin. It also stimulates pancreatic polypeptide and CCK release and promotes pancreatic exocrine secretion and growth (67). Meijers et al. (68) studied the effect of bombesin on the development of azaserine-induced pancreatic carcinoma in rats. In this study, 4 mo after azaserine was given, rats were treated with bombesin (10 gg/kg) 3 d a week for 8 mo by sc injection. Growth of the pancreas and the development of preneoplastic lesions and frank carcinomas was increased. The mechanism does not appear to involve increased secretion of CCK because the effect was not inhibited by the CCK antagonist lorglumide. The same results were obtained in a similar study by Douglas et al. (69). Volume 24, 1998

Gastrointestinal Hormones as Adjuvant Treatment The effect of bombesin on the growth of established pancreatic cancer has also been studied in the rat model (70). In cell culture, GRP increased incorporation of 3H-thymidine by azaserine-induced pancreatic cancer cells in a dose-dependent manner. GRP receptors were demonstrated on these cells by competitive binding assays with ]25I-GRP. Also, GRP (30 ~tg/kg/d) for 15 d by miniosmotic pumps, significantly increased tumor weight in rats bearing azaserine-induced, subcutaneously transplanted pancreatic carcinomas. In other studies, bombesin had an opposite effect on the development of BOP-induced pancreatic carcinoma in the Syrian hamster (71). Bombesin caused an increase in growth of the pancreas accompanied by a decrease in the number of preneoplastic and neoplastic ductular pancreatic lesions. As in the rat model, the CCK antagonist lorglumide (CR- 1409) did not influence the effect of bombesin on pancreatic carcinogenesis, indicating that this opposite effect in the hamster is also not mediated by CCK. By contrast, Szepeshazi et ai. (72) found that the bombesin antagonist RC-3095 had an inhibitory effect on the induction of pancreatic cancer in the hamster model. This effect was augmented by concomitant treatment with the somatostatin analog RC-160 (72). The effect of GRP on the growth of established pancreatic cancer has also been studied in the hamster model (73). GRP alone did not stimulate the growth of pancreatic cancer in this model. The GRP antagonist RC-3095 did inhibit the growth of established BOP-induced pancreatic ductal adenocarcinoma in the hamster. Bombesin has been shown, in one study, to inhibit the growth of human pancreatic adenocarcinoma xenografts in nude mice (74). The human pancreatic cancer cell line SKI was implanted in nude mice that received ip injections of bombesin (20 gg/kg) three times a day for 57 d. Pancreas weight was increased but final tumor weight was decreased by 46%. One possible explanation offered for this unexpected finding was a bombesin-induced release of somatostatin. We have examined the effect of bombesin on the growth of the human pancreatic carcinoma cell lines MIA PaCa-2 and Panc-1 and the hamster H2T cell line. Bombesin (10 -] L10-7M) inhibited the proliferation of all three of these cell lines in tissue culture. However, these data have not been submitted for International Journal of Pancreatology

175 publication because we were subsequently unable to reproduce these results.

Secretin Whereas CCK acts primarily as a stimulator of the acinar cells, secretin is a stimulant of the ductal and ductular cells (75). This suggests that secretin may act as a more potent cocarcinogen than CCK in the development of ductal adenocarcinoma. Howatson and Carter (76) examined the effect of exogenous secretin on ductal pancreatic carcinogenesis in the hamster. Secretin (20 U/kg) was given subcutaneously for 6 wk after BOP treatment. Secretin reduced the latency and increased the induction rate of tumor development when compared with the carcinogen given alone (76). Pour and Kazakoff(77) also examined the effect of secretin on pancreatic carcinogenesis in this model. They gave 100 U/kg by six injections, 30 min apart. One treatment group was given secretin before BOP, another after BOP, and a third was given half before and half after BOP. Secretin caused inhibition rather than promotion of induction of pancreatic cancer when it was given prior to or simultaneously with a single dose of BOP, but it had no effect on carcinogenesis when it was administered after BOP. They also examined a treatment schedule in which BOP and secretin were given weekly for 20 wk and found no effect on the incidence of pancreatic tumors (77). Smith et al. (61) has demonstrated that secretin (10-1~ significantly increased DNA synthesis as measured by incorporation of 3H-thymidine by SW-1990-cuitured human pancreatic cancer cells. Estival et al. (78) demonstrated the presence of secretin receptors on several human pancreatic cancers established as xenografts in athymic mice. Stimulation with secretin (10~M) caused an increase in cAMP (78).

Gastrin Watson et al. confirmed the presence of gastrin receptors (receptors that bind gastrin) on AR42J, a rat pancreatic acinar carcinoma, and examined the effect of CR2093, a gastrin receptor antagonist, on the growth of these cells as xenografts in nude mice. Mice were treated with gastrin, CR2093, or both. Gastrin alone ( 10 lag/d) administered by miniosmotic pumps, significantly promoted tumor growth. However, CR2093 alone (40 mg/kg/d) administered intravenously, had no effect. Interestingly, when Volume 24, 1998

176 gastrin and the antagonist were given together, tumor weight was significantly less than that of the untreated control (79).

EGF and Transforming Growth Factor-oc The mitogenic polypeptide EGF is known to exert atrophic effect on the exocrine pancreas, which is known to have EGF-Rs (80,81). Chester et al. (82) examined the effect of EGF on the incidence of pancreatic cancer in hamsters treated with BOP. Pancreatic adenocarcinomas were induced in hamsters by 19 weekly injections of BOP, and from wk 5 to 8, 5 Jag of EGF was given every 3 d by sc injection. The incidence of pancreatic cancer in the EGF-treated animals was significantly increased. The EGF-R is similar to the v-erbB oncogene product. Korc et al. (83) demonstrated that four human pancreatic carcinoma cell lines--Panc-1, T3M4, COLO 357, and UACC-462--overexpress the EGF-R gene. The overexpression of EGF-Rs was associated with structural or numerical alterations of chromosome 7, the chromosomal locus of the v-erbB oncogene. An overabundance of EGF-Rs caused by these chromosomal alterations may be involved in human pancreatic carcinogenesis. TGF-cc is a 50-amino acid polypeptide that is structurally similar to EGF whose actions are mediated through the EGF-R (84). Smith et al. (85) examined five human pancreatic cancer cell lines--AS PC- 1, T3M4, Panc-1, COLO 357, and MIA PaCa-2--for TGF-~ peptide and mRNA. All five cell lines were found to express TGF-oc mRNA. Picogram quantities of TGF-~ were detected only in media conditioned for 48 h by 106 ASPC-1 and T3M 4 cells. Specific competitive binding was demonstrated for EGF and TGF-~ on all five cell lines, indicating the presence of the EGF-R. The effect of EGF and TGF-ct on cell growth was tested in three of the cell lines. EGF and TGF-~ (1-10 ng/mL) exerted a dosedependent increase in colony formation of the ASPC-I, T3M4, and Panc-1 cell lines in soft agar. The investigators concluded that production of TGF-oc may be a mechanism whereby pancreatic cancer cells that overexpress the EGF-R obtain a growth advantage (85). We have examined five human pancreatic cancer cell lines--Capan-l, Capan-2, CAV, MIA PaCa-2, and Panc-l--for the presence of EGF-Rs using 125I-[Cys(Acm2~ 20-31 (Peninsula, Belmont CA) and determined the response of these International Journal of Pancreatology

Fisher et al. cell lines to EGF in tissue culture. We were unable to demonstrate EGF-Rs on any of these cell lines, and we observed no increase in the incorporation of 3H-thymidine when the cells were cultured in the presence of [Cys(Acm2~ I)]-EGF 20-31 (10-I i_10-7M) in our preliminary unpublished experiments.

Insulin Not many investigators have actually examined the effect of insulin on the growth of pancreatic cancer. Perhaps this is because it is so obvious that insulin is a potent trophic peptide for so many types of cells. Type II diabetes is a risk factor for pancreatic carcinoma. We have hypothesized that the hyperinsulinemia in type II diabetes promotes pancreatic cancer growth. Takeda and Escribano (86) found that insulin promoted the growth and colony formation of two human pancreatic carcinoma cell lines: BxPC-3 and SO J-6. We have examined six human pancreatic cancer cell lines--Capan-1, Capan-2, CFPAC-I, HS766T, MIA PaCa-1, and Panc-l--for insulin receptors by competitive binding with 125I-insulin. All six cell lines were found to possess specific cellsurface insulin receptors. Dose-dependent increases in DNA synthesis, as measured by incorporation of 3H-thymidine, were caused by adding insulin (10-12-10-8M) to the cell culture media (87).

Insulin-Like Growth Factor-I IGF-I is a polypeptide similar in structure to insulin that has been shown to stimulate cell growth directly. The MIA PaCa-2 human pancreatic cancer cell line has been shown to produce IGF-I, which stimulates the growth of this cell line in an autocrine fashion (88). We have examined five human pancreatic cancer cell lines--Capan-1, Capan-2, CAV, MIA PaCa-2, and Panc-l--for the presence of IGF-I receptors using 125I-IGF-I(Peninsula) and determined the response of these cell lines to IGF-I in tissue culture (unpublished data). We were able to demonstrate IGF-I receptors on the MIA PaCa-2 and Panc-I cell lines and observed a dose-dependent increase in the incorporation of 3H-thymidine when these cells were cultured in the presence of IGF-I (10 -l l_l 0-7M). These unpublished preliminary data support a role for IGF-I in the growth of pancreatic cancer.

Conclusions Although much work is still required, this impressive body of experimental data, generated by many Volume 24, 1998

Gastrointestinal Hormones as Adjuvant Treatment

investigators, over decades, provides compelling evidence that hormonal therapy for pancreatic cancer may be a clinically useful option in the future. It is clear that polypeptide hormones and their antagonists can promote and inhibit pancreatic carcinogenesis and the growth of some established pancreatic cancers in vitro and in vivo. The subset of pancreatic adenocarcinomas that may respond to gastrointestinal hormonal therapy is unknown. Although some studies in which no effect was observed have been published, countless other "negative" studies may have gone unreported, and perhaps repeated, because of the typical lack of interest in such data. There are multiple reasons for the apparent discrepancies in the published literature summarized herein. Different doses and timing of administration of the same hormone may have different effects on the cancer cell. Expression of receptors may be related to the degree of differentiation of the cancer cell, and all pancreatic cancer cells clearly do not possess the same receptors. Passage of cells in culture may alter their receptor expression. In addition, there may be several forms of a receptor for a given hormone that function differently. Hormones may act directly on cancer cells or indirectly by suppression or promotion of the release of other growth factors, making the total effect complicated. Although it is unlikely that these obstacles will be easily overcome, the studies summarized herein represent early steps of major importance in our understanding of the tremendously complex regulation of pancreatic cancer growth by gastrointestinal hormones. A strong foundation for the gastrointestinal hormonal therapy of pancreatic cancer has been laid by the summarized data. However, more investigations into the efficacy of this treatment must be conducted before clinical application is possible. Optimal clinical application of this approach will require a method to determine which patients have responsive tumors. Evaluation of excised pancreatic cancers for gastrointestinal hormone receptors may help define the responsive subset of patients in a manner similar to current strategies utilized successfully in the treatment of patients with breast cancer. Many therapeutic strategies deserve further inquiry. If gastrointestinal hormones bind to tumor cell surface receptors, can cytotoxic agents be linked to these peptides and thus be carried directly to the target cell? Can receptornegative pancreatic cancer cells be rendered responsive International Journal of Pancreatology

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to the growth-modulating effects of gastrointestinal hormones by transfecting the genes for their receptors? The experimental models presently established will certainly help answer these and other questions and contribute to the development of new therapeutic approaches with eventual clinical utility.

Acknowledgments This work was supported by the American Cancer Society, the Bremer Foundation, the National Cancer Institute, National Research Service Award CA-09338, the Division of Cancer Prevention and Control and Surgical Research Incorporated.

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