Proc. Nail. Acad. Sci. USAVol. 84, pp. 1112-1116, February 1987Medical Sciences

Treatment of nitrosamine-induced pancreatic tumors in hamsterswith analogs of somatostatin and luteinizinghormone-releasing hormone

[exocrine pancreatic cancer/N-nitrosobis(2-oxopropyl)amine/therapy with analogs of hypothalamic hormones]

J. I. PAZ BOUZA*tt, T. W. REDDING*, AND A. V. SCHALLY*t*Endocrine, Polypeptide and Cancer Institute, Veterans Administration Medical Center; and tDepartment of Medicine, Tulane University School of Medicine,New Orleans, LA 70146

Contributed by A. V. Schally, October 27, 1986

ABSTRACT Pancreatic ductal adenocarcinoma was in-duced in female Syrian golden hamsters by injecting N-nitrosobis(2-oxopropyl)amine (BOP) once a week at a dose of10 mg per kg of body weight for 18 weeks. Hamsters were thentreated with somatostatin analog D-Phe-Cys-Tyr-D-Trp-Lys-Val-Cys-Trp-NH2 (RC-160) or with [6-D-tryptophan]luteiniz-ing hormone-releasing hormone ([D-Trp6]LH-RH) delayed de-livery systems. Microcapsules of somatostatin analog RC-160,designed to release a dose of 5 ,sg/day, were injected twice amonth and microcapsules of [D-Trp]LH-RH§ calculated toliberate 25 ,ig per day, once a month. After 18 weeks of BOPadministration, the hamsters were divided into three groups of10-20 animals each. Group I consisted of untreated controls,group II was injected with RC-160, and groupm was injectedwith [D-Trp6]LH-RH. A striking decrease in tumor weight andvolume was obtained in animals treated with [D-Trp6]LH-RHor with the somatostatin analog RC-160. After 45 days oftreatment with either analog, the survival rate was significantlyhigher in groups It add III (70%), as compared with the controlgroup (35%). The studies, done by light microscopy, high-resolution microscopy, and electron microscopy, showed adecrease in the total number of cancer tells and changes in theepithelium, connective tissue, and cellular organelles in groupsII and III treated with the hypothalamic analogs as comparedto controls. These results in female hamsters with inducedductal pancreatic tumors confirm and extend our findings,obtained in male animals with transplanted tumors, that[D-Trp6]LH-RH and somatostatin analogs inhibit the growth ofpancreatic cancers.

New cases of pancreatic carcinomas diagnosed in the UnitedStates number 25,500 annually and cause >24,000 deaths peryear (1). The most common type of pancreatic cancer is theduct cell carcinoma, which constitutes ==90% of the pancre-atic exocrine tumors. The lack of knowledge about itsetiology, the increasing incidence of pancreatic cancer, andthe poor results from the different methods oftreatment havecreated a need for a better animal model of the human disease(2-5). In recent years, various animal models ofexperimentalpancreatic cancer have been developed (3, 6-10). The Syriangolden hamster has become the model of choice since,anatomically, it has lobulation of the pancreas comparable tothat of humans and tumors can develop spontaneously ormay be induced by carcinogenic agents. These tumors aremorphologically and histologically similar to human pancre-atic cancer (4-10).

Several classes of chemical substances have been used toinduce experimental pancreatic cancer. Nitrosamines (4, 5,10-12) are more specific for the induction of ductal carcino-

mas, while azaserine (8-10, 13, 14) will induce only the acinartype of tumors. Administration of large doses of the carcin-ogen N-nitrosobis(2-oxopropyl)amine (BOP) has beenshown to induce carcinomas of the pancreas, lungs, liver, andkidneys, but with low-dose regimens, pancreatic tumors canbe selectively produced (10, 12). The incidence of thepancreatic tumors induced by BOP in the Syrian goldenhamster model is -'90%.

Experimental and clinical findings suggest that it might bepossible to develop a hormonal therapy for cancer of thepancreas based on somatostatin analogs (15-17). Somato-statin and some of its analogs exert an inhibitory action on theexocrine and endocrine pancreas and suppress the release ofinsulin, glucagon, and the secretion or action of gastrin,secretin, cholecystokinin, and VIP. Secretin and cholecys-tokinin have been shown to produce hyperplasia andhypertrophy of the exocrine pancreas in addition to stimu-lating pancreatic secretion of enzymes and bicarbonate(17-20). Gastrin can also produce pancreatic hypertrophy.Studies on ductal pancreatic adenocarcinoma cell lines havedemonstrated that gastrointestinal hormones stimulate thegrowth ofthese malignant cells (21). In addition, the presenceof intracellular receptors for estrogen and androgen in pan-creatic cells indicates that sex hormones could exert someinfluence on the growth of normal and malignant pancreaticcells (22, 23). These findings suggest that pancreatic adeno-carcinoma might be sensitive to both gastrointestinal and sexhormones. Preliminary clinical results support this view (22,24). Previously, we showed that chronic administration ofsome somatostatin analogs or the agonist, [6-D-tryptophan]-luteinizing hormone-releasing hormone ([D-Trp6]LH-RH),inhibited the growth of transplanted pancreatic carcinomas inrat and hamster models (15, 16). In this study, we haveselectively induced pancreatic tumors in Syrian golden ham-sters by administration of low doses of BOP. We thenfollowed tumor progression and morphological and histolog-ical changes in the tumors and other organs by light andelectron microscopy during treatment with microcapsules of[D-Trp6]LH-RH and somatostatin analog RC-160.

MATERIALS AND METHODSFifty female Syrian golden hamsters (weighing 100-120 g)were obtained from NCI-Frederick Cancer Research Center(Frederick, MD). They were kept in a temperature-controlledroom with a 12-hr light/12-hr dark schedule and receivedpelleted diet and water ad libitum. BOP (Ash-Stevens,

Abbreviations: [D-Trp6]LH-RH, [6-D-tryptophan]luteinizing hor-mone-releasing hormone; BOP, N-nitrosobis(2-oxopropyl)amine;VIP, vasoactive intestinal peptide.tTo whom reprint requests should be addressed at: Veterans Ad-ministration Medical Center, 1601 Perdido Street, Room 7F100,New Orleans, LA 70146.

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Thie publication costs of this article were defrayed in part by page chargepayment. This article must therefore be hereby marked "advertisement"in accordance with 18 U.S.C. §1734 solely to indicate this fact.

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Detroit, MI) in sterile NaCl solution was injected s.c. once aweek for 18 weeks, at a dose of 10 mg per kg of body weight.Somatostatin analog RC-160 (D-Phe-C s-Tyr-D-Trp-Lys-Val-Cys-Trp-NH2) was synthesized by solid-phase methods(25). Microcapsules of [D-Trp6]LH-RH (16, 26) and somato-statin analog RC-160 in poly(DL-lactide-coglycolide) wereprepared by a phase-separation process by Cytotech(Martigny, Switzerland) and supplied by Debiopharm(Lausanne, Switzerland). Microcapsules of [D-Trp6]LH-RHin aliquots of 35 mg were designed to release a dose of 25 ,gper day for 30 days (16, 26). A prototype batch ofmicrocapsules of RC-160 in the amount of 7.5 mg wascalculated to liberate a dose of =5 ,ug per day for 15 days.Microcapsules of [D-Trp6]LH-RH and of RC-160 wereweighed directly in disposable syringes and suspended in 0.7ml of injection vehicle, containing 2% CM-cellulose and 1%Tween 20 in water. The suspensions of microcapsules of[D-Trp6]LH-RH were injected s.c. once a month and RC-160was injected every 15 days. The hamsters were observeddaily and weighed once a week during the administration ofBOP. After 18 weeks, all the hamsters had lost weight butnone had died, so 5 of them were subjected to laparotomy toconfirm the existence of tumors. The remaining hamsterswere divided into three groups of 10-20 animals each. GroupI consisted of untreated controls, group II was treated withRC-160, and group III was treated with [D-Trp6]LH-RH. Thetherapy with hypothalamic analogs was continued for 45days. Hamsters that died during the course of treatment wereautopsied and organs were fixed in 10% buffered formalin forhistological examination. At the conclusion of the experi-ment, the remaining hamsters were sacrificed by decapita-tion, trunk blood was collected, and serum was separated forRIA of progesterone and insulin.The tumors were removed, cleaned, and carefully mea-

sured and weighed. For light microscopy, the tissues werefixed in 10% buffered formalin and then embedded in paraf-fin. Serial sections were cut and stained with eitherhematoxylin and eosin, Masson trichrome, or periodic acidSchiff. Appropriate tumor sections were selected for high-resolution light microscopy and electron microscopy exam-ination. The latter specimens were fixed in paraformaldehydeand postfixed in osmium tetroxide according to standardmethods. The specimens were then dehydrated, embedded inplastic, and polymerized overnight in an oven. Severalsections (1-3 ,m thick) were obtained using an MT2 or JB4microtome and placed in sequence on glass slides. Onesection was stained with toluidine blue, while the others wereprocessed for electron microscopy (27).

All data are reported as the mean ± SEM. Statisticalanalyses were performed by using computer-assisted pro-

grams for Duncan's new multiple range test or Student's ttest.

RESULTS

Survival. Hamsters injected weekly with BOP lost weight,but the survival rate was 100% after 18 weeks. During the 45days of the treatment with [D-Trp6]LH-RH or RC-160, amarked difference in survival rate was observed among thegroups. The survival rate in the control group was 35%, butin the groups treated with either [D-Trp6]LH-RH microcap-sules or RC-160 it was 70%. During the last week oftreatment, control hamsters showed a marked decrease inbody weight; the animals that died showed the greatestweight losses.

Gross Morphology. All the hamsters in the control groupdeveloped pancreatic tumors after BOP. Metastasis wasobserved in the lungs in five animals (25%) and the liver infour animals (20%). Ten of these tumors were localized in thesplenic portion (50%), whereas six were found in the hepaticportion (30%) and four were in both portions (20%) of thepancreas. One tumor was localized in the head of thepancreas, and the liver in that animal presented a greenishcolor and the gallbladder was enlarged. Tumor volume of thecontrol group was 1360.7 ± 280 mm3 and tumor weight was880.3 ± 120 mg (Table 1).

In the group treated with microcapsules of [D-Trp6]LH-RH, only one of the hamsters did not have a pancreatictumor. Eight of these hamsters had tumors localized in thesplenic portion (40%), five in the hepatic portion (25%), fivein both portions (25%), and one was diffuse (5%). Metastasesto the lungs were present in three of the hamsters (15%),while two had metastasis to the liver (10%). The reduction intumor volume (324.7 ± 120 mm3; P < 0.01) and tumor weight(205.4 ± 70 mg; P < 0.001) in the group treated with[D-Trp6]LH-RH was significant when compared with con-trols (Table 1).

In the group treated with somatostatin analog RC-160, fiveof the hamsters had pancreatic tumors localized in the splenicpart (50%), while three had tumors in the hepatic part (30%)and two animals (20%) had diffuse tumors. There was asignificant reduction in tumor volume (659.2 ± 220 mm3; P <0.05) and tumor weight (430 ± 170 mg; P < 0.05) in hamsterstreated with somatostatin RC-160 as compared with thecontrol group. However, this response was about half of thatobserved with [D-Trp6]LH-RH (Table 1).

In the group treated with [D-Trp6]LH-RH, progesteronelevels (2.7 ± 0.1 ng/ml) were significantly decreased ascompared with controls (4.79 ± 0.8 ng/ml; P < 0.01). Levelsof insulin in the control group were 14.2 ± 3.2 microunits as

compared to 23.5 ± 29 microunits/ml (NS) in the RC-160

Table 1. The effects of treatment with somatostatin analog RC-160 and [D-Trp6]LH-RHmicrocapsules on tumor volumes and weights and survival rate in Syrian goldenhamsters with BOP-induced pancreatic cancer

No. of animals,survival rate Tumor Tumor

Treatment 18 weeks 24 weeks volume, mm3 weight, mg

Control 20 (100%) 7 (35%) 1360 ± 280 880 ± 120RC-160* 10 (100%) 7 (70%) 659 ± 220 434 ± 170

P < 0.05t P < 0.05t[D-Trp6]LH-RH§ 20 (100%) 14 (70%) 324 ± 120 205 ± 70

P < 0.0it P < 0.001tResults are means ± SEM. The therapy was initiated after 18 weeks of treatment with BOP and

continued for 45 days.*Microcapsules liberating 5 jig per day administered every 15 days for 45 days.tSignificance was calculated by Duncan's new multiple range test.*Significance was calculated by Student's t test.§Microcapsules liberating 25 gg per day administered every 30 days for 45 days.

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treated group and 34.4 ± 0.6 microunits/ml (P < 0.05) in theanimals given [D-Trp6]LH-RH. Increased levels of insulin inthe treated groups might reflect a better secretory activity ofpancreatic islets, because of smaller tumor volume as com-pared to controls. Analog RC-160 inhibits insulin releasemuch less than that of growth hormone and other hormones,and 15 days after the last injection ofmicrocapsules the levelsof insulin would be expected to recover.

Microscopic Morphology. All the tumors of the controlgroup were well-differentiated ductal cell adenocarcinomaswith columnar or cuboidal epithelial cells. These cells withhyperchromatic nuclei and pale cytoplasm showed areas of atypical papillary hyperplasia and goblet cell metaplasia (Fig.1). These tumor cells were supported on a scant stromashowing an inflammatory reaction with an infiltration com-prised ofpolymorphonuclear leukocytes. In the group treatedwith [D-Trp6]LH-RH, a glandular pattern was present withthe larger glands showing cuboidal or columnar epitheliawithout goblet cell metaplasia. Frequently, those glandsformed cysts. The stroma tissue was increased with aproliferation of the interstitial cells (Fig. 2). Tumors treatedwith so~natostatin analog RC-160 showed a glandular orcribriform pattern (Fig. 3). Sometimes these patterns werediffused and dispersed with atypical cells. Metastasis wasfound in the lungs and liver in all the groups, and thehistological characteristics were the same as those of thepancreatic tumoral cells. A tumoral infiltration of the dia-phragmatic muscle was found in one animal.

Ultrastructural Study. The tumoral cells in the controlgroup were cuboidal or columnar with irregular apical surfaceprojections, numerous microvilli, and dense glycocalyx. Thelateral membranes had interdigitations with adjacent cellsand junctional complexes near the luminal surface. Thecytoplasm showed pleomorphic mitochondria with variableand irregular cristae and electron-dense bodies. Shortcisternae of the granular endoplasmic recticulum free ribo-somes, lipid droplets, and small Golgi complex were ob-served. In the apical cytoplasm, mucigen granules werefound. The nucleus was irregular with prominent nucleoli andmarginated chromatin (Fig. 4).

In the group treated with [D-Trp6]LH-RH, an increase ofthe interdigitations with the adjacent cells of the granularendoplasmic reticulum and of the number and size of themitochondria was observed. Mucigen granules were de-creased. The nuclei were irregular with typical nuclear bodiesand marginated chromatin. The perinuclear cisternae of somecells were distended (Fig. 5).

FIG. 1. Histological section of pancreatic tumor from the controlgroup. Note glandular pattern with columnar epithelium and gobletcell metaplasia. (Hematoxylin and eosin; x 100.)

FIG. 2. Section of plastic-embedded pancreatic tumor from the[D-Trp6]LH-RH-treated group showing an increase of interstitialcells. (Toluidine blue; X200.)

The tumoral cells of the group treated with somatostatinanalog RC-160 showed an increase of the cytoplasmic organ-elles, numerous mitochondria, more abundant granularendoplasmic reticulum, free ribosomes, and well-developedGolgi complex. The nuclei exhibited prominent nucleoli andgranular nuclear bodies. The basal membrane showed vari-ous layers (Figs. 6 and 7).

DISCUSSIONThe high incidence of pancreatic tumors (98%) obtained inour study after the administration of the carcinogen BOP toSyrian golden hamsters suggests that this animal model iswell-suited for the study of pancreatic carcinomas (10-12).The low incidence of tumors in other organs may be due tothe dose regimen used (10-12). Furthermore, the metastaseswere found exclusively in lung and liver, and this differs fromfindings in other reports (10, 11). Adenomas were notencountered in this study, possibly because of the completetransformation of all the adenomas into adenocarcinomasduring the total experimental time interval (10-12). Morpho-logically, the adenocarcinomas were very similar to humanductal adenocarcinoma with histological and ultrastructuralcharacteristics previously reported by different authors (4,10, 12).The survival time of the hamsters after administration of

BOP was >18 weeks. The survival rate at 24 weeks was

FIG. 3. Histological section of pancreatic adenocarcinoma fromthe group treated with RC-160. Note diffuse and glandular pattern.(Hematoxylin and eosin; x 100.)

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FIG. 4. Electron micrograph of pancreatic adenocarcinoma fromthe control group showing numerous microvilli, dense glycocalyx,free ribosomes, pleomorphic mitochondria, and mucigen granules.The irregular nuclei have marginated chromatin and prominentnucleoli. (x1200.)

increased after treatment with [D-Trp6]LH-RH or with thesomatostatin analog RC-160 to 70%, as compared with the35% in the control group. In this study, the greatest reductionin tumor volume and weight was obtained after treatmentwith [D-Trp6]LH-RH, in accord with our prior work in malerats and hamsters with transplanted tumors (16). Sincechronic administration of [D-Trp6]LH-RH inhibits thepituitary-gonadal axis and creates a state of sex-hormonedeprivation, this suggests that pancreatic cancers may, atleast in part, be sex-hormone sensitive (15, 16, 22, 24). Inturn, this implies the existence of sex-hormone receptors inthe hamster pancreatic tumoral cells similar to those found inhumans and rats (22, 23). A significant reduction in tumorvolume and weight was also obtained following treatmentwith the microcapsules of somatostatin analog RC-160. Thebatch used was an early prototype of RC-160 microcapsules,and a better inhibition of growth of pancreatic tumors mightbe obtained with improved batches of microcapsules. Theseresults are in agreement with those obtained previously inhamsters and rats with pancreatic carcinomas followingtreatment with older (early) somatostatin analogs (16). The

FIG. 6. Electron micrograph of the tumoral cells from the grouptreated with RC-160, showing irregular nuclei with prominentnucleoli and typical nuclear bodies. The cytoplasm exhibits anincrease of organelles. (x 1200.)

decrease in volume and weight of pancreatic tumors may beexplained, in part, by the inhibitory action of somatostatinanalogs upon the release or action of gastrointestinal hor-mones, some of which have been implicated in the stimula-tion ofgrowth ofnormal and tumoral pancreatic cells (18-21).In addition, somatostatin and its analogs may act directly ontumor cells by suppressing cellular proliferation, inhibitingcentrosomal separation, and nullifying growth stimulationinduced by the epidermal growth factor (28). Other possiblemechanisms of action of somatostatin analogs on pancreaticcancer have been also proposed. Somatostatin and its analogsmay act directly by stimulation of dephosphorylation ofmembrane receptors (17, 28-30) and indirectly by inhibitingthe secretion of growth hormone, which in turn may lead toa decrease in tumor autocrine growth factors (17).The reduction in the tumoral cells and the increase in

connective tissue observed in pancreatic tumors treated with[D-Trp6]LH-RH are similar to those previously reported byour group in the Dunning rat prostatic cancer model followingtherapy with this analog (26). Comparable, but less intense,histological changes were found in pancreatic tumors treatedwith the somatostatin analog RC-160. A high degree ofcellular pleomorphism and, sometimes, the presence of

.,'.'At"

FIG. 5. Electron micrograph of the tumoral cells from the grouptreated with [D-Trp6]LH-RH, showing increase of interdigitations,junctional complex near the luminal surface, and few mucigengranules. (x3550.)

FIG. 7. Detail of electron micrograph in Fig. 6, showing distendedcisternae of granular endoplasmic reticulum and an increase ofmitochondria and Golgi complex. (x5200.)

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undifferentiated patterns have recently been described (31).The results obtained in our ultrastructural study were anal-ogous to those reported in other studies of pancreaticcarcinomas induced by nitrosamines (32, 33). The increase ofthe number and pleomorphism of mitochondria and granularendoplasmic reticulum with distension of cisternae observedin the treated groups may be an attempt by tumoral cells torepair the damage caused by the hormonal therapy. Theseresults in female hamsters with induced ductal pancreatictumors confirm and extend our findings, obtained in maleanimals with transplanted tumors, that [D-Trp6]LH-RH andsomatostatin analogs inhibit the growth of pancreatic can-cers. These data may be useful in the development ofhormonal therapy for pancreatic cancer based on somato-statin analogs and [D-Trp6]LH-RH.

We thank Dr. P. Orsolini (Cytotech-Debiopharm) for microcap-sules of RC-160, Dr. E. Hoffman and Dr. M. Abad for their help inultrastructural study, Martha Sampson for technical assistance, andDr. Parvis Pour for valuable advice on BOP lesions. The workdescribed in this paper was supported by National Institutes ofHealth Grants AM07467 and CA40077 (A.V.S.) and by the MedicalResearch Service of the Veterans Administration.

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