Somatostatin Analogues and Bombesin/Gastrin-Releasing Peptide Antagonist RC-3095 Inhibit the Growth of Human Glioblastomas in Vitro and in Vivo1

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[CANCERRESEARCH54, 5895-5901, November 15, 19941 Somatostatin Analogues and Bombesin/Gastrin-releasing Peptide Antagonist RC-3095 Inhibit the Growth of Human Glioblastomas in Vitro and in Vivo1

Jacek Pliiski, Andrew V. Schally,2 Gabor Halmos, Karoly Szepeshazi, and Kate Groot Endocrine,Polypeptideand Cancerinstitute, VeteransAffairsMedical Center,New Orleans,Louisiana70146and Sectionof ExperimentalMedicine,TulaneUniversitySchoolof MedIcine. New Orleans, Louisiana 70112

ABSTRACT The expression of receptors for EGFS1 and IGF-I and IGF-II in human brain tumors, including malignant gliomas, is also well established (4, We Investigated the effects of somatostatin analogues and a synthetic 8â€”15).Itthusappearsthat sex steroidsand growth factorsmight be bombethilgastrln-releasing peptide (GRP) antagonist on the growth of the involved in the proliferation of brain tumors. Some types of brain human m@Ilgnantgiloma cell lines U-87MG and U-373MG transplanted tumors, such as astrocytomas and meningiomas, contain significant to nude mice or cultured in vitro. Nude mice bearing s.c. Implanted U-87MG or U-373MG tumors were treated for 4 and 6 weeks, respec levels of high-affinity receptors for bombesin/GRP or somatostatin lively, with various somatostatin analogues or bombesin/GRP antagonist (16â€”23).Thesefindingssuggestthe needfor investigationstodeter RC-3095. Soniatostatin analogues RC-160, RC-160-ll, and RC-101-I, mine the effects of somatostatin analogues and bombesin/GRP given s.c@Indoses of 100 pg/@nlmal/day,Inhibited the growth of U-S7MG antagonists on the growth of brain tumors. xenografts as shown by more than 60% reduction In tumor volumes and Potent octapeptide analogues of somatostatin and pseudononapep 45%reductIonIntumorweightscomparedwiththecontrolgroup.Bomb tide antagonists of bombesin/GRP have been synthesized in our esIn@RP antagonist RC-3095, given s.c. at a dose of 20 pg@an1maltwice laboratory (24â€”26).Somatostatin analogues RC-160, RC-160-II, and daily, had the greatest inhibitory effect and decreased tumor volumes and RC-101-I are about 100 times more potent than somatostatin in tests weights by approximately 79% and 72%, respectively. The growth of for inhibition of GH release in rats, and possess a prolonged duration U-373MG xenografts was also significantly Inhibited by treatment with of action (25). [D-Tpi6,Leu13uI@CH2NH)'4]bombesin(6-14)(RC-3095)is analogue RC-160 or antagonist RC-3095. The mean survival time of nude mice, Inoculated orthotopically with U-S7MG cells Into the brain, was a short-chain pseudononapeptide bombesin antagonist (26). This asia significantly prolonged by 4.9 days by treatment with antagonist RC-3095. logue blocks GRP-stimulated amylase release from rat pancreatic acini, @ Serum levels In nnhnals bearing U-S7MG tumors, treated with binds to Swiss 3D fibroblasts and H-345 small-cell lung carcinoma cells, antagonist RC-3095 or somatostatin analogues, were decreased compared and inhibits GRP-stimulated growth of these cells in vitro (26). Various with controls. All three somatostatin analogues also reduced serum findings suggest that somatostatin analogues as well as bombesin/GRP growth hormone levels. Receptor analyses demonstrated high-aMnity antagonists may inhibit the growth of several cancers by interfering with binding sites for bombesin, somatostatin, and epidermal growth factor on the action, secretion, signal transmission, or receptors of endogenous membranes of U-87MG and U-373MG tumors. The concentration of growth factors (24â€”31).Previously, we have shown that both types of bindingites for epidermal growth factor on both tumors was significantly these peptide analogues can significantly inhibit the growth of gastric, deciaiiid after in vivo treatment with antagonist RC-3095 or the soma tostatin analogues. In studies in vitro, RC-3095, added to the culture pancreatic, mammary, prostatic, and lung cancers in vivo (27â€”31).This medium, significantly IHifihited the proliferation of U-S7MG and antitumor effect ofbombesin/GRP antagonist and somatostatin analogues U-373MG cells In the presence of GRP(14-27), as measured by cell nuns was associated with a significant down-regulation of EGF receptors on ber, but only a moderate suppression of growth of both cell lines was membranes of these tumors. observed with somatostatin analogue RC-160. These results demonstrate In this study, we have evaluated the effects of somatostatin ana that bosnbeslnlGRP antagonist RC-3095 and somatostatin analogues such logues RC-160, RC-160-II, and RC-101-I and bombesinlGRP antag as RC-160 can Inhibit the growth of human glioblastoma ecU lines omst RC-3095 on the growth of xenografts of the human malignant U-87MG and U-373MG in vim, as well as in vivo. Our work suggests the glioma cell lines U-87MG and U-373MG in athymic nude mice. To merit of fUrther Investigations of these analogues for the possible devel clarify the mechanisms of antitumor action of these agents, detailed opment of new approaches for treatments of patients with malignont receptor status and histological examinations were performed. We also examined the effects of RC-160 and RC-3095 on serum GH and gastrmnlevels because the growth of brain tumors may be promoted by INTRODUCTION GH through stimulation of IGF-I (32), as well as by gastrin, since Malignant astrocytomas or glioblastomas represent the most com evidence exists for the presence of gastrmn and gastrin receptors in mon type ofprimary braintumorin adults(1, 2). Morethanhalfof the human brain (33, 34). In addition, possible direct effects of RC-3095 glial tumors of adults are malignant astrocytomas (1, 2). Surgery, and RC-160 on proliferation of U-87MG and U-373MG cells in vitro radiation, and chemotherapy are of limited effectiveness in the treat were evaluatedin tissue cultures. ment of malignantgliomas and othertherapeuticapproachesmust be explored. That glioblastomas and other brain tumors are hormone MATERIALS AND METHODS sensitive is supported by epidemiological, clinical, and laboratory evidence (3â€”7).The presence of receptors for glucocorticoid, andro Peptides gen, and progesteronein glioblastomas has been documented(4â€”7). Somatostatin analogues D-Phe--C)@s-Tyr--D-Trp-Lys-Val-Cys--Trp-NH2

Received 6/20/94; accepted 9/27/94. (RC-160), N-acetyl-D-Phe-C@s-Tyr-D-Trp-Lys-Val--Cy@--Trp-NH2(RC Thecostsofpublicationofthisarticleweredefrayedinpartby thepaymentofpage 160-I! correspondingtoacetylatedRC-160),and N-acetyl-Phe-C@s-Phe-D charges.Thisarticlemustthereforebeherebymarkedadvertisementinaccordancewith 18 U.S.C Section 1734 solely to indicatethis fact. Trpâ€”Lysâ€”Thr--C@rs-Thr-NH2(RC-101-I),originally synthesized by us (35), 1 This work was supported by the Medical Research Service of the Veterans Affairs were made by classical synthesisby Novabiochem(Laufingen,Switzerland) [A. V. S.] and NIH GrantCA 40077.The contentsof this manuscriptaresolelythe responsibilityofthe authorsand do not necessarilyrepresenttheofficialviewsof the NatiOnal Cancer Institute. 3 The abbreviations used are: EOF, epidermal growth factor; CIRP, gastrin-releasing 2 To whom requests for reprints should be addressed, at VA Medical Center, 1601 peptide;IGF-I,insulin-likegrowthfactorl; OH,growthhormone;Tpi,2,3,4,9-tetrahydro PerdidoStreet,NewOrleans,LA70146. 1H-pyrido[3,4-b]indol-3-carboxylic acid; CNS, central nervous system. 5895

Downloaded from cancerres.aacrjournals.org on September 24, 2021. © 1994 American Association for Cancer Research. PEPTIDEANALOGUESIN GUOBLASTOMAS and supplied by Debiopharm S. A. (Lausanne, Switzerland). For the injections, RC-3095, injected s.c. twice daily at a dose of 20 p@g/animal;and(c) RC-160, the somatostatin analogues were dissolved in 0.1 Macetic acid and diluted with injected s.c. at a dose of 100 p@g/day/animal. saline containing 0.1â€”0.5%bovine serum albumin. Histological Procedures. The histological procedures were the same as Bombesin/GRP antagonist RC-3095 [D-Tpi6, Leu'3 L1,(CH2NH) described previously (28). The number of mitotic and apoptotic cells per 1000 Leu'4]bombesin(6â€”14),originally synthesized in our laboratory (26), was cellswasdetermined,andthepercentageareaofnecrosisintumorsectionswas madeby Asta Medica(Frankfurt/Main,Germany).Tpi is a conformationally examinedusingthepoint-coustingmethod(28),inwhichthecrossingpointsof an constrained secondary amine derivate of tryptophan. GRP(14-27) was synthe ocular net that hide or coincide with necrosis in various sections are counted. sized using solid-phase methods in our laboratory. RC-3095 and GRP(14-27) Radioimmunoassays. Serum levels of OH were determined by double were dissolved in 0.1% dimethylsulfoxide in saline. antibody radioimmunoassay using materials supplied by the National Hormone and Pituitary Program of the National Institute of Diabetes and Digestive and Animals Kidney Diseases. Interassay and intraassay coefficients of variation were less than 15% and 10%, respectively. Serum gastrin levels were measured by Athymic male NCrnzilnu nude mice, approximately 6 weeks old on arrival, double-antibody radioimmunoassay with a kit provided by Becton Dickinson were obtained from the National Cancer Institute (Bethesda, MD) and (Orangeburg, NY). The interassay variation was less than 7.5%, and the maintained under pathogen-limited conditions. intraassay variation was less than 4.0%. Receptor Assays. Receptorsforbombesin,somatostatin,andEGFon the Cell Lines membranes of U-87M0 and U-373-MG tumors were measured as described previously (28, 29, 35). The UGAND PC computerized curve-fitting program The U-87MG and U-373MG malignant glioma cell lines (astrocytomas, of Munson and Rodbard (37) was used to determine the types of receptor grade III; American Type Culture Collection, Rockville, MD) were grown as binding, dissociation constant (Kd) values, and the maximal binding capacity monolayers in Eagle's basal medium supplemented with 10% fetal bovine (Bm,,,3of receptors. serum, sodium pyruvate, and antibiotics and antimycotics. Cultures were incubated at 5% CO2 in air at 37Â°C. In Vitro Studies In Vivo Studies U-87MG and U-373MG cells from 70 to 80% confluent cultures were seeded into Costar 24-multiwell plates (Costar, Cambridge, MA) at a density Studies on Tumor Growth. In the first experiment, xenografts were mi of 5 X i0@cells/well and grown in Eagle's minimum essential medium hated by s.c. injection of 1 X i0@U-87MGcells into the right flanks of 5 male supplementedwith 1 mMsodium pyruvate and 5% fetal bovine serum. The cell mice. Tumors resulting after 4 weeks were aseptically dissected and mechan viability, measured by trypan blue dye exclusion, was more than 90%. After 48 ically minced; 3-mm3 pieces of tumor tissue were transplanted s.c. by trocar h (day 0), the medium was replaced with fresh medium containing RC-3095 at needle into 60 male animals under methoxyflurane anesthesia. Two weeks concentrations of 10 â€˜1105 M in combination with GRP(14-27) at a con after transplantation, when tumors had grown to a volume of approximately 25 centration of iO@ M.Proliferation ofcells exposed to RC-3095 in the presence mm3,the mice were randomized and divided into 5 experimental groups of 10 of GRP(14-27) was compared to that of cells which were cultured only with animals each and received the following treatment for 4 weeks: group 1, 0.1 M GRP(14-27).In initial experiments,the effect of iO@ M GRP(14-27)on acetic acid in saline; group 2, RC-160 at a dose of 100 @@day/animals.c.; growth of both cell lines was also evaluated. group 3, RC-160-II at a dose of 100 pg/day/animal s.c.; group 4, RC-101-I at In other experiments, RC-3095 and RC-160 in concentrations of 10â€•- a dose of 100 @.tg/day/animals.c.;and group 5, RC-3095 at a dose of 20 iO@ M were added to the culture medium and the proliferation of these cells @Wanimal,injecteds.c. twice daily. was compared to that of cells which were cultured in the absence of these In the second experiment, xenografts were initiated by s.c. injection of analogues. The cell number for each well was determined in a Coulter counter 1 X@ U-373MG cells into the right flanks of 5 male mice. Tumors resulting after detachment of the cells by trypsinization on day 4. in vitro experiments after 4 weeks were aseptically dissected and mechanically minced; 3-mm3 were performed 3 times with 6 replicates for each concentration. pieces of tumor tissue were transplanted s.c. by trocar needle into 40 male Statistical Methods. Statistical analyses of the tumor data were performed animals under methoxyflurane anesthesia. Two weeks after transplantation, using Duncan's new multiple range test. when tumors had grown to a volume of approximately 7 mm3, the mice were randomized and divided into 3 experimental groups of 10 animals each and received the following treatment for 6 weeks: group 1, 0.1% dimethyl sulfox RESULTS ide in saline; group 2, RC-160 at a dose of 100 @â€˜day/animals.c.;and group 3, RC-3095 at a dose of 20 @.&Wanimal,injecteds.c. twice daily. Effect of Peptide Analogues on Growth of Malignant Gliomas In both experiments,the tumorswere measuredonce a week. The tumor in Nude Mice. The effects of treatment with various peptide ana volumes and weights were measured in a blinded manner. Tumor volume was logues on body and tumor weights in nude mice implanted s.c. with calculated as length x width x height x 0.5236. Tumor volume doubling time malignant gliomas as well as final tumor volumes in both experiments was calculated between the start and the end of the treatment. At the end of are shown in Table 1. At the end of the experiments, there were no both experiments, mice were anesthetized with methoxyflurane, killed by significant differences in body weights between groups. decapitation, trunk blood was collected for analyses, body weights were In experiment 1, all three somatostatin analogues significantly recorded, and various organs were removed and weighed. Tumors were cleaned and weighed and samples were taken for histology and receptor suppressed growth of U-87MG tumors. After 4 weeks, the mean studies. tumor volume was significantly (P < 0.01) reduced in groups receiv Survival Test. U-87MG cells were inoculated into the brains of 6-week ing RC-160, RC-160-ll, and RC-1O1-I to 598.6 Â± 123.9 mm@, old athymic male NCrniilnu nude mice. Mice were anesthetized with methoxy 448.5 Â±91.2 mm3, and 398.0 Â±126.7 mm3, respectively, as com flurane (Metofane; Pitman-Moore, Mundelein, IL). A midline incision was pared with that of the control group (1599.0 Â±250.1 mm3) (Fig. 1; made over the anterioraspect of the craniumand the scalp retractedto the Table 1). Tumor volume doubling time was significantly prolonged by right. A guarded 26-gauge needle was used to drill a hole in the skull 3 mm to all three somatostatin analogues. Treatment with RC-101-I extended the right of the midline, just anterior to the coronal suture, and 3â€”4-mmdeep. the tumor doubling time to 8.0 Â±1.6 days compared with that of the A Hamiltonsyringe (Reno, NV) was used to inject 10 ,d of 0.9% sterile control group, which had a tumor doubling time of 4.6 Â±0.1 days. sodium chloride containing about 10@ U-87MG glioblastoma cells. The scalp The fmal tumor weights were reduced by 45â€”61%in the groups was closed with surgical skin staples. Control mice were treated identically, treated with RC-160, RC-160-II, or RC-1O1-I as compared with those except that their intracranial injections contained no tumor cells. Treatment was startedon the day of inoculationof glioblastomascells and continued of the controls (Table 1). throughout the life span of the animals. The mice were divided into 3 groups Therapy with bombesin/GRP antagonist RC-3095 appeared to be (8 animals/group): (a) control tumorous animals, saline injections only; (b) the most effective and resulted in the greatest inhibition of tumor 5896

Table1 E;flectoftreatmentwithvariouspeptideanaloguesonbodyweightandtumor Effect of RC-3095 and RC-160 on Survival Time. The life span w;lwne in nude mice with xenografts of human malignant glioma of mice, inoculated orthotopically with U-87MG cells, was prolonged cell lines U-87MG and U-373MG significantly (P < 0.01) by giving them bombesin/GRP antagonist (mm3)InitialFinalExperiment volume Treatment weight weight RC-3095 (Fig. 3). The mean survival time of mice in the control and groupBody (g)Tumor (g)Tumor RC-3095-treated groups were 41.2 Â± 0.7 and 46.1 Â± 0.6 days, 1:(U-87M0)Control24.1 respectively. Thus, treatment with RC-3095 increased the mean sur vival time by 4.9 days. The treatment with somatostatin analogue 250.1â€•RC-309522.0Â±0.8Â°1.8 Â±0.3Â°28.2 Â±2.5Â°1599.0 Â± Â±RC-16022.4 Â±1.30.5 Â±02b26.3 Â±5.1328.2 RC-160 also prolonged the mean survival time to 44.2 Â±1.8 days (not 123.9â€•RC-160-ll22.3Â±0.7'@1.0 Â±0.2'@28.1 Â±3.4598.6 Â± shown), but this extension of survival time was not statistically 91.2â€•RC-101-I21.0 Â±0.70.8 Â±0.2â€•21.5 Â±2.2448.5 Â± 126.7â€•Experiment Â±1.20.7 Â±03b25.4 Â±3.6398.0 Â± significant compared to the control group. 2:(U-373MG)Control25.4 Histological Findings. Histologically, the U-87MG tumors were highly cellular and contained very little stroma. The cells were po 63.2RC-16024.3 Â±0.70.2 Â±0.088.5 Â±1.9167.2 Â± lygonal or elongated and the oval-shaped and chromatin-rich nuclei 8.6cRC-309523.0 Â±2.10.035 Â±0.01â€•7.5 Â±1.336.7 Â± Â±1.10.025 Â±0.01â€•6.9 Â±0.719.3 Â±5.1c were surrounded by broad, light eosinophilic and finely granular a Mean Â±SE cytoplasms. Some of the tumors contained necrotic areas with inflam b p < o.oi vs. control. matory cell infiltration. The number of mitotic cells decreased and that C p < of apoptotic cells increased in the treated tumors, but the difference was not statistically significant from that of controls. The ratio of apoptotic to 2000 mitotic indices was significantly (P < 0.01) higher only in the group receiving RC-101-I (2.67 Â±0.8versus 0.84 Â±0.2in controls). F') 1800 0â€”0 :Control E The U-373-MG tumors consisted of elongated cells arranged in E 1600 .â€”. :RCâ€”3095 @â€”@:RCâ€”16O curving bundles. The cells had narrow eosinophilic cytoplasms and Li 1400 Aâ€”A :RCâ€”160â€”Il long, rod-shaped nuclei. The number of mitotic cells decreased and -j 1200 0 Dâ€”D:RCâ€”1O1 â€”I > 1000 a: 0 800 250 I- 600 0â€”0 Control /5 F') 400 E â€¢â€”â€¢:RCâ€”160 E 200 :RCâ€”3095 UI ,_!!@@@::z4====:==:@ . Li

0 i 2 3 4 -J 0 WEEKS OF TREATMENT > a: 100 Fig. 1. Tumor volumes in nude mice with s.c. transplanted U-87M0 glioblastomas 0 during treatment with bombesin/GRP antagonist RC-3095, given s.c. at a dose of 20 Iâ€” gag/animaltwice daily, and somatostatin analogues RC-160, RC-160-II, and RC-101-l, 50 given s.c@in doses of 100 gig/animal/day. Treatment was started when the tumors measuredapproximately25mm3andlastedfor 4 weeks.Verticalbars,SE. Â°P<0.01 wrsa&ccontrol. The inhibition was significant after 2 weeks of treatment for RC-3095, RC-160-ll,andRC-101-I. 2 3 4 5 6

WEEKSOFTREATMENT weight and volume (Fig. 1; Table 1). Administration of RC-3095 Fig. 2. Tumor volumes in nude mice with s.c. transplanted U-373M0 glioblastomas significantly (P < 0.01) inhibited tumor growth from day 14 until the during treatment with bombesin/GRP antagonist RC-3095, given s.c. at a dose of 20 ILg/arnmaltwice daily, and somatostatin analogue RC-160, given s.c. at a dose of 100 end of the experiment (Fig. 1; Table 1). Tumor volume doubling time j@g/animaI/day.Treatmentwas started when the tumors measured approximately 7 mm3 in mice receiving RC-3095 was extended to 8.3 Â±1.0 days. The mean and lasted for 6 weeks. Verticalbars, SE. P < 0.01 versuscontrol.The inhibitionwas tumor weight was reduced significantly (P < 0.01) by about 72% in significant after 1 week of treatment for RC-160 and RC-3095. the group treated with RC-3095 compared with that of the control group (Fable 1). In experiment 2, therapy with somatostatin analogue RC-160 or bombesin/GRP antagonist RC-3095 inhibited the growth of w 0 U-373M0 tumors (Fig. 2; Table 1). The mean tumor volume and 0 â€” 0 :Control 0 â€¢â€”S: RCâ€”3095 weight were significantly (P < 0.01) reduced in animals receiving z RC-160 for 6 weeks to 36.7 Â± 8.6 mm@ and 0.035 Â± 0.01 g, > > a: respectively, compared with those in the control group (167.2 Â±63.2 :2 mm3 and 0.2 Â±0.08 g, respectively, Table 1). Vt Therapy with bombesin/GRP antagonist RC-3095 was even more 0 a: effective as in experiment 1, and produced a greater inhibition of w tumor volume and weight than RC-160 (Fig. 2; Table 1). The final tumor volume and weight were reduced in the group treated with z RC-3095 by about 89% and 88%, respectively, compared with those in the control group (Table 1). Tumor doubling time was prolonged by DAYSOF TREATMENT RC-3095 to 40.6 Â±8.6 days compared to 10.4 Â±0.7 days for the Fig. 3. Survival times of nude mice, inoculated orthotopically with U-87M0 glioblas control group. tomacells,duringtreatmentwithbombesin/ORPantagonistRC-3095. 5897

that of apoptotic cells increased, but the ratio of apoptotic to mitotic 100. indices was not significantly changed by the treatments. Serum Hormone Levels. The levels of serum gastrin and OH in 90. nude mice bearing U-87MG glioma treated with peptide analogues in 80. the first experiment are shown in Table 2. Gastrmnlevels in animals treated with the somatostatin analogues or bombesin/GRP antagonist â€” 70. @ RC-3095 were significantly reduced compared with the levels of 60. controls. Serum OH in animals treated with somatostatin analogues Ui @ RC-160, RC-160-II, and RC-101-I was greatly decreased (P < 0.01) 50. compared with that of controls. There were no changes in serum OH z -J 40. -J levels after treatment with RC-3095. UI Receptor Findings. The binding characteristics of receptors for C.) 30 bombesin/ORP, somatostatin, and EGF in U-87M0 and U-373M0 20 .â€”. U-87M6 tumors were analyzed following treatment with peptide analogues in aâ€”.U.373MG both experiments, and the results on the EGF receptors are presented 10 in Table 3. In experiment 1, receptor assays on U-87M0 tumor 0 membranes showed high-affinity binding sites for bombesin/ORP -11 -10 -9 -8 -7 -6 -5 (Kd, 6.1 Â± 0.5 nM), somatostatin (Kd, 7.1 Â± 2.1 nM), and EGF (Kd, 0.9 Â±0.1 nM).The concentration of receptors for bombesin/GRP was LOGCONCENTRATiON(M) significantly (P < 0.01) reduced by treatment with bombesin/GRP Fig. 4. Influence of bombesin/GRP antagonist RC-3095 at i0â€•-i05 Mconcentra antagonist RC-3095 to 172.7 Â±10.3 fmol/mg protein as compared to tions on proliferation of U-87MG and U-373M0 glioblastoma cells in the presence of iO@ MORP(14-27).Theresultsare expressedas percentageof the numberof cells that in the control group (392.7 Â±27.8 fmol/mg protein). This may be found after the addition of RC-3095 in concentrations of 10 11105 Min the presence due to receptor occupation by this analogue. The binding capacity of of @MGRP(14-17)comparedtothenumberofccllsfoundwithGRP(14-27)alone, EOF receptors was significantly (P < 0.01) decreased after treatment which was accepted as 100%. Statistical significance was calculated using Duncan's newmultiplerangetest. Verticalbars, SE. @P<0.01 versusgrouptreatedonlywith with somatostatin analogues RC-160, RC-160-II, and RC-101-I or iO@ MGRP(14-27). bombesin/ORP antagonist RC-3095 (Table 3). Therapy with the so matostatin analogues somewhat increased the binding capacity of receptors for somatostatin on membranes of U-87M0 tumors, but this high-affinity binding sites for bombesin/ORP (Kd, 1.3 Â±0.2 mi), increase was not statistically significant when compared to that of the somatostatin (Kd, &4 Â±1.1 ntis),and EGF (Kd, 0.86 Â±0.1 mi) (Table controls. The binding capacity (Bm@) Ofsomatostatin receptors in the 3). A significant (P < 0.01) reduction in binding capacity of bomb control group was 706.7 Â±155.0 fmol/mg protein. In experiment 2, esin/ORP receptors was again observed after treatment with RC-3095. receptor assays on membranes of U-373M0 tumors also demonstrated The binding capacity (Bm@) of bombesin/GRP receptors in the con trol tumors was 788.0 Â±18.2 fmol/mg protein, but only 530.0 Â±24.7 fmol/mg protein in tumors treated with RC-3095. Somatostatin ana humanmalignantTable2 Serumgastrinand GHlevelsin nudemicewithxenograftsofthe analoguesTreatmentglioma cell line U-87MG after treatment with various peptide logue RC-160 and bombesin/ORP antagonist RC-3095 significantly (P < 0.01) decreased the binding capacity of EOF receptors in mem hormonegroup Gastrin Growth (ng/ml)Control (pg/mi) branes of this tumor, the latter analogue causing a greater reduction as 4.9Â°RC-3095 167.5 Â±12.0â€• 17.0 Â± in experiment 2 (Table 3). No changes in binding capacity and affinity 6.5RC-160 130.2 Â±14.8â€• 19.5 Â± of somatostatin receptors occurred after treatment with RC-3095 or 2.4cRC-160-II 99.5 Â±10.8c 7.2 Â± RC-160 on U-373M0 glioblastomas cells in vitro. 0.2cRC-101-I 98.6 Â±34C 3.9 Â± 95.8Â±74C 3.8Â±0.7'@ In Vilro Studies. The proliferationof U-373M0 and U-87M0 a Mean Â±SE. glioblastoma cells in vitro monitored by cell number could be only bp<005 moderately stimulated by about 15% (P < 0.05) and 11%, respec C p < o.oi vs. control. tively, as compared with the controls, by the addition of ORP(14-24) at i0@ Mconcentration to the culture medium (not shown). This poor Table3U-87MGand Bindingcharacteristicsof receptorsfor EGF in membranesof stimulation by exogenous ORP could be due to production of endog U-373MG human malignantvariouspeptide gliomas after in vivo treatmetu with enous bombesin-like peptides by these tumors. Bombesin/ORP antag analoguesEGFB,@Group omst RC-3095, added alone to the culture medium at i0@, 10_6, and io@Mconcentrations,significantly(P<0.01)decreasedthenumber of U-87M0 cells by 14%,30%, and 37%, respectively,as compared protein)Experiment K,,(aM) (fmol/mg to that of controls. At lower doses of RC-3095, the decrease in cell 1:(U-87MG)Control number was not statistically significant. In contrast, RC-3095 signif 14.6RC-3095 0.9 Â±0.1Â° 250.3 Â± icantly (P < 0.01) suppressed the proliferation of U-373M0 glioblas 9.8cRC-160 0.4 Â±02b 69.7 Â± toma cells at concentrations of M,but not at higher doses. 14.1'@RC-l60-II 1.0 Â±0.1 130.3 Â± @ 17.3cRC-101-I 1.0 Â±0.2 139.7 Â± In the presence of 10â€•, 10_b, and 10_8 M RC-3095, the 8.1cExperiment 1.1Â±0.1 141.0Â± number of U-373M0 cells was reduced by 33%, 25%, 27%, and 21%, 2:(U-373MG)Control respectively, as compared to cells not exposed to this antagonist. 3.9RC-160 0.86 Â±0.1 244.8 Â± Bombesin antagonist RC-3095 significantly inhibited the prohuer 113.4Â±1.6cRC-3095 0.58Â±0.1 0.69 Â±0.2 87.2 Â±12.2c ation of U-87M0 and U-373M0 cells in vitro at 10_h1@10_5 M a Binding characteristics were obtained from 10-point displacement experiments in concentrations in the presence of (10@ M)GRP(14-27) as shown in triplicate tubes. Significance was calculated with Duncan's new multiple range test. All Fig. 4. At 10_8 Mconcentration of RC-3095, the number of U-87MG valuesrepresentmeanÂ±SEof 2â€”3experiments,eachdoneintriplicate. and U-373MG cells was decreased by about 40% and 61%, respec bp < o.os. C p < 0.01 vs. control. tively (Fig. 4). In the presence of (10@ M) RC-3095, the number of 5898

U-87MG and U-373M0 cells was reduced by approximately 43% and results suggest that bombesin and GRP may function by up 67%, respectively, as compared to cells cultures only with iO@ M regulating EOF receptors and that antagonist RC-3095 prevents GRP(14-27). this up-regulation (45). Bombesin/ORP antagonists may also block Somatostatin analogue RC-160 significantly suppressed the prolif early cellular events that precede calcium mobilization and stim eration of U-87M0 and U-373MG glioblastoma cell lines at 106 and ulation of mitogenesis (46). i05 M concentrations (not shown). At lower doses of RC-160, the Chronic administration of our potent somatostatin analogues also decrease in cell number was not statistically significant as compared significantly inhibited the growth of U-87MG and U-373MG cell to U-87MG andU-373-MO cells not exposed to this analogue.In the lines xenografted into nude mice. The number of mitotic cells de presence of 10_6 or i0@ M RC-160, the number of U-373M0 cells creased and that of apoptotic cells increased in the treated tumors, but was significantly (P < 0.01) reduced by approximately 38% and 37%, the ratio of apoptotic to mitotic indices was significantly higher only respectively. In the case of the U-87MG cell line, a somewhat smaller in the group receiving RC-101-I. Antineoplastic actions of somatosta but significant (P < 0.05) decrease in cell number of 13 and 15%, tin analogues appear to involve multiple mechanisms. A significant respectively, was found at both concentrations of RC-160. fall in OH levels induced by somatostatin analogues could, through mechanisms involving suppression of endogenous growth factors such as IOF-I and IGF-II, be of major importance for the inhibition of DISCUSSION tumor growth (24). In our study, serum OH levels in mice treated with In this study, we demonstrated a significant inhibitory effect of RC-160, RC-160-II, or RC-101-I were decreased by more than 58% bombesin/GRP antagonist RC-3095 on proliferation of U-87MG and as compared with those of control mice. Membrane receptors for U-373MG cell lines in vitro as well as on the growth of xenografts of IGF-I and specific mRNA for IOF-I and IGF-II were found in human these malignant gliomas in athymic nude mice. RC-3095 also pro CNS, suggesting that these growth factors may play a role in the longed the survival time of nude mice inoculated orthotopically with growth and maturation of neural tissue (47â€”49).In addition, it has U-87MG cells. We have shown that inhibition of the growth of both been shown that meningiomas and gliomas possess specific IOF glioblastomas was accompanied by a decrease in the concentration of receptors and that exposure to IGFs can promote the proliferation of bombesin/GRP receptors on membranes of these tumors. Our find these cells in culture (32, 50), suggesting that IOFs may play a role in ings, indicating the presence of high affinity-binding sites for bomb the development and progression of certain CNS tumors. On the basis esin/GRP on membranes of both tumors, are in agreement with results of our receptor assay results, which indicate the presence of high previously reported by other groups demonstrating the expression of affinity receptors for somatostatin on tumor membranes, analogues of high-affinity receptors for bombesin/ORP in rat and human glioblas somatostatin could also directly inhibit the growth of glioblastoma toma cell lines (16â€”18).Inour study, the proliferationof both glio cells. Somatostatin and its analogues stimulate tyrosine phosphatase blastoma cell lines was only moderately stimulated by addition of and promote the dephosphorylation of EGF receptors (19, 51). In our 5M GRP(14-27) to the culture medium. This observation could be study, somatostatin analogues produced a great reduction in the con tentatively explained by an autocrmne or paracrine production of centration of EGF receptors on membranes of both glioblastoma bombesin/GRP-like peptides by the glioblastoma cells, thereby caus tumors. Thus the sensitivity of U-87MG and U-373MG tumor cells to ing a nearlymaximumstimulationof cell growth.Bombesin/GRP-Iike transforming growth factor a and EOF could have been decreased in peptides were shown to stimulate the growth of small-cell lung our study. Inhibition of tumor growth may also be brought about by carcinoma, prostatic, mammary, and pancreatic cancer lines (38â€”41), the blockade of gastrin production, since gastrin and gastrin receptors and this growth could be inhibited by bombesin/ORP antagonists (38, were demonstrated in human brain (33, 34). However, the physiolog 39, 42). The inhibition of U-87M0 and U-373M0 tumor growth by ical role of gastrin in the brain, the presence of gastrin receptors in bombesin/GRP antagonist RC-3095 might be partially brought about brain tumors, as well as the influence of gastrin on brain tumor growth by blockade and/or down-regulation of bombesin/GRP receptors on remain to be elucidated in future studies. U-87MG and U-373MG cells. We have also shown that inhibition of In contrast to our data, Reubi et a!. (22, 23) reported that highly growth of various cancers, including pancreatic, prostatic, mammary, undifferentiated glial tumors, such as glioblastomas, do not express gastric, and lung by antagonist RC-3095, was associated with a major somatostatin receptors. This discrepancy may be partially explained decrease in EGF receptor levels on tumor membranes (27â€”31). by more recent studies, which demonstrated the existence of several Thus, bombesin/GRP antagonists may act locally by various mech somatostatin receptor subtypes (19â€”21).Molecular cloning revealed anisms which result in a reduction in the available binding sites for the presence of five structurally related integral membrane glycopro EGF. EGF receptors have been shown to be present in various teins, that are pharmacologically distinct high-affinity somatostatin brain tumors such as meningiomas and glia-derived tumors (4, 8, receptors with different regional distributions and functions (19â€”21). 11â€”15).A positive relationship was found between EOF receptor Pharmacological studies on the characteristics of somatostatin recep levels, phosphokinase activity, and the degree of malignancy of tors showed that the different subtypes exhibit major differences in astrocytomas (1 1). In primary cell cultures, EGF was reported to their affinities for structural analogues of somatostatin (19â€”21).For stimulate the proliferation of rat astrocytic glial cells and human example, SMS 201â€”995,asomatostatin analogue that is used to treat glioma cells derived from surgically obtained glioma specimens acromegaly as well as carcinoid syndrome, binds strongly to the (13, 43). somatostatin receptor subtype SSTR2, whereas receptor subtype The exact molecular mechanism of action of bombesin/GRP antag SSTR1 has low affinity for this analogue (19, 21). The methodologies onists ofEGF receptors is still not well understood. Bombesin initiates used by Reubi et a!. (22, 23) in their studies on glioblastomas included a series of intracellularsignals which cause an increase in inositol in vitro receptor-binding analyses on homogenates and receptor au 1,4,5-triphosphate, a mobilization of Ca2@, and diacylglycerol pro toradiography on tissue sections, using preferentially as radioligands duction, leading to activation of protein kinase C (29, 42, 44). Acti a somatostatin-28 analogue or a somatostatin octapeptide analogue vation of protein kinase C causes phosphorylation of EGF receptors (â€˜@â€˜I-labeledTyr@-SMS201-995) (22, 23). In our study, â€˜251-labeled on threonineresidues.Bombesin andORPwere shown to enhancethe RC-160 was used as a tracer in radioreceptor assays. Thus, the phosphorylation of EOF receptors, and antagonist RC-3095 inhibited divergent results on the concentration of somatostatin receptors in these effects in various cancer lines and cancer specimens (45). These glioblastomas between these two studies may be due to different 5899

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Downloaded from cancerres.aacrjournals.org on September 24, 2021. © 1994 American Association for Cancer Research. Somatostatin Analogues and Bombesin/Gastrin-releasing Peptide Antagonist RC-3095 Inhibit the Growth of Human Glioblastomas in Vitro and in Vivo

Jacek Pinski, Andrew V. Schally, Gabor Halmos, et al.

Cancer Res 1994;54:5895-5901.

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