Proc. Natl. Acad. Sci. USAVol. 93, pp. 12513-12518, October 1996Medical Sciences

A tumor-selective somatostatin analog (TT-232) with strong invitro and in vivo antitumor activity

(apoptosis/cell proliferation/tyrosine kinase/xenograft)

Gy. KERI*t, J. ERCHEGYI*, A. HORVA,TH*, I. MEZ6*, M. IDEI*, T. VANTus*, A. BALOGH*, ZS. VADA&SZ*, Gy.BOK6NYI*, J. SEPRODI*, I. TEPLAN*, 0. CSuKAt, M. TEJEDAt, D. GAALt, ZS. SZEGEDI§, B. SZENDE§,C. ROZEI, H. KALTHOFFII, AND A. ULLRICH***Joint Research Organization of the Hungarian Academy of Sciences and Semmelweis University Medical School, Department of Medical Chemistry, MolecularBiology and Pathobiochemistry, Budapest, 8. POB 260, Hungary 1444; tNational Institute of Oncology, Budapest, Hungary 1112; 1st Institute of Pathology andExperimental Cancer Research, Semmelweis University Medical School, Budapest, Hungary 1085; Institut National de la Sante et de la Recherche M6dicale U410 Faculte de Medicine, Paris, France 75870; Department of General Surgery, University Hospital, Kiel, Germany; and **Max-Planck Institut fOir Biochemie,Martinsried, Germany 82152

Communicated by Tibor Farkas, Hungarian Academy of Sciences, Szeged, Hungary, July 29, 1996 (received for review January 20, 1996)

ABSTRACT We report a series of new in vitro and in vivodata proving the selective antitumor activity ofour somatosta-tin structural derivative, TT-232. In vitro, it inhibited theproliferation of 20 different human tumor cell lines in therange of 50-95% and induced a very strong apoptosis. In vivoTT-232 was effective on transplanted animal tumors (Colon26, B16 melanoma, and S180 sarcoma) and on human tumorxenografts. Treatment of MDA-MB-231 human breast cancerxenografted in mice with low submaximal doses of TT-232[0.25 and 0.5 mg/kg of body weight (b.w.)] caused an average80% decrease in the tumor volume resulting in 301% tumor-freeanimals surviving for longer than 200 days. Treatment ofprostate tumor (PC-3) xenografted animals with 20 mg/kg ofb.w. of TT-232 for 3 weeks resulted in 60% decrease in tumorvolume and 100% survival even after 60 days, while 80% ofnontreated animals perished. We have demonstrated thatTT-232 did not bind to the membrane preparation of ratpituitary and cortex and had no antisecretory activity. TT-232was not toxic at a dose of 120 mg/kg of b.w. in mice. Long-termincubation (24 h) of tumor cells with TT-232 caused signifi-cant inhibition oftyrosine kinases in good correlation with theapoptosis-inducing effect. The level of p53 or KU86 did notchange following TT-232 treatment, suggesting a p53-independent apoptotic effect. Preincubation of human breastcancer cells (MDA-MB-453) with TT-232 for 2 h decreased thegrowth factor receptor autophosphorylation. All of these datasuggest that TT-232 is a promising and selective antitumoragent.

Somatostatin, a natural tetradecapeptide, inhibits both thegrowth hormone release (1) and various other endocrinesecretions (i.e., glucagon, insulin, and gastrin) (2, 3). It inhibitsor regulates several cell functions as well, and it has also beendiscovered to be an important endogen antiproliferative agent(4-8). In recent years, many potent somatostatin analogs havebeen developed as antisecretory and antiproliferative hor-mones (Sandostatin or Octreotide of Sandoz, Somatuline ofBiomeasure, and RC-160 of Debiopharm), acting longer thanthe native hormone, and they have been used in the treatmentof hormone dependent tumors, but their administration asantitumor agents is limited because of their side effects (9-13).Lots of efforts have been made to obtain a tumor selectivesomatostatin analog but without any significant success.

Previously we reported a unique, potent tumor-selectivesomatostatin analog of a five-residue ring structure: D-Phe-Cys-Tyr-D-Trp-Lys-Cys-Thr-NH2 (TT-232). This analog had

practically no growth hormone (GH) release inhibitory activityeither in superfused rat pituitary cells or in rats in vivo. TT-232showed a strong antiproliferative effect both in vitro and in vivo(14-16) and induced apoptosis in a pancreatic tumor cell line(17) as demonstrated for other somatostatin analogs (18, 19).This compound has a special structure, its backbone behavingvery differently from that observed in the GH active analogs(20). TT-232 inhibited tyrosine kinase activity of some humancolon tumor cell lines, and this inhibition correlated well withthe inhibition of cell proliferation (14). When incubating colontumor cells with this peptide for short periods of time (10-100min), no significant inhibition of tyrosine kinases was ob-served, but a significant stimulation of tyrosine phosphatasescould be detected with a biphasic dose dependency (21).TT-232 has been tested on 60 various human tumor cell linesby the National Cancer Institute of the National Institutes ofHealth. It was found that at a higher dose (100 ,M) TT-232had a very strong antiproliferative effect on 58 cell lines, whileTT-248, one of our "traditional type" somatostatin analogs,which in other respect showed similar biological effects to thatof RC-160 or Sandostatin had practically no effect on the samecell lines (unpublished results). Somatostatins activate proteinkinase C and stimulate the inositol triphosphate level in colontumor cells following a long term incubation (22, 23). So it maybe concluded that TT-232 interacts with various signalingpathways in tumor cells, and the interrelationship of thesepathways may determine the antiproliferative effect via theinduction of apoptosis and the inhibition of mitosis. In vivo,TT-232 has been very effective in the Lewis lung tumor,spleen-liver metastasis model in mice, causing a 70% inhibitionin the number of metastasis (17).To test the possibility whether TT-232 could be a tumor-

selective drug, several new in vitro and in vivo studies, as wellas mechanism of action studies have been carried out, and theresults of these studies are presented here. The objective ofthese studies were to investigate what kind of tumor cells couldbe the primary target of TT-232, to investigate the selectivityof TT-232 considering the traditional somatostatin-like ef-fects, and to investigate the interrelationship of tyrosine kinaseinhibitory, anfiproliferative, and apoptosis-inducing effect ofTT-232. The other objectives of these studies were to investi-gate the in vivo antitumor effect of TT-232.

MATERIALS AND METHODSPeptides. D-Phe-Cys-Tyr-D-Trp-Lys-Cys-Thr-NH2 (TT-232)

and 3-Asp(Ind)-C s-Tyr-D-Trp-Lys-Val-Cys-Thr-NH2 (TT-

Abbreviations: GH, growth hormone; b.w., body weight.tTo whom reprint requests should be addressed.

12513

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

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248) were synthesized in our laboratory as described earlier(14). D-Phe-Cys-Tyr-D-Trp-Lys-Val-Cys-Trp-NH2 (RC-160)(24) was kindly supplied by Debiopharm, Lausanne, Switzer-land, and D-Phe-Cys-Phe-D-Trp-Lys-Thr-Cys-Thr(ol) San-dostatin (25) was a generous gift of Sandoz Pharmaceutical.Tumor Cell Culture. The cells were obtained from Ameri-

can Type Culture Collection and cultured with RPMI medium1640 or Leibovitz's L-15 medium supplemented with 10% fetalcalf serum (all from SEBAK, Aidenbach, Germany). The cellswere kept at 37°C in a humidified atmosphere of 5% CO2 and95% air.

In Vitro Antitumor Assays. Approximately 350,000 cellswere cultured in 2 ml of culture medium in a well (6-well plateswere used, Greiner, Nurtingen, Germany) and incubated for24 h with different doses of TT-232 in the absence of fetal calfserum. After trypsinization and dispersion, the cells were dyedwith trypan blue and counted in a hemocytometer.

Study of Apoptosis. Tumor cells (105/ml) were treated withvarious doses of TT-232 for 8-24 h. Cell count was determinedregularly at 24, 48, and 72 h. Triplicate determinations per doseand time points were performed both in cases of treated anduntreated cell cultures. Three coverslips per dose and timepoint were fixed in ethanol/acetic acid (3:1) and stained withhematoxylin and eosin. The specific morphology of apoptoticcells (nuclear pyknosis or karyorrhexis, cytoplasmic shrinkage,apoptotic bodies in the cytoplasm of nonapoptotic tumor cells)was identified by the hematoxylin and eosin staining and byroutine transmission electromicroscopic investigation. Theratio of apoptotic and mitotic figures was determined bycounting 500 cells. Immunohistochemical reaction usingApop-Tag kit (26) (Oncor) was applied to detect in situ the new3'-OH DNA ends characteristic to apoptosis.

Tyrosine Kinase Activity Assay. The assay was carried outas described earlier (27) using a synthetic peptide substrate(E11G1). The results were expressed as incorporated pmol of32p per mg of protein per min.Animals and Experimental Procedures for in Vivo Tests.

Colon 26 tumor fragments (from Karolinska Institute, Stock-holm) were s.c. transplanted into BALB/C inbred mice; B16melanoma cell suspension (also from Karolinska Institute) wastransplanted into BDF1 hybrid mice; S180 sarcoma tumorfragments (Chester Beatty Cancer Research Institute, Lon-don) were transplanted into Swiss outbred mice. Ten animalswere in each group (21-23 g). The i.p. treatment with differentdoses of TT-232 [15, 150 and 750 ,ug/kg body weight (b.w.)twice a day] was initiated 1 day after the tumor transplantationand continued till the first animal perished. Lifetime changeswere compared with nontreated tumor-bearing animals, andthe size of solid tumors was measured with a caliper two orthree times weekly. Tumor volume was calculated using theformula V = a2 X b X ir/6 where a means the shorter diameter,which is perpendicular to b the longest diameter of a tumor(28).Human MDA-MB-231 breast and PC-3 prostate tumor cell

lines obtained from American Type Culture Collection werexenografted in immunodeficient mice. Female 6-7-week-oldinbred CBA/Ca mice from our specified pathogen-free breed-ing were used. Pieces of adequate tumor tissue (1-1.5 mm3)were transplanted s.c. Development of immunosuppression innormal CBA/Ca mice was carried out by thymectomy, wholebody irradiation (9.5 Gy), and bone marrow transplantation.Two or three weeks after tumor transplantation, tumors hadgrown to a volume of approximately 0.1 cm3, the mice wererandomized and divided into experimental groups (10 mice pergroup). TT-232 was administered in a saline vehicle containing0.1% acetic acid s.c. on the side opposite of the tumor once ortwice daily at doses of 0.25, 0.5, 5, 10, and 20 mg/kg of b.w.Treatments of breast and prostate tumor-bearing animals withTT-232 were started on days 21 and 18, respectively, aftertumor transplantation. Drug administration was applied con-

tinuously for 30 and 24 days, respectively. Tumors weremeasured two or three times weekly, and their volumes werecalculated as described above. The effect of TT-232 treatmentson the changes in lifetime was compared with that of thenontreated tumor-bearing animals as well.

Gastric Acid Secretion. Experiments were carried out onmale Wistar rats (Iffa Credo) weighing 250-350 g providedwith a Thomas cannula. Gastric juice was collected in 20-minfractions. The volume was measured, and the acid concentra-tion and output were calculated after titration to pH 7 with 0.01M NaOH. The basal level was taken as the mean of the threefirst 20-min samples. After the basal collection period (t = 0),a venous infusion of RC-160 (2 ,tg/kg), TT-232 (1.78 p,g/kg),TT-248 (2.28 ,ug/kg), or NaCl was performed for 3 h in a tailvein. One hour later (t = 60 min), while continuing the peptideinfusion, gastric secretion was stimulated by a venous step-doseinfusion of pentagastrin (0.25; 1 and 4 ,ug/kg) each for 40 min.

Study of Phosphotyrosine Content of Tumor Cells. MDA-MB-453 attached cells (2 x 105) (obtained from AmericanType Culture Collection) were treated with TT-232 or San-dostatin (0.1, 1, or 30 ,ug/ml) for 2 h and then scraped in 200,ul of lysis buffer (50 mM Hepes- 150 mM NaCl/1.5 mMMgCl2/1 mM EGTA/10% glycerol/1% Triton X-100/1 mMphenylmethyl sulfonyl fluoride/10 ,ug/ml aprotinin/100 mMNaF/ 40 mM sodiumvanadate/ 5 mM Na4P207; all fromSigma). After 10 min on ice, the samples were centrifuged at10,000 x g for 5 min. Seventy microliters of the supernatantswas loaded to 7-15% gradient SDS-polyacrylamide gels, andelectrophoresis was carried out as described (29). Then elec-trotransfer of proteins on nitrocellulose was performed with asemi-dry blotting apparatus. The homogeneity of the proteintransfer was controlled by Ponceau red staining. Phosphoty-rosine content was revealed with a rabbit polyclonal antibody(at a 1:2000 dilution, 4 h). After the incubation with the secondantibody (coupled to peroxidase, 1:20,000, 1 h) we used theimmunochemiluminescent (ECL, Amersham) technique tovisualize the bands of phosphotyrosine.

Toxicology Studies. Toxicology tests were carried out onmale and female 6-7-week-old inbred CBA/Ca mice (22-25 g)from our specified pathogen-free breeding. The effect ofdifferent doses of TT-232 on survival and changes of bodyweights was tested for 2 weeks. The weight of different organs(heart, kidney, spleen, lung, liver, thymus, and uterus) wasmeasured after 1- and 18-day treatments, respectively. Hema-tological studies, the analysis of bone marrow and cytologystudies of different organs have been conducted as well.Binding Studies. Binding studies were similar to that de-

scribed before (30). Approximately 30 pug of membrane pro-tein was incubated with 30-50,000 cpm of [125I-Tyr3]octreotidewithout or with excess of unlabeled [Tyr3]Octreotide or TT-232.

Stability Studies. The TT-232 content of the samples andformation of degradation products were followed by HPLCsimilarly to that described before (31).

RESULTSIn Vitro Antiproliferative Effect ofTT-232 on Human Tumor

Cell Lines. Table 1 summarizes the results. TT-232 showed atleast 50% antiproliferative effect on the examined cell lines;however, around 90% inhibition was achieved on breast car-cinoma, prostate tumor, pancreatic, leukemia, melanoma, andgastric tumor cell lines. The effect of TT-232 and RC-160 wascompared on the proliferation of PC-3 and HT-29 tumor celllines. In a representative experiment on PC-3 cell line, TT-232at 30 ,ug/ml dose showed a 90 ± 2% decrease in the numberof tumor cells, but RC-160 resulted in only a 29 ± 2% decrease.On HT-29 cell line, the inhibition caused by TT-232 was about72 ± 5%, and by RC-160 it was only 25 ± 3% when both

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Table 1. In vitro antiproliferative effect of TT-232 on differenttumor cell lines

Tumor cell line

1. MCF7 breast (16)*2. BSMZ breast3. SK-BR-3 breast4. MDA-MB breast5. PC-3 prostate6. DU 145 prostate

(14)*7. SW620 colon (14)*8. HT-29 colon (14)*9. COLO 205 colon

10. A-549 lung11. Lewis Lung

Carcinoma12. P818 pancreatic13. P818-4 pancreatic

(17)*14. HT-58 lymphoma15. K-562 leukemia16. WM 938

melanoma17. Ml melanoma18. SK-OV-3 ovary19. 4-1ST gastric20. M27 rat carcinoma

Inhibition of cellproliferation (%)

87 ± 1281 ± 569± 644± 690± 2

59± 369± 672± 554± 759± 6

97± 596± 2

90± 484± 898± 3

81 ± 791 ± 255 ± 595 ± 567 ± 11

The applied dose of TT-232 was 20-30 ,ug/ml, and the treatmentlasted for 24 h.*These data have already been published. The inhibitory potency ofTT-232 is shown around the IC50 values of TT-232 on each cell line.IC50 values were between 20 and 40 ,uM and were determined inseveral but at least in two independent experiments. Values are themean ± SD.

peptides were applied at the same 30 ,ug/ml dose and cells weretreated for 24 h.

In Vivo Antitumor Effect of TT-232. TT-232 strongly inhib-ited the tumor growth of transplanted Colon 26 tumor reach-ing a 70% inhibition at the low submaximal dose of 750 ,tg/kgof b.w. of TT-232. Even at the dose of 15 ,ug/kg of b.w. morethan 60% inhibition was achieved by the 24th day relative tocontrol animals (Fig. 1).

Transplanted B16 melanoma was also inhibited with about50 ± 8% by 750 ,ug/kg of b.w. of TT-232 after 19 days. A

10 11 12 13 14 15 16 17 18 19 20 21 22 23 24 25

days after tumor transplantation

FIG. 1. Effect of different doses of TT-232 on the growth oftransplanted Colon 26 tumor in mice by measuring tumor volume for24 days. Ten animals were treated in each group; the control exper-iment was accomplished with 10 mice as well. Tumor volume wascalculated as described in the Materials and Methods section. Resultsare the mean ± SD (P < 0.01).

smaller dose (15 ,tg/kg of b.w.) resulted in 25 ± 7% inhibitionof tumor growth.

S180 sarcoma tumor growth was inhibited with about 50 ±10% by 750 p,g/kg of b.w. of TT-232.Treatment for 30 days with 0.25 and 0.5 mg of TT-232 per

kg of b.w. once a day significantly decreased the xenograftedMDA- MB-231 tumor volume, and the complete regression oftumors was observed at some animals in both dose groups. Fig.2 illustrates the tumor growth curves of five mice in the 0.5mg/kg of b.w. dose group, where about 80% tumor growthinhibition was observed compared with the control animals.TT-232 treatment for 30 days resulted in two tumor-freesurviving animals for longer than 200 days out of eight mice atthe dose of 0.25 mg/kg of b.w. and three out of eight mice inthe 0.5 mg/kg of b.w. dose group.

In the literature to perform the in vivo assays, a very broaddose range of the various somatostatin analogs has been used(ranging from 25-1500 jig/mice = 1.25-60 mg/kg of b.w.). Inthe very unresponsive PC-3 xenograft we used higher doses ofTT-232 (5, 10, and 20 mg/kg of b.w.), and at 20 mg/kg of b.w.dose, a 60% inhibition of tumor growth was achieved after 3weeks of treatment (Fig. 3). Lower doses of TT-232 inhibitedthe tumor growth less. Treatment with 20 mg/kg of b.w. ofTT-232 for 3 weeks resulted in 100% survival 60 days posttumor transplantation, while in the control group the survivalwas only 20% (one mouse out of five). No difference was foundin the percentage of survival among the different, but lowerdose groups; all groups showed 60% survival.

Toxicology Studies with TT-232. Further in vivo studieswhere higher doses of TT-232 were to be applied (about 10-20mg/kg of b.w.) necessitated toxicology studies with TT-232.These experiments showed that a lethal dose for TT-232 couldnot be determined because beyond a small and temporary bodyweight loss (10% compared with the control), no death wasobserved even when this peptide was applied at a dose of 120mg/kg of b.w. A significant growth in the weight of uterus(about 70%) and a decrease in the weight of lungs (about 30%)could be observed. The weight of other organs was not affectedby the treatment with different doses up to 120 mg/kg ofTT-232. The hematological parameters and the qualitativeblood picture were not affected either. TT-232 even at 120mg/kg of b.w. dose had no effect on the number and thequalitative picture of the bone marrow cells. Histologicalstudies of different organs from TT-232 treated mice did notshow significant modification in the tissues.

Studies Proving the Selectivity of TT-232. Previously wehave reported that in vitro 10-8M of TT-232 did not inhibit theGH releasing hormone-induced GH release, and in in vivoassays in rats no decrease of initial serum GH levels was

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FIG. 3. Effect of different doses of TT-232 on the growth ofxenografted PC-3 human prostate carcinoma in mice by measuringtumor volume after 24 days. Each group contained five mice. Resultsare the mean ± SD (P < 0.01).

observed by TT-232 at the applied doses of 1 and 4 ,tg/100 g(16).

Binding studies with TT-232 have also been carried out andcompared with the binding of [Tyr3]octreotide. The binding of[125I-Tyr3]octreotide to the membrane preparation of rat pi-tuitary (maximum binding capacity was 2614 fmol/mg ofmembrane protein) could be displaced in a dose-dependentmanner with unlabeled [Tyr3]octreotide but not with TT-232.Similar results were obtained with the rat cortex membranepreparation (data not shown). The fact that TT-232 did notbind either to the pituitary or to the brain receptors supportsthe previous finding that it has no effect on GH release.

It has been reported that somatostatin inhibits the secretionof gastric acid (2). We found that TT-232 did not inhibit thesecretion of gastric acid; meanwhile results shown in Fig. 5indicate that RC-160 and TT-248 two "traditional type"somatostatin analogs induced significant inhibition (90%) ofgastric acid secretion when administered concurrently withdifferent doses (0.25 ,tg/kg; 1 ,tg/kg; 4 ,tg/kg) of pentagastrin.Mechanism of Action Studies with TT-232. In the present

work, we have demonstrated that TT-232 induces apoptosis(programmed cell death) in human colon (HT-29 and SW620),pancreatic (P818), leukemia (K-562), melanoma (WM 938/B,M-1, EP), and lymphoma (HT-58) tumor cell lines. Theoccurrence of apoptosis was followed by light- and electron-microscopic as well as by immunohistochemical studies. In arepresentative experiment, the apoptotic index increased

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about 70 times in M-1 melanoma, 60 times in K-562 leukemia,13 times in P818 pancreatic cells, seven times in HT-29 humancolon tumor cells, and three times in PC-3 prostate tumor cellsafter 48 h incubation with 10 ,ug/ml of TT-232. In case ofWMmelanoma and EP melanoma cells, five and four times increaseof apoptotic index has been observed after 24 h of incubationwith the same dose of TT-232 (Fig. 5). In some of the abovedescribed cell lines, further increasing the dose of TT-232 andthe time of the incubation cell lysis occurred, which is probablydue to a so far unknown mechanism resulting in destruction ofthe structure of cell membranes as a consequence of apoptosis.We have investigated the effect of 10 and 30 ,ug/ml TT-232 onthe level of wild-type p53 protein in P818 pancreatic and inHT-29 colon tumor cells and found no alterations in p53 level.We also investigated the effect of TT-232 on the translocationof the KU86 protein from the cytosol to the nucleus (KU86 isa nuclear somatostatin receptor associated with p53) andfound no significant. effect, suggesting that TT-232 inducesapoptosis on a p53-independent way.TT-232 strongly inhibited tyrosine kinases (75%) after long

term incubation (24 h) in SW620 colon tumor cell lines (Fig.6), this effect correlates well with the antiproliferative andapoptosis inducing effect of TT-232.

In MDA-MB-453 human breast cancer cells, different doses(0.1, 1, or 30 ,tg/ml) of TT-232 and Sandostatin resulted in adecrease in the phosphotyrosine content of the proteins. At 30,tg/ml dose, no bands of phosphotyrosine could be visualizedat the immunoblot analysis in the case of TT-232, whileSandostatin also caused a decrease in the phosphotyrosinecontent but to a much smaller extent.

Stability Studies with TT-232. The stability of TT-232, bothin solid (lyophilized) form and in aqueous solution, wasinvestigated during storage at different temperatures. Samplesstored for various time periods were analyzed for TT-232content as well as for degradation products using HPLCmethods (31). TT-232 was stable at least for 36 days in solid(lyophilized) form kept at 22°C. In sterile aqueous solution itwas stable at least for 40 days when kept in refrigerator, for 35days at ambient temperature, and for 2 days at 37°C.

DISCUSSIONA somatostatin structural derivative (TT-232) has been devel-oped in our laboratory with strong antiproliferative effect butno GH release inhibitory activity (14-16). In the presentpaper, we report a series of new in vitro and in vivo studiesdemonstrating that TT-232 is the first tumor-selective and

K-562 PC-3 SW620 WM* HT-29 M- 1 EP* P-818

Peptide infusion

FIG. 4. Effect of equimolar doses of RC-160, TT 232, and TT-248on basal and pentagastrin stimulated gastric acid concentration inconscious rats. Each peptide was infused for 3 h. Results are themean ± SEM in groups of 10 treated rats and 24 controls (NaCl).

Tumor cell lines

FIG. 5. Apoptotic cell number of various cultured tumor cellstreated with 10 ,ug/ml of TT-232 after 48 h of treatment, * in case ofWM and EP melanomas data derive from 24 h of treatment. Resultsare the mean ± SD, (P < 0.001).

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FIG. 6. Dose dependence of TT-232 stimulated tyrosine kinaseactivity on SW620 colon tumor cells. SW620 cells were incubated withvarious concentrations of TT-232 for 24 h at 37°C. Each value isrepresentative of at least three separate experiments. The control(100%) value denotes a tyrosine kinase activity of 0.137 32P/min mgof protein; *, P < 0.05; **, P < 0.005; ***, P < 0.001.

potent somatostatin analog that inhibits the tyrosine kinasesignaling pathways and induces apoptosis, so it has greatpharmaceutical perspectives. There are several small molec-ular tyrosine kinase inhibitors as potential drugs described inthe literature, but their application is still limited because ofthe lack of selectivity (32).TT-232 has been selected from a big series of somatostatin

analogs on the bases of structure-activity relationship studiesregarding tyrosine kinase inhibitory and antiproliferative ef-fect versus GH release inhibition (16). In the present work,tyrosine kinase inhibitory studies were performed in humancolon tumor cells where tyrosine kinase inhibition following a

long term preincubation with the peptide correlated well withthe dramatic inhibition of cell proliferation and the inductionof apoptosis. We demonstrated now that TT-232 inducesapoptosis in a series of cell lines suggesting that this peptideexerts its direct antitumor activity via signal transductionmodulation and the induction of apoptosis.

Recently, the role of apoptosis in the regression of hormone-treated tumors has been widely studied (18, 33). The role oftyrosine kinase inhibition in the induction of apoptosis hasbeen well demonstrated as well, while our recent studiesproved that an epidermal growth factor receptor-selectivetyrosine kinase inhibitor induced a nonapoptotic programmedcell death but a nonselective tyrosine kinase inhibitor inducedapoptosis (34). Our finding that TT-232 results in a decreaseof phosphotyrosine content in MDA MB-453 breast cancer

cells, which contain Her3 receptor [the third member of theepidermal growth factor receptor family (35)], supports our

concept that TT-232 exerts its antiproliferative effect via theinduction of programmed cell death. It has been reported inthe literature that the nuclear protein KU86, which regulatesp53 expression and apoptosis, is a somatostatin receptor (36).We found that TT-232 had no effect on p53 and KU86,suggesting that its apoptotic effect is mediated independent ofp53 (unpublished results).The TT-232-induced inhibition of the growth-promoting

tyrosine kinase signal could be coupled to the induction of theregular cell cycle with an apoptotic end. The long term tyrosinekinase inhibition and the previously found short term tyrosinephosphatase activation might be two separate effects of TT-232. Since we have demonstrated that TT-232 does not bind tothe rat pituitary or the cortex, where all the known somatosta-tin receptor subtypes are expressed (37), and has no GH

release inhibitory and antisecretory activity, we believe thatthe antiproliferative and apoptosis inducing effect of TT-232is mediated through a still unknown receptor. Known soma-tostatin analogs like RC-160 or Sandostatin exert their anti-proliferative effect through the STR2 receptor, which is alsopresent in the pituitary (38). We are currently working on theisolation of the TT-232 receptor from tumor cells with affinitychromatography.

In in vivo experiments, relatively low doses of TT-232 havebeen applied, although our in vitro data show that for thedramatic antiproliferative and apoptosis-inducing effect, acritical dose-which also depends on the cell type-has to bereached. TT-232 had strong in vivo antitumor activity even atrelatively low doses, which give us very good perspectives forthe application of higher doses, reaching the critical thresholddose for the induction of apoptosis. Toxicity studies clearlydemonstrated that TT-232 does not show toxic side effectseven at a dose of 120 mg/kg of b.w.

In conclusion, TT-232 has good perspectives as a potent andselective antitumor drug.

We acknowledge the antiproliferative assays performed by NationalCancer Institute of the National Institutes of Health, MitshubishiKasei (Tokyo), the binding studies performed by J. Hofland and S. W.J. Lamberts (Department of Internal Medicine III. University Hos-pital Dijkzigt, Rotterdam, The Netherlands), and the skillful assistanceof M6nika Torma and Em6ke Tovari. This work was supported byresearch grants from the Hungarian National Research Fund (OTKA6335, OTKA 7722), National Committee for Technological Develop-ment (OMFB 0717), ETT-01 053/93, Biomedl/PECO (277).

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