J Cancer Res Clin Oncol (1991) 117:416-420

C~ncer ~esearch Clinical 9

017152169100072R

9 Springer-Verlag 1991

Effects of insulin and somatostatin on the growth and the colony formation of two human pancreatic cancer cell lines * Y. Takeda and M. J. Escribano Laboratoire d'Immunochimie, Institut de Recherches Scientifiques sur le Cancer, C.N.R.S., Bo~te postal n~

F-94801 Villejuif, France

Received 21 January 1991/Accepted 8 April 1991

Summary. The effects of insulin and somatostatin on the growth and the colony formation of two human pancreatic cancer cell lines, BxPC-3 and SOJ-6, were studied. The BxPC-3 cell line (American Type Culture Collection no. CRL 1687) was derived from a moderately differentiated pancreatic adenocarcinoma. The SO J-6 cell line is a subclone of SOJ that was initiated from ascites of a welldifferentiated pancreatic adenocarcinoma. Both cell lines express fetoacinar pancreatic antigen, an antigen that might be associated with early transformation stages. However, these lines have different proliferation and tumoral powers. SO J-6 cells showed an almost twofold higher division rate over BxPC-3 cells when cultured in RPMI-1640 medium containing 10% fetal bovine serum. The tumorigenic degree of SO J-6 cells, as assessed by tumor growth in nude mice, was about three times greater than that of BxPC-3. The in vitro growth of BxPC-3 cells was significantly promoted by insulin, and was slightly inhibited by somatostatin, whereas the growth of SO J-6 cells was not influenced by these hormones. Using a clonogenic assay in soft agar, the average ratio of colony numbers formed by SO J-6 and BxPC-3 was about 10/1, indicating a good correlation between the colony formation and tumorigenic degree in vivo. In this test, the number of colonies formed by BxPC-3 cells was increased about twofold in insulin-supplemented medium. On the other hand, somatostatin inhibited the colony formation by a factor of four to six. However, no hormonal modulation of the colony formation of SO J-6 cells was observed. Our data show that pancreatic cancer cell lines respond differently to pancreatic hormones, and suggest that this may be correlated to a tumour stage or a tumour type. * This work was financed in part with grant 6394 Association pour la Recherche sur le Cancer (ARC) (Villejuif). Y. Takeda was supported by a fellowship from the ARC Abbreviations: FAP antigen, fetoacinar pancreatic antigen; FBS, fetal bovine serum; PBS, phosphate-buffered saline; RPMI-1640, Roswell Park Memorial Institute's Medium 1640; CEA, carcinoembryonic antigen; CA 19-9, carbohydrate antigen 19-9 Offprint requests to: M.J. Escribano

Key words: Pancreas cancer Growth - Colony formation

Insulin - Somatostatin

Introduction Hormonal regulation of pancreatic cancer has been reported, indicating that this could lead to new therapeutic modalities for this incurable disease. The higher incidence of pancreatic cancer in males than in females in humans (Faivre et al. 1990) and in azaserin-treated rats (Longnecker 1990), and the antagonistic effects of testosterone and oestrogens in this animal model (Lhoste et al. 1987) favour sex hormone modulation. Pancreatic hormones could also play an important role in pancreatic cancer. Antitumour activities of somatostatin and its analogues on chemically induced pancreatic adenocarcinoma in hamsters (Redding and Schally 1984; Paz-Bouza et al. 1987; Zalatnai and Schally 1989), as well as some benefits of this hormone in humans (Gonzalez-Barcena et al. 1986) have been documented. The complexity of the transformation process and the heterogeneity of cell populations suggest that the mechanisms of somatostatin action and the predictable differential sensitivity of cancer cells will not be understood unless studies at the cellular level are conducted. Fetoacinar pancreatic (FAP) antigen, characterized by the murine monoclonal antibody J28, may be involved in acinar cell transformation (Albers et al. 1990), which in turn could be a first step in the generation of some types of pancreatic adenocarcinoma (Bockman 1981; Flaks et al. 1982; Roebuck et al. 1984; Escribano et al. 1985; Carre-Llopis et al. 1987; Eriguchi et al. 1987). A recent study showed that the expression of FAP antigen was restricted to a few pancreatic cell lines and that there was an association between the histogenesis o f the tumour of origin and FAP positivity in the derived cells.

417 M o r e o v e r , the cell line richest in F A P (BxPC-3) was positively a c t i v a t e d by insulin as this h o r m o n e a l l o w e d survival o f the cells in serum-free m e d i u m ( M a z o et al. 1991). T h e a i m o f this study was to see w h e t h e r s o m a t o s t a t i n w o u l d exert an i n h i b i t o r y effect in this insulin-sensitive cell line. W e also studied a n o t h e r F A P - e x p r e s s i n g cell line (SO J-6), which s h o w e d e n h a n c e d t u m o r i g e n i c i t y and was o b t a i n e d by s u b c l o n i n g the F A P - n e g a t i v e cell line, S O J (Fujii et al. 1990). T h e effects o f insulin and s o m a t o s t a t i n on the g r o w t h and the degree o f t u m o r i g e n i c i t y o f these lines are described here.

tion and replaced by freshly made medium on the 4th and 7th days. All experiments were done in duplicate.

Materials and methods

Clonogenic assay. This was done in semi-solid agar cell cultures (Macpherson and Montagnier 1964) using cells at the exponential growth phase. Cultures were prepared as follows: the 9.6-cm / wells on six multiwell culture plates, were layered with 3 ml 0.5% agar gel (feeder layer) made in the appropriate nutrient medium; RPMI1640 and 10% FBS for the standard conditions, supplemented with 10 gg/ml insulin or 5 gg/ml somatostatin. The gel was allowed to solidify at 37 ~ C in a humidified CO2 incubator and then 1.5 ml cell suspension in 0.3% agar was added. Cells in 0.3% agar were conditioned in the same medium as the feeder layer. This procedure was performed using different cell concentrations; 9.4 x 10 2, 9.4 x 103, or 9.4 x 104 cells/well. The cells were then cultured at 37~ C in a humidified CO2 incubator and the number of colonies reaching at least 2-3 mm in size was counted until day 13 of the incubation. All experiments were done in triplicate.

Hormones. Insulin from bovine pancreas and synthetic somatostatin were purchased from Sigma (Saint Louis, Mo., USA). Cell lines. The human pancreatic cell line BxPC-3 was provided by American Type Culture Collection (CRL 1687). This cell line was initiated from a moderately differentiated adenocarcinoma in the body of the pancreas by needle biopsy during operation. The cells are oval to round, forming homogeneous confluent colonies in RPMI-1640 medium with a population doubling time of 48-60 h (Tan et al. 1986). SO J-6 is a subline of SOJ that was obtained after several subcloning procedures. The parental SOJ cell line has been recently established from ascites of a patient with a well-differentiated adenocarcinoma in the body of the pancreas at the Institute of Medical Science, the University of Tokyo, Japan. In RPMI-1640 medium, SOJ cells grow as epithelial-like, mucin-producing cells with a population doubling time of 50-70 h. This cell line is positive for carcinoentoryonic antigen (CEA), carbohydrate antigen (CA 19-9) and DU-PAN-2, and is negative for FAP antigen (Fujii et al. 1990). Cell cultures. Cells were cultured in 25-cm z tissue-culture flasks using RPMI-1640 medium (Biochrom KG, FRG) supplemented with 10% heat-inactivated fetal bovine serum (FBS; Schott, FRG), 2 mM glutamine (Gibco) and 100 IU/ml penicillin/0.1 mg/ml streptomycin (Gibco). Cultures were grown in a humidified incubator at 37~ C in an atmosphere of 95% air and 5% CO2. For continuous maintenance in culture, cells were detached by incubation in a solution containing 0.25% trypsin (Gibco) and 0.02% EDTA disodium salt (Gibco), and washed with a solution of 0.02% EDTA. The culture medium was changed every 3 or 4 days by aspiration.

Tumours transplanted to nude mice. Swiss nude mice (nu/nu, female, 4 5 weeks old) were provided by IFFA-CREDO, France. Cells cultured for 1 week in 80-cm 2 tissue-culture flasks were trypsinized, centrifuged at 1000 rpm for 5 rain, washed with PBS, and resuspended in PBS at the concentration of 20 x 106 cells/ml. A sample of 0.5 ml cell suspension (l 0 x 106 cells) was subcutaneously injected into the flank of each often animals (five for each cell line). Turnout formation was checked by palpation and palpable tumours were measured for their perpendicular diameters with vernier calipers weekly. The arithmetic mean of the two diameters was designated as tumour size and used for expression of a tumour growth curve.

Results Character&tics o f the S O J-6 cell line In R P M I m e d i u m s u p p l e m e n t e d with 10% F B S , SOJ-6 cells were s p i n d l e - s h a p e d a n d grew in clusters with a pavement-like m o r p h o l o g y with a population doubling time o f 40 50 h. I m m u n o c y t o l o g y using a c e t o n e - f i x e d cells s h o w e d a positive r e a c t i o n for C E A , C A 1 9 - 9 a n d F A P antigens. F A P was localized to the c y t o p l a s m a n d to the p e r i n u c l e a r area (Fig. 1).

lmmunocytology for FAP antigen. This was done in acetone-fixed cells using the murine monoclonal antibody J28 and fluoresceinconjugated anti-(mouse IgG) antibodies as described previously (Mazo et al. 1991). Growth curves. Cells grown to confluence were suspended by trypsinization and centrifuged at 1000 rpm for 5 min, washed with phosphate-buffered saline (PBS) pH 7.4, and resuspended in the fresh standard medium. Cell viability was assessed by trypan blue exclusion and cell counting was performed with a haemocytometer. Cell growth curves were established in the standard conditions, that is in RPMI-1640 medium containing 10% FBS, and in the same medium supplemented with 10 gg/ml or 20 Ixg/ml insulin or 2.5 gg/ ml or 5 Ixg/ml somatostatin. For every medium, 2 ml cell suspension (105 cells/ml) was plated in 9.6-cm 2 wells of multiwell plates. After 2, 4, 7 and 10 days of culture, cells were resuspended by trypsin, washed with PBS, stained with trypan blue, and counted. To ensure viability in long-term cultures, the medium was discarded by aspira-

Fig. 1. Immunofluorescence (SO J-6 for fetoacinar pancreatic antigen) (original magnification: x 400)

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Fig. 3. Growth curves of the tumour grafted in nude mice (the arithmetic mean of tumour diameters). Each point represents the mean _+ SE for five mice

Growth in vitro SO J-6 cells grew faster than BxPC-3 in culture. An approximately twofold higher growth rate was observed during the exponential growth phase, that is until the 7th day o f culture (Fig. 2).

Tumour formation in nude mice The injection of 10 • 106 SOJ-6 cells into athymic Swiss (nu/nu) mice resulted in production of palpable tumours in all animals (n = 5) in a very short time ( ~ 6 days). Tumours reached more than 5 m m in size after 7 days. Under the same conditions, BxPC-3 cells gave rise to slowly growing tumours of relatively small size (1.8 m m after 7 days) (Fig. 3).

Effect of insulin and somatostatin on cell growth Proliferation of BxPC-3 cells in culture was p r o m o t e d by insulin, especially during the exponential growth phase. Significant differences were observed in media supplemented with 10 gg/ml insulin after 4 and 7 days and with 20 gg/ml after 4 days of culture. As much as 69% increase

Fig. 4. Effect of insulin (A) and somatostatin (B) on the growth of BxPC-3 cell line. 2 ml cell suspension (10 s cells/ml) was plated in a 9.6-cm2 well of a multiwell plate. Cells were cultured in (A) 9 the standard medium (RPML1640 + 10% FBS); A - - - , supple~ mented with 10 gg/ml insulin; f t . . , supplemented with 20 gg/ml insulin; (B) o - - , the standard medium (RPMI-1640 + 10% FBS); A - - - , supplemented with 2.5 gg/ml somatostatin; I - . , supplemented with 5 gg/ml somatostatin. Each point represents the mean + SE for two cultures

in growth was observed after 4 days, using 20 gg/ml insulin (Fig. 4A). Addition of 4 mg/ml and 6 mg/ml glucose to insulin-supplemented medium has no influence in the insulin stimulation effect (data not presented). In the medium supplemented with 5 gg/ml somatostatin, about 20% inhibition was seen in 7-day cultures. A similar result was obtained using 2.5 ~tg/ml somatostatin (Fig. 4 B). G r o w t h of SO J-6 in insulin-supplemented medium was almost the same as under the standard conditions (Fig. 5 A). Similarly, somatostatin had no significant effect in the growth curve o f this cell line (Fig. 5 B).

Effect of insulin and somatostatin on the colony formation Using 9.4 • 10 2 cells/well, no colony formation was observed. On the contrary, plating of 9.4 x 10 4 cells resulted in confluent monolayers. The best results were obtained after 10 and 13 days of incubation using 9.4 x 10 3 cells/

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Fig. 5. Effect of insulin (A) and somatostatin (B) on the growth of

the SOJ-6 cell line. Conditions and symbols are the same as those in Fig. 4

well. Results of this experiment are given in Table 1. It can be seen that: (a) the number of colonies in SO J-6 cultures is on average a b o u t ten times higher than that in BxPC-3; (b) in BxPC-3 cells, the number of colonies in insulin-supplemented medium is about twice as high compared with the standard conditions, whereas it is diminished by a factor of four to six in the presence of somatostatin; (c) in SO J-6 cells, the number of colonies is not significantly affected by either of the hormones; (d) the validity of the assay is emphasised by the comparable values in triplicate experiments.

In this study we addressed the question of whether isolated pancreatic cells would show different responses to somatostatin, which is a potential inhibitor for pancreatic cancer. The predictable activating effect of insulin was studied in parallel. Our choice of BxPC-3 and SO J-6 cell lines was guided by the expression of F A P antigen, since this antigen is probably linked to early steps of the transformation process. The observation that insulin allows survival of BxPC3 cells in serum-free medium (Mazo et al. 1991) suggested that this pancreatic h o r m o n e would be essential for the growth of this cell line. In this paper, evidence for a growth factor activity of insulin on BxPC-3 cells was obtained from enhanced cell proliferation in R P M I / F B S supplemented with insulin. Activation of cell proliferation by insulin has been reported to be directly correlated to glucose consumption, as this h o r m o n e increases glucose uptake by normal and tumoral cells (Lieberman and Ove 1959). N o modification of the proliferation was noted on adding increased glucose amounts, probably because the glucose concentration in RPMI-1640 medium (2 mg/ml) is large enough to ensure cell growth even in the presence o f insulin (Popiela and M o o r e 1989). In similar experiments, somatostatin showed a small, yet not significant, inhibition of cell growth. The antagonistic action of the two hormones was, however, clearly demonstrated in experiments leading to estimation of colony formation in soft agar. In this technique, a good correlation between the number of colonies and the tumorigenicity was found in polyoma-transformed cells (Macpherson and Montagnier (1964). By comparing the t u m o u r formation in vivo with clonogenic power, we found the same correlation for BxPC-3 and SO J-6 cells. This very simple and reliable technique allows comparison of results as a function of culture conditions, which would be difficult in the more widely used turnout growth assay in nude mice. SO J-6 cells were insensitive to insulin, since b o t h the growth rate in vitro and the ability to f o r m colonies in soft agar, were unmodified in insulin-supplemented cultures when c o m p a r e d with cultures performed in the standard conditions. Somatostatin had no significant in-

Table 1. Colony formation in soft agar a Cell line

B • PC-3

SOJ-6

Medium b

Std Ins Som Std Ins Som

10 days

13 days

Ac

B~

Cc

Mean

A

B

C

Mean

3 3 0 36 35 23

2 7 0 22 30 21

4 7 2 37 35 31

3.0 5.7 0.7 31.7 33.3 25.0

4 12 0 80 52 73

7 15 1 55 48 53

6 10 2 61 60 74

5.7 12.3 1.0 65.3 53.3 66.7

" Values are numbers of colonies in individual wells plated with 9.4 x 103 cells. Only colonies equal to or greater than 2-3 mm in diameter were counted

b Std, standard medium: RPMI-1640+10% FBS; Ins, medium supplemented with I0 mg/ml insulin; Som, medium supplemented with 5 gg/ml somatostatin c A, B, C are individual six-well plates

420 h i b i t o r y effect o n this cell g r o w t h in vitro. Similarly, this h o r m o n e h a d a l m o s t n o influence on the c o l o n y f o r m a t i o n p o w e r o f SO J-6 cells. W e c o n c l u d e t h a t p a n c r e a t i c c a n c e r cells r e s p o n d differently to p a n c r e a t i c h o r m o n e s , in g o o d a g r e e m e n t w i t h results o n the h o r m o n a l t r e a t m e n t o f cells g r o w n in a t h y mic mice ( P o s t o n et al. 1990). T h e b e h a v i o u r in vitro a n d in vivo o f SO J-6 cells indicates t h a t this n e w l y e s t a b l i s h e d cell line is m o r e m a l i g n a n t t h a n BxPC-3. T h e r e f o r e , the inverse r e l a t i o n s h i p between m a l i g n a n c y a n d h o r m o n e sensitivity is the s e c o n d c o n c l u s i o n o f o u r study. A l s o , we n o t e d a c o n c o m i t a n t sensitivity to insulin a n d s o m a t o s t a t i n with either no a c t i o n (SO J-6 cells) o r a n a n t a g o n i s t i c effect (BxPC-3 cells). A c t i v a t i o n o f c a n c e r cells b y insulin a n d i n h i b i t i o n b y s o m a t o s t a t i n are in a g r e e m e n t with the f u n c t i o n o f these h o r m o n e s in the normal pancreas. T h e presence o f h o r m o n e r e c e p t o r s is one o f the p a r a m e t e r s o f the benefits o f sex h o r m o n e t h e r a p y on m a m m a r y a n d p r o s t a t i c cancers. A c i n a r cell f u n c t i o n is directly r e g u l a t e d b y islet h o r m o n e s t h r o u g h a p o r t a l syst e m t h a t c o n v e y s islet b l o o d to a c i n a r cells. O n the o t h e r h a n d , there is no b l o o d s t r e a m f r o m the exocrine to the e n d o c r i n e p a n c r e a s ( W i l l i a m s a n d G o l d f i n e 1985). M o r e o v e r , e n d o c r i n e h o r m o n e r e c e p t o r s in exocrine p a n creatic cells h a v e been d o c u m e n t e d ( M o s s n e r et al. 1984; S a k a m o t o et al. 1984). It is c o n c e i v a b l e t h a t the inhibit o r y a c t i o n o f s o m a t o s t a t i n w o u l d be m e d i a t e d b y specific receptors, a n d t h a t the presence o f such r e c e p t o r s m i g h t d e p e n d on a t u r n o u t stage o r a t u m o u r type. O u r results stress the need for f u r t h e r studies a t the cellular level in o r d e r to shed m o r e light o n the m e c h a nism o f s o m a t o s t a t i n action. This w o u l d a p p l y to o t h e r p u t a t i v e beneficial h o r m o n e s , i.e. steroids ( L o n g n e k e r 1990). T h e a p p l i c a t i o n o f h o r m o n e t h e r a p y to p a n c r e a t i c c a n c e r m i g h t d e p e n d o n a t u m o u r stage o r a t u m o u r type. A n d this deserves p a r t i c u l a r a t t e n t i o n for this a l m o s t fatal disease.

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Escribano MJ, Carre-Llopis A, Loridon-Rosa B (1985) Expression of oncofetal pancreatic antigens in hamster adult pancreas during experimental carcinogenesis. Br J Cancer 51:187-193 Faivre J, Bedenne L, Arveux P, Klepping C (1990) Epi-d6miologie descriptive du cancer du pancr6as. Bull Cancer 77:39-46 Flaks B, Moore MA, Flaks A (1982) Ultrastructual analysis of pancreatic carcinogenesis: V. Changes in differentiation of acinar cells during chronic treatment with N-nitrosobis(2-hydroxyprophyl) amine. Carcinogenesis 3:485-498 Fujii Y, Sekiguchi M, Shiroko Y, Shimizu H, Sugawara I, Hasumi K, Eriguchi M, Ikeuchi T, Uchida H (1990) Establishment and characterization of human pancreatic adeno-carcinoma cell line SOJ producing carcinoembryonic antigen and carbohydrate antigen 19-9. Hum Cell 3:31-36 Gonzalez-Barcena D, Rangel-Garcia NW, Perez-Sanchez PL et al. (1986) Response to D-Trp-6-LH-RH in advanced adeno-carcinoma of pancreas. Lancet 2:154 Lhoste EF, Roebuck BD, Stern JE, Longnecker DS (1987) Effect of orchiectomy and testosterone on the early stages of azaserine-induced pancreatic carcinogenesis in the rat. Pancreas 2:38-43 Lieberman I, Ove P (1959) Growth factors for mammalian cells in culture. J Biol Chem 234:2754 Longnecker DS (1990) Experimental pancreatic cancer: role of species, sex and diet. Bull Cancer 77:27-37 Macpherson I, Montagnier L (1964) Agar suspension cultures for the selective assay of cells transformed by polyoma virus. Virology 23:291-294 Mazo A, Fujii Y, Shimotake J, Escribano MJ (1991) Expression of FAP protein in the pancreatic human tumor cell line BxPC-3. Pancreas 6:37-45 Mossner J, Logsdon CD, Goldfine ID, Williams JA (1984) Regulation of pancreatic acinar cell insulin receptors by insulin. Am J Physiol 247:155-160 Paz-Bouza JI, Redding TW, Schally AV (1987) Treatment of nitrosamine-induced pancreatic tumors in hamsters with analogs of somatostatin and luteinizing hormone-releasing hormone. Proc Natl Acad Sci USA 84:1112-1116 Popiela H, Moore W (1989) Proliferation ofimmunocytochemically identified islet beta cells in culture: effect of growth factors, hormones, and nutrients. Pancreas 4:244-248 Poston G J, Townsend Jr CM, Rajaraman S, Thompson JC, Singh P (1990) Effect of somatostatin and tamoxifen on the growth of human pancreatic cancers in nude mice. Pancreas 5:151 157 Redding TW, Schally AV (1984) Inhibition of growth of pancreatic carcinomas in animal models by analogs of hypothalamic hormones. Proc Natl Acad Sci USA 81:248 252 Roebuck BD, Baumgartner K J, Thron CD (1984) Characterization of two populations of pancreatic atypical acinar cell foci induced by azaserine in the rat. Lab Invest 50:141-146 Sakamoto C, Goldfine ID, Williams JA (1984) The somatostatin receptor on isolated acinar cell plasma membranes. J Biol Chem 259:9623-9627 Tan MH, Nowak N J, Loor R, Ochi H, Sandberg AA, Lopes C, Pickren JW, Bzrjian R, Douglass HO, Chu TM (1986) Characterization of a new primary human pancreatic tumor line. Cancer Invest 4:15-23 Williams JA, Goldfine ID (1985) The insulin-pancreatic acinar axis. Diabetes 34:980-986 Zalatnai A, Schally AV (1989) Treatment of N-nitrosobis(2-oxopropyl)amine-induced pancreatic cancer in Syrian golden hamsters with D-Trp-6-LH-RH and somatostatin analogue RC-160. Cancer Res 49:1810 1815