GASTROENTEROLOGY

1990;98:1666-1674

Growth Effects of Regulatory Peptides on Human Pancreatic Cancer Lines PANC-1 and MIA PaCa-2 RALF-MARCO

LIEHR,

GEORGE

MELNYKOVYCH,

and TRAVIS E. SOLOMON Departments of Medicine and Physiology, Kansas University Medical Center, Kansas City, Kansas: and Research Service, Kansas CiW Veterans Administration Medical Center, Kansas City, Missouri

Several studies have reported effects of gastrointestinal regulatory peptides on growth of experimentally induced pancreatic neoplasms and human cancer cell lines. The growth of human pancreatic cancer lines PAN&l and MIA PaCa-2 was characterized in vitro, and the effects of cholecystokinin, bombesin, insulin, epidermal growth factor, secretin, vasoactive intestinal peptide, and somatostatin were determined. Fetal bovine serum was required for initiation of growth in both cell lines. Growth effects of peptides were determined by incubating cells with peptides in serum-free medium after a 72-h preincubation in 10% serum-supplemented medium alone. Epidermal growth factor (3.4 x lo-’ M) and insulin (1O-6 M) significantly (p < 0.001) increased growth of both cell lines as determined by increases in deoxyribonucleic acid and protein. Bombesin, secretin, vasoactive intestinal peptide, and somatostatin (all 10-O M) did not affect growth of either cell line. Neither cholecystokinin-8 nor [Thr4, Nle’] cholecystokinin-8 altered growth in concentrations from 10-12-10-6 M. Anchorage-dependent clonogenic growth of both cell lines was also not altered by cholecystokinin-8. Cholecystokinin added to cultures was degraded by separate effects of serum and cells. Addition of cholecystokinin-8 to cultures every 8 h maintained cholecystokinin levels but did not alter cell growth. These data support roles for epidermal growth factor and insulin as growth factors for human pancreatic cancer cell lines.

The following were purchased: Dulbecco’s modified Eagle’s medium (DMEM, No. 430-X00] from GIBCO, Grand

ecently it has been reported that some gastrointestinal regulatory peptides may act as growth modulators of pancreatic cancer in vivo and in vitro (for review, see 1).Cholecystokinin (CCK) was found to augment the effects of chemical carcinogens in rat and

Abbreviations used in this paper: BSA, bovine serum albumin; CCK, cholecystokinin; CCK6, cholecystokinin octapeptide; DMEM, Dulbecco’s modified Eagle’s medium: DMSO, dimethyl sulfoxide; dt, doubling time; FBS, fetal bovine serum; IGF-I, insulinlike growth factor I; PBS, phosphate-buffered saline. @ 1996 by the American Gastroenterological Association 0016-5065/90/$3.00

R

hamster pancreas in some experiments (2-4) and to stimulate protein secretion in a rat acinar cell carcinoma in vitro (5). Bombesin has been shown to stimulate growth of chemically induced preneoplastic acinar cell lesions in rats (4,6). Epidermal growth factor (EGF) has been reported to augment growth in a number of human pancreatic carcinoma cell lines (7-10). In addition, somatostatin has been reported to inhibit growth of chemically induced pancreatic carcinomas in hamsters (ll), as well as in vitro growth of the MIA PaCa-2 human pancreatic cancer cell line (7). Therefore, it is possible that human pancreatic cancer may be a hormone-sensitive or hormone-dependent malignancy, and that its growth may be inhibited by either hormone deprivation or by administration of antagonists. In the present study, the effects of several gastrointestinal regulatory peptides on growth of two human pancreatic cancer cell lines of ductal origin, PANC-1 and MIA PaCa-2, were studied. To avoid serum factors that might mask effects of regulatory peptides, an experimental design was chosen that allowed the effects of various peptides on human pancreatic cancer cell growth to be studied in the absence of serum. Materials

and Methods

Materials

June 1990

PEPTIDE-INDUCED GROWTH OF PANCREATIC CANCER 1667

Island, N.Y.; defined fetal bovine serum (FBS) from HyClone Laboratories, Inc., Logan, Utah: streptomycin sulfate, penicillin-G, Hank’s balanced salt solution without calcium and magnesium, phosphate buffered saline (PBS], ethylenediaminetetraacetate (EDTA), dimethyl sulfoxide (DMSO), sodium butyrate, sodium deoxycholate, Giemsa stain, bovine serum albumin (BSA), benzimide (33258 Hoechst), and calf thymus DNA from Sigma Chemical Co., St. Louis, MO.; murine EGF, culture grade, and insulin from Collaborative Research, Waltham, MA; trypsin without calcium and magnesium from K.C. Biological, Inc., Lenexa, KS: synthetic secretin, bombesin, somatostatin, vasoactive intestinal peptide (VIP), and sulfated cholecystokinin octapeptide (CCK8) from Peninsula Laboratories, Inc., Belmont, Calif. [Thr4, Nle7] CCK9 was a gift from Professors Luis Moroder and Erich Wilnsch, Max Planck Institute of Biochemistry, and Dr. Winfried Kolbeck, Diamalt, Munich, F.R.G.

Cell Culture Human pancreatic carcinoma cell lines PANC-1 (passage no. 58) and MIA PaCa-2 (passage no. 125) were obtained from the American Type Culture Collection (Rockville, Md.). The PANC-1 cell line (12) was derived from an undifferentiated carcinoma with squamous epithelial morphology in the head of the pancreas, whereas the MIA PaCa-2 cell line (13) was originally derived from a carcinoma with a fibroblastic morphology occurring in the body and the tail of the pancreas. Cells were maintained at 37°C in a humidified atmosphere of 5% CO, and 95% air in DMEM supplemented with streptomycin sulfate (200 pgg/ ml), penicillin-G (200 U/ml], and 10% defined FBS. The serum was heat inactivated at 56°C for 30 min and kept frozen. To exclude variability in stock solutions of FBS, a single batch was used in all studies. Stock cultures were grown in flasks [Corning Tissue Culture Flask No. 25100; 25 cm’ growth area; Corning Glass Works, Corning, N.Y.) and passaged weekly no more than 25 times before being replaced by cell stocks kept in medium containing 10% DMSO and 10% FBS frozen in liquid nitrogen. Cells were harvested by treatment with 0.25% (wt/vol) trypsin and 0.2% (wt/vol) EDTA in Hank’s balanced salt solution for 10 min at 37°C centrifuged, and resuspended in DMEM. Medium in stock cultures was changed every 3 days. All procedures were carried out in a laminar flow hood.

Growth Characteristics PaCa-2 Cells

of PANG-1

and MIA

To determine the growth patterns and doubling times of both cell lines in the laboratory, 60,000 cells were plated on 60-mm dishes [Corning No. 25010; growth area, 21 cm*) in 3 ml of chemically defined medium (DMEM) containing different proportions of FBS or serum-free DMEM without other supplements. Every 24 h a triplicate of dishes was prepared for counting as described for stock cultures. Cell counts were performed using a Coulter Counter (Model ZBI, Coulter Electronics, Inc., Hialeah, Fla.) with a multichannel particle-size analyzer which provided detailed analysis of cell size distribution. Medium was not changed

during the experiment. the equation

Doubling time (dt) was calculated

by

dt = t/r where r is the multiplication

rate such that

r = 3.32 x log [(X,/X,)/(t, - tl)] where X, is the initial count and X, is the final count at a selected time [t, and tz). Effect of Peptides and MIA PaCa-2

on Proliferation Cells

of PANC-1

Because there was good correlation between cell number, protein content, and deoxyribonucleic acid (DNA] (r = 0.871, cell growth in these experiments was quantitated by measuring protein and DNA content in individual wells. Cells (60,000-80,000 per well] were plated in triplicate using 6-well plates [Falcon, No. 3046; 9.5-cm2 growth area; Becton Dickinson Labware, Lincoln Park, N.J.). Both cell lines were grown in 2 ml of DMEM containing 10% FBS for 72 h to allow for attachment and establishment of exponential growth. Cell cultures were then rinsed; serum-free medium was added, and the cells were incubated in the presence of each peptide for another 72 h (total exposure time to experimental factors, 6 days). The experiment was ended by removing the medium. Cells were carefully rinsed 3 times with warm PBS and then lysed using 0.5% (wt/vol) sodium deoxycholate and sonicated. A 200-~1 aliquot of cell ultrasonicate was taken for protein determination according to the method of Lowry et al. (14) using BSA diluted in doubledistilled water as a standard. For DNA determination, a loo-p1 aliquot of cell lysate was taken. The DNA concentration was determined fluorometrically by measuring the interaction of benzimide (33258 Hoechst) and DNA, using calf thymus DNA as a standard (15). When cultures were harvested, cell growth was still logarithmic. Medium was not changed during the second incubation in serum-free medium with or without peptides. Peptide stock solutions were prepared in 154 mM NaCl containing 0.2% (wt/vol) BSA. Sterility was provided by filtering the stock solutions through a 0.2~pm nitrocellulose filter (Acrodisc, Gelman Sciences Inc., Ann Arbor, Mich.). Concentrations of CCK in stock solutions were measured by radioimmunoassay, as will be described. Peptides were added to cultures in 20-~1 portions to produce final concentrations as follows: CCK8, 10~ ’ M; [Th?, Nle7JCCK9, lo-” M-10m6 M; bombesin, lOmeM; somatostatin, 1Om8M; secretin, lOmaM; VIP, 1Om8M; EGF. 3.4 x 10m9M; and insulin, 1Om6M. Control cultures received the same volume of 154 mM NaCl and 0.2% BSA without peptides. Anchorage-Dependent Cloning Efficiency PANC-1 and MIA PaCa-2 Cells

of

In a second approach to determine whether human pancreatic cancer cells in vitro show changes in growth in the presence of CCK8, PANC-1 and MIA PaCa-2 cells were also tested for their clonogenic potential. A small inoculum

1666

LIEHR ET AL.

GASTROENTEROLOGY Vol. 96, No. 6

of cells (PAN&l, 1000 cells/well; MIA PaCa-2, 500 cells/ well] was plated onto 6-well plates (9.5 cm2 growth area) in triplicate in 2 ml DMEM containing 1% FBS and incubated at 37°C for 12 days without changing the medium. Three different concentrations of CCKB were added (10-‘“-10~8 M) to some wells every 3 days. After incubation, colonies were stained with 2% (wt/vol) Giemsa in 0.01 M phosphate buffer (pH 7.0) and counted. One colony was defined as a cluster of 50 cells or more.

Determination Concentrations

of Cholecystokinin in Cell Cultures /----.

Concentrations of CCK8 in cell culture supernatants, culture media, and fetal bovine serum were measured using a highly sensitive and specific radioimmunoassay as previously described (16). Antiserum R016, raised in rabbits, binds to the carboxyl terminus of CCK and recognizes all molecular forms of CCK from CCK8-CCK39 equally (crossreactivity with gastrin, tl%]. Using CCK8 as a standard, the assay detects 0.5 pM CCK8 and has an ID,, of 11 pM. To determine the effects on CCK8 concentrations of serum-free DMEM and DMEM containing 1% and 10% FBS, CCK8 (90 PM] was added to 2 ml of medium without cells and incubated as previously described. The medium was sampled for radioimmunoassay after 6, 12, 24, 48. 72, and 96 h. In a second set of experiments, PANC-1 and MIA PaCa-2 cells were preincubated in 10% serum-supplemented medium for 72 h, then changed to serum-free DMEM containing 1.7 nM CCK8. The CCK concentrations were assayed in the culture supernatants after 6, 12, 24, 48, and 72 h.

Statistical

10% FBS

MIA PoCa-2 1% FBS

TIME (DAYS)

Figure 1. Growth curves of PANC-1 (A) and MIA PaCa-2 cells (B) cultured with varying concentrations of FBS. Cells were grown in the presence of 1% or 10% FBS or in serum-free (SF) medium for 4-6 days. SF,, indicates that cells were incubated in medium with 10% FBS (W)for 2 days (MIA PaCa-2) or 3 days (PANC-1), then incubated in serum-free medium for 3 days (0). At indicated times, cells were harvested and counted. Points are means of triplicate values taken from a representative experiment.

Evaluation

Values represent mean f SE. Data from experiments with gastrointestinal peptides were analyzed by two-way analysis of variance with a randomized block design using a multiple general linear model (171. Differences were considered to be significant when p tO.05. Data from cloningefficiency experiments were analyzed by analysis of variance using multiple comparisons as described by ScheffB

(181.

Results Growth Characteristics PaCa-2 Cells

of PANG1

and MIA

PANC-1 and MIA PaCa-2 cells showed similar changes in growth when incubated with different concentrations of FBS (Figure 1). Maximal growth of both cell lines occurred when cells were cultured in 10% serum-supplemented medium. Neither cell line grew in serum-free medium alone; only a few cells attached, and the cell number per culture constantly decreased.

When CCK8 (lo-’ M) was added to medium containing 1% and 100/o serum, no changes in growth measurements were observed in either cell line (data not shown]. Therefore, growth characteristics were determined for cells preincubated in medium containing 10% serum for 48 h or 72 h and then incubated in serum-free medium for 72 h. Growth rates of PANC-1 and MIA PaCa-2 cells decreased compared with those in 1% and 10% serum-supplemented medium (Figure l), but cells remained viable as judged by morphological appearance, surface attachment, and dye exclusion. Furthermore, there were significant increases in protein and DNA content in PANC-1 and MIA PaCa2 cell cultures 72 h after changing to serum-free medium (Figure 2). Cells grown in medium containing 1% FBS and cells preincubated in 20% FBS-supplemented medium and then switched to serum-free medium had longer doubling times than those grown continuously in 10% FBS (Table 1).

PEPTIDE-INDUCED GROWTH OF PANCREATIC CANCER

* * *

I

1L al

2

-

PANC-

5

5 n

a -

1

lop9 M CCK8. No significant differences between control and CCK-treated cultures were observed at any time (Figure 4). None of the other gastrointestinal peptides altered growth parameters of either PAN&l or MIA PaCa-2 cells when administered alone [Tables 2 and 3). Anchorage-Dependent Cloning Efficiency of PANC-I and MIA PaCa-2 Cells

._c

n-

1669

MIA PaCa-2

Figure 2. Protein and DNA contents of PANG1 and MIA PaCa-8 cell cultures after incubation in serum-free medium. Both cell lines were incubated for 72 h in 10% FBS-supplemented medium, and protein and DNA levels were determined in a subset of cultures (= initial). Another subset was incubated in serum-free medium for a further 72 h, and protein and DNA contents were measured. Results are expressed as percentages of initial values and are means + SE from 8-10 experiments in triplicate. *Significantly different from initial cultures (p < 0.001) by analysis of variance.

Effects of Peptides on Proliferation and MIA PaCa-2 Cells

of PANC-1

Growth rates of PANC-1 and MIA PaCa-2 cells were increased by exposure to EGF and insulin (Figure 3). The presence of EGF significantly increased DNA and protein content of PANC-1 cultures by 59ci; and 44% above control levels (p < 0.001) and in MIA PaCa-2 cultures by 1870 and 17’70 (p < 0.001; Figure 3). The presence of insulin significantly increased DNA and protein content of PANC-1 cultures by 76% and 56% above control level (p < 0.001); identical increases (76% and 56%) were observed in the MIA PaCa-2 cultures (p < 0.001; Figure 31. The magnitude of growth stimulation by EGF and insulin was similar in PANC-1 cells; however, EGF caused smaller effects than insulin in MIA PaCa-2 cells. A wide range of CCK8 and [Thr4, Nle7] CCK9 concentrations (10-l’ M-10m6 M] did not significantly increase the protein or DNA content of cultures in either cell line after 72 h (Tables 2 and 3). To determine whether a CCK-mediated growth effect occurred at earlier times, protein content per culture was measured after 6, 12, 24, 48, and 72 h incubation with

To detect slight changes in growth, cloning efficiency was examined using a very small inoculum of cells and counting the number of colonies after 12 days of incubation. There were no significant differences in cloning efficiency between the control group and cultures incubated with three different CCK8 concentrations (Table 4). A significant increase in the number of colonies was found after incubation of both cell lines with 10% FBS (p < O.OOl),indicating that a growth response could be measured with this culture system. Degradation of Cholecystokinin Culture System

Levels in Cell

To determine whether rapid degradation of CCK in the culture system accounted for the lack of growth response to this peptide, CCK concentrations were measured in culture medium alone and in the presence of cells. The CCK concentration in undiluted FBS from the batch used in the present study was 2.5 pM. After adding a single concentration of CCK8 (90 PM) to DMEM containing 100/FFBS, CCK immunoreactivity decreased by 40% after 6 h and almost completely disappeared after 48 h (Figure 5). The CCK concentrations also decreased in lo/c FBS-supplemented medium, but there was still approximately 4OYcof the initial concentration remaining after 72 h, whereas in serum-free medium almost 70% of the

Table I. Doubling Times of PANG-1 and MIA Pa&-2

Cells Modified Eagle’s Different ,4mounts of Fetal

Incubated in Dulbecco’s Medium Containing Bovine Serum SF

1% FBS

10% FBS

SF,,

PANG1

NV

65.8

36.2

68.8

MIA PaCa-2

NV

34.9

23.0

62.2

Cell line

Values are doubling times in hours calculated using the mean cell number per triplicate during log-phase of a representative experiment. Cells were incubated in serum-free (SF] medium and in medium containing 1% and 10% FBS for 4-6 days. Both cell lines were also preincubated in 10% FBS-supplemented medium for 2 days [MIA PaCa-2] and 3 days (PANGlj and then grown in serum-free medium (SF,,). NV, not viable.

1670

GASTROENTEROLOGY

LIEHR ET AL.

i?Z!

G 5 +

40

5 \

30

T V

20

Q z n

10

bated with CCK8 in serum-free medium, the pattern of change in CCK concentrations was different than in serum-free medium alone (Figure 5). Similar CCK concentrations were seen after 24 h in cultures with serum-free medium alone or serum-free medium and cells. Thereafter, CCK concentrations decreased in the presence of PANC-1 and MIA PaCa-2 cells compared with serum-free medium alone. These decreases amounted to a reduction of CCK levels of about 30% at 48 h and a 60% reduction at 72 h in serum-free medium in the presence of cells compared with serum-free medium in the absence of cells. Thus, at 7.2h, less than 10% of the initially added CCK8 was present (Figure 5). To counteract the loss of CCK in the culture system, CCK8 (860 PM) was added every 8 h for 48 h, and changes in protein and DNA were determined. Again, growth parameters were not affected after 24 h and 48 h, even though CCK levels in culture supernatants of both cell lines were maintained or even increased with time [Figure 6).

Control *

0

*

*

Vol. 98. No. 6

*

Discussion PANC-

1

MIA PaCa-r

Figure 3. Levels of DNA (A) and protein (II] in PANG1 and MIA PaCa-2 cultures after 72-h incubation in serum-free medium in the presence of EGF (3.4 x 10m9 M) or insulin (lo-’ M). Values are means f SE from 6-15 experiments. *Significantly different from control (p < 0.001)by analysis of variance. Control group values are indicated by hatched bars, peptide-treated groups by open bars.

initial concentration was detectable. When CCK was incubated with serum-free medium, the concentration of immunoreactive CCK decreased slightly within the first 12 h and then remained constant until the end of the experiment (Figure 5). When PANC-1 and MIA PaCa-2 cells were incu-

The results of this study show that EGF and insulin stimulated the proliferation of two human pancreatic cancer cell lines, whereas CCK, bombesin, secretin, VIP, and somatostatin had no effect on cell proliferation under the conditions of these experiments. Epidermal growth factor has been reported to stimulate pancreatic growth when administered in vivo (191, to increase cell proliferation and potentiate CCKmediated amylase release in mouse pancreatic acinar cells in vitro (20,211, and to be necessary for the maintenance of rat acinar cells in serum-free medium (22). It has also been reported to nearly double the incidence of carcinogen-induced pancreatic cancers in hamsters (23). The effects of EGF on human pancre-

Table 2. Growth Effects of Various Peptides on PANC-1 Cell line CCKB 10m9 M Protein (fig/culture) Control Peptide

Peptide

10-l’ M

10 *M

lo-” M

Bombesin lo-* M

Secretin 10-O M

VIP lo-@ M

ss 10 ‘M

232 t 3

266 k 2

266 + 2

259 + 3

266 + 2

249 + 5

257 k 2

258 + 3

267 + 6

PI 230 + 3

(31 271 -+ 1

(3) 262 + 6

1151 262 + 4

(31 277 + 7

(151 244 + 8

(151 260 + 3

(151 262 + 2

(151 257 ? 11

(31

(31

PI DNA (ccg/culture) Control

CCK9 10-l’ M

PI

(31

PI

PI

PI

(91

14.7 f 2.7

12.1 k 0.8

12.1 + 0.8

15.5 _t 1.0

12.1 + 0.8

16.3 + 0.6

17.8 + 0.5

18.1 + 0.6

17.9 + 0.5

(51 15.0 + 3.3

(31 13.3 k 1.5

(31 13.9 -+ 0.7

PI 15.4 + 0.8

(31 11.7 + 0.5

PI 14.3 * 0.3

(91 17.9 * 0.3

PI 18.3 k 0.8

(91 17.8 + 0.6

('31

(31

(31

(61

Values are means * SE of the number of experiments given in parentheses. FBS for 72 h. then incubated in serum-free medium for 72 h. SS, somatostatin. measured in the absence (Control) and presence (Peptide) of each substance.

(31

(61

(61

(61

(61

Cells were first incubated in DMEM supplemented with 10% For each experimental condition, protein and DNA levels were

June 1990

PEPTIDE-INDUCED

GROWTH OF PANCREATIC CANCER

1671

Table 3. Growth Effects of Various Peptides on MIA PaCa-2 Cell line CCK9

CCK8 10 I’M Protein (fig/culture) Control

284 + 3 (9) 281 + 5

Peptide DNA (,rg/culture) Control

VIP 10 ‘M

SS lo-* M

322 + 2

323 k 2

326 k 2

(151 325 +z3

(151 322 k 3

(151 327 + 4

I31

301 + 13 (15) 286 t 20 (91

(91

(91

(91

24.5 * 0.5

25.7 + 1.1

28.2 -t0.4

28.3 + 0.6

29.2

(91 27.9 + 0.8

(91 29.1 t 0.8

(91 27.5 k 0.7

lo-* M

10~6M

317 + 2

317 k 2 (3) 333 ? 6 I31

323 ZL2

317 i 2

(151 326 + 3

I31 332 k 5

(31

(91

337 r 3 (31

23.0 t 2.6

24.5 + 0.5

23.9 -t 2.4

Secretin lo-’ M

lo-” M

(31

(61 Peptide

Bombesin lOmaM

10~” M

25.9 ? 0.4 (31

(61

24.5 + 0.5 (31 25.5 k 1.3

(91

26.8 f 0.7 191

27.1 + 0.7

(31

(61

(31 26.3 _t0.5 (31

191

22.1 ? 2.5 (61

161

I61

k

0.9

(61

Values are means k SE of the number of experiments in parentheses. Cells were incubated as described in Table 2. SS, somatostatin. For each experimental condition, protein and DNA were measured in the absence (Control) and presence (Peptide) of each substance.

atic cancer growth in vitro have been studied in three established cell lines (7,8,24) and in cancer cells obtained from patients (9). Both cell lines used in the present study have been shown to have receptors for EGF 1:8,10).Liebow et al. (7) report a growth response to EGF in MIA PaCa-2 cells that was complete within 18 h and that occurred only in serum-free medium. A similar observation is reported by Korc and Magun PANCm

m

1

Control CCK 8 10-gM

MIA PoCa-2

0

6h

12h

24h

48h

72h

Figure 4. Time course of protein content in PANG1 (A) and MIA PaCa-r? cultures (B) in the presence of CCK8. Time 0 indicates the end of 73-h preincubation, at which time 10% FBS-supplemented medium was replaced by serum-free medium containing 10e9 M CCKO. Data are means + SE of 3 experiments.

[lo), who were also able to unmask a growthpromoting effect of EGF on PANC-1 cells when the serum concentration was lowered to 0.1%. The current data confirm these results. However, Gamou et al. (24) report that EGF did not elicit any proliferative response in UCVA-1 cells, a human pancreatic cancer cell line expressing a high number of EGF receptors (241. There are few studies concerning the effects of insulin on pancreatic cancer in vivo and in vitro. Exogenously administered insulin has been reported to inhibit growth of carcinogen-induced malignant pancreatic lesions in Syrian golden hamsters (25). In contrast, insulin has been shown to exert a trophic effect on AR42J cells, a rat pancreatic carcinoma line maintained in vitro (26). To the authors’ knowledge, there are no previous reports of a direct growthpromoting effect of insulin on PANC-1 and MIA PaCa-2 cells cultured in serum-free medium. In the current study, stimulation of growth with insulin was observed at a concentration of 10e6 M. It is known that at high concentrations, insulin can directly interact with receptors for insulinlike growth factor (IGF) I (27). Therefore, the effect observed in the present study may be mediated through interaction of insulin with IGF-I receptors. However, in AR42J cells, insulin acts through an insulin receptor, and no IGF-I receptors were detectable [26). Receptor-binding studies would be necessary to evaluate the presence and specificity of insulin and IGF receptors on PANC-1 and MIA PaCa-2 cells. The important role of CCK as a regulator of exocrine pancreatic growth (28) suggests that it may influence the growth of pancreatic cancer as well. Evidence for this hypothesis is based on recent observations that the CCK analog cerulein enhanced growth of a transplantable carcinogen-induced pancreatic cancer in hamsters (291, and that CCK augmented the carcinogenic@ of chemicals in rat and hamster models (2-4). However, other investigators report no ef-

1672

GASTROENTEROLOGY

LIEHR ET AL.

Vol. 98, No. 6

Table 4. Effects of Cholecystokinin Octapeptide on Cloning Efficiency of PANG1 and MIA PaCa-2 Cells CCK8

PANG1 MIA PaCa-2

Control

10-l’ M

10--gM

10 *M

10% FBS

81.8 + 2.6 (61 22.3 * 2.8 (31

83.0 + 5.8 (3) 18.0 -r 2.5 (31

83.0 -+2.5 (31 19.0 k 4.9 (31

84.7 + 5.3 (31 21.3 + 4.2 (3)

183.3 t 5.6” 131 58.0 +-3.6” (31

Values are means + SE of the number of colonies counted after 12 days of incubation in 1% FBS alone, with CCK added every 3 days, or in 10% FBS. Number of experiments are given in parentheses. “Significantly different from control (p < 0.001) by analysis of variance.

fects of CCK on tumor induction (30,311 or even a protective effect of CCK (32). No detailed studies are available documenting a regulatory function of CCK on human pancreatic cancer growth in vitro. The lack of response to CCK in the present study may have been a result of the absence of CCK receptors on PAN&l and MIA PaCa-2 cells. Although 100&I

.\, y 80- bo\ .\

‘Z

‘ .-r

60-

Cl

?5 Y

40-

\

9==8T 0.

0-O

A-A

MIA PaCa-2 PANC-1 ---__ SF -’

0

2007

T

??

B 8h

24 h

48 h

72 h

Figure 5. Changes in CCK8 concentration in different culture media in the absence of cells (A) and in serum-free medium in the presence of PANC-1 and MIA PaCa-2 cells (B). Ninety-picomolar CCKB was added to serum-free DMEM and DMEM containing 1% and 10% FBS and incubated at 37% for 72 h. PANC-1 and MIA PaCa-2 cells were preincuhated for 72 h in 10% serum-supplemented medium, then changed to serum-free medium containing 1.7 nM CCKB. At indicated times, medium and culture supernatants were assayed for CCK levels. Data expressed as percentage of the initial CCK concentration and means f SE of a representative experiment in triplicate. (----) Concentration of CCK in serum-free medium in the absence of cells; SF, serum-free medium.

Estival et al. (33) postulated the presence of CCK receptors on a human pancreatic cancer cell line, the existence of such receptors was only indirectly proven by measuring increased levels of cGMP after incubation of tissue with the CCK analog cerulein. It is important to consider that neoplastic transformation has been shown to cause alterations in cell membrane structure and receptor expression in rat and human pancreatic cancer cells (34,35). Cholecystokinin octapeptide was degraded in serumfree medium in the presence of cells; this was most likely caused by the action of peptidases released by the cells, as has been shown recently for another human pancreatic cancer cell line (36). However, when CCK was added every 8 h to counteract its degradation, growth of PANC-1 and MIA PaCa-2 cells was not affected. This lack of growth response could be caused not only by the absence of CCK receptors but also by functional abnormalities either at the level of the receptor or at postreceptor pathways in PANC-1 and MIA PaCa-2 cells. Increases of intracellular calcium and cyclic adenosine monophosphate (CAMP) levels are considered initiators of mechanisms resulting in growth. Both calcium- and CAMP-dependent pathways have been shown to be altered in pancreatic carcinomas. Warren (5) reports that the inability of rat pancreatic carcinoma cells to respond to CCK was the result of an insensitivity to extracellular calcium. The lack of CAMP elevation in response to secretin binding to its receptor in transplanted hamster carcinomas suggests a deficiency of the CAMP-dependent protein kinase system (37). Further studies are necessary to examine whether CCK receptors exist on pancreatic cancer cells, and if so, whether the receptors and their intracellular mediators are functionally intact. Somatostatin is known to have inhibitory effects on the growth of normal exocrine pancreas (38), experimental pancreatic cancer (11,39), and two human pancreatic cancer lines maintained as xenografts in nude mice (40). Somatostatin receptors have been characterized on AR42J cells (41). MIA PaCa-2 cells respond to exogenous somatostatin with dephosphorylation of membrane proteins, suggesting the existence of specific receptors (42). In contrast, somatostatin receptors were not detectable in 12 human pancreatic

PEPTIDE-INDUCED

June 1990

El

PANC-1

24 h m

MIA PaCa-2

48 h

Protein

GROWTH

OF PANCREATIC

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hypothesis that gastrointestinal hormones have such a direct modulatory function. The conflicting results reported in the literature may reflect the heterogeneity of pancreatic cancer, with cell lines derived from tumor subpopulations showing different biological and pharmacological properties and various stages of differentiation (48). Therefore, it cannot be completely excluded that other human pancreatic cancer lines that have retained growth responses to gastrointestinal regulatory peptides will be defined in the future. References 1. Townsend CM, Singh P, Thompson JC. Gastrointestinal hormones and gastrointestinal and pancreatic carcinomas. Gastroenterology 1986;91:1002-1006. 2. Satake K, Mukai R, Kato Y, Umeyama K. Effects of cerulein on the normal pancreas and on experimental pancreatic carcinoma

in the Syrian golden hamster. Pancreas 1986;1:246-253. Howatson AG, Carter DC. Pancreatic carcinogenesis-enhancement by cholecystokinin in the hamster-nitrosamine model. Br J Cancer i985;5i:io7- 114. 4. Lhoste EF, Longnecker DS. Effect of bombesin and caerulein on early stages of carcinogenesis induced by azaserine in the rat pancreas. Cancer Res 1987;47:3273-3277. 5. Warren JR. Secretagogue response in rat pancreatic acinar carcinoma. J Nat1 Cancer Inst 1982;69:969-974. 6. Douglas BR, Woutersen RA, Jansen JBMJ, DeJong AJL, Rovati LC, Lamers CBHW. Influence of cholecystokinin antagonist on the effects of cholecystokinin antagonist on the effects of cholecystokinin and bombesin on azaserine-induced lesions in rat pancreas. Gastroenterology 1989;96:462-469. 7. Liebow C, Hierowski M, duSapin K. Hormonal control of pancreatic cancer growth. Pancreas 1986;1:44-48. a. Smith JJ. Derynck R, Korc M. Production of transforming growth factor 01in human pancreatic cancer cells: evidence for a superagonist autocrine cycle. Proc Nat1 Acad Sci USA 1987;84: 7567-7570. 9. Hamburger AW, White CP, Brown RB. Effect of epidermal growth factor on proliferation of human tumor cells in soft agar. J Nat1 Cancer Inst 1981;67:825-830. 10. Korc M, Magun BE. Binding and processing of epidermal growth factor in PANC-1 human pancreatic carcinoma cells. Life Sci i985;36:1849-1855. 11. Paz-Bouza JI, Redding TW. Schally AV. Treatment of nitrosamine-induced pancreatic tumors in hamsters with analogues of somatostatin and luteinizing hormone-releasing hormone. Proc Nat1 Acad Sci USA 1987:84:1112-1116. 12. Lieber M, Mazzetta J. Nelson-Rees W, Kaplan M, Todaro G. Establishment of a continuous tumor-cell line (PANC-1) from a human carcinoma of the exocrine pancreas. Int J Cancer 1975;15:741-747. 13. Yunis AA, Arimura GK, Russin DJ. Human pancreatic carcinoma (MIA PaCa-2) in continuous culture: sensitivity to asparaginase. Int J Cancer 1977;19:128-135. 14. Lowry OH, Rosebrough NJ, Farr AL, Randall RJ. Protein measurement with the Folin phenol reagent. J Biol Chem 1951;193:265-275. 15 Labarca C, Paigen K. A simple, rapid, and sensitive DNA assay procedure. Anal Biochem 1980:102:344-352. 16 Turkelson CM, Dale WE, Reidelberger R. Solomon TE. Development of cholecystokinin radioimmunoassay using synthetic CCK-10 as immunogen. Regul Peptides l986;15:205-217. 17 Wilkinson L. SYSTAT: the system for statistics. Evanston, Ill.: Systat, Inc., 1986. 3.

PANC-1

MIAPaCa-2

24 h

PANC-1

MIAPaCa-2

48 h

Figure 6. Protein and DNA contents of PANC-1 and MIA PaCa-2 cultures, incubated as previously described, after adding CCK8 (860 PM) every 8 h for 48 h. Protein and DNA contents of individual wells were determined after 24 h and 48 h and expressed as percentage of controls (B). Controls were cells incubated without CCK (----, 100% of controls). At the same time, CCK concentrations in culture supernatants of both cell lines were determined (A). Results are means + SE of 6 experiments.

adenocarcinomas obtained at surgery (43). Because somatostatin may exert its inhibitory effects only in combination with other peptides, the present results therefore cannot exclude such an indirect mechanism of somatostatin. Evidence for this idea is based on the observation that in MIA PaCa-2 cell cultures somatostat-in alone had no effect on growth, whereas the combination of EGF and somatostatin resulted in a significant inhibition of EGF-induced growth (7). Bombesin has been shown to induce pancreatic growth in vivo (44) and to stimulate growth of preneoplastic acinar cell lesions in rats (4,6). In contrast, bombesin is reported to inhibit growth of a human pancreatic cancer xenografted into mice (45). Secretin has been shown to act as a cocarcinogen in a chemically induced pancreatic cancer in hamsters (46). Vasoactive intestinal peptide has been reported to inhibit growth of a hamster pancreatic carcinoma but not MIA PaCa-2 transplanted into hamsters (47). Secretin, VIP, and bombesin had no effects on any of the growth parameters in the current study. The present results agree with data from studies of normal mouse acinar cells in vitro, which also did not show any growth response to VIP, secretin, or bombesin (21). In conclusion, the present results indicate a role for EGF and insulin in promoting growth of human pancreatic cancer in vitro but do not support the

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18. Scheffe HA. A method for judging all possible contrasts in the

analysis of variance. Biometrika 1953;40:87-104. 19. Dembinski A, Gregory H, Konturek SJ, Polanski M. Trophic action of epidermal growth factor on the pancreas and gastroduodenal mucosa in rats. J Physiol (Lond) 1982;325:35-42. 20. Logsdon CD. Stimulation of pancreatic acinar cell growth by CCK, epidermal growth factor, and insulin in vitro. Am J Physiol 1986;251:G487-G494. 21. Logsdon CD, Williams JA. Pancreatic acini in short-term culture: regulation by EGF, carbachol, insulin, and corticosterone. Am J Physiol1983;244:G675-G682. 22. Brannon PM, Hirschi K, Korc M. Effects of epidermal growth factor, insulin and insulin-like growth factor I on rat pancreatic acinar cells cultured in serum-free medium. Pancreas 1988;3:4148. 23. Chester JF, Gaissert HA, Ross JS. Malt RA. Pancreatic cancer in the Syrian hamster induced by N-nitrosobis(2-oxopropyl)-amine: cocarcinogenic effect of epidermal growth factor. Cancer Res 1986;46:2954-2957. 24. Gamou S, Kim YS, Shimizu N. Different responses to EGF in two human carcinoma cell lines, A431 and UCVA-1, possessing high numbers of EGF receptors. Mol Cell Endocrinol 1984;37: 205-213. 25. Pour PM, Stepan K. Modification of pancreatic carcinogenesis in the hamster model. VIII. Inhibitory effect of exogenous insulin. J Nat1 Cancer Inst 1984;72:1205-1208. 26. Mossner J, Logsdon CD, Goldfine ID, Williams JA. Do insulin and the insulin like growth factors (IGFs) stimulate growth of the exocrine pancreas? Gut 1987;28(Suppl1]:51-55. 27. Massague J, Czech MP. The subunit structures of two distinct receptors for insulin-like growth factors I and II and their relationship to the insulin receptor. J Biol Chem 1982;257:50385045. 28. Solomon TE, Vanier M, Morisset J. Cell site and time course of DNA synthesis in pancreas after caerulein and secretin. Am J Physiol1983;245:G99-G105. 29 Townsend CM, Franklin RB, Watson LC, Glass EJ, Thompson JC. Stimulation of pancreatic cancer growth by caerulein and secretin. Surg Forum 1981;32:228-229. 30 Pour PM, Lawson T, Helgeson S, Donnelly T, Stepan K. Effect of cholecystokinin on pancreatic carcinogenesis in the hamster model. Carcinogenesis 1988;9:597-601. A, Dawiskiba S. Ihse I. Studies of the effect of 31. Andren-Sandberg cerulein administration on experimental pancreatic carcinogenesis. Stand J Gastroenterol1984;19:122-128. 32. Johnson FE, LaRegina MC, Martin SA, Bashiti HM. Cholecystokinin inhibits pancreatic and hepatic carcinogenesis. Cancer

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36. Yamaguchi N, Kawai K. Acid protease secreted from human pancreatic carcinoma cell line HPC-YT into serum-free, chemically defined medium. Cancer Res 1986;46:5353-5359. 37. Scarpelli DG, Rao MS. Transplantable ductal adenocarcinoma of the Syrian hamster pancreas. Cancer Res 1979;39:452-458. 38. Morisset J, Genik P, Lord A, Solomon TE. Effects of chronic administration of somatostatin on rat exocrine pancreas. Regul Pept 1982;4:49-58. 39 Redding TW, Schally AV. Inhibition of growth of pancreatic carcinomas in animal models by analogues of hypothalamic hormones. Proc Nat1 Acad Sci USA 1984;81:248-252. 40 Upp JR, Olson D, Poston GJ, Alexander RW, Townsend CM, Thompson JC. Inhibition of growth of two human pancreatic adenocarcinomas in vivo by somatostatin analog SMS 201-995. Am J Surg 1988;155:29-35. 41 Viguerie N, Tahiri-Jouti N, Esteve J-P, Clerc P, Logsdon C, Svoboda M, Susini C, Vaysse N, Ribet A. Functional somatostatin receptors on a rat pancreatic acinar cell line. Am J Physiol 1988;255:G113-G120, 42. Hierowski MT, Liebow C, duSapin K, Schally AV. Stimulation by somatostatin of dephosphorylation of membrane proteins in 1pancreatic cancer MIA PaCa-2 cell line. FEBS Lett 1985:179:252256. 43. Reubi JC, Horisberger U, Essed CE, Jeekel J, Klijn JGH, Lamberts SWJ. Absence of somatostatin receptors in human exocrine pancreatic adenocarcinomas. Gastroenterology 1988;95: 760-763. 44. Solomon TE, Petersen

H, Elashoff J. Grossman MI. Effects of chemical messenger peptides on pancreatic growth in rats. In: Miyoshi A, ed. Gut peptides. Tokyo: Kodansha, 1979:213-219. 45. Alexander RW, Upp JR, Poston GJ, Townsend CM, Singh P, Thompson JC. Bombesin inhibits growth of human pancreatic adenocarcinoma in nude mice. Pancreas 1988;3:297-302. 46. Howatson AG, Carter DC. Pancreatic carcinogenesis: effect of secretin in the hamster-nitrosamine model. J Nat1 Cancer Inst 1987:78:101-105. 47. Poston GJ, Yao CZ, Upp JR, Alexander RW. Townsend CM, Thompson JC. Vasoactive intestinal peptide inhibits the growth of hamster pancreatic cancer but not human pancreatic cancer in vivo. Pancreas 1988;3:439-443. 48. Hollande E, Trocheris De St.-Front V, Louet-Hermitte P, Bara J. Pequignot J, Estival A, Clemente F. Differentiation features of human pancreatic tumor cells maintained in nude mice and in culture: immunocytochemical and ultrastructural studies. Int J Cancer 1984;34:177-185,

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of the human exocrine pancreas: Presence of secretin and caerulein receptors. Biochem Biophys Res Commun 1981;102:1336-1341, 34. Jamieson JD, Ingber DE, Muresan V, Hull BE, Sarras MP, Maylie-Pfenniger MF, Iwanij V. Cell surface properties of normal, differentiating, and neoplastic pancreatic acinar cells. Cancer 1981;47:1516-1525. 35. Kim YS, Tsao D, Hicks J. McIntyre LJ. Surface membrane glycoproteins of cultured human pancreatic cancer cells. Cancer 1981;47:1590-1596.

Received August 4,1989.Accepted December 12,1989. Address requests for reprints to: Travis Solomon, M.D., Ph.D. Research Service (1511,Kansas City VA Medical Center, 4801 Linwood Boulevard, Kansas City, Missouri 64128. This research was supported by the Medical Research Service of the Veterans Administration and by NIH grant DK 35152. R.-M. Liehr was supported by grant Li 427/1-l from the Deutsche Forschungsgemeinschaft. Technical assistance was provided by Judy Turkelson.