Corticotropin-releasing factor stimulates cyclic AMP, arachidonic acid release, and growth of lung cancer cells

Peptides, Vol. 15, No. 2, pp. 281-285, 1994 Copyright© 1994ElsevierScienceLtd Printedin the USA.All rightsreserved 0196-9781/94$6.00 + .00

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Corticotropin-Releasing Factor Stimulates Cyclic AMP, Arachidonic Acid Release, and Growth of Lung Cancer Cells T E R R Y W. M O O D Y , *l F A R A H ZIA,* R A J E S H V E N U G O P A L , * L O U I S Y. K O R M A N , t A L L A N L. G O L D S T E I N * A N D M I R E L A F A G A R A S A N *

*Department of Biochemistry and Molecular Biology, The George Washington University School of Medicine and Health Sciences, Washington, DC 20037 and ?Gastroenterology Section, V.A. Medical Center, Washington, DC 20422 Received 25 May 1993 MOODY, T. W., F. ZIA, R. VENUGOPAL, L. Y. KORMAN, A. L. GOLDSTEIN AND M. FAGARASAN. Corticotropinreleasingfactor stimulates cyclic AMP, arachidonic acid release, and growth of lung cancer cells. PEPTIDES 15(2) 281-285, 1994.--The effects of corticotropin-releasing factor (CRF) on human lung cancer cell lines was investigated. Corticotropinreleasing factor increased the cAMP levels in a dose-dependent manner; CRF (100 nM) elevated the cAMP levelsapproximately elevenfoldusing NCI-H345 cellsand increasedthe gastrin-releasingpeptide (GRP) secretion rate by approximately 70%. Similarly, sauvagine, a structural analogue of CRF, elevated the cAMP levels with a half-maximal effective dose (EDso) of 20 nM. The increase in cAMP caused by CRF and sauvagine was reversed by a-helical CRF(9-41). Corticotropin-releasing factor had no effect on cytosolic calcium but stimulated [3H]arachidonic acid release from NCI-HI299 cells with an EDs0 of 30 nM. The increase in [3H]arachidonicacid release caused by 100 nM CRF was significantlyreversed by 1 or 10 ~M a-helical CRF(9-41). Also, CRF stimulated the clonal growth of NC1-H345 and H720 cells and the growth increase caused by CRF was reversed by a-helical CRF(9-41 ). These data suggest that CRF may be a regulatory peptide in lung cancer. CRF

Lung cancer

cAMP

GRP secretion

Arachidonic acid

CORTICOTROPIN-RELEASING factor (CRF) is a 41 amino acid peptide initially isolated from ovine hypothalami (26). Corticotropin-releasing factor is synthesized in paraventricular nucleus of the hypothalamus in the form a a high molecular weight precursor protein and is released from nerve terminals in the external layer of the median eminence (15,22,24,25). Corticotropin-releasing factor administration provokes stress-like responses including activation of the sympathetic nervous system with consequential increases in plasma epinephrine, norepinephrine, and glucose resulting in increased heart rate and mean arterial blood pressure (4,28). Also, CRF alters feeding behavior and memory (8,11,13,21 ), Corticotropin-releasing factor causes release of ACTH and/3-endorphin from the anterior pituitary, and ACTH, in turn, stimulates the release of glucocorticoids from the adrenal cortex, creating a negative feedbak loop whereby circulating levels of glucocorticoids can suppress the release of both CRF and ACTH (27). High-affinity binding sites for CRF have been identified in the rat brain and anterior pituitary (5,6,29). [~25I][Tyr°]CRFbinds with high affinity to anterior pituitary membranes and is crosslinked to a 75 kDa protein (10). Corticotropin-releasing factor

stimulates adenylate cyclase activity in the anterior pituitary, and the increase in cAMP caused by CRF is reversed by the putative CRF antagonist a-helical CRF(9-4 l) (1,3). Previously, we found that numerous peptide receptors are present in small cell lung cancer (SCLC) cell lines including bombesin/gastrin-releasingpeptide (BN/GRP), cholecystokinin, neurotensin, arginine vasopressin, and bradykinin receptors (17). The receptors are coupled to a guanine nucleotide binding protein that stimulates phosphatidylinositol turnover (19) resulting in elevated cytosolic calcium (Ca2+). Also, SCLC and non-SCLC (NSCLC) cell lines have vasoactive intestinal peptide (VIP) receptors that are coupled to a stimulatory guanine nucleotide binding protein (14). Vasoactive intestinal peptide elevates cAMP in NSCLC cell line NCI-H 1299 and SCLC cell line NCI-H345. Here the effects of CRF on lung cancer cell lines were investigated. METHOD The SCLC cell lines NCI-H345 and H720 were cultured in serum-supplemented medium (RPMI-1640 containing 10%

Requests for reprints should be addressed to Dr. Terry W. Moody, NCI, BPRB, 9610 Medical Ctr. Dr., Rockville, MD 20850.

281

282

M O O D Y ET AL. TABLE 1 EFFECT OF AGENTS ON NCI-H345 CELLS Agent

Relative c A M P (%)

Secretionof GRP (%)

None CRF(100 nM) Forskolin (50 uM) PACAP-38 (100 nM)

100_+ 10 1144 _+ 98 1605 _+ 141 850 _+ 72

100 +_ 7 167 + 11 250 _+ 23 200 _+ 18

SCLC cells were treated with agents for 5 min at 37°C. The buffer was S1T containing 1% BSA and 100/~M IBMX. The cAMP and GRP were determined by radioimmunoassay. The mean value _+ SE of four determinations is indicated.

heat-inactivated fetal calf serum) in a humidified atmosphere of 5% CO2 and 95% air at 37°C (2). The NSCLC cell line NCIH 1299 was cultured in RPMI- 1640 containing 10% heat-inactivated fetal bovine serum. When a monolayer formed, the adherent cells were washed with PBS and treated with trypsin/ EDTA. The cells were pelleted and resuspended in serum-supplemented medium. Routinely the cells were passed 1/1 weekly and experiments were conducted when the cells were in exponential growth phase. Cyclic A M P was assayed by radioimmunoassay (12). Cell lines NCI-H345 were harvested and resuspended in SIT medium containing 1% bovine serum albumin (BSA), 1 mg/ml bacitracin,

and 100 # M isobutyl-methyl-xanthine (IBMX). After 5 min the reaction was quenched by the addition of an equal volume (0.5 ml) of ethanol. The samples were mixed and frozen at 80°C until assay. The samples were vortexed and an aliquot of supernatant was removed and added to 100 ul of 50 m M sodium acetate (pH 6.2). The sample (25/~1) was acetylated at 4°C by adding 10 #1 oftriethylamine followed by 5 #l of acetic anhydride. Then 200/A of goat anti-cAMP antibody was added followed by 8000 cpm of [~251]-2-succinyl(tyrosine methyl ester)-cAMP. After incubation tbr 16 h at 4°C, I ml of charcoal suspension (2 mg/ml Norit-A charcoal in 100 m M phosphate buffer, pH 6.3, which contains 0.25% BSA) was added, the tubes vortexed, and centrifuged at 1500 × g for 10 rain. An aliquot (1 ml) of supernatant, which contains radiolabeled antigen-antibody complex, was counted in a g a m m a counter. Also, it was investigated i f C R F analogues altered the intracellular Ca 2+. The SCLC cells were loaded with Fura 2AM. The cytosolic Ca 2' was determined using a Perkin-Elmer spectroflourometer (16). Gastrin-releasing peptide radioimmunoassays were conducted to assay for secreted G R P (20). NCI-H345 cells were treated with C R F and after 5 rain the medium was centrifuged and the supernatant was saved. The medium was added to G R P antiserum (1:100,000 dilution) in PBS containing 0.25% BSA. Then [~251]GRP (5000 cpm) was added, and after 16 h at 4°C, the antigen-antibody complex was isolated. Normal rabbit sera was added (1:200) followed by goat anti-rabbit sera (1:10) and 12% polyethylene glycol. The sample was centrifuged at 1000 × g for 15 min, and the supernatant was removed and the pellet counted in a g a m m a counter.

120

100

80

E 60 =E 0 40

20

0 1

I

|

1

I

I

l

-10

-9

-8

-7

-6

-5

(Peptide),

Log

M

FIG. 1. Cyclic AMP. The cAMP was determined as a function of sauvagine (ll), CRF (O), and ,~-helical CRF(9-41) (V1) concentration. The mean value _+SE of four determinations is indicated. This data is representative of two other experiments.

CRF RECEPTORS AND LUNG CANCER

283

TABLE 2 EFFECT OF CRF ANALOGUES ON NCI-H345 cAMP

m

o

E

Peptide

cAMP (fmol)

None CRF(I #M) Sauvagine (1/~M) a-Helical CRF(9-41) (1 #M) CRF + a-helical CRF(9-41)

12+ 2 113 ___38 106 _ 22 17 +_ 5 42 _+ 13

t,,I--

"6200 (D U~ 0

m

m L_ 1 0 0

NCI-H345 cells (5 X 105) were exposed to 1 ml of SIT medium for 5 min and 25 #1 of medium was assayed for cAMP. The mean value _+ SD of four determinations is indicated.

tY"

__..// / /

I -10

I -8 (CRF),

I -6 Log M

FIG. 2. GRP secretion dose-response curve. NCI-H345 cells (5 × 10s) were incubated in 1 ml of SIT medium for 5 min and 400 ~1 was assayed for immunoreactive GRP. The secreted GRP was determined as a function of CRF concentration. The mean value _ SE of four determinations is indicated. This experiment is representative of two others.

Lung cancer cells were assayed for arachidonic acid release (7). NCI-H1299 cells (5 × 104) were placed in 24-well plates coated with h u m a n fibronectin (20 #g). After a monolayer of cells formed (5 days), ([3H]-5,6,8,9,11,12,14,15)arachidonic acid (2.5 X 10 ~ cpm) was added. After 16 h the cells were washed twice in 1 ml of SIT m e d i u m containing 0.2% fatty acid-free bovine serum albumin (SIT/BSA). New medium was added containing CRF-like peptides. After 40 min, 100 ~tl of media was removed from each well and placed in a scintillation vial; scintillation fluid was added and the sample was counted in a B-counter.

Growth assays were conducted using NCI-H345 or NCI-H720 and the agarose cloning system. The base layer consisted of 3 ml of 0.5% agarose in SIT medium containing 5% fetal bovine serum in six-well plates (16). The top layer consisted of 3 ml ot SIT medium in 0.3% agarose, peptide, and 2 × 104 single viable cells. For each cell line and peptide concentration, triplicate wells were plated. After 2 weeks, 1 ml of 0.1% p-iodonitrotetrazolium violet was added and after 16 h at 37°C the plates were screened for colony formation. The number of viable colonies larger than 120 # m in diameter were counted. RESULTS

The effects of C R F on c A M P levels was investigated. Table 1 shows that 100 n M C R F caused an elevenfold increase in the c A M P levels. Also, forskolin increased the c A M P sixteenfold whereas PACAP-38 stimulated the c A M P eightfold. Sauvagine, a 40 amino acid peptide isolated from frog skin that has sequence homology with CRF, increased the c A M P in a dose-dependent manner, and the half-maximal effective dose (EDso) was approximately 20 n M (Fig. 1). Corticotropin-releasing factor was slightly less potent with an EDs0 of 30 nM, whereas a-helical CRF(9-41) did not elevate cAMP. Table 2 shows that 10 nM

B

A

100 nMCRF

100 nMBN

.C

D

100 nMCCK-8

100 nM NT

2,,. o

cO 0 X

w

,

F

°'n4

F 3mln 4

F

3min 4

W 3mln--~

Time,min FIG. 3. Cytosolic calcium. CRF (100 nM) (A) had no effect on the cytosolic Ca 2÷ of NCI-H345 cells whereas the cytosolic Ca2÷ was increased by 100 nM BN, CCK-8, or NT (B-D). This experiment is representative of three others.

284

MOODY ET AL. TABLE 3 10

EFFECT OF CRF ANALOGUES ON ARACHIDONIC ACID RELEASE Addition

cpm

None CRF (1000 nM) Sauvagine (1000 nM) a-Helical CRF(9-41) (1000 riM)

140 + 12 933 _+ 120" 866 _+ 136t 135 _+ 27

The mean value _+SD of four determinations is indicated. *p <0.01. -[p < 0.05.

A

_9.

10

B

o

J -//

L

I

I

- 1 0 '-9 -8

I

-7

7,/

I -6

(CFF), Log M

CRF significantly elevated the cAMP approximately tenfold and 1 uM a-helical CRF(9-41 ) inhibited the increase in cAMP caused by CRF. Similar data were obtained using NCI-H720 cells. Table 1 shows that that 100 nM CRF increased the secretion rate of BN/GRP by 67%, whereas forskolin and PACAP-38 increased the secretion rate by 150% and 100%, respectively. Figure 2 shows that 10 ~0CRF had little effect on GRP secretion whereas 10 8 or 10-6 M CRF increased the GRP secretion rate: the basal secretion rate was 80 fmol. Corticotropin-releasing factor had no effect on cytosolic Ca 2+ (Fig. 3). In contrast, 100 nM BN, cholecystokinin (CCK-8), or neurotensin (NT) strongly elevated cytosolic Ca 2+ using NCIH345 cells. Using NCI-H 1299 cells, CRF stimulated [3H]arachidonic acid release. Table 3 shows that 1 uM CRF or sauvagine stimulated [3H]arachidonic acid release sevenfold, whereas a-helical CRF(941) had no effect. Corticotropin-releassing factor stimulated [3H]aracidonic acid release in a dose-dependent manner and the EDs0 was 30 nM (Fig. 4); CRF(9-41) reversed the increase in [3H]arachidonic acid release caused by 100 nM CRF and the EDs0 was 5 uM. Also, the effects on SCLC growth were investigated. Table 4 shows that 10 nM CRF increased the number of NCI-H720 colonies threefold. Similarly, 10 n M sauvagine increased the number of NCI-H720 colonies twofold, a-Helical CRF(9-41) ( 1 #M) did not alter the number of basal colonies but inhibited the increase in colony number caused by 10 n M CRF. Similar data were obtained using NCI-H345 cells. DISCUSSION

This manuscript demonstrates that CRF elevates cAMP, causes [3H]arachidonic acid release, and stimulates the growth of lung cancer cells. Therefore, CRF receptors may be present on lung cancer cells. Corticotropin-releasing factor and sauvagine elevated the cAMP in a dose-dependent manner in NCI-H345 cells. The EDs0 for sauvagine was 20 nM. Similarly, CRF elevated cAMP levels with an EDs0 of 30 n M in NCI-H345. Previously, CRF was demonstrated to stimulate adenylate cyclase activity in rat cortex membranes with an EDs0 of 2 n M (3). Also, CRF and sauvagine inhibited binding to CRF receptors using rat brain midbrain membranes with ICs0 values of 20 and 10 n M (3). These data suggest that CRF and sauvagine bind to SCLC and rat brain receptors with similar affinity. Furthermore, SCLC CRF receptors may be coupled to a stimulatory guanine nucleotide binding protein that stimulates adenylate cyclase. Previously, using NCI-H345 cells, we found that 1 #M VIP elevated cAMP levels tenfold and that VIP increased the secretion

~ -8

I -7

I -6

i -5

(CRF(9.-41)), Log M

FIG. 4. [3H]Arachidonicacid release. (A): [3H]arachidonicacid release was determined in the presence of increasing concentrations of CRF; *p < 0.05, *~rp < 0.01 relative to control. (B): [3H]arachidonicacid release was determined in the presence of 100 nM CRF and increasing doses of c~-helicalCRF(9-41). The mean value _+ SE of four determinations is indicated: *p < 0.05, **p < 0.01 relative to 100 nMCRF.

rate of BN/GRP (12). Similarly, 10 or 1000 nM CRF increased the secretion rate of BN/GRP by approximately 70%. Using HPLC techniques, however, NCI-H345 cell extracts and conditioned media contain mostly GRP(14-27), a GRP metabolite that has appreciable biological activity (T. Moody, unpublished). These data suggest that protein kinase A may phosphorylate protein substrates resulting in exocytosis of SCLC granules that contain BN/GRP. Similarly, forskolin and PACAP, a structural analogue of VIP, elevated the cAMP levels in NCI-H345, resulting in increased secretion of BN/GRP. Previously, CRF was demonstrated to release fl-endorphin from rat primary neurointermediate pituitary lobe cells with an EDs0 of 3 nM (27), Corticotropin-releasing factor had no effect on NCI-H345 cytosolic Ca 2+ levels. Bombesin, NT, or CCK-8 elevated cytosolic Ca 2+ levels in Fura-2AM-loaded NCI-H345 cells. These data suggest that BN/GRP, NT, and CCK-8, but not CRF, may cause phosphatidylinositol turnover and that the resulting inositol1,4,5-trisphosphate may release Ca 2+ from intracellular pools. Corticotropin-releasing factor stimulated [3H]arachidonic acid release from NCI-H1299 cells. Similarly, sauvagine, but not a-helical CRF(9-41 ), stimulated [3H]arachidonic acid release and the EDs0 for CRF was 30 nM. In contrast, the increase in [3H]arachidonic acid release caused by 100 n M C R F was reversed

TABLE 4 EFFECT OF CRF ANALOGUES ON SCLC G R O W T H Peptide

NCI-H720

None CRF (10 nM) Sauvagine (10 nM) a-Helical CRF(9-41) (1 uM) CRF + c~-helicalCRF(9-41)

27 _+ 3 70 _+6* 51 _+7t 33 _+7 37 + 6

The mean number of viable NCI-H720 colonies _+SE of three determinationsis indicated. *p <0.01. t P < 0.05.

CRF RECEPTORS AND LUNG CANCER

285

by a-helical CRF(9-41) and the EDso was 5 ~M. These data suggest that C R F binds with higher affinity to the C R F receptor than does a-helical CRF(9-41). Also, SCLC C R F receptors may stimulate phospholipase A2 activity, resulting in increased arachidonic acid from lung cancer cells. Corticotropin-releasing factor (10 riM) stimulated the clonal growth of NCI-H720 cells approximately twofold. Similarly, 10 n M sauvagine stimulated the growth of NCI-H345; 1000 n M ahelical CRF(9-41) had no effect, a-Helical CRF(9-41) reversed the increase in the clonal growth caused by 10 n M CRF. Previously, we found that 100 n M VIP significantly stimulated SCLC growth and that a VIP receptor antagonist, VIP hybrid, strongly inhibited colony formation (18). Also, VIP hybrid inhibited [125I]VIP binding to SCLC cells and the increase in c A M P caused by VIP. Therefore, VIP hybrid functions as a VIP receptor

antagonist whereas a-helical CRF(9-41) functions as a C R F receptor antagonist. Vasoactive intestinal peptide m R N A and VIP immunoreactivity are also present in SCLC cells (9). Also, [~25I]VIP binds with high affinity to almost all SCLC cell lines examined (23). These data suggest that VIP may function as an autocrine growth factor in lung cancer. It remains to be determined if C R F is synthesized in and secreted from lung cancer cells. In summary, because C R F stimulates cAMP, causes [3H]arachidonic acid release, and stimulates lung cancer growth it may function as a regulatory peptide in lung cancer. ACKNOWLEDGEMENTS This research was presented in part at the 14th Annual Winter Neuropeptide Conference in Breckenridge, CO and is supported in part by NCI grant CA-53477.

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15. Liao, N.: Vaudry, H.: Pelletier, G. Neuroanatomical connections between corticotropin-releasing factor (CRF) and somatostatin (SRIF) nerve endings and thyrotropin-releasing hormone (TRH) neurons in the paraventricular nucleus of rat hypothalamus. Peptides 13:677-680:1992. 16. Mahmoud, S.; Staley, J.; Taylor, J.; et al. (Psi-13,14)Bombesin analogues inhibit growth of small cell lung cancer in vitro and in vivo. Cancer Res. 51:1798-1802; 1991. 17. Moody, T. W.; Cuttitta, F. Growth factor and peptide receptors in small cell lung cancer. Life Sci. 52:1161-1173: 1993. 18. Moody, T. W.; Koros, A. M. C.; Reubi, J. C.; Naylor, P. H.; Goldstein, A. L. VIP analogues inhibit small cell lung cancer growth. Biomed. Res. 13(2):131-136; 1993. 19. Moody, T. W.; Murphy, A.; Mahmoud, S.; Fiskum, G. Bombesinlike peptides elevate cytosolic calcium in small cell lung cancer cells. Biochem. Biopfiys. Res. Commun. 147:189-195; 1987. 20. Moody, T. W.; Pert, C. B.; Gazdar, A. F.; Carney, D. N.; Minna, J. D. High levels of intracellular bombesin characterize human small cell lung carcinoma. Science 214:1246-1248; 1981. 21. Moreley, J. E.: Levine, A. S. Corticotropin-releasing factor, grooming and ingestive behavior. Life Sci. 31:1459-1464: 1982. 22. Olshowka, J. A.; O'Donohue, T. L.; Mueller, G. P.; Jacobowitz, D.M. The distribution of corticotropin-releasing factor-like immunoreactive neurons in rat brain. Peptides 3:995-1015: 1982. 23. Shaffer, M. M.; Carney, D. N.: Korman, L. Y.; Lebovic, G. S.; Moody, T. W. High affinity binding of VIP to human lung cancer cell lines. Peptides 8:1101-1106; 1987. 24. Shibahara, S.; Morimoto, Y.; Furutani, Y.; et al. The isolation and sequence analysis of the human corticotropin-releasing factor precursor gene. EMBO J. 2:775-780: 1983. 25. Swanson, L. W.: Sawchenko, P. E.; Rivier, J.; Vale, W. W. Organization of ovine corticotropin-releasing factor immunoreactive cells and fibers in the rat brain: An immunohistochemical study. Neuroendocrinology 36:165-186; 1983. 26. Vale, W.; Spiess, J.; Rivier, C.; Rivier, J. Characterization of a 41residue ovine hypothalamic peptide that stimulates secretion ofcorticotropin and beta-endorphin. Science 213:1394-1397; 1981. 27. Vale, W. W.; Rivier, C.; Brown, M. R.; et al. Chemical and biological characterization ofcorticotropin-releasing factor. Recent Prog. Horm. Res. 39:245-270; 1983. 28. Valentino, R. J.; Foote, S. L.; Aston-Jones, G. Corticotropin-releasing factor activates noradrenergic neurons of the locus coeruleus. Brain Res. 270:363-367; 1983. 29. Wynn, P. C.; Hauger, R. L.; Holmes, M. C.; Millan, M. A.; Cart, K. J.; Aguilera, G. Brain and pituitary receptors for corticotropinreleasing factor: Localization and differential regulation after adrenalectomy. Peptides 5:1077-1084; 1984.