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Endocrinology Vol. 143, No. 3 807-813
Copyright © 2002 by The Endocrine Society

CRH-ACTH-POMC-ADRENAL

CRH Inhibits Cell Growth of Human Endometrial Adenocarcinoma Cells via CRH-Receptor 1-Mediated Activation of cAMP-PKA Pathway

Grazia Graziani, Lucio Tentori, Ilaria Portarena, Marcella Barbarino, Giuseppe Tringali, Giacomo Pozzoli and Pierluigi Navarra

Institute of Pharmacology, Catholic University Medical School (G.T., G.P., M.B., P.N.), Rome 00168, Italy; Pharmacology and Medical Oncology Section, Department of Neuroscience, University of Rome "Tor Vergata" (G.G., L.T., I.P.), Rome 00133, Italy

Address all correspondence and requests for reprints to: Prof. Pierluigi Navarra, Institute of Pharmacology, Catholic University Medical School, Largo Francesco Vito 1-00168 Rome, Italy. E-mail: . pnavarra{at}rm.unicatt.it


   Abstract
Top
 Abstract
Introduction
 Materials and Methods
 Results
 Discussion
 References
 
CRH produced by human endometrial cells exerts decidualizing activity via an autocrine mechanism mediated via CRH-R1 receptors. We postulated that such activity exerted by CRH on normal endometrial cells might translate into an antiproliferative action on endometrial-derived malignancies, provided that neoplastic cells maintain the expression of CRH receptors. In this light, here we investigated the possible antiproliferative effects of CRH in an adenocarcinoma cell line derived from human endometrium.

CRH induces time- and concentration-dependent inhibition of Ishikawa cell growth, the maximal effect (50% inhibition) being achieved after 3 d of treatment with 10-7 M CRH. A decrease in telomerase activity, which paralleled tumor growth inhibition, was also observed in CRH-treated samples. The antiproliferative effect was confirmed by colony-formation assay for long-term survival. This effect was counteracted in a concentration-dependent manner by both {alpha}-helical CRH and astressin; the former also showed intrinsic inhibitory activity. These findings suggested the involvement of CRH-R1 receptor subtype; this hypothesis was confirmed by RNase protection analysis showing the expression of human CRH-R1 mRNA. Experiments with the PKA inhibitor 14–22 amide and forskolin, as well as the measurement of intracellular cAMP, suggested the downstream involvement of cAMP-PKA pathway in CRH-induced inhibition of Ishikawa cell growth.


   Introduction
Top
 Abstract
 Introduction
Materials and Methods
 Results
 Discussion
 References
 
IT IS CURRENTLY agreed that CRH produced by placental trophoblast cells plays a crucial role in steroid regulation of the human fetoplacental unit (1); a well established theory also correlates the production of placental CRH with the duration of labor and the timing of parturition (2, 3). Less is known, at this time, about the function of CRH in the female reproductive system under nonpregnant conditions. CRH gene expression and synthesis have been detected in epithelial and stromal cells of the human endometrium (4, 5). In the latter, CRH gene is expressed both in the proliferative and secretory phase of menstrual cycle (5). These authors also showed that the CRH-R1 receptor gene is expressed in normal stromal cells (5). Actually, in in vitro experimental models CRH demonstrated differentiating activity toward human endometrial cells that express R1 receptor subtype; in fact, these cells, under the influence of CRH, undergo decidualization (6). Such process seems to be subsequent to the activation of the cAMP pathway (6, 7). Taken collectively, this evidence indicates that CRH likely contributes to the control of normal endometrial function under physiological conditions.

The gene expression and biosynthesis of CRH have also been demonstrated in a tumor cell line derived from the human endometrium, namely adenocarcinoma Ishikawa cells (4). However, the possible role of this peptide in the control of tumor cell proliferation has not been investigated yet.

Here we postulate that the above-described decidualizing activity exerted by CRH on normal endometrial cells might translate into an antiproliferative action on endometrial-derived malignancies, provided that neoplastic cells maintain the expression of CRH receptors as well as normal signal transduction mechanisms. Within this conceptual framework, in this study we investigated the antiproliferative effects of CRH on Ishikawa cells, including the study of receptor-operated mechanisms and signal transduction pathways.


   Materials and Methods
Top
 Abstract
 Introduction
 Materials and Methods
Results
 Discussion
 References
 
Cell line and culture conditions
The human endometrial adenocarcinoma Ishikawa cell line was kindly provided by Prof. Gigliola Sica (Institute of Histology, Catholic University Medical School, Rome, Italy). Cells were cultured in DMEM (Life Technologies, Inc., Paisley, Scotland, UK) supplemented with 10% FCS (Life Technologies, Inc.), 2 mM L-glutamine, 100 U/ml penicillin, and 100 µg/ml streptomycin (Flow Laboratories, Mc Lean, VA), at 37 C in a 5% CO2 humidified atmosphere.

Drug treatment and cell growth analysis
CRH and {alpha}-helical CRH were purchased from Sigma (St. Louis, MO). The peptide stock solution (10-5 M) was prepared by dissolving both CRH and {alpha}-helical CRH in 0.01 N HCl/0.1% BSA in PBS (vehicle) and stored at -20 C. Astressin (8) was dissolved in 0.01 M HCl/0.1% BSA in distilled water and stored at -20 C. Forskolin and 3-isobutyl-1methylxanthine (IBMX) (Sigma), and the PKA inhibitor 14–22 amide (Calbiochem, La Jolla, CA) were dissolved in distilled water. All peptides and drugs were diluted to working concentrations in incubation medium.

Cells were cultured in flasks (Falcon, Becton Dickinson \|[amp ]\| Co. Labware, Oxnard, CA) (8 x 105 cells/flask). Adherent cells were treated with graded concentration of CRH (10-9-10-7 M) or with drug solvent only. After 24 h, the peptide was added again to cell culture. Cells were then incubated at 37 C for 3 d and growth was evaluated, every 24 h, by counting cells in quadruplicate. Cell viability was determined by trypan blue dye exclusion. When peptide antagonists, PKA inhibitor, or forskolin were used, agents were added to cell culture 20 min before CRH treatment, whereas IBMX was added 30 min before CRH.

Cell proliferation was also evaluated using a Promega Corp. kit, according to the manufacturer’s instructions (CellTiter 96 Aqueous One Solution Cell Proliferation Assay, Promega Corp., Madison, WI). The assay utilizes the novel tetrazolium compound 3-(4,5-dimethylthiazol-2-yl)-5-(3-carboxymethoxyphenyl)2-(4-sulfophenyl)-2H-tetrazolium, inner salt (MTS). Briefly, cells were seeded in 96-well plates (eight wells for each group, 2.5 x 103/well in 0.1 ml). At the end of the incubation period, 20 µl of MTS reagent were added to each well and, 4 h later, plates were read at 490 nm using a Multiscanner microplate reader (Labsystems Oy, Multiscan Bichromatic, Helsinki, Finland).

Long-term cell survival was determined by means of colony-forming assay. Cells (1.6 x 103) were seeded into 10-cm plastic Petri dishes to allow colony formation. After 10 d, untreated and drug-treated colonies were fixed and stained with rhodamine B basic violet 10 (ICN Biomedicals, Inc., Aurora, OH). Survival was calculated as percentage of control.

Evaluation of telomerase activity
The telomeric repeat amplification protocol (TRAP) assay, based on PCR amplification of telomerase extension products, was performed as previously described (9) with minor modifications (10). Extracts were prepared by lysing the cells in ice-cold extraction buffer [0.5% NP-40, 10 mM Tris-HCl (pH 7.5), 1 mM MgCl2, 1 mM EGTA, 0.25 mM sodium deoxycholate, 150 mM NaCl, 10% glycerol, 5 mM ß-mercaptoethanol, 0.1 mM 4-(2-aminoethyl)-benzene-sulfonyl fluoride hydrochloride]. Four microliters of cell extracts, corresponding to 2 x 102 of untreated control, were used for TRAP assay. The telomerase reaction was carried out in 40 µl of the reaction mixture consisting of 20 mM Tris-HCl (pH 8.3), 68 mM KCl, 1.5 mM MgCl2, 1 mM EGTA, 0.05% Tween 20, 0.1 µg of TS primer (5'-AATCCGTCGAGCAGAGTT-3'), 0.1 µM T4 gene 32 protein and 50 µM of each deoxynucleotide triphosphate. Samples were incubated at room temperature for 15 min to allow telomerase to extend TS primer. The reaction was stopped in ice and 2 U of Taq DNA polymerase, 0.16 µl of {alpha}[32P]deoxy-CTP (3,000 Ci/mmol, NEN Life Science Products, Boston, MA) and 0.1 µg of CX oligonucleotide (5'-CCCTTACCCTTACCCTTACCCTAA-3') were added to each single PCR tube. Amplification of the telomeric products was performed by PCR (94 C 30 sec; 50 C 30 sec; 72 C 1 min; 31 cycles). After the TRAP assay, 40 µl of the PCR were separated on a 10% nondenaturing polyacrylamide gel. Subsequently, gels were fixed and exposed to x-ray films (Kodak, Rochester, NY) at -80 C. The signal of the telomeric ladder was quantified by bidimensional densitometry using a Bio-Rad Laboratories, Inc. (Richmond, CA) scanning apparatus (Imaging densitometer, GS-670; Molecular Analyst software), and each value was corrected for the background (i.e. lane relative to lysis buffer).

RNase protection assay
To measure CRH-R1 mRNA expression, a 522-bp fragment of the human CRH-R1 cDNA encoding the N-terminal 174 amino acids was amplified by PCR using the full-length human CRH-R1 as template (11) and the following oligonucleotide primers: sense, 5'-GATCGGATCCATGGGAGGGCACCCGCAGCTC-3'; and antisense, 5'-GATCGAATTCTAGCTGGACCACGAACCAGGT-3'. The PCR-amplified fragment was subcloned into the BamHI/EcoRI sites of pBluescript II SK (+/-) (Stratagene, La Jolla, CA) and sequenced for verification. The plasmid DNA (pGP-1) was linearized with BamHI, and a 605-nucleotide antisense of which 522 nucleotides would be protected by hybridization to the CRH-R1 transcript, was synthesized using T7 RNA polymerase in the presence of [{alpha}-32P]UTP (800 Ci/mmol). Human glyceraldehyde-3-phosphate dehydrogenase (hGAPDH) was used as internal loading control. Linearized plasmid cDNA (pTRI, Ambion, Inc., Austin, TX), that included a 316-bp fragment derived from exons 5–8 of human GAPDH was digested with StyI, and an antisense riboprobe of 244 nucleotides, resulting in a protected fragment of 134 nucleotides, was synthesized with SP6 RNA polymerase and [{alpha}-32P]UTP. RNase protection analyses were performed (12) by hybridizing 25 µg total RNA in 24 µl deionized formamide plus 6 µl hybridization buffer containing 5 x 105 cpm CRH-R1 and 8,000 cpm GAPDH riboprobes. After heating at 80 C for 5 min, the samples were hybridized at 45 C for 15 h and subsequently digested by RNase (200 µg/ml RNase A and 350 U/ml RNase T1) at 24 C for 60 min. The samples were then resolved on 5% polyacrylamide-8 M urea gels and visualized by autoradiography as shown (see Fig. 5Go).



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  		Figure 5. Expression of CRH-R1 mRNA in Ishikawa cells. A representative autoradiogram of RNase protection assay of CRH-R1 mRNA is shown; total RNA isolated from the cells was hybridized with the antisense probe specific to human CRH-R1, hCRH-R1 (5 x 105 cpm), and hGAPDH (8,000 cpm). The protected fragments were resolved on a 5% polyacrilamide 8 M urea gel.

 
Measurement of intracellular cAMP
In experiments for the measurement of cAMP, incubation media were discarded, cells were resuspended in 250 µl of Tris/EDTA buffer (50 mM solution at pH 7.5 containing 4 mM EDTA) and sonicated for 30 sec with a Labsonic sonicator (B. Braun Biotech International, Melsungen, Germany). Subsequently, the samples were heated for 10 min in a boiling water bath to coagulate proteins, and centrifuged. After 15-min centrifugation at 12,000 rpm and 4 C, supernatants were collected and assayed for cAMP using a commercial RIA kit (TRK 432; Amersham Pharmacia Biotech, Little Chalfont, UK).

Producer’s instructions were modified to improve sensitivity of the assay. Briefly, 100 µl of unknown or standard (the latter in the range 0.06–16 pmol/tube) were incubated for 2 h at 4 C with 50 µl of tracer (3H-cAMP) and 100 µl of binding protein. Separation of bound from free cAMP was carried out with 100 µl of charcoal; the tubes were shaken and then centrifuged for 8 min at 4 C. Supernatants were decanted into 10 ml of scintillation fluid with 1 ml of water. Radioactivity was measured by liquid-scintillation counting.

Statistical analysis
Data were expressed as individual values or the means ± 1 SEM of (n) replicates per group. Data were analyzed by ANOVA and post hoc Newman-Keul test for multiple comparisons among group means, or by t test where appropriate, using a Prism computer program (GraphPad Software, Inc., San Diego, CA). Differences were considered statistically significant if P < 0.05.


   Results
Top
 Abstract
 Introduction
 Materials and Methods
 Results
Discussion
 References
 
Growth inhibitory effects of CRH in adenocarcinoma cells
Ishikawa cells were exposed in vitro to concentrations of CRH ranging from 10-10-10-7 M. Cells were then counted at daily intervals during a 72-h period of culture. The results, illustrated in Fig. 1Go, indicate that the peptide was capable of inhibiting cell proliferation, with respect to untreated or vehicle-treated controls, in a time- and dose-dependent manner. CRH growth inhibitory activity was confirmed using MTS, an assay based on the measurement of cell metabolism that is commonly adopted to evaluate cell growth (Fig. 2Go). The antiproliferative effect of CRH was not associated with induction of apoptosis, as indicated by flow cytometry analysis of hypodiploid DNA content (data not shown). The influence of CRH on long-term survival was assessed by colony-formation assay. The results, illustrated in Fig. 2Go, showed that the treatment with 10-7 M CRH markedly reduced the ability of Ishikawa cells to form colonies.



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  		Figure 1. Cell growth analysis of Ishikawa cells treated with CRH. Cells were exposed to graded concentrations of CRH (10-10-10-7 M) or to solvent only (vehicle) and cell growth was evaluated in terms of number of viable cells. Each symbol value represents the mean of cell counts performed in quadruplicate. Bars, SEM. The results are representative of one out of three repeated experiments. Regression line analysis applied to growth inhibition of cells exposed to CRH (10-8-10-7 M) showed high statistically significant differences (P < 0.01) with respect to controls at all time points, whereas growth inhibition induced by 10-9 CRH was significantly different only at d 3 after CRH exposure (P < 0.01).

 


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  		Figure 2. Effects of CRH on cell metabolism evaluated by MTS assay and on long-term survival by colony-forming ability. Cells were treated with 10-7 M CRH and tested on d 3 and 5 using the MTS assay. Results are expressed as percentage of inhibition of the ability of drug treated cells to convert tetrazolium salt to colored formazan with respect to untreated control. For colony-forming ability test, untreated or drug-treated cells were seeded in Petri dishes. After 10 d, colonies were stained and counted. Results are expressed as percent inhibition of the number of colonies formed by CRH-treated cells with respect to untreated controls. The indicated means and the relative SEM (bars) were calculated following angular transformation of the percentages. Values represent the mean of three independent experiments (three plates per group of treatment in each experiment). The number of colonies formed in the presence of CRH was significantly lower (P < 0.01) with respect to untreated controls.

 
We previously demonstrated that suppression of telomerase activity can be considered an indicator of cell growth inhibition induced by anticancer drugs (13, 14, 15). Telomerase is a ribonucleoprotein complex dedicated to the maintenance of telomere length. Its activity is down-regulated in most normal human tissues and is reactivated in the vast majority of cancer cells, making it an almost ubiquitous tumor marker (16). Reduction of telomerase activity, with respect to untreated control, is observed in cell extracts corresponding to equal volumes of cultures that undergo growth inhibition upon drug treatment. In the present study, we adopted this approach to further confirm the antiproliferative effect of CRH. Indeed, the results of TRAP assay, performed at daily intervals up to 72 h of culture, indicated at all time points a reduction of telomerase activity in CRH-treated samples, with respect to untreated controls (Fig. 3Go).



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  		Figure 3. Analysis of residual telomerase activity in Ishikawa cells treated with CRH. Untreated or CRH (10-7 M) treated cells were harvested at 24, 48, and 72 h and equal volumes of total cell extracts were analyzed for telomerase activity by TRAP assay. As negative control, cell extract was replaced with an equal volume of lysis buffer. Densitometric analysis of telomeric ladder revealed a 50% decrease in enzymatic activity at d 1 and 2, whereas on d 3 a 30% reduction was observed.

 
Growth inhibitory effect of CRH is mediated by the activation of CRH-R1 receptor subtype
Experiments were conducted to investigate whether the antiproliferative effect of the peptide was mediated by specific receptors, and to assess which CRH-R subtype was involved. To address these issues we adopted a pharmacological approach based on the use of currently available receptor antagonists. Therefore, Ishikawa cells were treated with a fixed concentration of CRH (10-7 M) in the presence of graded concentrations of {alpha}-helical CRH (10-8-10-6 M). Figure 4AGo shows that {alpha}-helical CRH displayed an intrinsic inhibitory activity, but it was able to counteract the antiproliferative effect of CRH. These results are consistent with a partial agonist profile for {alpha}-helical CRH in this paradigm. Another series of experiments was carried out with the putative full CRH antagonist, astressin. The results indicate that astressin was devoid of any effect on cell growth per se, suggesting that the amounts of CRH produced by the cells (4) in the present experimental conditions are seemingly too low to display intrinsic antiproliferative activity because this would have been unmasked by astressin. The latter, instead, antagonized in a concentration-dependent manner growth inhibition induced by CRH addition to the culture medium (Fig. 4BGo).



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  		Figure 4. The effects of CRH receptor antagonists on CRH-induced inhibition of Ishikawa cell growth. The results are expressed as percentage of growth inhibition of peptide treated tumor cells with respect to untreated control (first column of each group: vehicle vs. control). Empty columns and hatched columns, percentage of inhibition at d 1 and d 2, respectively. {alpha}-Helical CRH antagonizes the growth inhibitory effects of CRH in a dose-dependent manner but displays intrinsic agonist activity (P < 0.01 vs. untreated control) (A), whereas astressin acts as a full antagonist of CRH growth inhibitory effect (B). Histograms represent the means ± SEM of four replicates per group. CRH-treated groups were always significantly different from vehicle treated controls (P < 0.01). * And **, P < 0.05 and P < 0.01 vs. CRH alone, respectively.

 
The finding of partial agonist activity by {alpha}-helical CRH together with full antagonist activity by astressin meet the pharmacological criteria that characterize the CRH-R1 receptor subtype (17, 18), thereby suggesting the involvement of this subtype in mediating the effect of CRH. Thus, to directly demonstrate the presence of this receptor subtype, the expression of CRH-R1 transcript in Ishikawa cells was assessed using RNase protection assay. Figure 5Go shows that indeed these cells express the gene encoding CRH-R1 protein under basal conditions.

CRH inhibition of tumor cell growth is mediated by the cAMP-PKA pathway.
The stimulation of CRH receptors is classically associated to the activation of cAMP-PKA signaling pathway (19). To test whether such mechanism is also involved in CRH-induced inhibition of Ishikawa cell growth, we used a cell-permeable PKA inhibitor, PKI 14–22 amide. This peptide inhibited in a concentration-dependent manner the effect of CRH at both time points tested (Fig. 6AGo). On the other side, forskolin, a direct activator of adenylyl cyclase, tended to synergize with CRH in inhibiting tumor cell growth. This PKA activator also showed a weak, although significant (P < 0.01), inhibitory effect when given alone (Fig. 6BGo).



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  		Figure 6. The involvement of cAMP-PKA pathway in cell growth inhibition induced by CRH. A PKA inhibitor, the cell-permeable peptide 14–22 amide, antagonizes the inhibitory effect of CRH on Ishikawa cell growth (A), whereas the adenylyl cyclase activator forskolin potentiates CRH inhibitory activity (B). Empty columns and solid columns, percentage of inhibition at d 1 and d 2, respectively (first column of each group: vehicle vs. control). Histograms represent the means ± SEM of four replicates per group. CRH-treated groups were always significantly different from vehicle-treated controls (P < 0.01). **, P < 0.01 vs. CRH alone.

 
In addition to pharmacological evidence, we also tested the ability of CRH to increase adenylyl cyclase activity and cAMP production in Ishikawa cells. Figure 7Go shows that CRH induces a concentration-dependent stimulation of cAMP production, which plateaus at 10-8 M. As expected, inhibition of phosphodiesterase activity by 10-4 M IBMX caused an average 3-fold increase in cAMP accumulation compared with controls without IBMX (Fig. 7Go).



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  		Figure 7. CRH increases cAMP production in Ishikawa cells. Symbols represent individual cAMP values, measured in duplicate. The results are representative of one out of three experiments. Experiments were conducted in the absence (empty columns) or in the presence of 10-4 M IBMX (hatched columns).

 

   Discussion
Top
 Abstract
 Introduction
 Materials and Methods
 Results
 Discussion
References
 
In the present study, we demonstrated that CRH is able to inhibit, in a dose- and time-dependent manner, tumor cell growth in a model of human endometrial adenocarcinoma. The antiproliferative effect of CRH is mediated by the activation of CRH-R1 receptor subtype, which is expressed under basal conditions by these tumor cells. We also showed that, in this model system, growth inhibition afforded by CRH appears to be mediated by the activation of cAMP-PKA pathway.

The role of CRH in the control of neoplastic disease has been scarcely investigated and remains controversial at present. The peptide has been shown to exert in vitro antiproliferative activity in a corticotropic murine tumor cell line, AtT-20 cells; this effect is mediated by CRH receptors because it is inhibited by specific antagonists (20, 21). Similarly to normal corticotrophs (12), AtT-20 cells express CRH-R1 receptors, whose stimulation by CRH causes the activation of adenylyl cyclase (21, 22); however, the antiproliferative effect of CRH did not appear to be mediated by this pathway (20). An antitumor effect of CRH was also observed in an intracerebral tumor model of a rat mammary carcinoma, that also expresses CRH-R1 receptors even though at a lesser extent with respect to the normal tissue counterpart (23). Similarly to AtT-20, inhibition of tumor growth induced by CRH was not associated to the activation of adenylyl cyclase; in this case, a mechanism involving the activation of nitric oxide pathway was described (23). These authors also reported that CRH reduces peritumoral brain edema in glioblastoma bearing rats (24). On the other side, CRH was found to stimulate angiogenesis within a human epithelial tumor (25), and the extent of CRH/CRH-R expression correlated with tumor progression in melanoma cells (26). Thus, from the analysis of available evidence, it emerges that CRH does not exert widespread antiproliferative activities, as it is, for example, in the case of another neuropeptide, somatostatin. On the contrary, any effect appears to be restricted to selected tissues and cell types, and to be dependent on the expression of specific receptors. To further address this issue, studies are currently in progress to analyze the antiproliferative effect of CRH in other tumors deriving from tissues expressing CRH receptors. In this regard, preliminary results showed that the peptide induced growth inhibition in a human glioblastoma multiform cell line (our manuscript in preparation).

The putative antitumor effect of CRH was investigated in tissues such as the mammary gland or the skin, where the physiological role of the peptide has not been fully elucidated yet. No data have been provided until now about the possible antiproliferative activity of CRH on the human endometrium, where the peptide exerts a well-established role in the control of various functions, both during pregnancy and under nonpregnant conditions (see Introduction). Here we report, for the first time, an antiproliferative effect of CRH in tumor cells derived from human endometrium. In normal tissue, the constitutive expression of both CRH and its receptor suggests a mechanism according to which CRH is secreted and acts on endometrial cells in an autocrine and paracrine fashion. Taken together, the above mechanism and the findings presented here lead to postulate that the peptide produced by endometrial tumor cells is secreted and acts locally to inhibit cell growth via the activation of the R1 receptor subtype. Indeed, Ishikawa cells produce and secrete detectable amounts of immunoreactive CRH (4). However, the expression of CRH-R in these cells was not reported until now, and this was an important point because the presence of receptor appears to be required for any antiproliferative activity of the peptide. Actually, preliminary pharmacological evidence suggested the presence of the CRH-R subtype. In particular, we confirmed here that {alpha}-helical CRH possesses partial agonist activity because it mimics the effects of CRH on Ishikawa cell growth while antagonizing CRH if the peptides are given together. After the original observation that {alpha}-helical CRH has intrinsic activity in stimulating ACTH release from the pituitary gland (27), the partial agonist activity of this peptide was demonstrated in various experimental paradigms, including cell electrophysiology (28, 29), prostanoid production (30), and neuroprotection (31), although all attempts to show that {alpha}-helical CRH by itself elicits cAMP accumulation in isolated cells in vitro have been unsuccessful (32, 33, 34).

What cascade of events links the activation of CRH-R1 receptors to the inhibition of cell growth? The signaling pathway identified in this study is that classically described for CRH receptors, CRH-R1 in particular, in physiological target tissues, involving the increase in intracellular cAMP production and the subsequent activation of PKA (12, 19). The latter event did not cause apoptosis in our model system. In the absence of cytotoxic phenomena, it might be postulated that CRH influences cell cycle by promoting differentiation in Ishikawa cells. This would be quite an obvious explanation because it envisages an extension of the physiological role of CRH in normal endometrium, which is specifically mediated by the activation of adenylyl cyclase (6, 7, 35).

In conclusion, here we have demonstrated a clear antiproliferative effect of CRH on Ishikawa tumor cells. Although the latter represents a well-established model system (36), the relevance of the present findings in human pathology needs to be verified with further preclinical investigation.


   Acknowledgments
 
We thank Prof. W. Vale and J. Rivier (Clayton Foundation Laboratories for Peptide Biology, Salk Institute, La Jolla, CA) for generously providing astressin.


   Footnotes
 
This study was supported by Fondi di Ateneo (2000–2001) (to P.N.), and in part by a grant from the Italian Association for Cancer Research. M.B. was supported by Sigma Tau Pharmaceutical Industries (Pomezia, Italy).

Abbreviations: CRH-R1, CRH receptor 1; GAPDH, glyceraldehyde-3-phosphate dehydrogenase; hGAPDH, human GAPDH; IBMX, 3-isobutyl-1methylxanthine; MTS, 3-(4,5-dimethylthiazol-2-yl)-5-(3-carboxymethoxyphenyl)2-(4-sulfophenyl)-2H-tetrazolium, inner salt; TRAP, telomeric repeat amplification protocol.

Received September 26, 2001.

Accepted for publication November 15, 2001.


   References
Top
 Abstract
 Introduction
 Materials and Methods
 Results
 Discussion
 References
 

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