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Original Studies |
Endocrine, Polypeptide, and Cancer Institute, Veterans Affairs Medical Center (I.C., A.V.S., J.L.V., K.G., P.A., R.B., G.H.), and Section of Experimental Medicine, Department of Medicine, Tulane University School of Medicine (I.C., A.V.S., J.L.V., R.B., G.H.), New Orleans, Louisiana 70112
Address all correspondence and requests for reprints to: Dr. Andrew V. Schally, Endocrine, Polypeptide, and Cancer Institute, Veteran Affairs Medical Center, 1601 Perdido Street, New Orleans, Louisiana 70112-1262.
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Abstract |
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Introduction |
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Insulin-like growth factors I and II (IGF-I and -II) stimulate cell growth in multiple tissues and in cancer cell lines, including human epithelial ovarian cancers (2, 3, 4, 5, 6, 7). Tumor cell growth in vivo may be enhanced by IGFs derived from serum or tumor stroma (5). In addition, tumor cells with functional IGF receptors may be able to enhance their own growth by synthesis of endogenous autocrine IGFs (8, 9). Much evidence supports the importance of the IGF system in the regulation of ovarian function and its implication in follicular development (10). IGFs bind to IGF type I and II receptors and also cross-react with human insulin receptor (5). The biological effects of IGF-I and IGF-II are primarily mediated by the IGF receptor type I, a transmembrane heterotetramer with tyrosine kinase activity (5, 9). The function of the mannose 6-phosphate/IGF-II receptor (M6P/IGFR-IIr) is less clear. This receptor exists as a single transmembrane chain with a small intracellular domain and does not possess tyrosine kinase activity, but plays a role in intracellular protein trafficking and the degradation of transforming growth factor-ß and IGF-II (5, 9). It has a much higher affinity for IGF-II than IGF-I and a negligible affinity for insulin. Recent evidence indicates that the M6P/IGFR-IIr gene may function as a tumor suppressor gene in liver, breast, and probably other tissues (11, 12, 13, 14).
GHRH antagonists inhibit the growth of various IGF- dependent cancers, such as prostatic, breast, renal, and lung carcinomas, and osteosarcomas (15, 16, 17, 18, 19, 20, 21). The antiproliferative action of GHRH antagonists can be ascribed in part to the suppression of GH release from the pituitary and the consequent decrease in the production of IGF-I in the liver (15). In contrast to IGF-I, serum concentrations of IGF-II are much less dependent on GH (15). IGF-II is synthesized by a wide variety of tissues, such as kidney, liver, and muscles (15). Various studies indicate that the autocrine/paracrine expression of IGF-II by tumor cells plays an important role in their proliferation (8, 9). A significant reduction in concentrations of IGF-I and/or IGF-II levels and messenger ribonucleic acid (mRNA) expression for IGF-II in osteosarcomas; renal cell carcinomas; nonsmall cell lung carcinomas, prostatic, mammary, colorectal, and pancreatic cancers; and brain tumors after treatment of nude mice with GHRH antagonists suggests a direct action of GHRH antagonists on the tumors (15, 16, 17, 18, 19, 20, 21). Recently, using a labeled antagonistic analog of GHRH, [125I]JV-1-42, as a new radioligand (22), we were able to detect high affinity, low capacity binding sites on MiaPaCa-2 human pancreatic, LNCaP prostatic, and CAKI-I renal cancer cells (22, 23). In addition, by RT-PCR we detected new splice variants of GHRH receptor mRNA in six different human cancer cell lines, including OV-1063 ovarian cancer cells (22, 23).
Somatostatin analogs inhibit the release of GH from the pituitary, which produces a decrease in hepatic IGF-I secretion. In addition, somatostatin analogs can interact directly with the somatostatin receptors on the membrane of the cancer cells. RC-160, a potent somatostatin analog synthesized in our laboratory, inhibits the growth of pancreatic, gastric, mammary, prostatic, small cell and nonsmall cell lung carcinomas, and brain tumors in experimental animals (24, 25, 26, 27, 28, 29, 30).
In this study we compared the antineoplastic effects of the GHRH antagonists MZ-5-156 and JV-1-36 and the somatostatin analog RC-160 in nude mice bearing xenografts of the human epithelial ovarian cancer cell line OV-1063. The effects of the treatment on mRNA for IGF-I and IGF-II and on IGF receptor types I and II in the tumors and serum levels of GH, IGF-I, and IGF-II were studied. The presence of binding sites and the expression of mRNA for GHRH receptors were also investigated.
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Materials and Methods |
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TopAbstractIntroductionMaterials and MethodsResultsDiscussionReferences |
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The somatostatin analog RC-160 (D-Phe-Cys-Tyr-D-Trp-Lys-Val-Cys-Trp-NH2) was made by classical synthesis by Novabiochem (Laufelfingen, Switzerland) and was supplied by Debiopharm (Lausanne, Switzerland). For daily injections, RC-160 was dissolved in 0.1% dimethyl sulfoxide in saline solution. Human (h) GHRH-(1–29)NH2 and GHRH antagonists MZ-5–156 ([PhAc-Tyr1,D-Arg2,Phe(4-Cl)6,Abu15,Nle27]hGHRH-(1–28) Agm) and JV-1–36 ([PhAc-Tyr1,D-Arg2,Phe(4-Cl)6,Arg9,Abu15,Nle27,D-Arg28,Har29]- hGHRH-(1–29)NH2) were synthesized by solid phase methods as previously described (31, 32). For daily injections, peptides were dissolved in 0.1% dimethylsulfoxide in sterile aqueous 10% propylene glycol (vehicle solution).
Animals
Six-week-old female athymic NCR/c (nu/nu) nude mice were purchased from the Frederick Cancer Research Facility of the NCI (Frederick, MD), housed in a laminar airflow cabinet under pathogen-free conditions with a 12-h light/12-h dark schedule, and fed autoclaved standard chow and water ad libitum. Their care was in accord with institutional guidelines.
Cell line
The human epithelial ovarian cancer cell line OV-1063 originated from a metastatic papillary cystadenocarcinoma of the ovary in a 57-yr-old woman and was obtained from American Type Culture Collection (Manassas, VA) (33). It was maintained in RPMI 1640 medium supplemented with 10% FBS, vitamins, antibiotics, and antimycotics as described previously (34). Cells were cultured in Costar T-75 flasks (Cambridge, MA) in a humidified atmosphere of 5% CO2 and 95% air at 37 C and passaged every 4–6 days using 0.25% trypsin-ethylenediamine tetraacetate (35). Complementary DNA (cDNA) from the LNCaP human prostate cancer cell line was used as a positive control (23).
In vivo studies
Xenografts of OV-1063 cells were initiated by sc injection of 107 cells into the right flanks of three female nude mice. The OV-1063 tumors resulting after 4 weeks were aseptically dissected and mechanically minced; 1-mm3 tumor pieces were transplanted sc by trocar needle into the right flanks of the mice. Three weeks after transplantation, when tumors reached a volume of approximately 60–70 mm3, mice were randomly divided into four experimental groups of six or seven animals each and received the following treatment as sc injections: group 1(control), vehicle solution; group 2, somatostatin analog RC-160 at a dose of 60 µg/day·animal; group 3, GHRH antagonist MZ-5-156 at a dose of 20 µg/day·animal; and group 4, GHRH antagonist JV-1-36 at a dose of 20 µg/day·animal. The treatment was continued for 25 days. Tumors were measured twice a week with microcalipers, and the tumor volume was calculated as length x width x height x 0.5236 (36). At the end of the experiment, mice were anesthetized with methoxyflurane (Metofane, Pitman-Moore, Mundelein, IL) and killed by decapitation, and trunk blood was collected. The serum was separated and analyzed by RIA. Body weights were recorded, and various organs were removed and weighed. Tumors were dissected, cleaned, and weighed, and samples were stored at -70 C for molecular biology analysis.
RIAs for GH, IGF-I, and IGF-II
Serum GH was determined using materials provided by Dr. A. F. Parlow (NIDDK’s National Hormone and Pituitary Program, Torrance, CA): mouse GH 10783B for standard iodination and antirat GH-RIA-5/AFP-411S. The methods used for determination of IGF-I and IGF-II levels in serum after acid-ethanol cryoprecipitation were described previously (16, 17, 20, 37).
RNA extraction
Total RNA was extracted from human OV-1063 tumors and LNCaP human prostate cancer cells using RNAzol B (Tel-Test, Friendswood, TX) according to the manufacturer’s instructions (23). Polyadenylated [poly(A)+] RNA was also purified from total RNA using oligo(deoxythymidine)-cellulose (MicroPolyAPure mRNA isolation kit, Ambion, Inc., Austin, TX).The RNA pellets were suspended in 50 µL 10 mmol/L Tris/1 mmol/L ethylenediamine tetraacetate buffer (pH 8.0) and quantified spectrophotometrically at 260 nm. The optical density ratios (260 nm/280 nm) of the RNA preparations were greater than 1.8.
RT-PCR
One microgram of total RNA (for hGAPDH, IGF-I, IGF-II, and IGF receptors I and II) or poly(A)+ RNA (for GHRH receptor) was used in a test tube containing 10 mmol/L Tris-HCl (pH 8.3), 50 mmol/L KCl, 5 mmol/L MgCl2, 1 mmol/L of each deoxynucleoside triphosphate, 1 U ribonuclease inhibitor, and 2.5 µmol/L random hexamer primers (for hGAPDH, IGF-I, IGF-II, and IGF receptors I and II) or 2.5 mmol/L oligo(deoxythymidine) (GHRH receptor) in a final volume of 19 µL ribonuclease-free deionized distilled water. The mixture was heated for 10 min at 65 C and quenched on ice, then 2.5 U Moloney murine leukemia virus reverse transcriptase (Perkin-Elmer Corp., Norwalk, CT) in 1 µL were added for a total reaction volume of 20 µL. The mixture was incubated at room temperature for 10 min and then at 42 C for 1 h. The reaction was ended by heating at 95 C for 5 min and quenching on ice. The PCR amplification of the cDNAs for human glyceraldehyde-3-phosphate dehydrogenase (hGAPDH), IGF-I, IGF-II, IGF receptors I and II, and GHRH receptors was performed as follows. One to 5 µL cDNA were amplified in a 50-µL solution containing 10 mmol/L Tris-HCI (pH 8.3), 50 mmol/L KCI, 1.7 mmol/L MgCl2 (for hGAPDH, IGF-I, IGF-II, and IGF receptors I and II) or 2 mmol/L MgCl2 (for GHRH receptor), 200 µmol/L of each deoxy-NTP, 2.5 U Taq DNA polymerase, and 0.4 µmol/L of each primer. The primers used were 5'-TCCTCTGACTTCAACAGCGACACC-3' and 5'-TCTCTCTTCCTCTTGTGCTCTTGG-3' for hGAPDH (38), 5'-AAATCAGCAGTCTTCCAACC-3' and 5'-CTTCTGGGTCTTGGGCATGT-3' for IGF-I (10), 5'-AGTCGATGCTGGTGCTTCTCACCTTCTTGGC-3' and 5'-TGCGGCAGTTTTGCTCACTTCCGATTGCTGG-3' for IGF-II (17), 5'-AACCACGAGGCTGAGAAGCT-3' and 5'-CAGCATAATCACCAACCCTC-3' for IGF-I receptor (10), 5'-TCA ACA- TCT GTGGAAGTGTG-3' and 5'-GAATAGAGAAGTGTCCGGATCGGA- GTC-3' for IGF-II receptor (12), and 5'-CGCCACCATGACCAACTTCAGC-3' and 5'-CACGTGCCAGTGAAGAGCACGG-3' for GHRH receptor (23). PCR consisted of 1 cycle at 95 C for 3 min, 58 C for 1 min, and 72 C for 1 min and subsequently 26 (hGAPDH), 30 (IGF-II, IGF receptors I and II), or 35 (IGF-I) cycles of 95 C for 35 s, 58 C for 40 s, and 72 C for 40 s or 35 cycles of 95 C for 30 s, 60 C for 30 s, and 72 C for 45 s for GHRH receptor using a Robocycler 40 system (Stratagene, La Jolla, CA). For the multiplex PCRs, the cDNA for each target gene was amplified simultaneously with the cDNA for hGAPDH after supplementation of the primers for hGAPDH at the appropriate cycle at 95 C. For the detection of IGF-I, after the first round of PCR, 1 µL PCR product was subjected to a second round of PCR consisting of 28 cycles. All other amplification parameters for the second round of PCR amplification were similar to those described above for the first round of PCR amplification. The number of cycles was determined in preliminary experiments to be within the exponential range of PCR amplification. Aliquots of PCR-amplified product were resolved by electrophoresis on a 1.8% agarose gel, stained with ethidium bromide, and visualized under UV light. For quantitation of PCR-amplified product a scanning densitometer (model GS-700, Bio-Rad Laboratories, Inc., Richmond, CA) was used, coupled with Bio-Rad Laboratories, Inc., personal computer analysis software. Each RT-PCR experiment was repeated at least 3 times, and similar results were obtained. The levels of expression of mRNA for IGF-II, IGF-I, and IGF-II receptors for each sample tested were normalized vs. the corresponding mRNA levels of hGAPDH.
In vitro studies
The effects of GHRH, IGF-I, IGF-II, RC-160, and JV-1-36 on the growth of OV-1063 cells cultured in vitro were evaluated by crystal violet assay as described previously with a few modifications (39). Cells were seeded into 96-well microplates (Falcon, Becton Dickinson and Co., Lincoln Park, NJ). After 24 h, the culture medium was removed and replaced with RPMI 1640 containing 2% FBS (serum reduced) and IGF-I or IGF-II in concentrations of 5–25 ng/mL or 10-7–10-5 mol/L RC-160, hGHRH-(1–29)NH2, and JV-1-36. Controls received medium only. After an incubation period of 69–73 h, the medium was removed, and 1% glutaraldehyde was added for 15 min to fix the cells. The glutaraldehyde was removed, replaced with PBS, and kept at 4 C until staining. The PBS was then decanted from all plates, replaced with an aqueous solution of crystal violet (0.02%; Sigma, St. Louis, MO), and incubated for 30 min. The plates were decanted and washed to remove noninternalized stain, then the crystal violet was extracted with 70% ethanol. The OD at 600 nm of each well was measured using a Beckman Coulter, Inc. (Palo Alto, CA), plate reader. The %T/C was calculated, where T is the optical density (OD600nm) of treated cultures, and C is the OD600nm of control cultures x 100. The data presented are expressed as the mean ± SE of six to eight replicate experiments. Each experiment was repeated three or four times, and similar results were obtained.
GHRH receptor binding studies
Receptors for GHRH on OV-1063 tumors from the control group were characterized by radioligand competition assay. Preparation of tumor membrane fractions and receptor binding studies of GHRH were performed as reported in detail previously (22, 40).
Statistical analyses
Data are expressed as the mean ± SE. Statistical analyses were performed using Duncan’s new multiple range test and Student’s two-tailed t test. All P values are based on two-sided hypothesis testing (41).
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Results |
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After 25 days of treatment, the OV-1063 tumor volumes in the
groups receiving GHRH antagonists MZ-5-156 and JV-1-36 were 928.0
± 234.2 and 647.1 ± 185.2 mm3,
respectively, corresponding to decreases of 58.3% (P
< 0.05) and 70.9% (P < 0.01), respectively, compared
with the control value of 2223.6 ± 374.7
mm3 (Table 1
and
Fig. 1). The volume of tumors in the
group treated with the somatostatin analog RC-160 was also decreased by
60.6% to 875.5 ± 153.4 mm3
(P < 0.01 vs. the controls) 25 days after
the initiation of the therapy (Table 1 and Fig. 1). The reduction in
final tumor weight was 70.1% (P < 0.01) in the group
receiving MZ-5–156, 73.9% (P < 0.01) in nude mice
given JV-1-36, and 60.4% (P < 0.01) in the group
injected with RC-160 compared with the control group (Table 1).
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Effects of GHRH antagonists MZ-5-156 and JV-1-36 and somatostatin analog RC-160 on GH, IGF-I, and IGF-II levels in serum of OV-1063 tumor-bearing mice
As shown in Table 2, serum levels of
GH were significantly (P < 0.05) reduced to 4.3
± 0.9 ng/mL in the group treated with somatostatin analog RC-160 and
to 3.6 ± 0.5 and 3.2 ± 0.3 ng/mL, respectively, in the
groups injected with MZ-5-156 and JV-1-36 compared with the controls
(7.3 ± 1.69 ng/mL). The serum levels of IGF-I and IGF-II are
shown in Table 2. There were no significant differences in serum levels
of IGF-II between the mice receiving the peptide analogs and the
controls. However, administration of RC-160 significantly reduced the
serum level of IGF-I to 87.9 ± 5.7 ng/mL (P <
0.05) compared with the controls (110.4 ± 6.0 ng/mL). Serum
levels of IGF-I were not affected significantly by treatment with
JV-1-36 and MZ-5-156.
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The expression of mRNA for IGF-I, IGF-II, and IGF receptors I and
II in OV-1063 tumors was analyzed by RT coupled with multiplex PCR
analysis in three randomly selected tumors from each group. Using
specific primers for human IGF-I, we could not find the expression of
IGF-I mRNA in OV-1063 tumors. Preliminary experiments, in which the
target genes were coamplified with hGAPDH over a range of cycles,
indicated the number of cycles in which the PCR is in the
dose-dependent phase (Fig. 2). In
contrast, we detected high levels of mRNA for IGF-II in OV-1063 tumors
(Fig. 3). Multiplex RT-PCR analysis and
densitometric quantification revealed that mRNA levels for IGF-II in
tumors from mice treated with MZ-5-156 and JV-1-36 were decreased by
22% (not significant) and 52% (P < 0.05),
respectively, compared with those in tumors from untreated mice. A
slight and insignificant decrease of 8% was also found in mRNA levels
of IGF-II after treatment with RC-160 (Fig. 3).
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). Levels of mRNA for IGF-I receptor
were increased by 38%, 36%, and 25% in the groups treated with
RC-160, JV-1-36, and MZ-5-156, respectively, compared with the control
value, but none of these differences was statistically significant.
Densitometric analysis of RT-PCR products also revealed that mRNA
levels for IGF-II receptor were significantly increased by 60%
(P = 0.05) in the group treated with MZ-5-156 compared
with the control value. Slight and nonsignificant increases of 19% and
42% were also found in the levels of mRNA for IGF-II receptor in the
tumors of mice receiving JV-1-36 and RC-160, respectively.
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Using complete displacement analyses with 125I-labeled [His1,Nle27]hGHRH-(1–32)NH2 as radioligand (40), we could not detect the presence of pituitary-type GHRH receptors in OV-1063 ovarian cancer samples. However, ligand competition assays using the 125I-labeled GHRH antagonist JV-1-42 as radioligand (22) revealed the presence of high affinity (Kd = 0.78 ± 0.11 nmol/L) binding sites for GHRH, with a maximal binding capacity of 50.5 ± 9.89 fmol/mg protein in the membrane fraction of OV-1063 tumors from the control group.
The expression of mRNA for GHRH receptor was evaluated by RT-PCR. Using
gene-specific primers for amplifying cDNAs for splice variant 1
(23) of the GHRH receptor, we detected a product of the
expected size of 147 bp in both OV-1063 tumors and the LNCaP prostatic
cancer cell line (Fig. 5). This PCR
product corresponded to a fragment of nucleotide sequence 699–845 of
the splice variant 1 for GHRH receptors (23). This
sequence is also found in cDNA of human pituitary GHRH receptors, but
the first 334 nucleotides of splice variant 1 are completely different
from those in pituitary (23).
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As shown in Fig 6, IGF-I at a
concentration of 25 ng/mL produced an 11% increase in the
proliferation of OV-1063 cells compared with the control value
(P < 0.05). Exposure of OV-1063 cells cultured
in vitro to IGF-II at concentrations of 5, 15, and 25 ng/mL
significantly stimulated cell proliferation by 7% (P
< 0.05), 14% (P < 0.001), and 12%
(P < 0.001), respectively.
hGHRH-(1–29)NH2 at
10-7–10-5
mol/L also stimulated the proliferation of OV-1063 cells cultured
in vitro by 10–30% (P < 0.05) compared
with that of cells exposed to medium alone (Fig. 7). The GHRH antagonist JV-1-36 at a
concentration of 10-5
mol/L inhibited the growth of OV-1063 cells in vitro by 98%
(P < 0.001; Fig. 7), whereas at a concentration of
10-6 or
10-7 mol/L it had
virtually no effect on proliferation. RC-160 did not affect OV-1063
cell growth at concentrations of
10-7–10-5
mol/L.
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Discussion |
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TopAbstractIntroductionMaterials and MethodsResultsDiscussionReferences |
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The findings of this study demonstrate that GHRH antagonists such as MZ-5-156 and JV-1-36 and the somatostatin analog RC-160 can inhibit the growth of human ovarian epithelial carcinoma cell line, OV-1063, xenografted into nude mice. The mechanism of action of the GHRH antagonists is not fully clarified (15). The present in vivo and in vitro studies shed additional light on the tumor inhibitory effects of these peptides in ovarian cancer. Previous studies showed that both GHRH antagonists and somatostatin analogs exert their antitumor action in part by an indirect mechanism that involves the suppression of the GH release from the pituitary and subsequently the inhibition of the hepatic production of IGF-I (15, 28, 29). The IGF system plays an important role in the growth of ovarian tumors, and thus, inhibition of the IGF axis has been proposed for the treatment of ovarian cancers (4, 7, 42). In the present study exposure of OV-1063 cells cultured in vitro to IGF-I at 25 ng/mL resulted in a statistically significant (P < 0.05) stimulation of cell proliferation. Thus, the strong antitumor action of RC-160, and to a lesser extent that of MZ-5-156 and JV-1-36, could be attributed at least partially to inhibition of the GH-IGF axis. A significant decrease in serum levels of GH was noted in all of the treated groups, but surprisingly and in contrast with RC-160, no significant reduction in the levels of IGF-I in serum could be observed after treatment with MZ-5-156 and JV-1-36. However, this observation should be interpreted with caution, because the IGF-I levels observed could have been affected by the time of blood sampling, and the variation in IGF-I levels in serum was relatively high (43).
In addition to the indirect mechanism, the antagonistic analogs of GHRH can inhibit the proliferation of tumor cells by interfering with the autocrine/paracrine production of IGF-I and IGF-II or by blocking the effects of locally produced GHRH (15, 44, 45, 46, 47). In the case of OV-1063 human ovarian epithelial cell carcinoma, the antitumor effects of the GHRH antagonists MZ-5-156 and JV-1-36 appear to be exerted mainly by a direct action. The expression of the GHRH gene by OV-1063 tumor xenografts and human ovarian cancer specimens has recently been demonstrated by Kahan et al. (45). In our experiment, hGHRH-(1–29)NH2 significantly stimulated the growth of OV-1063 cells in vitro by 10–30% (P < 0.05). Thus, it is possible that locally produced GHRH acts as an autocrine growth factor in this ovarian cancer, and that GHRH antagonists exert their antiproliferative effect by blocking the action of GHRH on tumor cells. This situation would be similar to that recently found in the case of small cell lung carcinomas (46, 47). Exposure of OV-1063 cells cultured in vitro to JV-1-36 at 10-5 mol/L nearly completely inhibited cell proliferation (98% inhibition; P < 0.001). A nonspecific toxic effect of JV-1-36 at this concentration is not likely, as the chemically related hGHRH-(1–29)NH2 at equimolar concentration stimulated cell proliferation. In addition, no evidence of toxicity was found in any of the models tested using JV-1-36 and other GHRH antagonists with similar structures. IGF-II is produced by OV-1063 cells in vitro and in vivo and acts by an autocrine mechanism that requires the interaction with IGF receptors type I and probably type II, both of which are expressed by OV-1063 cells (44). Exposure of OV-1063 cells cultured in vitro to IGF-II at 5–25 ng/mL resulted in a significant stimulation of cell proliferation, confirming that this cell line depends on IGF-II production. This stimulatory effect of IGF-II on the proliferation of OV-1063 cells was probably underestimated because of the endogenous production and secretion of IGF-II (44). RT-PCR analysis showed that tumors of mice treated with GHRH antagonists had reduced levels of mRNA for IGF-II compared with the controls. Therefore, suppression of the autocrine production of IGF-II may have contributed to the tumor inhibition observed in these animals.
It cannot be excluded that the antitumor action of GHRH antagonists is due in part to an indirect mechanism involving suppression of the GHRH/GH/IGF-I axis. However, the findings that GHRH stimulates, whereas GHRH antagonist JV-1-36 strongly inhibits, the growth of OV-1063 cells cultured in vitro emphasize the importance of the direct action of GHRH antagonists on OV-1063 cells. This would be in agreement with recent findings that GHRH stimulates the release of cAMP in OV-1063 ovarian carcinoma and other cell lines in vitro (48, 49). A strong suppression of IGF-II mRNA expression in OV-1063 cancers after treatment with JV-1-36 also points to a likely direct effect of GHRH antagonist on tumors. Using the radiolabeled GHRH antagonist JV-1-42, the presence of high affinity, low capacity binding sites for GHRH was detected in the membrane fraction of OV-1063 tumors. Previous investigations in our laboratory of the receptors for GHRH antagonists in human tumors indicated that they are different from the pituitary GHRH receptors (15, 22, 23). Our recent work demonstrated the presence of distinct binding sites for GHRH analogs and the expression of splice variants of GHRH receptor mRNA in several human cancer cell lines, including OV-1063 human ovarian cancer cells (22, 23). These splice variants of GHRH receptors may mediate the antiproliferative actions of GHRH antagonists (22, 23).
These tumoral GHRH receptors are distinct from the pituitary-type GHRH receptors or receptors for the other peptides of the secretin/glucagon family (22, 23). Our studies showed that all the splice variants of the GHRH receptor have a retained intronic sequence at their 5'-end and lack the first three exons (22, 23). We assume that the lack of the first three exons observed in splice variant 1 of tumoral GHRH receptors (22, 23) could result in a truncation of the N- terminal extracellular domain of the GHRH receptor isoform, which may cause the observed differences compared with the pituitary-type GHRH receptor.
In the present study we detected the mRNA expression for GHRH receptors in OV-1063 tumors grown in nude mice. However, we found only the expression of splice variant 1 for GHRH receptors, but not the other splice variants previously demonstrated by us in various human cancer models (22, 23). This could be due to the fact that the expression of mRNA for GHRH receptors in OV-1063 tumors was weaker than that in LNCaP prostate cancer. The major part of the cDNA sequence (nucleotides 77–1383) of splice variant 1 shows more than 99% identity with the corresponding sequence of pituitary GHRH receptor cDNA, but the first 334 nucleotides of splice variant 1 are completely different.
In addition to the potential binding of the GHRH antagonists to the proteins encoded by these splice variants of the GHRH receptor mRNA, an additional possibility exists as an explanation for the direct effects of these peptide analogs on OV-1063 and other tumors. GHRH is a member of the family of peptides that includes glucagon, secretin, vasoactive intestinal peptide (VIP), gastric inhibitory peptide, and pituitary adenylate cyclase-activating peptide (17). These peptides present significant amino acid sequence homology. The human and rat GHRH receptors are homologous to secretin and VIP receptor proteins (17). GHRH antagonists suppress not only the stimulatory effect of GHRH, but also that of VIP on the cAMP production of various cancer cells (48, 49).
The levels of mRNA for IGF-I receptor were not significantly affected by the GHRH antagonists, JV-1-36 and MZ-5-156, or the somatostatin analog RC-160, but an increase in mRNA levels for IGF-II receptor was observed in all treated groups; the greatest elevation was produced by MZ-5-156. IGF-II receptor functions in the degradation of IGF-II; the activation of transforming growth factor-ß, which is a potent growth inhibitor for most cell types; and the intracellular trafficking of lysosomal enzymes (11, 14). The expression of IGF-II receptor is frequently reduced in hepatocellular carcinomas, and a high incidence of loss of heterozygosity at this site was observed in liver and breast tumors (11, 12, 13, 14). These observations led to the suggestion that the IGF-II receptor gene may function as a tumor suppressor gene (11, 12, 13, 14). Within this context, the observation that the antitumor effects of the GHRH antagonists and the somatostatin analog were accompanied by an induction of mRNA for the IGF-II receptor is not unexpected. It is interesting that although both GHRH antagonists significantly inhibited tumor growth, they produced different effects on mRNA expression. JV-1-36 had a greater inhibitory effect on IGF-II mRNA levels, whereas MZ-5-156 significantly stimulated mRNA for IGF receptor II. Both peptide analogs are specific GHRH antagonists on the pituitary. However, several structurally homologous receptors, such as those for VIP-like peptides, pituitary adenylate cyclase-activating peptide, gastric inhibitory peptide, or even for some members of this family of receptors not yet identified, are probably expressed on cancer cells. Considering that GHRH antagonists may cross-react with various receptors on tumors for which they possess distinct affinities, diverse responses of the cancer cells after exposure to different antagonists cannot be excluded. Although the results were statistically significant, these findings should be interpreted with caution because a limited number of samples was tested.
Antiproliferative action directly on the cancer cells has also been proposed for the analogs of somatostatin, after interaction with specific receptors on the cell membrane (15, 29). It appears that in the case of OV-1063 cells, this mechanism is less important than the indirect action that involves suppression of the GH/IGF-I axis, because RC-160 failed to inhibit the growth of OV-1063 cells cultured in vitro.
In conclusion, our findings demonstrate that GHRH antagonists such as MZ-5-156 and JV-1-36 and the somatostatin analog RC-160 suppress the growth of OV-1063 human ovarian epithelial cell carcinoma. Our work suggests the merit of further investigation of these analogs for the possible treatment of ovarian carcinomas.
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Acknowledgments |
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Footnotes |
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Received November 9, 1999.
Revised May 31, 2000.
Revised January 18, 2001.
Accepted January 26, 2001.
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A. Havt, A. V. Schally, G. Halmos, J. L. Varga, G. L. Toller, J. E. Horvath, K. Szepeshazi, F. Koster, K. Kovitz, K. Groot, et al. The expression of the pituitary growth hormone-releasing hormone receptor and its splice variants in normal and neoplastic human tissues PNAS, November 29, 2005; 102(48): 17424 - 17429. [Abstract] [Full Text] [PDF]
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L. B. Jaeger, W. A. Banks, J. L. Varga, and A. V. Schally Antagonists of growth hormone-releasing hormone cross the blood-brain barrier: A potential applicability to treatment of brain tumors PNAS, August 30, 2005; 102(35): 12495 - 12500. [Abstract] [Full Text] [PDF]
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G. Keller, A. V. Schally, K. Groot, G. L. Toller, A. Havt, F. Koster, P. Armatis, G. Halmos, M. Zarandi, J. L. Varga, et al. Effective treatment of experimental human non-Hodgkin's lymphomas with antagonists of growth hormone-releasing hormone PNAS, July 26, 2005; 102(30): 10628 - 10633. [Abstract] [Full Text] [PDF]
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 J. B. Engel, G. Keller, A. V. Schally, G. L. Toller, K. Groot, A. Havt, P. Armatis, M. Zarandi, J. L. Varga, and G. Halmos Inhibition of Growth of Experimental Human Endometrial Cancer by an Antagonist of Growth Hormone-Releasing Hormone J. Clin. Endocrinol. Metab., June 1, 2005; 90(6): 3614 - 3621. [Abstract] [Full Text] [PDF]
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Z. Rekasi, T. Czompoly, A. V. Schally, F. Boldizsar, J. L. Varga, M. Zarandi, T. Berki, R. A. Horvath, and P. Nemeth Antagonist of growth hormone-releasing hormone induces apoptosis in LNCaP human prostate cancer cells through a Ca2+-dependent pathway PNAS, March 1, 2005; 102(9): 3435 - 3440. [Abstract] [Full Text] [PDF]
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 Z. Szereday, A. V. Schally, J. L. Varga, C. A. Kanashiro, F. Hebert, P. Armatis, K. Groot, K. Szepeshazi, G. Halmos, and R. Busto Antagonists of Growth Hormone-Releasing Hormone Inhibit the Proliferation of Experimental Non-Small Cell Lung Carcinoma Cancer Res., November 15, 2003; 63(22): 7913 - 7919. [Abstract] [Full Text] [PDF]
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H. Kiaris, I. Chatzistamou, A. V. Schally, G. Halmos, J. L. Varga, H. Koutselini, and A. Kalofoutis Ligand-dependent and -independent effects of splice variant 1 of growth hormone-releasing hormone receptor PNAS, August 5, 2003; 100(16): 9512 - 9517. [Abstract] [Full Text] [PDF]
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G. Halmos, A. V. Schally, T. Czompoly, M. Krupa, J. L. Varga, and Z. Rekasi Expression of Growth Hormone-Releasing Hormone and Its Receptor Splice Variants in Human Prostate Cancer J. Clin. Endocrinol. Metab., October 1, 2002; 87(10): 4707 - 4714. [Abstract] [Full Text] [PDF]
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R. Busto, A. V. Schally, J. L. Varga, M. O. Garcia-Fernandez, K. Groot, P. Armatis, and K. Szepeshazi The expression of growth hormone-releasing hormone (GHRH) and splice variants of its receptor in human gastroenteropancreatic carcinomas PNAS, September 3, 2002; 99(18): 11866 - 11871. [Abstract] [Full Text] [PDF]
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H. Kiaris, A. V. Schally, R. Busto, G. Halmos, S. Artavanis-Tsakonas, and J. L. Varga Expression of a splice variant of the receptor for GHRH in 3T3 fibroblasts activates cell proliferation responses to GHRH analogs PNAS, January 1, 2002; (2002) 12590999. [Abstract] [Full Text] [PDF]
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H. Kiaris, A. V. Schally, R. Busto, G. Halmos, S. Artavanis-Tsakonas, and J. L. Varga Expression of a splice variant of the receptor for GHRH in 3T3 fibroblasts activates cell proliferation responses to GHRH analogs PNAS, January 8, 2002; 99(1): 196 - 200. [Abstract] [Full Text] [PDF]
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