Expression of growth hormone-releasing hormone (GHRH) and splice variants of GHRH receptors in human experimental prostate cancers (original) (raw)
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Breast Cancer Research and Treatment, 2003
Antagonists of GHRH inhibit the growth of various human tumors, including prostate cancer, but the tumoral receptors mediating the antiproliferative effect of GHRH antagonists have not been clearly identified. Recently, we demonstrated that human cancer cell lines express splice variants (SVs) of receptors for GHRH, of which SV1 exhibits the greatest similarity to the pituitary GHRH receptors. In this study we investigated the expression of GHRH and SVs of GHRH receptor and the binding characteristics of the GHRH receptor isoform in 20 surgical specimens of organ-confined and locally advanced human prostatic adenocarcinomas. The mRNA expression of GHRH and SVs of GHRH receptor was investigated by RT-PCR. The affinity and density of receptors for GHRH were determined by ligand competition assays based on bind-ing of 125 I-labeled GHRH antagonist JV-1-42 to tumor membranes. Twelve of 20 tumors (60%) exhibited specific, high affinity binding for JV-1-42, with a mean dissociation constant (K d ) of 0.81 nmol/liter and a mean maximal binding capacity of 185.2 fmol/mg membrane protein. The mRNA of SV1 was detected in 13 of 20 (65%) prostate cancer specimens and was consistent with the presence of GHRH binding. RT-PCR analyses also revealed the expression of mRNA for GHRH in 13 of 15 (86%) prostatic carcinoma specimens examined. The presence of GHRH and its tumoral receptor SVs in prostate cancers suggests the possible existence of an autocrine mitogenic loop. The antitumor effects of GHRH antagonists in prostate cancer could be exerted in part by interference with this local GHRH system. (J Clin Endocrinol Metab 87: 4707-4714, 2002) Abbreviations: B max , Maximal binding capacity; hGHRH, human GHRH; PACAP, pituitary adenylate cyclase-activating polypeptide; SCLC, small cell lung cancer; SV, splice variant; VIP, vasoactive intestinal peptide.
Background: Hypothalamic GH-releasing hormone (GHRH) regulates GH release from the pituitary, but an ectopic production of GHRH has been detected in various non-hypothalamic tissues, especially cancers. Objective: To investigate whether endometrial tumors produce GHRH. Methods: Twenty-four endometrioid, three serous papillary (SP), three mixed type endometrioid/ serous papillary adenocarcinomas and one malignant mixed Mü llerian tumor (MMMT) were assessed for GHRH immunoreactivity by the polyclonal anti-rabbit antibody SV95 and for the expression of GHRH mRNA by in situ hybridization using an oligonucleotide probe. Results: Increased GHRH immunoreactivity was detected in 15 out of 24 (63%) of the endometrioid tumors, including two out of three (66%) of the mixed type endometrioid/serous adenocarcinomas but not in the three SP or the MMMT tumor. Cytoplasmic staining was detected in all positive cases, while in three of them strong nuclear localization of GHRH was also revealed. In situ hybridization indicated the presence of GHRH mRNA in six cases, all characterized as positive for GHRH immunoreactivity. Conclusion: GHRH is expressed in a subset of endometrial tumors, of the endometrioid type in particular. A paracrine/autocrine role for GHRH in the development of the disease should be considered.
Molecular and Cellular Endocrinology, 2004
Various hormones and growth factors have been implicated in progression of prostate cancer, but their role and the underlying molecular mechanism(s) involved remain poorly understood. In this study, we investigated the role of human growth hormone (GH) and its receptor (GHR) in human prostate cancer. We first demonstrated mRNA expression of GHR and of its exon 9-truncated isoform (GHR tr ) in benign prostate hyperplasia (BPH) and prostate adenocarcinoma patient tissues, as well as in LNCaP, PC3 and DU145 human prostate cancer cell lines. GHR mRNA levels were 80% higher and GHR tr only 25% higher, in the carcinoma tissues than in BPH. Both isoforms were also expressed in LNCaP and PC3 cell lines and somewhat less so in DU145 cells. The LNCaP cell GHR protein was further characterized, on the basis of its M r of 120 kDa, its binding to two different GHR monoclonal antibodies, its high affinity and purely somatogenic binding to 125 I-hGH and its ability to secrete GH binding protein, all characteristic of a functional GHR. Furthermore, GH induced rapid, time-and dose-dependent signaling events in LNCaP cells, including phosphorylation of JAK2 tyrosine kinase, of GHR itself and of STAT5A (JAK2-STAT5A pathway), of p42/p44 MAPK and of Akt/PKB. No effect of GH (72 h) could be shown on basal or androgen-induced LNCaP cell proliferation nor on PSA secretion. Interestingly, however, GH caused a rapid (2-12 h) though transient striking increase in immunoreactive androgen receptor (AR) levels (≤5-fold), followed by a slower (24-48 h) reduction (≤80%), with only modest parallel changes in serine-phosphorylated AR. In conclusion, the GH-induced activation of signaling pathways, its effects on AR protein in LNCaP cells and the isoform-specific regulation of GHR in prostate cancer patient tissues, suggest that GH, most likely in concert with other hormones and growth factors, may play an important role in progression of human prostate cancer.
Proceedings of the National Academy of Sciences, 2003
The antiproliferative effects of an antagonist of growth hormonereleasing hormone (GHRH) JV-1-38 were evaluated in nude mice bearing s.c. xenografts of LNCaP and MDA-PCa-2b human androgen-sensitive and DU-145 androgen-independent prostate cancers. In the androgen-sensitive models, JV-1-38 greatly potentiated the antitumor effect of androgen deprivation induced by surgical castration, but was ineffective when given alone. Thus, in castrated animals bearing MDA-PCa-2b cancers, the administration of JV-1-38 for 35 days virtually arrested tumor growth (94% inhibition vs. intact control, P < 0.01; and 75% vs. castrated control, P < 0.05). The growth of LNCaP tumors was also powerfully suppressed by JV-1-38 combined with castration (83% inhibition vs. intact control, P < 0.01; and 68% vs. castrated control, P < 0.05). However, in androgen-independent DU-145 cancers, JV-1-38 alone could inhibit tumor growth by 57% (P < 0.05) after 45 days. In animals bearing MDA-PCa-2b and LNCaP tumors, the reduction in serum prostatespecific antigen levels, after therapy with JV-1-38, paralleled the decrease in tumor volume. Inhibition of MDA-PCa-2b and DU-145 cancers was associated with the reduction in the expression of mRNA and protein levels of vascular endothelial growth factor. The mRNA expression for GHRH receptor splice variants was found in all these models of prostate cancer. Our results demonstrate that GHRH antagonists inhibit androgen-independent prostate cancers and, after combination with androgen deprivation, also androgensensitive tumors. Thus, the therapy with GHRH antagonist could be considered for the management of both androgen-dependent or -independent prostate cancers.
Histology and histopathology, 2005
Growth hormone releasing hormone receptor (GHRH-R) mRNA and protein was first localized to the anterior pituitary gland, consequent with the action of its ligand on GH synthesis and release. Subsequent studies found GHRH-R also expressed in the hypothalamus and in systemic tissues including those of the reproductive system. In the present work, we studied the distribution of GHRH-R in human reproductive system of males and females by immunohistochemical method. GHRH-R immunostaining was localized in male reproductive system: Leydig cells, Sertoli and basal germ cells of the seminiferous tubules and prostate secretory cells. GHRH-R immunostaining was also demonstrated in the ovary: oocytes, follicular cells, granulosa, thecal and corpus luteum cells. Endometrial glands, placenta and normal mammary glands also showed GHRH-R immunostaining. Our results demonstrate the localization of GHRH-R in the reproductive system, which may mediate the direct action of GHRH in these tissues. Moreov...
Proceedings of The National Academy of Sciences, 2005
Various attempts to detect human pituitary growth hormonereleasing hormone receptor (pGHRH-R) in neoplastic extrapituitary tissues have thus far failed. Recently, four splice variants (SVs) of GHRH-R have been described, of which SV1 has the highest structural homology to pGHRH-R and likely plays a role in tumor growth. The aim of this study was to reinvestigate whether human tumors and normal human extrapituitary tissues express the pGHRH-R and to corroborate our previous findings on its SVs. Thus, we developed a real-time PCR method for the detection of the mRNA for the pGHRH-R, its SVs, and the GHRH peptide. Using real-time PCR, Western blotting, and radioligand-binding assays, we detected the mRNA for pGHRH-R and pGHRH-R protein in various human cancer cell lines grown in nude mice and in surgical specimens of human lung cancers. The expression of mRNA for SVs of pGHRH-R and GHRH was likewise found in xenografts of human non-Hodgkin's lymphomas, pancreatic cancer, glioblastoma, smallcell lung carcinomas, and in human nonmalignant prostate, liver, lung, kidney, and pituitary. Western blots showed that these normal and malignant human tissues contain SV1 protein and immunoreactive GHRH. Our results demonstrate that some normal human tissues and tumors express mRNA and protein for the pGHRH-R and its splice variants. These findings confirm and extend the concept that GHRH and its receptors play an important role in the pathophysiology of human cancers.
International Journal of Cancer, 2013
New approaches are needed to the therapy of advanced prostate cancer. This study determined the effect of growth hormonereleasing hormone (GHRH) antagonists, JMR-132 and JV-1-38 on growth of PC3 tumors as well as on angiogenesis and metastasis through the evaluation of various factors that contribute largely to the progression of prostate cancer. Human PC3 androgen-independent prostate cancer cells were injected subcutaneously into nude mice. The treatment with JMR-132 (10 lg/day) or JV-1-38 (20 lg/day) lasted 41 days. We also evaluated the effects of JMR-132 and JV-1-38 on proliferation, cell adhesion and migration in PC-3 cells in vitro. Several techniques (Western blot, reverse transcription polymerase chain reaction, immunohistochemistry, ELISA and zymography) were used to evaluate the expression levels of GHRH receptors and its splice variants, GHRH, vascular endothelial growth factor (VEGF), hypoxia inducible factor (HIF)-1a, metalloproteinases (MMPs) 22 and 29, b-catenin and E-cadherin. GHRH antagonists suppressed the proliferation of PC-3 cells in vitro and significantly inhibited growth of PC3 tumors. After treatment with these analogues, we found an increase in expression of GHRH receptor accompanied by a decrease of GHRH levels, a reduction in both VEGF and HIF-1a expression and in active forms of MMP-2 and MMP-9, a significant increase in levels of membrane-associated b-catenin and a significant decline in E-cadherin. These results support that the blockade of GHRH receptors can modulate elements involved in angiogenesis and metastasis. Consequently, GHRH antagonists could be considered as suitable candidates for therapeutic trials in the management of androgen-independent prostate cancer.
Proceedings of the National Academy of Sciences
The effects of the growth hormone-releasing hormone (GHRH) agonist MR409 on various human cancer cells were investigated. In H446 small cell lung cancer (SCLC) and HCC827 and H460 (non-SCLC) cells, MR409 promoted cell viability, reduced cell apoptosis, and induced the production of cellular cAMP in vitro. Western blot analyses showed that treatment of cancer cells with MR409 up-regulated the expression of cyclins D1 and D2 and cyclin-dependent kinases 4 and 6, down-regulated p27kip1, and significantly increased the expression of the pituitary-type GHRH receptor (pGHRH-R) and its splice-variant (SV1). Hence, in vitro MR409 exerts agonistic action on lung cancer cells in contrast to GHRH antagonists. However, in vivo, MR409 inhibited growth of lung cancers xenografted into nude mice. MR409 given s.c. at 5 μg/day for 4 to 8 weeks significantly suppressed growth of HCC827, H460, and H446 tumors by 48.2%, 48.7%, and 65.6%, respectively. This inhibition of tumor growth by MR409 was accomp...
Human renal cell carcinoma expresses distinct binding sites for growth hormone-releasing hormone
Proceedings of The National Academy of Sciences, 2000
Antagonists of growth hormone-releasing hormone (GHRH) inhibit the proliferation of various human cancers in vitro and in vivo by mechanisms that include apparent direct effects through specific binding sites expressed on tumors and that differ from pituitary human GHRH (hGHRH) receptors. In this study, GHRH antagonist JV-1-38 (20 g͞day per animal s.c.) inhibited the growth of orthotopic CAKI-1 human renal cell carcinoma (RCC) by 83% and inhibited the development of metastases to lung and lymph nodes. Using ligand competition assays with 125 I-labeled GHRH antagonist JV-1-42, we demonstrated the presence of specific high-affinity (K d ؍ 0.25 ؎ 0.03 nM) binding sites for GHRH with a maximal binding capacity (B max) of 70.2 ؎ 4.1 fmol͞mg of membrane protein in CAKI-1 tumors. These receptors bind GHRH antagonists preferentially and display a lower affinity for hGHRH. The binding of 125 I-JV-1-42 is not inhibited by vasoactive intestinal peptide (VIP)-related peptides sharing structural homology with hGHRH. The receptors for GHRH antagonists on CAKI-1 tumors are distinct from binding sites detected with 125 I-VIP (Kd ؍ 0.89 ؎ 0.14 nM; B max ؍ 183.5 ؎ 2.6 fmol͞mg of protein) and also have different characteristics from GHRH receptors on rat pituitary as documented by the insignificant binding of [His 1 , 125 I-Tyr 10 ,Nle 27 ]hGHRH(1-32)NH2. Reverse transcription-PCR revealed the expression of splice variants of hGHRH receptor in CAKI-1 RCC. Biodistribution studies demonstrate an in vivo uptake of 125 I-JV-1-42 by the RCC tumor tissue. The presence of specific receptor proteins that bind GHRH antagonists in CAKI-1 RCC supports the view that distinct binding sites that mediate the inhibitory effect of GHRH antagonists are present on various human cancers.