Ana M. Bajo

Growth hormone-releasing hormone (GHRH) antagonists inhibit the proliferation of androgen- dependent and -independent prostate cancers

The antiproliferative effects of an antagonist of growth hormone-releasing 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 prostate-specific 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 androgen-sensitive tumors. Thus, the therapy with GHRH antagonist could be considered for the management of both androgen-dependent or -independent prostate cancers.

Bombesin antagonists inhibit proangiogenic factors in human experimental breast cancers.

The overexpression of angiogenic factors such as vascular endothelial growth factor (VEGF), fibroblast growth factor (FGF) and insulin-like growth factors (IGFs) plays a role in the migration and proliferation of endothelial cells in many cancers. Consequently, we investigated the effects of bombesin/gastrin-releasing peptide (GRP) antagonists on the expression of these angiogenic factors, the activities of matrix metalloproteinases (MMPs)-2 and -9, as well as the vascular density in MDA-MB-435 human oestrogen-independent breast cancers. Nude mice bearing orthotopic xenografts of MDA-MB-435 breast cancers were treated with bombesin/GRP antagonists for 6 weeks. Daily administration of 20 μg of RC-3095 or 10 μg of RC-3940-II significantly decreased the weight of MDA-MB-435 cancers by 44 and 53%, respectively. The inhibition of tumour growth was associated with a substantial reduction in the expression of mRNA and protein levels of basic fibroblast growth factor (bFGF), IGF-II and VEGF-A in the tumours. Both bombesin/GRP antagonists significantly decreased the vessel density of the tumours by about 37%, as shown by immunohistochemical detection of vessels on tumour slides. Gelatinolytic activities, detected by zymography, revealed a 33–46% reduction in MMP-9 activity after the treatment with either antagonist. In vitro studies revealed that MDA-MB-435 cells secrete bFGF, IGF-II and VEGF-A, and the secretion of these factors is inhibited by RC-3095 and RC-3940-II. This study demonstrates the antiangiogenic effect of bombesin/GRP antagonists RC-3095 and RC-3940-II, and underscores their possible therapeutic application for treatment of breast cancers.

Bombesin antagonists inhibit growth of MDA-MB-435 estrogen-independent breast cancers and decrease the expression of the ErbB-2/HER-2 oncoprotein and c-jun and c-fos oncogenes

Previous studies showed that antagonists of bombesin (BN)/gastrin-releasing peptide (GRP) inhibit the growth of various cancers by interfering with the growth-stimulatory effects of BN-like peptides and down-regulating epidermal growth factor receptors on tumors. Because the overexpression of the human epidermal growth factor receptor-2 (ErbB-2/HER-2/neu) oncogene plays a role in the progression of many breast cancers, we investigated whether BN/GRP antagonists can affect HER-2 in mammary tumors. Female nude mice bearing orthotopic xenografts of MDA-MB-435 human estrogen-independent breast cancers were treated daily with BN/GRP antagonists RC-3095 (20 μg) or RC-3940-II (10 μg) for 6 weeks. The expression of BN/GRP receptors on tumors was analyzed by reverse transcription–PCR and immunoblotting. We also evaluated whether the mRNA expression for the c-jun and c-fos oncogenes is affected by the therapy. Both BN/GRP antagonists significantly inhibited growth of MDA-MB-435 cancers; RC-3095 reduced tumor volume by 40% and RC-3940-II by 65%. The GRP receptors (subtype 1) were detected in MDA-MB-435 tumors, showing that they mediate the inhibitory effect of the antagonists. Tumor inhibition was associated with a substantial reduction in the expression of mRNA and protein levels of the ErbB/HER receptor family as well as with a decrease in the expression of c-jun and c-fos oncogenes. BN/GRP antagonists RC-3940-II and RC-3095 could be considered for endocrine therapy of estrogen-independent breast cancers that express members of the ErbB/HER receptor family and the c-jun and c-fos oncogenes.

Preclinical evaluation of therapeutic effects of targeted cytotoxic analogs of somatostatin and bombesin on human gastric carcinomas

New modalities are necessary for the treatment of patients with unresectable gastric carcinoma. The authors investigated whether receptors for somatostatin and bombesin were present in human gastric carcinoma lines and tested the antitumor effects of cytotoxic somatostatin analog AN‐238 and cytotoxic bombesin conjugate AN‐215.

Nuclear localization of vasoactive intestinal peptide (VIP) receptors in human breast cancer

Vasoactive intestinal peptide (VIP) and its receptors (VPACs) are involved in proliferation, survival, and differentiation in human breast cancer cells. Its mechanism of action is traditionally thought to be through specific plasma membrane receptors. There is compelling evidence for a novel intracrine mode of genomic regulation by G-protein-coupled receptors (GPCRs) that implies both endocytosis and nuclear translocation of peripheral GPCR and/or the activation of nuclear-located GPCRs by endogenously-produced, non-secreted ligands. Regarding to VPAC receptors, which are GPCRs, there is only a report suggesting them as a dynamic system for signaling from plasma membrane and nuclear membrane complex. In this study, we show that VPAC(1) receptor is localized in cell nuclear fraction whereas VPAC(2) receptor presents an extranuclear localization and its protein expression is lower than that of VPAC(1) receptor in human breast tissue samples. Both receptors as well as VIP are overexpressed in breast cancer as compared to non-tumor tissue. Moreover, we report the markedly nuclear localization of VPAC(1) receptors in estrogen-dependent (T47D) and independent (MDA-MB-468) human breast cancer cell lines. VPAC(1) receptors are functional in plasma membrane and nucleus as shown by VIP stimulation of cAMP production in both cell lines. In addition, VIP increases its own intracellular and extracellular levels, and could be involved in the regulation of VPAC(1)-receptor traffic from the plasma membrane to the nucleus. These results support new concepts on function and regulation of nuclear GPCRs which could have an impact on development of new therapeutic drugs.

Effects of long-term treatment with the luteinizing hormone-releasing hormone (LHRH) agonist Decapeptyl and the LHRH antagonist Cetrorelix on the levels of pituitary LHRH receptors and their mRNA expression in rats

The effects of depot formulations of the luteinizing hormone-releasing hormone (LHRH) agonist Decapeptyl (25 μg/day) for 30 days or LHRH antagonist Cetrorelix pamoate (100 μg/day) for 30 days and daily injections of 100 μg of Decapeptyl for 10 days on the expression of mRNA for pituitary LHRH receptor (LHRH-R) and the levels of LHRH-R protein were evaluated in rats. Serum sex steroid concentrations and the weights of the reproductive organs were greatly reduced in all groups treated with analogs, demonstrating an efficient blockade of the pituitary–gonadal axis. Decapeptyl microcapsules elevated serum LH in female rats, but decreased it in male rats. LHRH-R mRNA expression in female pituitaries was reduced to 41% and 56–65% on days 10 and 30, respectively, whereas LHRH-R protein was 64% of control on day 10 and returned to pretreatment levels on day 30. Decapeptyl microcapsules reduced LHRH-R mRNA expression in male pituitaries to 58% on day 30 but not LHRH-R protein. Daily injections of Decapeptyl caused a desensitization of LH responses in female rats, while raising LHRH-R mRNA expression in female rats by 23% and LHRH-R protein levels by 119%. Cetrorelix pamoate reduced serum LH in female rats and diminished LHRH-R mRNA to 30% and 26% and LHRH-R protein to 57% and 48% on days 10 and 30, respectively. Elevated LHRH-R protein levels of ovariectomized rats were reduced after 10-day treatment with Cetrorelix or 100 μg/day Decapeptyl. Thus, changes in the mRNA expression after treatment with Cetrorelix, but not always Decapeptyl, paralleled those of LHRH-R protein. The inhibitory effect of Cetrorelix on serum LH, pituitary LHRH-R mRNA, and LHRH-R protein was greater than that of Decapeptyl.

Vasoactive intestinal peptide (VIP) induces transactivation of EGFR and HER2 in human breast cancer cells

We analyzed the cross-talk between receptors for vasoactive intestinal peptide (VIP) and the human epidermal growth factor family of tyrosine kinase receptors (HER) in oestrogen-dependent (T47D) and oestrogen-independent (MDA-MB-468) human breast cancer cells. VIP treatment slowly increased the expression levels of EGFR but it rapidly augmented phosphorylation of EGFR and HER2 in both cell lines. This pattern of HERs transactivation was blocked by the specific VIP antagonist JV-1-53, supporting the direct involvement of VIP receptors in formation of P-EGFR and P-HER2. VIP-induced transactivation was also abolished by H89 (protein kinase A inhibitor), PP2 (Src inhibitor) or TAPI-1 (inhibitor of matrix metalloproteases), following a differential pattern. These results shed a new light on the specific signalling pathways involved in EGFR/HER2 transactivation by VPAC receptors and suggest the potential usefulness of VIP receptor antagonists together with current antibodies against EGFR/HER2 and/or tyrosine kinase inhibitors for breast cancer therapy.

Vasoactive intestinal peptide enhances growth and angiogenesis of human experimental prostate cancer in a xenograft model

We show that vasoactive intestinal peptide (VIP) exerts trophic and proangiogenic activities in experimental prostate cancer in vivo. Nude mice were subcutaneously injected with Matrigel impregnated with LNCaP prostate cancer cells. Cell treatment with 100 nM VIP for 1h before xenograft resulted in increased tumor growth after 8 and, more remarkably, 15 days of injection. The same occurred with the mRNA expression of the main angiogenic factor, vascular endothelial growth factor (VEGF), as shown by real-time RT-PCR quantification. The proangiogenic activity of VIP was further established by showing increases of hemoglobin levels, Masson trichromic staining, and immunohistochemical CD34 staining in tumors excised 15 days after subcutaneous injection of VIP-treated cells as compared to control conditions. All these parameters indicate that VIP increases vessel formation. This xenograft model is a useful tool to study in vivo the effects of VIP-related peptides in tumor growth and development of blood supply as well as their therapeutical potential in prostate cancer.

Vasoactive intestinal peptide (VIP) induces malignant transformation of the human prostate epithelial cell line RWPE-1.

The carcinogenic potential of vasoactive intestinal peptide (VIP) was analyzed in non-tumor human prostate epithelial cells (RWPE-1) and in vivo xenografts. VIP induced morphological changes and a migratory phenotype consistent with stimulation of expression/activity of metalloproteinases MMP-2 and MMP-9, decreased E-cadherin-mediated cell-cell adhesion, and increased cell motility. VIP increased cyclin D1 expression and cell proliferation that was blocked after VPAC(1)-receptor siRNA transfection. Similar effects were seen in RWPE-1 tumors developed by subcutaneous injection of VIP-treated cells in athymic nude mice. VIP acts as a cytokine in RWPE-1 cell transformation conceivably through epithelial-mesenchymal transition (EMT), reinforcing VIP role in prostate tumorigenesis.

Ontogeny of vasoactive intestinal peptide receptors in rat ventral prostate

1. The stimulatory effect of VIP on rat prostatic adenylyl cyclase changes during postnatal development. It peaks at 2 months (peripubertal period), remains in a plateau between 3 and 12 months (adult period), and decreases at 24 months (old period). 2. The stimulation of rat prostatic adenylyl cyclase by the beta-adrenergic agonist isoproterenol follows a pattern rather similar to that of VIP. 3. The values of VIP binding capacity correlate well with those observed for adenylyl cyclase between 1 and 12 months, whereas there appears to exist a great number of uncoupled VIP receptors at 0.5 and 24 months. 4. The apparent molecular mass (51 kDa) of the rat prostatic VIP-receptor complex remains unaltered during ontogenic development.