Antagonist of Growth Hormone-Releasing Hormone Receptor MIA-690 Suppresses the Growth of Androgen-Independent Prostate Cancers.

The development of resistance remains the primary challenge in treating castration-resistant prostate cancer (CRPC). GHRH receptors (GHRH-R), which are coupled to G-proteins (GPCRs), can mediate EGFR transactivation, offering an alternative pathway for tumour survival. This study aimed to evaluate the effects of the GHRH-R antagonist MIA-690, in combination with the EGFR inhibitor Gefitinib, on cell viability, adhesion, gelatinolytic activity, and the cell cycle in advanced prostate cancer PC-3 cells. The findings demonstrate a synergistic effect between MIA-690 and Gefitinib, leading to the inhibition of cell viability, adhesion, and metalloprotease activity. Cell cycle analysis suggests that both compounds induce cell cycle arrest, both individually and in combination. Furthermore, similar effects of the GHRH-R antagonist MIA-690 combined with Gefitinib were observed in PC-3 tumours developed by subcutaneous injection in athymic nude mice 36 days post-inoculation. These results indicate that combined therapy with a GHRH-R antagonist and an EGFR inhibitor exerts a stronger antitumor effect compared to monotherapy by preventing transactivation between EGFR and GHRH-R in CRPC.

Urinary excretion of parathyroid hormone-related protein correlates with renal function in control rats and rats with cisplatin nephrotoxicity.

Parathyroid hormone-related protein (PTHrP) and its receptor are abundantly expressed throughout the renal parenchyma, where PTHrP exerts a modulatory action on renal function. PTHrP upregulation is a common event associated with the mechanism of renal injury and repair. However, no study has yet explored the putative excretion of PTHrP in urine, including its potential relationship with renal function. In the present study, we tested this hypothesis by studying the well-known rat model of acute renal injury induced by the chemotherapeutic agent cisplatin. Using Western blot analysis, we could detect a single protein band, corresponding to intact PTHrP, in the urine of both control and cisplatin-injected rats, whose levels were significantly higher in the latter group. PTHrP was detected in rat urine by dot blot, and its quantification with two specific ELISA kits showed that, compared with control rats, those treated with cisplatin displayed a significant increase in urinary PTHrP (expressed as the PTHrP-to-creatinine ratio or 24-h excretion). In addition, a positive correlation between urinary PTHrP excretion and serum creatinine was found in these animals. In conclusion, our data demonstrate that PTHrP is excreted in rat urine and that this excretion is higher with the decrease of renal function. This suggests that urinary PTHrP levels might be a renal function marker.

Growth hormone-releasing hormone antagonists abolish the transactivation of human epidermal growth factor receptors in advanced prostate cancer models.

Growth hormone-releasing hormone (GHRH) and its receptors have been implicated in a variety of cellular phenotypes related with tumorigenesis process. Human epidermal growth factor receptor family members (HER) such as EGFR and HER2 are involved in mitogenic signaling pathways implicated in the progression of prostate cancer. We analyzed the cross-talk between GHRH and EGF receptors in prostate cancer. The effects of GHRH in HER signaling were evaluated on human androgen-independent PC3 prostate cancer cells in vitro and GHRH antagonist in vitro and in nude mice xenografts of PC3 prostate cancer. Time-course studies indicated that GHRH had a stimulatory activity on both the expression of EGFR and HER2. GHRH analogues, JMR-132 and JV-1-38, endowed with antagonistic activity for GHRH receptors, abrogated the response to GHRH in PC3 cells. GHRH stimulated a rapid ligand-independent activation of EGFR and HER2 involving at least cAMP/PKA and Src family signaling pathways. GHRH also stimulated a slow ligand-dependent activation of EGFR and HER2 involving an extracellular pathway with an important role for ADAM. Preliminary results also revealed an increase of mRNA for GHRH and GHRH receptor induced by EGF. The inhibition of tumor growth, in vivo, was associated with a substantial reduction in the expression of mRNA and protein levels of EGFR and HER2 in the tumors. GHRH antagonist JV-1-38, significantly decreased the phosphorylated Src levels. The cross-talk between HER and GHRH-R may be impeded by combining drugs acting upon GHRH receptors and HER family members in human advanced prostate cancer.

Inhibitory effects of antagonists of growth hormone-releasing hormone on growth and invasiveness of PC3 human prostate cancer.

New approaches are needed to the therapy of advanced prostate cancer. This study determined the effect of growth hormone-releasing 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 µg/day) or JV-1-38 (20 µg/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)-1α, metalloproteinases (MMPs) -2 and -9, ß-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-1α expression and in active forms of MMP-2 and MMP-9, a significant increase in levels of membrane-associated ß-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.

Antitumoral effects of vasoactive intestinal peptide in human renal cell carcinoma xenografts in athymic nude mice.

We studied antitumor effect of VIP in human renal cell carcinoma (RCC) (A498 cells xenografted in immunosuppressed mice). VIP-treated cells gave resulted in p53 upregulation and decreased nuclear ß-catenin translocation and NFκB expression, MMP-2 and MMP-9 activities, VEGF levels and CD-34 expression. VIP led to a more differentiated tubular organization in tumours and less metastatic areas. Thus, VIP inhibits growth of A498-cell tumours acting on the major issues involved in RCC progression such as cell proliferation, microenvironment remodelling, tumour invasion, angiogenesis and metastatic ability. These antitumoral effects of VIP offer new therapeutical possibilities in RCC treatment.

Overexpression of vasoactive intestinal peptide receptors and cyclooxygenase-2 in human prostate cancer. Analysis of potential prognostic relevance.

Vasoactive intestinal peptide (VIP) is a potent inductor of cyclooxygenase-2 (COX-2) expression in human prostate cancer cell lines. There are conflicting data regarding the role of COX-2 in the progression of this disease. Here we examined the expression of VIP receptors (VPAC1 and VPAC2) and COX-2 in prostate cancer specimens. Correlations among protein levels and various clinicopathological factors and prognosis of patients were statistically analyzed. For these purposes, formaldehyde-fixed, paraffin-embedded prostate tissue specimens from 63 patients with prostate cancer and 9 control samples were used. The expression of VPAC1 and VPAC2 receptors and COX-2 was analyzed at mRNA levels by quantitative reverse transcriptase-PCR. The corresponding expression at protein level was studied by immunohistochemistry, scored as negative, weak, moderate, or strong, and correlated with different clinicopathological factors by means of multivariate analysis. 88% of prostate cancer tissues overexpressed VPAC1-receptor at mRNA level, 72% VPAC2-receptor and 77% COX-2. Simultaneous overexpression of the three genes was seen in 52% of patients. Similar overexpression patterns were observed at protein level. The correlation between VPAC1 and VPAC2 receptor protein levels was statistically significant. However, no significant correlations existed among protein levels of VPAC receptors and COX-2 with patient age, prostate-specific antigen (PSA) levels, tumor stage, Gleason score and survival time. The overexpression of VPAC1 and VPAC2 receptors and COX-2 in cancer tissue gives them a potential role as targets for diagnosis of prostate cancer but results do not support a clear value as biomarkers for the clinical prognosis of this disease.

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.

Control of the annual testicular cycle of the marbled-newt by p53, p21, and Rb gene products.

p53, p21, and Rb are proteins with an important role in cell-cycle control. The expression and distribution of these gene products and the apoptotic rate were studied in the marbled-newt testis along the annual cycle to know the role of these factors in the control of spermatogenesis and glandular tissue formation. The study was carried out using Western blot analysis and immunohistochemistry. The results differed from those, previously reported in mammals showing constant spermatogenesis. Greater expression of p53 and p21 was found in the quiescence period and was detected in PCGs (primordial germ cells), spermatogonia, follicular, interstitial cells, and glandular tissue. Greater expression of Rb and phosph-Rb was present in the proliferation period, in PCGs, and spermatogonia. Apoptosis was only detected in secondary spermatogonia (quiescence and spermiogenesis periods) and primary spermatocytes (proliferation and spermiogenesis periods). In the quiescence period, the increase in p53 expression activates p21 expression, which inhibits Rb phosphorylation and arrests the cell cycle in G1. In the proliferation period and, in a lesser degree, in the spermiogenesis period, the expressions of p53 and p21 decrease and phosph-Rb increases, enhancing cell proliferation. These gene products do not seem to be related to apoptosis.