Stimulation of neuroendocrine differentiation in prostate cancer cells by GHRH and its blockade by GHRH antagonists.

Prostate cancer is the second leading cause of cancer-related deaths among men in developed countries. Neuroendocrine prostate cancer, in particular, is associated with an aggressive phenotype and a poor prognosis. Neuroendocrine cells produce and secrete peptide hormones and growth factors in a paracrine/autocrine manner which promote the progression of the disease. Recent studies have demonstrated that extracellular vesicles or exosomes are released by prostate cancer cells, supporting the spread of prostate cancer. Hence, the aim of this study was to investigate the effect of growth hormone-releasing hormone (GHRH) on neuroendocrine differentiation (NED) in the androgen-dependent prostate cancer cell line LNCaP and the molecular mechanisms underlying these effects. GHRH induced an increase in the percentage of neurite-bearing cells and in the protein levels of Neuron-Specific Enolase. Both effects were blocked by the GHRH receptor antagonist MIA-690. In addition, pretreatment of these cells with the calcium chelator BAPTA, the EGFR inhibitor AG-1478 or the HER2 inhibitor AG-825 reduced the effect of GHRH, suggesting that the GHRH-induced stimulation of NED involves calcium channel activation and EGFR/HER2 transactivation. Finally, PC3-derived exosomes led to an increase in NED, cell proliferation and cell adhesion. Altogether, these findings suggest that GHRH antagonists should be considered for in the management of neuroendocrine prostate cancer.

Anticancer Activity of Dendriplexes against Advanced Prostate Cancer from Protumoral Peptides and Cationic Carbosilane Dendrimers.

The interaction of neuropeptides, vasoactive intestinal peptide (VIP), or growth hormone-releasing hormone (GHRH), with a cationic carbosilane dendrimer forms dendriplexes with antitumoral behavior in advanced prostate cancer cells PC3. At the concentrations used for dendriplexes formation, the free peptides were protumoral and prometastatic in advanced prostate cancer, while dendrimer only showed low cytotoxicity, but did not avoid the metastatic behavior of PC3 cells. However, these nanoplexes favored also cell adhesion and avoided cell migration. Also, the dendriplexes were not toxic for no tumoral prostate cells (RPWE-1) or fibroblasts. The use of labeled GHRH peptide (rhodamine labeled) and a dendrimer (fluorescein labeled) allowed us to observe that both systems reach the intracellular milieu after dendriplex formation. The treatment of PC3 cells with the nanoplexes reduced expression of vascular endothelial growth factor (VEGF) and cyclic adenosine monophosphate (cAMP). Molecular modeling analysis highlights the important contribution of the carbosilane framework in the stabilization of the dendriplex, since dendrimer interacts with a peptide region where hydrophobic amino acids are presented.

Growth hormone-releasing hormone (GHRH) promotes metastatic phenotypes through EGFR/HER2 transactivation in prostate cancer cells.

The involvement of growth hormone-releasing hormone (GHRH) in several relevant processes that contribute to prostate cancer progression was analyzed. Firstly, we evaluated GHRH effects on cell proliferation and adhesion in human cancer prostate cell lines, LNCaP and PC3, by using specific assays (BrdU incorporation and collagen adhesion). The expression levels of the main marker molecules of these processes were measured by RT-PCR, Western blotting and zymography assays. GHRH increased both cell proliferation and proliferating cell nuclear antigen (PCNA) levels in LNCaP cells and in PC3 cells; however, such a rise was faster in the PC3 cells that represent the most aggressive stage of prostate cancer. Furthermore, GHRH significantly reduced cell adhesion and E-cadherin levels in LNCaP and PC3 cells and up-regulated the total and nuclear expression of ß-catenin in PC3 cells. In addition, we assessed cell cycle, cell migration and VEGF secretion in PC3 cells. GHRH augmented the number of cells in G2/M-phase but diminished that corresponding to G1-phase. Cell-cycle specific markers were evaluated since GHRH effects may be related to their differential expression; we observed a decrease of p53, p21, and Bax/Bcl2 ratio. Furthermore, GHRH increased the expression of CD44, c-myc and cyclin D1, MMP-2 and MMP-9 activity, and VEGF secretion. We also observed that EGFR and/or HER2 transactivation is involved in cell adhesion, cell migration and VEGF secretion produced by GHRH. Consequently, present results define GHRH as a proliferative, anti-apoptotic and migratory agent in prostate cancer.

Growth hormone-releasing hormone induced transactivation of epidermal growth factor receptor in human triple-negative breast cancer cells.

Triple-negative breast cancer (TNBC) is a subset of breast cancers which is negative for expression of estrogen and progesterone receptors and human epidermal growth factor receptor-2 (HER2). Chemotherapy is currently the only form of treatment for women with TNBC. Growth hormone-releasing hormone (GHRH) and epidermal growth factor (EGF) are autocrine/paracrine growth factors in breast cancer and a substantial proportion of TNBC expresses receptors for GHRH and EGF. The aim of this study was to evaluate the interrelationship between both these signaling pathways in MDA-MB-468 human TNBC cells. We evaluated by Western blot assays the effect of GHRH on transactivation of EGF receptor (EGFR) as well as the elements implicated. We assessed the effect of GHRH on migration capability of MDA-MB-468 cells as well as the involvement of EGFR in this process by means of wound-healing assays. Our findings demonstrate that in MDA-MB-468 cells the stimulatory activity of GHRH on tyrosine phosphorylation of EGFR is exerted by two different molecular mechanisms: i) through GHRH receptors, GHRH stimulates a ligand-independent activation of EGFR involving at least cAMP/PKA and Src family signaling pathways; ii) GHRH also stimulates a ligand-dependent activation of EGFR implicating an extracellular pathway with an important role for metalloproteinases. The cross-talk between EGFR and GHRHR may be impeded by combining drugs acting upon GHRH receptors and EGFR family members. This combination of GHRH receptors antagonists with inhibitors of EGFR signalling could enhance the efficacy of both types of agents as well as reduce their doses increasing therapeutic benefits in management of human breast cancer.

Anti-proliferative and pro-apoptotic effects of GHRH antagonists in prostate cancer.

Growth hormone-releasing hormone (GHRH) and its receptors have been implicated in the progression of various tumors. In vitro and in vivo studies have demonstrated that GHRH antagonists inhibit the growth of several cancers. GHRH antagonists, JMR-132 and JV-1-38 inhibit the growth of androgen-independent prostate tumors. Here we investigated the involvement of GHRH antagonists in proliferative and apoptotic processes. We used non-tumoral RWPE-1 and tumoral LNCaP and PC3 human prostatic epithelial cells, as well as an experimental model of human tumor PC3 cells. We evaluated the effects of JMR-132 and JV-1-38 antagonists on cell viability and proliferation in the three cell lines by means of MTT and BrdU assays, respectively, as well as on cell cycle and apoptotic process in PC3 cells. The expression levels of PCNA, p53, p21, CD44, Cyclin D1, c-myc, Bax and Bcl2 were determined in both in vivo and in vitro models by means of Western-blot and RT-PCR. GHRH antagonists suppressed cell proliferation and decreased the levels of the proliferation marker, PCNA, in the three cell lines and in PC3 tumor. GHRH antagonists led to an increase of cells in S-phase and a decrease in G1 and G2/M phases, and induced S-phase arrest and increase of apoptotic cells. The effects of GHRH-antagonists on cell cycle could be due to the changes observed in the expression of p21, p53, Bax, Bcl2, CD44, Cyclin D1, c-myc and caspase 3. Present results confirm and extend the role of GHRH antagonists as anti-proliferative and pro-apoptotic molecules in prostate cancer.

VIP induces NF-κB1-nuclear localisation through different signalling pathways in human tumour and non-tumour prostate cells.

The nuclear factor κB (NF-κB) is a powerful activator of angiogenesis, invasion and metastasis. Transactivation and nuclear localisation of NF-κB is an index of recurrence in prostate cancer. Vasoactive intestinal peptide (VIP) exerts similar effects in prostate cancer models involving increased expression of vascular endothelial growth factor (VEGF) and cyclooxygenase-2 (COX-2) which are related to NF-κB transactivation. Here we studied differential mechanisms of VIP-induced NF-κB transactivation in non-tumour RWPE-1 and tumour LNCaP and PC3 human prostate epithelial cells. Immunofluorescence studies showed that VIP increases translocation of the p50 subunit of NF-κB1 to the nucleus, an effect that was inhibited by curcumin. The signalling transduction pathways involved are different depending on cell transformation degree. In control cells (RWPE1), the effect is mediated by protein kinase A (PKA) activation and does not implicate extracellular signal-regulated kinase (ERK) or phosphoinositide 3-kinase (PI3-K) pathways whereas the opposite is true in tumour LNCaP and PC3 cells. Exchange protein directly activated by cAMP (EPAC) pathway is involved in transformed cells but not in control cells. Curcumin blocks the activating effect of VIP on COX-2 promoter/prostaglandin E2 (PGE2) production and VEGF expression and secretion. The study incorporates direct observation on COX-2 promoter and suggests that VIP effect on VEGF may be indirectly mediated by PGE2 after being synthesised by COX-2, thus amplifying the initial signal. We show that the signalling involved in VIP effects on VEGF is cAMP/PKA in non-tumour cells and cAMP/EPAC/ERK/PI3K in tumour cells which coincides with pathways mediating p50 nuclear translocation. Thus, VIP appears to use different pathways for NF-κB1 (p50) transactivation in prostate epithelial cells depending on whether they are transformed or not. Transformed cells depend on pro-survival and pro-proliferative signalling pathways involving ERK, PI3-K and cAMP/EPAC which supports the potential therapeutic value of these targets in prostate cancer.

Signalling pathways involved in antitumoral effects of VIP in human renal cell carcinoma A498 cells: VIP induction of p53 expression.

Vasoactive intestinal peptide (VIP) decreases cell proliferation through PI3K signalling and prevents tumour progression in clear renal cell carcinoma (RCC). Here we analyzed the signalling pathways that mediate such VIP effects by using human RCC A498 cells. The effects of treatment with 1 µM VIP and/or specific protein kinase inhibitors such as H89, Wortmannin and PD98059 were studied by cell adhesion assay, ELISA of VEGF165 and ROS production assays. Semiquantitative RT-PCR and western blot were performed to study p53 expression. VIP increased cell adhesion and ROS production, and decreased VEGF165 secretion through PI3K signalling. Moreover, VIP increased nuclear expression of tumour suppressor p53. VIP effects could be blocked by cell incubation with a specific p53 inhibitor, cyclin pifithrin-α hydrobromide (CPFT-αH). In conclusion, this study provides a p53-dependent mechanism by which VIP regulates cell proliferation in RCC development. It supports a potential usefulness of VIP in new therapies of RCC.

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.

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.

Vasoactive intestinal peptide induces oxidative stress and suppresses metastatic potential in human clear cell renal cell carcinoma.

Molecular mechanisms involved in progression of clear-cell renal-cell carcinomas (ccRCCs) are poorly understood. A common genetic mutation found in ccRCC is the loss of the von Hippel-Lindau (VHL) gene, which contributes to cancer progression and metastasis. We investigated VIP effects on metastatic and angiogenic factors in human VHL-null A498 ccRCC and HK2 renal cells. VIP increased adhesion but decreased expression of metalloproteinases, MMP2 and MMP9, as well as cell migration and VEGF expression and secretion in A498 but not in HK2 cells. VIP enhanced ROS levels and decreased nuclear levels of ß-catenin and NFκB p50-subunit in A498 cells, suggesting neuropeptide involvement in the observed decrease of metastatic ability in clear-cell carcinoma. VIP effects in A498 cells were blocked by the VPAC(1/2)-receptor antagonist JV-1-53. In conclusion, present data point to a role of VIP in preventing invasion and metastasis in ccRCCs and support its potential therapeutic usefulness in this disease.

Antioxidant activity of vasoactive intestinal peptide in HK2 human renal cells.

Oxidative stress is a major mediator of tissue and cell injuries. The injury in chronic nephrotic syndrome, acute renal failure, myeloma kidney injury and other kidney diseases is initiated by oxidative stress. We have previously demonstrated that vasoactive intestinal peptide (VIP) acts as an antiproliferative agent in renal cancer cells. This study was designed to evaluate the renoprotective activity of VIP against H(2)O(2)-induced oxidative damage in a proximal tubule kidney cell line (human, non-tumor, HK2 cells) in order to investigate the potential usefulness of this peptide in the treatment of oxidative-stress related kidney diseases. HK2 cell viability was assessed by 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyl tetrazolium bromide assay. Propidium iodide was used to identify cells undergoing apoptosis. Western blotting was performed with anti-Bcl-2, anti-Bax and anti-formyl peptide receptor (low-affinity variant FPRL-1) monoclonal antibodies whereas 2,7-dichlorofluorescein diacetate was used for measurement of levels of intracellular reactive oxygen species (ROS). HK2 cells were injured with H(2)O(2) in order to induce apoptosis: the effect was time- and dose-dependent. VIP increased the levels of the antiapoptotic protein Bcl-2 and decreased those of the proapoptotic protein Bax. VIP decreased the intracellular ROS levels reached by H(2)O(2)-induced oxidative stress. VIP effect on ROS levels involved FPLR-1 but not VPAC(1,2) receptors as evidenced by the use of the respective antagonists WRW4 and JV-1-53. Thus, VIP protects HK2 cells from apoptosis by increasing Bcl-2 levels and this effect is initiated through FPLR1 receptor. In conclusion, VIP might exert a renoprotective effect by the suppression of oxidative stress.

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.

Vasoactive intestinal peptide (VIP) inhibits human renal cell carcinoma proliferation.

Clear renal cell carcinoma (cRCC) is an aggressive and fatal neoplasm. The present work was undertaken to investigate the antiproliferative potential of vasoactive intestinal peptide (VIP) exposure on non-tumoral (HK2) and tumoral (A498, cRCC) human proximal tubular epithelial cell lines. Reverse transcription and semiquantitative PCR was used at the VIP mRNA level whereas enzyme immunoanalysis was performed at the protein level. Both renal cell lines expressed VIP as well as VIP/pituitary adenylate cyclase-activating peptide (VPAC) receptors whereas only HK2 cells expressed formyl peptide receptor-like 1 (FPRL-1). Receptors were functional, as shown by VIP stimulation of adenylyl cyclase activity. Treatment with 0.1µM VIP (24h) inhibited proliferation of A498 but not HK2 cells as based on a reduction in the incorporation of [(3)H]-thymidine and BrdU (5'-Br-2'-deoxyuridine), PCNA (proliferating-cell nuclear antigen) expression and STAT3 (signal transducer and activator of transcription 3) expression and activation. VPAC(1)-receptor participation was established using JV-1-53 antagonist and siRNA transfection. Growth-inhibitory response to VIP was related to the cyclic adenosine monophosphate (cAMP)/exchange protein directly activated by cAMP (EPAC)/phosphoinositide 3-kinase (PI3-K) signaling systems as shown by studies on adenylate cyclase stimulation, and using the EPAC-specific compound 8CPT-2Me-cAMP and specific kinase inhibitors such as H89, wortmannin and PD98059. The efficacy of VIP on the prevention of tumor progression was confirmed in vivo using xenografted athymic mouse. These actions support a potential role of this peptide and its agonists in new therapies for cRCC.

RNA interference-directed silencing of VPAC1 receptor inhibits VIP effects on both EGFR and HER2 transactivation and VEGF secretion in human breast cancer cells.

We used small-interference RNA (siRNA) to explore the mechanisms of some vasoactive intestinal peptide (VIP) actions on human breast cancer cells. Transfection of estrogen-dependent (T47D) and estrogen-independent (MDA-MB-468) breast cancer cells with VPAC(1)-receptor siRNA completely abolished VIP stimulatory effect on secretion of the main angiogenic factor, vascular endothelial growth factor (VEGF), and transactivation of epidermal growth factor receptor (EGFR or HER1) and HER2, two members of HER family of tyrosine-kinase receptors. The silencing procedure suggested the involvement of EGFR and HER2 transactivation in VIP-stimulated VEGF secretion. It was further supported by blocking tyrosine kinase activity by the selective HER inhibitors AG-1478 (EGFR) and AG-825 (HER2). Results give value to the specific signaling of VIP through VPAC(1) receptor in human breast cancer cells and support the potential use of VPAC(1)-receptor antagonists in combined targeted therapies for breast cancer. Molecular therapies involving RNA interference of VPAC(1)-receptor expression could also be considered.

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.

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.

Regulation of HER expression and transactivation in human prostate cancer cells by a targeted cytotoxic bombesin analog (AN-215) and a bombesin antagonist (RC-3095).

Bombesin (BN) and gastrin-releasing peptide (GRP) have been shown to stimulate the growth of human prostate cancer in vivo and in vitro by mechanisms initiated by binding of the peptide to BN/GRP receptor (GRPR). GRPR is overexpressed in a variety of human cancers, including human prostatic carcinoma. This led us to evaluate the effectiveness of blocking GRPR and of chemotherapy targeted to GRPR in androgen-dependent (LNCaP) and androgen-independent (PC-3) prostate cancer cells, which exhibit different features of disease progression. Thus, we used a cytotoxic BN/GRP analog, AN-215, consisting of 2-pyrrolinodoxorubicin (AN-201) linked to BN-like carrier peptide, and a BN/GRP receptor antagonist, RC-3095. Semiquantitative RT-PCR and Western blotting revealed that mRNA and protein levels for GRPR increased in prostate cancer cells as compared with nonneoplastic RWPE-1 cells. Immunofluorocytochemistry and Western blot assays revealed that AN-215 was the most effective analog decreasing both the expression of epidermal growth factor receptor family members and the activation of epidermal growth factor receptor and HER-2, which are associated to a poor prognosis. Furthermore, analogs targeted to BN/GRP receptors, AN-215 and RC-3095, blocked the effect of BN on cell growth in RWPE-1, LNCaP and PC-3 cells. These findings shed light on the mechanisms of action of these analogs and support the view that the use of AN-215 and RC-3095 for blocking BN/GRP receptors for targeted therapy may be of benefit for treatment of advanced prostate cancer.

Multifunctional role of VIP in prostate cancer progression in a xenograft model: suppression by curcumin and COX-2 inhibitor NS-398.

We used an in vivo model of human experimental prostate cancer in order to shed a new light on the effects of vasoactive intestinal peptide (VIP) on tumor growth as well as its pro-metastatic potential in this disease. We used nude mice subcutaneously injected with prostate cancer androgen-independent PC3 cells for 30 days. The regulatory role of VIP on cyclooxygenase-2 (COX-2) and vascular endothelial growth factor (VEGF) expression as well as on matrix metalloproteinase-2 and 9 (MMP-2 and 9) activities was examined. A selective COX-2 inhibitor, NS-398, and curcumin were used to block VIP effects. Xenografts of VIP-treated PC3 prostate cancer cells in nude mice gave tumors that grew significantly faster than those in the untreated group. It is conceivably a result of both the trophic effect of VIP on prostate cancer cells and the proangiogenic action of the neuropeptide in the growing tumor. We show the overexpression at mRNA and/or protein levels of VIP, its main receptor VPAC(1), the major angiogenic factor VEGF, and the pro-inflammatory enzyme COX-2 as well as the increased activity of MMP-2 and 9 in tumors derived from VIP-treated PC3 cells as compared with control group. The overexpression of the above biomarkers was suppressed in tumors derived from VIP-treated PC3 cells that had been previously incubated with curcumin or NS-398. Thus, the potential therapeutic role of curcumin and selective COX-2 inhibitors in combination with available VIP antagonists should be considered in prostate cancer therapy as supported by their inhibitory activities on tumor cell growth.

Vasoactive intestinal peptide behaves as a pro-metastatic factor in human prostate cancer cells.

BACKGROUND: There is little known on the involvement of vasoactive intestinal peptide (VIP) in the metastatic cascade of human prostate cancer, that is, cell proliferation, cell-cell adhesion, extracellular-matrix degradation, and migration/invasion. Here we evaluated the expression of related biomarker proteins (cyclin D1, metalloproteinases MMP-2 and MMP-9, and E-cadherin) in human androgen-dependent (LNCaP) and independent (PC3) prostate cancer cells. METHODS: Reverse transcriptase (RT)-polymerase chain reaction (PCR), gelatin zymography, Western blotting, confocal immunofluorescence microscopy, and assays on cell proliferation, adhesion, wound-healing, migration and random homing were performed. RESULTS: VIP increased cell proliferation and cyclin D1 expression whereas it decreased cell adhesion and E-cadherin expression in LNCaP and PC3 cells. VIP enhanced the gelatinolytic activity of MMP-2 and MMP-9. Semiquantitative RT-PCR assays showed that VIP stimulated mRNA levels of these MMPs and suppressed mRNA levels of its inhibitory protein RECK. VIP promoted cell invasion and migration, and the responses were faster according to the most aggressive status in cancer progression (androgen-independence). The involvement of nuclear factor-kappaB (NF-kappaB) was demonstrated since the anti-inflammatory agent curcumin blocked VIP effects on the above biomarkers in both cell lines. CONCLUSIONS: Taken together, these results and the presence of kappaB sites on gene promoter of cyclin D1, MMPs and, possibly, E-cadherin suggest that VIP may act as a cytokine in an early metastatic stage of human prostate cancer through the NF-kappaB/MMPs-RECK/E-cadherin system. Our findings may help to define novel targets and agents with potential usefulness in prostate cancer therapy.

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.