Induction of more aggressive tumoral phenotypes in LNCaP and PC3 cells by serum exosomes from prostate cancer patients.

Prostate cancer (PCa) is the second most frequent and sixth most fatal cancer in men worldwide. Despite its high prevalence, our understanding of its etiology and the molecular mechanisms involved in the progression of the disease is substantially limited. In recent years, the potential participation of exosomes in this process has been suggested. Therefore, we aim to study the effect of exosomes isolated from the serum of patients with PCa on various cellular processes associated with increased tumor aggressiveness in two PCa cell lines: LNCaP-FGC and PC3. The exosomes were isolated by filtration wand ultracentrifugation. Their presence was confirmed by immunodetection of specific markers and their size distribution was analyzed by Dynamic Light Scattering (DLS). The results obtained demonstrated that serum exosomes from PCa patients increased migration of PC3 cells and neuroendocrine differentiation of LNCaP-FGC cells regardless of the grade of the tumor. PCa serum exosomes also enhanced the secretion of enzymes related to invasiveness and resistance to chemotherapeutics, such as extracellular matrix metalloproteases 2 and 9, and gamma-glutamyltransferase in both cell lines. Altogether, these findings support the pivotal participation of exosomes released by tumoral cells in the progression of PCa. Future studies on the molecular mechanisms involved in the observed changes could provide crucial information on this disease and help in the discovery of new therapeutic targets.

Tumorigenic transformation of human prostatic epithelial cell line RWPE-1 by growth hormone-releasing hormone (GHRH).

BACKGROUND: Growth hormone-releasing hormone (GHRH) and its receptors have been implicated in the progression of various tumors. In this study, we analyzed the carcinogenetic potential of exposure to GHRH of a nontumor human prostate epithelial cell line (RWPE-1) as well as its transforming effect in a xenograft model. METHODS: We performed cell viability, cell proliferation, adhesion and migration assays. In addition, metalloprotease (MMP)-2 activity by means gelatin zymography, GHRH-R subcellular location using confocal immunofluorescence microscopy and vascular endothelial growth factor (VEGF) levels by enzyme-linked immunoassay were assessed. Besides, we developed an in vivo model in order vivo model to determine the role of GHRH on tumorigenic transformation of RWPE-1 cells. RESULTS: In cell cultures, we observed development of a migratory phenotype consistent with the gelatinolytic activity of MMP-2, expression of VEGF, as well as E-cadherin-mediated cell-cell adhesion and increased cell motility. Treatment with 0.1 µM GHRH for 24 h significantly increased cell viability and cell proliferation. Similar effects of GHRH were seen in RWPE-1 tumors developed by subcutaneous injection of GHRH-treated cells in athymic nude mice, 49 days after inoculation. CONCLUSIONS: Thus, GHRH appears to act as a cytokine in the transformation of RWPE-1 cells by mechanisms that likely involve epithelial-mesenchymal transition, thus reinforcing the role of GHRH in tumorigenesis of prostate.

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.

Growth hormone-releasing hormone receptor antagonists modify molecular machinery in the progression of prostate cancer.

BACKGROUND: Therapeutic strategies should be designed to transform aggressive prostate cancer phenotypes to a chronic situation. To evaluate the effects of the new growth hormone-releasing hormone receptor (GHRH-R) antagonists: MIA-602, MIA-606, and MIA-690 on processes associated with cancer progression as cell proliferation, adhesion, migration, and angiogenesis. METHODS: We used three human prostate cell lines (RWPE-1, LNCaP, and PC3). We analyzed several molecules such as E-cadherin, ß-catenin, Bcl2, Bax, p53, MMP2, MMP9, PCNA, and VEGF and signaling mechanisms that are involved on effects exerted by GHRH-R antagonists. RESULTS: GHRH-R antagonists decreased cell viability and provoked a reduction in proliferation in LNCaP and PC3 cells. Moreover, GHRH-R antagonists caused a time-dependent increase of cell adhesion in all three cell lines and retarded the wound closure with the highest value with MIA-690 in PC3 cells. GHRH-R antagonists also provoked a large number of cells in SubG0 phase revealing an increase in apoptotic cells in PC3 cell line. CONCLUSIONS: Taken all together, GHRH-R antagonists of the MIAMI series appear to be inhibitors of tumor progression in prostate cancer and should be considered for use in future therapeutic strategies on this malignancy.

Hydrogen bonding and anticancer properties of water-soluble chiral p-cymene Ru(II) compounds with amino-oxime ligands.

The investigation of the hydrogen-bonding effect on the aggregation tendency of ruthenium compounds [(η6-p-cymene)Ru(κNHR,κNOH)Cl]Cl (R = Ph (1a), Bn (1b)) and [(η6-p-cymene)Ru(κ2NH(2-pic),κNOH)][PF6]2 (1c), [(η6-p-cymene)Ru(κNHBn,κNO)Cl] (2b) and [(η6-p-cymene)Ru(κNBn,κ2NO)] (3b), has been performed by means of concentration dependence 1H NMR chemical shifts and DOSY experiments. The synthesis and full characterization of new compounds 1c, [(η6-p-cymene)Ru(κNPh,κ2NO)] (3a) and 3b are also reported. The effect of the water soluble ruthenium complexes 1a-1c on cytotoxicity, cell adhesion and cell migration of the androgen-independent prostate cancer PC3 cells have been assessed by MTT, adhesion to type-I-collagen and recovery of monolayer wounds assays, respectively. Interactions of 1a-1c with DNA and human serum albumin have also been studied. Altogether, the properties reported herein suggest that ruthenium compounds 1a-1c have considerable potential as anticancer agents against advanced prostate cancer.

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.

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.

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.

Carbohydrate effect of novel arene Ru(II) phenanthroline-glycoconjugates on metastatic biological processes.

Novel water-soluble half-sandwich ruthenium(II) polypyridyl-glycoconjugates [Ru(p-cymene)Cl{N-(1,10-phenanthroline-5-yl)-ß-glycopyranosylamine}][Cl] (glycopyranosyl = d-glucopyranosyl (1), D-mannopyranosyl (2), L-rhamnopyranosyl (3) and l-xylopyranosyl (4)) have been synthesized and fully characterized. Their behaviour in water under physiological conditions has been studied by nuclear magnetic resonance spectroscopy, revealing their hydrolytic stability. Interactions of the novel compounds with duplex-deoxiribonucleic acid (dsDNA) were investigated by different techniques and the results indicate that, under physiological pH and saline conditions, the metal glycoconjugates bind DNA in the minor groove and/or through external, electrostatic interactions, and by a non-classical, partial intercalation mechanism in non-saline phosphate buffered solution. Effects of compounds 1-4 on cell viability have been assessed in vitro against two human cell lines (androgen-independent prostate cancer PC-3 and non-tumorigenic prostate RWPE-1), showing moderate cytotoxicities, with IC50 values higher than those found for free ligands [N-(1,10-phenanthroline-5-yl)-ß-glycopyranosylamine] (glycopyranosyl = d-glucopyranosyl (a), D-mannopyranosyl (b), L-rhamnopyranosyl (c) and l-xylopyranosyl (d)) or corresponding metal-aglycone. Cell viability was assayed in the presence and absence of the glucose transporters (GLUTs) inhibitor [N4-{1-(4-cyanobenzyl)-5-methyl-3-(trifluoromethyl)-1H-pyrazol-4-yl}-7-fluoroquinoline-2,4-dicarboxamide] (BAY-876), and the results point to a negligible impact of the inhibition of GLUTs on the cytotoxicity caused by Ru(II) compounds 1-4. Remarkably, glycoconjugates 1-4 potently affect the migration pattern of PC-3 cells, and the wound healing assay evidence that the presence of the carbohydrate and the Ru(II) center is a requisite for the anti-migratory activity observed in these novel derivatives. In addition, derivatives 1-4 strongly affect the matrix metalloproteinase MMP-9 activities of PC-3 cells, while proMMP-2 and especially proMMP-9 were influenced to a much lesser extent.

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.

Cyclopentadienyl ruthenium(II) carbosilane metallodendrimers as a promising treatment against advanced prostate cancer.

In searching for efficient and selective antitumour drugs, a new family of carbosilane metallodendrimers functionalized with [Ru(η5-C5H5)(PTA)Cl] (PTA = 1,3,5-triaza-7-phosphatricyclo-[3.3.1.1] decane) is reported. Experiments of the biophysical characterization showed an ability to interact with biological membranes, as well as with proteins (e.g. human serum albumin) without affecting their usual biological activity. These metallodendrimers possessed potent and selective anticancer activity in vitro in a panel of tumour cell lines. Importantly, the first generation metallodendrimer, bearing 4 Ru(II) complexes, was remarkably active towards resistant prostate cancer cells, inhibiting both cell proliferation and metastasis to bone tissues. Such promising antitumour activity can be further improved when given with docetaxel, with in vitro cytotoxicity being in the nanomolar range. Furthermore, its intravenous administration to an advanced prostate cancer mice model inhibited tumour growth up to 25% and 45% when given 10 mg/kg/week and 7.5 mg/kg/4-5 days, respectively.

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.

Water soluble, optically active monofunctional Pd(ii) and Pt(ii) compounds: promising adhesive and antimigratory effects on human prostate PC-3 cancer cells.

New water soluble, enantiopure palladium and platinum compounds RN-[M{(1S,4R)-κNOH,κ2NH(2-pic)}Cl]Cl and SN-[M{(1R,4S)-κNOH,κ2NH(2-pic)}Cl]Cl (2-pic = 2-picolyl, M = Pd 1 and 1', Pt 2 and 2', respectively), and heterometallic Pd/Ti [(η5-C5H5)2Ti{(1S,4R)-κON,κ2NH(2-pic)}(PdCl)]Cl (3) have been synthesized. These novel compounds were fully characterized by NMR spectroscopy and CHN elemental analysis and 1, 1', 2 and 2' were further evaluated by polarimetry, ultra-violet and circular dichroism spectroscopy. The aqueous stability of novel compounds was studied by NMR spectroscopy under physiological conditions and the new species detected under such conditions have been characterized by NMR techniques and HR-ESI-MS (High-Resolution Electrospray Ionization Mass Spectrometry). Compound-DNA interactions have been investigated for the palladium and platinum compounds by equilibrium dialysis, Fluorescence Resonance Energy Transfer (FRET) DNA melting assays and viscometric titrations, revealing a better binding affinity and ability to affect duplex DNA of the palladium compounds. Metal derivatives have been tested in vitro against three cancer (prostate PC-3, cervical HeLa and breast MCF-7) and one non-tumorigenic (human prostate RWPE-1) cell lines. The highest anticancer activities were shown by palladium compounds 1 and 1' in all cancer lines, although their toxicity was lower than that found for cisplatin. Most importantly, the effect of the compounds on the cell adhesion and migration of the androgen-independent prostate cancer PC-3 cells has been assessed, and the efficacy of Pd enantiomers to affect the invasive phenotype of PC-3 cells has been demonstrated.

Insight into the antitumor activity of carbosilane Cu(ii)-metallodendrimers through their interaction with biological membrane models.

Current cancer therapies present serious drawbacks including severe side-effects and development of drug resistance. Strategies based on nanosized metallodrugs combine the structural diversity and non-classical modes of action of metal complexes with the selectivity arising from the unique interaction of nanoparticles with biological membranes. A new family of water-soluble copper(ii) carbosilane metallodendrimers was synthesized and characterized as a nanotechnological alternative to current therapies. The interactions occurring over time between the dendrimers, at different generations (G0 to G2) and with different Cu(ii) counter-ions (nitrate vs. chloride), and cell-membrane models (cethyl-trimethylammonium bromide (CTAB) micelles and lecithin liposomes) were investigated using a computer-aided analysis of the electron paramagnetic resonance (EPR) spectra. The EPR analysis provided structural and dynamical information on the systems indicating that the increase in generation and the change of the Cu(ii) contra-ion - from nitrate to chloride - produce an increased relative amount and strength of interaction of the dendrimer with the model membranes. Interestingly, the stabilization effect produced a lower toxicity towards cancer cells. The cytotoxic effect of Cu(ii) metallodendrimers was verified by an in vitro screening in a selection of tumor cell lines, revealing the impact of multivalency on the effectivity and selectivity of the metallodrugs. As a proof-of-concept, first-generation dendrimer G1-Cu(ONO2)2 was selected for in-depth in vitro and in vivo antitumor evaluation towards resistant prostate cancer. The Cu(ii)-metallodendrimers produced a significant tumor size reduction with no signs of toxicity during the experiment, confirming their promising potential as anticancer metallodrugs.

Biological evaluation of water soluble arene Ru(II) enantiomers with amino-oxime ligands.

New water soluble, enantiopure arene ruthenium compound SRuSN-(1R,4S)-[(η6-p-cymene)Ru{ĸNH(Bn),ĸNOH}Cl]Cl (Bn = benzyl, 1a') has been synthesized. The novel compound along with that previously described RRuRN-(1S,4R)-[(η6-p-cymene)Ru{ĸNH(Bn),ĸNOH}Cl]Cl (1a) was evaluated by polarimetry, ultra-violet and circular dichroism spectroscopy. The structure of novel ruthenium derivative 1a' was determined by single crystal X-ray crystallography. Both enantiomers have been tested against several cancer cell lines in vitro: prostate PC-3, lung A-549, pancreas MIA PaCa-2, colorectal HCT-116, leukemia Jurkat and cervical HeLa. Both enantiomers are active and versatile cytotoxic agents, showing IC50 values from 2 to 12 times lower than those found for cisplatin in the different cell lines evaluated. The mechanism of cell death induced by the metal compounds was analyzed in A-549 and Jurkat cell lines. Derivatives 1a and 1a' induced apoptotic cell death of A-549 cells while dose-dependent cell death mechanisms have been found in the Jurkat cell line. Compound-DNA interactions have been investigated by equilibrium dialysis, Fluorescence Resonance Energy Transfer (FRET) melting assays and viscometric titrations, revealing moderate binding affinity of 1a and 1a' towards duplex DNA. Finally, the efficacy of 1a in a preliminary in vivo assay of PC-3 xenografts in nude mice has been evaluated, resulting in a promising inhibition of tumor growth by 45%. Analysis of tumor tissue also showed a significant decrease of levels of crucial molecules in the invasive phenotype of PC-3 cells.

Vasoactive intestinal peptide induces cyclooxygenase-2 expression through nuclear factor-kappaB in human prostate cell lines Differential time-dependent responses in cancer progression.

The effect of vasoactive intestinal peptide (VIP) on cyclooxygenase-2 (COX-2) expression was analyzed in human prostate non-neoplastic (RWPE-1) as well as cancer androgen-dependent (LNCaP) and independent (PC3) cells. The three cell lines expressed VIP mRNA and VIP peptide, as measured by RT-PCR and immunochemistry, which supports an autocrine/paracrine action of VIP in the prostate gland. VIP levels were progressively higher from non-neoplastic to androgen-dependent and independent cells. Real-time RT-PCR and Western-blotting showed that VIP stimulated both COX-2 mRNA and protein expression in a faster manner as prostate cancer stage progressed (i.e. RWPE1

Transactivation of HER2 by vasoactive intestinal peptide in experimental prostate cancer: Antagonistic action of an analog of growth-hormone-releasing hormone.

Receptors for vasoactive intestinal peptide (VIP) and the human epidermal growth factor family of tyrosine kinase receptors (HER) are potent promoters of cell proliferation, survival, migration, adhesion and differentiation in prostate cancer cell lines. In this study, we analyzed the cross-talk between both classes of receptors through the regulation of HER2 transactivation and expression by VIP. Three growth-hormone-releasing hormone analogs endowed with antagonistic activity for VIP receptors (JV-1-51, -52, and -53) abrogated the autocrine/paracrine stimuli of VIP on androgen-independent PC3 cells in the absence or the presence of 10% fetal bovine serum. Semiquantitative and real-time quantitative RT-PCR together with Western blotting showed increased expression levels of both mRNA and proteins for HER2 and HER3 in PC3 and androgen-dependent LNCaP prostate cancer cells as compared to non-neoplastic RWPE-1 cells. VIP (100 nM) stimulated the expression levels of both HER2 and HER3 in PC3 cells in a time-dependent manner. Whereas these effects were relatively slow, VIP rapidly (0.5 min) increased HER2 tyrosine phosphorylation. This pattern of HER transactivation was blocked by H89, a protein kinase A (PKA) inhibitor, as well as by the specific VIP antagonist JV-1-53, indicating the involvement of VIP receptors and PKA activity in phosphorylated HER2 formation. These findings support the merit of further studies on the potential usefulness of VIP receptor antagonists and both HER2 antibodies and tyrosine kinase inhibitors for prostate 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) increases vascular endothelial growth factor (VEGF) expression and secretion in human breast cancer cells.

Previous studies have shown that vasoactive intestinal peptide (VIP) and its receptors (VPAC(1) and VPAC(2) receptors) are involved in promotion and growth of many human tumours including breast cancer. Here we investigated whether VIP regulates the expression of the main angiogenic factor, vascular endothelial cell growth factor (VEGF) in human oestrogen-dependent (T47D) and oestrogen-independent (MDA-MB-4687) breast cancer cells. Semiquantitative and quantitative real-time RT-PCRs were used at mRNA level whereas enzyme immunoanalysis was performed at protein level. Both cancer cell lines expressed VIP and VPAC(1) (but not VPAC(2)) receptors that were functional as shown by VIP stimulation of adenylate cyclase activity. VIP induced VEGF expression at both mRNA and protein levels following a time-dependent pattern. The responses were faster in T47D than in MDA-MB-468 cells. The observed VIP regulation of VEGF expression appears to be modulated at least by the cAMP/protein kinase A (PKA) and the phosphoinositide 3-kinase (PI3-K) signalling systems as shown by studies of adenylate cyclase stimulation and using specific kinase inhibitors such as H89 and wortmannin. These actions suggest a proangiogenic potential of VIP in breast cancer.