RESUMEN
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.
Asunto(s)
Receptores ErbB/genética , Regulación Neoplásica de la Expresión Génica , Hormona Liberadora de Hormona del Crecimiento/fisiología , Activación Transcripcional , Neoplasias de la Mama Triple Negativas/metabolismo , Línea Celular Tumoral , Movimiento Celular , Receptores ErbB/metabolismo , Femenino , Humanos , Metaloproteinasas de la Matriz/metabolismo , Transducción de Señal , Neoplasias de la Mama Triple Negativas/genética , Neoplasias de la Mama Triple Negativas/patología , Familia-src Quinasas/metabolismoRESUMEN
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.
Asunto(s)
Núcleo Celular/metabolismo , Subunidad p50 de NF-kappa B/metabolismo , Transducción de Señal/efectos de los fármacos , Péptido Intestinal Vasoactivo/farmacología , Línea Celular , Curcumina/farmacología , AMP Cíclico/metabolismo , Proteínas Quinasas Dependientes de AMP Cíclico/metabolismo , Ciclooxigenasa 2/genética , Ciclooxigenasa 2/metabolismo , Dinoprostona/metabolismo , Quinasas MAP Reguladas por Señal Extracelular/metabolismo , Factores de Intercambio de Guanina Nucleótido/metabolismo , Humanos , Masculino , Fosfatidilinositol 3-Quinasas/metabolismo , Próstata/citología , Próstata/metabolismo , Neoplasias de la Próstata/metabolismo , Neoplasias de la Próstata/patología , Receptores de Péptido Intestinal Vasoactivo/metabolismo , Activación Transcripcional/efectos de los fármacos , Factor A de Crecimiento Endotelial Vascular/metabolismoRESUMEN
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.
Asunto(s)
Carcinoma de Células Renales/genética , Proteína p53 Supresora de Tumor/biosíntesis , Factor A de Crecimiento Endotelial Vascular/genética , Péptido Intestinal Vasoactivo/administración & dosificación , Carcinoma de Células Renales/tratamiento farmacológico , Carcinoma de Células Renales/patología , Línea Celular Tumoral , Proliferación Celular/efectos de los fármacos , AMP Cíclico/metabolismo , Humanos , Fosfatidilinositol 3-Quinasas/genética , Especies Reactivas de Oxígeno/metabolismo , Transducción de Señal/efectos de los fármacosRESUMEN
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.
Asunto(s)
Carcinoma de Células Renales/metabolismo , Regulación Neoplásica de la Expresión Génica , Neoplasias Renales/metabolismo , Péptido Intestinal Vasoactivo/farmacología , Transporte Activo de Núcleo Celular , Animales , Línea Celular Tumoral , Núcleo Celular/metabolismo , Humanos , Masculino , Ratones , Ratones Desnudos , Metástasis de la Neoplasia , Trasplante de Neoplasias , beta Catenina/metabolismoRESUMEN
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.
Asunto(s)
Antineoplásicos/farmacología , Carcinoma de Células Renales/tratamiento farmacológico , Neoplasias Renales/tratamiento farmacológico , Estrés Oxidativo/efectos de los fármacos , Péptido Intestinal Vasoactivo/farmacología , Cadherinas/metabolismo , Carcinoma de Células Renales/secundario , Adhesión Celular/efectos de los fármacos , Línea Celular Tumoral/efectos de los fármacos , Movimiento Celular , Humanos , Neoplasias Renales/patología , Metaloproteinasa 2 de la Matriz/metabolismo , Metaloproteinasa 9 de la Matriz/metabolismo , Subunidad p50 de NF-kappa B/metabolismo , Invasividad Neoplásica , Especies Reactivas de Oxígeno/metabolismo , Factor A de Crecimiento Endotelial Vascular/metabolismo , beta Catenina/metabolismoRESUMEN
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.
Asunto(s)
Antineoplásicos/farmacología , Hormona Liberadora de Hormona del Crecimiento/análogos & derivados , Hormona Liberadora de Hormona del Crecimiento/antagonistas & inhibidores , Metástasis de la Neoplasia/tratamiento farmacológico , Neovascularización Patológica/tratamiento farmacológico , Neoplasias de la Próstata/tratamiento farmacológico , Sermorelina/análogos & derivados , Animales , Cadherinas/metabolismo , Adhesión Celular , Línea Celular Tumoral , Proliferación Celular , Hormona Liberadora de Hormona del Crecimiento/farmacología , Humanos , Subunidad alfa del Factor 1 Inducible por Hipoxia/metabolismo , Masculino , Metaloproteinasa 2 de la Matriz/metabolismo , Metaloproteinasa 9 de la Matriz/metabolismo , Ratones , Ratones Desnudos , Invasividad Neoplásica , Neoplasias de la Próstata/patología , Distribución Aleatoria , Receptores de Neuropéptido/metabolismo , Receptores de Hormona Reguladora de Hormona Hipofisaria/metabolismo , Sermorelina/farmacología , Factores de Crecimiento Endotelial Vascular/metabolismo , Ensayos Antitumor por Modelo de Xenoinjerto , beta Catenina/metabolismoRESUMEN
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.
Asunto(s)
Antioxidantes/farmacología , Estrés Oxidativo/efectos de los fármacos , Péptido Intestinal Vasoactivo/farmacología , Antioxidantes/metabolismo , Apoptosis/efectos de los fármacos , Supervivencia Celular/efectos de los fármacos , Células Cultivadas , Relación Dosis-Respuesta a Droga , Humanos , Peróxido de Hidrógeno/antagonistas & inhibidores , Peróxido de Hidrógeno/farmacología , Especies Reactivas de Oxígeno/metabolismo , Relación Estructura-Actividad , Factores de Tiempo , Péptido Intestinal Vasoactivo/metabolismoRESUMEN
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.
Asunto(s)
Carcinoma de Células Renales/patología , Neoplasias Renales/patología , Péptido Intestinal Vasoactivo/metabolismo , Animales , Carcinoma de Células Renales/genética , Línea Celular Tumoral , Proliferación Celular/efectos de los fármacos , Transformación Celular Neoplásica/patología , AMP Cíclico/metabolismo , Regulación Neoplásica de la Expresión Génica/efectos de los fármacos , Humanos , Espacio Intracelular/efectos de los fármacos , Espacio Intracelular/metabolismo , Neoplasias Renales/genética , Ratones , Ratones Desnudos , ARN Mensajero/genética , ARN Mensajero/metabolismo , Receptores de Péptido Intestinal Vasoactivo/genética , Receptores de Péptido Intestinal Vasoactivo/metabolismo , Receptores de Tipo I del Polipéptido Intestinal Vasoactivo/metabolismo , Factor de Transcripción STAT3/genética , Factor de Transcripción STAT3/metabolismo , Transducción de Señal/efectos de los fármacos , Péptido Intestinal Vasoactivo/genética , Péptido Intestinal Vasoactivo/farmacología , Ensayos Antitumor por Modelo de XenoinjertoRESUMEN
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.