Nutrigenomic approach to study the potential role of walnut polyphenols and their human metabolites in cancer prevention and treatment

Abstract

Epidemiologic studies and clinical trials have demonstrated consistent benefits of walnut (Juglans regia L.) consumption. Walnuts have been described as a rich source of polyphenols with a broad array of different structures. However, an accurate screening of its complete phenolic profile was lacking. Using liquid chromatography coupled with electrospray ionization hybrid linear trap quadrupole-Orbitrap mass spectrometry (LC–LTQ-Orbitrap) a comprehensive identification of phenolic compounds in walnuts was performed. A total of 120 compounds were identified, with ellagitannins, ellagic acid and its derivatives, as major polyphenols in walnuts.

After consumption, ellagitannins are hydrolyzed to release ellagic acid, which is converted by gut microflora to urolithin A (UA) and urolithin B (UB). Ellagitannins and their metabolites have been shown to slow down cancer development and progression. The identification of dietary agents that may modulate molecular pathways related to cancer is of great interest. Therefore, the main aim of this study was to determine gene expression changes induced by urolithins using a nutrigenomic approach.

We investigated the effects of urolithins on the expression of prostate specific antigen and the androgen receptor in a prostate cancer cell model. The mRNA and protein levels of both prostate specific antigen and androgen receptor were down-regulated after incubating LNCaP cells with urolithins A and B. Transient transfection of PC-3 cells with a luciferase construct driven by the PSA-promoter containing three androgen response elements showed that urolithins inhibited AR-mediated PSA expression at the transcriptional level. Electrophoretic Mobility Shift Assays revealed that urolithins decreased the androgen receptor binding to its consensus Response Element. In addition, we performed a genomic analysis to study the effect of UA on whole genome expression in LNCaP cells. CDKN1A, a node gene determined by Biological Association Networks (BANs) using the list of differentially expressed genes, was further validated; its promoter activity, mRNA and protein levels were significantly up-regulated.

The role urolithins have in the modulation of the cell cycle and apoptosis induction was also explored. Cell cycle was measured by flow cytometry, and apoptosis was determined by caspase 3/7 activation and using the rhodamine method. Cell cycle analyses showed an increase at G1, we also observed an induction of apoptosis and caspase 3/7 activation and this effect correlated with a decrease in the anti-apoptotic protein Bcl-2.

Our results suggest that urolithins A and B attenuate the function of the AR by repressing its expression. A down-regulation of AR and PSA mRNA and protein levels could provoke an interruption of the interaction between PSA and AR, with a proven role in prostate cancer development and progression. In addition a clear effect of urolithin A on whole genome expression in a prostate cancer cell model was observed. The significant up regulation of p21, which has a known role in cell cycle and apoptosis, correlated with an increased percentage of cells in the G1 phase of the cell cycle, furthermore, the multi-targeted effects of urolithins resulted in an induction of apoptosis. The aforementioned results indicate a potential role of walnuts as a chemo-preventive and/or chemo-therapeutic agent for prostate cancer.

Estudios clínicos y epidemiológicos han demostrado que existen múltiples beneficios sobre la salud por incluir nueces (Juglans regia L.) en la dieta. Existe una amplia evidencia de la relación de la dieta y componentes específicos de nuestros alimentos con vías moleculares importantes en el desarrollo del cáncer. La etiología del cáncer es multifactorial, una variedad de factores genéticos, dietéticos y de estilo de vida juegan un rol fundamental en el desarrollo y progresión de esta enfermedad. La identificación de los mecanismos moleculares involucrados en la carcinogénesis y su relación con factores de riesgo modificables genera una base para el desarrollo de estrategias específicas de prevención. La nutrigenómica es un enfoque nuevo en la nutrición que busca explorar la interacción gen-dieta y el posible papel que esta interacción juega en el desarrollo de enfermedades. Utilizando una aproximación nutrigenómica, el objetivo de esta tesis doctoral es analizar el efecto de las urolitinas A y B, metabolitos del polifenol mayoritario en nueces, sobre vías moleculares clave en el desarrollo y progresión del cáncer de próstata.

Nuestros resultados sugieren que las urolitinas A y B atenúan la función del receptor de andrógeno al inhibir su expresión. La disminución de los niveles de mRNA y proteína del receptor de andrógeno y su diana el antígeno prostático especifico podría causar una interrupción en la interacción entre ambas proteínas, las cuales se sabe juegan un papel en el desarrollo y progresión del cáncer de próstata. Además, a través de un estudio genómico funcional se pudo comprobar el claro efecto que ejerce la urolitina A sobre la expresión génica en un modelo de cáncer de próstata. La urolitina A causó una sobrexpresión de p21, proteína reguladora del ciclo celular, lo cual se correlacionó a un aumento en el porcentaje de células en la fase G1 del ciclo celular. Asimismo, el efecto de las urolitinas sobre múltiples dianas, incluyendo Bcl-2, caspasas 3 y 7 y p21, resultaron en la inducción de apoptosis.

En resumen, el estudio de la relación entre dieta y cáncer a través de la aplicación de nuevas tecnologías ligadas a enfoques novedosos como la nutrigenómica podría permitir desarrollar recomendaciones y estrategias de prevención efectivas.