A

Abstract:  Current evidence on diet-cancer interaction remains incomplete, particularly in oral carcinoma (OC). This study aimed to evaluate the relationship among daily saturated, monounsaturated and polyunsaturated fatty acids (FAs) intake and eating patterns in people with / without OC, during a minimum period of five years, and its possible relationship with SNP TP53-R72P.  A retrospective case-control study (n = 79), matched by sex and age (21-85 years old), was carried out. Dietary information was collected using a quantitative food frequency survey validated for Cordoba population. TP53-R72P alleles were determined using conventional PCR. The Mann–Whitney U test and logistic regression were applied to assess the association between case/control status and FAs intake adjusted by alcohol/tobacco, respectively. Dietary patterns were identified by principal component factor analysis. The intake of saturated FAs such as myristic (p = 0.037), palmitic (p = 0.004), stearic (p = 0.009), arachidic (p = 0.012), monounsaturated such as palmitoleic (p = 0.013), oleic (p = 0.002) and polyunsaturated as linoleic (p = 0.0006) and alpha-linoleic (p = 0.003) was higher in cases. Omega6/omega3 ratio was significantly higher in cases (p = 0.003). The mutated C allele was more frequent in cases (p = 0.0061) than controls. All surveyed patients with OC were heterozygous for TP53-R72. In addition, they presented a high consumption of total fats in relation to controls. The studied population presented a dietary pattern described as “Western diet”;  related with a high daily intake of meat, eggs and alcohol; it was remarkable an association between red meat consumption and the presence of OC (p = 0.015). This diet has a high amount of omega-6 FAs, which leads to an increase in arachidonic acid and its derivatives, than have been related to the risk of OC. The presence of mutated TP53-R72P allele in OC patients is related to the loss of ability to induce apoptosis. The presence of both risk factors, diet and presence of a mutated polymorphic variant, could linked to an increaserisk of developing OC.Resumen:  La evidencia actual sobre la interacción dieta-cáncer sigue siendo incompleta, particularmente en el carcinoma oral (CO). El objetivo del estudio fue evaluar la relación entre la ingesta dietaria de ácidos grasos (AGs) saturados, monoinsaturados y poliinsaturados y patrones de alimentación en personas con/sin CO, durante un período mínimo previo de cinco años al momento de la encuesta; y su posible relación con la presencia del polimorfismo TP53-R72P. Se realizó un estudio retrospectivo de casos/controles (n=79), apareados por sexo y edad (21-85 años). Se recolectaron datos de un cuestionario cuantitativo de frecuencia alimentaria validado para la población cordobesa. TP53-R72P se genotipificó por PCR alelo específica. Se aplicaron la prueba Mann-Whitney y regresión logística para evaluar la asociación entre casos/controles y la ingesta de AGs ajustada por alcohol/tabaco, respectivamente. Los patrones alimentarios se identificaron por análisis factorial de componentes principales. La ingesta de AGs saturados como mirístico (p=0.037), palmítico (p=0.004), esteárico (p=0.009), araquídico (p=0.012), monoinsaturados como palmitoleico (p=0.013), oleico (p=0.002) y poliinsaturados como linoleico (p=0.0006) y alfa-linoleico (p=0.003) fue mayor en los casos. La relación omega-6/omega-3 fue significativamente mayor en los casos (p=0.003).  De los sujetos genotipificados, el alelo mutado C fue más frecuente en los casos (p=0.0061) que en los controles. De los sujetos encuestados y con genotipificación TP53-R72P se observó que todos los pacientes con CO eran heterocigotos y, además, presentaron un alto consumo de grasas en relación a los controles. La población total presentó un patrón alimentario reconocido como dieta occidental asociada a una ingesta diaria elevada en carnes, huevos y alcohol de la población estudiada; siendo notorio una asociación entre el consumo de carnes rojas y la presencia de CO (p=0.015). Esta dieta presenta una alta cantidad de AGs omega-6, lo que conduce a un aumento del ácido araquidónico y sus derivados, los cuales se han relacionado con el riesgo de CO. La presencia del alelo mutado TP53-R72P en pacientes con CO está relacionado con la pérdida de capacidad de conducir a la célula a apoptosis. La presencia de ambos factores de riesgo, dieta y presencia de variante polimórfica mutada, podría incrementar el riesgo de desarrollar CO.

A

Abstract:  Pancreatic ductal adenocarcinoma (PDCA) is one of the most aggressive and lethal cancers in the western world with a very poor survival. A characteristic pathway in the initiation of PDAC is the activation of the lipid-modified Sonic Hedgehog (SHH) ligand. Polyunsaturated fatty acids (PUFAs) are natural ligands of the transcription factor Gamma Peroxisome Proliferator Activated Receptor (PPARγ), which is also key to the SHH metabolic network. However, it is unknown how PUFAs regulate the SHH signaling pathway and PPARγ involved in the development of PDAC. Here we evaluated the effect of ω-3 and ω-6 PUFAs on SHH and PPARγ activation on tumor progression employing the human pancreatic cancer line PANC-1 in-vitro and in KPC knock-in transgenic mice in-vivo. PANC-1 cells were treated with PUFAs: arachidonic acid (ω-6, AA), eicosapentaenoic acid (ω-3, EPA) or docosahexaenoic acid (ω-3, DHA). Animals were fed with a semisynthetic diet with corn oil (ω-6) or fish oil (ω-3). The mRNA was analyzed by qPCR, proteins by Western Blot and cell viability of PANC-1 by Resazurin. Gas Chromatography was used to analyze the PUFAs profile of the PANC-1 cells and KPC mice tumors. Tumor volume was measured using a caliper, the fibrotic index by histologic assessment (Masson staining) and SHH by Immunochemistry. Data were analyzed by ANOVA. In PANC-1 cells the results showed that DHA reduced SHH gene and protein expression, increased PPARγ expression levels (p<0.05) and reduced cell viability in a dose-dependent manner (p<0.0001). Membrane lipid profile in PANC-1 and in KPC tumor cells correlated with pure and dietary PUFAs treatment respectively. The ω-3 significantly reduced tumor size (p<0.05), stromal desmoplasia (p<0.01) and SHH expression (p<0.05). The data obtained demonstrate that ω-3 PUFAs could modulate pancreatic tumor progression through PPARγ activation and SHH regulation promoting changes in the tissue environment affecting tumor growth.Resumen:  El adenocarcinoma de páncreas ductal (ACPD) es uno de los cánceres más mortales entre todas las neoplasias del mundo occidental con escasa sobrevida. Una vía característica en la iniciación de ACPD es la activación del ligando Sonic Hedgehog (SHH), modificado por lipidos. Los ácidos grasos poliinsaturados (AGPs)  también son ligandos naturales del factor de transcripción Receptor Gama Activado por Proliferadores de Peroxisomas gama (PPARγ) que además, es clave de la red metabólica de SHH. Sin embargo se desconoce como los AGPs regulan la vía de señalización de SHH y el factor PPARγ involucrados en el desarrollo del ACPD. Aquí evaluamos el efecto de AGPs ω-3 y ω-6 sobre la activación de SHH y PPARγ en la progresión tumoral empleando la línea de cáncer pancreático humano PANC-1 in-vitro y en ratones transgénicos knock-in KPC in-vivo. Las PANC-1 fueron tratadas con  AGPs: ácido araquidónico (ω-6, AA), ácido eicosapentanoico (ω-3, EPA) o ácido docosahexaenoico (ω-3, DHA). Los animales fueron alimentados con una dieta semisintética con aceite de maíz (ω-6) o aceite de pescado (ω-3). Se analizó el  ARNm por qPCR,  las proteinas por Western Blot y la viabilidad en PANC-1 mediante Resazurina. Se analizaron los datos por ANOVA.  En PANC-1 y en tumores de ratones KPC se evaluó el perfil de AGPs por cromatografía gaseosa. Se analizó el volumen tumoral usando calimetro, el índice fibrótico en cortes histológicos (tinción Masson) y SHH por Inmunohistoquimica. Los resultados indicaron que en PANC-1, el DHA redujo la expresión génica  y proteica de  SHH, incrementó los niveles de expresión de PPARγ  (p<0.05) y redujo la viabilidad celular de manera dosis-dependiente (p<0.0001). El perfil lipídico de membranas en PANC-1 y células tumorales KPC se correlacionó con  el tratamiento de AGPs puros y dietarios respectivamente. Los ω-3 redujeron significativamente el tamaño tumoral (p<0.0452), la desmoplasia estromal (p<0.0082) y la expresión de SHH (p<0.0433). Los datos obtenidos demuestran que los AGPs ω-3 podrían modular la progresión tumoral pancreática a través de la activación de PPARγ y de la regulación de SHH promoviendo cambios en el ambiente tisular afectando el crecimiento tumoral.

A

Published evidence suggests a strong association between nutrition and the development and maintenance of tumoral processes. In Córdoba, a greater adherence to dietary patterns (DP) provided by carbohydrates- and saturated fatty acids-rich food, represented mainly by fructose (F) and palmitic acid (PA), is positively associated with the risk of colorectal and breast cancer. However, the cellular mechanisms by which these components promote tumor aggressiveness are not fully understood. Our objective was to evaluate in vitro the role of tumor-associated fibroblasts (CAFs) as mediators of the proliferative effects of F and PA. F88 cell line, corresponding to human breast cancer CAFs, was grown in DMEM with 10% FBS and stimulated with F 40 mM, AP 250 uM, combinations of both (F+PA) or their respective vehicles, for 24 hours (characteristic concentrations of DP in Cordoba). Subsequently, supernatants were removed, total proteins were quantified and stored at -80 °C for assays. Breast epithelial tumor cells of the MCF7 line were cultured in DMEM medium with 10% FBS and then stimulated with conditioned media (from F88 cells) for 24 hours. As controls, conditioned media from vehicle-treated cells were used. Any of the stimuli did not cause statistically significant changes in F88 cell proliferation. F + AP combination did induce a strong phenotypic change, with greater development of proteinopoietic and secretory organelles when observed by transmission electron microscopy. In addition, stimuli with PA and F + PA produced a decrease in the expression of Fibroblast Activation Protein (FAP) by western blot. On the other hand, MCF7 cells when stimulated with conditioned media from F88 treated with F + PA showed an increase in cell proliferation (ANOVA, p <0.05), determined by incorporation of bromodeoxyuridine and cell count. However, there were no significant differences between F and PA individually. These results suggest a pathogenic effect of food rich in F and PA on tumor proliferation in breast cancer, which would be mediated by CAFs. Numerosas evidencias señalan una fuerte asociación entre componentes nutricionales y el desarrollo y mantenimiento de procesos tumorales. En Córdoba, se describió que la mayor adherencia a patrones dietarios (PD) provistos por alimentos ricos en carbohidratos y ácidos grasos saturados, representados principalmente por fructosa (F) y ácido palmítico (AP) respectivamente, se asocia positivamente al riesgo de cáncer colorrectal y de mama. Sin embargo, no se conocen completamente los mecanismos celulares por los cuales estos componentes promoverían mayor agresividad tumoral. Nuestro objetivo fue evaluar in vitro el rol de los fibroblastos asociados a tumores (CAFs) como mediadores de posibles efectos proliferativos de F y AP. Para ello, células de la línea F88, correspondientes a CAFs de cáncer mamario humano fueron crecidas en medio DMEM con 10% SFB y estimuladas con F 40 mM, AP 250 uM, combinaciones de ambos (F+AP) o sus respectivos vehículos, por 24 horas (concentraciones características de PD cordobeses). Posteriormente, se retiraron sobrenadantes, se cuantificaron proteínas totales y se guardaron a -80 °C para los ensayos posteriores. Células tumorales epiteliales de mama de la línea MCF7 fueron cultivadas en medio DMEM con 10% SFB y posteriormente estimuladas con los medios condicionados (provenientes de las células F88) por 24 horas. Como controles, se utilizaron los medios condicionados de células tratadas con vehículo. Los estímulos no provocaron cambios estadísticamente significativos en la proliferación celular de las F88, aunque la combinación F+AP sí indujo un fuerte cambio fenotípico, con mayor desarrollo de organelas proteinopoiéticas y secretorias al ser observadas por microscopía electrónica de transmisión. Además, los estímulos con AP y F+AP produjeron una disminución en la expresión de Fibroblast Activation Protein (FAP) por western blot.  Por otro lado, las células MCF7 al ser estimuladas con medios condicionados provenientes de F88 tratadas con F+AP mostraron un incremento en la proliferación celular (ANOVA, p<0,05), determinada mediante incorporación de bromodeoxiuridina y conteo celular. No obstante, no hubo diferencias significativas entre F y AP en forma individual. Estos resultados sugieren un efecto patogénico de alimentos ricos en F y AP sobre la proliferación tumoral en el cáncer de mama, que sería mediado por CAFs.

A

Abstract:  Glioblastomas (GBs) are the most common primary tumors of the central nervous system. The first-line treatment is surgical resection complemented with chemo and radiotherapy, but due to its late diagnosis and its infiltrative characteristic the survival is low. The chemotherapy used in most GBs is Temozolomide (TMZ). As a second line, other chemotherapeutic agents and oncolytic virotherapy are used. It is known that omega-6 polyunsaturated fatty acids (PUFAs) have a high therapeutic potential in GBs treatment since they exhibit tumoricidal activity without significant side effects. In the present work we study the effects of two omega-6 PUFAs: arachidonic acid (AA, 20:04) and gamma linolenic acid (GLA, 18:03) associated with TMZ on cell viability and proliferation and their relationship with p53 expression in a murine glioblastoma cell line (GL26). The cells were treated with: AA, GLA, TMZ, AA + TMZ or GLA + TMZ [10, 20, 30, 40, 50 and 100 µM] and ethanol (control). We evaluate cell viability by fluorimetry with resazurin; cell proliferation with Ki67 and p53 expression by immunocytochemistry. Lipid cell profile by gas chromatography (GC) and generation of eicosanoids by high pressure liquid chromatography (HPLC). Statistical analysis was performed using the Kruskal Wallis test. Our results show that AA + TMZ and GLA + TMZ significantly reduce the viability compared to TMZ (p <0.05). GLA, TMZ and GLA + TMZ significantly reduce the expression of Ki67 (p <0.01) and increase the expression of p53 (p <0.1) with respect to the control. The percentages of AA and GLA are higher in GL26 cell membranes treated with AA and GLA respectively in relation to cells treated with TMZ. GLA reduce the release of 12-HHT compared to the control. TMZ combined with GLA exerts an antiproliferative effect by decreasing the release of 12-HHT and increasing the expression of p53.Resumen:  Los gliobastomas (GBs) son los tumores primarios más frecuentes del sistema nervioso central. El tratamiento de primera línea es la resección quirúrgica  complementada con quimio y radioterapia pero por su diagnóstico tardío y su poder infiltrativo la supervivencia es baja. El quimioterápico usado en la mayoría de los GBs es la Temozolomida (TMZ). Como segunda línea se emplean otros quimioterapéuticos y viroterapia oncolítica. Se conoce que los ácidos grasos poliinsaturados (AGPs) de la familia omega-6 tienen un alto potencial terapéutico en el tratamiento de GBs ya que exhiben actividad tumoricida sin presentar  efectos secundarios significativos. En el presente trabajo estudiamos los efectos de dos AGPs omega-6: el ácido araquidónico (AA, 20:04) y el ácido gama linolénico (GLA, 18:03) asociados a TMZ sobre la viabilidad y proliferación celular y su relación con la expresión de p53 en una línea celular de glioblastoma murina (GL26). Las células fueron tratadas con: AA, GLA, TMZ, AA+TMZ o GLA+TMZ [10, 20, 30, 40, 50 y 100 µM] y etanol  (control).   Evaluamos viabilidad celular por fluorimetría con resazurina; proliferación celular con Ki67 y expresión de p53 por inmunocitoquímica; perfil celular lipídico por cromatografía de gases (GC) y generación de eicosanoides por cromatografía líquida de alta presión (HPLC). El análisis estadístico se realizó mediante la prueba de Kruskal Wallis. Nuestros resultados mostraron que AA+TMZ y GLA+TMZ redujeron significativamente la viabilidad respecto a TMZ (p<0.05). GLA, TMZ y GLA+TMZ redujeron significativamente la expresión de KI67 (p<0.01) y aumentaron la expresión de p53 (p<0.1) con respecto al control. Los porcentajes de AA y GLA fueron mayores en membranas de células GL26 tratadas con AA y GLA respectivamente en relación a las tratadas con TMZ.  GLA redujo la liberación del eicosanoide 12-HHT respecto al control. La TMZ combinada con GLA ejerce un efecto antiproliferativo disminuyendo la liberación de 12-HHT y aumentando la expresión de p53.

Treatment of Peritoneal Carcinomatosis by Targeted Delivery of the Radio-Labeled Tumor Homing Peptide 213Bi-DTPA-[F3]2 into the Nucleus of Tumor Cells

BACKGROUND: Alpha-particle emitting isotopes are effective novel tools in cancer therapy, but targeted delivery into tumors is a prerequisite of their application to avoid toxic side effects. Peritoneal carcinomatosis is a widespread dissemination of tumors throughout the peritoneal cavity. As peritoneal carcinomatosis is fatal in most cases, novel therapies are needed. F3 is a tumor homing peptide which is internalized into the nucleus of tumor cells upon binding to nucleolin on the cell surface. Therefore, F3 may be an appropriate carrier for alpha-particle emitting isotopes facilitating selective tumor therapies. PRINCIPAL FINDINGS: A dimer of the vascular tumor homing peptide F3 was chemically coupled to the alpha-emitter (213)Bi ((213)Bi-DTPA-[F3](2)). We found (213)Bi-DTPA-[F3](2) to accumulate in the nucleus of tumor cells in vitro and in intraperitoneally growing tumors in vivo. To study the anti-tumor activity of (213)Bi-DTPA-[F3](2) we treated mice bearing intraperitoneally growing xenograft tumors with (213)Bi-DTPA-[F3](2). In a tumor prevention study between the days 4-14 after inoculation of tumor cells 6x1.85 MBq (50 microCi) of (213)Bi-DTPA-[F3](2) were injected. In a tumor reduction study between the days 16-26 after inoculation of tumor cells 6x1.85 MBq of (213)Bi-DTPA-[F3](2) were injected. The survival time of the animals was increased from 51 to 93.5 days in the prevention study and from 57 days to 78 days in the tumor reduction study. No toxicity of the treatment was observed. In bio-distribution studies we found (213)Bi-DTPA-[F3](2) to accumulate in tumors but only low activities were found in control organs except for the kidneys, where (213)Bi-DTPA-[F3](2) is found due to renal excretion. CONCLUSIONS/SIGNIFICANCE: In conclusion we report that (213)Bi-DTPA-[F3](2) is a novel tool for the targeted delivery of alpha-emitters into the nucleus of tumor cells that effectively controls peritoneal carcinomatosis in preclinical models and may also be useful in oncology

Successful Expansion but Not Complete Restriction of Tropism of Adeno-Associated Virus by In Vivo Biopanning of Random Virus Display Peptide Libraries

Targeting viral vectors to certain tissues in vivo has been a major challenge in gene therapy. Cell type-directed vector capsids can be selected from random peptide libraries displayed on viral capsids in vitro but so far this system could not easily be translated to in vivo applications. Using a novel, PCR-based amplification protocol for peptide libraries displayed on adeno-associated virus (AAV), we selected vectors for optimized transduction of primary tumor cells in vitro. However, these vectors were not suitable for transduction of the same target cells under in vivo conditions. We therefore performed selections of AAV peptide libraries in vivo in living animals after intravenous administration using tumor and lung tissue as prototype targets. Analysis of peptide sequences of AAV clones after several rounds of selection yielded distinct sequence motifs for both tissues. The selected clones indeed conferred gene expression in the target tissue while gene expression was undetectable in animals injected with control vectors. However, all of the vectors selected for tumor transduction also transduced heart tissue and the vectors selected for lung transduction also transduced a number of other tissues, particularly and invariably the heart. This suggests that modification of the heparin binding motif by target-binding peptide insertion is necessary but not sufficient to achieve tissue-specific transgene expression. While the approach presented here does not yield vectors whose expression is confined to one target tissue, it is a useful tool for in vivo tissue transduction when expression in tissues other than the primary target is uncritical

A Novel Fibronectin Binding Motif in MSCRAMMs Targets F3 Modules

BBK32 is a surface expressed lipoprotein and fibronectin (Fn)-binding microbial surface component recognizing adhesive matrix molecule (MSCRAMM) of Borrelia burgdorferi, the causative agent of Lyme disease. Previous studies from our group showed that BBK32 is a virulence factor in experimental Lyme disease and located the Fn-binding region to residues 21-205 of the lipoprotein.Studies aimed at identifying interacting sites between BBK32 and Fn revealed an interaction between the MSCRAMM and the Fn F3 modules. Further analysis of this interaction showed that BBK32 can cause the aggregation of human plasma Fn in a similar concentration-dependent manner to that of anastellin, the superfibronectin (sFn) inducing agent. The resulting Fn aggregates are conformationally distinct from plasma Fn as indicated by a change in available thermolysin cleavage sites. Recombinant BBK32 and anastellin affect the structure of Fn matrices formed by cultured fibroblasts and inhibit endothelial cell proliferation similarly. Within BBK32, we have located the sFn-forming activity to a region between residues 160 and 175 which contains two sequence motifs that are also found in anastellin. Synthetic peptides mimicking these motifs induce Fn aggregation, whereas a peptide with a scrambled sequence motif was inactive, suggesting that these motifs represent the sFn-inducing sequence.We conclude that BBK32 induces the formation of Fn aggregates that are indistinguishable from those formed by anastellin. The results of this study provide evidence for how bacteria can target host proteins to manipulate host cell activities

A multi-element psychosocial intervention for early psychosis (GET UP PIANO TRIAL) conducted in a catchment area of 10 million inhabitants: study protocol for a pragmatic cluster randomized controlled trial

Multi-element interventions for first-episode psychosis (FEP) are promising, but have mostly been conducted in non-epidemiologically representative samples, thereby raising the risk of underestimating the complexities involved in treating FEP in 'real-world' services