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Procesos oncogénicos como proliferación incontrolable, resistencia apoptótica, aumento de mecanismos angiogénicos y evasión inmune son regulados generalmente por factores de transcripción, como HIF-1. Por tanto, se ha señalado a esta molécula como un blanco terapéutico prometedor. Para explorar esta posibilidad, un oligonucleótido tipo “señuelo” dirigido a HIF-1α (ODN) fue diseñado para evaluar su eficiencia en esquema tanto monoterapéutico, como en combinación con dos agentes quimioterapéuticos en un modelo in vitro de cáncer de seno. Después de comprobar, mediante citometría de flujo e inmunofluorescencia, la localización del blanco, el señuelo fue transfectado en la línea celular MDA-MB-231. Se estableció la IC-50 de HIF-1α ODN, Cisplatino y Taxol con el método de Resazurina. Mecanismos de muerte celular fueron evaluados con el método de TUNEL. Por último, se estableció el índice de combinación (IC) de cada uno de los quimio-agentes en combinación con el ODN. Se evidencio que HIF-1α ODN causa un efecto citotóxico en MDA-MB-231 de hasta un 90% hacia las 72h pos-tratamiento. Este efecto no se observa tanto en los controles del ensayo, como en el cultivo primario de células no tumorales (FIBRO), siendo este agente altamente selectivo hacia células tumorales, al activar mecanismos pro-apoptóticos. A su vez, HIF-1α ODN potencializa la actividad tumorogénica de Cisplatino y Taxol en la línea celular tumoral. Por tanto, HIF-1α ODN demostró tener actividad selectiva potencialmente antitumoral, al disminuir la proliferación celular e inducir apoptosis; optimizando de forma sinérgica, la eficacia de fármacos quimioterapéuticos de alto espectro, en tratamientos combinados.

Resumen Introducción. Los fibroblastos gingivales (FGs) son células del tejido conjuntivo gingival que han tomado en los últimos años una relevancia promisoria por su probable utilización en la terapia celular, dadas sus capacidades de multipotencialidad y de autorrenovación. Objetivo. Conocer y describir el impacto de la ausencia en la suplementación de Suero Fetal Bovino (SFB) en la supervivencia de fibroblastos gingivales en cultivos. Materiales y métodos. Fibroblastos gingivales fueron aislados de tejido gingival de pacientes sanos y cultivados en medios de cultivos DMEM (Dulbecco's Modified of Eagle Medium) en ausencia y suplementados con 0.2% de SFB a 37°C en una atmósfera húmeda con 5% de CO2. Se llevó a cabo una evaluación morfológica, de supervivencia y proliferación de los FGs, así como la identificación mediante la técnica de inmunofluorescencia de marcadores del citoesqueleto celular como la actina y mitocondrias. Resultados. Los FGs cultivados en ausencia y con suplementación de 0.2% de SFB evidenciaron una forma fusiforme, con núcleos ovalados y numerosas prolongaciones citoplasmáticas durante el tiempo de cultivo. Un leve aumento en la proliferación de FGs fue observado en aquellas células en contacto con el medio DMEM+0.2% de SFB comparadas con el medio donde estuvo ausente la suplementación. El inmunomarcaje de la actina y las mitocondrias dejó en evidencia que la ausencia y suplementación a 0.2% de SFB no afectó su localización en los FGs evaluados. Conclusión. Los fibroblastos gingivales sobreviven y proliferan en ausencia de SFB, conservando sus características morfológicas celulares. Abstract Introduction. Gingival fibroblasts (GF) are cells of gingival connective tissue that have taken promising relevance in recent years due to their probable use in cell therapy, given their multipotencial and self-renewal capabilities. Objective. To know and to describe the impact of the absence of Fetal Bovine Serum (FBS supplementation on the survival of gingival fibroblasts in cultures. Materials and methods. Gingival fibroblasts were isolated from gingival tissue of healthy patients and cultured in DMEM (Dulbecco's Modified of Eagle Medium) culture media in absence and supplemented with 0.2% FBS at 37 ° C in a humid atmosphere with 5% CO2. A morphological evaluation, survival and proliferation of GF were carried out, as well as the identification by the immunofluorescence technique of cellular cytoskeleton markers such as actin and mitochondria. Results. The GF grown in the absence and with supplementation of 0.2% FBS showed a fusiform shape, with oval nuclei and numerous cytoplasmic extensions during the culture time. A slight increase in the proliferation of GF was observed in those cells in contact with the DMEM medium +0.2% FBS compared to the medium where the supplementation was absent. Immunostaining of actin and mitochondria showed that the absence and supplementation to 0.2% of FBS did not affect its location in the evaluated. Conclusion. Gingival fibroblasts survive and proliferate in the absence of FBS, preserving their cellular morphological characteristics.

The synthetic compound fasentin has been described as a modulator of GLUT-1 and GLUT-4 transporters, thus inhibiting glucose uptake in some cancer cells. Endothelial glucose metabolism has been recently connected to angiogenesis and it is now an emerging topic in scientific research. Indeed, certain compounds with a known effect on glucose metabolism have also been shown to inhibit angiogenesis. In this work we tested the capability of fasentin to modulate angiogenesis in vitro and in vivo. We show that fasentin inhibited tube formation in endothelial cells by a mechanism that involves a negative effect on endothelial cell proliferation and invasion, without affecting other steps related to the angiogenic process. However, fasentin barely decreased glucose uptake in human dermal microvascular endothelial cells and the GLUT-1 inhibitor STF-31 failed to inhibit tube formation in these cells. Therefore, this modulatory capacity on endothelial cells function exerted by fasentin is most likely independent of a modulation of glucose metabolism. Taken together, our results show a novel biological activity of fasentin, which could be evaluated for its utility in cancer and other angiogenesis-dependent diseases.

Objective: The role of diffusion-weighted MRI in differential diagnostics and predicting the survival of patients with spine metastases was studied. Methods: The study included data from MRI and morphological studies of 23 patients with spine metastases. Results: The values obtained for the apparent diffusion coefficient (ADC) of tumors were compared with their histological type, cell density and Ki-67 proliferation index. The effect of ADC values on overall patient survival was also assessed. A reliable inverse correlation was established between ADC values and Ki-67 proliferation index for various types of spine metastases (r=-0.753, p=0.017). The dependence of ADC values and overall survival of patients with metastases in the spine is shown. Conclusion: The technique of diffusion-weighted MRI can be used as part of a comprehensive assessment in the preoperative planning of surgical treatment, and as a prognostic factor of overall survival for this group of patients. Level of Evidence II. Prognostic retrospective study, RESUMO Objetivos: O papel da RM ponderada por difusão nos diagnósticos diferenciais e na previsão da sobrevida dos pacientes com metástases da coluna vertebral foram analisados. Métodos: O estudo incluiu dados da RM e estudos morfológicos de 23 pacientes com metástases da coluna vertebral. Resultados: Os valores obtidos para o coeficiente de difusão aparente (ADC) dos tumores foram comparados com seu tipo histológico, densidade celular e índice de proliferação Ki-67. Além disso, avaliou-se o efeito dos valores de ADC na sobrevida global dos pacientes. Foi estabelecida uma correlação inversa confiável entre os valores de ADC e o índice de proliferação Ki-67 para diversos tipos de metástases da coluna vertebral (r =-0,753, p=0,017). A dependência dos valores de ADC e a sobrevida global dos pacientes com metástases na coluna vertebral é demostrada. Conclusão: A técnica da RM ponderada por difusão pode ser utilizada como parte de uma avaliação abrangente do planejamento pré-operatório do tratamento cirúrgico e como fator prognóstico da sobrevida global para esse grupo de pacientes. Nível de Evidência II. Estudo retrospectivo prognóstico. RESUMEN Objetivos: Fueron analizados el papel de la RM ponderada por difusión en los diagnósticos diferenciales y en la previsión de la sobrevida de los pacientes con metástasis de la columna vertebral. Métodos: El estudio incluyó datos de RM y estudios morfológicos de 23 pacientes con metástasis de la columna vertebral. Resultados: Los valores obtenidos para el coeficiente de difusión aparente (ADC) de los tumores se compararon con su tipo histológico, densidad celular e índice de proliferación Ki-67. Además, se evaluó el efecto de los valores de ADC en la sobrevida global de los pacientes. Se estableció una correlación inversa confiable entre los valores de ADC y el índice de proliferación Ki-67 para diversos tipos de metástasis de la columna vertebral (r =-0,753, p=0,017). Es demostrada la dependencia de los valores de ADC y la sobrevida global de los pacientes con metástasis en la columna vertebral. Conclusión: La técnica de RM ponderada por difusión puede utilizarse como parte de una evaluación integral de la planificación preoperatoria del tratamiento quirúrgico y como un factor pronóstico de la sobrevida global para este grupo de pacientes. Nivel de Evidencia II. Estudio retrospectivo pronóstico.

Simple Summary The treatment of oral squamous cell carcinoma (OSCC) represents a significant problem worldwide. Among cancers with the highest incidence, OSCC renders one of the worst prognoses. Therefore, novel prognostic biomarkers and therapeutic tools to tackle OSCC are urgently needed. Somatostatin-analogues (SSA) are an invaluable therapeutic option in the treatment of several cancers. We aimed to determine the expression levels of all somatostatin-receptors (SSTs) in OSCC, compared to adjacent healthy control tissues, to analyze the relationship of SSTs expression with key clinical and histopathological data, and to explore the direct in vitro effect of different SSAs on OSCC cancer cells. Our findings highlight a potential role of SST2 as a good prognostic biomarker for recurrence and metastasis in OSCC and unveil that SSA exerts antitumoral effects on OSCC cells, providing a relevant clinical conclusion, which should be soon tested for their use in humans. Abstract Oral squamous cell carcinoma (OSCC) incidence has increased by 50% over the last decade. Unfortunately, surgery and adjuvant radiotherapy and chemotherapy are still the mainstream modality of treatment, underscoring the need for alternative therapies. Somatostatin-analogues (SSA) are efficacious and safe treatments for a variety of tumors, but the presence of somatostatin-receptors (SSTs) and pharmacological effects of SSA on OSCC are poorly known. In this study, we demonstrated that SST2 and SST3 levels were significantly higher in OSCC, compared to adjacent healthy control tissues. SST2 expression was associated with less regional metastasis and a lower recurrence rate. Moreover, SST2 was elevated in OSCC and associated with histopathological good prognosis factors, such as high peritumoral inflammation, smaller depth of invasion, and expansive vs. infiltrative front of tumor invasion. Importantly, treatment with different SSA (octreotide, lanreotide, and pasireotide) significantly reduced cell-proliferation in OSCC primary cell cultures. Altogether, this study demonstrated that SST2 is overexpressed in OSCC vs. healthy tissues and could represent a novel prognostic biomarker, since its expression is associated with tumors that show better prognostic factors and less recurrent rate. Moreover, our data unveil clear antitumoral effects of SSAs on OSCC, opening new avenues to explore their potential as targeting therapy to OSCC.

© The Author(s) 2021., [Background]: Pancreatic ductal adenocarcinoma (PDAC) is a highly lethal cancer, requiring novel treatments to target both cancer cells and cancer stem cells (CSCs). Altered splicing is emerging as both a novel cancer hallmark and an attractive therapeutic target. The core splicing factor SF3B1 is heavily altered in cancer and can be inhibited by Pladienolide-B, but its actionability in PDAC is unknown. We explored the presence and role of SF3B1 in PDAC and interrogated its potential as an actionable target. [Methods]: SF3B1 was analyzed in PDAC tissues, an RNA-seq dataset, and publicly available databases, examining associations with splicing alterations and key features/genes. Functional assays in PDAC cell lines and PDX-derived CSCs served to test Pladienolide-B treatment effects in vitro, and in vivo in zebrafish and mice. [Results]: SF3B1 was overexpressed in human PDAC and associated with tumor grade and lymph-node involvement. SF3B1 levels closely associated with distinct splicing event profiles and expression of key PDAC players (KRAS, TP53). In PDAC cells, Pladienolide-B increased apoptosis and decreased multiple tumor-related features, including cell proliferation, migration, and colony/sphere formation, altering AKT and JNK signaling, and favoring proapoptotic splicing variants (BCL-XS/BCL-XL, KRASa/KRAS, Δ133TP53/TP53). Importantly, Pladienolide-B similarly impaired CSCs, reducing their stemness capacity and increasing their sensitivity to chemotherapy. Pladienolide-B also reduced PDAC/CSCs xenograft tumor growth in vivo in zebrafish and in mice. [Conclusion]. SF3B1 overexpression represents a therapeutic vulnerability in PDAC, as altered splicing can be targeted with Pladienolide-B both in cancer cells and CSCs, paving the way for novel therapies for this lethal cancer., This work has been supported by Spanish Ministry of Economy [MINECO; BFU2016–80360-R (to JPC)] and Ministry of Science and Innovation [MICINN; PID2019-105201RB-I00 (to JPC), PID2019-105564RB-I00 (to RML)]. Instituto de Salud Carlos III, co-funded by European Union (ERDF/ESF, “Investing in your future”) [FIS Grants PI17/02287 and PI20/01301 (to MDG), PI18/00757 (to BS,Jr); DTS Grant DTS20/00050 (to RML)); Postdoctoral Grant Sara Borrell CD19/00255 (to AIC); Predoctoral contract FI17/00282 (to EAP)]. Coordinated grant (GC16173694BARB) from the Fundación Asociación Española Contra el Cáncer (AECC) (to BS,Jr). Spanish Ministry of Universities [Predoctoral contracts FPU14/04290 (to SPA); FPU16/06190 (to VHA); FPU18/02275 (to RBE)]. Boehringer Ingelheim Fonds travel grant (EAP). Junta de Andalucía (BIO0139). GETNE2016 and GETNE2019 Research grants (to JPC); and CIBERobn. Associazione Italiana Ricerca Cancro (AIRC 5×1000 n. 12182); Fondazione Italiana Malattie Pancreas – Ministero Salute [FIMPCUP_J38D19000690001]; Fondazione Cariverona: Oncology Biobank Project “Antonio Schiavi” (prot.203885/2017).

Resumen Introducción. Se ha demostrado que el caseinato de sodio y sus componentes (caseínas α, β y κ) inhiben la proliferación de la línea celular hematopoyética de ratón 32D clone 3 (32Dcl3) e inducen su diferenciación hacia macrófagos. Se sabe que la caseína α induce la producción de IL-1β y que esta última citocina inhibe la proliferación celular mediante la producción del factor de necrosis tumoral alfa (TNF-α), pero se desconoce si el caseinato de sodio y las caseínas inducen la producción de TNF y si este es el responsable de la inhibición de la proliferación. Objetivo. Evaluar si el caseinato de sodio y las caseínas α, β y κ inhiben la proliferación de la línea celular 32Dcl3 mediante la producción de TNF-α. Materiales y métodos. Se usaron diferentes concentraciones de caseinato de sodio y de las caseínas α, β y κ en las células 32Dcl3. Posteriormente, se evaluaron la viabilidad celular mediante una prueba con el MTT [3-(4,5-dimetiltiazol-2-ilo)-2,5-difeniltetrazol], la inducción de apoptosis con la citometría de flujo y la síntesis del TNF-α con el ELISA. Además, se hicieron pruebas de neutralización con anti-TNF-α en células 32Dcl3 tratadas con caseinato de sodio y caseína α, y se evaluó la proliferación celular. Resultados. Se encontró que el caseinato de sodio y las caseínas α, β y κ reducían la proliferación de la línea celular 32Dcl3 sin afectar la viabilidad, y que solo el caseinato y la caseína α inducían la apoptosis y la liberación al medio de TNF-α. La proliferación de células 32Dcl3 tratadas con caseinato y caseína α se restableció al usar anticuerpos anti-TNF-α. Conclusión. El TNF-α fue el principal responsable de la inhibición de la proliferación en las células 32Dcl3 tratadas con caseinato de sodio o caseína α. Abstract Introduction: Sodium caseinate (CS) and its components (alpha-casein, beta-casein, and kappa-casein) have been shown to inhibit the proliferation of the mouse hematopoietic 32D clone 3 (32Dcl3) cell line and induce its differentiation into macrophages. It is well-known that alpha-casein induces IL-1β production and that this cytokine inhibits the proliferation via the production of tumor necrosis factor alpha (TNF-alpha), but it is not known if CS and the caseins inhibit the proliferation via TNF-alpha production. Objective: To evaluate if CS and alpha-casein, beta-casein and kappa-casein inhibit the proliferation on 32Dcl3 cell line via TNF-alpha. Materials and methods: We used different concentrations of CS, alpha-casein, beta-casein and kappa-casein in 32Dcl3 cells to evaluate cell proliferation. We assessed cell viability by MTT, induction to apoptosis by flow cytometry, and TNF-alpha synthesis by ELISA. Additionally, we performed anti-TNF-alpha neutralization assays on 32Dcl3 cells treated with CS and alpha-casein and we evaluated proliferation. Results: The results showed that CS, alpha-casein, beta-casein, and kappa-casein reduced proliferation of the 32Dcl3 cell line without affecting the viability and that only CS and alpha-casein induced apoptosis and the release of TNF-alpha. The 32Dcl3 cells treated with CS and alpha-casein reestablished their proliferation by using anti-TNF-alpha antibodies. Conclusion: TNF-alpha was the main responsible for the inhibition of proliferation in 32Dcl3 cells treated with CS or alpha-casein.

La formación de nuevas células se genera a partir de células preexistentes a través de una serie ordenada de eventos denominada ciclo celular. El control de este es fundamental para la integridad del genoma, por lo que hay múltiples proteínas regulando este proceso. Actualmente, se conoce que el complejo Mantenimiento de Minicromosoma (MCM2-7) tiene un rol esencial en la replicación del ADN, específicamente involucrado en la proliferación, durante el ciclo celular. La identificación inmunohistoquimica de las proteínas de este complejo a nivel tisular, podría ser una herramienta muy útil, para usarlos como biomarcadores y así comprender uno de los mecanismos biológicos que se ven afectados en el cáncer. Nuestro objetivo es realizar una revisión del complejo MCM2-7, ya que estas proteínas como se ha descrito, podrían comportarse como buenos marcadores biológicos de proliferación celular, y así poder realizar un buen diagnóstico, pronóstico y futuros blancos terapéuticos de aquellas lesiones principalmente neoplásicas, principalmente las que asientan a nivel de la mucosa bucal. New cells are formed from preexisting cells through an ordered series of events called cell cycle. As the control of this cycle is fundamental for genome integrity, multiple proteins regulate this process. We currently know that the Minichromosome maintenance (MCM2-7) complex has a major role in DNA replication in the cell cycle, in particular regarding proliferation. The immunohistochemical identification of the proteins in this complex on tissues may be useful, as they could be used as biomarkers and would help us understand one of the biological mechanisms affected in cancer processes. Our aim is to collect the existing evidence regarding the members of the MCM2-7 complex, since these proteins could be effective biological cell proliferation markers, which would help practitioners make accurate diagnosis, prognosis and future therapeutic targets of lesions that are mainly neoplastic, especially in the buccal mucosa.

The study of cancer metabolism is regaining center stage and becoming a hot topic in tumor biology and clinical research, after a period where such kind of experimental approaches were somehow forgotten or disregarded in favor of powerful functional genomic and proteomic studies [...]

The seeds of Euphorbia lathyris have been used in traditional medicine to treat various medical conditions. However, neither all of their active biocompounds nor the molecular mechanisms underlying their therapeutic effects have been described. A new ethanolic extract of defatted flour from mature seeds of Euphorbia lathyris showed a high total polyphenol content and significant antioxidant activity. Chromatographic analysis showed that esculetin, euphorbetin, gaultherin, and kaempferol-3-rutinoside were the most abundant polyphenolic bioactive compounds. Antiproliferative assays showed a high and selective antitumor activity against colon cancer cell lines (T84 and HCT-15). In addition, a significant antiproliferative activity against glioblastoma multiforme cells was also demonstrated. Its mechanism of action to induce cell death was mediated by the overexpression of caspases 9, 3, and 8, and by activation of autophagy. Interestingly, a reduction in the migration capacity of colon cancer cells and a significant antiangiogenic effect on human umbilical vein endothelial cells were also demonstrated. Finally, the extract significantly reduced the subpopulations of cancer stem cells. This extract could be the basis to develop new therapeutic strategies for the treatment of colon cancer, although further experiments will be necessary to determine its in vivo effects., Granada University (project PSE/17/002), Ministerio de Ciencia e Innovación (project RTC-2017-6540-1), CTS-107 and AGR145 Groups

Hepatocellular carcinoma (HCC) represents 80% of the primary hepatic neoplasms. It is the sixth most frequent neoplasm, the fourth cause of cancer-related death, and 7% of registered malignancies. Sorafenib is the first line molecular targeted therapy for patients in advanced stage of HCC. The present study shows that Sorafenib exerts free radical scavenging properties associated with the downregulation of nuclear factor E2-related factor 2 (Nrf2)-regulated thioredoxin 1 (Trx1) expression in liver cancer cells. The experimental downregulation and/or overexpression strategies showed that Trx1 induced activation of nitric oxide synthase (NOS) type 3 (NOS3) and S-nitrosation (SNO) of CD95 receptor leading to an increase of caspase-8 activity and cell proliferation, as well as reduction of caspase-3 activity in liver cancer cells. In addition, Sorafenib transiently increased mRNA expression and activity of S-nitrosoglutathione reductase (GSNOR) in HepG2 cells. Different experimental models of hepatocarcinogenesis based on the subcutaneous implantation of HepG2 cells in nude mice, as well as the induction of HCC by diethylnitrosamine (DEN) confirmed the relevance of Trx1 downregulation during the proapoptotic and antiproliferative properties induced by Sorafenib. In conclusion, the induction of apoptosis and antiproliferative properties by Sorafenib were related to Trx1 downregulation that appeared to play a relevant role on SNO of NOS3 and CD95 in HepG2 cells. The transient increase of GSNOR might also participate in the deactivation of CD95-dependent proliferative signaling in liver cancer cells., Graphical abstract Graphical Abstract. Sorafenib reduced ROS/RNS cytoplasmic concentrations which were associated with decreased Nrf2 nuclear translocation, Trx1 expression/activity, and SNO–NOS3 expression and NO intracellular generation in HepG2. The reduction of SNO–NOS3 expression and NO generation might also be influenced by the induction of transient GSNOR expression/activity and reduced NOS3 expression by Sorafenib. The diminution of SNO-CD95 expression by Sorafenib involved reduction of caspase-8 activity and cell proliferation, and increased downstream caspase-3 activity in liver cancer cells.Image 1, Highlights • Sorafenib induces mitochondrial ROS generation, but also acts as nucleophilic scavenger. • Sorafenib reduces Nrf2-depenent Trx1 expression, and SNO–NOS3 and SNO-CD95 ratios. • Sorafenib-related antitumoral in vivo activity involves diminution of Trx1 and SNO-CD95.

This article belongs to the Special Issue Trends in Molecular Research for Transplantation Immunology., Regulatory T cells (Tregs) are essential for the maintenance of tolerance to self and non-self through cell-intrinsic and cell-extrinsic mechanisms. Peripheral Tregs survival and clonal expansion largely depend on IL-2 and access to co-stimulatory signals such as CD28. Engagement of tumor necrosis factor receptor (TNFR) superfamily members, in particular TNFR2 and DR3, contribute to promote peripheral Tregs expansion and sustain their survival. This property can be leveraged to enhance tolerance to allogeneic transplants by tipping the balance of Tregs over conventional T cells during the course of immune reconstitution. This is of particular interest in peri-transplant tolerance induction protocols in which T cell depletion is applied to reduce the frequency of alloreactive T cells or in conditioning regimens that allow allogeneic bone marrow transplantation. These conditioning regimens are being implemented to limit long-term side effects of continuous immunosuppression and facilitate the establishment of a state of donor-specific tolerance. Lymphopenia-induced homeostatic proliferation in response to cytoreductive conditioning is a window of opportunity to enhance preferential expansion of Tregs during homeostatic proliferation that can be potentiated by agonist stimulation of TNFR., This work has been supported by grant FIS PI13/00029 (Fondo de Investigaciones Sanitarias, Ministry of Health, Spanish Government and co-funded by European Union ERDF/ESF, “Investing in your future”), LE093U13 and Unit of Excellence Research UIC #012 (Department of Education of the Regional Government, Junta de Castilla y Leon), and Gerencia Regional de Salud (BIO/01/15) to JIRB. It was also funded by FIS PI16/00002 (Instituto de Salud Carlos III and co-funded by European Union ERDF/ESF, “Investing in your future”) and Gerencia Regional de Salud GRS963/A/2014, GRS1142/A/2015 and GRS 1505/A/2017 to M.L.R.G. This work has been supported with funding from the Spanish National Network CIBER-ONC (oncology research) CB16/12/00480 to JAPS. PS is supported by the Swiss National Science Foundation (grant 31003A-176526).

Glutaminase (GA) catalyzes the first step in mitochondrial glutaminolysis playing a key role in cancer metabolic reprogramming. Humans express two types of GA isoforms: GLS and GLS2. GLS isozymes have been consistently related to cell proliferation, but the role of GLS2 in cancer remains poorly understood. GLS2 is repressed in many tumor cells and a better understanding of its function in tumorigenesis may further the development of new therapeutic approaches. We analyzed GLS2 expression in HCC, GBM and neuroblastoma cells, as well as in monkey COS-7 cells. We studied GLS2 expression after induction of differentiation with phorbol ester (PMA) and transduction with the full-length cDNA of GLS2. In parallel, we investigated cell cycle progression and levels of p53, p21 and c-Myc proteins. Using the baculovirus system, human GLS2 protein was overexpressed, purified and analyzed for posttranslational modifications employing a proteomics LC-MS/MS platform. We have demonstrated a dual targeting of GLS2 in human cancer cells. Immunocytochemistry and subcellular fractionation gave consistent results demonstrating nuclear and mitochondrial locations, with the latter being predominant. Nuclear targeting was confirmed in cancer cells overexpressing c-Myc- and GFP-tagged GLS2 proteins. We assessed the subnuclear location finding a widespread distribution of GLS2 in the nucleoplasm without clear overlapping with specific nuclear substructures. GLS2 expression and nuclear accrual notably increased by treatment of SH-SY5Y cells with PMA and it correlated with cell cycle arrest at G2/M, upregulation of tumor suppressor p53 and p21 protein. A similar response was obtained by overexpression of GLS2 in T98G glioma cells, including downregulation of oncogene c-Myc. Furthermore, human GLS2 was identified as being hypusinated by MS analysis, a posttranslational modification which may be relevant for its nuclear targeting and/or function. Our studies provide evidence for a tumor suppressor role of GLS2 in certain types of cancer. The data imply that GLS2 can be regarded as a highly mobile and multilocalizing protein translocated to both mitochondria and nuclei. Upregulation of GLS2 in cancer cells induced an antiproliferative response with cell cycle arrest at the G2/M phase.

A high adherence to a Mediterranean diet has been related to numerous beneficial effects in human health, including a lower incidence and mortality of prostate cancer (PCa). Olive oil is an important source of phenolic bioactive compounds, mainly hydroxytyrosol (HT), of this diet. Be-cause of the growing interest of this compound and its derivatives as a cancer chemopreventive agent, we aimed to compare the in vitro effect of HT isolated from olive mill wastewaters and five semisynthetic alkyl ether, ester, and nitro-derivatives against prostate cancer (PCa) cell lines. The effect in cell proliferation was determined in RWPE-1, LNCaP, 22Rv1, and PC-3 cells by resazurin assay, the effect in cell migration by wound healing assay, and tumorsphere and colony formation were evaluated. The changes in key signaling pathways involved in carcinogenesis were assessed by using a phosphorylation pathway profiling array and by Western blotting. Antiproliferative effects of HT and two lipophilic derivatives [hydroxytyrosyl acetate (HT-Ac)/ethyl hydroxytyrosyl ether (HT-Et)] were significantly higher in cancerous PC-3 and 22Rv1 cells than in non-malignant RWPE-1 cells. HT/HT-Ac/HT-Et significantly reduced migration capacity in RWPE-1 and PC-3 and prostatosphere size and colony formation in 22Rv1, whereas only HT-Ac and HT-Et reduced these functional parameters in PC-3. The cytotoxic effect in 22Rv1 cells was correlated with modifications in the phosphorylation pattern of key proteins, including ERK1/2 and AKT. Consistently, HT-Ac and HT-Et decreased p-AKT levels in PC-3. In sum, our results suggest that HT and its lipophilic derivatives could be considered as potential therapeutic tools in PCa. Junta de Andalucía PI-0152-2019, BIO-0139, RH-0084-2020 Ministerio de Economía y Competitividad PID2019-105564RB-I00, PID2019-105201RB-I00, FPU17/00263, FPU16/06190, FPU18/02485

The scaffolds and their interaction with mesenchymal stem cells are objects of study in bioengineering and tissue repair. Mechanisms such as surface adhesion, proliferation, viability, and cytotoxicity are essential for the development of therapies. The present study analyzed the influence of platelet-rich fibrin (PRF) in viability, cytotoxicity, and proliferation of stem cells from human exfoliated deciduous teeth (SHED) exposed to bovine biomaterial surfaces. The studied groups were divided and analyzed as follows: (S) only SHED as control Group; (SB) SHED + biomaterial; (SBP) SHED + biomaterial + PRF. Analyses of cells seeded in 24-well plates were performed after 24, 48 and 72 hours. Individual groups were subjected to viability, cytotoxicity and cell proliferation tests using neutral red, MTT and crystal violet, respectively; and in the 72-hour group, scanning electron microscopy (SEM) was performed to record cell ultra-morphology. Data were submitted to statistical analysis by two-factor ANOVA with a significance level of 5%. The results demonstrated a better performance in the viability/cytotoxicity and proliferation of stem cells for the group (SBP) in comparison to the group (SB) and the group (S). The applied statistical tests showed that the biomaterial factor, time, and interaction between them gave rise to results with statistical significance. SHED submitted to bovine biomaterial were more viable, proliferative and with lower toxicity when associated with PRF. PRF seemed to activate the metabolism of stem cells in culture, indicating that such an association can bring an effective benefit in clinical outcome. El marco y su interacción con las células madres mesenquimales son objetos de estudio en bioingeniería y reparación de tejidos. Mecanismos como la adhesión superficial, la proliferación, la viabilidad y la citotoxicidad son fundamentales para el desarrollo de terapias. El presente estudio analizó la influencia de la fibrina rica en plaquetas (PRF) sobre la viabilidad, citotoxicidad y proliferación de células madre mesenquimales de dientes primarios humanos exfoliados (SHED) en contacto con biomateriales bovinos. Los grupos estudiados se dividieron y analizaron de la siguiente manera: (S) solo SHED como grupo de control; (SB) SHED + biomaterial; (SBP) SHED + biomaterial + PRF. Se realizaron análisis de células sembradas en placas de 24 pocillos después de 24, 48 y 72 horas. Los grupos individuales se sometieron a pruebas de viabilidad, citotoxicidad y proliferación celular utilizando rojo neutro, MTT y violeta cristal, respectivamente, y en el grupo de 72 horas, se realizó microscopía electrónica de barrido (SEM) para registrar la morfología celular. Los datos se sometieron a análisis estadístico mediante ANOVA de dos factores con un nivel de significancia del 5%. Los resultados demostraron un mejor desempeño en la viabilidad / citotoxicidad y proliferación de células madre para el grupo (SBP) en comparación con el grupo (SB) y el grupo (S). Las pruebas estadísticas aplicadas mostraron que el factor biomaterial, el tiempo y la interacción entre ellos dieron lugar a resultados con significación estadística, las SHED sometidas a biomaterial bovino fueron más viables, proliferativos y con menor toxicidad cuando se asociaron con PRF. PRF parecio activar el metabolismo de las células madres en cultivo, lo que indica que tal asociación puede aportar un beneficio efectivo en los resultados clínicos. O arcabouço e a sua interação com as células-tronco mesenquimais são objetos de estudo em bioengenharia e reparo de tecidos. Mecanismos como adesão superficial, proliferação, viabilidade e citotoxicidade são fundamentais para o desenvolvimento de terapias. O presente estudo analisou a influência da fibrina rica em plaquetas (PRF) na viabilidade, citotoxicidade e proliferação de células-tronco mesenquimais de dentes decíduos humanos esfoliados (SHED) em contato com biomaterial de origem bovina. Os grupos estudados foram divididos e analisados ​​da seguinte forma: (S) apenas SHED como Grupo controle; (SB) SHED + biomaterial; (SBP) SHED + biomaterial + PRF. As análises das células semeadas em placas de 24 poços foram realizadas após 24, 48 e 72 horas. Grupos individuais foram submetidos a testes de viabilidade, citotoxicidade e proliferação celular usando vermelho neutro, MTT e cristal violeta, respectivamente, e no grupo de 72 horas foi realizada a microscopia eletrônica de varredura (MEV) para registrar a ultra morfologia celular. Os dados foram submetidos à análise estatística por ANOVA de dois fatores com nível de significância de 5%. Os resultados demonstraram um melhor desempenho na viabilidade / citotoxicidade e proliferação de células-tronco para o grupo (SBP) em relação ao grupo (SB) e ao grupo (S). Os testes estatísticos aplicados mostraram que o fator biomaterial, o tempo e a interação entre eles deram origem a resultados com significância estatística. As SHED submetidas ao biomaterial bovino se apresentaram mais viáveis, proliferativas e com menor toxicidade quando associadas à PRF. A PRF pareceu ativar o metabolismo das células-tronco em cultivo, indicando que tal associação pode trazer um benefício efetivo nos desfechos clínicos.

Cigarette smoke compromises the salivary glands, altering saliva. Cotinine is the most effective biomarker to assess tobacco consumption, and passive smokers are characterized by serum cotinine levels above 2.1 ng/mL. This study aimed to evaluate the effect of exposure to cigarette smoke on rats submandibular glands. The morphology, the proliferative cells of intercalated ducts (immunohistochemistry for KI-67) and its correlation with serum cotinine levels were analyzed. Wistar rats were grouped: control (C); inhalation of smoke for 30 days (T1) and 60 days (T2). Data were analyzed with the Levene variance homogeneity test, Spearman's test, ANOVA and Tukey's post-test, with significance p

TRIM32 is a E3 ubiquitin -ligase containing RING, B-box, coiled-coil and six C-terminal NHL domains. Mutations involving NHL and coiled-coil domains result in a pure myopathy (LGMD2H/STM) while the only described mutation in the B-box domain is associated with a multisystemic disorder without myopathy (Bardet-Biedl syndrome type11), suggesting that these domains are involved in distinct processes. Knock-out (T32KO) and knock-in mice carrying the c.1465G > A (p.D489N) involving the NHL domain (T32KI) show alterations in muscle regrowth after atrophy and satellite cells senescence. Here, we present phenotypical description and functional characterization of mutations in the RING, coiled-coil and NHL domains of TRIM32 causing a muscle dystrophy. Reduced levels of TRIM32 protein was observed in all patient muscle studied, regardless of the type of mutation (missense, single amino acid deletion, and frameshift) or the mutated domain. The affected patients presented with variable phenotypes but predominantly proximal weakness. Two patients had symptoms of both muscular dystrophy and Bardet-Biedl syndrome. The muscle magnetic resonance imaging (MRI) pattern is highly variable among patients and families. Primary myoblast culture from these patients demonstrated common findings consistent with reduced proliferation and differentiation, diminished satellite cell pool, accelerated senescence of muscle, and signs of autophagy activation., Supported in part by grants from the Health Institute Carlos III and FEDER a way to achieve Europe (PI16–01843 to CP, JR15/00042 to MC-S), the Fundación Progreso y Salud, Junta de Andalucía (PI-0085-2016 to JLN-G), and Australian National Health and Medical Research Council (NHMRC) Fellowships (APP1122952 and APP1117510 to GR and NGL).

Introduction: Gliomas are characterized by rapid proliferation and aggressive invasion into normal surrounding brain tissue. In medical laboratories, the in vitro wound healing assay stands out as a simple, easy, inexpensive and affordable method to evaluate cell migration and proliferation. Objective: To standardize the in vitro wound healing assay using antimicrotubule drugs as positive controls. Methods: U87MG glioma cells were seeded at different densities and, after 24 h, the monolayer was scratched using different micropipette tip size to create a gap with no cells. The cells were then treated with colchicine and paclitaxel in culture medium with the presence or absence of fetal bovine serum. The wound was photographed with the aid of an inverted microscope and the wound area was measured using the Image J software. Results: Better defined edges scratches and monolayer with approximately 90% confluence were obtained at 1.5 and 2 x 105 cells/well density. The width and area of the scratch were, respectively, 948 pm/2.193221 mm2; 964 pm/2.266 mm2 and 1448 pm/3.221 mm2 to 10. 200 and 1000 pl micropipette tips. Colchicine inhibited wound closure by 12.6% or 3.4%, both in the presence or absence of serum; paclitaxel 2.4 and 6.7% respectively. Conclusion: Under standardized conditions, colchicine and paclitaxel proved to be efficient positive controls into the in vitro wound healing assay. RESUMEN Introducción: Gliomas se caracterizan por rápida proliferación e invasion agresiva del tejido cerebral normal circundante. En laboratorios médicos, el ensayo de cierre de herida - una prueba in vitro - se destaca por ser un método simple, fácil, de bajo costo y accesiblepara evaluar la migración y la proliferación celular. Objetivo: Estandarizar el ensayo de cierre de herida usando agentes anti-microtúbulos como control positivo. Métodos: Las células de glioma U87MG fueron sembradas en diferentes concentracionesy, después de 24 horas, la monocamadafue rayada con punteras de diferentes tamanos para crear una hendidura sin células. Las células fueron entonces tratadas con colchicina y paclitaxel, en medio con o sin suero fetal bovino. La ranura fue fotografiada con la ayuda de un microscopio invertido, y el área de la ranura fue medida mediante el programa Image J. Resultados: Ranuras con bordes más bien-definidos y monocamada con alrededor de 90% de confluencia se obtuvieron con 1,5 y 2 x 105 células/pozo. La anchura y el área de las ranuras obtenidas fueron, respectivamente, 948 p.m/2,193 mm2; 964p.m/2,266mm2; y 1448p.m/3,221 mm2 para las punteras de 10,200y 1000pl. La colchicina inhibió el cierre de las ranuras en 12,6% o 2,4%, tanto en presencia como en ausencia de suero; el paclitaxel, 3,4% y 6,7%, respectivamente. Conclusión: En condiciones estandarizadas, colchicina y paclitaxel pueden ser usados como control positivo en el ensayo de cierre de herida in vitro. RESUMO Introdução: Gliomas são caracterizados por terem rápida proliferação e invasão agressiva no tecido cerebral circundante normal. Em laboratórios médicos, o ensaio de ranhura - um teste in vitro - destaca-se por ser um método simples, fácil, barato e acessível para avaliar a migração e a proliferação celular. Objetivo: Padronizar o ensaio de ranhura, utilizando drogas antimicrotúbulos como controles positivos. Métodos: As células de glioma U87MG foram semeadas em diferentes concentrações e, após 24 horas, a monocamada foi arranhada usando ponteiras de diferentes tamanhos para criar uma fenda sem células. As células foram então tratadas com colchicina e paclitaxel, com meio em ausência ou presença de soro fetal bovino. A ranhura foi fotografada com auxílio de microscópio invertido, e a área da ranhura foi medida por meio do programa Image J. Resultados: Ranhuras com bordas mais bem definidas e monocamada com aproximadamente 90% de confluência foram obtidas com 1,5 e 2 x 105 células/poço. A largura e a área das ranhuras obtidasforam, respectivamente, 948pm/2,193 mm2;964pm/2,266mm2; e 1448 pm/3,221 mm2 para as ponteiras de 10, 200 e 1000 pl. A colchicina inibiu o fechamento das ranhuras em 12,6% ou 2,4%, tanto na presença quanto na ausência de soro; o paclitaxel, em 3,4% e 6,7%, respectivamente. Conclusão: Em condições padronizadas, colchicina e paclitaxel podem ser usados como controles positivos eficientes no teste in vitro de ranhura.

This research was funded by (1) the Ministry of Economy and Competitiveness and European Regional Development Fund [Project MAT2017-85999-P MINECO/AEI/FEDER/UE], (2) University of Granada/Regional Government of Andalusia Research Fund from Spain and European Regional Development Fund (A-BIO-157-UGR-18/FEDER). This research is part of Manuel Toledano-Osorio’s PhD research study., Polymeric membranes are employed in guided bone regeneration (GBR) as physical barriers to facilitate bone in-growth. A bioactive and biomimetic membrane with the ability to participate in the healing and regeneration of the bone is necessary. The aim of the present study was to analyze how novel silicon dioxide composite membranes functionalized with zinc or doxycycline can modulate the osteoblasts' proliferation, differentiation, and expression of selected antigenic markers related to immunomodulation. Nanostructured acrylate-based membranes were developed, blended with silica, and functionalized with zinc or doxycycline. They were subjected to MG63 osteoblast-like cells culturing. Proliferation was assessed by MTT-assay, differentiation by evaluating the alkaline phosphatase activity by a spectrophotometric method and antigenic phenotype was assessed by flow cytometry for selected markers. Mean comparisons were conducted by one-way ANOVA and Tukey tests (p < 0.05). The blending of silica nanoparticles in the tested non-resorbable polymeric scaffold improved the proliferation and differentiation of osteoblasts, but doxycycline doped scaffolds attained the best results. Osteoblasts cultured on doxycycline functionalized membranes presented higher expression of CD54, CD80, CD86, and HLA-DR, indicating a beneficial immunomodulation activity. Doxycycline doped membranes may be a potential candidate for use in GBR procedures in several challenging pathologies, including periodontal disease., European Commission MAT2017-85999-P A-BIO-157-UGR-18/FEDER, Ministry of Economy and Competitiveness MAT2017-85999-P, University of Granada/Regional Government of Andalusia Research Fund from Spain A-BIO-157-UGR-18/FEDER

The aim of this study was to determine the optimal nanopore diameter of titanium nanostructured surfaces to improve human gingival fibroblast (hGF) response, with the purpose of promoting gingiva integration to dental implant abutments. Two TiO2 nanoporous groups with different diameters (NP-S 48 nm and NP-B 74 nm) were grown on Ti foils using an organic electrolyte containing fluoride by electrochemical oxidation, varying the applied voltage and the interelectrode spacing. The surfaces were characterized by scanning electron microscope (SEM), atomic force microscopy (AFM), and contact angle. The hGF were cultured onto the different surfaces, and metabolic activity, cytotoxicity, cell adhesion, and gene expression were analyzed. Bigger porous diameters (NP-B) were obtained by increasing the voltage used during anodization. To obtain the smallest diameter (NP-S), apart from lowering the voltage, a lower interelectrode spacing was needed. The greatest surface area and number of peaks was found for NP-B, despite these samples not being the roughest as defined by R-a. NP-B had a better cellular response compared to NP-S. However, these effects had a significant dependence on the cell donor. In conclusion, nanoporous groups with a diameter in the range of 74 nm induce a better hGF response, which may be beneficial for an effective soft tissue integration around the implant., This research was funded by the Osteology Foundation (13-069), the Ministerio de Educacion Cultura y Deporte (contract to M.A.L.-G; FPU15/03412), the Instituto de Salud Carlos III (contract to J.M.R; CP16/00124), and the Ministerio de Empleo y Seguridad Social with the Sistema de Garantia Juvenil (contract to M.d.M.F.-C.).

The radioprotective potential of histamine on healthy tissue has been previously demonstrated. The aims of this work were to investigate the combinatorial effect of histamine or its receptor ligands and gamma radiation in vitro on the radiobiological response of 2 breast cancer cell lines (MDA-MB-231 and MCF-7), to explore the potential molecular mechanisms of the radiosensitizing action and to evaluate the histamine-induced radiosensitization in vivo in a triple negative breast cancer model. Results indicate that histamine significantly increased the radiosensitivity of MDA-MB-231 and MCF-7 cells. This effect was mimicked by the H1R agonist 2-(3-(trifluoromethyl)phenyl)histamine and the H4R agonists (Clobenpropit and VUF8430) in MDA-MB-231 and MCF-7 cells, respectively. Histamine and its agonists enhanced radiation-induced oxidative DNA damage, DNA double-strand breaks, apoptosis and senescence. These effects were associated with increased production of reactive oxygen species, which correlated with the inhibition of catalase, glutathione peroxidase and superoxide dismutase activities in MDA-MB-231 cells. Histamine was able also to potentiate in vivo the anti-tumoral effect of radiation, increasing the exponential tumor doubling time. We conclude that histamine increased radiation response of breast cancer cells, suggesting that it could be used as a potential adjuvant to enhance the efficacy of radiotherapy. Fil: Martinel Lamas, Diego José. Universidad de Buenos Aires. Facultad de Farmacia y Bioquímica; Argentina. Pontificia Universidad Católica Argentina "Santa María de los Buenos Aires". Instituto de Investigaciones Biomédicas. Consejo Nacional de Investigaciones Científicas y Técnicas. Oficina de Coordinación Administrativa Houssay. Instituto de Investigaciones Biomédicas; Argentina Fil: Cortina, Jorge E.. Universidad de Buenos Aires. Facultad de Farmacia y Bioquímica; Argentina Fil: Ventura, Clara. Universidad de Buenos Aires. Facultad de Farmacia y Bioquímica; Argentina. Consejo Nacional de Investigaciones Científicas y Técnicas; Argentina Fil: Sterle, Helena Andrea. Universidad de Buenos Aires. Facultad de Farmacia y Bioquímica. Departamento de Química Biológica; Argentina. Pontificia Universidad Católica Argentina "Santa María de los Buenos Aires". Instituto de Investigaciones Biomédicas. Consejo Nacional de Investigaciones Científicas y Técnicas. Oficina de Coordinación Administrativa Houssay. Instituto de Investigaciones Biomédicas; Argentina Fil: Valli, Eduardo. Pontificia Universidad Católica Argentina "Santa María de los Buenos Aires". Instituto de Investigaciones Biomédicas. Consejo Nacional de Investigaciones Científicas y Técnicas. Oficina de Coordinación Administrativa Houssay. Instituto de Investigaciones Biomédicas; Argentina. Universidad de Buenos Aires. Facultad de Farmacia y Bioquímica. Departamento de Química Biológica; Argentina Fil: Balestrasse, Karina Beatriz. Universidad de Buenos Aires. Facultad de Farmacia y Bioquímica. Departamento de Química Biológica; Argentina. Consejo Nacional de Investigaciones Científicas y Técnicas. Oficina de Coordinación Administrativa Parque Centenario. Instituto de Investigaciones en Biociencias Agrícolas y Ambientales. Universidad de Buenos Aires. Facultad de Agronomía. Instituto de Investigaciones en Biociencias Agrícolas y Ambientales; Argentina Fil: Blanco, Horacio Alejandro. Gobierno de la Ciudad de Buenos Aires. Hospital Municipal de Oncología "Marie Curie"; Argentina Fil: Cremaschi, Graciela Alicia. Consejo Nacional de Investigaciones Científicas y Técnicas. Oficina de Coordinación Administrativa Parque Centenario. Instituto de Investigaciones en Biociencias Agrícolas y Ambientales. Universidad de Buenos Aires. Facultad de Agronomía. Instituto de Investigaciones en Biociencias Agrícolas y Ambientales; Argentina. Universidad de Buenos Aires. Facultad de Farmacia y Bioquímica; Argentina Fil: Rivera, Elena Susana. Universidad de Buenos Aires. Facultad de Farmacia y Bioquímica; Argentina Fil: Medina, Vanina Araceli. Consejo Nacional de Investigaciones Científicas y Técnicas. Oficina de Coordinación Administrativa Parque Centenario. Instituto de Investigaciones en Biociencias Agrícolas y Ambientales. Universidad de Buenos Aires. Facultad de Agronomía. Instituto de Investigaciones en Biociencias Agrícolas y Ambientales; Argentina. Universidad de Buenos Aires. Facultad de Farmacia y Bioquímica; Argentina

15 Páginas; 6 Figuras, Several important biological activities have been attributed to the pentacyclic triterpene ursolic acid (UA), being its antitumoral effect extensively studied in human adenocarcinomas. In this work, we focused on the efficacy and molecular mechanisms involved in the antitumoral effects of UA, as single agent or combined with doxorubicin (DXR), in human soft tissue sarcoma cells. UA (5–50 μM) strongly inhibited (up to 80%) the viability of STS cells at 24 h and its proliferation in soft agar, with higher concentrations increasing apoptotic death up to 30%. UA treatment (6–9 h) strongly blocked the survival AKT/GSK3β/β-catenin signalling pathway, which led to a concomitant reduction of the anti-apoptotic proteins c-Myc and p21, altogether resulting in the activation of intrinsic apoptosis. Interestingly, UA at low concentrations (10–15 μM) enhanced the antitumoral effects of DXR by up to 2-fold, while in parallel inhibiting DXR-induced AKT activation and p21 expression, two proteins implicated in antitumoral drug resistance and cell survival. In conclusion, UA is able to induce intrinsic apoptosis in human STS cells and also to sensitize these cells to DXR by blocking the AKT signalling pathway. Therefore, UA may have beneficial effects, if used as nutraceutical adjuvant during standard chemotherapy treatment of STS., Research was supported by the Institute of Health Carlos III through the subprogramme “CIBERobn (Centro de Investigación Biomédica en Red de la Fisiopatología de la Obesidad y Nutrición)”. VHV was supported by a predoctoral fellowship from ‘Govern de les Illes Balears, Conselleria d’Educació, Cultura i Universitats’ under a program of joint financing with the European Social Fund.

A nuclear power source in the cell DNA is packaged onto nucleosomes, the principal component of chromatin. This chromatin must be remodeled to allow gene transcription, DNA replication, and DNA repair machineries access to the enclosed DNA. Chromatin-remodeling complexes require high levels of cellular energy to do their job. Wright et al. show that the energy needed to remodel chromatin can be derived from a source, poly-ADP-ribose, in the cell nucleus, rather than by diffusion of ATP from mitochondria in the cytoplasm, the usual powerhouse of the cell. Poly-ADP-ribose is converted to ADP-ribose and then to ATP, which can be used to fuel chromatin remodeling within the nucleus. Science , this issue p. 1221

Endocannabinoids participate in the control of neurogenesis, neural cell death and gliosis. The pharmacological effect of the fatty acid amide hydrolase (FAAH) inhibitor URB597, which limits the endocannabinoid degradation, was investigated in the present study. Cell proliferation (phospho-H3(+) or BrdU(+) cells) of the main adult neurogenic zones as well as apoptosis (cleaved caspase-3(+)), astroglia (GFAP(+)), and microglia (Iba1(+) cells) were analyzed in the hippocampus, hypothalamus and striatum of rats intraperitoneally treated with URB597 (0.3 mg/kg/day) at one dose/4-days resting or 5 doses (1 dose/day). Repeated URB597 treatment increased the plasma levels of the N-acylethanolamines oleoylethanolamide, palmitoylethanolamide and arachidonoylethanolamine, reduced the plasma levels of glucose, triglycerides and cholesterol, and induced a transitory body weight decrease. The hippocampi of repeated URB597-treated rats showed a reduced number of phospho-H3(+) and BrdU(+) subgranular cells as well as GFAP(+), Iba1(+) and cleaved caspase-3(+) cells, which was accompanied with decreased hippocampal expression of the cannabinoid CB1 receptor gene Cnr1 and Faah. In the hypothalami of these rats, the number of phospho-H3(+), GFAP(+) and 3-weeks-old BrdU(+) cells was specifically decreased. The reduced striatal expression of CB1 receptor in repeated URB597-treated rats was only associated with a reduced apoptosis. In contrast, the striatum of acute URB597-treated rats showed an increased number of subventricular proliferative, astroglial and apoptotic cells, which was accompanied with increased Faah expression. Main results indicated that FAAH inhibitor URB597 decreased neural proliferation, glia and apoptosis in a brain region-dependent manner, which were coupled to local changes in Faah and/or Cnr1 expression and a negative energy context. Grant sponsor: 7th Framework Programme of European Union; Grant number: HEALTH-F2-2008-223713, REPROBESITY to FR and BL; Grant sponsor: Ministerio de Ciencia e Innovación; Grant numbers: SAF2010-19087, SAF 2010-20521; Grant sponsor: Instituto de Salud Carlos III, Ministerio de Economía y Competitividad, UE-ERDF; Grant number: CP12/03109; Grant sponsor: Red de Trastornos Adictivos; Grant numbers: RD12/0028/0001, RD12/0028/0009; Grant sponsor: CIBERobn; Grant sponsor: Plan Nacional Sobre Drogas, Ministerio de Sanidad y Consumo; Grant number: PNSD2010/143; Grant sponsor: Consejería de Economía, Innovación y Ciencia, Junta de Andalucía, UE/ERDF; Grant numbers: CTS-433, P-11-CVI-07637; Grant sponsor: Consejería de Salud, Junta de Andalucía; Grant numbers: PI0232/2008, PI0029/2008, SAS111224; Grant sponsor: Fundació La Marató de TV3; Grant number: 386/C/2011; Grant sponsor: German Research Foundation DFG; Grant number: FOR629, project CP2 to BL. JS, FP, and AS hold “Miguel Servet” research contracts from the National System of Health, ISCIII (Grant numbers: CP12/03109, CP14/00212, CP14/00173 respectively).

Hexachlorobenzene (HCB) is a widespread environmental pollutant, and a liver tumor promoter in rodents. Depending on the particular cell lines studied, exposure to these compounds may lead to cell proliferation, terminal differentiation, or apoptosis. The aryl hydrocarbon receptor (AhR) is a ligand-activated transcription factor that is involved in drug and xenobiotic metabolism. AhR can also modulate a variety of cellular and physiological processes that can affect cell proliferation and cell fate determination. The mechanisms by which AhR ligands, both exogenous and endogenous, affect these processes involve multiple interactions between AhR and other signaling pathways. In the present study, we examined the effect of HCB on cell proliferation and AhR expression, using an initiation-promotion hepatocarcinogenesis protocol in rat liver and in the human-derived hepatoma cell line, HepG2. Female Wistar rats were initiated with a single dose of 100. mg/kg of diethylnitrosamine (DEN) at the start of the experiment. Two weeks later, daily dosing of 100. mg/kg HCB was maintained for 10 weeks. Partial hepatectomy was performed 3 weeks after initiation. The number and area of glutathione S-transferase-P (GST-P)-positive foci, in the rat liver were used as biomarkers of liver precancerous lesions.Immunohistochemical staining showed an increase in proliferating cell nuclear antigen (PCNA)-positive cells, along with enhanced AhR protein expression in hepatocytes within GST-P-positive foci of (DEN HCB) group, when compared to DEN. In a similar manner, Western blot analysis demonstrated that HCB induced PCNA and AhR protein expression in HepG2 cells. Flow cytometry assay indicated that the cells were accumulated at S and G2/M phases of the cell cycle. HCB increased cyclin D1 protein levels and ERK1/2 phosphorylation in a dose-dependent manner. Treatment of cells with a selective MEK1 inhibitor, prevented HCB-stimulatory effect on PCNA and cyclinD1, indicating that these effects are mediated by ERK1/2. Pretreatment with an AhR antagonist, prevented HCB-induced PCNA protein levels, ERK1/2 phosphorylation and alterations in cell cycle distribution. These results demonstrate that HCB-induced HepG2 proliferation and cell cycle progression depend on ERK1/2 phosphorylation which is mediated by the AhR.Our results provide a clue to the molecular events involved in the mechanism of action of HCB-induced hepatocarcinogenesis. Fil: de Tomaso Portaz, Ana Clara. Universidad de Buenos Aires. Facultad de Medicina. Departamento de Bioquímica Humana. Cátedra de Química Biologica; Argentina. Consejo Nacional de Investigaciones Científicas y Técnicas. Oficina de Coordinación Administrativa Houssay; Argentina Fil: Romero Caimi, Vanesa Giselle. Universidad de Buenos Aires. Facultad de Medicina. Departamento de Bioquímica Humana. Cátedra de Química Biologica; Argentina Fil: Sánchez, Marcela. Universidad de Buenos Aires. Facultad de Medicina. Departamento de Bioquímica Humana. Cátedra de Química Biologica; Argentina Fil: Chiappini, Florencia Ana. Consejo Nacional de Investigaciones Científicas y Técnicas. Oficina de Coordinación Administrativa Houssay; Argentina. Universidad de Buenos Aires. Facultad de Medicina. Departamento de Bioquímica Humana. Cátedra de Química Biologica; Argentina Fil: Randi, Andrea Silvana. Universidad de Buenos Aires. Facultad de Medicina. Departamento de Bioquímica Humana. Cátedra de Química Biologica; Argentina. Consejo Nacional de Investigaciones Científicas y Técnicas. Oficina de Coordinación Administrativa Houssay; Argentina Fil: Kleiman, Diana Leonor. Universidad de Buenos Aires. Facultad de Medicina. Departamento de Bioquímica Humana. Cátedra de Química Biologica; Argentina. Consejo Nacional de Investigaciones Científicas y Técnicas. Oficina de Coordinación Administrativa Houssay; Argentina Fil: Alvarez, Laura. Universidad de Buenos Aires. Facultad de Medicina. Departamento de Bioquímica Humana. Cátedra de Química Biologica; Argentina. Consejo Nacional de Investigaciones Científicas y Técnicas. Oficina de Coordinación Administrativa Houssay; Argentina

Background: Cell adhesion is a process that involves the interaction between the cell membrane and another surface, either a cell or a substrate. Unlike experimental tests, computer models can simulate processes and study the result of experiments in a shorter time and lower costs. One of the tools used to simulate biological processes is the cellular automata, which is a dynamic system that is discrete both in space and time. Method: This work describes a computer model based on cellular automata for the adhesion process and cell proliferation to predict the behavior of a cell population in suspension and adhered to a substrate. The values of the simulated system were obtained through experimental tests on fibroblast monolayer cultures. Results: The results allow us to estimate the cells settling time in culture as well as the adhesion and proliferation time. The change in the cells morphology as the adhesion over the contact surface progress was also observed. The formation of the initial link between cell and the substrate of the adhesion was observed after 100 min where the cell on the substrate retains its spherical morphology during the simulation. The cellular automata model developed is, however, a simplified representation of the steps in the adhesion process and the subsequent proliferation. Conclusion: A combined framework of experimental and computational simulation based on cellular automata was proposed to represent the fibroblast adhesion on substrates and changes in a macro-scale observed in the cell during the adhesion process. The approach showed to be simple and efficient. info:eu-repo/semantics/publishedVersion

Apoptosis can be triggered in two different ways, through the intrinsic or the extrinsic pathway. The intrinsic pathway is mediated by the mitochondria via the release of cytochrome C while the extrinsic pathway is prompted by death receptor signals and bypasses the mitochondria. These two pathways are closely related to cell proliferation and survival signaling cascades, which thereby constitute possible targets for cancer therapy. In previous studies we introduced two plant derived isomeric flavonoids, flavone A and flavone B which induce apoptosis in highly tumorigenic cancer cells of the breast, colon, pancreas, and the prostate. Flavone A displayed potent cytotoxic activity against more differentiated carcinomas of the colon (CaCo-2) and the pancreas (Panc28), whereas flavone B cytotoxic action is observed on poorly differentiated carcinomas of the colon (HCT 116) and pancreas (MIA PaCa). Apoptosis is induced by flavone A in better differentiated colon cancer CaCo-2 and pancreatic cancer Panc 28 cells via the intrinsic pathway by the inhibition of the activated forms of extracellular signal-regulated kinase (ERK) and pS6, and subsequent loss of phosphorylation of Bcl-2 associated death promoter (BAD) protein, while apoptosis is triggered by flavone B in poorly differentiated colon cancer HCT 116 and MIA PaCa pancreatic cancer cells through the extrinsic pathway with the concomitant upregulation of the phosphorylated forms of ERK and c-JUN at serine 73. These changes in protein levels ultimately lead to activation of apoptosis, without the involvement of AKT. Incluye bibliografia

Neoblasts are totipotent cells, solely responsible for the proliferation and maturation of tissues in free-living flatworms. Similar cells have been isolated from parasitic flatworms such as Echinococcus. Taenia solium causes human taeniasis (intestinal) and cysticercosis in humans and pigs. Brain infection with larvae (cysts) of T. solium results in neurocysticercosis which is hyperendemic in Peru, and its treatment is associated with serious neurological symptoms. The proliferative capacity and development stages of T. solium have not been described and the neoblasts of this parasite have not been characterized We looked for cell proliferation in T. solium cysts collected from an infected pig, which were identified when replicating and incorporating bromodeoxyuridine nucleotide detected with a monoclonal antibody. A stable cell line of neoblasts would be useful for systematic in vitro studies on drug efficacy and the biology of T. solium. Los neoblastos son células totipotentes, únicas responsables de la proliferación y maduración de tejidos en platelmintos de vida libre. Células similares se han aislado en platelmintos parásitos como Echinococcus. Taenia solium causa la teniasis humana (intestinal) y la cisticercosis en humanos y cerdos. La infección del cerebro con larvas (quistes) de T. solium resulta en neurocisticercosis, hiperendémica en el Perú; su tratamiento se asocia a síntomas neurológicos graves. La capacidad proliferativa y el desarrollo de los estadios de T. solium aún no se describen, y no se ha caracterizado los neoblastos de este parásito. Se buscó células proliferativas en quistes de T. solium colectados de un cerdo infectado, que fueron identificadas al replicarse e incorporar el nucleótido bromodesoxiuridina, detectado con un anticuerpo monoclonal. Una línea celular estable de neoblastos sería útil para estudios sistemáticos in vitro sobre eficacia de drogas y sobre la biología de T. solium.

Antecedentes La diafonía entre la señalización de Hedgehog (HH) y otras cascadas de transducción de señales se ha estudiado ampliamente en diferentes tipos de cáncer. En el neuroblastoma, se sabe que la señalización de mTOR / S6K1 tiene un papel en el desarrollo de esta enfermedad y la evidencia reciente también implica la vía HH. Además, se ha demostrado que la quinasa S6K1 fosforila GLI1, el efector de la señalización de HH, promoviendo la actividad transcripcional de GLI1 y la función oncogénica en el adenocarcinoma esofágico. En este estudio, examinamos la posible interacción de la señalización S6K1 y GLI1 en neuroblastoma. Métodos Se utilizaron las reducciones de siRNA para suprimir la expresión de S6K1 y GLI1, y los efectos de siRNA se validaron mediante PCR en tiempo real y transferencia Western. El análisis de proliferación celular se realizó con el ensayo de incorporación de EdU. El análisis citotóxico con concentraciones crecientes de inhibidores de PI3K / mTOR y GLI, individualmente y en combinación, se utilizó para determinar la respuesta al fármaco. Resultados Aunque la eliminación de S6K1 o GLI1 reduce la proliferación celular de las células de neuroblastoma, hay poco efecto de S6K1 en la expresión de ARNm y proteína GLI1 y en la capacidad de GLI1 para activar genes diana. No se observa fosforilación detectable de GLI1 antes o después de la eliminación de S6K1. La sobreexpresión de GLI1 no puede rescatar la proliferación reducida provocada por la caída de S6K1. Además, los inhibidores de la señalización de PI3K / mTOR y GLI redujeron el crecimiento celular del neuroblastoma, pero no se detectaron efectos inhibidores del crecimiento adicionales cuando se combinaron las dos clases de fármacos. Conclusión Nuestros resultados demuestran que el impacto de la quinasa S6K1 en las células de neuroblastoma no está mediado por la modulación de la expresión / actividad de GLI1. The crosstalk between Hedgehog (HH) signaling and other signal transduction cascades has been extensively studied in different cancers. In neuroblastoma, mTOR/S6K1 signaling is known to have a role in the development of this disease and recent evidence also implicates the HH pathway. Moreover, S6K1 kinase has been shown to phosphorylate GLI1, the effector of HH signaling, promoting GLI1 transcriptional activity and oncogenic function in esophageal adenocarcinoma. In this study, we examined the possible interplay of S6K1 and GLI1 signaling in neuroblastoma. Methods siRNA knockdowns were used to suppress S6K1 and GLI1 expression, and the siRNA effects were validated by real-time PCR and Western blotting. Cell proliferation analysis was performed with the EdU incorporation assay. Cytotoxic analysis with increasing concentrations of PI3K/mTOR and GLI inhibitors, individually and in combination, was used to determine drug response. Results Although knockdown of either S6K1 or GLI1 reduces the cellular proliferation of neuroblastoma cells, there is little effect of S6K1 on the expression of GLI1 mRNA and protein and on the capacity of GLI1 to activate target genes. No detectable phosphorylation of GLI1 is observed prior or following S6K1 knockdown. GLI1 overexpression can not rescue the reduced proliferation elicited by S6K1 knockdown. Moreover, inhibitors of PI3K/mTOR and GLI signaling reduced neuroblastoma cell growth, but no additional growth inhibitory effects were detected when the two classes of drugs were combined. Conclusion Our results demonstrate that the impact of S6K1 kinase on neuroblastoma cells is not mediated through modulation of GLI1 expression/activity.

Objetivos. Evaluar el efecto cicatrizante del extracto hidroetanólico de Piper aduncum, en una línea celular de fibroblastos Dermales Adultos Humanos (hDFa). Materiales y métodos. El extracto se obtuvo mediante extracción sólido-líquido, fue concentrado y liofilizado. Se purificaron las proteínas del extracto mediante cromatografía líquida de alta eficacia de fase reversa (RP-HPLC); las proteínas fueron identificadas por espectrometría de masas en tándem de péptidos trípticos y se analizaron por MALDI-TOF-TOF en un espectrómetro de masa ABSciex4800. Los valores de concentración efectiva media (EC50), concentración inhibitoria media (IC50), y el porcentaje de proliferación celular; fueron determinados por ensayos con sales de tetrazolio (MTT). La migración celular se evaluó mediante la "técnica de rayado". Se analizó la expresión de factores de crecimiento mediante el ensayo de reacción en cadena de la polimerasa con transcriptasa reversa a tiempo real (RT- qPCR). Resultados. La línea hDFa evidenció un IC50 de 200 µg/mL con el extracto, el valor de EC50 fue 103,5 µg/mL. En el ensayo de proliferación, la proteína K2; mostró mayor actividad en la proliferación respecto de otros tratamientos (1 µg/mL). En el ensayo de migración de fibroblastos, la proteína K2 mostró mayor actividad (50 µg/mL). La expresión relativa del factor de crecimiento derivado de plaquetas (PDGF) se incrementó 8,6 veces respecto al control, en presencia de la proteína K2. Conclusiones. El extracto hidroetanólico, de Piper aduncum, así como las proteínas que contiene, incrementaron la proliferación y migración de fibroblastos dermales humanos (hDFa); así mismo, aumentaron la expresión de factores de crecimiento que intervienen en el proceso de cicatrización. Objectives. To evaluate the healing effect of a Piper aduncumethanol-water extract on an adult human dermal fibroblast cell line (hDFa). Materials and Methods. After obtaining the extract via solid-liquid extraction, concentration, and lyophilization, extract proteins were purified using reverse phase high-performance liquid chromatography, identified using tandem mass spectrometry of tryptic peptides, and analyzed using MALDI-TOF-TOF on an ABSciex4800 mass spectrometer. Half maximum effective concentration values (EC50), half maximum inhibiting concentration (IC50), and percentages of cell proliferation were determined using tetrazolium salt assays. Cell migration was evaluated using a "scratch assay". Growth factor expression in cells was analyzed via quantitative real-time reverse transcription polymerase chain reaction. Results. Against the hDFa cell line, the extract had an IC50 of 200 μg/mL and EC50 of 103.5 µg/mL. In the proliferation assay, protein K2 (obtained from the extract) exhibited increased proliferative activity relative to other treatments (1 µg/mL); this agent also exhibited increased activity (50 µg/mL) in the fibroblast migration assay. Furthermore, the relative expression of platelet-derived growth factor increased by 8.6-fold in the presence of K2 protein relative to the control. Conclusions. The hydroethanolic extract of Piper aduncum and its component proteins increased the proliferation and migration of hDFa and increased the expression of growth factors involved in the healing process.

The therapeutic effect of doxorubicin (DXR) in the treatment of soft tissue sarcomas (STS) is limited by its toxicity and the development of multidrug resistance (MDR), the latter mainly induced by high expression of efflux pumps (e. g., P-glycoprotein [P-gp]). Therefore, the search for alternative therapies, which sensitize these tumors to chemotherapy while maintaining a low toxicity profile, is a rational approach. We assessed efficacy and molecular mechanisms involved in the antiproliferative effects of the tyrosine kinase inhibitors, nilotinib and imatinib, as single agents or in combination with DXR, in human synovial sarcoma SW982 and leiomyosarcoma SK-UT-1 cells. As single compound nilotinib (1-10 mu M) was more potent than imatinib inhibiting the growth of SK-UT-1 and SW982 cells by 33.5-59.6%, respectively. Importantly, only nilotinib synergized the antitumoral effect of DXR (0.05-0.5 mu M) by at least 2-fold, which clearly surpassed the mere sum of effects according to isobolographic analysis. Moreover, nilotinib in combination with DXR had a sustained effect on cell number (-70.3+/-5.8%) even 12 days after withdrawal of drugs compared to DXR alone. On the molecular level, only nilotinib fully blocked FBS-induced ERK1 and p38 MAPK activation, hence, reducing basal and DXR-induced up-regulation of P-gp levels. Moreover, efflux activity of the MDR-related proteins P-gp and MRP-1 was inhibited, altogether resulting in intracellular DXR retention. In high-risk STS tumors 53.8% and 15.4% were positive for P-gp and MRP-1 expression, respectively, with high incidence of P-gp in synovial sarcoma (72.7%). In summary, nilotinib exhibits antiproliferative effects on cellular models of STS and sensitizes them to DXR by reverting DXR-induced P-gp-mediated MDR and inhibiting MRP-1 activity, leading to a synergistic effect with potential for clinical treatment., Research was supported by grants Jose Ma Buesa del grupo GEIS and the Asociacion Espanola contra el Cancer (Junta de Baleares) and by Grand EC10-150 awarded by the Spanish Ministry of Health in 2010 (Independent Clinical Research Call). V. H. V. is supported by a PhD fellowship from the Conselleria d'Economia, Hisenda i Innovacio (Govern de les Illes Balears). The funders had no role in study design, data collection and analysis, decision to publish, or preparation of the manuscript.

Formal Correction: Error in Funding Posted by PLoS_ONE_Group on 29 Nov 2011 at 22:27 GMT The complete funding information is: "This work was supported by grants from the Spanish Ministry of Education and Science (MEC SEJ2007-61187, MICINN PSI2010-16160 to LJS), Programme for Stabilization of Researchers and Research Technician and Intensification of Research Activity in the National Health System (I3SNS Programme to GET), the Human Frontier Science Programme (to JC, FRDF), Fund for Health Research, Carlos III Health Institute (FIS 02/1643, FIS PI07/0629, FIS PI10/02514 to GET), Red de Trastornos Adictivos RTA (RD06/001 to FRDF), Andalusian Ministry of Health and of Innovation, Science and Enterprise (CTS065 to GET; CTS433 to FRDF) and the US National Institutes of Health MH051699 and MH01723 (to JC). The author E. Castilla-Ortega has a FPU Grant from the Spanish Ministry of Education (AP-2006-02582). The funders had no role in the study design, data collection and analysis, decision to publish, or preparation of the manuscript." Journal Article; Research Support, N.I.H., Extramural; Research Support, Non-U.S. Gov't; BACKGROUND The lysophosphatidic acid LPA₁ receptor regulates plasticity and neurogenesis in the adult hippocampus. Here, we studied whether absence of the LPA₁ receptor modulated the detrimental effects of chronic stress on hippocampal neurogenesis and spatial memory. METHODOLOGY/PRINCIPAL FINDINGS Male LPA₁-null (NULL) and wild-type (WT) mice were assigned to control or chronic stress conditions (21 days of restraint, 3 h/day). Immunohistochemistry for bromodeoxyuridine and endogenous markers was performed to examine hippocampal cell proliferation, survival, number and maturation of young neurons, hippocampal structure and apoptosis in the hippocampus. Corticosterone levels were measured in another a separate cohort of mice. Finally, the hole-board test assessed spatial reference and working memory. Under control conditions, NULL mice showed reduced cell proliferation, a defective population of young neurons, reduced hippocampal volume and moderate spatial memory deficits. However, the primary result is that chronic stress impaired hippocampal neurogenesis in NULLs more severely than in WT mice in terms of cell proliferation; apoptosis; the number and maturation of young neurons; and both the volume and neuronal density in the granular zone. Only stressed NULLs presented hypocortisolemia. Moreover, a dramatic deficit in spatial reference memory consolidation was observed in chronically stressed NULL mice, which was in contrast to the minor effect observed in stressed WT mice. CONCLUSIONS/SIGNIFICANCE These results reveal that the absence of the LPA₁ receptor aggravates the chronic stress-induced impairment to hippocampal neurogenesis and its dependent functions. Thus, modulation of the LPA₁ receptor pathway may be of interest with respect to the treatment of stress-induced hippocampal pathology. Yes

Objective To evaluate the effects of celecoxib and rosiglitazone on the implantation and growth of endometriotic-like lesions in a murine model of endometriosis. Design Prospective experimental study. Setting Animal research and laboratory facility. Animal(s) Two-month-old female BALB/c mice. Intervention(s) Surgically induced endometriosis in female BALB/C mice; 28 days of treatment with celecoxib, rosiglitazone, or their combination; counting, measuring, excising, and fixing lesions. Main Outcome Measure(s) Immunohistochemical examination for proliferating cell nuclear antigen (PCNA), CD31, and CD34 to assess cell proliferation and vascularization, with the terminal deoxynucleotidyl transferase dUTP nick end labeling (TUNEL) technique for apoptosis evaluation. Result(s) Celecoxib and the combined treatment (celecoxib and rosiglitazone) statistically significantly reduced the mean number of lesions established per mouse, and all treatments diminished the implant volume. In addition, cell proliferation within the implants was statistically significantly reduced, and apoptosis was statistically significantly enhanced by all treatments. Also, we found that all treatments diminished the vascularized area in the lesion. Conclusion(s) These results are promising and reveal that celecoxib and rosiglitazone, combined or separately, have a beneficial effect on overall endometriotic growth.

Journal Article; Research Support, Non-U.S. Gov't; In autoimmune type 1 diabetes mellitus, proinflammatory cytokine-mediated apoptosis of beta-cells has been considered to be the first event directly responsible for beta-cell mass reduction. In the Bio-Breeding (BB) rat, an in vivo model used in the study of autoimmune diabetes, beta-cell apoptosis is observed from 9 wk of age and takes place after an insulitis period that begins at an earlier age. Previous studies by our group have shown an antiproliferative effect of proinflammatory cytokines on cultured beta-cells in Wistar rats, an effect that was partially reversed by Exendin-4, an analogue of glucagon-like peptide-1. In the current study, the changes in beta-cell apoptosis and proliferation during insulitis stage were also determined in pancreatic tissue sections in normal and thymectomized BB rats, as well as in Wistar rats of 5, 7, 9, and 11 wk of age. Although stable beta-cell proliferation in Wistar and thymectomized BB rats was observed along the course of the study, a decrease in beta-cell proliferation and beta-cell mass from the age of 5 wk, and prior to the commencement of apoptosis, was noted in BB rats. Exendin-4, in combination with anti-interferon-gamma antibody, induced a near-total recovery of beta-cell proliferation during the initial stages of insulitis. This highlights the importance of early intervention and, as well, the possibilities of new therapeutic approaches in preventing autoimmune diabetes by acting, initially, in the insulitis stage and, subsequently, on beta-cell regeneration and on beta-cell apoptosis. Yes

Producción Científica, Aims: Vascular smooth muscle cell (VSMC) proliferation is involved in cardiovascular pathologies associated with unwanted arterial wall remodelling. Coordinated changes in the expression of several K+ channels have been found to be important elements in the phenotypic switch of VSMCs towards proliferation. We have previously demonstrated the association of functional expression of Kv3.4 channels with proliferation of human uterine VSMCs. Here, we sought to gain deeper insight on the relationship between Kv3.4 channels and cell cycle progression in this preparation. Methods and results: Expression and function of Kv3.4 channels along the cell cycle was explored in uterine VSMCs synchronized at different checkpoints, combining real-time PCR, western blotting, and electrophysiological techniques. Flow cytometry, Ki67 expression and BrdU incorporation techniques allowed us to explore the effects of Kv3.4 channels blockade on cell cycle distribution. We found cyclic changes in Kv3.4 and MiRP2 mRNA and protein expression along the cell cycle. Functional studies showed that Kv3.4 current amplitude and Kv3.4 channels contribution to cell excitability increased in proliferating cells. Finally, both Kv3.4 blockers and Kv3.4 knockdown with siRNA reduced the proportion of proliferating VSMCs. Conclusion: Our data indicate that Kv3.4 channels exert a permissive role in the cell cycle progression of proliferating uterine VSMCs, as their blockade induces cell cycle arrest after G2/M phase completion. The modulation of resting membrane potential (VM) by Kv3.4 channels in proliferating VSMCs suggests that their role in cell cycle progression could be at least in part mediated by their contribution to the hyperpolarizing signal needed to progress through the G1 phase., Ministerio de Sanidad, Consumo y Bienestar Social - Instituto de Salud Carlos III (grants R006/009 and PI041044), Ministerio de Ciencia, Innovación y Universidades (grants BFU2004-05551 and BFU2007-61524), Junta de Castilla y León (grant GR242)

Producción Científica, Vascular smooth muscle cells (VSMCs) perform diverse functions that can be classified into contractile and synthetic (or proliferating). All of these functions can be fulfilled by the same cell because of its capacity of phenotypic modulation in response to environmental changes. The resting membrane potential is a key determinant for both contractile and proliferating functions. Here, we have explored the expression of voltage-dependent K+ (Kv) channels in contractile (freshly dissociated) and proliferating (cultured) VSMCs obtained from human uterine arteries to establish their contribution to the functional properties of the cells and their possible participation in the phenotypic switch. We have studied the expression pattern (both at the mRNA and at the protein level) of Kvα subunits in both preparations as well as their functional contribution to the K+ currents of VSMCs. Our results indicate that phenotypic remodeling associates with a change in the expression and distribution of Kv channels. Whereas Kv currents in contractile VSMCs are mainly performed by Kv1 channels, Kv3.4 is the principal contributor to K+ currents in cultured VSMCs. Furthermore, selective blockade of Kv3.4 channels resulted in a reduced proliferation rate, suggesting a link between Kv channels expression and phenotypic remodeling., Ministerio de Sanidad, Consumo y Bienestar Social - Instituto de Salud Carlos III (grants R006/009. FS041139-0 and PI041044), Ministerio de Ciencia, Innovación y Universidades (grants BFU2004-05551 and BFU2007-61524), Junta de Castilla y León (grant GR242)

Journal Article; Research Support, Non-U.S. Gov't; Soy extracts have been claimed to be neuroprotective against brain insults, an effect related to the estrogenic properties of isoflavones. However, the effects of individual isoflavones on obesity-induced disruption of adult neurogenesis have not yet been analyzed. In the present study we explore the effects of pharmacological administration of daidzein, a main soy isoflavone, in cell proliferation, cell apoptosis and gliosis in the adult hippocampus of animals exposed to a very high-fat diet. Rats made obese after 12-week exposure to a standard or high-fat (HFD, 60%) diets were treated with daidzein (50 mg kg(-1)) for 13 days. Then, plasma levels of metabolites and metabolic hormones, cell proliferation in the subgranular zone of the dentate gyrus (SGZ), and immunohistochemical markers of hippocampal cell apoptosis (caspase-3), gliosis (GFAP and Iba-1), food reward factor FosB and estrogen receptor alpha (ERα) were analyzed. Treatment with daidzein reduced food/caloric intake and body weight gain in obese rats. This was associated with glucose tolerance, low levels of HDL-cholesterol, insulin, adiponectin and testosterone, and high levels of leptin and 17β-estradiol. Daidzein increased the number of phospho-histone H3 and 5-bromo-2-deoxyuridine (BrdU)-ir cells detected in the SGZ of standard diet and HFD-fed rats. Daidzein reversed the HFD-associated enhanced immunohistochemical expression of caspase-3, FosB, GFAP, Iba-1 and ERα in the hippocampus, being more prominent in the dentate gyrus. These results suggest that pharmacological treatment with isoflavones regulates metabolic alterations associated with enhancement of cell proliferation and reduction of apoptosis and gliosis in response to high-fat diet. Yes