Natural killer cells efficiently target multiple myeloma clonogenic tumor cells.

The multiple myeloma (MM) landscape has changed in the last few years, but most patients eventually relapse because current treatment modalities do not target clonogenic stem cells, which are drug-resistant and can self-renew. We hypothesized that side population (SP) cells represent myeloma clonogenic stem cells and, searching for new treatment strategies, analyzed the anti-myeloma activity of natural killer (NK) cells against clonogenic cells. Activated and expanded NK cells (NKAE) products were obtained by co-culturing NK cells from MM patients with K562-mb15-41BBL cell line and characterized by flow cytometry. Functional experiments against MM cells were performed by Eu-TDA release assays and methylcellulose clonogenic assays. Side population was detected by Dye Cycle Violet labeling and then characterized by flow cytometry and RNA-Seq. Self-renewal capacity was tested by clonogenic assays. Sorting of both kind of cells was performed for time-lapse microscopy experiments. SP cells exhibited self-renewal potential and overexpressed genes involved in stem cell metabolism. NK cells from MM patients exhibited dysregulation and had lower anti-tumor potential against clonogenic cells than healthy donors' NK cells. Patients' NK cells were activated and expanded. These cells recovered cytotoxic activity and could specifically destroy clonogenic myeloma cells. They also had a highly cytotoxic phenotype expressing NKG2D receptor. Blocking NKG2D receptor decreased NK cell activity against clonogenic myeloma cells, and activated NK cells were able to destroy SP cells, which expressed NKG2D ligands. SP cells could represent the stem cell compartment in MM. This is the first report describing NK cell activity against myeloma clonogenic cells.

Glucocorticoid-dependent expression of IAP participates in the protection against TNF-mediated cytotoxicity in MCF7 cells.

BACKGROUND: Glucocorticoid receptor (GR) activation has been associated with breast cancer cell survival in vitro. Glucocorticoid (GC)-dependent protection against tumor necrosis factor (TNF)-induced cell death has been well characterized in MCF7 luminal A breast cancer cells. The GR activates a variety of protective mechanisms, such as inhibitors of apoptosis proteins (IAPs). However, the relative contribution of the GR-dependent expression of IAPs in the protection of cell death has not, to our knowledge, been evaluated. METHODS: MCF7 cells were used for all experiments. GR was activated with cortisol (CORT) or dexamethasone (DEX) and inhibited with mifepristone (RU486). Cell viability was determined in real-time with the xCELLigence™ RTCA System and at specific endpoints using crystal violet stain. The mRNA levels of the eight members of the IAP family were measured by qRT-PCR. The protein levels of GR, PR, ERα, HER2, PARP1, c-IAP1 and XIAP were evaluated by Western blot analysis. The knockdown of c-IAP1 and XIAP was accomplished via transient transfection with specific siRNAs. GR activation was verified by a gene reporter assay. Via the cBioportal interphase we queried the mRNA levels of GR and IAPs in breast cancer tumors. RESULTS: RU486 significantly inhibited the anti-cytotoxic effect of both GCs. PARP1 processing was diminished in the presence of both GCs. The combined treatments of GCs + TNF increased the relative mRNA levels of Survivin>c-IAP1 > NAIP>Apollon>XIAP>Ts-IAP > ML-IAP > c-IAP2. Additionally, GR mRNA content increased with the combined treatments of GCs + TNF. Sustained levels of the proteins c-IAP1 and XIAP were observed after 48 h of the combined treatments with GCs + TNF. With c-IAP1 and XIAP gene silencing, the GC-mediated protection was diminished. In the breast tumor samples, the GR mRNA was coexpressed with Apollon and XIAP with a Pearson coefficient greater than 0.3. CONCLUSIONS: The effect of GCs against TNF-mediated cytotoxicity involves increased mRNA expression and sustained protein levels of c-IAP1 and XIAP. The antagonist effects of RU486 and the qRT-PCR results also suggest the role of the GR in this process. This finding may have clinical implications because the GR and IAPs are expressed in breast tumor samples.

Effect of soluble factors derived from ZR 75.30 breast cancer cells on endothelial activation.

In this study, we analyzed soluble factors secreted by two Estrogen Receptor Positive (ER-α) human breast cancer cell lines, ZR 75.30 (luminal B) and MCF7 (luminal A), and evaluated their effect on endothelial activation. The composition of tumoral soluble factors (TSFs) was analyzed by ELISA (Bio-Plex). TSFs from ZR 75.30 cells expressed higher levels of TNF, IFN-γ, IL-6, and IL-8 compared to TSFs from MCF-7 cells. TSFs from ZR 75.30 cells induced a pro-adhesive phenotype in human umbilical vein endothelial cells (HUVECs), as characterized by increased monocytic cell adhesion, adhesion molecule expression and NF-κB activation and decreased IκB-α expression. Conversely, TSFs from MCF-7 cells exerted none of these effects on HUVECs. We then added TNF, IFN-γ, IL-6 or IL-8 alone or in combination with TSFs from MCF-7 cells to HUVECs. Only the combinations that included TNF induced endothelial activation. A neutralizing antibody against IL-1ß (this cytokine was not measured in the ELISA) had a modest blocking effect on cellular adhesion or the expression of adhesion molecules induced by TSFs from ZR 75.30 cells in HUVECs. However neutralizing antibodies against TNF, IFN-γ, IL-6 or IL-8 had no effect. Our results suggest that although TNF is an inducer of endothelial cell activation, it is not the only molecule that is responsible for this effect in TSFs from ZR 75.30 cells.

Pro-adhesive phenotype of normal endothelial cells responding to metastatic breast cancer cell conditioned medium is linked to NFκB-mediated transcriptomic regulation.

Tumor microenvironment is an important promoter of tumorigenesis in all forms of breast cancer and has been associated with the risk of metastasis in the different breast cancer subtypes including the more frequent luminal subtypes that encompass 60% of cancer patients. Adhesive properties of endothelial cells (ECs) are strikingly affected during cancer cell dissemination and are related to functional changes of adhesion receptors. The contribution of tumor secreted factors to tumor­EC adhesion represents a therapeutic opportunity for breast cancer metastasis. Conditioned medium (CM) of tumor cells can be used as a model to study the role of the secreted molecules to the tumor microenvironment. We explored transcriptomic changes associated to a pro­adhesive phenotype in primary human umbilical vein endothelial cells (HUVECs) treated with CM of the breast cancer cell line ZR75.30 or with TNF for 3 h. Selected genes were used to validate the microarray through RT­qPCR. The bioinformatic analysis identified NFκB as the main regulator of the pro-adhesive phenotype and this was confirmed by pharmacological inhibition of NFκB pathway with BAY 11­7085. The changes induced by ZR75.30­CM mimic those promoted by TNF and display changes in the expression of genes related to inflammatory response, wound healing, extracellular matrix, cytokines, metabolism and cell communication. Despite the abundance of G­CSF, IL­8, IL­6 and VEGF in the ZR75.30­CM and the confirmed activation of STAT3 and VEGFR2 pathways, our results suggest dominance of NFκB as a central controller of the transcriptomic response of ECs to breast cancer cells leading to expression of cell adhesion receptors.

Estudio sobre muerte súbita en deportistas 1985-1995 / Study of sudden death in sportsman 1985-1995

La relación entre el corazón y sus cambios con el ejercicio ha sido motivo de estudios desde finales del siglo XIX y desde entonces varios autores como Niemela, Kirsch y Linzbach analizaron la muerte de deportistas encontrando en la mayor parte de los casos alteraciones en el corazón, en las coronarias y un componente arterioesclerótico importante. Esto originó el síndrome del Corazón del Deportista, el cual consiste en la adaptación fisiológica que hace el músculo cardíaco ante el aumento de trabajo que genera el ejercicio, y se refleja en una hipertrofia global y bradicardia con presión arterial normal. La muerte súbita en deportistas es la que ocurre instantáneamente durante la actividad o minutos después de la misma. Filipedes fue el primer deportista que murió después de correr una Maratón en Atenas en el año 490 A.C. Se ha observado que los deportistas fallecidos menores de 30 años de edad, generalmente tienen un problema cardiovascular de carácter congénito y los mayores de 30 años tienen un componente arterioesclerçotico severo. Tomando en cuenta estos conceptos los Doctores Mayela Valerio Hernández, Alma Reyes Guzmán y Lempira Escobar Yanes, analizaron 10 años de autopsias entre enero de 1985 y julio de 1995 en los cuales se analizaron 19.359 autopsias y de estas, 33 casos correspondieron a muerte súbita en deportistas. En todos los casos se descartó el trauma y se hizo estudio histológico de los tejidos. El año que más casos presentó fue 1994 y la mayoría de ellos procedía de San José. De estos 33 casos, 32 fueron hombres, solo una mujer, 31 eran de raza blanca y 2 de raza negra. El mayor grupo de edad se ubicó entre 26-30 años con 10 casos. El más joven de todos tenía 12 años y el mayor 62 años, para una edad promedio de 29 años. El deporte más practicado fue el fútbol, acorde con nuestra cultura, seguido del atletismo, natación, ciclismo, baloncesto, motocross. Según la causa de muerte, 26 casos fueron de origen cardíaco (72 por ciento), 5 de origen cerebral (15 por ciento) y 2 por origen vascular (6 por ciento). Confirmando los datos de la literatura al respecto, en los casos menores de 30 años (11 casos), se encontraron como causa de muerte en orden decreciente: cardiomiopatía isquémica, vasculopatía hipertrófica, miocarditis inespecífica, comunicación intrauricular, miocardiopatía idiopática, displasia fibromuscular, alteraciones en la conformación de las coronarias y solo dos personas tenían antecedentes de tabaquismo y alcoholismo...