Metabolic and mitochondrial remodelling in cisplatin resistance: studies on ovarian cancer cells and derived cytoplasmic hybrids

The onset of resistance to cisplatin still limits its use in the chemotherapy of ovarian cancer and, despite several mechanisms of resistance have been discovered, they are not exhaustive. The aim of this study was to identify which other pathways are exploited by cancer cells to escape cisplatin cytotoxicity, to possibly prevent or overcome the phenomenon with new pharmacological approaches. The increase of anaerobic glycolysis, even in the presence of oxygen (Warburg effect), is the first observation indicating the alteration of energetic metabolism used by tumor cells as a strategy to adapt and grow independently from the availability of the substrate. This evidence suggested us to investigate the hypothesis that a similar metabolic strategy might be of relevance in resistance to cisplatin. Recently it has been shown that only approximately 1% of intracellular platinum is bound to nuclear DNA, while the great majority of the intracellular drug is available to interact with other nucleophilic sites including but not limited to phospholipids, cytosolic, cytoskeletal and membrane proteins, RNA and mitochondrial DNA. mtDNA, unlike nDNA, does not possess efficient repair systems and is therefore more susceptible to the onset of mutations often associated to cancer development, loss of tumor suppressor, activation of oncogenes and mitochondrial dysfunctions often related with an increase of glycolytic activity. Therefore, the aim of this study was to investigate the energetic metabolism and the mitochondrial function of cisplatin-resistant (C13) and sensitive (2008) ovarian cancer cells with different experimental approaches. Results revealed that resistant cells present a significant reduced respiratory chain activity correlated to a lower mitochondrial mass, altered mitochondrial morphology as well as a metabolomic profile typical of a lipogenic phenotype. To investigate the role of mtDNA and nDNA in the mitochondrial and metabolic remodeling of cisplatin-resistant line, cancer cells (2008-C13) were used to generate transmitochondrial hybrids (H2008-HC13). Mitochondrial DNA of parental and hybrid cells was sequenced, showing similar, almost non pathological, polymorphisms. Interestingly, investigating the energetic metabolism and the mitochondrial structure of hybrids, no differences were observed between H2008 and HC13. These data demonstrated that the metabolic reprogramming of C13 cells was not dependent from mtDNA, but was controlled by nuclear factors. Having regard to these data, the activity of some nuclear transcription factors (HIF-1α, and c-Myc) involved in the metabolic reprogramming of tumor cells, has been evaluated and it has been highlighted a different expression of some of their target genes involved in the glycolytic flux. Finally, the metabolic profile of 2008-C13 cells has been outlined by LC-MS which evidenced some interesting differences in aminoacids, phospholipids and antioxidants content

Boswellia serrata Preserves Intestinal Epithelial Barrier from Oxidative and Inflammatory Damage

Aminosalicylates, corticosteroids and immunosuppressants are currently the therapeutic choices in inflammatory bowel diseases (IBD), however, with limited remission and often serious side effects. Meanwhile complementary and alternative medicine (CAM) use is increasing, particularly herbal medicine. Boswellia serrata is a traditional Ayurvedic remedy with anti-inflammatory properties, of interest for its usefulness in IBDs. The mechanism of this pharmacological potential of Boswellia serrata was investigated in colonic epithelial cell monolayers exposed to H2O2 or INF-\u3b3+TNF-\u3b1, chosen as in vitro experimental model of intestinal inflammation. The barrier function was evaluated by the transepithelial electrical resistance (TEER) and paracellular permeability assay, and by the tight junction proteins (zonula occludens-1, ZO-1 and occludin) immunofluorescence. The expression of phosphorylated NF-\u3baB and reactive oxygen species (ROS) generation were determined by immunoblot and cytofluorimetric assay, respectively. Boswellia serrata oleo-gum extract (BSE) and its pure derivative acetyl-11-keto-\u3b2-boswellic acid (AKBA), were tested at 0.1-10 \u3bcg/ml and 0.027\u3bcg/ml, respectively. BSE and AKBA safety was demonstrated by no alteration of intestinal cell viability and barrier function and integrity biomarkers. H2O2 or INF-\u3b3+TNF-\u3b1 treatment of Caco-2 cell monolayers significantly reduced TEER, increased paracellular permeability and caused the disassembly of tight junction proteins occludin and ZO-1. BSE and AKBA pretreatment significantly prevented functional and morphological alterations and also the NF-\u3baB phosphorylation induced by the inflammatory stimuli. At the same concentrations BSE and AKBA counteracted the increase of ROS caused by H2O2 exposure. Data showed the positive correlation of the antioxidant activity with the mechanism involved in the physiologic maintenance of the integrity and function of the intestinal epithelium. This study elucidates the pharmacological mechanisms mediated by BSE, in protecting intestinal epithelial barrier from inflammatory damage and supports its use as safe adjuvant in patients affected by IBD

Protective effects of \u3c8 taraxasterol 3-O-myristate and arnidiol 3-O-myristate isolated from Calendula officinalis on epithelial intestinal barrier

The triterpene esters psi taraxasterol-3-O-myristate (1) and amidio1-3-O-myristate (2) were tested for their ability to protect epithelial intestinal barrier in an in vitro model. Their effects on ROS production and on trans-epithelial resistance were investigated on CaCo-2 cell monolayers both in basal and stress -induced conditions. Both compounds were able to modulate the stress damage induced by H2O2 and INF gamma + TNF alpha, showing a potential use as model compounds for the study of new therapeutic agents for intestinal inflammations

Respuestas estructurales y dieta de Chironomidae en bañados de desborde fluvial

En el presente trabajo se estudiaron parámetros estructurales (diversidad, riqueza, equitabilidad) y funcionales (dieta) de la taxocenosis de Chironomidae, con el objetivo de analizar si existen diferencias entre dos arroyos bonaerenses con bañados de desborde fluvial sometidos a diferente uso del suelo. Se seleccionaron los arroyos; Chubichaminí, con ganadería extensiva y Del Gato con agricultura intensiva y, se identificó un total de 12 géneros, con dominancia de la subfamilia Chironominae. Rheotanytarsus fue el género más abundante en el Chubichaminí mientras que Parachironomus en el Del Gato. Chubichaminí presentó mayor riqueza, diversidad de Shannon y densidad (p < 0.05, ANOVA de dos vías). El análisis de contenidos digestivos permitió establecer un mayor consumo de detritos con respecto a los demás ítems alimenticios en todos los géneros. El método de ordenamiento aplicado (MDS) permitió agrupar a los organismos según su alimentación, no pudiendo establecerse una diferenciación entre sitios de muestreo.Instituto de Limnología "Dr. Raúl A. Ringuelet

Effects of Boswellia Serrata Roxb. and Curcuma longa L. in an In Vitro Intestinal Inflammation Model Using Immune Cells and Caco-2

Inflammatory bowel diseases, which consist of chronic inflammatory conditions of the colon and the small intestine, are considered a global disease of our modern society. Recently, the interest toward the use of herbal therapies for the management of inflammatory bowel diseases has increased because of their effectiveness and favourable safety profile, compared to conventional drugs. Boswellia serrata Roxb. and Curcuma longa L. are amongst the most promising herbal drugs, however, their clinical use in inflammatory bowel diseases is limited and little is known on their mechanism of action. The aim of this work was to investigate the effects of two phytochemically characterized extracts of B. serrata and C. longa in an in vitro model of intestinal inflammation. Their impact on cytokine release and reactive oxygen species production, as well as the maintenance of the intestinal barrier function and on intestinal mucosa immune cells infiltration, has been evaluated. The extracts showed a good protective effect on the intestinal epithelium at 1 µg/mL, with TEER values increasing by approximately 1.5 fold, compared to LPS-stimulated cells. C. longa showed an anti-inflammatory mechanism of action, reducing IL-8, TNF-α and IL-6 production by approximately 30%, 25% and 40%, respectively, compared to the inflammatory stimuli. B. serrata action was linked to its antioxidant effect, with ROS production being reduced by 25%, compared to H₂O₂-stimulated Caco-2 cells. C. longa and B. serrata resulted to be promising agents for the management of inflammatory bowel diseases by modulating in vitro parameters which have been identified in the clinical conditions

Cisplatin resistance can be curtailed by blunting Bnip3-mediated mitochondrial autophagy

Cisplatin (CDDP) is commonly used to treat a multitude of tumors including sarcomas, ovarian and cervical cancers. Despite recent investigations allowed to improve chemotherapy effectiveness, the molecular mechanisms underlying the development of CDDP resistance remain a major goal in cancer research. Here, we show that mitochondrial morphology and autophagy are altered in different CDDP resistant cancer cell lines. In CDDP resistant osteosarcoma and ovarian carcinoma, mitochondria are fragmented and closely juxtaposed to the endoplasmic reticulum; rates of mitophagy are also increased. Specifically, levels of the mitophagy receptor BNIP3 are higher both in resistant cells and in ovarian cancer patient samples resistant to platinum-based treatments. Genetic BNIP3 silencing or pharmacological inhibition of autophagosome formation re-sensitizes these cells to CDDP. Our study identifies inhibition of BNIP3-driven mitophagy as a potential therapeutic strategy to counteract CDDP resistance in ovarian carcinoma and osteosarcoma

A multicenter, randomized, controlled trial on short-term feasibility and impact on functional capacity, symptoms and neurohumoral activation

RE-START is a multicenter, randomized, prospective, open, controlled trial aiming to evaluate the feasibility and the short- and medium-term effects of an earlystart AET program on functional capacity, symptoms and neurohormonal activation in chronic heart failure (CHF) patients with recent acute hemodynamic decompensation. Study endpoints will be: 1) safety of and compliance to AET; 2) effects of AET on i) functional capacity, ii) patient- reported symptoms and iii) AET-induced changes in beta-adrenergic receptor signaling and circulating angiogenetic and inflammatory markers. Two-hundred patients, randomized 1:1 to training (TR) or control (C), will be enrolled. Inclusion criteria: 1) history of systolic CHF for at least 6 months, with ongoing acute decompensation with need of intravenous diuretic and/or vasodilator therapy; 2) proBNP >1000 pg/ml at admission. Exclusion criteria: 1) ongoing cardiogenic shock; 2) need of intravenous inotropic therapy; 3) creatinine >2.5 mg/dl at admission. After a 72-hour run-in period, TR will undergo the following 12-day early-start AET protocol: days 1-2: active/passive mobilization (2 sessions/day, each 30 minutes duration); days 3-4: as days 1-2 + unloaded bedside cycle ergometer (3 sessions/day, each 5-10 minutes duration); days 5-8: as days 1-2 + unloaded bedside cycle ergometer (3 sessions/day, each 15-20 minutes duration); days 9-12: as days 1-2 + bedside cycle ergometer at 10-20 W (3 sessions/day, each 15-20 minutes duration). During the same period, C will undergo the same activity protocol as in days 1-2 for TR. All patients will undergo a 6- minWT at day 1, 6, 12 and 30 and echocardiogram, patient- reported symptoms on 7-point Likert scale and measurement of lymphocyte G protein coupled receptor kinase, VEGF, angiopoietin, TNF alfa, IL-1, IL-6 and eNOS levels at day 1, 12 and 30

Caracterización estructural y funcional de los macroinvertebrados en los bañados de desborde fluvial del área pampeana

En este estudio se caracteriza el ensamble de macroinvertebrados en bañados de desborde fluvial de cuatro arroyos pampeanos bonaerenses con diferentes actividades en el uso del suelo (intensa horticultura y ganadería extensiva) desde un punto de vista estructural y funcional. Se aplicaron índices bióticos, ecológicos, análisis multivariados y se analizaron los ensambles de oligoquetos y quironómidos. Los mayores valores diversidad y riqueza taxonómica de macroinvertebrados correspondieron al bañado de Chubichaminí (H´= 1,9; S=19,25). Este arroyo presentó aguas ligeramente poluídas, siendo aceptable en términos de calidad del agua. El análisis multivariado ordenó a los sitios de los bañados Del Gato y Carnaval con la mayor carga de nutrientes vinculados a los taxa más tolerantes como los dípteros Stratiomyidae, Ephydridae y nematodes, oligoquetos y gasterópodos Physidae. La mayor diversidad y riqueza de oligoquetos se registró en los bañados del Cajaravillas y Chubichaminí en los meses cálidos y las diferencias encontradas se debieron principalmente a Limnodrilus hoffmeisteri Claparède, 1862. El ensamble de quironómidos estuvo conformado por 24 taxones pertenecientes a Chironominae, Orthocladiinae y Tanypodinae. El género Chironomus Meigen fue más abundante en el A° Del Gato, correspondiendo principalmente a C. calligraphus Goeldi, asociado a ambientes con evidente impacto antrópico. Los sitios estudiados mostraron diferentes patrones en la proporción de grupos funcionales alimentarios sin una clara relación con el uso del suelo. Los índices aplicados en el ensamble de macroinvertebrados resultaron efectivos para evaluar el estado ecológico de los bañados analizados.In this study, the assemblage of aquatic macroinvertebrates in the riverine wetlands (RW) of four streams in the Pampean region of Buenos Aires with different activities in land use (intensive horticulture and extensive livestock) is characterized from a structural and functional point of view. Biotic and ecological indices and multivariate analyses were applied, and the assemblages of oligochaetes and chironomids were analyzed. The highest values of diversity and taxonomic richness of macroinvertebrates corresponded to the Chubichaminí RW (H´= 1.9; S = 19.25). This RW showed slightly polluted waters, being acceptable in terms of water quality. The multivariate analysis ordered the sites of Del Gato and Carnaval RWs with the highest nutrient load associated with more tolerant taxa such as the dipterans Stratiomyidae, Ephydridae, nematodes, oligochaetes and Physidae gastropods. The greatest diversity and richness of oligochaetes were recorded in the Cajaravillas and Chubichaminí RW in the warm months being the differences mainly due to Limnodrilus hoffmeisteri Claparède, 1862. The chironomid assemblage consisted of 24 taxa belonging to Chironominae, Orthocladiinae and Tanypodinae. The genus Chironomus Meigen was more abundant in Del Gato RW, corresponding mainly to C. calligraphus Goeldi, associated with environments with evident anthropic impact. The RWs studied showed different patterns in the proportion of functional feeding groups without a clear relation with land use. The indexes applied to the macroinvertebrate assemblage were effective in evaluating the ecological status of the analysed RWsInstituto de Limnología "Dr. Raúl A. Ringuelet