Efecto de nuevas terapias anti-ERBB-2 y ácido retinoico en el movimiento de células de cáncer de mama humano

El carcinoma de mama se clasifica en subtipos moleculares de acuerdo a lapresencia/ausencia de receptores de estrógenos (RE), receptor de progesterona y el receptor 2 del factor de crecimiento epidérmico humano (ErbB-2, también conocido como HER-2). Los tumores que presentan sobreexpresión de HER-2 son más agresivos y metastásicos, debido a que la señalización a través de este receptor contribuye a la transformación oncogénica, teniendo las pacientes mal pronóstico. Se han desarrollado nuevas estrategias anti-HER-2 que se utilizan actualmente en la clínica, un anticuerpo monoclonal que se une al dominioextracelular de HER-2 llamado trastuzumab (Tz) o Herceptin®. A pesar de este progreso, entre el 40-60% de los pacientes no responden a la terapia odesarrollan resistencia a la misma. Todo esto, lleva a la necesidad de desarrollar nuevos enfoques terapéuticos basados en la combinación de distintas drogas. Los retinoides, y principalmente el ácido retinoico (AR) han sido propuestos en el tratamiento adyuvante del carcinoma de mama por su capacidad para inhibir el crecimiento e inducir la diferenciación morfológica o fenotípica. Su actividad antitumoral está mediada principalmente por losreceptores de ácido retinoico (RAR), que pertenecen a la superfamilia de receptores nucleares. El proceso de adhesión y migración celular es crítico para que la célula se propague, invada y realice metástasis en otro sitio. Moesin induce la despolimerización de la actina y su translocación hacia el borde de la membrana celular, siendo responsable de la formación de complejos corticales de actina. Otra proteína clave es la quinasa de adhesión focal (FAK), la cual participa en el ensamblaje y desensamblaje de los complejos de adhesiones focales, reorganizándolos en la dirección de la migración; su sobreexpresión esta correlacionada con tumores más agresivos. La hipótesis de este proyecto es que la combinación de trastuzumab (Tz) y ácido retinoico (AR) tiene un efecto sinérgico en la disminución de la viabilidad, adhesión y migración de células de cáncer de mama humano HER-2+/RAR+ mediante modificaciones en la expresión/localización de proteínas vinculadas en el movimiento celular como moesin y FAK. Específicamente proponemos 1) Evaluar el efecto de terapias anti-ErbB-2 en combinación o no con el AR en la viabilidad, adhesión y migración de células de cáncer de mama. 2) Investigar la morfología de células tumorales mamarias, mediante el estudio del citoesqueleto actínico luego de los tratamientos con nuevas terapias anti-ErbB-2 en combinación o no con AR. 3) Identificar el posible mecanismo molecular por el cual las terapias anti-ErbB-2regularían/modularían, la expresión de proteínas vinculadas en procesos claves del movimiento celular como moesin, FAK y el receptor HER-2. Se utilizaron las líneas celulares de cáncer de mama humano SKBR-3 (RE-/HER-2+/RAR+) y BT-474 (RE+/HER-2+/RAR+) para un 90 análisis comparativo, ya que se ha demostrado que el tratamiento con Tz tiene un menor efecto en pacientes que presentan tumores RE+ comparado con las que son RE-. Se realizaron tratamientos durante 72 horas con Tz 1 y 10 μg/ml, AR 10-6 M y la combinación de ambas drogas y luego ensayos de MTT, análisis de interacción farmacológica, inmunofluorescencia, ensayos de adhesión, migración e invasión celular, silenciamientos con ARN de interferencia, uso del inhibidor de FAK y western blot. Se demostró que eltratamiento durante 72 horas con distintas dosis de Tz (0,1 ? 1 ? 10 ? 100 μg/ml) y de AR (10-8 ? 10-7 ? 10-6 ? 10-5 M) disminuye la proliferación celular de manera dosis-dependiente en ambas líneas celulares (Fig. A). En la línea SKBR-3 el tratamiento con Tz 1 ? 10 ? 100 μg/ml en combinación con AR 10-6 M el efecto es sinérgico (Fig. B). Por otro lado, en la línea celular BT-474 el tratamiento con Tz 10 ? 100 μg/ml en combinación con AR 10-6 M ejerce un efectosinérgico a diferencia del tratamiento con Tz 1 μg/ml combinado con AR 10-6 M que genera un efecto aditivo en la disminución de la proliferación celular. El índice de reducción de dosis (DRI), obtenido para la línea SKBR-3 indica que dentro de las combinaciones sinérgicas Tz 1 ?10 μg/ml + AR 10-6 M podemos disminuir 10 veces la dosis de Tz logrando el mismo efecto mientras que para la combinación Tz 100 μg/ml + AR 10-6 M la reducción de la dosis de Tz es de 100 veces (Fig. C). En la línea BT-474, en la combinación Tz 10 μg/ml + AR 10-6 M, la dosis de Tz utilizada es 10 veces menor cuando se lo usa dentro de ésta combinación sinérgica y para la combinación Tz 100 μg/ml + AR 10-6 M, el DRI indica que dentro de este escenario sinérgico el efecto es 100 veces más potente comparado con Tz sólo. La coadministración de las drogas disminuyó con lostratamientos. Mediante inmunofluorescencia se observó que Tz 10 μg/ml, AR 10-6 M y la combinación de ambas drogas, induce la translocación de FAK desde el citosol al núcleo en ambas líneas celulares (Fig. E). Además, se observó una distribución granular del receptor HER-2 luego de los tratamientos combinados (Fig. F). Mediante microscopía confocal pudimos confirmar que en las células control HER-2 se encuentra en la membrana celular y la coadministración de ambas drogas induce la internalización de éstos, en mayor medida en la línea SKBR-3. Los tratamientos combinados de AR y Tz disminuyen fuertemente la expresión de FAK, Moesin y HER-2 (Fig. G). En conclusión, en modelos celulares de cáncer de mama HER-2+/RAR+, la coadministración de Tz y AR, ejercen un efecto sinérgico en la disminución de la proliferación celular, lo que permite reducir las dosis utilizadas, disminuyendo la toxicidad mientras se mantiene la eficacia.Además la coadministración de Tz y AR inducen la relocalización de FAK al núcleo y la disminución de la adhesión/migración/invasión celular en mayor medida en la línea RE-. La internalización y posterior degradación del receptor HER-2 tendría un efecto benéfico al desactivar la cascada de activación de éstas vías altamente oncogénicas. La combinación Tz más AR resultó en una potente disminución de la expresión de las proteínas esenciales para la migración, FAK/Moesin y del receptor HER-2. Estos hallazgos sugieren que la coadministración de ambas drogas, en pacientes con este tipo de cáncer podría contribuir a mejorar su pronóstico y a disminuir los efectos adversosde la terapia debido a que las dosis de Tz aplicadas serían menores gracias al efecto adyuvante de AR.Fil: Vanderhoeven, F.. No especifíca;Fil: Flamini, Marina Ines. Consejo Nacional de Investigaciones Científicas y Técnicas. Centro Científico Tecnológico Conicet - Mendoza. Instituto de Medicina y Biología Experimental de Cuyo; Argentin

EXTRA-NUCLEAR SIGNALING OF ESTROGEN RECEPTOR TO BREAST AND ENDOMETRIAL CANCER CELL MOVEMENT AND INVASION THROUGH THE ACTIN CYTOSKELETON

Estrogen and selective estrogen receptor modulators (SERMs) differentially impact endometrial and breast cancer cell function, however, the biological basis of these differences is not established. Deregulated cell adhesion to the extracellular matrix, cell movement and invasion are related to several disorders, such as endometriosis, endometrial and breast cancer metastasis. Remodeling of the actin cytoskeleton is required to achieve cell adhesion and movement. Estrogen receptor (ER) regulates actin and cell membrane remodeling through extra-nuclear signaling cascades. The aim of the present study was to characterize the effects of raloxifene (RAL), tamoxifen (TAM) or of 17β-estradiol (E2) with or without RAL or TAM on ER+T47-D breast cancer cell and on endometrial cell line Ishikawa or human endometrial stromal cells (ESC) cytoskeletal remodeling, migration and invasion. Our findings show that, when given alone, RAL induces a weak actin cytoskeleton remodeling in breast cancer cells, with the formation of specialized cell membrane structures implicated in cell motility and interaction with the extracellular matrix. However, in the presence of physiological amounts of estradiol, that potently drives breast cancer cell cytoskeletal remodeling and motility, RAL displays a powerful inhibitory effect on estrogen-promoted cell migration and invasion. These actions are plaid through an interference of RAL with an extra-nuclear signaling cascade involving G proteins and the RhoA-associated kinase, ROCK-2, linked to the recruitment of the cytoskeletal controller, moesin. Hence, in the presence of E2, RAL acts as an ER antagonist on a set of cellular events implicated with the process of breast cancer migration and metastasis. Furthermore, we show that administration E2 and TAM to Ishikawa or to ESC also results in remodeling of actin fibers and cell membrane. This is linked to rapid phosphorylation on moesin and enhanced migration and invasion of normal and Ishikawa cells. On the contrary RAL alone does not result in moesin activation or actin remodeling in endometrial cells. When endometrial cells are exposed to E2 in the presence of TAM or RAL, both SERMs interfere with the recruitment of moesin, with the remodeling of the cytoskeleton, and with cell movement and migration induced by E2. The differential actions of E2, TAM and RAL are linked to a distinct modulation of the extra- nuclear signaling of ER to G proteins and to the Rho-associated kinase. These findings increase our understanding of the actions of estrogen and SERMs in breast cancer and endometrial cells and highlight potential molecular targets to interfere with the estrogen-related altered cell adhesion encountered in endometrial disorders or breast cancer progression and/or metastasis induced by estrogens in postmenopausal women

Rapid estrogen and progesterone signaling to dendritic spine formation via cortactin/wave1-ARP2/3 complex

Synaptic plasticity is the neuronal capacity to modify the function and structure of dendritic spines (DS) in response to neuromodulators. Sex steroids, particularly 17β-estradiol (E2) and progesterone (P4), are key regulators in the control of DS formation through multiprotein complexes including WAVE1 protein, and are thus fundamental for the development of learning and memory. Objectives: The aim of this work was to evaluate the molecular switch Cdk5 kinase/PP2A phosphatase in the control of WAVE1 protein(phosphorylation/dephosphorylation) and the regulation of WAVE1 and cortactin to the Arp2/3 complex, in response to rapid treatments with E2 and P4 in cortical neuronal cells. Results: Rapid treatment with E2 and P4 modified neuronal morphology and significantly increased the number of DS. This effect was reduced by the use of a Cdk5 inhibitor (Roscovitine). In contrast, inhibition of PP2A with PP2A DN construct significantly increased DS formation, evidencing the participation of kinase/phosphatase in the regulation of WAVE1 in DS formation induced by E2 and P4. Cortactin regulates DS formation via Src and PAK1 kinase induced by E2 and P4. Both cortactin and WAVE1 signal to Arp2/3 complex to synergistically promote actin nucleation. Conclusion: These results suggest thatE2 and P4 dynamically regulate neuron morphology through non-genomic signaling via cortactin/WAVE1-Arp2/3 complex. The control of these proteins is tightly orchestrated by phosphorylation, where kinases and phosphatases are essential for actin nucleation and, finally, DS formation. This work provides a deeper understanding of the biological actions of sex steroids in the regulation of DS turnover and neuronal plasticity processes.Fil: Uzair, Ivonne Denise. Consejo Nacional de Investigaciones Científicas y Técnicas. Centro Científico Tecnológico Conicet - Mendoza. Instituto de Medicina y Biología Experimental de Cuyo; ArgentinaFil: Flamini, Marina Ines. Consejo Nacional de Investigaciones Científicas y Técnicas. Centro Científico Tecnológico Conicet - Mendoza. Instituto de Medicina y Biología Experimental de Cuyo; ArgentinaFil: Sanchez, Angel Matias. Consejo Nacional de Investigaciones Científicas y Técnicas. Centro Científico Tecnológico Conicet - Mendoza. Instituto de Medicina y Biología Experimental de Cuyo; Argentin

Molecular Actions of Thyroid Hormone on Breast Cancer Cell Migration and Invasion via Cortactin/N-WASP

The thyroid hormone triiodothyronine (T3) plays a fundamental role in growth regulation, differentiation, metabolism and cellular movement. These processes are particularly important considering that deregulation of T3 levels could promote abnormal responsiveness of mammary epithelial cells, which may lead to the development and progression of breast cancer (BC). Once cells migrate and invade different tissues, BC metastasis is the main cause of cancer-related death because it is particularly difficult to revert this multistep process. Cell migration integrates several steps that induce changes in cell structure and morphology to promote BC cell invasion. These sequential steps include actin cytoskeleton remodeling, focal adhesion complex formation and, finally, the turnover of branched actin filament networks. In this article, we demonstrate that T3 has the ability to modify the Epithelial-Mesenchymal Transition process. In addition, we show that T3 induces actin cytoskeleton reorganization, triggers focal adhesion formation and, as a consequence, promotes actin nucleation via non-genomic pathway. These events are specifically modulated by T3 via integrin αvβ3 to FAK/paxillin/cortactin/N-WASP/Arp2/3 complex signaling pathway, increasing cell adhesion, migration and invasion of T-47D BC cells. We suggest that T3 influences the progression of tumor metastasis by controlling signaling pathways that converge in cell motility. This knowledge is crucial for the development of novel therapeutic strategies for BC treatment

Molecular basis of LH action on breast cancer cell migration and invasion via kinase and scaffold proteins

Breast cancer (BC) is a major public health problem affecting women worldwide. Approximately 80% of diagnosed cases are hormone-dependent breast cancers. These hormones are known to stimulate tumor development and progression. In this setting, tentative evidence suggests that luteinizing hormone (LH) may also play a role in tumors. In BC cells that express functional LH receptors (LHR), this hormone regulates cell migration and invasion by controlling several kinases that activate actin cytoskeletal proteins. In this article, we show that LH induces phosphorylation of paxillin and its translocation toward the plasmatic membrane, where focal adhesion complexes are assembled. This process is triggered via a rapid extra-gonadal LHR signaling to Src/FAK/paxillin, which results in the phosphorylation/activation of the nucleation promoter factors cortactin and N-WASP. As a consequence, Arp2/3 complexes induce actin polymerization, essential to promote cell adhesion, migration, and invasion, thus enhancing metastatic spread of tumoral cells. Our findings provide relevant information about how gonadotrophins exert their action in BC. This information helps us understand the extragonadal effects of LH on BC metastasis. It may provide new perspectives for therapeutic treatment, especially for women with high serum levels of gonadotrophins.Fil: Mondaca, Joselina Magali. Consejo Nacional de Investigaciones Científicas y Técnicas. Centro Científico Tecnológico Conicet - Mendoza. Instituto de Medicina y Biología Experimental de Cuyo; ArgentinaFil: Uzair, Ivonne Denise. Consejo Nacional de Investigaciones Científicas y Técnicas. Centro Científico Tecnológico Conicet - Mendoza. Instituto de Medicina y Biología Experimental de Cuyo; ArgentinaFil: Castro Guijarro, Ana Carla. Consejo Nacional de Investigaciones Científicas y Técnicas. Centro Científico Tecnológico Conicet - Mendoza. Instituto de Medicina y Biología Experimental de Cuyo; ArgentinaFil: Flamini, Marina Ines. Consejo Nacional de Investigaciones Científicas y Técnicas. Centro Científico Tecnológico Conicet - Mendoza. Instituto de Medicina y Biología Experimental de Cuyo; ArgentinaFil: Sanchez, Angel Matias. Consejo Nacional de Investigaciones Científicas y Técnicas. Centro Científico Tecnológico Conicet - Mendoza. Instituto de Medicina y Biología Experimental de Cuyo; Argentin

Retinoic acid reduces migration of human breast cancer cells: role of retinoic acid receptor beta

Breast cancer is the most common malignancy in women and the appearance of distant metastases produces the death in 98% of cases. The retinoic acid receptor b (RARb) is not expressed in 50% of invasive breast carcinoma compared with normal tissue and it has been associated with lymph node metastasis. Our hypothesis is that RARb protein participates in the metastatic process. T47D and MCF7 breast cancer cell lines were used to perform viability assay, immunobloting, migration assays, RNA interference and immunofluorescence. Administration of retinoic acid (RA) in breast cancer cells induced RARb gene expression that was greatest after 72 hrs with a concentration 1 lM. High concentrations of RA increased the expression of RARb causing an inhibition of the 60% in cell migration and significantly decreased the expression of migration-related proteins [moesin, c-Src and focal adhesion kinase (FAK)]. The treatment with RARa and RARc agonists did not affect the cell migration. On the contrary, the addition of the selective retinoid RARb-agonist (BMS453) significantly reduced cell migration comparable to RA inhibition. When RARb gene silencing was performed, the RA failed to significantly inhibit migration and resulted ineffective to reduce moesin,c-Src and FAK expressions. RARb is necessary to inhibit migration induced by RA in breast cancer cells modulating the expression of proteins involved in cell migration.Fil: Flamini, Marina Ines. Consejo Nacional de Investigaciones Científicas y Técnicas. Centro Científico Tecnológico Conicet - Mendoza. Instituto de Medicina y Biología Experimental de Cuyo; ArgentinaFil: Gauna, Gisel Valeria. Consejo Nacional de Investigaciones Científicas y Técnicas. Centro Científico Tecnológico Conicet - Mendoza. Instituto de Medicina y Biología Experimental de Cuyo; Argentina. Universidad Nacional de Cuyo. Facultad de Ciencias Médicas; ArgentinaFil: Sottile Fleury, Mayra Lis. Consejo Nacional de Investigaciones Científicas y Técnicas. Centro Científico Tecnológico Conicet - Mendoza. Instituto de Medicina y Biología Experimental de Cuyo; ArgentinaFil: Nadin, Silvina Beatriz. Consejo Nacional de Investigaciones Científicas y Técnicas. Centro Científico Tecnológico Conicet - Mendoza. Instituto de Medicina y Biología Experimental de Cuyo; ArgentinaFil: Sanchez, Angel Matias. Consejo Nacional de Investigaciones Científicas y Técnicas. Centro Científico Tecnológico Conicet - Mendoza. Instituto de Medicina y Biología Experimental de Cuyo; ArgentinaFil: Vargas Roig, Laura Maria. Consejo Nacional de Investigaciones Científicas y Técnicas. Centro Científico Tecnológico Conicet - Mendoza. Instituto de Medicina y Biología Experimental de Cuyo; Argentin

Bioactivity, proximate, mineral and volatile profiles along the flowering stages of Opuntia microdasys (Lehm.): defining potential applications

Opuntia spp. flowers have been traditionally used for medical purposes, mostly because of their diversity in bioactive molecules with health promoting properties. The proximate, mineral and volatile compound profiles, together with the cytotoxic and antimicrobial properties were characterized in O. microdasys flowers at different maturity stages, revealing several statistically significant differences. O. microdasys stood out mainly for its high contents of dietary fiber, potassium and camphor, and its high activities against HCT15 cells, Staphylococcus aureus, Aspergillus versicolor and Penicillium funiculosum. The vegetative stage showed the highest cytotoxic and antifungal activities, whilst the full flowering stage was particularly active against bacterial species. The complete dataset has been classified by principal component analysis, achieving clearly identifiable groups for each flowering stage, elucidating also the most distinctive features, and comprehensively profiling each of the assayed stages. The results might be useful to define the best flowering stage considering practical application purposes.The authors are grateful to Fundação para a Ciência e a Tecnologia (FCT, Portugal) for financial support to CIMO (strategic project PEst-OE/AGR/UI0690/2011) and ALIMNOVA research group (UCM-GR35/10A). J.C.M. Barreira and R.C. Calhelha thank FCT, POPH-QREN and FSE for their grants (SFRH/BPD/72802/2010 and SFRH/BPD/68344/2010, respectively). The Serbian Ministry of Education is also acknowledged for the Science and Technological Development Grant No. 173032

Bioactivity, hydrophilic, lipophilic and volatile compounds in pulps and skins of Opuntia macrorhiza and Opuntia microdasys fruits

Opuntia genus includes several species able to grow in arid regions and known for producing delicate fruits, which are far from being thoroughly characterized. Herein, fruits from O. macrorhiza and O. microdasys were divided in skins and pulps (without including seeds) and studied for chemical composition, individual phytochemicals and bioactivity. The major volatiles were camphor and ethyl acetate, while citric acid was the main organic acid. The fatty acids detected in highest percentages were linoleic acid (skins) and lauric acid (pulps); α- tocopherol was the major isoform of vitamin E. Quercetin-3-O-rutinoside and quercetin-O-(deoxyhexoside-rutinoside) were the main phenolics in fruit skins of O. macrorhiza and O. microdasys (respectively), similarly to piscidic acid in O. macrorhiza pulp (O. microdasys pulp showed no quantifiable compounds). Betanin and isobetanin were the major betalains. All samples were antioxidant (particularly O. macrorhiza), but antimicrobial activity was only detected in skins. Cytotoxicity was low in all cases. Overall, these fruits proved to be potential new ingredients for food or pharmaceutical related applications, adding value to these natural species able to grow in arid environments.Authors are grateful to the Foundation for Science and Technology (FCT, Portugal) and FEDER under Programme PT2020 for financial support to CIMO (UID/AGR/00690/2013), C2TN (UID/Multi/04349/ 2013), L. Barros, J. Barreira and R. Calhelha contracts. This work is funded by the European Structural and Investment Funds (FEEI) through the Regional Operational Program North 2020, within the scope of Project Mobilizador ValorNatural®. Authors also thank FEDER Interreg España-Portugal programme for financial support through the project 0377_Iberphenol_6_E.info:eu-repo/semantics/publishedVersio

Drospirenone increases endothelial nitric oxide synthesis via a combined action on progesterone and mineralocorticoid receptors

BACKGROUND: Progestins have actions on the cardiovascular system, which depend on the structure as well as on receptor binding characteristics. Drospirenone (DRSP) is a progestin that uniquely interferes with the signaling of the mineralocorticoid receptor (MR). Hormone therapy containing DRSP results in blood pressure reduction in hypertensive post-menopausal women. METHODS: We describe the effects of DRSP on endothelial nitric oxide (NO) synthesis and compare them with those of progesterone (P) and of medroxyprogesterone acetate (MPA). In addition, we herein tested the relevance of the anti-mineralocorticoid activity of DRSP for NO synthesis. RESULTS: DRSP results in rapid activation of the endothelial NO synthase (eNOS) through mitogen-activated protein kinases and phosphatidylinositol 3-kinase as well as in enhanced eNOS expression. These actions depend on P receptor. When the cells are exposed to aldosterone, a reduction of eNOS expression is found that is antagonized by DRSP. This action is not shared by P or MPA. In addition, DRSP does not interfere with the induction or activation of eNOS induced by estradiol, as opposed to MPA. CONCLUSIONS: DRSP acts on endothelial cells via a combined action through the P and MRs. These results help to interpret the anti-hypertensive effects of hormonal therapies containing DRSP

Chemical Composition and Biological Activities of Oregano and Lavender Essential Oils

Folk medicine uses wild herbs, especially from the Lamiaceae family, such as oregano and lavender, in the treatment of many diseases. In the present study, we investigated the antibacterial activity of the essential oils of Origanum glandulosum Desf. and Lavandula dentata L. against multidrug- resistant Klebsiella pneumoniae strains. The chemical composition of essential oils and their effect on the ultrastructure of the tested bacteria and on the release of cellular components that absorb at 260 nm were studied. Furthermore, the cytotoxicity and the production of reactive oxygen species in human lymphocytes treated with essential oils were evaluated. Thymol (33.2%) was the major constituent in O. glandulosum, and β-pinene (17.3%) was the major constituent in L. dentata. We observed ultrastructural damage in bacteria and increased release of cellular material. Furthermore, ROS production in human lymphocytes treated with essential oils was lower than in untreated lymphocytes and no cytotoxicity was observed. Therefore, the essential oils of lavender and oregano could be used as a source of natural antibacterial and antioxidant agents with potential pharmacological applications