Apoptotic cell death in mammary adenocarcinoma cells is prevented by soluble factors present in the target organ of metastasis

Target organ of metastasis determines the fate of metastasis. The soluble factors released from one or more cell types in the new stroma may influence growth and survival of metastatic cells. In the present study, we used conditioned media from the kidney, liver and lung, the latter being the target organ of metastasis of murine mammary adenocarcinoma cell lines LM3, LMM3 and F3II, to assess whether the soluble factors released from these organs could modulate in vitro survival of these cell lines after apoptosis-inducing treatments and to investigate the mechanisms involved in this effect. We demonstrate that conditioned medium from lung, but not from liver or kidney, promotes survival of these cells after doxorubicin, cisplatin, agonistic anti-Fas antibody and serum withdrawal treatments. Furthermore, LMM3 cells treated with lung conditioned medium after doxorubicin exposure maintained their tumorigenic capacity and metastatic potential. Neither IGF nor EGF could promote survival but, surprisingly, TGF-β could reduce sensitivity of LMM3 cells to doxorubicin in vitro. Doxorubicin treatment induced Bax expression and down-regulated Bcl-2 expression. In contrast, lung conditioned medium increased Bcl-2 expression and inhibited doxorubicin-mediated Bcl-2 down-regulation. Neither of those treatments alone modified Bcl-xL expression, although co-treatment induced a 3- to 5-fold increase of its expression. These results suggest that the lung microenvironment could promote metastasis of these adenocarcinoma cell lines by increasing survival of metastatic cells, possibly by modulation of Bcl-2 protein family expression.Fil: Ladeda, Virginia. Universidad de Buenos Aires. Facultad de Medicina. Instituto de Oncología "Ángel H. Roffo"; ArgentinaFil: Adam, Alejandro P.. Universidad de Buenos Aires. Facultad de Medicina. Instituto de Oncología "Ángel H. Roffo"; ArgentinaFil: Puricelli, Lydia Ines. Universidad de Buenos Aires. Facultad de Medicina. Instituto de Oncología "Ángel H. Roffo"; Argentina. Consejo Nacional de Investigaciones Científicas y Técnicas; ArgentinaFil: Bal De Kier Joffé, Elisa. Universidad de Buenos Aires. Facultad de Medicina. Instituto de Oncología "Ángel H. Roffo"; Argentin

Generation and characterization of human insulin-releasing cell lines

<p>Abstract</p> <p>Background</p> <p>The in vitro culture of insulinomas provides an attractive tool to study cell proliferation and insulin synthesis and secretion. However, only a few human beta cell lines have been described, with long-term passage resulting in loss of insulin secretion. Therefore, we set out to establish and characterize human insulin-releasing cell lines.</p> <p>Results</p> <p>We generated ex-vivo primary cultures from two independent human insulinomas and from a human nesidioblastosis, all of which were cultured up to passage number 20. All cell lines secreted human insulin and C-peptide. These cell lines expressed neuroendocrine and islets markers, confirming the expression profile found in the biopsies. Although all beta cell lineages survived an anchorage independent culture, none of them were able to invade an extracellular matrix substrate.</p> <p>Conclusion</p> <p>We have established three human insulin-releasing cell lines which maintain antigenic characteristics and insulin secretion profiles of the original tumors. These cell lines represent valuable tools for the study of molecular events underlying beta cell function and dysfunction.</p

Tumor Cell Phenotype Is Sustained by Selective MAPK Oxidation in Mitochondria

Mitochondria are major cellular sources of hydrogen peroxide (H2O2), the production of which is modulated by oxygen availability and the mitochondrial energy state. An increase of steady-state cell H2O2 concentration is able to control the transition from proliferating to quiescent phenotypes and to signal the end of proliferation; in tumor cells thereby, low H2O2 due to defective mitochondrial metabolism can contribute to sustain proliferation. Mitogen-activated protein kinases (MAPKs) orchestrate signal transduction and recent data indicate that are present in mitochondria and regulated by the redox state. On these bases, we investigated the mechanistic connection of tumor mitochondrial dysfunction, H2O2 yield, and activation of MAPKs in LP07 murine tumor cells with confocal microscopy, in vivo imaging and directed mutagenesis. Two redox conditions were examined: low 1 µM H2O2 increased cell proliferation in ERK1/2-dependent manner whereas high 50 µM H2O2 arrested cell cycle by p38 and JNK1/2 activation. Regarding the experimental conditions as a three-compartment model (mitochondria, cytosol, and nuclei), the different responses depended on MAPKs preferential traffic to mitochondria, where a selective activation of either ERK1/2 or p38-JNK1/2 by co-localized upstream kinases (MAPKKs) facilitated their further passage to nuclei. As assessed by mass spectra, MAPKs activation and efficient binding to cognate MAPKKs resulted from oxidation of conserved ERK1/2 or p38-JNK1/2 cysteine domains to sulfinic and sulfonic acids at a definite H2O2 level. Like this, high H2O2 or directed mutation of redox-sensitive ERK2 Cys214 impeded binding to MEK1/2, caused ERK2 retention in mitochondria and restricted shuttle to nuclei. It is surmised that selective cysteine oxidations adjust the electrostatic forces that participate in a particular MAPK-MAPKK interaction. Considering that tumor mitochondria are dysfunctional, their inability to increase H2O2 yield should disrupt synchronized MAPK oxidations and the regulation of cell cycle leading cells to remain in a proliferating phenotype

A basic phospholipase A2 from Bothrops diporus venom inhibits cancer cell adhesion and migration

The vast majority of snakebite envenomings in northeastern Argentina are caused by Bothrops diporus. Thevenom of this species causes local tissue damage characterized by myonecrosis, hemorrhage, blistering, andedema. In the present study, we isolated a basic PLA 2 from B. diporus venom, and examined its potentialadhesion and migration inhibition effects on a tumoral cell line.Purification was made by a two-step procedure: ion exchange (HiTrapSP XL-AKTAprime) and gel filtrationchromatography (Sephadex G-75). Enzyme non-cytotoxic concentrations on a murine tumoral epithelial cellline (LM3) were selected for adhesion and migration inhibition assays. In order to evaluate cell adhesion, LM3cells (3×10 4 /well) were preincubated for 30 min at 37°C with PLA 2 (1.25-10 μg/mL) or culture medium (DMEM5% FBS-100% adhesion) and then added to 96-well plates. After 1.5 h, adherent cells were fixed and stainedwith crystal violet and the percentage of cell adhesion was determined. Cell migration was measured bywound-healing assay. Cells were grown in a 6-well plate and a wound was created by a sterile pipette tip.Culture medium alone or PLA 2 (0.125-0.25 μg/mL) were added to the cells and incubated for 24 h. Woundwidths were measured and percentage of cell migration was calculated.Results indicate that the isolated enzyme PLA 2 induces a dose-dependent inhibition of cell adhesion andmigration. The lowest dose assayed (1.25 μg/mL) evidenced a 20% of adhesion inhibition effect and with 10μg/mL, 60% of cells didn´t adhere to the substrate. On the other hand, the wound scratch in control cells wascompletely closed after 24 h of incubation. However, treatment with 0.125 and 0.25 μg/mL of PLA 2 resulted inthe suppression of cell migration in a concentration-dependent manner, decreasing respect to controls by 8and 55% respectively.Although more studies are needed, these findings demonstrate the therapeutic potential of this basic PLA 2isolated from B. diporus venom.Fil: Sasovsky, Daniela Jaqueline. Consejo Nacional de Investigaciones Científicas y Técnicas. Centro Científico Tecnológico Conicet - Nordeste. Instituto de Química Básica y Aplicada del Nordeste Argentino. Universidad Nacional del Nordeste. Facultad de Ciencias Exactas Naturales y Agrimensura. Instituto de Química Básica y Aplicada del Nordeste Argentino; ArgentinaFil: Galarza, Laura. Consejo Nacional de Investigaciones Científicas y Técnicas. Centro Científico Tecnológico Conicet - Nordeste. Instituto de Química Básica y Aplicada del Nordeste Argentino. Universidad Nacional del Nordeste. Facultad de Ciencias Exactas Naturales y Agrimensura. Instituto de Química Básica y Aplicada del Nordeste Argentino; ArgentinaFil: Leiva, Laura Cristina Ana. Consejo Nacional de Investigaciones Científicas y Técnicas. Centro Científico Tecnológico Conicet - Nordeste. Instituto de Química Básica y Aplicada del Nordeste Argentino. Universidad Nacional del Nordeste. Facultad de Ciencias Exactas Naturales y Agrimensura. Instituto de Química Básica y Aplicada del Nordeste Argentino; ArgentinaFil: Bal de Kier Joffé, Elisa D.. Universidad de Buenos Aires. Facultad de Medicina. Instituto de Oncología "Ángel H. Roffo"; ArgentinaFil: Bustillo, Soledad. Consejo Nacional de Investigaciones Científicas y Técnicas. Centro Científico Tecnológico Conicet - Nordeste. Instituto de Química Básica y Aplicada del Nordeste Argentino. Universidad Nacional del Nordeste. Facultad de Ciencias Exactas Naturales y Agrimensura. Instituto de Química Básica y Aplicada del Nordeste Argentino; Argentina7th International Toxinology MeetingOxfordInglaterraUniversity of OxfordLibPubMedi

Expression pattern of glypican-3 -GPC3- during human embryonic and fetal development

Glypicans represent a family of cell surface proteoglycans. Loss-of-function mutations in the human glypican-3 (GPC3) gene results in the Simpson-Golabi- Behmel syndrome, characterized by severe malformations and pre- and postnatal overgrowth. Because the expression of GPC3 during human embryonic and fetal periods remains largely unknown, we investigated by immunohistochemistry its pattern of expression during four periods of human development covering the embryonic period (P1) from 5 to 8 weeks of development, and the fetal periods (P2, P3 and P4) from 9 to 28 weeks of development. Hepatocytes were homogeneously positive for GPC3 during the four periods while pancreatic acini and ducts showed a rather high staining only during P1. GPC3 was also detected in several kidney structures and in the genital system where the sex cords were weakly positive in P1 and P2. In later developmental stages the male’s genital system expressed GPC3 while the female’s did not. While the mesenchyme in the limbs showed positive staining in P1, GPC3 was not detected during the following stages. The mesenchymal tissue localized between the most caudal vertebrae was also positive in P1. A strong GPC3 signal was observed in neurons of the spinal cord and dorsal root ganglia in P2 and P3, while the brain was negative. In sum our studies revealed that GPC3 expression is highly tissue- and stage-specific during human development. The expression pattern of GPC3 is consistent with the abnormalities seen in the Simpson- Golabi-Behmel syndrome

Autophagy Protects from Trastuzumab-Induced Cytotoxicity in HER2 Overexpressing Breast Tumor Spheroids.

Multicellular tumor spheroids represent a 3D in vitro model that mimics solid tumor essential properties including assembly and development of extracellular matrix and nutrient, oxygen and proliferation gradients. In the present study, we analyze the impact of 3D spatial organization of HER2-overexpressing breast cancer cells on the response to Trastuzumab. We cultured human mammary adenocarcinoma cell lines as spheroids with the hanging drop method and we observed a gradient of proliferating, quiescent, hypoxic, apoptotic and autophagic cells towards the inner core. This 3D organization decreased Trastuzumab sensitivity of HER2 over-expressing cells compared to monolayer cell cultures. We did not observe apoptosis induced by Trastuzumab but found cell arrest in G0/G1 phase. Moreover, the treatment downregulated the basal apoptosis only found in tumor spheroids, by eliciting protective autophagy. We were able to increase sensitivity to Trastuzumab by autophagy inhibition, thus exposing the interaction between apoptosis and autophagy. We confirmed this result by developing a resistant cell line that was more sensitive to autophagy inhibition than the parental BT474 cells. In summary, the development of Trastuzumab resistance relies on the balance between death and survival mechanisms, characteristic of 3D cell organization. We propose the use of spheroids to further improve the understanding of Trastuzumab antitumor activity and overcome resistance

Autophagy in BT474 cells.

<p>(a) Western blot (WB) analysis of LC3 in 2D and 3D cultures after 24 h treatment with Tz. (b) Autophagic flux analyzed by WB in cell monolayers treated with Tz (1μg/ml) and 5 nM Bafilomycin A1 (BAF) (c) Cellular distribution of autophagosomes with LC3 stain. Arrows point to autophagosomes. Images show representative zones of BT474 cell monolayers (200x). (d) Immunofluorescence for LC3 (green) and Propidium iodide (PI, red) in spheroids treated for 15 days with Tz (50 μg/ml) or control IgG. Magnified images correspond to the zones limited by the white squares in the left photographs (600x).</p

Study of spheroid subpopulations.

<p>Spheroids were treated with 50 μg/ml Tz or control IgG for 15 days. (a) H&E (200x); immunofluorescence for pHER2 and HER2, HIF-1α (600x) and cleaved caspase 3 (400x). (b) Immunohistochemistry for Ki67 and quantification of Ki67+ cells (*p<0.05). (c) Confocal analysis of p27 (200x). Filled white arrows correspond to nuclear staining and black filled arrows correspond to cytoplasmatic staining.</p