Autophagy-related protein LC3 and Beclin-1 in the first trimester of pregnancy

Autophagy is a degradation process that acts in response to environmental stressors. Recently, autophagy has been detected in normal term, preeclamptic and intrauterine growth-restricted placentas. The object of this work was to investigate the presence of autophagy in first trimester voluntary interruption of pregnancy placental villi by the expression of autophagy-related proteins, light chain 3 (LC3), and Beclin-1. In first trimester placental villi laser scanning confocal microscopy (LSCM) analysis revealed LC3 and Beclin-1 immunoreactivity prevalently located in villous cytotrophoblasts. Using LSCM, LC3, and Beclin-1 were localized to the cytoplasm of the trophoblast layer in human full-term placentas. Beclin-1 expression and LC3 activation were confirmed by western blotting. These data emphasize that autophagy activation is different among cytotrophoblasts and syncytiotrophoblasts depending on the gestational age and thus we speculate that autophagy might play a prosurvival role throughout human pregnancy

FTS and 2-DG induce pancreatic cancer cell death and tumor shrinkage in mice

The Ras inhibitor S-trans-trans farnesylthiosalicylic acid (FTS) inhibits active Ras, which controls cell proliferation, differentiation, survival, and metabolism. FTS also inhibits HIF1α expression in cancer cells, leading to an energy crisis. The synthetic glucose analog 2-deoxy-D-glucose (2-DG), which inhibits glycolysis, is selectively directed to tumor cells that exhibit increased glucose consumption. The 2-DG enters tumor cells, where it competes with glucose for glycolytic enzymes. In cancer models, as well as in human phase 1 trials, 2-DG inhibits tumor growth without toxicity. We postulated that under normoxic conditions, tumor cells treated with FTS would be more sensitive than normal cells to 2-DG. We show here that combined treatment with FTS and 2-DG inhibited cancer cell proliferation additively, yet induced apoptotic cell death synergistically both in vitro and in vivo. The induced apoptosis was inferred from QVD-OPH inhibition, an increase in cleaved caspase 3, and loss of survivin. FTS and 2-DG when combined, but not separately, also induced an increase in fibrosis of the tumor tissue, chronic inflammation, and tumor shrinkage. Overall, these results suggest a possible new treatment of pancreatic tumors by the combined administration of FTS and 2-DG, which together induce pancreatic tumor cell death and tumor shrinkage under non-toxic conditions

Evidence of Distinct Tumour-Propagating Cell Populations with Different Properties in Primary Human Hepatocellular Carcinoma

Increasing evidence that a number of malignancies are characterised by tumour cell heterogeneity has recently been published, but there is still a lack of data concerning liver cancers. The aim of this study was to investigate and characterise tumour-propagating cell (TPC) compartments within human hepatocellular carcinoma (HCC).After long-term culture, we identified three morphologically different tumour cell populations in a single HCC specimen, and extensively characterised them by means of flow cytometry, fluorescence microscopy, karyotyping and microarray analyses, single cell cloning, and xenotransplantation in NOD/SCID/IL2Rγ/⁻ mice.The primary cell populations (hcc-1, -2 and -3) and two clones generated by means of limiting dilutions from hcc-1 (clone-1/7 and -1/8) differently expressed a number of tumour-associated stem cell markers, including EpCAM, CD49f, CD44, CD133, CD56, Thy-1, ALDH and CK19, and also showed different doubling times, drug resistance and tumorigenic potential. Moreover, we found that ALDH expression, in combination with CD44 or Thy-1 negativity or CD56 positivity identified subpopulations with a higher clonogenic potential within hcc-1, hcc-2 and hcc-3 primary cell populations, respectively. Karyotyping revealed the clonal evolution of the cell populations and clones within the primary tumour. Importantly, the primary tumour cell population with the greatest tumorigenic potential and drug resistance showed more chromosomal alterations than the others and contained clones with epithelial and mesenchymal features.Individual HCCs can harbor different self-renewing tumorigenic cell types expressing a variety of morphological and phenotypical markers, karyotypic evolution and different gene expression profiles. This suggests that the models of hepatic carcinogenesis should take into account TPC heterogeneity due to intratumour clonal evolution

Postnatal vasculogenesis: identification, growth, and function of endothelial progenitors cells from peripheral blood mononuclear cells

To identify best growth conditions for the in vitro differentiation of human peripheral blood mono-nuclear cells (PBMNCs) into endothelial progenitor cells (EPCs), PBMNCs of healthy volunteers were cultured on fibronectin as follows: M199 with VEGF, bFGF, and IGF-I; M199 with bovine retina-derived growth supplement (RDGS); human umbilical vein endothelial cell (HUVEC) conditioned medium; DiI-stained PBMNCs with HUVECs (1:4 ratio) in M199 with RDGS (cocultures system); PHA burst (10μL/106 cells) for 24 h and culture in M199 with RDGF. EPCs were identified by FACS using mAbs for endothelial markers. EPCs migration was determined using a modified Boyden chamber assay and VEGF as chemoattractant. Matrigel was used to assess in vitro angiogenesis capability. Spindle-shaped and attached cells sprouted with growth factors, differentiating in EPCs within 2 weeks and forming cobblestone-like monolayers within 3 weeks. With RDGS, numerous large cell clusters appeared within 1 week but the number of clusters decreased during culture. After stimulation with PHA, many cells clusters and some adherent EC-like cells were observed within 7 days; their number and size increased within 14 days. With HUVEC conditioned medium, spindle-shaped cells were observed after 10 days. FACS confirmed the endothelial phenotype. Cocultures induced the appearance of double-labeled EPCs expressing endothelial antigens starting from 7 days. EPCs were able to migrate in response to VEGF and to incorporate in capillary-like structures formed by HUVECs on Matrigel. PBMNCs are able to differentiate in EPCs when stimulated with appropriate culture conditions. The contact with mature endothelial cells (ECs) makes this process quicker