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In recent years, studies of cancer development and recurrence have been influenced by the cancer stem cells (CSCs)/cancer-initiating cells (CICs) hypothesis. According to this, cancer is sustained by highly positioned, chemoresistant cells with extensive capacity of self renewal, which are responsible for disease relapse after chemotherapy. Growth of cancer cells as three-dimensional non-adherent spheroids is regarded as a useful methodology to enrich for cells endowed with CSC-like features. We have recently reported that cell cultures derived from malignant pleural effusions (MPEs) of patients affected by adenocarcinoma of the lung are able to efficiently form spheroids in non-adherent conditions supplemented with growth factors. By expression profiling, we were able to identify a set of genes whose expression is significantly upregulated in lung tumor spheroids versus adherent cultures. One of the most strongly upregulated gene was stearoyl-CoA desaturase (SCD1), the main enzyme responsible for the conversion of saturated into monounsaturated fatty acids. In the present study, we show both by RNA interference and through the use of a small molecule inhibitor that SCD1 is required for lung cancer spheroids propagation both in stable cell lines and in MPE-derived primary tumor cultures. Morphological examination and image analysis of the tumor spheroids formed in the presence of SCD1 inhibitors showed a different pattern of growth characterized by irregular cell aggregates. Electron microscopy revealed that the treated spheroids displayed several features of cellular damage and immunofluorescence analysis on optical serial sections showed apoptotic cells positive for the M30 marker, most of them positive also for the stemness marker ALDH1A1, thus suggesting that the SCD1 inhibitor is selectively killing cells with stem-like properties. Furthermore, SCD1-inhibited lung cancer cells were strongly impaired in their in vivo tumorigenicity and ALDH1A1 expression. These results suggest that SCD1 is a critical target in lung cancer tumor-initiating cells.

Microgravity exerts dramatic effects on cell morphology and functions, by disrupting cytoskeleton and adhesion structures, as well as by interfering with biochemical pathways and gene expression. Impairment of cells behavior has both practical and theoretical significance, given that investigations of mechanisms involved in microgravity-mediated effects may shed light on how biophysical constraints cooperate in shaping complex living systems. By exposing breast cancer MDA-MB-231 cells to simulated microgravity (~0.001 g), we observed the emergence of two morphological phenotypes, characterized by distinct membrane fractal values, surface area, and roundness. Moreover, the two phenotypes display different aggregation profiles and adherent behavior on the substrate. These morphological differences are mirrored by the concomitant dramatic functional changes in cell processes (proliferation and apoptosis) and signaling pathways (ERK, AKT, and Survivin). Furthermore, cytoskeleton undergoes a dramatic reorganization, eventually leading to a very different configuration between the two populations. These findings could be considered adaptive and reversible features, given that, by culturing microgravity-exposed cells into a normal gravity field, cells are enabled to recover their original phenotype. Overall these data outline the fundamental role gravity plays in shaping form and function in living systems.

Previous investigations demonstrated that melatonin exerts an oncostatic action on estrogen-responsive breast cancer, both in vitro and in vivo. Nevertheless, the pro-apoptotic effect of melatonin is still a matter of debate. An experimental study was undertaken to focus on melatonin-related apoptosis and to identify the apoptotic pathways involved. Whole cell-count, flow-cytometry analysis and proteins involved in apoptotic pathways [p53, p73, murine double minute 2 (MDM2), caspases-9,-7,-6, cleaved-poly ADP ribose polymerase (PARP), Bcl-2, Bax and apoptotic inducing factor (AIF)] were investigated in human MCF-7 breast cancer cells treated with physiological (1 nM) concentration of melatonin. Melatonin exerts a significant growth-inhibitory effect on MCF-7 cells, becoming evident after 72 hr and thereafter increasing linearly up to 144 hr. In this model, the growth-inhibition is transforming growth factor beta 1 (TGFbeta1)-dependent and it might be reversed by adding an anti-TGFbeta1 antibody. Melatonin induces a significant rise in apoptotic rate, at both 24 and 96 hr. The anti-TGFbeta1 antibody almost completely suppresses melatonin-related late apoptosis; however, early apoptosis is unaffected. Early programmed cell death is associated with a significant increase in the p53/MDM2 ratio and in AIF release, without modifications in caspase activity or cleaved-PARP levels. Activated caspases-9 and -7 and cleaved-PARP increased significantly at 96 hr, concomitantly with a down-regulation of the Bcl-2/Bax ratio. These data suggest that two distinct apoptotic processes are triggered by melatonin in MCF-7 cells: an early, TGFbeta1 and caspase-independent response, and a late apoptotic TGFbeta1-dependent process in which activated-caspase-7 is likely to be the terminal effector.

Previous studies have shown that proteins extracted from Zebrafish embryo share some cytostatic characteristics in cancer cells. Our study was conducted to ascertain the biological properties of this protein network. Cancer cell growth and apoptosis were studied in Caco2 cells treated with embryonic extracts. Cell proliferation was significantly inhibited in a dose-dependent manner. Cell-cycle analysis in treated cells revealed a marked accumulation in the G(2)/M phase preceding induction of apoptosis. Embryo proteins induced a significant reduction in FLIP levels, and increased caspase-3 and caspase-8 activity as well as the apoptotic rate. Increased phosphorylated pRb values were obtained in treated Caco2 cells: the modified balance in pRb phosphorylation was associated with an increase in E2F1 values and c-Myc over-expression. Our data support previous reports of an apoptotic enhancing effect displayed by embryo extracts, mainly through the pRb/E2F1 apoptotic pathway, which thus suggests that Zebrafish embryo proteins have complex anti-cancer properties.

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