Pierpaolo Coluccia

B-vitamin deprivation induces hyperhomocysteinemia and brain S-adenosylhomocysteine, depletes brain S-adenosylmethionine, and enhances PS1 and BACE expression and amyloid-β deposition in mice

Etiological and molecular studies on the sporadic form of Alzheimers disease have yet to determine the underlying mechanisms of neurodegeneration. Hyperhomocysteinemia is associated with Alzheimers disease, and has been hypothesized to promote neurodegeneration, by inhibiting brain methylation activity. The aim of this work was to determine whether a combined folate, B12 and B6 dietary deficiency, would induce amyloid-beta overproduction, and to study the mechanisms linking vitamin deficiency, hyperhomocysteinemia and amyloidogenesis in TgCRND8 and 129Sv mice. We confirmed that B-vitamin deprivation induces hyperhomocysteinemia and imbalance of S-adenosylmethionine and S-adenosylhomocysteine. This effect was associated with PS1 and BACE up-regulation and amyloid-beta deposition. Finally, we detected intraneuronal amyloid-beta and a slight cognitive impairment in a water maze task at a pre-plaque age, supporting the hypothesis of early pathological function of intracellular amyloid. Collectively, these findings are consistent with the hypothesis that abnormal methylation in association with hyperhomocysteinemia may contribute to Alzheimers disease.

Evidence for a biphasic apoptotic pathway induced by melatonin in MCF-7 breast cancer cells.

Abstract:  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 (TGFβ1)‐dependent and it might be reversed by adding an anti‐TGFβ1 antibody. Melatonin induces a significant rise in apoptotic rate, at both 24 and 96 hr. The anti‐TGFβ1 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, TGFβ1 and caspase‐independent response, and a late apoptotic TGFβ1‐dependent process in which activated‐caspase‐7 is likely to be the terminal effector.

Vascular endothelial growth factor increases the migration and proliferation of smooth muscle cells through the mediation of growth factors released by endothelial cells

BACKGROUND Vascular endothelial growth factor (VEGF), a highly specific chemotactic and mitogenic factor for vascular endothelial cells (EC), appears to be involved in the development of atherosclerosis. The purpose of our study was to assess if VEGF might indirectly stimulate SMC migration and proliferation in a EC-SMC coculture system, through the mediation of growth factors released by EC. METHODS Bovine aortic SMC were cocultured with bovine aortic EC treated with hrVEGF, to assess SMC proliferation and migration. The release and mRNA expression of basic fibroblast growth factor (bFGF) and transforming growth factor beta(1) (TGFbeta(1)) were assessed by ELISA and PCR analysis. RESULTS hrVEGF (10 ng/ml), added to EC cocultured with SMC, induced a significant increase in tritiated thymidine uptake by SMC as compared to controls (P < 0.01) and a significant increase in SMC migration in respect to control (27%; P < 0.01). EC stimulated with hrVEGF increased the release and the expression of bFGF and decreased the release and the expression of TGFbeta(1) with a statistically significant difference in respect to controls (P < 0.001). CONCLUSIONS VEGF indirectly stimulates SMC proliferation and migration through the modulation of bFGF and TGFbeta(1) released by EC.

Nicotine stimulates proliferation and inhibits apoptosis in colon cancer cell lines through activation of survival pathways

BACKGROUND Colorectal cancer is one of the leading causes of cancer-related death throughout the world, and the risk to develop this malignant disease seems to be associated with long-term cigarette smoking. Nicotine, one of the major components of cigarette smoking, can stimulate cell proliferation and suppress apoptosis both in normal cells and in several human cancer cell lines derived from various organs. However, although nicotine appears to have a role in stimulating cell proliferation of colon cancer cells, there is no information on its role in inhibiting apoptosis in these cells. MATERIALS AND METHODS Human colorectal cancer cell lines Caco-2 and HCT-8 were treated with 1 μM nicotine alone or in combination with 1 μM α-BTX in complete or in serum free medium. Cell proliferation and apoptosis were determined by cell count performed with a cell counter and by cytofluorimetric assay respectively. PI3K/Akt and PKC/ERK1/2 pathways, survivin, and P-Bcl2 (Ser70) were investigated by Western blot analysis. RESULTS Nicotine induced an increase in cell proliferation and a decrease of apoptosis in Caco-2 and HCT-8 cells. Both cell growth and apoptosis appear to be mediated by α7-nicotinic acetylcholine receptors, since treatment with α-Bungarotoxin inhibited these processes. Nicotine induced a statistically significant increase in the expression of PI3K and in P-Akt/Akt ratio as well as in the expression of PKC, ERK1/2, survivin, and P-Bcl2 (Ser70) in both cell lines. CONCLUSIONS Nicotine, contained in cigarette smoking, could participate in colon cancer development and progression by stimulating cell proliferation and suppressing physiological apoptosis.

Stearoyl-CoA desaturase-1 is a key factor for lung cancer-initiating cells

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.

Zebrafish embryo proteins induce apoptosis in human colon cancer cells (Caco2)

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 G2/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.

Apoptosis-inducing factor and caspase-dependent apoptotic pathways triggered by different grape seed extracts on human colon cancer cell line Caco-2

Consumption of grape seed extract (GSE) is widely marketed as a dietary supplement and is considered safe for human health. Nevertheless, the analytical composition of GSE from different grape cultivars, growing in special agronomic constraints, differs greatly in flavan-3-ols content. The major concern with GSE studies is a lack of availability of uniformly standardised preparations, which raises an important question whether different GSE samples have comparable activity and trigger the same mechanisms of action on a given biological system. Therefore, it is tempting to speculate that GSE, obtained from different cultivars, could exert differentiated anticancer effects. The focus of the present study is to determine the selective biological efficacy of GSE obtained from three different sources on the human colon cancer cell line Caco-2. Irrespective of its source, high doses of GSE induced a significant inhibition on Caco-2 cell growth. Moreover, apoptosis was enhanced through both caspase-dependent and caspase-independent mechanisms, leading to an early apoptosis-inducing factor release and, further, to a dramatic increase in caspase 7 and 3 activity. However, a significant difference in apoptotic rates induced by the three grape sources clearly emerged when treating cancer cells with low and intermediate GSE concentrations (25 and 50 microg/ml).

Nicotine Inhibits Apoptosis and Stimulates Proliferation in Aortic Smooth Muscle Cells Through a Functional Nicotinic Acetylcholine Receptor

Atherosclerosis and neointimal hyperplasia formation are induced by alterations in the homeostatic balance between cell growth and cell death. Apoptosis is a physiological cell death process that, when deregulated, may be involved in many pathological conditions. Cigarette smoking is a primary risk factor for vascular disease and nicotine seems to exert its atherogenic effects in part through the increase of smooth muscle cell (SMC) proliferation. The aim of this study was to investigate the effect of nicotine on SMC apoptosis. Nicotine added for 24 and 72 h to serum deprived cell cultures resulted in a decrease of apoptotic SMCs. The inhibition was direct and not mediated by platelet-derived growth factor, basic fibroblast growth factor, and transforming growth factor beta(1), autocrinally released by nicotine-treated SMCs, because it was not influenced by addition of specific neutralizing antibodies. Apoptosis inhibition as well as the proliferation increase, and basic fibroblast growth factor expression on nicotine-treated SMCs were blocked by nicotinic acetylcholine receptor antagonists, including alpha-bungarotoxin, a competitive antagonist of alpha subunits of nicotinic receptor. In conclusion, we propose that nicotine could lead to the increase of neointimal SMCs in vascular lesions by inducing the inhibition of physiological SMC apoptosis and the increase of SMC proliferation. We also showed that nicotine signaling occurs as a result of activation of the classical nicotine receptor pathways.

FLIP is expressed in mouse testis and protects germ cells from apoptosis

AbstractApoptosis control in adult testis is crucial to achieve normal spermatogenesis. In this study c-FLIP, an apoptosis-modulating protein, was investigated. In Western blot and immunohistochemical analyses, the 55 KDa c-FLIP long isoform (c-FLIPL) was found to be expressed strongly in spermatocytes and spermatids, at low levels in spermatogonia and at almost undetectable levels in Sertoli cells. This expression pattern was confirmed by Northern blot analyses. Further experiments carried out on GC-1spg germ cell line revealed that reducing c-FLIPL expression increases Fas-dependent apoptosis. Conversely, restoring c-FLIPL expression reduces this response to control levels. Caspase-10 expression was found to match c-FLIPL expression pattern; further, caspase-10 activation upon anti-Fas treatment inversely correlated with c-FLIPL expression. Finally, TUNEL staining of seminiferous tubules incubated with anti-Fas antibody showed that apoptosis occurs mostly in basally located germ cells, indicating that such cells, expressing low levels of c-FLIPL, are sensitive to Fas-mediated apoptosis.These data indicate for the first time that c-FLIPL might control germ cell apoptosis and caspase activity in the adult testis.

Phenotypic Switch Induced by Simulated Microgravity on MDA-MB-231 Breast Cancer 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.