Alessandra Cucina

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.

Shear stress induces transforming growth factor–beta1 release by arterial endothelial cells ☆

BACKGROUND Myointimal hyperplasia is a common complication after vascular reconstruction. Increasing shear stress has been shown to reduce formation of myointimal hyperplasia. The aims of our study were (1) to analyze the correlation between shear stress and release of transforming growth factor (TGF)-beta 1 by endothelial cells and (2) to determine the effect of TGF-beta 1 on smooth muscle cell proliferation. METHODS Bovine arterial endothelial cells were subjected to increasing shear stress in an in vitro serum-free system. The release of TGF-beta 1 by endothelial cells was assessed by enzyme-linked immunosorbent assay and Western blot analysis. The effect of TGF-beta 1 on the proliferation of the subconfluent monolayer of bovine smooth muscle cells was determined by tritiated thymidine uptake. RESULTS Shear stress induced a significant increase of the release of TGF-beta 1 by endothelial cells (p < 0.001). This phenomenon was proportional to the level of shear stress. The amount of TGF-beta 1 released by endothelial cells subjected to shear stress had a significant inhibitory effect on growth rate and tritiated thymidine uptake of smooth muscle cells. CONCLUSIONS On the basis of the results of our study, we conclude that increasing shear stress induces release of TGF-beta 1 by arterial endothelial cells in a concentration that has a clear inhibitory effect on smooth muscle cell proliferation. This phenomenon could explain the inhibitory effect of increasing shear stress on the formation of myointimal hyperplasia.

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.

Melatonin and vitamin D3 synergistically down-regulate Akt and MDM2 leading to TGFβ-1-dependent growth inhibition of breast cancer cells.

Abstract:  Melatonin and vitamin D3 inhibit breast cancer cell growth and induce apoptosis, but they have never been combined as a breast cancer treatment. Therefore, we investigated whether their association could lead to an enhanced anticancer activity. In MCF‐7 breast cancer cells, melatonin together with vitamin D3, induced a synergistic proliferative inhibition, with an almost complete cell growth arrest at 144 hr. Cell growth blockade is associated to an activation of the TGFβ‐1 pathway, leading to increased TGFβ‐1, Smad4 and phosphorylated‐Smad3 levels. Concomitantly, melatonin and D3, alone or in combination, caused a significant reduction in Akt phosphorylation and MDM2 values, with a consequent increase of p53/MDM2 ratio. These effects were completely suppressed by adding a monoclonal anti‐TGFβ‐1 antibody to the culture medium. Taken together, these results indicate that cytostatic effects triggered by melatonin and D3 are likely related to a complex TGFβ‐1‐dependent mechanism, involving down‐regulation of both MDM2 and Akt‐phosphorylation.

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.

Shear stress induces changes in the morphology and cytoskeleton organisation of arterial endothelial cells

OBJECTIVES The aim of this study was to determine the changes in the morphology and cytoskeleton organisation of endothelial cells (EC) determined by exposure to a laminar flow. Cultured EC were exposed to a wall shear stress of 6 dyne/cm2 for 24 hours. CHIEF OUTCOME MEASURES The morphology of EC was analysed by light and scanning electron microscopy. The organisation of the cytoskeleton was determined by double fluorescence labeling with antibody anti-vimentin, anti-vinculin, anti-tubulin, and with rhodamine-labeled phalloidin. RESULTS EC exposed to laminar flow become round-shaped with decreased area of adhesion to the substrate. There was a clear reorganisation of the cytoskeleton after exposure to shear stress; the distribution of actin changed from a stress fibre pattern to a more diffuse membrane-associated distribution. These changes in shape and cytoskeleton organisation were reversible after a 48-hour resting period. CONCLUSIONS EC respond to laminar flow in a predictable manner and these findings may be correlated to the functional changes of EC observed after exposure to shear stress.

Modulation of arterial smooth muscle cell growth by haemodynamic forces

To define the correlation between flow dynamics and the proliferation of arterial smooth muscle cells (SMCs), bovine arterial SMC were subjected to increasing laminar flow shear stress in an in vitro system. Smooth muscle cells were seeded in a fibronectin-coated polystyrene cylinder at 5 x 10(5) cells/tube. The experimental groups were subjected to increasing shear stress (3, 6, 9 dyn cm-2) for a 24-h period. The control group was subjected to similar incubation conditions without flow. Shear stress reduced significantly (p less than 0.01) the 24-h incorporation of tritiated thymidine and cell proliferation. This effect was proportional to the level of shear stress and was still evident 24 h after flow cessation. Flow cytometry demonstrated a lower percentage of SMCs in S-phase with increasing shear stress. Extrapolation of these findings to the clinical setting might explain how unphysiological shear stress can predispose to the abnormal proliferation rate of SMCs and early plaque formation.

Shear Stress Influences the Release of Platelet Derived Growth Factor and Basic Fibroblast Growth Factor by Arterial Smooth Muscle Cells

OBJECTIVES To determine the correlation between haemodynamic forces and the release of two mitogens for smooth muscle cells (SMC): Platelet Derived Growth Factor (PDGF) and basic Fibroblast Growth Factor (bFGF). METHODOLOGY Bovine aortic smooth muscle cells were seeded on fibronectin coated polystyrene cylinders and allowed to reach confluence. The cells were subjected to a laminar flow of 50 cc/min (3 dyne/cm2), 100 cc/min (6 dyne/cm2) and 150 cc/min (9 dyne/cm2) in an in vitro system. Control cells were subjected to similar incubation conditions without flow. PRINCIPAL RESULTS Shear stress increased the release of mitogens by SMC. The release of mitogens was proportional to the level of shear stress and was still evident 24 hours after flow cessation. Conditioned serum-free medium from SMC subjected to shear stress increased tritiated thymidine uptake in Swiss 3T3 fibroblasts 13-fold as compared to conditioned serum-free medium from control SMC not subjected to shear stress (p < 0.01) and threefold as compared to standard control (p < 0.001). Addition of an excess of anti-PDGF antibody reduced the mitogenic activity of the conditioned medium by 30% (p < 0.01). Addition of an excess of anti-bFGF antibody reduced the mitogenic activity of the conditioned medium by 60% (p < 0.01). CONCLUSIONS Increasing shear stress promotes the release of both PDGF and bFGF from arterial SMC in culture and is a possible explanation for atherosclerosis formation.

Antiproliferative and apoptotic effects triggered by grape seed extract (GSE) versus epigallocatechin and procyanidins on colon cancer cell lines

Grape seed extract has been proven to exert anticancer effects on different tumors. These effects are mainly ascribed to catechin and procyanidin content. Analytical studies demonstrated that grape seed extract composition is complex and it is likely other components could exert biological activities. Using cell count and flow cytometry assays, we evaluated the cytostatic and apoptotic effects produced by three different grape seed extracts from Italia, Palieri and Red Globe cultivars, on Caco2 and HCT-8 colon cancer cells. These effects were compared to those induced by epigallocatechin and procyanidins, alone or in association, on the same cell lines. All the extracts induced growth inhibition and apoptosis in Caco2 and HCT-8 cells, along the intrinsic apoptotic pathway. On both cell lines, growth inhibition induced by Italia and Palieri grape seed extracts was significantly higher than that it has been recorded with epigallocatechin, procyanidins and their association. In Caco2 cells, the extract from Red Globe cultivar was less effective in inducing growth inhibition than procyanidins alone and in association with epigallocatechin, whereas, in HCT-8 cells, only the association of epigallocatechin and procyanidins triggers a significant proliferation decrease. On both cell lines, apoptosis induced by Italia, Palieri and Red Globe grape seed extracts was considerably higher than has been recorded with epigallocatechin, procyanidins and their association. These data support the hypothesis by which other compounds, present in the grape seed extracts, are likely to enhance the anticancer effects.

Molecular mechanisms of melatonin's inhibitory actions on breast cancers

Melatonin is involved in many physiological functions and it plays an important role in many pathological processes as well. Melatonin has been shown to reduce the incidence of experimentally induced cancers and can significantly inhibit the growth of some human tumors, namely hormone-dependent cancers. The anticancer effects of melatonin have been observed in breast cancer, both in in vivo with models of chemically induced rat mammary tumors, and in vitro studies on human breast cancer cell lines. Melatonin acts at different physiological levels and its antitumoral properties are supported by a set of complex, different mechanisms of action, involving apoptosis activation, inhibition of proliferation, and cell differentiation.