Fabrizio D’Anselmi

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.

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.

Beyond the Oncogene Paradigm: Understanding Complexity in Cancerogenesis

In the past decades, an enormous amount of precious information has been collected about molecular and genetic characteristics of cancer. This knowledge is mainly based on a reductionistic approach, meanwhile cancer is widely recognized to be a ‘system biology disease’. The behavior of complex physiological processes cannot be understood simply by knowing how the parts work in isolation. There is not solely a matter how to integrate all available knowledge in such a way that we can still deal with complexity, but we must be aware that a deeply transformation of the currently accepted oncologic paradigm is urgently needed. We have to think in terms of biological networks: understanding of complex functions may in fact be impossible without taking into consideration influences (rules and constraints) outside of the genome. Systems Biology involves connecting experimental unsupervised multivariate data to mathematical and computational approach than can simulate biologic systems for hypothesis testing or that can account for what it is not known from high-throughput data sets. Metabolomics could establish the requested link between genotype and phenotype, providing informations that ensure an integrated understanding of pathogenic mechanisms and metabolic phenotypes and provide a screening tool for new targeted drug.

Nicotine increases survival in human colon cancer cells treated with chemotherapeutic drugs.

Cigarette smoking is implicated in the development of colon cancer. Furthermore, nicotine increases cell proliferation and inhibits apoptosis through α7-nicotinic acetylcholine receptor (α7-nAChR) activation in human colon carcinoma cells. An open issue is whether nicotine interfere with colorectal cancer pharmacological treatment, by inhibiting drug-mediated apoptosis. To assess this hypothesis, we evaluated nicotine effect on Caco-2 and HCT-8 colon cancer cells, treated with 5-Fluorouracil (5-FU) and Camptothecin (CPT), chemotherapeutics commonly utilized as adjuvant treatment of colon cancer. Nicotine decreased anti-proliferative and pro-apoptotic effects exerted by chemotherapeutics on both cell lines. These effects partially reverted by exposure to α-bungarotoxin (α-BTX), an inhibitor of α7-nAChR. Nicotine addition to Caco-2 and HCT-8, treated with 5-FU or CPT, decreased the cleavage of substrate of caspase 3 and 7, poly-ADP-ribose polymerase (PARP). Moreover, P-ERK/ERK ratio was modified by nicotine addition to 5-FU and CPT treated cells in an opposite manner. However, when co-administrating PD98059, an ERK phosphorylation inhibitor, an increased apoptosis was observed. In Caco-2 and HCT-8 nicotine reverted 5-FU and CPT apoptotic effects through AKT phosphorylation, as demonstrated by apoptotic increase in presence of LY294002, an AKT phosphorylation inhibitor. Nicotine interfered with colorectal cancer pharmacological treatment in vitro by inhibiting apoptosis induced by chemotherapeutic drugs. Nicotine anti-apoptotic effects were exerted through ERK and AKT pathway activation.

Grape seed extract suppresses MDA-MB231 breast cancer cell migration and invasion.

Background and aimBreast cancer remains a leading cause of mortality among women. In metastasis, cascade migration of cancer cells and invasion of extracellular matrix (ECM) represent critical steps. Urokinase-type plasminogen activator (uPA), as well as metalloproteinases MMP-2 and MMP-9, strongly contribute to ECM remodelling, thus becoming associated with tumour migration and invasion. In addition, the high expression of cytoskeletal (CSK) proteins, as fascin, has been correlated with clinically aggressive metastatic tumours, and CSK proteins are thought to affect the migration of cancer cells. Consumption of fruits and vegetables, characterized by high procyanidin content, has been associated to a reduced mortality for breast cancer. Therefore, we investigated the biological effect of grape seed extract (GSE) on the highly metastatic MDA-MB231 breast cancer cell line, focusing on studying GSE ability in inhibiting two main metastatic processes, i.e., cell migration and invasion.MethodsAfter MDA-MB231 breast cancer cells stimulated with GSE migration and invasion were evaluated by means of trans-well assays and uPA as well as MMPs activity was detected by gelatin zymography. Fascin, β-catenin and nuclear factor-κB (NF-κB) expression were determined using western blot technique. β-Catenin localization was observed by confocal microscopy.ResultsWe observed that high concentrations of GSE inhibited cell proliferation and apoptosis. Conversely, low GSE concentration decreased cell migration and invasion, likely by hampering β-catenin expression and localization, fascin and NF-κB expression, as well as by decreasing the activity of uPA, MMP-2 and MMP-9.ConclusionsThese results make GSE a powerful candidate for developing preventive agents against cancer metastasis.

Microenvironment Promotes Tumor Cell Reprogramming in Human Breast Cancer Cell Lines

The microenvironment drives mammary gland development and function, and may influence significantly both malignant behavior and cell growth of mammary cancer cells. By restoring context, and forcing cells to properly interpret native signals from the microenvironment, the cancer cell aberrant behavior can be quelled, and organization re-established. In order to restore functional and morphological differentiation, human mammary MCF-7 and MDA-MB-231 cancer cells were allowed to grow in a culture medium filled with a 10% of the albumen (EW, Egg White) from unfertilized chicken egg. That unique microenvironment behaves akin a 3D culture and induces MCF-7 cells to produce acini and branching duct-like structures, distinctive of mammary gland differentiation. EW-treated MDA-MB-231 cells developed buds of acini and duct-like structures. Both MCF-7 and MDA-MB-231 cells produced β-casein, a key milk component. Furthermore, E-cadherin expression was reactivated in MDA-MB-231 cells, as a consequence of the increased cdh1 expression; meanwhile β-catenin – a key cytoskeleton component – was displaced behind the inner cell membrane. Such modification hinders the epithelial-mesenchymal transition in MDA-MB-231 cells. This differentiating pathway is supported by the contemporary down-regulation of canonical pluripotency markers (Klf4, Nanog). Given that egg-conditioned medium behaves as a 3D-medium, it is likely that cancer phenotype reversion could be ascribed to the changed interactions between cells and their microenvironment.

P1-385: Methylation regulates amyloid β production: Relevance of S-adenosylmethionine/S-adenosylhomocysteine ratio in mice fed with B vitamin deprived diet

Cl, I, ClO4 , NO3 , PO4 , SO4 2and heparin, PO4 , SO4 2and heparin showed greater effect during the nucleation phase and early elongation phase compared to the others, and the greatest effect was obtained by SO4 . However, TTR aggregated similarly in the presence of different cations. For L55P TTR, distinct structural features could be observed at different stages of aging, following the order of TTR tetramer, irregular aggregates, protofibrils and mature fibers along the aging process. On the other hand, we found TTR induced increase in intracellular Ca within one minute of application (Ca max 40 6% and pD2 5.3 0.1 M). In addition, studying the intracellular Ca increase by L55P at different stages of ageing indicated that intracellular Ca increase was significantly enhanced by TTR aged for 7 d compared to TTR aged for 0 and 3 d (P 0.01), while intracellular Ca increase was significantly diminished by TTR aged for 14 d (P 0.05). Conclusions: TTR aggregation is mainly mediated by hydrophobic interactions. Anionic interactions are involved in TTR aggregation and proteoglycans might be an important factor in TTR aggregation and extracellular deposition. Disturbance of intracellular Ca is an initial event in TTR-induced cytotoxicity. Protofibrils might be the most cytotoxic species followed by irregular aggregates and TTR tetramers, with mature fibers being the least cytotoxic.

P4-054: Effects of folate, B12 and B6 deprivation on methylation metabolism and amyloid processing in human neuroblastoma and glioblastoma cells

Background: The accumulation of Amyloid beta peptide (A ) in senile plaques is widely believed to play a central role in Alzheimer’s disease (AD). A peptides ( 1-40 and 1-42), derive from the proteolytic processing of the Amyloid Precursor Protein (APP). The exact mechanism by which these peptides trigger neuronal death is not well defined. However, the disturbance of the calcium homeostasis and the activation of caspases and c-Jun N-terminal kinase (JNK) are factors involved in the death process induced by A . It has recently been demonstrated that the JNK scaffold protein, JIP-1, interacts with the cytoplasmic domain of APP, suggesting that JIP-1 and JNK may play important roles in the metabolism of APP. It has also been established that APP is phosphorylated at Thr 668 by JNK. This phosphorylation in the cytoplasmic domain of APP may result in a regulation of the APP processing. Objective(s): We examined here the role of JNK in AD pathogenesis using a specific cell-permeable JNK inhibitor peptide, D-JNKI-1, and two different in vitro models: primary cortical neurons and H4-15x cells, stably transfected with human APP carrying Swedish mutation. Methods: Cortical neurons and H4 cells were treated with increasing concentrations of D-JNKI-1 for 24h and western blot analysis of secreted APP (APPs) was conducted on both culture media and cell lysates. Results: We observed a dose-response effect of D-JNKI-1, with a powerful reduction in APPs production. In fact, in cortical neurons, D-JNKI-1 (6 M) reduced by about 80% the level of APPs in both lysates and media, and this also correlated with a decrease in the media of A fragments (40%). In the H4-15x using 80 M D-JNKI-1 we obtained 70% reduction in APPs production and 45% decrease of A fragments. Conclusions: In both cellular models D-JNKI-1 prevented phosphorylation of APP at Thr 668 in a dose-dependent way. These data indicate an important role of JNK signalling pathway in the regulation of APP metabolism: in our models the proteolytic production of APPs and 1-40/1-42 was strongly reduced by the application of a specific JNK inhibitor that prevented the APP phosphorylation.

P4-055: Evidence for a methylation-based molecular mechanism of hyperhomocysteinemia in AD onset leading to intracellular amyloid deposition in mice

Background: The accumulation of Amyloid beta peptide (A ) in senile plaques is widely believed to play a central role in Alzheimer’s disease (AD). A peptides ( 1-40 and 1-42), derive from the proteolytic processing of the Amyloid Precursor Protein (APP). The exact mechanism by which these peptides trigger neuronal death is not well defined. However, the disturbance of the calcium homeostasis and the activation of caspases and c-Jun N-terminal kinase (JNK) are factors involved in the death process induced by A . It has recently been demonstrated that the JNK scaffold protein, JIP-1, interacts with the cytoplasmic domain of APP, suggesting that JIP-1 and JNK may play important roles in the metabolism of APP. It has also been established that APP is phosphorylated at Thr 668 by JNK. This phosphorylation in the cytoplasmic domain of APP may result in a regulation of the APP processing. Objective(s): We examined here the role of JNK in AD pathogenesis using a specific cell-permeable JNK inhibitor peptide, D-JNKI-1, and two different in vitro models: primary cortical neurons and H4-15x cells, stably transfected with human APP carrying Swedish mutation. Methods: Cortical neurons and H4 cells were treated with increasing concentrations of D-JNKI-1 for 24h and western blot analysis of secreted APP (APPs) was conducted on both culture media and cell lysates. Results: We observed a dose-response effect of D-JNKI-1, with a powerful reduction in APPs production. In fact, in cortical neurons, D-JNKI-1 (6 M) reduced by about 80% the level of APPs in both lysates and media, and this also correlated with a decrease in the media of A fragments (40%). In the H4-15x using 80 M D-JNKI-1 we obtained 70% reduction in APPs production and 45% decrease of A fragments. Conclusions: In both cellular models D-JNKI-1 prevented phosphorylation of APP at Thr 668 in a dose-dependent way. These data indicate an important role of JNK signalling pathway in the regulation of APP metabolism: in our models the proteolytic production of APPs and 1-40/1-42 was strongly reduced by the application of a specific JNK inhibitor that prevented the APP phosphorylation.