Microcalcification morphological descriptors and parenchyma fractal dimension hierarchically interact in breast cancer: A diagnostic perspective.

INTRODUCTION: Herein, we propose a Systems Biology approach aimed at identifying quantitative morphological parameters useful in discriminating benign from malignant breast microcalcifications at digital mammography. MATERIALS AND METHODS: The study includes 31 patients in which microcalcifications had been detected during XR mammography and were further confirmed by stereotactic (XR-guided) biopsies. Patients were classified according to the BIRADS (Breast Imaging-Reporting and Data System), along with their parenchyma fractal dimension and biopsy size. A geometrical-topological characterization of microcalcifications was obtained as well. RESULTS: The 'size of biopsy' was the parameter endowed with the highest discriminant power between malignant and benign lesions thus confirming the reliability of surgeon judgment. The quantitative shape evaluation of both lesions and parenchyma allowed for a promising prediction of the BIRADS score. The area of lesions and parenchyma fractal dimension show a complex distribution for malignant breast calcifications that are consistent with their qualitative morphological pattern. Fractal dimension analysis enables the user to obtain reliable results as proved by its efficiency in the prediction of the morphology of breast cancer. CONCLUSION: By reconstructing a phase-space distribution of biophysical parameters, different patterns of aggregation are recognized corresponding to different calcium deposition patterns, while the combination of tissue and microcalcification morphological descriptors provide a statistically significant prediction of tumour grade. CLINICAL RELEVANCE: The development of an automated morphology evaluation system can help during clinical evaluation while also sketching mechanistic hypotheses of microcalcification generation.

Systems Biology-Driven Hypotheses Tested In Vivo: The Need to Advancing Molecular Imaging Tools.

Processing and interpretation of biological images may provide invaluable insights on complex, living systems because images capture the overall dynamics as a "whole." Therefore, "extraction" of key, quantitative morphological parameters could be, at least in principle, helpful in building a reliable systems biology approach in understanding living objects. Molecular imaging tools for system biology models have attained widespread usage in modern experimental laboratories. Here, we provide an overview on advances in the computational technology and different instrumentations focused on molecular image processing and analysis. Quantitative data analysis through various open source software and algorithmic protocols will provide a novel approach for modeling the experimental research program. Besides this, we also highlight the predictable future trends regarding methods for automatically analyzing biological data. Such tools will be very useful to understand the detailed biological and mathematical expressions under in-silico system biology processes with modeling properties.

Inositol induces mesenchymal-epithelial reversion in breast cancer cells through cytoskeleton rearrangement.

Inositol displays multi-targeted effects on many biochemical pathways involved in epithelial-mesenchymal transition (EMT). As Akt activation is inhibited by inositol, we investigated if such effect could hamper EMT in MDA-MB-231 breast cancer cells. In cancer cells treated with pharmacological doses of inositol E-cadherin was increased, ß-catenin was redistributed behind cell membrane, and metalloproteinase-9 was significantly reduced, while motility and invading capacity were severely inhibited. Those changes were associated with a significant down-regulation of PI3K/Akt activity, leading to a decrease in downstream signaling effectors: NF-kB, COX-2, and SNAI1. Inositol-mediated inhibition of PS1 leads to lowered Notch 1 release, thus contributing in decreasing SNAI1 levels. Overall, these data indicated that inositol inhibits the principal molecular pathway supporting EMT. Similar results were obtained in ZR-75, a highly metastatic breast cancer line. These findings are coupled with significant changes on cytoskeleton. Inositol slowed-down vimentin expression in cells placed behind the wound-healing edge and stabilized cortical F-actin. Moreover, lamellipodia and filopodia, two specific membrane extensions enabling cell migration and invasiveness, were no longer detectable after inositol addiction. Additionally, fascin and cofilin, two mandatory required components for F-actin assembling within cell protrusions, were highly reduced. These data suggest that inositol may induce an EMT reversion in breast cancer cells, suppressing motility and invasiveness through cytoskeleton modifications.

Paradoxical E-cadherin increase in 5FU-resistant colon cancer is unaffected during mesenchymal-epithelial reversion induced by γ-secretase inhibition.

AIM: Presenilin-1 (PS1), the main component of γ-secretase activity support a key role during Epithelial-Mesenchymal Transition (EMT) and chemoresistance acquisition by triggering a complex sequence of molecular events, including E-cadherin down-regulation. However, we hypothesize that EMT and chemoresistance should be deemed separate processes in HCT-8 colon cancer cells. MAIN METHODS: HCT-8 and HCT-8FUres invasion was evaluated by trans-well assay. uPA activity was detected by zymography. Prostaglandin E2 levels were quantified using an ELISA kit. E-cadherin FL and CTF2, PS1, Notch1, Cyclin D1, COX2, SNAI1 and α-SMA expression were determined using Western blot technique. ß-Catenin localization was observed by confocal microscopy. Cell apoptosis was evaluated by cytofluorimetric assay, and measurement of caspase-3 and cl-PARP. γ-Secretase activity was inhibited by DAPT, a γ-secretase inhibitor. KEY FINDINGS: Chemoresistant HCT-8 underwent EMT that can be efficiently reversed by inhibiting PS1 activity, leading thus to a normalization of mostly of the pivotal features showed by the invasive cancer phenotype. Indeed, we observed decreased SNAI1 and Notch 1 activation, altogether with reduced E-cadherin cleavage. Concomitantly, resistant HCT-8 invasiveness was almost completely abolished. However, such reversion was not followed by any increase in apoptotic rate, not by changes in E-cadherin levels. Indeed, despite HCT-8FUres underwent an undeniable EMT, full-length E-cadherin levels were found remarkably higher than those observed in wild HCT-8. SIGNIFICANCE: High E-cadherin concentration in presence of enhanced γ-secretase activity is incontestably a paradoxically result, highlighting that E-cadherin loss is not a pre-requisite for EMT. Additionally, EMT and chemoresistance acquisition in HCT-8 should be considered as distinct processes.

Multiwalled carbon nanotube buckypaper induces cell cycle arrest and apoptosis in human leukemia cell lines through modulation of AKT and MAPK signaling pathways.

MWCNT buckypaper (BP) shows physico-chemical and mechanical properties that make it potentially useful as a substrate in nano-bio interface research including in tissue engineering. When used as a scaffold material, BP comes into contact with host cells and surrounding tissues; therefore it is critical to determine its biocompatibility and interaction with living systems. The aim of this study was to investigate BP effects on cell growth, apoptosis and reactive oxygen species (ROS) production in three human leukemia cell lines HL-60, U-937 and K-562. BP was able to induce both the reduction of cell proliferation, associated with an arrest in G0/G1 phase of cell cycle and the increase of apoptosis in leukemic cell lines, thus exerting both cytostatic and cytotoxic effects. The growth inhibitory effect was likely mediated by the decrease of cyclins D, E, A, B1 levels and CDK4 expression; meanwhile, the apoptotic effect, not mediated by ROS production, was presumably due to the combined action of the survival and pro-apoptotic AKT and MAPK signal transduction pathways. These results raised the issue of biocompatibility of MWCNT BP for the creation of carbon nanotubes based scaffolds to utilize as prostheses in tissue engineering.

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.

Melatonin down-regulates MDM2 gene expression and enhances p53 acetylation in MCF-7 cells.

Compelling evidence demonstrated that melatonin increases p53 activity in cancer cells. p53 undergoes acetylation to be stabilized and activated for driving cells destined for apoptosis/growth inhibition. Over-expression of p300 induces p53 acetylation, leading to cell growth arrest by increasing p21 expression. In turn, p53 activation is mainly regulated in the nucleus by MDM2. MDM2 also acts as E3 ubiquitin ligase, promoting the proteasome-dependent p53 degradation. MDM2 entry into the nucleus is finely tuned by two different modulations: the ribosomal protein L11, acts by sequestering MDM2 in the cytosol, whereas the PI3K-AkT-dependent MDM2 phosphorylation is mandatory for MDM2 translocation across the nuclear membrane. In addition, MDM2-dependent targeting of p53 is regulated in a nonlinear fashion by MDM2/MDMX interplay. Melatonin induces both cell growth inhibition and apoptosis in MCF7 breast cancer cells. We previously reported that this effect is associated with reduced MDM2 levels and increased p53 activity. Herein, we demonstrated that melatonin drastically down-regulates MDM2 gene expression and inhibits MDM2 shuttling into the nucleus, given that melatonin increases L11 and inhibits Akt-PI3K-dependent MDM2 phosphorylation. Melatonin induces a 3-fold increase in both MDMX and p300 levels, decreasing simultaneously Sirt1, a specific inhibitor of p300 activity. Consequently, melatonin-treated cells display significantly higher values of both p53 and acetylated p53. Thus, a 15-fold increase in p21 levels was observed in melatonin-treated cancer cells. Our results provide evidence that melatonin enhances p53 acetylation by modulating the MDM2/MDMX/p300 pathway, disclosing new insights for understanding its anticancer effect.

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

BACKGROUND AND AIM: Breast 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. METHODS: After 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, ß-catenin and nuclear factor-κB (NF-κB) expression were determined using western blot technique. ß-Catenin localization was observed by confocal microscopy. RESULTS: We observed that high concentrations of GSE inhibited cell proliferation and apoptosis. Conversely, low GSE concentration decreased cell migration and invasion, likely by hampering ß-catenin expression and localization, fascin and NF-κB expression, as well as by decreasing the activity of uPA, MMP-2 and MMP-9. CONCLUSIONS: These results make GSE a powerful candidate for developing preventive agents against cancer metastasis.

Shape in migration: quantitative image analysis of migrating chemoresistant HCT-8 colon cancer cells.

Unsuccessful cytotoxic anticancer treatments may contribute to tumor morphologic instability and consequent tissue invasion, promoting the selection of a more malignant phenotype. Indeed, morphological changes have been demonstrated to be more pronounced in strongly vs. weakly metastatic cells. By means of normalized bending energy, we have previously quantitatively defined the link between cell shape modifications and the acquisition of a more malignant phenotype by 5-FU-resistant colon cancer cells (HCT-8FUres). Such changes were significantly correlated with an increase in motility speed. Herein, we propose a method to quantitatively analyze the shape of wild and chemoresistant HCT-8 migration front cells during wound healing assay. We evaluated the reliability of parameters (area/perimeter ratio [A/p], circularity, roundness, fractal dimension, and solidity) in describing the biological behavior of the two cell lines, enabling hence in distinguishing the chemoresistant line from the other one. We found solidity index the parameter that better described the difference between chemoresistant and wild cells. Moreover, solidity is able to capture the differences between chemoresistant and wild cells at each time point of the migration process. Indeed, motility speed was found to be inversely correlated with solidity, a quantitative index of cell deformability. Deformability is an outstanding hallmark of the process leading to metastatic spread; consequently, solidity may be considered a marker of acquired metastatic property.

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.

TCam-2 seminoma cells exposed to egg-derived microenvironment modify their shape, adhesive pattern and migratory behaviour: a molecular and morphometric analysis.

Seminoma is one of the most common Testicular Germ Cell Tumours that originates during embryonic development due to an alteration of the local niche that in turn results in a delayed or blocked differentiation of Primordial Germ Cells. The block of differentiation is actually a common way to develop cancer disease as postulated by the "embryonic rest theory of cancer". In agreement with this theory different studies have demonstrated that embryonic cues display the capacity of reprogramming aggressive cancer cells towards a less aggressive phenotype. Herein we investigate the ability of a culture medium added with 10% egg albumen (EW, Egg White) to modulate seminoma cell phenotype and behaviour, by ensuring a proper set of morphogenetic signals. We chose to use the TCam-2 seminoma cell line that has been established as the only available cell line, obtained from a primary testicular seminoma. EW is able to: 1) modify TCam-2 cell spreading rate and cell-substrate adhesion without affecting proliferation and survival indexes; 2) modulate TCam-2 actin distribution pattern increasing cortical localization of actin filaments; 3) increase TCam-2 cell-cell junction capability; 4) decrease both chemo-sensitive and collective TCam-2 migratory behaviour. According to these observations morphometric fractal analysis revealed the ability of EW to increase Circularity and Solidity parameters and, consequently, to decrease Fractal dimension. Prompted by these observations we hypothesize that EW treatment could rescue, at least in part, the neoplastic-metastatic behaviour of seminoma cells.

Theoretical aspects of Systems Biology.

The natural world consists of hierarchical levels of complexity that range from subatomic particles and molecules to ecosystems and beyond. This implies that, in order to explain the features and behavior of a whole system, a theory might be required that would operate at the corresponding hierarchical level, i.e. where self-organization processes take place. In the past, biological research has focused on questions that could be answered by a reductionist program of genetics. The organism (and its development) was considered an epiphenomenona of its genes. However, a profound rethinking of the biological paradigm is now underway and it is likely that such a process will lead to a conceptual revolution emerging from the ashes of reductionism. This revolution implies the search for general principles on which a cogent theory of biology might rely. Because much of the logic of living systems is located at higher levels, it is imperative to focus on them. Indeed, both evolution and physiology work on these levels. Thus, by no means Systems Biology could be considered a 'simple' 'gradual' extension of Molecular Biology.

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 ß-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 ß-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.