Francesca Aredia

Poly(ADP-ribose): A signaling molecule in different paradigms of cell death

Poly(ADP-ribosylation) results from the conversion of NAD(+) into ADP-ribose and the following addition of ADP-ribose units to form polymers, further bound to acceptor proteins; once post-translationally ADP-ribosylated, proteins could change their function in basic processes. Poly(ADP-ribosylation) is activated under critical situations represented by DNA damage and cellular stress, and modulated in different paradigms of cell death. The hallmarks of the main death processes, i.e. apoptosis, parthanatos, necroptosis and autophagy, will be described, focusing on the role of poly(ADP-ribose) as a signaling molecule.

Manipulation of autophagy in cancer cells: an innovative strategy to fight drug resistance

Autophagy is a catabolic process activated by stress conditions and nutrient deprivation, to which it reacts by promoting the degradation of damaged organelles and misfolded/aggregated proteins, as well as generating new energetic pools. Paradoxically, in cancer cells, which signal the dangerous microenvironment occurring during clinical therapies, autophagy could promote their proliferation and sustain drug resistance. Special attention is given to autophagy manipulation in order to counteract drug resistance of cancer cells. This article describes the basic properties of autophagy and focuses on the strategies of manipulating it.

Morphological Features of Organelles during Apoptosis: An Overview

An apoptotic program leading to controlled cell dismantling implies perturbations of nuclear dynamics, as well as changes affecting the organelle structure and distribution. In human cancer cells driven to apoptosis by different stimuli, we have recently investigated the morphological properties of several organelles, including mitochondria, lysosomes, endoplasmic reticulum and Golgi apparatus. In this review, we will discuss the body of evidence in the literature suggesting that organelles are generally relocated and/or degraded during apoptosis, irrespectively of the apoptogenic stimulus and cell type.

Effect of new berberine derivatives on colon cancer cells

The natural alkaloid berberine has been recently described as a promising anticancer drug. In order to improve its efficacy and bioavailability, several derivatives have been designed and synthesized and found to be even more potent than the lead compound. Among the series of berberine derivatives we have produced, five compounds were identified to be able to heavily affect the proliferation of human HCT116 and SW613-B3 colon carcinoma cell lines. Remarkably, these active compounds exhibit high fluorescence emission property and ability to induce autophagy.

Fluorescence properties of the Na+/H+exchanger inhibitor HMA (5-(N,N-hexamethylene)amiloride) are modulated by intracellular pH

HMA (5-(N,N-hexamethylene)amiloride), which belongs to a family of novel amiloride derivatives, is one of the most effective inhibitors of Na+/H+ exchangers, while uneffective against Na+ channels and Na+/Ca2+ exchangers. In this study, we provided evidence that HMA can act as a fluorescent probe. In fact, human retinal ARPE19 cells incubated with HMA show an intense bluish fluorescence in the cytoplasm when observed at microscope under conventional UV-excitation conditions. Interestingly, a prolonged observation under continuous exposure to excitation lightdoes not induce great changes in cells incubated with HMA for times up to about 5 min, while an unexpected rapid increase in fluorescence signal is observed in cells incubated for longer times. The latter phenomenon is particularly evident in the perinuclear region and in discrete spots in the cytoplasm. Since HMA modulates intracellular acidity, the dependence of its fluorescence properties on medium pH and response upon irradiation have been investigated in solution, at pH 5.0 and pH 7.2. The changes in both spectral shape and amplitude emission indicate a marked pH influence on HMA fluorescence properties, making HMA exploitable as a self biomarker of pH alterations in cell studies, in the absence of perturbations induced by the administration of other exogenous dyes.

Involvement of PARPs in cell death.

Poly(ADP-ribosylation), an NAD dependent reaction culminating in the formation of ADP-ribose monomers, and their following polymerization, is activated as an emergency process in crucial situations such as DNA damage and cellular stress; due to this crucial function, the modulation of poly(ADP-ribosylation) during cell death has been investigated. This review will describe the properties of poly(ADP-ribose) as a signalling molecule in different paradigms of cell death, i.e.apoptosis, parthanatos, necroptosis and autophagy.

Multiple effects of the Na+/H+ antiporter inhibitor HMA on cancer cells

Amiloride derivatives are a class of new promising chemotherapeutic agents. A representative member of this family is the sodium–hydrogen antiporter inhibitor HMA (5-(N,N-hexamethylene amiloride), which has been demonstrated to induce cellular intracytosolic acidification and cell death through the apoptotic pathway(s). This work aims at characterizing drug response of human cancer cell lines to HMA. After a first screening revealing that HMA interferes with cancer cell survival, we focused our attention on SW613-B3 colon carcinoma cells, which are intrinsically resistant to a panel of drugs. Searching for the activation of canonical apoptosis, we found that this process was abortive, given that the final steps of this process, i.e. PARP-1 cleavage and DNA ladder, were not detectable. Thus, we addressed caspase-independent paradigms of cell death and we observed that HMA promotes the induction of the LEI/L-DNase II pathway as well as of parthanatos. Finally, we explored the possible impact of autophagy of cell response to HMA, providing the evidence that autophagy is activated in our experimental system. On the whole, our results defined the biochemical reactions triggered by HMA, and elucidated its multiple effects, thus adding further complexity to the intricate network leading to drug resistance.

An innovative cell microincubator for drug discovery based on 3D silicon structures

We recently employed three-dimensional (3D) silicon microstructures (SMSs) consisting in arrays of 3 µm-thick silicon walls separated by 50 µm-deep, 5 µm-wide gaps, as microincubators for monitoring the biomechanical properties of tumor cells. They were here applied to investigate the in vitro behavior of HT1080 human fibrosarcoma cells driven to apoptosis by the chemotherapeutic drug Bleomycin. Our results, obtained by fluorescence microscopy, demonstrated that HT1080 cells exhibited a great ability to colonize the narrow gaps. Remarkably, HT1080 cells grown on 3D-SMS, when treated with the DNA damaging agent Bleomycin under conditions leading to apoptosis, tended to shrink, reducing their volume and mimicking the normal behavior of apoptotic cells, and were prone to leave the gaps. Finally, we performed label-free detection of cells adherent to the vertical silicon wall, inside the gap of 3D-SMS, by exploiting optical low coherence reflectometry using infrared, low power radiation. This kind of approach may become a new tool for increasing automation in the drug discovery area. Our results open new perspectives in view of future applications of the 3D-SMS as the core element of a lab-on-a-chip suitable for screening the effect of new molecules potentially able to kill tumor cells.

Label-free optical detection of cells grown in 3D silicon microstructures

We demonstrate high aspect-ratio photonic crystals that could serve as three-dimensional (3D) microincubators for cell culture and also provide label-free optical detection of the cells. The investigated microstructures, fabricated by electrochemical micromachining of standard silicon wafers, consist of periodic arrays of silicon walls separated by narrow deeply etched air-gaps (50 μm high and 5 μm wide) and feature the typical spectral properties of photonic crystals in the wavelength range 1.0-1.7 μm: their spectral reflectivity is characterized by wavelength regions where reflectivity is high (photonic bandgaps), separated by narrow wavelength regions where reflectivity is very low. In this work, we show that the presence of cells, grown inside the gaps, strongly affects light propagation across the photonic crystal and, therefore, its spectral reflectivity. Exploiting a label-free optical detection method, based on a fiberoptic setup, we are able to probe the extension of cells adherent to the vertical silicon walls with a non-invasive direct testing. In particular, the intensity ratio at two wavelengths is the experimental parameter that can be well correlated to the cell spreading on the silicon wall inside the gaps.

A New Cell-Selective Three-Dimensional Microincubator Based on Silicon Photonic Crystals

In this work, we show that vertical, high aspect-ratio (HAR) photonic crystals (PhCs), consisting of periodic arrays of 5 µm wide gaps with depth of 50 µm separated by 3 µm thick silicon walls, fabricated by electrochemical micromachining, can be used as three-dimensional microincubators, allowing cell lines to be selectively grown into the gaps. Silicon micromachined dice incorporating regions with different surface profiles, namely flat silicon and deeply etched PhC, were used as microincubators for culturing adherent cell lines with different morphology and adhesion properties. We extensively investigated and compared the proliferative behavior on HAR PhCs of eight human cell models, with different origins, such as the epithelial (SW613-B3; HeLa; SW480; HCT116; HT29) and the mesenchymal (MRC-5V1; CF; HT1080). We also verified the contribution of cell sedimentation into the silicon gaps. Fluorescence microscopy analysis highlights that only cell lines that exhibit, in the tested culture condition, the behavior typical of the mesenchymal phenotype are able to penetrate into the gaps of the PhC, extending their body deeply in the narrow gaps between adjacent silicon walls, and to grow adherent to the vertical surfaces of silicon. Results reported in this work, confirmed in various experiments, strongly support our statement that such three-dimensional microstructures have selection capabilities with regard to the cell lines that can actively populate the narrow gaps. Cells with a mesenchymal phenotype could be exploited in the next future as bioreceptors, in combination with HAR PhC optical transducers, e.g., for label-free optical detection of cellular activities involving changes in cell adhesion and/or morphology (e.g., apoptosis) in a three-dimensional microenvironment.