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

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

Analysis of ERK3 intracellular localization: dynamic distribution during mitosis and apoptosis

Extracellular signal-regulated kinases (ERK) 1, 2 and 3 are involved in cell proliferation and differentiation, and apoptosis; although ERK1/2 have been widely studied, limited knowledge on ERK3 is available. The present work aimed at investigating ERK3 distribution during cell cycle and apoptosis in human tumor HeLa cells. The analysis performed by double immunofluorescence and immunoelectron microscopy experiments revealed that during interphase ERK3 is mainly resident in the nucleoplasm in association with ribonuclear proteins involved in early pre-mRNA splicing, it undergoes cell cycle-dependent redistribution and, during apoptosis, it remains in the nucleus in the form of massive nuclear aggregates, then moves to the cytoplasm and is finally extruded

Analysis of ERK3 intracellular localization: dynamic distribution during mitosis and apoptosis

Extracellular signal-regulated kinases (ERK) 1, 2 and 3 are involved in cell proliferation and differentiation, and apoptosis; although ERK1/2 have been widely studied, limited knowledge on ERK3 is available. The present work aimed at investigating ERK3 distribution during cell cycle and apoptosis in human tumor HeLa cells. The analysis performed by double immunofluorescence and immunoelectron microscopy experiments revealed that during interphase ERK3 is mainly resident in the nucleoplasm in association with ribonuclear proteins involved in early pre-mRNA splicing, it undergoes cell cycle-dependent redistribution and, during apoptosis, it remains in the nucleus in the form of massive nuclear aggregates, then moves to the cytoplasm and is finally extruded.</p

Molecular features of the cytotoxicity of an NHE inhibitor: evidence of mitochondrial alterations, ROS overproduction and DNA damage

Background: NH exchangers (NHEs) play a crucial role in regulating intra/extracellular pH, which is altered in cancer cells, and are therefore suitable targets to alter cancer cell metabolism in order to inhibit cell survival and proliferation. Among NHE inhibitors, amiloride family members are commonly used in clinical practice as diuretics; we focused on the amiloride HMA, reporting a net cytotoxic effect on a panel of human cancer cell lines; now we aim to provide new insights into the molecular events leading to cell death by HMA. Methods: Colon cancer cell lines were treated with HMA and analysed with: morphological and cellular assays for cell viability and death, and autophagy; biochemical approaches to evaluate mitochondrial function and ROS production; in situ detection of DNA damage; molecular tools to silence crucial autophagy/necroptosis factors. Results: HMA affects cellular morphology, alters mitochondrial structure and function, causes an increase in ROS, which is detrimental to DNA integrity, stimulates poly(ADP-ribose) synthesis, activates RIPK3-dependent death and triggers autophagy, which is unable to rescue cell survival. These features are hot points of an intricate network of processes, including necroptosis and autophagy, regulating the homeostasis between survival and death. Conclusion: Our results allow the identification of multiple events leading to cell death in cancer cells treated with HMA. The here-defined intricate network activated by HMA could be instrumental to selectively target the key players of each pathway in the attempt to improve the global response to HMA. Our data could be the starting point for developing a newly designed targeted therapy

Morphological Features of Organelles during Apoptosis: An Overview

cell

Autophagy and Cancer

Autophagy is a housekeeping survival mechanism with a protective function against stress conditions. However, when stress severity or duration increases, it may promote cell death. Paradoxically, autophagy favors cancer development, since cancer cells could enhance their proliferation potential (thus becoming able to resist anticancer therapy) thanks to the energetic supply provided by organelle degradation typically driven by autophagy following a stepwise pathway. The main actors of the autophagic machinery as well as the features shared with apoptosis will be described. Special attention will be paid to the effects of autophagy manipulation

Reconstruction of cell distribution in 3D silicon microstructures by label-free optical detection

In this work, we report the results recently obtained with an innovative microsystem for human cell studies that combines the functionality of a highly ordered, silicon microstructure as 3D incubator with a label-free analytical approach. This system is suitable for monitoring distribution of cells, for example with a mesenchymal phenotype, that are able to stretch their bodies inside the narrow and deeply etched gaps of the 3D micromachined structure and to grow adherent to vertical surfaces. We demonstrate that the extension of the cell body inside the gaps can be recovered with a label-free optical detection method based on IR spectral reflectivity measurements performed with an all-fiberoptic configuration. © 2014 AEIT