Two coffins and a funeral: early or late caspase activation determines two types of apoptosis induced by DNA damaging agents.

Cell cytoskeleton makes profound changes during apoptosis including the organization of an Apoptotic Microtubule Network (AMN). AMN forms a cortical structure which plays an important role in preserving plasma membrane integrity during apoptosis. Here, we examined the cytoskeleton rearrangements during apoptosis induced by camptothecin (CPT), a topoisomerase I inhibitor, in human H460 and porcine LLCPK-1α cells. Using fixed and living cell imaging, we showed that CPT induced two dose- and cell cycle-dependent types of apoptosis characterized by different cytoskeleton reorganizations, time-dependent caspase activation and final apoptotic cell morphology. In the one referred as "slow" (~h) or round-shaped, apoptosis was characterized by a slow contraction of the actinomyosin ring and late caspase activation. In "slow" apoptosis the γ-tubulin complexes were not disorganized and microtubules were not depolymerized at early stages. In contrast, "fast" (~min) or irregular-shaped apoptosis was characterized by early caspase activation followed by full contraction of the actinomyosin ring. In fast apoptosis γ-tubulin complexes were disorganized and microtubules were initially depolymerized. However, after actinomyosin contraction, microtubules were reformed adopting a cortical but irregular disposition near plasma membrane. In addition to distinctive cytoskeleton reorganization kinetics, round and irregular-shaped apoptosis showed different biological properties with respect to AMN maintenance, plasma membrane integrity and phagocytes response. Our results suggest that the knowledge and modulation of the type of apoptosis promoted by genotoxic agents may be important for deciding a better therapeutic option and predicting the immune response in cancer treatment.

Apoptotic cells subjected to cold/warming exposure disorganize apoptotic microtubule network and undergo secondary necrosis.

Apoptotic microtubule network (AMN) is organized during apoptosis, forming a cortical structure beneath the plasma membrane which plays a critical role in preserving cell morphology and plasma membrane integrity. The aim of this study was to examine the effect of cold/warming exposure on apoptotic microtubules and plasma membrane integrity during the execution phase of apoptosis. We demonstrated in camptothecin-induced apoptotic H460 cells that cold/warming exposure disorganized apoptotic microtubules and allowed the access of active caspases to the cellular cortex and the cleavage of essential proteins in the preservation of plasma membrane permeability. Cleavage of cellular cortex and plasma membrane proteins, such as α-spectrin, paxilin, focal adhesion kinase and calcium ATPase pump (PMCA-4) involved in cell calcium extrusion resulted in increased plasma permeability and calcium overload leading apoptotic cells to secondary necrosis. The essential role of caspase-mediated cleavage in this process was demonstrated because the addition of the pan-caspase inhibitor z-VAD during cold/warming exposure that induces AMN depolymerization avoided the cleavage of cortical and plasma membrane proteins and prevented apoptotic cells to undergo secondary necrosis. Likewise, apoptotic microtubules stabilization by taxol during cold/warming exposure also prevented cellular cortex and plasma membrane protein cleavage and secondary necrosis. Furthermore, microtubules stabilization or caspase inhibition during cold/warming exposure was also critical for proper phosphatidylserine externalization and apoptotic cell clearance by macrophages. These results indicate that cold/warming exposure of apoptotic cells induces secondary necrosis which can be prevented by both, microtubule stabilization or caspase inhibition.

Effect of diol isomer/water mixtures on the stability of Zn-MOF-74.

The stability of metal-organic frameworks is a key factor in many applications in some fields that require working under harsh conditions. It is known that a large number of MOFs are vulnerable to humid air. It means that when they are exposed to water, a structural collapse of the crystal happens. In this work, Molecular Dynamics simulations using a reactive force field have been performed to study the stability of MOF-74 against the adsorption of catechol, resorcinol and hydroquinone in the presence of water. We reproduced the water instability of Zn-MOF-74 and we studied the resistance of the structure. Our simulations showed that the three isomers generate a volume change in the framework but the structural collapse does not happen. In contrast, for water-isomer mixtures, there is structural collapse. Not only do catechol, resorcinol and hydroquinone not behave as stabilizing agents but they do enhance the hydration effect on the structure.

Synthesis and Characterization of Elongated-Shaped Silver Nanoparticles as a Biocompatible Anisotropic SERS Probe for Intracellular Imaging: Theoretical Modeling and Experimental Verification.

Progress in the field of biocompatible SERS nanoparticles has promising prospects for biomedical applications. In this work, we have developed a biocompatible Raman probe by combining anisotropic silver nanoparticles with the dye rhodamine 6G followed by subsequent coating with bovine serum albumin. This nanosystem presents strong SERS capabilities in the near infrared (NIR) with a very high (2.7 × 107) analytical enhancement factor. Theoretical calculations reveal the effects of the electromagnetic and chemical mechanisms in the observed SERS effect for this nanosystem. Finite element method (FEM) calculations showed a considerable near field enhancement in NIR. Using density functional quantum chemical calculations, the chemical enhancement mechanism of rhodamine 6G by interaction with the nanoparticles was probed, allowing us to calculate spectra that closely reproduce the experimental results. The nanosystem was tested in cell culture experiments, showing cell internalization and also proving to be completely biocompatible, as no cell death was observed. Using a NIR laser, SERS signals could be detected even from inside cells, proving the applicability of this nanosystem as a biocompatible SERS probe.

Amitriptyline induces mitophagy that precedes apoptosis in human HepG2 cells.

Systemic treatments for hepatocellular carcinoma (HCC) have been largely unsuccessful. This study investigated the antitumoral activity of Amitriptyline, a tricyclic antidepressant, in hepatoma cells. Amitriptyline-induced toxicity involved early mitophagy activation that subsequently switched to apoptosis. Amitriptyline induced mitochondria dysfunction and oxidative stress in HepG2 cells. Amitriptyline specifically inhibited mitochondrial complex III activity that is associated with decreased mitochondrial membrane potential (∆Ψm) and increased reactive oxygen species (ROS) production. Transmission electron microscopy (TEM) studies revealed structurally abnormal mitochondria that were engulfed by double-membrane structures resembling autophagosomes. Consistent with mitophagy activation, fluorescence microscopy analysis showed mitochondrial Parkin recruitment and colocalization of mitochondria with autophagosome protein markers. Pharmacological or genetic inhibition of autophagy exacerbated the deleterious effects of Amitriptyline on hepatoma cells and led to increased apoptosis. These results suggest that mitophagy acts as an initial adaptive mechanism of cell survival. However persistent mitochondrial damage induced extensive and lethal mitophagy, autophagy stress and autophagolysome permeabilization leading eventually to cell death by apoptosis. Amitriptyline also induced cell death in hepatoma cells lines with mutated p53 and non-sense p53 mutation. Our results support the hypothesis that Amitriptyline-induced mitochondrial dysfunction can be a useful therapeutic strategy for HCC treatment, especially in tumors showing p53 mutations and/or resistant to genotoxic treatments.