A surprising and unexpected biomineralization process was observed during toxicological assessment of carbon nanoparticles on Paracentrotus lividus (sea urchin) pluteus larvae. The larvae activate a process of defense against external material, by incorporating the nanoparticles into microstructures of aragonite similarly to pearl oysters. Aiming at a better understanding of this phenomenon, the larvae were exposed to increasing concentrations of carbon nanoparticles and the biomineralization products were analyzed by electron microscopy, x-ray diffraction and Raman spectroscopy. In order to evaluate the possible influence of Sp-CyP-1 expression on this biomineralization process by larvae, analyses of gene expression (Sp-CyP-1) and calcein labeling were performed. Overall, we report experimental evidence about the capability of carbon nanoparticles to induce an increment of Sp-CyP-1 expression with the consequent activation of a biomineralization process leading to the production of a new pearl-like biomaterial never previously observed in sea urchins.

Cell deaths in Photodynamic therapy, that is an anticancer therapy requiring exposure of cells or tissue to photosensitizing drug followed by irradiation with visible light of the appropriate wavelength, occur by the efficient induction of apoptotic as well as non-apoptotic cell deaths, like necrosis and autophagy, or by a combination of the three mechanisms. However, the exact role of autophagy in Photodynamic therapy is still a matter of debate. To this purpose, we investigated the induction of autophagy in HeLa cells photosensitized with Rose Bengal Acetate (RBAc). After incubation with Rose Bengal Acetate (10-5 M), HeLa cells were irradiated for 90 seconds (green LED DPL 305, emitting at 530 ± 15 nm in order to obtain 1.6 J/cm2 as total light dose) and allowed to recover for 72 h. Induction of autophagy and apoptosis was observed with peaks at 8 h and 12 h after irradiation respectively for autophagy and apoptosis. Autophagy was detected by biochemical (Western Blot of LC3B protein) and morphological criteria (TEM, cytochemistry). In addition, the pan-caspases inhibitor z-VAD was not able to completely prevent cell deaths. The simultaneous onset of apoptosis and autophagy following Rose Bengal Acetate Photodynamic therapy is of remarkable interest in consideration of the findings that autophagy can result in class II presentation of antigens and thus explain why low dose Photodynamic therapy can yield anti-tumour vaccines.

The new concept of Immunogenic Cell Death (ICD), associated with Damage Associated Molecular Patterns (DAMPs) exposure and/or release, is recently becoming very appealing in cancer treatment. In this context, PhotoDynamic Therapy (PDT) can give rise to ICD and to immune response upon dead cells removal. The list of PhotoSensitizers (PSs) able to induce ICD is still short and includes Photofrin, Hypericin, Foscan and 5-ALA. The goal of the present work was to investigate if Rose Bengal Acetate (RBAc), a powerful PS able to trigger apoptosis and autophagy, enables photosensitized HeLa cells to expose and/or release pivotal DAMPs, i.e. ATP, HSP70, HSP90, HMGB1, and calreticulin (CRT), that characterize ICD. We found that apoptotic HeLa cells after RBAc-PDT exposed and released, early after the treatment, high amount of ATP, HSP70, HSP90 and CRT; the latter was distributed on the cell surface as uneven patches and co-exposed with ERp57. Conversely, autophagic HeLa cells after RBAc-PDT exposed and released HSP70, HSP90 but not CRT and ATP. Exposure and release of HSP70 and HSP90 were always higher on apoptotic than on autophagic cells. HMGB1 was released concomitantly to secondary necrosis (24 h after RBAc-PDT). Phagocytosis assay suggests that CRT is involved in removal of RBAc-PDT generated apoptotic HeLa cells. Altogether, our data suggest that RBAc has all the prerequisites (i.e. exposure and/or release of ATP, CRT, HSP70 and HSP90), that must be verified in future vaccination experiments, to be considered a good PS candidate to ignite ICD. We also showed tha CRT is involved in the clearance of RBAc photokilled HeLa cells. Interestingly, RBAc-PDT is the first cancer PDT protocol able to induce the translocation of HSP90 and plasma membrane co-exposure of CRT with ERp57.

Silver nanostructures were successfully synthesized through a simple and “green” method using saccharides as reducing and capping agent. Transmission electron microscopy (TEM) and UV–Vis absorption were used to certify the quality of the silver nanoparticles obtained: first, size and dispersion. In this work Silver NanoParticles (AgNPs) cytotoxicity related to saccharides capping (Glucose and Glucose-Sucrose) was explored. Human epitheloid cervix carcinoma cells (HeLa) were used for cytotoxicity test. The cells were incubated with increasing AgNPs number/cell and HeLa cells viability was monitored for a period of 48 h compared with the positive and negative controls. We observed that the toxicity increases in a incubation time and AgNPs number/cell related manner. In addition, the AgNP-G are more toxic than AgNP-GS, suggesting that AgNPs citotoxicity could depend on the capping agent. in HeLa cells the highest AgNP-G number/cell induces , cell deathsoon after 1 hr of incubation; conversely the lowest AgNP-GS number/cell induces cell proliferation.

In a previous work we showed that 6mT SMF interferes with monocyte/macrophage 12-Otetradecanoylphorbol- 13-acetate (TPA) -induced differentiation of promonocytes (U937 cells) and monocytes (THP-1 cells), in this work we investigate whether in the same cells and under the same conditions phagocytosis of apoptotic cells is influenced by 6 mT SMF exposure. Fluid phase endocytosis and phagocytosis of latex particles were also analysed for comparison. The results indicate that SMF exposure, whose influence was greater at the late stages of the macrophage differentiation (THP1 > U937 cells), has effects on phagocytosis but not on fluid phase endocytosis. Phagocytosis index and rate decreased under SMF exposure while the number of latex particles bound to the plasma membrane of TPA-differentiated U937 and THP1 cells increased. Conversely, phagocytosis rate of apoptotic cells increased under SMF exposure while the number of apoptotic cells bound to the plasma membrane of TPA-differentiated U937 and THP1 cells and Raw 264.7 macrophages decreased. Even in conditions of inability to phagocytose, i.e. non-differentiated U937 and THP-1 cells, the SMF exposure enhanced the number of cell surface bound apoptotic cells and latex beads.

Rose Bengal Acetate PhotoDynamic Therapy (RBAc-PDT) induced multiple cell deaths pathways in HeLa cells through ROS generation. The onset of apoptosis, autophagy and of the different apoptotic pathways were timed by determining the levels of caspases, Bcl 2 family, Hsp 70, LC3, Grp78 and phospho-eIF2α proteins. Four different apoptotic pathways plus autophagy were sequentially initiated by RBAc-PDT: intrinsic, extrinsic, caspase 12- dependent and caspase independent as reflected by peaks of the relative caspases, 9, 8, 3 and 12. Autophagy, revealed by the formation of acidic autophagosomes and by increased Light Chain 3-II (LC3BII) expression was conspicuous at 8 h post-PDT. In our system, autophagy had a pro-death role, since its inhibitor, 3-MethylAdenine (3-MA), significantly augmented cell viability. The increase of cleaved caspase 12 was consequent to the increase of Grp78 and phospho-eIF2α proteins, suggesting Endoplasmic Reticulum. Regulation of the intrinsic pathway of apoptosis was under the control of Bcl-2 family (i.e. soon after irradiation Bcl2 decreased, Bax and tBid increased) and of Hsp70 proteins (peak at 12-18h post-PDT). Interestingly, inhibition of one pathway, i.e.caspase-9 (Z-LEHD-FMK), caspase-8 (Z-IETD-FMK), pan-caspases (Z-VAD-FMK), autophagy (3-MA) and necrosis (Nec-1), did not impair the activation of the others, suggesting the independent onset of the different apoptotic and autophagic pathways in a not subordinated fashion.