No abstract

Scavenger Receptor B1 (SR-B1), also known as HDL receptor, is involved in cellular cholesterol uptake. Stratum corneum (SC), the outermost layer of the skin, is composed of more than 25% cholesterol. Several reports support the view that alteration of SC lipid composition may be the cause of impaired barrier function which gives rise to several skin diseases. For this reason the regulation of the genes involved in cholesterol uptake is of extreme significance for skin health. Being the first shield against external insults, the skin is exposed to several noxious substances and among these is cigarette smoke (CS), which has been recently associated with various skin pathologies. In this study we first have shown the presence of SR-B1 in murine and human skin tissue and then by using immunoblotting, immunoprecipitation, RT-PCR, and confocal microscopy we have demonstrated the translocation and the subsequent lost of SR-B1 in human keratinocytes (cell culture model) after CS exposure is driven by hydrogen peroxide (H 2 O 2 ) that derives not only from the CS gas phase but mainly from the activation of cellular NADPH oxidase (NOX). This effect was reversed when the cells were pretreated with NOX inhibitors or catalase. Furthermore, CS caused the formation of SR-B1-aldheydes adducts (acrolein and 4-hydroxy-2-nonenal) and the increase of its ubiquitination, which could be one of the causes of SR-B1 loss. In conclusion, exposure to CS, through the production of H 2 O 2 , induced post-translational modifications of SR-B1 with the consequence lost of the receptor and this may contribute to the skin physiology alteration as a consequence of the variation of cholesterol uptake.

Glioblastoma (GB) has a poor prognosis, despite current multimodality treatment. Beside surgical resection, adjuvant ionizing radiation (IR) combined with Temozolomide (TMZ) drug administration is the standard therapy for GB. This currently combined radio-chemotherapy treatment resulted in glial tumor cell death induction, whose main molecular death pathways are still not completely deciphered. In this study, the autophagy process was investigated, and in vitro modulated, in two different GB cell lines, T98G and U373MG (known to differ in their radiosensitivity), after IR or combined IR/TMZ treatments. T98G cells showed a high radiosensitivity (especially at low and intermediate doses), associated with autophagy activation, assessed by Beclin-1 and Atg-5 expression increase, LC3-I to LC3-II conversion and LC3B-GFP accumulation in autophagosomes of irradiated cells; differently, U373MG cells resulted less radiosensitive. Autophagy inhibition, using siRNA against BECN1 or ATG-7 genes, totally prevented decrease in viability after both IR and IR/TMZ treatments in the radiosensitive T98G cells, confirming the autophagy involvement in the cytotoxicity of these cells after the current GB treatment, contrary to U373MG cells. However, rapamycin-mediated autophagy, that further radiosensitized T98G, was able to promote radiosensitivty also in U373MG cells, suggesting a role of autophagy process in enhancing radiosensitivity. Taken together, these results might enforce the concept that autophagy-associated cell death might constitute a possible adjuvant therapeutic strategy to enhance the conventional GB treatment.

Glioblastoma (GBM) remains the most aggressive and lethal brain tumor due to its molecular heterogeneity and high motility and invasion capabilities of its cells, resulting in high resistance to current standard treatments (surgery, followed by ionizing radiation combined with Temozolomide chemotherapy administration). Locus amplification, gene overexpression, and genetic mutations of epidermal growth factor receptor (EGFR) are hallmarks of GBM that can ectopically activate downstream signaling oncogenic cascades such as PI3K/Akt/mTOR pathway. Importantly, alteration of this pathway, involved also in the regulation of autophagy process, can improve radioresistance in GBM cells, thus promoting the aggressive phenotype of this tumor. In this work, the endogenous EGFR expression profile and autophagy were modulated to increase radiosensitivity behavior of human T98G and U373MG GBM cells. Our results primarily indicated that EGFR interfering induced radiosensitivity according to a decrease of the clonogenic capability of the investigated cells, and an effective reduction of the in vitro migratory features. Moreover, EGFR interfering resulted in an increase of Temozolomide (TMZ) cytotoxicity in T98G TMZ-resistant cells. In order to elucidate the involvement of the autophagy process as pro-death or pro-survival role in cells subjected to EGFR interfering, the key autophagic gene ATG7 was silenced, thereby producing a transient block of the autophagy process. This autophagy inhibition rescued clonogenic capability of irradiated and EGFR-silenced T98G cells, suggesting a pro-death autophagy contribution. To further confirm the functional interplay between EGFR and autophagy pathways, Rapamycin-mediated autophagy induction during EGFR modulation promoted further impairment of irradiated cells, in terms of clonogenic and migration capabilities. Taken together, these results might suggest a novel combined EGFR-autophagy modulation strategy, to overcome intrinsic GBM radioresistance, thus improving the efficacy of standard treatments. J. Cell. Physiol. 229: 1863-1873, 2014. © 2014 Wiley Periodicals, Inc.

The interplay between a non-lethal autophagic response and apoptotic cell death is still a matter of debate in cancer cell biology. In the present study performed on human melanoma cells, we investigate the role of basal or stimulated autophagy in cisplatin-induced cytotoxicity, as well as the contribution of cisplatin-induced activation of caspases 3/7 and conventional calpains. The results show that, while down-regulating Beclin-1, Atg14 and LC3-II, cisplatin treatment inhibits the basal autophagic response, impairing a physiological pro-survival response. Consistently, exogenously stimulated autophagy, obtained with trehalose or calpains inhibitors (MDL-28170 and calpeptin), protects from cisplatin-induced apoptosis, and such a protection is reverted by inhibiting autophagy with 3-methyladenine or ATG5 silencing. In addition, during trehalose-stimulated autophagy, the cisplatin-induced activation of calpains is abrogated, suggesting the existence of a feedback loop between the autophagic process and calpains. On the whole, our results demonstrate that in human melanoma cells autophagy may function as a beneficial stress response, hindered by cisplatin-induced death mechanisms. In a therapeutic perspective, these findings suggest that the efficacy of cisplatin-based polychemotherapies for melanoma could be potentiated by inhibitors of autophagy.