Resveratrol-induced apoptosis depends on the lipid kinase activity of Vps34 and on the formation of autophagolysosomes

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Knock-Down of Cathepsin D Affects the Retinal Pigment Epithelium, Impairs Swim-Bladder Ontogenesis and Causes Premature Death in Zebrafish

The lysosomal aspartic protease Cathepsin D (CD) is ubiquitously expressed in eukaryotic organisms. CD activity is essential to accomplish the acid-dependent extensive or partial proteolysis of protein substrates within endosomal and lysosomal compartments therein delivered via endocytosis, phagocytosis or autophagocytosis. CD may also act at physiological pH on small-size substrates in the cytosol and in the extracellular milieu. Mouse and fruit fly CD knock-out models have highlighted the multi-pathophysiological roles of CD in tissue homeostasis and organ development. Here we report the first phenotypic description of the lack of CD expression during zebrafish (Danio rerio) development obtained by morpholino-mediated knock-down of CD mRNA. Since the un-fertilized eggs were shown to be supplied with maternal CD mRNA, only a morpholino targeting a sequence containing the starting ATG codon was effective. The main phenotypic alterations produced by CD knock-down in zebrafish were: 1. abnormal development of the eye and of retinal pigment epithelium; 2. absence of the swim-bladder; 3. skin hyper-pigmentation; 4. reduced growth and premature death. Rescue experiments confirmed the involvement of CD in the developmental processes leading to these phenotypic alterations. Our findings add to the list of CD functions in organ development and patho-physiology in vertebrates

Patented Biomarkers for the Early Detection of Ovarian Cancer

Ovarian cancer is responsible for the highest fatality rate among the gynecologic malignancies, and there is a great urgency in defining screening tests for its early detection. Presently, CA125 is the only serum marker recommended for early detection (in combination with transvaginal ultrasonography) in hereditary syndromes and for differential diagnosis in suspicious pelvic mass. However, given the complexity of the pathogenesis and of the tumor-host interaction, it is unlikely that a single serum biomarker bears sufficient information for the specific discrimination of ovarian cancer at its very early stage form other non-malignant pelvic lesions. Consistently, CA125 has demonstrated its utility for monitoring the therapy and prognosis, but owing to its scarce sensitivity and specificity it is not recommended for screening of ovarian cancer in asymptomatic patients. Recently, the introduction of high-throughput multiplex technologies, that allow to measure simultaneously a large number of molecules in the femtomolar range of concentration in the serum, has led to the definition of panels of biomarkers for the early detection of ovarian cancer. Here, we review the latest patents in this field

Chelation of Lysosomal Iron Protects Dopaminergic SH-SY5Y Neuroblastoma Cells from Hydrogen Peroxide Toxicity by Precluding Autophagy and Akt Dephosphorylation

In human neuroblastoma SH-SY5Y cells, hydrogen peroxide (H(2)O(2), 200\u3bcM) rapidly (< 5 min) induced autophagy, as shown by processing and vacuolar relocation of light chain 3(LC3). Accumulation of autophagosome peaked at 30 min of H(2)O(2) exposure. The continuous presence of H(2)O(2) eventually (at > 60 min) caused autophagy-dependent annexin V-positive cell death. However, the cells exposed to H(2)O(2) for 30 min and then cultivated in fresh medium could recover and grow, despite ongoing autophagy. H(2)O(2) rapidly (5 min) triggered the formation of dichlorofluorescein-sensitive HO(\ub7)-free radicals within mitochondria, whereas the mitochondria-associated oxidoradicals revealed by MitoSox (O(2)(\ub7-)) became apparent after 30 min of exposure to H(2)O(2). 3-Methyladenine inhibited autophagy and cell death, but not the generation of HO(\ub7). Genetic silencing of beclin-1 prevented bax- and annexin V-positive cell death induced by H(2)O(2), confirming the involvement of canonical autophagy in peroxide toxicity. The lysosomotropic iron chelator deferoxamine (DFO) prevented the mitochondrial generation of both HO(.) and O(2)(\ub7-) and suppressed the induction of autophagy and of cell death by H(2)O(2). Upon exposure to H(2)O(2), Akt was intensely phosphorylated in the first 30 min, concurrently with mammalian target of rapamycin inactivation and autophagy, and it was dephosphorylated at 2 h, when > 50% of the cells were dead. DFO did not impede Akt phosphorylation, which therefore was independent of reactive oxygen species (ROS) generation but inhibited Akt dephosphorylation. In conclusion, exogenous H(2)O(2) triggers two parallel independent pathways, one leading to autophagy and autophagy-dependent apoptosis, the other to transient Akt phosphorylation, and both are inhibited by DFO. The present work establishes HO(\ub7) as the autophagy-inducing ROS and highlights the need for free lysosomal iron for its production within mitochondria in response to hydrogen peroxide

Inhibition of PI3k Class III-Dependent Autophagy Prevents Apoptosis and Necrosis by Oxidative Stress in Dopaminergic Neuroblastoma Cells

Hydrogen peroxide (H(2)O(2)) is an extremely reactive oxidoradical that is normally produced as a by-product of the mitochondrial activity and also under several metabolic stress conditions. Autophagy, a lysosomal degradation pathway, is triggered by oxidative stress as a defensive response. How autophagy and death pathways are coordinated in cells subjected to oxidative stress is still poorly understood. In human neuroblastoma SH-SY5Y cells, 200microM H(2)O(2) rapidly induced the formation of LC3-positive autophagic vacuoles and of beclin1-Vps34 double-positive macroaggregates. Vacuolar LC3 and beclin1 aggregates did not form when oxidative stress was performed in cells pretreated with 3-methyladenine (3MA), an inhibitor of Vps34, or infected with a recombinant adenovirus expressing a dominant-negative mutant of Vps34. H(2)O(2) provoked the permeabilization of lysosomes (at 30 min) and of mitochondria, the concomitant oligomerization of bax, and eventually (at 2 h), cell death in about 50% of the cell culture. Inactivation of Vps34-dependent autophagy in oxidative-stressed cells abrogated lysosome leakage, bax activation, and caspase-dependent apoptosis and conferred protection for as long as 16 h. Inhibition of caspase activity (by ZVAD-fmk) did not trigger an alternative cell death pathway but rather afforded complete protection from oxidative toxicity, despite the ongoing generation of oxidoradicals and the cellular accumulation of autophagic vacuoles and of leaking lysosomes. On long-term (16 h) exposure to H(2)O(2), signs of necrotic cell death became apparent in LC3-positive cells, which could be prevented by ZVAD-fmk. The present data highlight the pivotal role of autophagy in H(2)O(2)-induced cell death in dopaminergic neuroblastoma cells

Resveratrol induces cell death in colorectal cancer cells by a novel pathway involving lysosomal cathepsin D

In human colorectal cancer cells, the polyphenol resveratrol (RV) activated the caspase-dependent intrinsic pathway of apoptosis. This effect was not mediated via estrogen receptors. Pepstatin A, an inhibitor of lysosomal cathepsin D (CD), not (2S,3S)-trans-epoxysuccinyl-L-leucylamido-3-methylbutane ethyl ester, an inhibitor of cathepsins B and L, prevented RV cytotoxicity. Similar protection was attained by small interference RNA-mediated knockdown of CD protein expression. RV promoted the accumulation of mature CD, induced lysosome leakage and increased cytosolic immunoreactivity of CD. Inhibition of CD or its post-transcriptional down-regulation precluded Bax oligomerization, permeabilization of mitochondrial membrane, cytosolic translocation of cytochrome c, caspase 3 activation and terminal deoxinucleotidyl transferase-mediated dUTP-biotin nick end labeling positivity occurring in RV-treated cells. The present study identifies the lysosome as a novel target of RV activity and demonstrates a hierarchy of the proteolytic pathways involved in its cytotoxic mechanism in which the lysosomal CD acts upstream of the cytosolic caspase activation. Our data indicate that metabolic, pharmacologic or genetic conditions affecting CD expression and/or activity could reflect on the sensitivity of cancer cells to RV

Folding, activity and targeting of mutated human cathepsin D that cannot be processed into the double-chain form

The precursor of human cathepsin D (CD) is converted into the single-chain and the double-chain active polypeptides by subsequent proteolysis reactions taking place in the endosomal-lysosomal compartment and involving specific aminoacid sequences. We have mutagenized the region of aminoacids (comprising the beta-hairpin loop) involved in the latter proteolytic maturation step and generated a mutant CD that cannot be converted into the mature double-chain form. This mutant CD expressed in rodent cells reaches the lysosome and is stable as single-chain polypeptide, bears high-mannose type sugars, binds to pepstatin A and is enzymatically active, indicating that it is correctly folded. The present work provides new insights on the aminoacid region involved in the terminal processing of human CD and on the function of the processing beta-hairpin loop