1,720,977 research outputs found
Autophagy: a self-eating process in cell physiology and pathology [Autofagia: un processo fisiopatologico di autodigestione cellulare]
No full textMacroautophagy, commonly referred to as autophagy, is a self-degradation process through which virtually all eukaryotic cells sequester cytoplasmic components (macromolecules, but also entire organelles or microorganisms) into de novo formed, double-membrane vescicles (autophagosomes) and degrade them after lysosomal fusion. The degradation products released from lysosomes are recycled into metabolic and biosynthetic pathways. Autophagy, normally occurring for ordinary organelle and protein turnover, can be enhanced in stress conditions as an adaptive, survival response to microenvironmental and intracellular noxia, including glucose, amino acid or growth factors withdrawal, hypoxia, oxidative stress, mitochondrial or organelle dysfunction, infections, and cytotoxic drugs. However, an excessive or long-lasting activation of autophagy can culminate in a peculiar mode of cell death. An imbalanced autophagy (either unrestrained or deficient) plays a role in the pathogenesis of different diseases, including cancer, neurodegenerative diseases, cardiovascular and autoimmune pathologies. In tumor cells, however, autophagy proves to play an ambiguous dual role (protective or cytotoxic), possibly depending on tumor cell type, genetic background and tumor stage
Hydrogel containing L-valine residues as a platform for cisplatin chemotherapy
No full textTwo acrylic hydrogels, of low cross-linking content and carrying the l-valine residues, were synthesized
and studied as a platform to load and release the chemotherapeutic agent cisplatin. The platinum(II)-
complex species showed a well-defined stoichiometric ratio in which two carboxylate groups of the
collapsing gel coordinate a metal center; this was confirmed by FT-IR spectra. When loaded in water,
a zero-order release rate of platinum(II)-species was shown in the physiologic solution (PBS, pH 7.40)
for more than one week. Moreover, the amount of platinum(II)-species released from the hydrogel may
be improved either by the cross-linking degree and by the temperature. Any increase of the cross-links
results in a decreased slope of the straightline Pt(II)/gel (mg/g) versus time, whereas the increasing
temperature results in a greater amount of platinum(II)-species in solution. The chemical- and swellingcontrolled
release are the main mechanisms supervising the whole release process. On the other hand,
the loading of cisplatin and temsirolimus in DMF showed a characteristic two phase releasing pattern;
the initial burst effect was always followed by the zero-order release rate for a week. In this case only a
swelling-controlled mechanism was mainly invoked. The cytotoxic activity towards Me665/2/21 human
melanoma cell line, afforded by the cisplatin-loaded hydrogel, was close and in some cases higher compared
to the native cisplatin at the same concentration; an interesting synergy in term of cytotoxicity was
observed when a combined treatment of temsirolimus and cisplatin was used, although temsirolimus
exerted only a moderate inhibition of cell proliferation
Regulation of autophagy in the uterus: from physiological processes to endometriosis and uterine fibroids
No full textEndometriosis and uterine fibroids are 2 common gynecological disorders. Their pathophysiology remains unclear, although several
different risk factors have been identified, including genetic predisposition, hormones, and proinflammatory cytokines. The present review
outlines the experimental evidence showing the role of autophagy in the physiology of the endometrium and myometrium and
emerging evidence that autophagy may contribute to the onset of endometriosis and uterine fibroids. The full definition of the role of autophagy is likely to lead to effective new pharmacologic treatments for these disorders
Hypoxia response in glioblastoma cells: effect of trehalose on macropinocytosis, autophagy and cell survival
Full text linkIn glioblastoma multiforme, the most malignant brain tumor in adults, the hypoxic milieu is believed to contribute to tumor aggressiveness and resistance to therapy. Here, human glioblastoma U373-MG and T98G cells were exposed to hypoxia (1 % oxygen) or normoxia (18 % oxygen), and treated with trehalose, a natural disaccharide increasingly studied for its therapeutic potential in cancer. In all samples under hypoxia, HIF-1 alpha stabilization was accompanied by a decrease in Nrf2 and p62/SQSTM1 proteins; redox imbalance also occurred, as documented by increased levels of ROS and parallel lowering of glutathione. Trehalose treatment increased Nrf2 and p62 proteins under normoxia, an effect lost or downsized under hypoxia. Differently, under both oxygen concentrations, trehalose increased glutathione content, consistently with its antioxidant role. In U373-MG cells, trehalose induced remarkable macropinocytosis under hypoxia, albeit less than under normoxia; on the contrary, in autophagy-proficient T98G cells, trehalose further increased the autophagic process under hypoxia compared to normoxia. As regards long-term cell fate (evaluated as colony-forming capacity), hypoxia only proved to be a favorable condition for T98G cells. However, in both cell lines, trehalose treatment significantly and dose-dependently decreased clonogenic capacity under normoxia and hypoxia; in particular, the long-lasting stimulation of macropinocytosis in U373-MG cells induced extensive cell death by methuosis. Overall, our findings suggest that trehalose-induced macropinocytosis or autophagy could also play a tumour-suppressive role in the hypoxic tumor milieu that characterizes glioblastoma, making its synergy with conventional chemotherapy and radiotherapy worth exploring
Antiproliferative Effect of Rottlerin on Sk-Mel-28 Melanoma Cells
Full text linkMelanoma is the most aggressive and chemoresistant form of skin cancer. Mutated, constitutively active B-RAF is believed to play a crucial role, although the selective B-RAF inhibition has shown poor clinical success, since phenomena of resistance usually occur, likely arising from additional genetic aberrations, such as loss of function of p53 and PTEN, overexpression of cyclin D1, hyperactivation of NF-κB, and downregulation of p21/Cip1. Since all of them are present in the Sk-Mel-28 melanoma cells, this cell line could be an ideal, albeit hard to study, model to develop new therapeutic strategies. In the current study, we tested the cytostatic action of Rottlerin on Sk-Mel-28 melanoma cells, on the basis of the known Rottlerin effects on the main proliferative signaling pathways. We presented evidence that the drug inhibits cell growth by an Akt- and p21/Cip1-independent mechanism, involving the dual inhibition of ERK and NF-κB and downregulation of cyclin D1. In addition, we found that Rottlerin increases ERK phosphorylation, but, surprisingly, this resulted in decreased ERK activity. Pull-down experiments, using Rottlerin-CNBr-conjugated Sepharose beads, revealed that Rottlerin binds to ERK, independently from its phosphorylation status. This direct interaction could in part explain the paradoxical blockage of ERK downstream signaling and growth arrest. We would like to dedicate this paper to the memory of our friend and colleague, prematurely deceased, Claudia Torricelli, who actively contributed to this project
Lipid peroxidation and antioxidant systems in the liver injury produced by glutathione depleting agents
No full textThe mechanisms of the liver damage produced by three glutathione (GSH) depleting agents, bromobenzene, allyl alcohol and diethylmaleate, was investigated. The change in the antioxidant systems represented by α-tocopherol (vitamin E) and ascorbic acid were studied under conditions of severe GSH depletion. With each toxin liver necrosis was accompanied by lipid peroxidation that developed only after severe depletion of GSH. The hepatic level of vitamin E was decreased whenever extensive lipid peroxidation developed. In the case of bromobenzene intoxication, vitamin E decreased before the onset of lipid peroxidation. Changes in levels of the ascorbic and dehydroascorbic acid indicated a redox cycling of vitamin C with the oxidative stress induced by all the three agents. Such a change of the redox state of vitamin C (increase of the oxidized over the reduced form) may be an index of oxidative stress preceding lipid peroxidation in the case of bromobenzene. In the other cases, such a change is likely to be a consequence of lipid peroxidation. Experiments carried out with vitamin E deficient or supplemented diets indicated that the pathological phenomena occurring as a consequence of GSH depletion depend on hepatic levels of vitamin E. In vitamin E deficient animals, lipid peroxidation and liver necrosis appeared earlier than in animals fed the control diet. Animals fed a vitamin E supplemented diet had an hepatic vitamin E level double that obtained with a commercial pellet diet. In such animals, bromobenzene and allyl alcohol had only limited toxicity and diethylmaleate none in spite of comparable hepatic GSH depletion. Thus, vitamin E may largely modulate the expression of the toxicity by GSH depleting agents. © 1990
Cross-talk between calpain and caspase-3/-7 in cisplatin-induced apoptosis of melanoma cells: a major role of calpain inhibition in cell death protection and p53 status
No full textThe contribution of different proteolytic systems, in particular calpains and effector caspases, in apoptotic cell death is still controversial. In this paper, we show that during cisplatin-induced apoptosis of human metastatic melanoma cells, calpain activation, as measured in intact cells by two different fluorescent substrates, is an early event, taking place well before caspase-3/-7 activation, and progressively increasing during 48 h of treatment. Such activation appears to be independent from any intracellular calcium imbalance; in fact, an increase of cytosolic calcium along with emptying of the reticular stores occur only at very late stages, uniquely in frankly apoptotic, detached cells. Calpain activation proves to be an early and crucial event in the apoptotic machinery, as demonstrated by the significant protection of cell death in samples co-treated with the calpain inhibitors, MDL 28170, calpeptin and PD 150606, where a variable but significant reduction of both caspase-3/-7 activity and cell detachment is observed. Consistently, such a protective effect can be at least partially due to the impairment of cisplatin-induced p53 activation, occurring early in committed, preapoptotic cells. Furthermore, in late apoptotic cells, calpain activity is also responsible for the formation of a novel p53 proteolytic fragment (approximate to 26 kDa), whose function is so far to be elucidated
Cleavage of Bcl-2 in oxidant- and cisplatin-induced apoptosis of human melanoma cells
No full textAlthough the anti-apoptotic effect of Bcl-2 is well established, the role of Bcl-2 in tumour response to therapy and drug resistance is still unclear. The posttranslational modifications of Bcl-2 are likely involved in the control of the apoptotic pathway. In the present study we have investigated the role of Bcl-2 in cellular response to oxidative stress (hydrogen peroxide) and cisplatin using a clone of human metastatic melanoma, which, in spite of Bcl-2 (over)expression, exhibited a moderate chemosensitivity. With both treatments melanoma cells died through an apoptotic process, associated with detachment of cells from the monolayer. In the floating apoptotic cells generated by either hydrogen peroxide or cisplatin, along with morphological and biochemical features of apoptosis, we detected a significant Bcl-2 cleavage, yielding the Bax-like fragment of 23 kDa. Preincubation of cells with the caspase-3/-7 inhibitor DEVD-CHO completely suppressed Bcl-2 cleavage, thus confirming that such a specific proteolysis requires activation of caspase-3/-7. The oxidant- and cisplatin-induced processing of Bcl-2 documented in the present study may represent a regulatory mechanism to circumvent the survival function of Bcl-2 upon apoptosis triggering and to enhance apoptotic response. Since the Bcl-2 cleavage should be regarded as a pro-apoptotic event, Bcl-2 expression is expected to increase susceptibility to apoptosis. Thus, such a pathway could be exploited to improve the efficacy of cytotoxic therapy of melanomas expressing Bcl-2
Lipid peroxidation, protein thiols and calcium homeostasis in bromobenzene-induced liver damage
No full textThe mechanisms of bromobenzene hepatotoxicity in vivo were studied in mice. The relationships among glutathione (GSH) depletion, lipid peroxidation, loss of protein thiols, disturbed calcium homeostasis and liver necrosis were investigated. Liver necrosis (as estimated by the serum glutamate-pyruvate transaminase (SGPT) level) appeared between 9 and 12 hr and increased at 18 hr. Lipid peroxidation which was already detectable at 6 hr in some animals, increased thereafter showing a good correlation with the severity of liver necrosis. Despite a quite fast depletion of hepatic GSH, a significant decrease in protein thiols could be observed at 12-18 hr only. Loss of protein thiols in both whole liver and subcellular fractions (microsomes and mitochondria) was correlated with lipid peroxidation. Also a good inverse correlation was seen between lipid peroxidation and the calcium sequestration activity of liver microsomes and mitochondria. The treatment of mice with desferrioxamine (DFO) after bromobenzene-intoxication completely prevented lipid peroxidation, loss of protein thiols and liver necrosis in the animals sacrificed 15 hr after poisoning. When, however, the animals were examined at 24 hr, although the general correlation between lipid peroxidation and liver necrosis was held, in some animals (about 30% of the survivors) elevation of SGPT was observed in the virtual absence of lipid peroxidation. It seems likely therefore that the liver damage seen during the first phase of bromobenzene-intoxication is strictly related to lipid peroxidation. It is, however, possible that in some animals in which for some reason lipid peroxidation does not develop, another mechanism of liver necrosis unrelated to lipid peroxidation occurs at later times. © 1987
The autophagy inducer trehalose stimulates macropinocytosis in NF1-deficient glioblastoma cells
Full text linkBACKGROUND: Glioblastoma is a highly aggressive brain tumor. A big effort is required to find novel molecules which can cross the blood–brain barrier and efficiently kill these tumor cells. In this perspective, trehalose (α-glucopyranosyl‐[1→1]‐α‐d‐glucopyranoside), found in various dietary sources and used as a safe nutrient supplement, attracted our attention for its pleiotropic effects against tumor cells. METHODS: Human glioblastoma cell lines U373-MG and T98G were exposed to trehalose and analyzed at different time points. Cell proliferation was evaluated at medium term, and clonogenic capacity and cell morphology were evaluated at long term. Western blot was used to evaluate biochemical markers of autophagy (also measured in cells co-treated with EIPA or chloroquine), and mTOR, AMPK and ERK 1/2 signalling. Macropinocytosis was evaluated morphologically by bright-field microscopy; in cells loaded with the fluorescein-conjugated fluid-phase tracer dextran, macropinocytic vacuoles were also visualized by fluorescence microscopy, and the extent of macropinocytosis was quantified by flow cytometry. RESULTS: The long-term effect of trehalose on U373-MG and T98G cell lines was impressive, as indicated by a dramatic reduction in clonogenic efficiency. Mechanistically, trehalose proved to be an efficient autophagy inducer in macropinocytosis-deficient T98G cells and an efficient inducer of macropinocytosis and eventual cell death by methuosis in U373-MG glioblastoma cells, proved to be poorly responsive to induction of autophagy. These two processes appeared to act in a mutually exclusive manner; indeed, co-treatment of U373-MG cells with the macropinocytosis inhibitor, EIPA, significantly increased the autophagic response. mTOR activation and AMPK inhibition occurred in a similar way in the two trehalose-treated cell lines. Interestingly, ERK 1/2 was activated only in macropinocytosis-proficient U373-MG cells harbouring loss-of-function mutations in the negative RAS regulator, NF1, suggesting a key role of RAS signalling. CONCLUSIONS: Our results indicate that trehalose is worthy of further study as a candidate molecule for glioblastoma therapy, due to its capacity to induce a sustained autophagic response, ultimately leading to loss of clonogenic potential, and more interestingly, to force macropinocytosis, eventually leading to cell death by methuosis, particularly in tumor cells with RAS hyperactivity. As a further anticancer strategy, stimulation of macropinocytosis may be exploited to increase intracellular delivery of anticancer drugs
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