Evaluation of the anticancer effects exerted by 5-fluorouracil and heme oxygenase-1 inhibitor hybrids in HTC116 colorectal cancer cells.

Colon cancer remains a clinical challenge in industrialised countries. Its treatment with 5-Flurouracil (5-FU) develops many side effects and resistance. Thus, several strategies have been undertaken so far, including the use of drug cocktails and polypharmacology. Heme oxygenase-1 (HO-1) is an emerging molecular target in the treatment of various cancers. We recently demonstrated that a combination of HO-1 inhibitors with 5-FU and the corresponding hybrids SI1/17, SI1/20, and SI1/22, possessed anticancer activity against prostate and lung cancer cells. In this work, we evaluated these hybrids in a model of colon cancer and found that SI1/22 and the respective combo have greater potency than 5-FU. Particularly, compounds inhibit HO-1 activity in cell lysates, increase ROS and the expression of HO-1, SOD, and Nrf2. Moreover, we observed a decrease of pro-caspase and an increase in cleaved PARP-1 and p62, suggesting apoptotic and autophagic cell death and potential application of these drugs as anticancer agents.

Cytotoxic Effect Induced by Sicilian Oregano Essential Oil in Human Breast Cancer Cells.

Origanum vulgare L. is an aromatic plant that exerts antibacterial, antioxidant, anti-inflammatory, and antitumor activities, mainly due to its essential oil (EO) content. In this study, we investigated the possible mechanism underlying the in vitro antitumor activity of EO extracted by hydrodistillation of dried flowers and leaves of Origanum vulgare L. grown in Sicily (Italy) in MDA-MB-231 and MCF-7 breast cancer cell lines. Gas chromatography-mass spectrometry analysis of Oregano essential oil (OEO) composition highlighted the presence of twenty-six major phytocompounds, such as p-cymene, γ-terpinene, and thymoquinone p-acetanisole. OEO possesses strong antioxidant capacity, as demonstrated by the DPPH test. Our studies provided evidence that OEO reduces the viability of both MCF-7 and MDA-MB-231 cells. The cytotoxic effect of OEO on breast cancer cells was partially counteracted by the addition of z-VAD-fmk, a general caspase inhibitor. Caspases and mitochondrial dysfunction appeared to be involved in the OEO-induced death mechanism. Western blotting analysis showed that OEO-induced activation of pro-caspases-9 and -3 and fragmentation of PARP decreased the levels of Bcl-2 and Bcl-xL while increasing those of Bax and VDAC. In addition, fluorescence microscopy and cytofluorimetric analysis showed that OEO induces a loss of mitochondrial membrane potential in both cell lines. Furthermore, we tested the effects of p-cymene, γ-terpinene, thymoquinone, and p-acetanisole, which are the main components of OEO. Our findings highlighted that the effect of OEO on MDA-MB-231 and MCF-7 cells appears to be mainly due to the combination of different constituents of OEO, providing evidence of the potential use of OEO for breast cancer treatment.

Foodomics-Based Approaches Shed Light on the Potential Protective Effects of Polyphenols in Inflammatory Bowel Disease.

Inflammatory bowel disease (IBD) is a chronic and progressive inflammatory disorder affecting the gastrointestinal tract (GT) caused by a wide range of genetic, microbial, and environmental factors. IBD is characterized by chronic inflammation and decreased gut microbial diversity, dysbiosis, with a lower number of beneficial bacteria and a concomitant increase in pathogenic species. It is well known that dysbiosis is closely related to the induction of inflammation and oxidative stress, the latter caused by an imbalance between reactive oxygen species (ROS) production and cellular antioxidant capacity, leading to cellular ROS accumulation. ROS are responsible for intestinal epithelium oxidative damage and the increased intestinal permeability found in IBD patients, and their reduction could represent a potential therapeutic strategy to limit IBD progression and alleviate its symptoms. Recent evidence has highlighted that dietary polyphenols, the natural antioxidants, can maintain redox equilibrium in the GT, preventing gut dysbiosis, intestinal epithelium damage, and radical inflammatory responses. Here, we suggest that the relatively new foodomics approaches, together with new technologies for promoting the antioxidative properties of dietary polyphenols, including novel delivery systems, chemical modifications, and combination strategies, may provide critical insights to determine the clinical value of polyphenols for IBD therapy and a comprehensive perspective for implementing natural antioxidants as potential IBD candidate treatment.

MCL1 Inhibition Overcomes the Aggressiveness Features of Triple-Negative Breast Cancer MDA-MB-231 Cells.

Triple-Negative Breast Cancer (TNBC) is a particularly aggressive subtype among breast cancers (BCs), characterized by anoikis resistance, high invasiveness, and metastatic potential as well as Epithelial-Mesenchymal Transition (EMT) and stemness features. In the last few years, our research focused on the function of MCL1, an antiapoptotic protein frequently deregulated in TNBC. Here, we demonstrate that MCL1 inhibition by A-1210477, a specific BH3-mimetic, promotes anoikis/apoptosis in the MDA-MB-231 cell line, as shown via an increase in proapoptotic markers and caspase activation. Our evidence also shows A-1210477 effects on Focal Adhesions (FAs) impairing the integrin trim and survival signaling pathways, such as FAK, AKT, ERK, NF-κB, and GSK3ß-inducing anoikis, thus suggesting a putative role of MCL1 in regulation of FA dynamics. Interestingly, in accordance with these results, we observed a reduction in migratory and invasiveness capabilities as confirmed by a decrease in metalloproteinases (MMPs) levels following A-1210477 treatment. Moreover, MCL1 inhibition promotes a reduction in EMT characteristics as demonstrated by the downregulation of Vimentin, MUC1, DNMT1, and a surprising re-expression of E-Cadherin, suggesting a possible mesenchymal-like phenotype reversion. In addition, we also observed the downregulation of stemness makers such as OCT3/4, SOX2, NANOG, as well as CD133, EpCAM, and CD49f. Our findings support the idea that MCL1 inhibition in MDA-MB-231 could be crucial to reduce anoikis resistance, aggressiveness, and metastatic potential and to minimize EMT and stemness features that distinguish TNBC.

Oncogenic BRAF and p53 Interplay in Melanoma Cells and the Effects of the HDAC Inhibitor ITF2357 (Givinostat).

Oncogenic BRAF mutations have been widely described in melanomas and promote tumour progression and chemoresistance. We previously provided evidence that the HDAC inhibitor ITF2357 (Givinostat) targets oncogenic BRAF in SK-MEL-28 and A375 melanoma cells. Here, we show that oncogenic BRAF localises to the nucleus of these cells, and the compound decreases BRAF levels in both the nuclear and cytosolic compartments. Although mutations in the tumour suppressor p53 gene are not equally frequent in melanomas compared to BRAF, the functional impairment of the p53 pathway may also contribute to melanoma development and aggressiveness. To understand whether oncogenic BRAF and p53 may cooperate, a possible interplay was considered in the two cell lines displaying a different p53 status, being p53 mutated into an oncogenic form in SK-MEL-28 and wild-type in A375 cells. Immunoprecipitation revealed that BRAF seems to preferentially interact with oncogenic p53. Interestingly, ITF2357 not only reduced BRAF levels but also oncogenic p53 levels in SK-MEL-28 cells. ITF2357 also targeted BRAF in A375 cells but not wild-type p53, which increased, most likely favouring apoptosis. Silencing experiments confirmed that the response to ITF2357 in BRAF-mutated cells depends on p53 status, thus providing a rationale for melanoma-targeted therapy.

A Deadly Liaison between Oxidative Injury and p53 Drives Methyl-Gallate-Induced Autophagy and Apoptosis in HCT116 Colon Cancer Cells.

Methyl gallate (MG), which is a gallotannin widely found in plants, is a polyphenol used in traditional Chinese phytotherapy to alleviate several cancer symptoms. Our studies provided evidence that MG is capable of reducing the viability of HCT116 colon cancer cells, while it was found to be ineffective on differentiated Caco-2 cells, which is a model of polarized colon cells. In the first phase of treatment, MG promoted both early ROS generation and endoplasmic reticulum (ER) stress, sustained by elevated PERK, Grp78 and CHOP expression levels, as well as an upregulation in intracellular calcium content. Such events were accompanied by an autophagic process (16-24 h), where prolonging the time (48 h) of MG exposure led to cellular homeostasis collapse and apoptotic cell death with DNA fragmentation and p53 and γH2Ax activation. Our data demonstrated that a crucial role in the MG-induced mechanism is played by p53. Its level, which increased precociously (4 h) in MG-treated cells, was tightly intertwined with oxidative injury. Indeed, the addition of N-acetylcysteine (NAC), which is a ROS scavenger, counteracted the p53 increase, as well as the MG effect on cell viability. Moreover, MG promoted p53 accumulation into the nucleus and its inhibition by pifithrin-α (PFT-α), which is a negative modulator of p53 transcriptional activity, enhanced autophagy, increased the LC3-II level and inhibited apoptotic cell death. These findings provide new clues to the potential action of MG as a possible anti-tumor phytomolecule for colon cancer treatment.

Hypertrophy and ER Stress Induced by Palmitate Are Counteracted by Mango Peel and Seed Extracts in 3T3-L1 Adipocytes.

A diet rich in saturated fatty acids (FAs) has been correlated with metabolic dysfunction and ROS increase in the adipose tissue of obese subjects. Thus, reducing hypertrophy and oxidative stress in adipose tissue can represent a strategy to counteract obesity and obesity-related diseases. In this context, the present study showed how the peel and seed extracts of mango (Mangifera indica L.) reduced lipotoxicity induced by high doses of sodium palmitate (PA) in differentiated 3T3-L1 adipocytes. Mango peel (MPE) and mango seed (MSE) extracts significantly lowered PA-induced fat accumulation by reducing lipid droplet (LDs) and triacylglycerol (TAGs) content in adipocytes. We showed that MPE and MSE activated hormone-sensitive lipase, the key enzyme of TAG degradation. In addition, mango extracts down-regulated the adipogenic transcription factor PPARγ as well as activated AMPK with the consequent inhibition of acetyl-CoA-carboxylase (ACC). Notably, PA increased endoplasmic reticulum (ER) stress markers GRP78, PERK and CHOP, as well as enhanced the reactive oxygen species (ROS) content in adipocytes. These effects were accompanied by a reduction in cell viability and the induction of apoptosis. Interestingly, MPE and MSE counteracted PA-induced lipotoxicity by reducing ER stress markers and ROS production. In addition, MPE and MSE increased the level of the anti-oxidant transcription factor Nrf2 and its targets MnSOD and HO-1. Collectively, these results suggest that the intake of mango extract-enriched foods in association with a correct lifestyle could exert beneficial effects to counteract obesity.

The Histone Deacetylase Inhibitor ITF2357 (Givinostat) Targets Oncogenic BRAF in Melanoma Cells and Promotes a Switch from Pro-Survival Autophagy to Apoptosis.

Histone deacetylase inhibitors (HDACI) are epigenetic compounds that have been widely considered very promising antitumor agents. Here, we focus on the effects of the pan-HDAC inhibitor ITF2357 (Givinostat) in comparison with SAHA (Vorinostat) in melanoma cells bearing BRAF V600E oncogenic mutation. Our results indicate both ITF2357 and SAHA dose-dependently reduce the viability of BRAF-mutated SK-MEL-28 and A375 melanoma cells. The comparison of IC50 values revealed that ITF2357 was much more effective than SAHA. Interestingly, both inhibitors markedly decreased oncogenic BRAF protein expression levels, ITF2357 being the most effective compound. Moreover, the BRAF decrease induced by ITF2357 was accompanied by a decrease in the level of phospho-ERK1/2. The inhibitor of upstream MEK activity, U0126, reduced ERK1/2 phosphorylation and dramatically potentiated the antitumor effect of ITF2357, exacerbating the reduction in the BRAF level. ITF2357 stimulated an early pro-survival autophagic response, which was followed by apoptosis, as indicated by apoptotic markers evaluation and the protective effects exerted by the pan-caspase inhibitor z-VADfmk. Overall, our data indicate for the first time that ITF2357 targets oncogenic BRAF in melanoma cells and induces a switch from autophagy to classic apoptosis, thus representing a possible candidate in melanoma targeted therapy.

Production of CSSi013-A (9360) iPSC line from an asymptomatic subject carrying an heterozygous mutation in TDP-43 protein.

Amyotrophic Lateral Sclerosis (ALS) is a fatal disease affecting both upper and lower motoneurons. The transactive response DNA binding protein (TARDBP) gene, encoding for TDP-43, is one of the most commonly mutated gene associated with familial cases of ALS (10%). We generated a human induced pluripotent stem cell (hiPSC) line from the fibroblasts of an asymptomatic subject carrying the TARDBP p.G376D mutation. This mutation is very rare and was described in a large Apulian family, in which all ALS affected members are carriers of the mutation. The subject here described is the first identified asymptomatic carrier of the mutation.

ALS monocyte-derived microglia-like cells reveal cytoplasmic TDP-43 accumulation, DNA damage, and cell-specific impairment of phagocytosis associated with disease progression.

BACKGROUND: Amyotrophic lateral sclerosis (ALS) is a multifactorial neurodegenerative disease characterised by the loss of upper and lower motor neurons. Increasing evidence indicates that neuroinflammation mediated by microglia contributes to ALS pathogenesis. This microglial activation is evident in post-mortem brain tissues and neuroimaging data from patients with ALS. However, the role of microglia in the pathogenesis and progression of amyotrophic lateral sclerosis remains unclear, partly due to the lack of a model system that is able to faithfully recapitulate the clinical pathology of ALS. To address this shortcoming, we describe an approach that generates monocyte-derived microglia-like cells that are capable of expressing molecular markers, and functional characteristics similar to in vivo human brain microglia. METHODS: In this study, we have established monocyte-derived microglia-like cells from 30 sporadic patients with ALS, including 15 patients with slow disease progression, 6 with intermediate progression, and 9 with rapid progression, together with 20 non-affected healthy controls. RESULTS: We demonstrate that patient monocyte-derived microglia-like cells recapitulate canonical pathological features of ALS including non-phosphorylated and phosphorylated-TDP-43-positive inclusions. Moreover, ALS microglia-like cells showed significantly impaired phagocytosis, altered cytokine profiles, and abnormal morphologies consistent with a neuroinflammatory phenotype. Interestingly, all ALS microglia-like cells showed abnormal phagocytosis consistent with the progression of the disease. In-depth analysis of ALS microglia-like cells from the rapid disease progression cohort revealed significantly altered cell-specific variation in phagocytic function. In addition, DNA damage and NOD-leucine rich repeat and pyrin containing protein 3 (NLRP3) inflammasome activity were also elevated in ALS patient monocyte-derived microglia-like cells, indicating a potential new pathway involved in driving disease progression. CONCLUSIONS: Taken together, our work demonstrates that the monocyte-derived microglia-like cell model recapitulates disease-specific hallmarks and characteristics that substantiate patient heterogeneity associated with disease subgroups. Thus, monocyte-derived microglia-like cells are highly applicable to monitor disease progression and can be applied as a functional readout in clinical trials for anti-neuroinflammatory agents, providing a basis for personalised treatment for patients with ALS.

Parthenolide and Its Soluble Analogues: Multitasking Compounds with Antitumor Properties.

Due to its chemical properties and multiple molecular effects on different tumor cell types, the sesquiterpene lactone parthenolide (PN) can be considered an effective drug with significant potential in cancer therapy. PN has been shown to induce either classic apoptosis or alternative caspase-independent forms of cell death in many tumor models. The therapeutical potential of PN has been increased by chemical design and synthesis of more soluble analogues including dimethylaminoparthenolide (DMAPT). This review focuses on the molecular mechanisms of both PN and analogues action in tumor models, highlighting their effects on gene expression, signal transduction and execution of different types of cell death. Recent findings indicate that these compounds not only inhibit prosurvival transcriptional factors such as NF-κB and STATs but can also determine the activation of specific death pathways, increasing intracellular reactive oxygen species (ROS) production and modifications of Bcl-2 family members. An intriguing property of these compounds is its specific targeting of cancer stem cells. The unusual actions of PN and its analogues make these agents good candidates for molecular targeted cancer therapy.

Prognostic Role of CSF ß-amyloid 1-42/1-40 Ratio in Patients Affected by Amyotrophic Lateral Sclerosis.

The involvement of ß-amyloid (Aß) in the pathogenesis of amyotrophic lateral sclerosis (ALS) has been widely discussed and its role in the disease is still a matter of debate. Aß accumulates in the cortex and the anterior horn neurons of ALS patients and seems to affect their survival. To clarify the role of cerebrospinal fluid (CSF) Aß 1-42 and Aß 42/40 ratios as a potential prognostic biomarker for ALS, we performed a retrospective observational study on a cohort of ALS patients who underwent a lumbar puncture at the time of the diagnosis. CSF Aß 1-40 and Aß 1-42 ratios were detected by chemiluminescence immunoassay and their values were correlated with clinical features. We found a significant correlation of the Aß 42/40 ratio with age at onset and Mini Mental State Examination (MMSE) scores. No significant correlation of Aß 1-42 or Aß 42/40 ratios to the rate of progression of the disease were found. Furthermore, when we stratified patients according to Aß 1-42 concentration and the Aß 42/40 ratio, we found that patients with a lower Aß 42/40 ratio showed a shorter survival. Our results support the hypothesis that Aß 1-42 could be involved in some pathogenic mechanism of ALS and we suggest the Aß 42/40 ratio as a potential prognostic biomarker.

A Deletion of the Nuclear Localization Signal Domain in the Fus Protein Induces Stable Post-stress Cytoplasmic Inclusions in SH-SY5Y Cells.

Mutations in Fused-in-Sarcoma (FUS) gene involving the nuclear localization signal (NLS) domain lead to juvenile-onset Amyotrophic Lateral Sclerosis (ALS). The mutant protein mislocalizes to the cytoplasm, incorporating it into Stress Granules (SG). Whether SGs are the first step to the formation of stable FUS-containing aggregates is still unclear. In this work, we used acute and chronic stress paradigms to study the SG dynamics in a human SH-SY5Y neuroblastoma cell line carrying a deletion of the NLS domain of the FUS protein (homozygous: ΔNLS-/-; heterozygous: ΔNLS+/-). Wild-type (WT) cells served as controls. We evaluated the subcellular localization of the mutant protein through immunoblot and immunofluorescence, in basal conditions and after acute stress and chronic stress with sodium arsenite (NaAsO2). Cells were monitored for up to 24 h after rescue. FUS was expressed in both nucleus and cytoplasm in the ΔNLS+/- cells, whereas it was primarily cytoplasmic in the ΔNLS-/-. Acute NaAsO2 exposure induced SGs: at rescue,>90% of ΔNLS cells showed abundant FUS-containing if compared to less than 5% of the WT cells. The proportion of FUS-positive SGs remained 15-20% at 24 h in mutant cells. Cycloheximide did not abolish the long-lasting SGs in mutant cells. Chronic exposure to NaAsO2 did not induce significant SGs formation. A wealth of research has demonstrated that ALS-associated FUS mutations at the C-terminus facilitate the incorporation of the mutant protein into SGs. We have shown here that mutant FUS-containing SGs tend to fail to dissolve after stress, facilitating a liquid-to-solid phase transition. The FUS-containing inclusions seen in the dying motor neurons might therefore directly derive from SGs. This might represent an attractive target for future innovative therapies.

WIN55,212-2-Induced Expression of Mir-29b1 Favours the Suppression of Osteosarcoma Cell Migration in a SPARC-Independent Manner.

WIN55,212-2 (WIN) is a synthetic agonist of cannabinoid receptors that displays promising antitumour properties. The aim of this study is to demonstrate that WIN is able to block the migratory ability of osteosarcoma cells and characterize the mechanisms involved. Using wound healing assay and zymography, we showed that WIN affects cell migration and reduces the activity of the metalloproteases MMP2 and MMP9. This effect seemed to be independent of secreted protein acidic and rich in cysteine (SPARC), a matricellular protein involved in tissue remodeling and extracellular matrix deposition. SPARC release was indeed prevented by WIN, and SPARC silencing by RNA interference did not influence the effect of the cannabinoid on cell migration. WIN also increased the release of extracellular vesicles and dramatically upregulated miR-29b1, a key miRNA that modulates cell proliferation and migration. Interestingly, reduced cell migration was observed in stably miR-29b1-transfected cells, similarly to WIN-treated cells. Finally, we show the absence of SPARC in the extracellular vesicles released by osteosarcoma cells and no changes in SPARC level in miR-29b1 overexpressing cells. Overall, these findings suggest that WIN markedly affects cell migration, dependently on miR-29b1 and independently of SPARC, and can thus be considered as a potential innovative therapeutic agent in the treatment of osteosarcoma.

ALS-linked FUS mutations confer loss and gain of function in the nucleus by promoting excessive formation of dysfunctional paraspeckles.

Mutations in the FUS gene cause amyotrophic lateral sclerosis (ALS-FUS). Mutant FUS is known to confer cytoplasmic gain of function but its effects in the nucleus are less understood. FUS is an essential component of paraspeckles, subnuclear bodies assembled on a lncRNA NEAT1. Paraspeckles may play a protective role specifically in degenerating spinal motor neurons. However it is still unknown how endogenous levels of mutant FUS would affect NEAT1/paraspeckles. Using novel cell lines with the FUS gene modified by CRISPR/Cas9 and human patient fibroblasts, we found that endogenous levels of mutant FUS cause accumulation of NEAT1 isoforms and paraspeckles. However, despite only mild cytoplasmic mislocalisation of FUS, paraspeckle integrity is compromised in these cells, as confirmed by reduced interaction of mutant FUS with core paraspeckle proteins NONO and SFPQ and increased NEAT1 extractability. This results in NEAT1 localisation outside paraspeckles, especially prominent under conditions of paraspeckle-inducing stress. Consistently, paraspeckle-dependent microRNA production, a readout for functionality of paraspeckles, is impaired in cells expressing mutant FUS. In line with the cellular data, we observed paraspeckle hyper-assembly in spinal neurons of ALS-FUS patients. Therefore, despite largely preserving its nuclear localisation, mutant FUS leads to loss (dysfunctional paraspeckles) and gain (excess of free NEAT1) of function in the nucleus. Perturbed fine structure and functionality of paraspeckles accompanied by accumulation of non-paraspeckle NEAT1 may contribute to the disease severity in ALS-FUS.

Sicilian Litchi Fruit Extracts Induce Autophagy versus Apoptosis Switch in Human Colon Cancer Cells.

Litchi chinensis Sonnerat is a tropical tree whose fruits contain significant amounts of bioactive polyphenols. Litchi cultivation has recently spread in Sicily where the climate conditions are particularly favorable for this crop. Recent findings have shown that Litchi extracts display anti-tumor and pro-apoptotic effects in vitro, but the precise underlying mechanisms have not been fully elucidated. In this study, we report for the first time the effects of Sicilian litchi fruit extracts on colon cancer cells. The results indicated that litchi exocarp, mesocarp and endocarp fractions reduce the viability and clonogenic growth of HT29 cells. These effects were due to cell cycle arrest in the G2/M phase followed by caspase-dependent cell death. Interestingly, litchi exocarp and endocarp triggered a precocious autophagic response (16⁻24 h), which was accompanied by an increase in the level of autophagy related 1/autophagy activating kinase 1 (ATG1/ULK1), beclin-1, microtubule associated protein 1 light chain 3 (LC3)-II and p62 proteins. Autophagy inhibition by bafilomycin A1 or beclin-1 silencing increased cell death, thus suggesting that autophagy was initially triggered as a pro-survival response. Significant effects of Litchi extracts were also observed in other colon cancer cells, including HCT116 and Caco-2 cells. On the other hand, differentiated Caco-2 cells, a model of human enterocytes, appeared to be insensitive to the extracts at the same treatment conditions. High-Performance Liquid Chromatography⁻Electrospray Ionization-Quadrupole-Time-Of-Flight HPLC/ESI/Q-TOF evidenced the presence of some polyphenolic compounds, specifically in exocarp and endocarp extracts, that can account for the observed biological effects. The results obtained suggest a potential therapeutic efficacy of polyphenolic compounds purified from Sicilian Litchi fractions for the treatment of colon cancer. Moreover, our findings indicate that modulation of autophagy can represent a tool to improve the effectiveness of these agents and potentiate the anti-tumor response of colon cancer cells.

Palmitoylation is a post-translational modification of Alix regulating the membrane organization of exosome-like small extracellular vesicles.

BACKGROUND: Virtually all cell types have the capacity to secrete nanometer-sized extracellular vesicles, which have emerged in recent years as potent signal transducers and cell-cell communicators. The multifunctional protein Alix is a bona fide exosomal regulator and skeletal muscle cells can release Alix-positive nano-sized extracellular vesicles, offering a new paradigm for understanding how myofibers communicate within skeletal muscle and with other organs. S-palmitoylation is a reversible lipid post-translational modification, involved in different biological processes, such as the trafficking of membrane proteins, achievement of stable protein conformations, and stabilization of protein interactions. METHODS: Here, we have used an integrated biochemical-biophysical approach to determine whether S-palmitoylation contributes to the regulation of extracellular vesicle production in skeletal muscle cells. RESULTS: We ascertained that Alix is S-palmitoylated and that this post-translational modification influences its protein-protein interaction with CD9, a member of the tetraspanin protein family. Furthermore, we showed that the structural organization of the lipid bilayer of the small (nano-sized) extracellular vesicle membrane with altered palmitoylation is qualitatively different compared to mock control vesicles. CONCLUSIONS: We propose that S-palmitoylation regulates the function of Alix in facilitating the interactions among extracellular vesicle-specific regulators and maintains the proper structural organization of exosome-like extracellular vesicle membranes. GENERAL SIGNIFICANCE: Beyond its biological relevance, our study also provides the means for a comprehensive structural characterization of EVs.

ALS-Related Mutant FUS Protein Is Mislocalized to Cytoplasm and Is Recruited into Stress Granules of Fibroblasts from Asymptomatic FUS P525L Mutation Carriers.

BACKGROUND: Amyotrophic lateral sclerosis (ALS) shows a strong genetic basis, with SOD1, FUS, TARDBP, and C9ORF72 being the genes most frequently involved. This has allowed identification of asymptomatic mutation carriers, which may be of help in understanding the molecular changes preceding disease onset. OBJECTIVES: We studied the cellular expression of FUS protein and the effect of heat-shock- and dithiothreitol-induced stress in fibroblasts from FUS P525L mutation carriers, healthy controls, and patients with sporadic ALS. METHODS: Western blots and immunocytochemistry were performed to study the subcellular localization of FUS protein. Control and stressed cells were double stained with FUS and the stress marker TIA-R. RESULTS: Fibroblasts from healthy controls and sporadic ALS cases showed a prominent nuclear FUS expression. In the 2 FUS P525L mutation carriers, instead, most cells showed a protein localization in both nucleus and cytoplasm, or exclusively in the cytoplasm. Stress prompted the formation of cytoplasmic granules in all subjects and in sporadic ALS FUS mislocalization to the cytoplasm. Cytoplasmic FUS was recruited into stress granules, which showed a time-dependent decrease in all subjects. However, in the FUS P525L fibroblasts, the granules persisted longer, and they were more numerous than those detected in the cells from controls and sporadic ALS patients. CONCLUSIONS: We show that in fibroblasts of FUS P525L mutation carriers, FUS mislocalized to the cytoplasm where it redistributed into stress granules with likely a dose effect, i.e. a higher number of cells with granules, which persist longer, than in controls and ALS cases. These data represent an early molecular change occurring before ALS onset, suggesting a transient preaggregative state.

The secreted protein acidic and rich in cysteine is a critical mediator of cell death program induced by WIN/TRAIL combined treatment in osteosarcoma cells.

Secreted protein acidic and rich in cysteine (SPARC) is a multi-functional protein which modulates cell-cell and cell-matrix interactions. In cancer cells, SPARC behaves as a tumor promoter in a number of tumors, but it can also act as a tumor suppressor factor. Our previous results showed that the synthetic cannabinoid WIN55,212-2 (WIN), a potent cannabinoid receptor agonist, is able to sensitize osteosarcoma MG63 cells to TNF-related apoptosis-inducing ligand (TRAIL)-induced apoptosis which is accompanied with endoplasmic reticulum (ER)-stress induction and the increase in autophagic markers. In the present investigation, we studied the role of SPARC in WIN/TRAIL-induced apoptosis demonstrating that WIN increased the level of SPARC protein and mRNA in a time-dependent manner. This event was functional to WIN/TRAIL-dependent apoptosis as demonstrated by RNA interfering analysis which indicated that SPARC-silenced cells were less sensitive to cytotoxic effects induced by the combined treatment. Our experiments also demonstrate that SPARC interacts with caspase-8 thus probably favoring its translocation to plasma membrane and the activation of extrinsic apoptotic pathway. In conclusion, to the best of our knowledge, our results are the first to show that WIN-dependent increase in the level of SPARC plays a critical role in sensitizing osteosarcoma cells to TRAIL action.