Differentiation of glioblastoma stem cells promoted by miR-128 or miR-302a overexpression enhances senescence-associated cytotoxicity of axitinib.

Despite the intense global efforts towards an effective treatment of glioblastoma (GB), current therapeutic options are unsatisfactory with a median survival time of 12-15 months after diagnosis, which has not improved significantly over more than a decade. The high tumoral heterogeneity confers resistance to therapies, which has hindered a successful clinical outcome, GB remaining among the deadliest cancers. A hallmark of GB is its high recurrence rate, which has been attributed to the presence of a small subpopulation of tumor cells called GB stem-like cells (GSC). In the present work, the efficacy of a multimodal strategy combining microRNA (miRNA) modulation with new generation multitargeted tyrosine kinase inhibitors (imatinib and axitinib) was investigated aiming at tackling this subpopulation of GB cells. MiR-128 and miR-302a were selected as attractive therapeutic candidates on the basis of previous findings reporting that reestablishment of their decreased expression levels in GSC resulted in cell differentiation, which could represent a possible strategy to sensitize GSC to chemotherapy. Our results show that overexpression of miR-128 or miR-302a induced GSC differentiation, which enhanced senescence mediated by axitinib treatment, thus further impairing GSC proliferation. We also provided evidence for the capacity of GSC to efficiently internalize functionalized stable nucleic acid lipid particles, previously developed and successfully applied in our laboratory to target GB. Taken together, our findings will be important in the future design of a GB-targeted multimodal miRNA-based gene therapy, combining overexpression of miR-128 or miR-302a with axitinib treatment, endowed with the ability to overcome drug resistance.

Downregulation of long non-protein coding RNA MVIH impairs glioblastoma cell proliferation and invasion through an miR-302a-dependent mechanism.

Glioblastoma (GB) is the most frequent and malignant type of brain tumor, for which no effective therapy exists. The high proliferative and invasive nature of GB, as well as its acquired resistance to chemotherapy, makes this type of cancer extremely lethal shortly after diagnosis. Long non-protein coding RNAs (lncRNA) are a class of regulatory RNAs whose levels can be dysregulated in the context of diseases, unbalancing several physiological processes. The lncRNA associated with microvascular invasion in hepatocellular carcinoma (lncRNA-MVIH), overexpressed in several cancers, was described to co-precipitate with phosphoglycerate kinase 1 (PGK1), preventing secretion of this enzyme to the extracellular environment and promoting cell migration and invasion. We hypothesized that, by silencing the expression of lncRNA-MVIH, the secretion of PGK1 would increase, reducing GB cell migration and invasion capabilities. We observed that lncRNA-MVIH silencing in human GB cells significantly decreased glycolysis, cell growth, migration, and invasion and sensitized GB cells to cediranib. However, no increase in extracellular PGK1 was observed as a consequence of lncRNA-MVIH silencing, and therefore, we investigated the possibility of a mechanism of miRNA sponge of lncRNA-MVIH being in place. We found that the levels of miR-302a loaded onto RISC increased in GB cells after lncRNA-MVIH silencing, with the consequent downregulation of several miR-302a molecular targets. Our findings suggest a new mechanism of action of lncRNA-MVIH as a sponge of miR-302a. We suggest that lncRNA-MVIH knockdown may be a promising strategy to address GB invasiveness and chemoresistance, holding potential towards its future application in a clinical context.

MiR-200c-based metabolic modulation in glioblastoma cells as a strategy to overcome tumor chemoresistance.

Glioblastoma (GB) is the most aggressive and common form of primary brain tumor characterized by fast proliferation, high invasion and resistance to current standard treatment. The average survival rate post-diagnosis is 14.6 months, despite the aggressive standard post-surgery radiotherapy concomitant with chemotherapy with temozolomide (TMZ). Currently, efforts are being endowed to develop new and more efficient therapeutic approaches capable to overcome chemoresistance, inhibit tumor progression and improve overall patient survival rate. Abnormal microRNA (miRNA) expression has been correlated with chemoresistance, proliferation and resistance to apoptosis, which result from their master regulatory role of gene expression. Altered cell metabolism, favoring glycolysis, was identified as an emerging cancer hallmark and has been described in GB, thus offering a new target for innovative GB therapies. In this work, we hypothesized that a gene therapy-based strategy consisting of the overexpression of a miRNA downregulated in GB and predicted to target crucial metabolic enzymes might promote a shift of GB cell metabolism, decreasing the glycolytic dependence of tumor cells and contributing to their sensitization to chemotherapy with TMZ. The increase of miR-200c levels in DBTRG cells resulted in downregulation of messenger RNA of enzymes involved in bioenergetics pathways and impaired cell metabolism and mobility. In addition, miR-200c overexpression prior to DBTRG cell exposure to TMZ resulted in cell cycle arrest. Overall, our results show that miR-200c overexpression could offer a way to overcome chemoresistance developed by GB cells in response to current standard chemotherapy, providing an improvement to current GB standard treatment, with benefit for patient outcome.

Stearoyl CoA Desaturase-1 Silencing in Glioblastoma Cells: Phospholipid Remodeling and Cytotoxicity Enhanced upon Autophagy Inhibition.

Modulation of lipid metabolism is a well-established cancer hallmark, and SCD1 has been recognized as a key enzyme in promoting cancer cell growth, including in glioblastoma (GBM), the deadliest brain tumor and a paradigm of cancer resistance. The central goal of this work was to identify, by MS, the phospholipidome alterations resulting from the silencing of SCD1 in human GBM cells, in order to implement an innovative therapy to fight GBM cell resistance. With this purpose, RNAi technology was employed, and low serum-containing medium was used to mimic nutrient deficiency conditions, at which SCD1 is overexpressed. Besides the expected increase in the saturated to unsaturated fatty acid ratio in SCD1 silenced-GBM cells, a striking increase in polyunsaturated chains, particularly in phosphatidylethanolamine and cardiolipin species, was noticed and tentatively correlated with an increase in autophagy (evidenced by the increase in LC3BII/I ratio). The contribution of autophagy to mitigate the impact of SCD1 silencing on GBM cell viability and growth, whose modest inhibition could be correlated with the maintenance of energetically associated mitochondria, was evidenced by using autophagy inhibitors. In conclusion, SCD1 silencing could constitute an important tool to halt GBM resistance to the available treatments, especially when coupled with a mitochondria disrupter chemotherapeutic.

Glucosylceramide synthase silencing combined with the receptor tyrosine kinase inhibitor axitinib as a new multimodal strategy for glioblastoma.

A great deal of evidence revealing that lipid metabolism is drastically altered during tumorigenesis has been accumulated. In this work, glucosylceramide synthase (GCS) was targeted, using RNA interference technology (siRNAs), in U87 and DBTRG human glioblastoma (GBM) cells, as in both cell types GCS showed to be overexpressed with respect to normal human astrocytes. The efficacy of a combined therapy to tackle GBM, allying GCS silencing to the new generation chemotherapeutics sunitinib and axitinib, or to the alkylating drugs etoposide and temozolomide, is evaluated here for the first time. With this purpose, studies addressing GBM cell viability and proliferation, cell cycle and apoptosis were performed, which revealed that combination of GCS silencing with axitinib treatment represents a promising therapeutic approach. The reduction of cell viability induced by this combined therapy is proposed to be mediated by excessive production of reactive oxygen species. This work, identifying GCS as a key molecular target to increase GBM susceptibility to a new generation chemotherapeutic, opens windows to the development of innovative strategies to halt GBM recurrence after surgical resection.

MiR-144 overexpression as a promising therapeutic strategy to overcome glioblastoma cell invasiveness and resistance to chemotherapy.

Glioblastoma (GB) is the most aggressive and common form of primary brain tumor, characterized by fast proliferation, high invasion, and resistance to current standard treatment. The average survival rate post-diagnosis is only of 14.6 months, despite the aggressive standard post-surgery treatment approaches of radiotherapy concomitant with chemotherapy with temozolomide. Altered cell metabolism has been identified as an emerging cancer hallmark, including in GB, thus offering a new target for cancer therapies. On the other hand, abnormal expression levels of miRNAs, key regulators of multiple molecular pathways, have been correlated with pathological manifestations of cancer, such as chemoresistance, proliferation, and resistance to apoptosis. In this work, we hypothesized that gene therapy based on modulation of a miRNA with aberrant expression in GB and predicted to target crucial metabolic enzymes might impair tumor cell metabolism. We found that the increase of miR-144 levels, shown to be downregulated in U87 and DBTRG human GB cell lines, as well as in GB tumor samples, promoted the downregulation of mRNA of enzymes involved in bioenergetic pathways, with consequent alterations in cell metabolism, impairment of migratory capacity, and sensitization of DBTRG cells to a chemotherapeutic drug, the dichloroacetate (DCA). Taken together, our findings provide evidence that the miR-144 plus DCA combined therapy holds promise to overcome GB-acquired chemoresistance, therefore deserving to be explored toward its potential application as a complementary therapeutic approach to the current treatment options for this type of brain tumor.

Lauroylated Histidine-Enriched S413-PV Peptide as an Efficient Gene Silencing Mediator in Cancer Cells.

PURPOSE: This study aimed to endow the cell-penetrating peptide (CPP) S413-PV with adequate features towards a safe and effective application in cancer gene therapy. METHODS: Peptide/siRNA complexes were prepared with two new derivatives of the CPP S413-PV, which combine a lauroyl group attached to the N- or C-terminus with a histidine-enrichment in the N-terminus of the S413-PV peptide, being named C12-H5-S413-PV and H5-S413-PV-C12, respectively. Physicochemical characterization of siRNA complexes was performed and their cytotoxicity and efficiency to mediate siRNA delivery and gene silencing in cancer cells were assessed in the absence and presence of serum. RESULTS: Peptide/siRNA complexes prepared with the C12-H5-S413-PV derivative showed a nanoscale (ca. 100 nm) particle size, as revealed by TEM, and efficiently mediated gene silencing (37%) in human U87 glioblastoma cells in the presence of 30% serum. In addition, the new C12-H5-S413-PV-based siRNA delivery system efficiently downregulated stearoyl-CoA desaturase-1, a key-enzyme of lipid metabolism overexpressed in cancer, which resulted in a significant decrease in the viability of U87 cells. Importantly, these complexes were able to spare healthy human astrocytes. CONCLUSIONS: These encouraging results pave the way for a potential application of the C12-H5-S413-PV peptide as a promising tool in cancer gene therapy.

High-throughput screening uncovers miRNAs enhancing glioblastoma cell susceptibility to tyrosine kinase inhibitors.

Glioblastoma (GBM) is a deadly and therapy resistant malignant brain tumour, characterized by an aggressive and diffuse growth pattern, which prevents complete surgical resection. Despite advances in the identification of genomic and molecular alterations that fuel the tumour, average patient survival post-diagnosis remains very low (∼14.6-months). In addition to being highly heterogeneous, GBM tumour cells exhibit high adaptive capacity to targeted molecular therapies owing to an established network of signalling cascades with functional redundancy, which provides them with robust compensatory survival mechanisms. Here, we investigated whether a multimodal strategy combining multitargeted tyrosine kinase inhibitors (MTKIs) and microRNA (miRNA) modulation could overcome the signalling pathway redundancy in GBM and, hence, promote tumour cell death. By performing a high-throughput screening, we identified a myriad of miRNAs, including those belonging to the miR-302-3p/372-3p/373-3p/520-3p family, which coordinately act with the MTKI sunitinib to decrease GBM cell viability. Two members of this family, hsa-miRNA-302a-3p and hsa-miRNA-520 b, were found to modulate the expression of receptor tyrosine kinase mediators (including AKT1, PIK3CA and SOS1) in U87 and DBTRG human GBM cells. Importantly, administration of mimics of these miRNAs with sunitinib or axitinib resulted in decreased tumour cell proliferation and enhanced cell death, whereas no significant effect was observed when coupling miRNA modulation with temozolomide, the first-line drug for GBM therapy. Overall, our results provide evidence that combining the 'horizontal' inhibition of signalling pathways promoted by MTKIs with the 'vertical' inhibition of the downstream signalling cascade promoted by hsa-miR-302a-3p and hsa-miR-520 b constitutes a promising approach towards GBM treatment.

Gemini surfactants mediate efficient mitochondrial gene delivery and expression.

Gene delivery targeting mitochondria has the potential to transform the therapeutic landscape of mitochondrial genetic diseases. Taking advantage of the nonuniversal genetic code used by mitochondria, a plasmid DNA construct able to be specifically expressed in these organelles was designed by including a codon, which codes for an amino acid only if read by the mitochondrial ribosomes. In the present work, gemini surfactants were shown to successfully deliver plasmid DNA to mitochondria. Gemini surfactant-based DNA complexes were taken up by cells through a variety of routes, including endocytic pathways, and showed propensity for inducing membrane destabilization under acidic conditions, thus facilitating cytoplasmic release of DNA. Furthermore, the complexes interacted extensively with lipid membrane models mimicking the composition of the mitochondrial membrane, which predicts a favored interaction of the complexes with mitochondria in the intracellular environment. This work unravels new possibilities for gene therapy toward mitochondrial diseases.

A biophysical approach to menadione membrane interactions: relevance for menadione-induced mitochondria dysfunction and related deleterious/therapeutic effects.

Menadione (MEN), a polycyclic aromatic ketone, was shown to promote cell injury by imposing massive oxidative stress and has been proposed as a promising chemotherapeutic agent for the treatment of cancer diseases. The mechanisms underlying MEN-induced mitochondrial dysfunction and cell death are not yet fully understood. In this work, a systematic study was performed to unveil the effects of MEN on membrane lipid organization, using models mimicking mitochondrial membranes and native mitochondrial membranes. MEN was found to readily incorporate in membrane systems composed of a single phospholipid (phosphatidylcholine) or the lipids dioleoylphosphatidylcholine, dioleoylphosphatidylethanolamine and tetraoleoylcardiolipin at 1:1:1 molar ratio, as well as in mitochondrial membranes. Increased permeability in both membrane models, monitored by calcein release, seemed to correlate with the extent of MEN incorporation into membranes. MEN perturbed the physical properties of vesicles composed of dipalmitoylphosphatidylcholine or dipalmitoylphosphatidylethanolamine plus tetraoleoylcardiolipin (at 7:3 molar ratio), as reflected by the downshift of the lipid phase transition temperature and the emergence of a new transition peak in the mixed lipid system, detected by DSC. (31)P NMR studies revealed that MEN favored the formation of non-lamellar structures. Also, quenching studies with the fluorescent probes DPH and TMA-DPH showed that MEN distributed across the bilayer thickness in both model and native mitochondrial membranes. MEN's ability to promote alterations of membrane lipid organization was related with its reported mitochondrial toxicity and promotion of apoptosis, predictably involved in its anti-carcinogenic activity.

Application of thermoresponsive PNIPAAM-b-PAMPTMA diblock copolymers in siRNA delivery.

Gene knockdown has emerged as an important tool for cancer gene therapy as well as for viral infections and dominantly inherited genetic disorders. The generation of suitable siRNA delivery systems poses some challenges, namely, to avoid nuclease degradation, to surpass the cytoplasmic membrane, and to release the nucleic acids into the cytosol. Aiming at evaluating the ability of thermoresponsive block copolymers formed by units of N-isopropylacrylamide and of (3-acrylamidopropyl)trimethylammonium chloride to efficiently deliver siRNAs, an extensive study was performed with four different copolymers using a human fibrosarcoma cell line as cell model. The silencing ability and cytotoxicity of the generated copolymer-based siRNA delivery systems were found to be dependent on the cloud point of the polymer, which corresponds to the transition temperature at which the aggregation or precipitation of the polymer molecules becomes thermodynamically more favorable than their solubilization. In the present study, a system capable of delivering siRNAs efficiently, specifically and without presenting relevant cytotoxicity, even in the presence of serum, was developed. Confocal fluorescence experiments showed that the ability of the generated systems to silence the target gene is related to some extent to nucleic acid internalization, being also dependent on polymer/siRNA dissociation at 37 °C. Thus, a delicate balance between nucleic acid internalization and intracellular release must be met in order to reach an ideal knockdown efficiency. The special features and potential for manipulation of the N-isopropylacrylamide-based copolymers make them suitable materials for the design and synthesis of new and promising siRNA delivery systems.

S4(13)-PV cell-penetrating peptide induces physical and morphological changes in membrane-mimetic lipid systems and cell membranes: implications for cell internalization.

The present work aims to gain insights into the role of peptide-lipid interactions in the mechanisms of cellular internalization and endosomal escape of the S4(13)-PV cell-penetrating peptide, which has been successfully used in our laboratory as a nucleic acid delivery system. A S4(13)-PV analogue, S4(13)-PVscr, displaying a scrambled amino acid sequence, deficient cell internalization and drug delivery inability, was used in this study for comparative purposes. Differential scanning calorimetry, fluorescence polarization and X-ray diffraction at small and wide angles techniques showed that both peptides interacted with anionic membranes composed of phosphatidylglycerol or a mixture of this lipid with phosphatidylethanolamine, increasing the lipid order, shifting the phase transition to higher temperatures and raising the correlation length between the bilayers. However, S4(13)-PVscr, in contrast to the wild-type peptide, did not promote lipid domain segregation and induced the formation of an inverted hexagonal lipid phase instead of a cubic phase in the lipid systems assayed. Electron microscopy showed that, as opposed to S4(13)-PVscr, the wild-type peptide induced the formation of a non-lamellar organization in membranes of HeLa cells. We concluded that lateral phase separation and destabilization of membrane lamellar structure without compromising membrane integrity are on the basis of the lipid-driven and receptor-independent mechanism of cell entry of S4(13)-PV peptide. Overall, our results can contribute to a better understanding of the role of peptide-lipid interactions in the mechanisms of cell-penetrating peptide membrane translocation, helping in the future design of more efficient cell-penetrating peptide-based drug delivery systems.

Comparison of the efficiency of complexes based on S4(13)-PV cell-penetrating peptides in plasmid DNA and siRNA delivery.

The successful application of gene therapy approaches is highly dependent on the efficient delivery of nucleic acids into target cells. In the present study, new peptide-based nonviral systems were developed to enhance plasmid DNA and siRNA delivery, aiming at generating appropriate gene delivery and gene silencing tools for preclinical and clinical application. For this purpose, a new cell-penetrating peptide derived from the wild-type S4(13)-PV peptide was synthesized through the addition of a five-histidine tail to its N-terminus (H5-S4(13)-PV), and its ability to mediate gene expression and gene silencing was evaluated and compared to that of the wild-type peptide. The histidine-enriched peptide, H5-S4(13)-PV, proved to be generally more efficient and less toxic than the wild-type peptide in the delivery of plasmid DNA. In addition, complexes of H5-S4(13)-PV with siRNAs, but not of S4(13)-PV, were efficiently internalized by cells and presented high knockdown activity (63%). Interestingly, systems containing the S4(13)-PV or the H5-S4(13)-PV peptide exhibited superior biological activity when compared to those containing the reverse NLS or scrambled peptides, suggesting that both the cell-penetrating sequence and the NLS of the S4(13)-PV peptide influence the competence of binary and ternary complexes to accomplish nucleic acid delivery. In order to unravel the cancer therapeutic potential of formulations with the histidine-enriched peptide, their efficiency to mediate silencing of the oncogenic protein survivin was evaluated. As opposed to complexes with the wild-type peptide, H5-S4(13)-PV complexes showed the ability to promote a high survivin knockdown at the level of both protein (44%) and mRNA (73%), in HT1080 cells.

Rapeseed oil-rich diet alters hepatic mitochondrial membrane lipid composition and disrupts bioenergetics.

Diet is directly related with physiological alterations occurring at a cell and subcellular level. However, the role of diet manipulation on mitochondrial physiology is still largely unexplored. Aiming at correlating diet with alterations of mitochondrial membrane composition and bioenergetics, Wistar-Han male rats were fed for 11, 22 and 33 days with a rapeseed oil-based diet and mitochondrial bioenergetics, and membrane composition were compared at each time point with a standard diet group. Considerable differences were noticed in mitochondrial membrane lipid composition, namely in terms of fatty acyl chains and relative proportions of phospholipid classes, the modified diet inducing a decrease in the saturated to unsaturated molar ratio and an increase in the phosphatidylcholine to phosphatidylethanolamine molar ratio. Mass spectrometry lipid analysis showed significant differences in the major species of cardiolipin, with an apparent increased incorporation of oleic acid as a result of exposure to the modified diet. Rats fed the modified diet during 22 days showed decreased hepatic mitochondrial state 3 respiration and were more susceptible to Ca(2+)-induced transition pore opening. Rapeseed oil-enriched diet also appeared to promote a decrease in hydroperoxide production by the respiratory chain, although a simultaneous decrease in vitamin E content was detected. In conclusion, our data indicate that the rapeseed oil diet causes negative alterations on hepatic mitochondrial bioenergetics, which may result from membrane remodeling. Such alterations may have an impact not only on energy supply to the cell, but also on drug-induced hepatic mitochondrial liabilities.

Gemini surfactant dimethylene-1,2-bis(tetradecyldimethylammonium bromide)-based gene vectors: a biophysical approach to transfection efficiency.

Cationic liposomes have been proposed as biocompatible gene delivery vectors, able to overcome the barriers imposed by cell membranes. Besides lipids, other surfactant molecules have been successfully used in the composition of gene carriers. In the present work, we used a Gemini surfactant, represented by the general structure [C(14)H(29)(CH(3))(2)N(+)(CH(2))(2)N(+)(CH(3))(2)C(14)H(29)]2Br(-) and herein designated 14-2-14, to prepare cationic gene carriers, both as the sole component and in combination with neutral helper lipids, cholesterol and DOPE. The effectiveness of three Gemini-based formulations, namely neat 14-2-14, 14-2-14:Chol (1:1 molar ratio) and 14-2-14:Chol:DOPE (2:1:1 molar ratio), to mediate gene delivery was evaluated in DNA mixtures of +/- charge ratios ranging from 1/1 to 12/1. After ruling out cytotoxicity as responsible for the differences observed in the transfection competence, structural and physical properties of the vector were investigated, using several techniques. The size and surface charge density (zeta potential) of surfactant-based structures were determined by conventional techniques and the thermotropic behaviour of aqueous dispersions of surfactant/lipid/DNA formulations was monitored by fluorescence polarization of DPH and DPH-PA probes. The capacity of lipoplexes to interact with membrane-mimicking lipid bilayers was evaluated, using the PicoGreen assay and a FRET technique. Our data indicate inefficiency of the neat 14-2-14 formulation for gene delivery, which could result from the large dimensions of the particles and/or from its relative incompetence to release DNA upon interaction with anionic lipids. The addition of cholesterol or cholesterol and DOPE conferred to Gemini-based gene carrier transfection activity at specific ranges of +/- charge ratios. Fluorescence polarization data suggest that an order parameter within a specific range was apparently needed for complexes to display maximal transfection efficiency. The transfection-competent formulations showed to be efficiently destabilized by interaction with different anionic and zwitterionic bilayers, including those containing PS and cardiolipin. These data are discussed in terms of the potential of these formulations to address different intracellular targets.

Interaction of carbonylcyanide p-trifluoromethoxyphenylhydrazone (FCCP) with lipid membrane systems: a biophysical approach with relevance to mitochondrial uncoupling.

FCCP (carbonylcyanide p-trifluoromethoxyphenylhydrazone), a classical uncoupler of mitochondrial oxidative phosphorylation, is used in this study as a model to clarify how interactions of uncouplers with membrane lipid bilayers may influence membrane biophysics and their protonophoric activity itself. In order to disclose putative effects that may be important when considering using uncouplers for pharmacological purposes, an extensive characterization of FCCP membrane lipid interactions using accurate biophysical approaches and simple model lipid systems was carried out. Differential scanning calorimetry studies showed that FCCP molecules disturb lipid bilayers and favor lateral phase separation in mixed lipid systems. (31)P NMR assays indicated that FCCP alters the curvature elastic properties of membrane models containing non-bilayer lipids, favoring lamellar/H(II) transition, probably by alleviation of hydrocarbon-packing constraints in the inverted hexagonal phase. Taking advantage of FCCP quenching effects on the fluorescent probes DPH (1,6-diphenyl-1,3,5-hexatriene) and DPH-PA (3-(p-(6-phenyl)-1,3,5-hexatrienyl)phenylpropionic acid), it is demonstrated that FCCP distributes across the bilayer thickness in both a single and a ternary lipid system mimicking the inner mitochondrial membrane. This behavior is consistent with the ability of the compound to migrate through the thickness of the inner mitochondrial membrane, an event required for its protonophoric activity. Finally, the study of the membrane fluidity in different lipid systems, as reported by the rotational correlation time (θ) of DPH or DPH-PA, showed that the extension at which FCCP disturbs membrane properties associated with the dynamics and the order of lipid molecules depends on the lipid composition of the model lipid system assayed.

Exploratory Data Analysis of Cell and Mitochondrial High-Fat, High-Sugar Toxicity on Human HepG2 Cells.

Non-alcoholic steatohepatitis (NASH), one of the deleterious stages of non-alcoholic fatty liver disease, remains a significant cause of liver-related morbidity and mortality worldwide. In the current work, we used an exploratory data analysis to investigate time-dependent cellular and mitochondrial effects of different supra-physiological fatty acids (FA) overload strategies, in the presence or absence of fructose (F), on human hepatoma-derived HepG2 cells. We measured intracellular neutral lipid content and reactive oxygen species (ROS) levels, mitochondrial respiration and morphology, and caspases activity and cell death. FA-treatments induced a time-dependent increase in neutral lipid content, which was paralleled by an increase in ROS. Fructose, by itself, did not increase intracellular lipid content nor aggravated the effects of palmitic acid (PA) or free fatty acids mixture (FFA), although it led to an up-expression of hepatic fructokinase. Instead, F decreased mitochondrial phospholipid content, as well as OXPHOS subunits levels. Increased lipid accumulation and ROS in FA-treatments preceded mitochondrial dysfunction, comprising altered mitochondrial membrane potential (ΔΨm) and morphology, and decreased oxygen consumption rates, especially with PA. Consequently, supra-physiological PA alone or combined with F prompted the activation of caspase pathways leading to a time-dependent decrease in cell viability. Exploratory data analysis methods support this conclusion by clearly identifying the effects of FA treatments. In fact, unsupervised learning algorithms created homogeneous and cohesive clusters, with a clear separation between PA and FFA treated samples to identify a minimal subset of critical mitochondrial markers in order to attain a feasible model to predict cell death in NAFLD or for high throughput screening of possible therapeutic agents, with particular focus in measuring mitochondrial function.

Improving pollutants environmental risk assessment using a multi model toxicity determination with in vitro, bacterial, animal and plant model systems: The case of the herbicide alachlor.

Several environmental pollutants, including pesticides, herbicides and persistent organic pollutants play an important role in the development of chronic diseases. However, most studies have examined environmental pollutants toxicity in target organisms or using a specific toxicological test, losing the real effect throughout the ecosystem. In this sense an integrative environmental risk of pollutants assessment, using different model organisms is necessary to predict the real impact in the ecosystem and implications for target and non-target organisms. The objective of this study was to use alachlor, a chloroacetanilide herbicide responsible for chronic toxicity, to understand its impact in target and non-target organisms and at different levels of biological organization by using several model organisms, including membranes of dipalmitoylphosphatidylcholine (DPPC), rat liver mitochondria, bacterial (Bacillus stearothermophilus), plant (Lemna gibba) and mammalian cell lines (HeLa and neuro2a). Our results demonstrated that alachlor strongly interacted with membranes of DPPC and interfered with mitochondrial bioenergetics by reducing the respiratory control ratio and the transmembrane potential. Moreover, alachlor also decreased the growth of B. stearothermophilus and its respiratory activity, as well as decreased the viability of both mammalian cell lines. The values of TC50 increased in the following order: Lemna gibba < neuro2a < HeLa cells < Bacillus stearothermophilus. Together, the results suggest that biological membranes constitute a putative target for the toxic action of this lipophilic herbicide and point out the risks of its dissemination on environment, compromising ecosystem equilibrium and human health.

The effect of cationic gemini surfactants upon lipid membranes. An experimental and molecular dynamics simulation study.

Gemini surfactants possess interesting interfacial and aggregation properties that have prompted comprehensive studies and successful applications in a wide variety of fields. However, a systematic study on the effect of gemini tail and spacer length upon the organization of lipid membranes has not been presented so far. In this study, we analyze the action of dicationic alkylammonium bromide gemini surfactants on DPPC liposomes, the latter employed as a model of lipid membranes. Differential scanning calorimetry results indicate that the surfactants presenting shorter tails (12 carbons) induce a decrease in the overall order of the bilayer, while those with longer tails (16 and 18 carbons) lead to the formation of more ordered structures. The respective influence on the degree of lipid order transverse to the bilayer was additionally studied resorting to a detailed fluorescence anisotropy study. In this case, it is observed that among the shorter tail surfactants, those with longer spacers (6 and 10 carbons) are responsible for a more pronounced disrupting effect upon the membrane, especially close to the lipid polar heads. Molecular dynamics simulation supports the most important findings and provides insight into the mechanism that governs this interaction. Accordingly, the interplay between tail and spacer length accounts for the differential vertical positioning of the gemini molecules and atom-density in the core of the bilayer, that provide a rationale for the experimental observations.

Dual Imaging Gold Nanoplatforms for Targeted Radiotheranostics.

Gold nanoparticles (AuNPs) are interesting for the design of new cancer theranostic tools, mainly due to their biocompatibility, easy molecular vectorization, and good biological half-life. Herein, we report a gold nanoparticle platform as a bimodal imaging probe, capable of coordinating Gd3+ for Magnetic Resonance Imaging (MRI) and 67Ga3+ for Single Photon Emission Computed Tomography (SPECT) imaging. Our AuNPs carry a bombesin analogue with affinity towards the gastrin releasing peptide receptor (GRPr), overexpressed in a variety of human cancer cells, namely PC3 prostate cancer cells. The potential of these multimodal imaging nanoconstructs was thoroughly investigated by the assessment of their magnetic properties, in vitro cellular uptake, biodistribution, and radiosensitisation assays. The relaxometric properties predict a potential T1- and T2- MRI application. The promising in vitro cellular uptake of 67Ga/Gd-based bombesin containing particles was confirmed through biodistribution studies in tumor bearing mice, indicating their integrity and ability to target the GRPr. Radiosensitization studies revealed the therapeutic potential of the nanoparticles. Moreover, the DOTA chelating unit moiety versatility gives a high theranostic potential through the coordination of other therapeutically interesting radiometals. Altogether, our nanoparticles are interesting nanomaterial for theranostic application and as bimodal T1- and T2- MRI / SPECT imaging probes.