Intracellular GSH Levels Research Articles

Synergistic effect of buthionine sulfoximine on the chlorin e6-based photodynamic treatment of cancer cells

In this study, we investigated the synergistic effect of L-buthionine sulfoximine (BSO) on the chlorin e6 (Ce6)-based photodynamic therapy (PDT) of cancer cells. Among various cancer cells, HCT116 cells have highest intracellular L-glutathione (GSH) level and SNU478 cells showed the lowest GSH level. BSO alone showed negligible intrinsic cytotoxicity against CCD986sk cells. Since HCT116 and SNU478 cells showed the highest and the lowest intracellular GSH levels, respectively, those were used to test synergistic effect on the Ce6-based PDT. In the absence of light, BSO and Ce6 combination did not practically increase reactive oxygen species (ROS) in either of HCT116 or SNU478 cells, while light irradiation increased ROS level dose-dependently. 10μM BSO treatment significantly depleted total GSH level in cancer cells, i.e. total GSH level decreased to one-fourth of the control in HCT116 cells while it decreased to two-fifth of the control treatment at SNU478 cell. BSO showed synergistic effect on the ROS production in HCT116 cells while it has practically no benefits in ROS production of SNU478 cells. No synergistic effect was observed in viability of SNU478 cells because BSO itself was cytotoxic to SNU478 cells. However, BSO had negligible cytotoxicity against HCT116 cells and showed synergistic anticancer effect in combination with Ce6-based PDT. Furthermore, the addition of glutathione reduced ethyl ester (GSH-OEt), recovered intracellular GSH level, and cell viability with reduced the intracellular ROS level. We suggest that synergistic effect of BSO in the Ce6-based PDT should be considered with intrinsic intracellular GSH level of cancer cells.

Edaravone, a free radical scavenger, protects against ferroptotic cell death in vitro

Ferroptosis is characterized by an iron-dependent cell death with increased lipid peroxidation and is typically induced by either a decrease in glutathione (GSH) levels due to an insufficient supply of cysteine (Cys) or the inhibition of phospholipid hydroperoxide glutathione peroxidase (Gpx4). While lipid peroxides are the direct trigger for ferroptosis, the issue of how radical species involve in the cytocidal process remains unclear. To gain insights into this issue, we employed edaravone, a free radical scavenger that is clinically approved for the treatment of acute ischemic strokes and amyotrophic lateral sclerosis (ALS), against ferroptotic cell death caused by various situations, notably under cystine deprivation. We initially investigated the effects of edaravone on ferroptosis in mouse hepatoma Hepa 1–6 cells cultivated in cystine-free medium and found that edaravone largely suppressed ferroptosis. Ferroptosis that was induced in the cells by the use of inhibitors for xCT or Gpx4 was also suppressed by edaravone. Moreover, edaravone also suppressed ferroptosis in xCT-knockout mouse-derived embryonic fibroblasts, which usually die in normal cultivating conditions due to the depletion of intracellular Cys and GSH. Although the edaravone treatment had no effects on the intracellular levels of Cys and GSH, both of which remained low in Hepa 1–6 cells under conditions of cystine deprivation, the causative factors for ferroptosis, including ferrous iron and lipid peroxide levels, were significantly suppressed. Collectively, these results indicate that radical species produced at the initial stage of the cytocidal process under Cys-deprived conditions trigger ferroptosis and scavenging these radicals by edaravone represents a promising treatment.

Cyclodextrin based unimolecular micelles with targeting and biocleavable abilities as chemotherapeutic carrier to overcome drug resistance.

An amphiphilic star-shaped copolymer β-CD-g-PCL-SS-PEG-FA, consisting of a β-cyclodextrin (β-CD) core as well as grafted with bioreducible disulfide linkage in PCL-SS-PEG multiarms and targeting folic acid (FA) as end moiety, is designed with unimolecular micelles formation ability for targeted transport of chemotherapeutics to drug resistant tumor cells. Firstly, β-CD was utilized as core to growth PCL arms by ring-opening polymerization (ROP) of ε-CL, before disulfide terminal group transformation to render β-CD-g-PCL-SS-COOH. Secondly, α-hydroxy-ω-amine protected PEG (HO-PEG-NHBoc) was connected to β-CD-g-PCL-SS-COOH to obtain amphiphilic β-CD-g-PCL-SS-PEG, where PCL and PEG were connected via bioreducible disulfide bond. After deprotection of -Boc group, FA was introduced onto the distal end of block arms to obtain the desired β-CD-g-PCL-SS-PEG-FA copolymer. Because of highly branched core-shell amphiphilic structures, β-CD-g-PCL-SS-PEG-FA could act as unimolecular micelles. Interestingly, this unimolecular micelle could release the encapsulated drug in a glutathione (GSH) dependent manner due to disulfide linkage. More importantly, this unimolecular micelle could load doxorubicin (DOX) to promote its cellular uptake in multidrug resistance (MDR) protein overexpression tumor cells, by taking the advantage of FA targeting group and intracellular high GSH level in cancer cells. Together with satisfactory biocompatibility, this novel star-like β-CD-g-PCL-SS-PEG-FA unimolecular micelle could potentially be utilized as targeting nanocarriers in drug resistant cancer therapy.

Intracellular redox status during acute coronary syndrome

In Algeria, more than one million people have cardiovascular diseases including coronary syndrome, with 20,000 to 25,000 deaths each year. The aim of our study was to determine the oxidative stress markers and antioxidant defence system during acute coronary syndrome. This work was carried on 50 men with acute coronary syndrome admitted in cardiology department of Tlemcen University Hospital Center matched to 50 healthy controls. Erythrocyte levels of bimolecular oxidation (malondialdehyde, carbonylated proteins) and intracellular levels of free radicals (NO and O 2− ) were assessed. Furthermore, the erythrocyte activity of antioxidant enzymes (superoxide dismutase (SOD) and catalase) and the intracellular level of reduced glutathione (GSH) were evaluated. Compared to controls, patients with acute coronary syndrome had a significant higher level of intracellular MDA, carbonylated proteins and free radicals (O 2− and NO), while the intracellular level of GSH and the erythrocyte activity of antioxidant enzymes (SOD and catalase) were lower. In conclusion, acute coronary syndrome is associated with oxidative stress characterized by an oxidant/antioxidant imbalance with a concomitant decrease in antioxidant defence, associated to an increase in intracellular of free radical levels and markers of oxidation.

Intracellular cascade activated nanosystem for improving ER+ breast cancer therapy through attacking GSH-mediated metabolic vulnerability

Estrogen receptor-positive (ER+) breast carcinoma therapy faces the challenges of estrogen receptors heterogeneity and endocrine therapy resistance. Selectively attacking glutathione (GSH) biosynthesis which is the metabolic vulnerability of ER+ breast carcinoma could bypass conventional treatment limitations through blocking oxidative stress disorders-driven tumor cell proliferation. Herein, we developed drug-organics-inorganics self-assembled nanosystem (DFTA) with doxorubicin (DOX) as chemotherapeutic agent, ferric chloride (FeCl3) as ferroptosis inducer and tannic acid (TA) as activator of superoxide dismutase (SOD)-like reaction in intracellular cascade for the combined therapy in ER+ breast carcinoma. DFTA displayed a particle size of 106.4 ± 0.7 nm with flat irregular nanonetwork-like shape and predominant photothermal effect produced in the assembly process. The drug release from DFTA could be triggered by photothermal excitation efficiently. ELISA analysis showed that DFTA + laser group significantly reduced intracellular GSH level through reactive oxygen species (ROS)-produced intracellular oxidative stress cascade amplification and photothermal therapy (PT)-mediated ROS production. Furthermore, in vivo antitumor efficiency evaluation showed that the tumor inhibition ratio of DFTA + laser was as high as 93.38 % even though the dosage of iron and DOX reduced by about 9 times and 1.5 times respectively. In summary, our study established a high-efficiency nanosystem based on triple combination therapy of chemotherapy, ferroptosis and PT, which might be a promising nanosystem for effective ER+ breast carcinoma therapy.

MRP1 modulators synergize with buthionine sulfoximine to exploit collateral sensitivity and selectively kill MRP1-expressing cancer cells

Members of the ABC transporter family, particularly P-glycoprotein (P-gp, ABCB1), breast cancer resistance protein (BCRP, ABCG2) and multidrug resistance protein 1 (MRP1, ABCC1) are well characterized mediators of multidrug resistance, however their pharmacological inhibition has so far failed as a clinical strategy. Harnessing collateral sensitivity, a form of synthetic lethality where cells with acquired multidrug resistance exhibit hypersensitivity to unrelated agents, may be an alternative approach to targeting multidrug resistant tumour cells. We characterized a novel small molecule modulator that selectively enhanced MRP1-dependent efflux of reduced glutathione (GSH), an endogenous MRP1 substrate. Using cell lines expressing high levels of endogenous MRP1 from three difficult to treat cancer types—lung cancer, ovarian cancer and high-risk neuroblastoma—we showed that the MRP1 modulator substantially lowered intracellular GSH levels as a single agent. The effect was on-target, as MRP1 knockdown abolished GSH depletion. The MRP1 modulator was synergistic with the GSH synthesis inhibitor buthionine sulfoximine (BSO), with the combination exhausting intracellular GSH, increasing intracellular reactive oxygen species (ROS) and abolishing clonogenic capacity. Clonogenicity was rescued by the ROS scavenger N-acetylcysteine, implicating GSH depletion in the effect. The MRP1 modulator in combination with BSO also strongly sensitized cancer cells to MRP1-substrate chemotherapeutic agents, particularly arsenic trioxide, and was more effective than either the MRP1 modulator or BSO alone. GSH-depleting MRP1 modulators may therefore provide an enhanced therapeutic window to treat chemo-resistant MRP1-overexpressing pediatric and adult cancers.

ATF3 promotes erastin-induced ferroptosis by suppressing system Xc\u2013

The amino acid antiporter system Xc− is important for the synthesis of glutathione (GSH) that functions to prevent lipid peroxidation and protect cells from nonapoptotic, iron-dependent death (i.e., ferroptosis). While the activity of system Xc− often positively correlates with the expression level of its light chain encoded by SLC7A11, inhibition of system Xc− activity by small molecules (e.g., erastin) causes a decrease in the intracellular GSH level, leading to ferroptotic cell death. How system Xc− is regulated during ferroptosis remains largely unknown. Here we report that activating transcription factor 3 (ATF3), a common stress sensor, can promote ferroptosis induced by erastin. ATF3 suppressed system Xc−, depleted intracellular GSH, and thereby promoted lipid peroxidation induced by erastin. ATF3 achieved this activity through binding to the SLC7A11 promoter and repressing SLC7A11 expression in a p53-independent manner. These findings thus add ATF3 to a short list of proteins that can regulate system Xc− and promote ferroptosis repressed by this antiporter.

Effects of Hyaluronidase during In Vitro Maturation on Maturation and Developmental Competence in Porcine Oocytes

The aim of this study was to investigate effects of hyaluronidase during IVM on oocyte maturation, oxidative stress status, expression of cumulus expansion-related (PTX, pentraxin; GJA1, gap junction protein alpha 1; PTGS2, prostaglandin-endoperoxide synthase 2) and fatty acid metabolism-related (FADS1, delta-6 desaturase; FADS2, delta-5 desaturase; PPARα, peroxisome proliferator-activated receptor-alpha) mRNA, and embryonic development of porcine oocytes. The cumulus-oocyte complexes (COCs) were incubated with 0.1 mg/mL hyaluronidase for 44 h. Cumulus expansion was measured at 22 h after maturation. At 44 h after maturation, nuclear maturation, intracellular glutathione (GSH) and reactive oxygen species (ROS) levels were measured. Gene expression in cumulus cells was analyzed using real time PCR. The cleavage rate and blastocyst formation were evaluated at Day 2 and 7 after insemination. In results, expansion of cumulus cells was suppressed by treatment of hyaluronidase at 22 h after maturation. Intracellular GSH level was reduced by hyaluronidase treatment (p < 0.05). On the other hand, hyaluronidase increased ROS levels in oocytes (p < 0.05). Only PTGS2 mRNA was enhanced in COCs by hyaluronidase (p < 0.05). Population of oocytes reached at metaphase II stage was higher in control group than hyaluronidase treated group (p < 0.05). Both of cleavage rate and blastocyst formation were higher in control group than hyaluronidase group (p < 0.05). Our present results showed that developmental competence of porcine oocytes could be reduce by hyaluronidase via inducing oxidative stress during maturation process and it might be associated with prostaglandin synthesis. Therefore, we suggest that suppression of cumulus expansion of COCs could induce oxidative stress and decrease nuclear maturation via reduction of GSH synthesis and it caused to decrease developmental competence of mammalian oocytes.

PS1113 OLIGOCLONALITY OF T-CELL RECEPTOR AND THE MAIN SUBPOPULATION COMPOSITION IN PATIENTS WITH APLASTIC ANEMIA AT DIFFERENT STAGES OF THE DISEASE

Background: Diamond Blackfan anemia (DBA) is a rare congenital bone marrow failure disorder, characterized by severe hypoplastic anemia, associated congenital anomalies, and an increased risk of developing cancer. Based on the underlying mutations in ribosomal protein (RP) genes, DBA is classified as a ribosomopathy. While disruptions in ribosomal biogenesis would be expected to have global effects, hematological in DBA are primarely of the erythroid lineage. Moreover, since cellular determinants following ribosomal stress are still not well understood, and most functional studies are performed in models representing only the most frequently affected RP-genes, the development of novel therapeutic strategies for all DBA patients has been been hampered. Here we propose, for the first time, a novel untargeted metabolomics approach to study anemia in DBA patient cells. Metabolomics is the large-scale, unbiased study of metabolites and their interactions within a biological system, and a powerful functional tool, directly reflecting the underlying biochemical activity and the state of cells. The DBA-metabolome can be used to study commonly deregulated cellular pathways during erythroid development, irrespective of the underlying molecular defect. Aims: Defining a metabolic signature of RP-deficiency in DBA, will 1. Increase our understanding of erythroid-specific defects in DBA, and 2. Contribute to the identification of potential novel therapeutic targets. Methods: Quantification of metabolites was performed on dried blood spots from 6 DBA patients (RPS26, RPL5, RPL9, unknown) by direct infusion high resolution mass spectrometry (DI-HRMS) followed by an untargeted metabolomics pipeline. For annotation, the Human Metabolome Database (HMDB) was used. Results were compared with dried blood spots samples of 10 healthy controls. Results: Preliminary data from 6 DBA patients with different genotypes demonstrate a significant increase in intermediates of the pentose phosphate pathway (PPP) (Figure 1A). This indicates that the PPP is upregulated in red blood cells from DBA patients, regardless of the underlying molecular defect. In line with this we observed a significant increase in erythrocyte reduced glutathione (GSH) content in 9 DBA patients (RPS26, RPL5, RPL11, unknown), compared to 12 controls (Figure 1B). In red blood cells the PPP mainly serves to provide the cell with sufficient amounts of NADPH to maintain high intracellular levels of GSH, which is needed to protect the cell against oxidative damage. Our results therefore support a role for oxidative stress response in DBA pathophysiology irrespective of the underlying genotype. Figure 1. (A) Increase in metabolites involved in PPP in DBA patient cells. (B) Increased GSH concentration in DBA patient erythrocytes.Summary/Conclusion: DBS metabolomics can be used as a novel functional tool to investigate deregulated cellular pathways in DBA irrespective of the underlying molecular defect. This will contribute to increased insights in cell type specific phenomena and DBA pathophysiology in general.

PS1111 QUANTIFICATION OF METABOLITES IN DRIED BLOOD SPOTS OF DIAMOND BLACKFAN ANEMIA PATIENTS REVEALS NOVEL INSIGHTS IN DEREGULATED CELLULAR PATHWAYS INVOLVED IN HYPOPLASTIC ANEMIA

Background:Diamond Blackfan anemia (DBA) is a rare congenital bone marrow failure disorder, characterized by severe hypoplastic anemia, associated congenital anomalies, and an increased risk of developing cancer. Based on the underlying mutations in ribosomal protein (RP) genes, DBA is classified as a ribosomopathy. While disruptions in ribosomal biogenesis would be expected to have global effects, hematological in DBA are primarely of the erythroid lineage. Moreover, since cellular determinants following ribosomal stress are still not well understood, and most functional studies are performed in models representing only the most frequently affected RP‐genes, the development of novel therapeutic strategies for all DBA patients has been been hampered. Here we propose, for the first time, a novel untargeted metabolomics approach to study anemia in DBA patient cells. Metabolomics is the large‐scale, unbiased study of metabolites and their interactions within a biological system, and a powerful functional tool, directly reflecting the underlying biochemical activity and the state of cells. The DBA‐metabolome can be used to study commonly deregulated cellular pathways during erythroid development, irrespective of the underlying molecular defect.Aims:Defining a metabolic signature of RP‐deficiency in DBA, will 1. Increase our understanding of erythroid‐specific defects in DBA, and 2. Contribute to the identification of potential novel therapeutic targets.Methods:Quantification of metabolites was performed on dried blood spots from 6 DBA patients (RPS26, RPL5, RPL9, unknown) by direct infusion high resolution mass spectrometry (DI‐HRMS) followed by an untargeted metabolomics pipeline. For annotation, the Human Metabolome Database (HMDB) was used. Results were compared with dried blood spots samples of 10 healthy controls.Results:Preliminary data from 6 DBA patients with different genotypes demonstrate a significant increase in intermediates of the pentose phosphate pathway (PPP) (Figure 1A). This indicates that the PPP is upregulated in red blood cells from DBA patients, regardless of the underlying molecular defect. In line with this we observed a significant increase in erythrocyte reduced glutathione (GSH) content in 9 DBA patients (RPS26, RPL5, RPL11, unknown), compared to 12 controls (Figure 1B). In red blood cells the PPP mainly serves to provide the cell with sufficient amounts of NADPH to maintain high intracellular levels of GSH, which is needed to protect the cell against oxidative damage. Our results therefore support a role for oxidative stress response in DBA pathophysiology irrespective of the underlying genotype.Figure 1. (A) Increase in metabolites involved in PPP in DBA patient cells. (B) Increased GSH concentration in DBA patient erythrocytes.Summary/Conclusion:DBS metabolomics can be used as a novel functional tool to investigate deregulated cellular pathways in DBA irrespective of the underlying molecular defect. This will contribute to increased insights in cell type specific phenomena and DBA pathophysiology in general.image

Effects of sublethal concentrations of the antifouling biocide Sea-Nine on biochemical parameters of the marine polychaete Perinereis aibuhitensis

Sea-Nine™ 211 is an emerging biocide that has an adverse impact on aquatic environments. In this study, the marine polychaete Perinereis aibuhitensis was exposed to Sea-Nine (0.1, 1, and 10 μg L−1), and acute toxicity and biochemical responses such as changes in the intracellular contents of malondialdehyde (MDA) and glutathione (GSH) and enzymatic activities of catalase (CAT), superoxide dismutase (SOD), glutathione peroxidase (GPx), glutathione reductase (GR), glutathione S-transferase (GST), and acetylcholinesterase (AChE) were evaluated over a period of 14 d. Determined median lethal doses, LC50 were 268 μg L−1, 142 μg L−1, and 55 μg L−1 at 24 h, 96 h, and 14 d, respectively. The MDA content increased significantly in a dose- and time-dependent manner, indicative of lipid peroxidation-related oxidative damage. Significantly higher intracellular GSH levels and antioxidant defense-related enzyme (CAT, SOD, GPx, GR, and GST) activities were observed after exposure to 10 μg L−1 Sea-Nine. In contrast, Sea-Nine treatment significantly reduced AChE activity at the highest concentration of Sea-Nine used (10 μg L−1). Taken together, these results indicate that sublethal concentrations of Sea-Nine are toxic to marine polychaetes through potential lipid peroxidation, induction of oxidative stress, and modulation of the cholinergic system. Our results can contribute to biomonitoring of aquatic environments and ecotoxicological research through the measurements of polychaete cellular defenses against waterborne biocides.

Cystine/glutamate antiporter xCT (SLC7A11) facilitates oncogenic RAS transformation by preserving intracellular redox balance

The RAS family of proto-oncogenes are among the most commonly mutated genes in human cancers and predict poor clinical outcome. Several mechanisms underlying oncogenic RAS transformation are well documented, including constitutive signaling through the RAF-MEK-ERK proproliferative pathway as well as the PI3K-AKT prosurvival pathway. Notably, control of redox balance has also been proposed to contribute to RAS transformation. However, how homeostasis between reactive oxygen species (ROS) and antioxidants, which have opposing effects in the cell, ultimately influence RAS-mediated transformation and tumor progression is still a matter of debate and the mechanisms involved have not been fully elucidated. Here, we show that oncogenic KRAS protects fibroblasts from oxidative stress by enhancing intracellular GSH levels. Using a whole transcriptome approach, we discovered that this is attributable to transcriptional up-regulation of xCT, the gene encoding the cystine/glutamate antiporter. This is in line with the function of xCT, which mediates the uptake of cystine, a precursor for GSH biosynthesis. Moreover, our results reveal that the ETS-1 transcription factor downstream of the RAS-RAF-MEK-ERK signaling cascade directly transactivates the xCT promoter in synergy with the ATF4 endoplasmic reticulum stress-associated transcription factor. Strikingly, xCT was found to be essential for oncogenic KRAS-mediated transformation in vitro and in vivo by mitigating oxidative stress, as knockdown of xCT strongly impaired growth of tumor xenografts established from KRAS-transformed cells. Overall, this study uncovers a mechanism by which oncogenic RAS preserves intracellular redox balance and identifies an unexpected role for xCT in supporting RAS-induced transformation and tumorigenicity.

Discriminative Detection of Glutathione in Cell Lysates Based on Oxidase-Like Activity of Magnetic Nanoporous Graphene.

As the most abundant intracellular biothiol, glutathione (GSH) plays a central role in many cellular functions and has been proved to be associated with numerous clinical diseases. Nevertheless, it is still a challenge to detect GSH over other mercaptoamino acids owing to their similar structures and activities. In this paper, magnetic nanoporous graphene (MNPG) nanocomposites were prepared for the first time through partial combustion of graphene oxide (GO) and ferric chloride. Due to the combination of porous graphene and magnetic nanoparticles, the MNPG nanocomposites exhibited large specific surface area, fast mass, and electron transport kinetics, resulting in remarkable oxidase mimic activity and easy separation. On the basis of the inhibition effect of GSH on the MNPG-catalyzed oxidation of thiamine, a novel and simple method for fluorescence determination of GSH was established. The sensor displayed a good linear response in the range of 0.2-20 μM toward GSH with a limit of detection of 0.05 μM. High sensitivity and selectivity facilitated its practical application for discriminative detection of GSH levels in PC12 cell lysates. The presented assay will be a simple and powerful tool to monitor intracellular GSH levels for biomedical diagnosis. Furthermore, the MNPG nanocomposites will provide insights to construct nanoporous graphene-based hybrids and push forward the advancement of porous graphene for wide applications.

Non-invasive Use of Positron Emission Tomography to Monitor Diethyl maleate and Radiation-Induced Changes in System xC- Activity in Breast Cancer.

The system xC- transporter is upregulated in cancer cells in response to oxidative stress (OS). 5-[18F]fluoroaminosuberic acid ([18F]FASu) has been reported as a novel positron emission tomography (PET) imaging agent, targeting system xC-. The goal of this study was to evaluate the utility of [18F]FASu in monitoring cellular response to diethyl maleate (DEM) and radiation-induced OS fluctuations. [18F]FASu uptake by breast cancer cells was studied in correlation to OS biomarkers: glutathione (GSH) and reactive oxygen species (ROS), as well as transcriptional and translational levels of xCT (the functional subunit of xC-). System xC- inhibitor, sulfasalazine (SSZ), and small interfering RNA (siRNA) knockdown were used as negative controls. Radiotracer uptake was evaluated in three breast cancer models: MDA-MB-231, MCF-7, and ZR-75-1, at two-time points (1h and 16h) following OS induction. In vivo [18F]FASu imaging and biodistribution were performed using MDA-MB-231 xenograft-bearing mice at 16 and 24h post-radiation treatment. [18F]FASu uptake was positively correlated to intracellular GSH and SLC7A11 expression levels, and radiotracer uptake was induced both by radiation treatment and by DEM at time points longer than 3h. In an in vivo setting, there was no statistically significant uptake difference between irradiated and control tumors. [18F]FASu is a specific system xC- PET radiotracer and as such it can be used to monitor system xC- activity due to OS. As such, [18F]FASu has the potential to be used in therapy response monitoring by PET. Further optimization is required for in vivo application.

Lenvatinib-zinc phthalocyanine conjugates as potential agents for enhancing synergistic therapy of multidrug-resistant cancer by glutathione depletion

The therapeutic efficacy of targeted therapy is dramatically hindered by multidrug resistance (MDR) because of elevated GSH levels. Thus, depletion of intracellular GSH level is highly desirable for targeted-therapeutic agents to reverse tumor drug resistance. In this study, a photosensitive multifunctional conjugate ZnPc-C8-Len, in which lenvatinib (a VEGFR inhibitor) is linked to a photosensitizer ZnPc through an alkyl chains, was synthesized to realize photodynamic therapy to reverse multidrug resistance and enhanced antitumor therapy. Upon the irradiation, ZnPc-C8-Len could generate ROS to deplete intracellular GSH. The decreased GSH would enhance apoptotic cell death by Bcl-2/caspase 3 pathway and reduce expression of P-gp to reverse lenvatinib resistance. Moreover, through PEG2000-PLA2000 encapsulation, ZnPc-C8-Len NPs displayed significantly enhanced tumor accumulation and excellent in vivo antitumor activity. And the fluorescence characteristics of ZnPc-C8-Len could monitor the changes of nanoparticles in vivo in real time to guide when and where to conduct the subsequent therapy. As a result, conjugate ZnPc-C8-Len had an outstanding capability to enhance synergistic therapy of multidrug-resistant cancer by glutathione depletion. And the approach reported here provide a promising strategy in development of conjugate integrated targeted therapy with photodynamic therapy to reverse targeted drug multidrug resistance and enhance synergistic therapy.

EF24 Suppresses Cholangiocellular Carcinoma Progression, Inhibits STAT3 Phosphorylation, and Induces Apoptosis via ROS-Mediated Oxidative Stress.

Therapeutic options for advanced stage cholangiocellular carcinoma (CCC) are very limited as of today and patients carry an exceptionally poor overall prognosis. In recent years, increasing evidence has been accumulated to suggest that malignant cells widely show increased intrinsic ROS levels and exhibit altered redox profiles as compared to normal counterparts, opening up potential avenues for therapeutic intervention. This study provides preclinical experimental evidence of therapeutic activity of the curcumin analog EF24 in cholangiocarcinoma models. In CCC cell lines, EF24 inhibited cell viability and induced apoptosis through excessive ROS generation. Moreover, administration of EF24 led to depletion of total intracellular GSH levels, induced mitochondrial depolarization, and abrogated STAT3 phosphorylation. Of interest, these effects were readily averted by treating the cells with exogenous antioxidants such as N-acetyl cysteine (NAC) or glutathione monoethyl ester (GEE). In vivo, EF24, solubilized using a cyclodextrin formulation, significantly suppressed the growth of tumor xenografts without exhibiting any toxic adverse effects. Immunohistochemical analysis of extracted tumor tissues demonstrated reduced nuclear staining for Ki-67 and downregulation of phospho-STAT3 as well as strong staining for oxidative stress biomarker 8-OHdG. Therefore, the data presented here suggest EF24 as potential therapeutic compound against CCC which might act at least to some extent through ROS-induced oxidative damage, subsequently inducing apoptosis. Further evaluation of this approach should be carried out in future follow-up studies.

Endosulfan Induces Embryotoxicity in the Marine Medaka Oryzias javanicus

The present study aimed to investigate the embryotoxicity of endosulfan (ES) on marine medaka Oryzias javanicus embryos using several toxicity endpoints such as measurements of oxidative stress, DNA damage, cell death, and AChE enzyme activity upon different concentrations of waterborne ES. Fertilized marine medaka embryos (3 hpf) were exposed to three concentrations of ES (86.2, 172.4, and 862 µg L−1). Oxidative stress parameters [e.g. intracellular reactive oxygen species (ROS), malondialdehyde (MDA), and glutathione (GSH)], nitric oxide (NO) production, DNA damage, cell death, and acetylcholinesterase (AChE) activity were analyzed. The highest concentration 862 µg L−1 of ES significantly increased the intracellular ROS, MDA, GSH, and NO in the embryos compared to control groups. Endosulfan increased DNA damage and cell death in the marine medaka embryos during post fertilization period. In the case of AChE, a significant inhibition was detected in the 862 µg L−1 ES-treated medaka embryo. In addition, intracellular GSH level was increased by 172.2 µg L−1 ES exposure in medaka embryos. Our results suggest that ES may induce strong cell death through increase of oxidative stress and DNA damage. Inhibition of AChE would represent potential neurotoxicity on cholinergic system of marine medaka embryos. The parameters tested in this study would be a useful biomarker for risk assessment of ES as an early signal of its toxicity.

A polyphenol-rich extract from an oenological oak-derived tannin influences in vitro maturation of porcine oocytes

Tannins have been demonstrated to have antioxidant and various health benefit properties. The aim of this study was to determine the effect of an ethanol extract (TRE) of a commercial oenological tannin (Quercus robur toasted oak wood, Tan’Activ R®) on female gamete using an in vitro model of pig oocyte maturation (IVM) and examining nuclear maturation, cytoplasmic maturation, intracellular GSH and ROS levels and cumulus cell steroidogenesis.To this aim, during IVM performed in medium either supplemented (IVM A) or not supplemented (IVM B) with cysteine and β-mercaptoethanol, TRE was added at different concentrations (0, 1, 5, 10, 20 μg/ml).The addition of TRE at all the concentration tested to either IVM A or IVM B, did not influence oocyte nuclear maturation. When IVM was performed in IVM A, no effect was induced on cytoplasmic maturation by TRE at the concentration of 1, 5 and 10 μg/ml, while TRE 20 μg/ml significantly reduced the penetration rate after IVF (p < 0.05) and the blastocyst rate after parthenogenetic activation (p < 0.01). Oocyte maturation in IVM B, compared to IVM A group, decreased GSH (p < 0.001) and increased ROS (p < 0.01) intracellular levels and in turn impaired oocyte cytoplasmic maturation reducing the ability to sustain male pronuclear formation after IVM (p < 0.001) and the developmental competence after parthenogenetic activation (p < 0.001). TRE supplementation to IVM B significantly reduced ROS production (5, 10, 20 μg/ml TRE) to levels similar to IVM A group, and increased GSH levels (10, 20 μg/ml TRE) compared to IVM B (p < 0.05) without reaching those of IVM A group. TRE supplementation to IVM B at the concentrations of 1, 5 and 10 μg/ml significantly improved (p < 0.001) oocyte cytoplasmic maturation enhancing the ability to sustain male pronuclear formation without reaching, however, IVM A group levels.TRE addition at all the concentration tested to both IVM A and IVM B, did not induce any effect on E2 and P4 secretion by cumulus cells suggesting that the biological effect of the ethanol extract is not exerted thought a modulation of cumulus cell steroidogenesis.In conclusion, TRE, thanks to its antioxidant activity, was partially able to reduce the negative effect of the absence of cysteine and β-mercaptoethanol in IVM B, while TRE at high concentration in IVM A was detrimental for oocyte cytoplasmic maturation underlying the importance of maintaining a balanced redox environment during oocyte maturation.

PRIMA-1MET-induced neuroblastoma cell death is modulated by p53 and mycn through glutathione level

BackgroundNeuroblastoma is the most common extracranial solid tumor in children. This cancer has a low frequency of TP53 mutations and its downstream pathway is usually intact. This study assessed the efficacy of the p53 activator, PRIMA-1MET, in inducing neuroblastoma cell death.MethodsCellTiter 2.0 was used to study susceptibility and specificity of NB cell lines to PRIMA-1MET. Real-time PCR and western blot were used to assess the most common p53 transactivation targets. Induction of p53 and Noxa, and inhibition of Cas3/7, were used to assess impact on cell death after PRIMA-1MET treatment. Flow cytometry was used to analyze cell cycle phase and induction of apoptosis, reactive oxygen species, and the collapse of mitochondrial membrane potential.ResultsNeuroblastoma cell lines were at least four times more susceptible to PRIMA-1MET than were primary fibroblasts and keratinocyte cell lines. PRIMA-1MET induced cell death rapidly and in all cell cycle phases. Although PRIMA-1MET activated p53 transactivation activity, p53’s role is likely limited because its main targets remained unaffected, whereas pan-caspase inhibitor demonstrated no ability to prevent cell death. PRIMA-1MET induced oxidative stress and modulated the methionine/cysteine/glutathione axis. Variations of MYCN and p53 modulated intracellular levels of GSH and resulted in increased/decreased sensitivity of PRIMA-1MET. PRIMA-1MET inhibited thioredoxin reductase, but the effect of PRIMA-1MET was not altered by thioredoxin inhibition.ConclusionsPRIMA-1MET could be a promising new agent to treat neuroblastoma because it demonstrated good anti-tumor action. Although p53 is involved in PRIMA-1MET-mediated cell death, our results suggest that direct interaction with p53 has a limited role in neuroblastoma but rather acts through modulation of GSH levels.

NOX1/NADPH oxidase regulates the expression of multidrug resistance-associated protein 1 and maintains intracellular glutathione levels.

The involvement of superoxide-generating NADPH oxidase (NOX) in the cytotoxic effects of cigarette smoke extracts has been documented. However, the underlying molecular mechanisms and NOX isoform involved have not been fully clarified. Among the different NADPH oxidase isoforms identified so far, NOX1 and NOX4 were found to be expressed in rat H9c2 cardiomyocytes. When H9c2 cells were exposed to acrolein or methyl vinyl ketone (MVK), major toxic components of cigarette smoke extracts, a dose-dependentdecline in cell viability was observed. Unexpectedly, disruption of Nox1 as well as Nox4 significantly exacerbated cytotoxicity induced by acrolein or MVK. Compared with Nox4-disrupted cells, Nox1-disrupted cells were more vulnerable to acrolein and MVK at lower concentrations. Disruption of Nox1 markedly attenuated the levels of total and reduced glutathione (GSH) in H9c2 clones. Reduction in the cystine level in the culture medium to deplete intracellular GSH significantly exacerbated acrolein or MVK-induced cytotoxicity. Nox1 disruption neither attenuated the level of glutamate-cystine antiporter protein nor the activity of glutamate-cysteine ligase, both rate-limiting factors for GSH synthesis. On the other hand, increased expression of multidrug resistance-associated protein 1 (MRP1), which mediates glutathione efflux, was demonstrated in Nox1-disrupted cells. The augmented toxicity of acrolein and MVK in these cells was partially but significantly blunted in the presence of an MRP1 inhibitor, reversan. Taken together, these results show that NOX1/NADPH oxidase regulates the expression of MRP1 to maintain intracellular GSH levels in cardiomyocytes and protect against cytotoxic components of cigarette smoke extracts. A novel crosstalk between NOX1 and MRP1 was demonstrated in this study.