Intracellular GSH Levels Research Articles

Synthesis and biological evaluation of capsaicin analogues as antioxidant and neuroprotective agents.

Capsaicin and its analogues 3a-3q were designed and synthesized as potential new antioxidant and neuroprotective agents. Many analogues exhibited good antioxidant effects, and some showed more potent free radical scavenging activities than the positive drug quercetin (IC50 = 8.70 ± 1.75 μM for DPPH assay and 13.85 ± 2.87 μM for ABTS assay, respectively). The phenolic hydroxyl of capsaicin analogues was critical in determining antioxidant activity. Among these compounds, 3k displayed the most potent antioxidant activity. Cell vitality tests revealed that the representative compound 3k was good at protecting cells from H2O2-induced oxidative damage at low concentrations (cell viability increased to 90.0 ± 5.5% at 10 μM). In addition, the study demonstrated that 3k could reduce intracellular ROS accumulation and increase GSH levels to prevent H2O2-induced oxidative stress in SY5Y cells. In the mitochondrial membrane potential assay, 3k significantly increased the MMP level of SY5Y cells treated with H2O2 and played an anti-neuronal cell death role. These results provide a promising strategy to develop novel capsaicin analogues as potential antioxidant and neuroprotective agents.

Application of singlet oxygen-activatable nanocarriers to boost X-ray-induced photodynamic therapy and cascaded ferroptosis for breast cancer treatment.

Ferroptosis has appealing antitumor potential that is mainly based on the accumulation of lipid peroxide to a lethal level. The cytotoxic singlet oxygen (1O2) generated from nanoscale X-ray-induced photodynamic therapy (X-PDT) may facilitate glutathione (GSH) depletion and further activate ferroptosis. To realize combined X-PDT and ferroptosis, a nanocarrier (D-NPVR) was engineered with a hyperbranched copolymer with 1O2-sensitive linkers, where both the photosensitizer (verteporfin) and ferroptosis inducer RAS-selective lethal small molecule 3 (RSL3) were encapsulated. Upon X-ray radiation, D-NPVR could produce a large amount of 1O2 for apoptosis. Subsequently, 1O2 triggered D-NP dissociation by cleavage of 1,2-bis(2-hydroxyethylthio)ethylene bonds to boost payload release and decrease levels of intracellular GSH via thiol oxidation. Liberated RSL3 is a covalent inhibitor for glutathione peroxide 4 (GPX4), which is responsible for detoxifying lipid peroxides to lipid alcohols with GSH assistance, and both 1O2-induced GSH depletion and GPX4 inactivation thereby produced ferroptotic cell death. Tumor growth inhibition in murine 4T1 tumor-bearing mice demonstrated that D-NPVR produced pronounced therapeutic efficiency where ferroptosis induction was supported by the GPX4 content and expression. This study highlights the contribution of 1O2-sensitive nanocarriers for promoting the potency of combined X-PDT and ferroptosis.

Gold nanoparticle doped Cuhemin nanosheets with a remodeling tumor microenvironment for multiple radiotherapy sensitization.

Effective radiosensitizers are urgently needed due to the serious negative effects that high radiation doses might have. We created an integrated nano-system (Cuhemin-Au) made of Cuhemin nanosheets and Au nanoparticles (Au NPs) for sensitizing radiotherapy to solve this issue. This system can manifest enzyme-like activities to universally suppress the resistance pathways in breast cancer cells for amplifying radiation damage. Cuhemin-Au NPs increase the energy deposition of radiation owing to the high X-ray attenuation coefficient of Au. In addition, Cuhemin-Au has peroxidase (POD)-like and glucose oxidase (Gox)-like activity, and can also consume intracellular GSH, which can reduce intracellular GSH levels to reduce tumor cells' capacity to repair DNA and deplete intracellular glucose via their characteristic Gox-like catalytic activities, which can cause an increase in the oxidative stress and further produce H2O2. Cuhemin-Au then produced ˙OH, which upsets redox equilibrium and destroys mitochondria, leading to radiation sensitivity, after reacting with enough hydrogen peroxide in tumor cells. Cuhemin-Au combined with low dose RT (4 Gy) could significantly limit tumor development with fewer adverse effects, according to in vivo and in vitro experiments. This platform generated a fresh concept for the construction of a radiotherapy sensitization system and accomplished synergistic radiotherapy sensitization.

Antioxidant effect of ergothioneine on in vitro maturation of porcine oocytes.

Ergothioneine (EGT) is a natural amino acid derivative in various animal organs and is a bioactive compound recognized as a food and medicine. This study examined the effects of EGT supplementation during the in vitro maturation (IVM) period on porcine oocyte maturation and subsequent embryonic development competence after in vitro fertilization (IVF). Each EGT concentration (0, 10, 50, and 100 µM) was supplemented in the maturation medium during IVM. After IVM, nuclear maturation, intracellular glutathione (GSH), and reactive oxygen species (ROS) levels of oocytes were investigated. In addition, the genes related to cumulus function and antioxidant pathways in oocytes or cumulus cells were investigated. Finally, this study examined whether EGT could affect embryonic development after IVF. After IVM, the EGT supplementation group showed significantly higher intracellular GSH levels and significantly lower intracellular ROS levels than the control group. Moreover, the expression levels of hyaluronan synthase 2 and Connexin 43 were significantly higher in the 10 µM EGT group than in the control group. The expression levels of nuclear factor erythroid 2-related factor 2 (Nrf2) and NAD(P)H quinone dehydrogenase 1 (NQO1) were significantly higher in the oocytes of the 10 µM EGT group than in the control group. In the assessment of subsequent embryonic development after IVF, the 10 µM EGT treatment group improved the cleavage and blastocyst rate significantly than the control group. Supplementation of EGT improved oocyte maturation and embryonic development by reducing oxidative stress in IVM oocytes.

GSH-depleting metal-polyphenol-network nanoparticles with dual enzyme activities induce enhanced ferroptosis.

Ferroptosis is a non-apoptotic form of regulated cell death. The efficiency of ferroptosis is restrained in the tumor microenvironment (TME) by overexpression of glutathione (GSH) and insufficient production of hydrogen peroxide (H2O2). In this work, theranostic nanoparticles Ce-aMOFs@Fe3+-EGCG, termed MEFs, are developed by coating uniform Ce-based amorphous metal-organic frameworks (Ce-aMOFs) with epigallocatechin gallate (EGCG) and Fe3+. Fe3+ is chelated by the adjacent phenol hydroxyl groups in EGCG. In the tumor cell interior, overexpressed GSH and weak acidic medium degrade the coating to release Fe3+ and EGCG accompanied by exposure of Ce-aMOFs. Fe3+ and EGCG consume GSH along with turning Fe3+ into Fe2+. Ce-aMOFs act as a nanozyme possessing dual-enzymatic activities, i.e. superoxide dismutase (SOD)- and phosphatase-like activities. In the TME, Ce-aMOFs catalyze the conversion of endogenous superoxide (O2˙-) into H2O2, and Fe2+ catalyzes H2O2 to generate toxic hydroxyl radicals (˙OH), which may further induce tumor cell death through ferroptosis. In addition, the phosphatase-like activity of Ce-aMOFs may sustainably dephosphorylate NADPH and effectively inhibit intracellular biosynthesis of GSH. Therefore, MEFs ensure down-regulation of intracellular GSH levels and up-regulation of oxidative pressure, which enhance the ferroptosis effect.

Combination with vorinostat enhances the antitumor activity of cisplatin in castration-resistant prostate cancer by inhibiting DNA damage repair pathway and detoxification of GSH.

Like DNA methylation, histone modifications are considered important processes for epigenetic alterations in gene function, and abnormally high expression of histone deacetylases (HDACs) plays a key role in many human diseases. In addition to regulating the acetylation levels of histone and non-histone proteins and gene transcription, HDAC inhibitors as antitumor drugs can also affect the DNA damage repair (DDR) pathway in tumor cells. Prostate cancer (PCa) is one of the most heritable malignancies in which DDR pathway defects can be detected in a considerable proportion of cases. Such defects are more prevalent in castration-resistant prostate cancer (CRPC) and are highly enriched in metastatic lesions. There is currently evidence that DDR pathway-deficient PCa is associated with high-risk biological behaviors and response sensitivity to platinum-based chemotherapy. Platinum-based drugs have been used in multiple clinical trials as monotherapy or in combination with other chemotherapeutic agents for the treatment of CRPC. This study evaluated the combined anticancer effect of (cisplatin) CDDP and the HDAC inhibitors vorinostat (SAHA) on three androgen-dependent cell lines PC-3, DU-145, and C4-2B in vitro. The efficacy and safety of SAHA combined with CDDP in the treatment of CRPC were further verified through animal experiments. The combination of the two drugs increases cytotoxic effects by increasing DNA damage. Our results showed that the SAHA could not only reduce the expression of homologous recombinant repair proteins BRCA2, BRCA1, PARP1, and RAD51, but also decrease enzymes that Reduce the key enzymes of GSH biosynthesis, GSS and GCLC, and GSTP1 which can catalyze the binding of GSH to cisplatin. The intracellular GSH level also decreased with the increase of SAHA concentration, at the same time, the content of intracellular Pt element. The combination of CDDP and SAHA can produce synergistic anticancer effects in androgen-independent PCa cells in vitro and in vivo. Our results open up a new avenue for the effective treatment of CRPC. To optimize the chemotherapy regimen for patients with advanced PCa, it is necessary to further study the molecular mechanism of platinum drugs, HDAC inhibitors, and their combined action.

New insights into extracellular and intracellular redox status in COVID-19 patients

BackgroundThe imbalance of redox homeostasis induces hyper-inflammation in viral infections. In this study, we explored the redox system signature in response to SARS-COV-2 infection and examined the status of these extracellular and intracellular signatures in COVID-19 patients. MethodThe multi-level network was constructed using multi-level data of oxidative stress-related biological processes, protein-protein interactions, transcription factors, and co-expression coefficients obtained from GSE164805, which included gene expression profiles of peripheral blood mononuclear cells (PBMCs) from COVID-19 patients and healthy controls. Top genes were designated based on the degree and closeness centralities. The expression of high-ranked genes was evaluated in PBMCs and nasopharyngeal (NP) samples of 30 COVID-19 patients and 30 healthy controls. The intracellular levels of GSH and ROS/O2• − and extracellular oxidative stress markers were assayed in PBMCs and plasma samples by flow cytometry and ELISA. ELISA results were applied to construct a classification model using logistic regression to differentiate COVID-19 patients from healthy controls. ResultsCAT, NFE2L2, SOD1, SOD2 and CYBB were 5 top genes in the network analysis. The expression of these genes and intracellular levels of ROS/O2• − were increased in PBMCs of COVID-19 patients while the GSH level decreased. The expression of high-ranked genes was lower in NP samples of COVID-19 patients compared to control group. The activity of extracellular enzymes CAT and SOD, and the total oxidant status (TOS) level were increased in plasma samples of COVID-19 patients. Also, the 2-marker panel of CAT and TOS and 3-marker panel showed the best performance. ConclusionSARS-COV-2 disrupts the redox equilibrium in immune cells and the upper respiratory tract, leading to exacerbated inflammation and increased replication and entrance of SARS-COV-2 into host cells. Furthermore, utilizing markers of oxidative stress as a complementary validation to discriminate COVID-19 from healthy controls, seems promising.

Synergistic In Vitro Anticancer Toxicity of Pulsed Electric Fields and Glutathione

Despite continuous advancement in skin cancer therapy, the disease is still fatal in many patients, demonstrating the need to improve existing therapies, such as electrochemotherapy (ECT). ECT can be applied in the palliative or curative setting and is based on the application of pulsed electric fields (PEF), which by themselves exerts none to low cancer toxicity but become potently toxic when combined with low-dosed chemotherapeutics such as bleomycin and cisplatin. Albeit their favorable side-effect profiles, not all patients respond to standard ECT, and some responders experience tumor recurrence. To identify potential adjuvant or alternative agents to standard electrochemotherapy, we explored the possibility of combining PEF with a physiological compound, glutathione (GSH), to amplify anticancer toxicity. GSH is an endogenous antioxidant and is available as a dietary supplement. Surprisingly, neither GSH nor PEF mono treatment but GSH + PEF combination treatment exerted strong cytotoxic effects and declined metabolic activity in four skin cancer cell lines in vitro. The potential applicability to other tumor cells was verified by corroborating results in two leukemia cell lines. Strikingly, GSH + PEF treatment did not immediately increase intracellular GSH levels, while levels 24 h following treatment were enhanced. Similar tendencies were made for intracellular reactive oxygen species (ROS) levels, while extracellular ROS increased following combination treatment. ROS levels and the degree of cytotoxicity could be partially reversed by pre-incubating cells with the NADPH-oxidase (NOX) inhibitor diphenyleneiodonium (DPI) and the H2O2-degrading enzyme catalase. Collectively, our findings suggest a promising new "endogenous" drug to be combined with PEF for future anticancer research approaches.

Suppressed Expression of ASCT1 Contributes to the Maintenance of Leukemia Stemness Via the Enhancement of Antioxidant Capacity

Introduction: Acute myeloid leukemia (AML) is one of the most common hematological malignancies derived from self-renewing leukemic stem cells (LSCs). LSCs represent a small cell fraction harboring the self-renewal capacity and potential to give rise to mature leukemic cells and play a central role in the relapse of AML. In general, LSCs can be enriched within reactive oxygen species (ROS) low fraction. Glutathione (GSH), a major antioxidant small molecule, plays a central role in the maintenance of lower ROS levels; however, the molecular mechanisms of GSH maintenance in LSCs remain unclear. Result: First, we evaluated the intracellular GSH levels in the CD34+ immature fraction, in which LSCs can be enriched and CD34- mature AML cells from the identical patients (n=#). CD34+AML cells contained significantly higher levels of GSH. To clarify the molecular mechanisms how CD34+ primitive AML cells could maintain high GSH level, we analyzed the expression of cysteine transporters, because cysteine uptake is the rate-limiting step of GSH synthesis. In human, three amino acid transporters including ASCT1, ASC1 and xCT are known as cysteine/cystine transporters. The expression levels of ASC1 and xCT were not significantly different among the three cellular fractions of AML: the most immature CD34+CD38-, CD34+CD38+, and more differentiated CD34- cells. However, we found that CD34+CD38- LSCs completely lacked the expression of ASCT1 in all the cases examined (n=#). The expression of ASCT1 increased along with cellular differentiation; more differentiated CD34- AML cells expressed the higher ASCT1 expression than the most immature CD34+CD38- cells. We, therefore, hypothesized that the different expression pattern of ASCT1 might be associated with higher GSH levels in LSCs through the regulation of cellular cysteine levels. ASCT1 is known to transport neutral amino acids (alanine, serine, cysteine, threonine), no role for ASCT1 in leukemia has ever been investigated. We found that ASCT1 was highly expressed in M4/M5 monocytic leukemias according to the FAB classification. Analysis of beatAML RNA-seq data showed that patients with low ASCT1 expression had a worse prognosis. We found that primary AML cells with the higher ASCT1 expression had reduced intracellular GSH as compared to those with lower ASCT1 expression (n=#), suggesting the association of ASCT1 expression and GSH levels in primary AML cells. To test this hypothesis, we knocked down (KD) the expression of ASCT1 using shRNAs in cell lines. KD of ASCT1 induced an increase in intracellular GSH compared to control cells, suggesting that ASCT1 decreases intracellular GSH by transporting cysteine extracellularly. To clarify the function of ASCT1 in human AML-LSCs in vivo, we performed xenotransplantation experiments. The identical number of FACS-purified ASCT1+ and ASCT- AML cells from the identical patients were transplanted into the irradiated NOD.Cg-PrkdcscidIl2rgtm1Wjl/SzJ (NSG) mice. We found that ASCT1- AML cells efficiently reconstituted human AML in vivo, whereas ASCT1+ AML cells completely failed in the engraftment in vivo in all the cases tested, suggesting that LSCs exclusively reside within ASCT1- AML cellular fraction. These results collectively suggest that LSCs maintain low ASCT1 expression, which is one of the molecular machineries to keep intracellular GSH high. Furthermore, only the ASCT1- fraction reconstitute AML in immunodeficient mice, indicating that suppression of ASCT1 is important for the maintenance of leukemia stemness.

A purine derivative, paraxanthine, promotes cysteine uptake for glutathione synthesis

Purine derivatives such as caffeine and uric acid have neuroprotective activities and are negatively correlated with the incidence of both Alzheimer's disease and Parkinson's disease. We have reported that an increment of intracellular glutathione (GSH) via cysteine uptake in neuronal cells is one of the mechanisms by which caffeine and uric acid confer neuroprotection. Here, we investigated whether caffeine metabolites such as paraxanthine, theophylline, theobromine, 1,7-dimethyluric acid and monomethylxanthines would increase cysteine uptake in mouse hippocampal slices. The metabolites were administered to hippocampal slices for 30 min at doses of 0, 10, or 100 μM, and then cysteine was added for 30 min. Paraxanthine, a major metabolite of caffeine, increased cysteine content in the slices, whereas the other metabolites did not. In vitro treatment with paraxanthine promoted cysteine uptake and increased GSH in HEK293 cells. The paraxanthine-induced cysteine uptake was inhibited by an excitatory amino-acid carrier-1 (EAAC1) inhibitor, and H2O2-induced cell damage was prevented by the paraxanthine treatment of SH-SY5Y cells. These results suggest that paraxanthine, an active metabolite of caffeine, acts to increase intracellular GSH levels via EAAC1 leading to neuroprotection.

Monitoring and recovery of hyperglycaemia-induced endothelial dysfunction with rheopheresis in diabetic lower extremity ulceration with hyperviscosity.

Rheopheresis is an extracorporeal haematotherapy that improves haemorheological status by filtering proteins that enhance blood viscosity. It also has anti-inflammatory effects by removing inflammatory cytokines. Our study aims to examine the effects of rheopheresis on the endothelial status in diabetic lower extremity ulceration. In vitro experiments were performed in a HUVEC model to mimic hyperglycaemic stress. We determined the changes in gene expression levels of IL-6, IL-8, TNF-alpha, endothelin convertase enzyme, ET-1, and NO synthase, as well as the ROS and intracellular GSH levels upon hyperglycaemia. In in vivo studies, two rheopheresis procedures were performed on seven patients with diabetic lower extremity ulceration with hyperviscosity, and we measured the changes in plasma concentrations of ET-1, TXB2, SOD enzyme activity, and extracellular components of the glutathione pool depending on treatments. Our results showed that hyperglycaemia increases endothelial expression of inflammatory cytokines, ET-1, and endothelin convertase enzyme, while NO synthase was decreased. As a result of rheopheresis, we observed decreased ET-1 and TXB2 concentrations in the plasma and beneficial changes in the parameters of the glutathione pool. To summarize our results, hyperglycaemia-induced oxidative stress and endothelial inflammation can be moderated by rheopheresis in diabetic lower extremity ulceration with hyperviscosity.

ATF3 Regulates Osteogenic Function by Mediating Osteoblast Ferroptosis in Type 2 Diabetic Osteoporosis.

Osteoporosis is a complication of type 2 diabetes, and it is characterized by reduced bone mass, augmented bone fragility, and increased risk of fracture, thus reducing patient quality of life, especially in the elderly. Ferroptosis has been implicated in the pathological process of type 2 diabetic osteoporosis (T2DOP), but the specific underlying mechanisms remain largely unknown. This study clarified the role of activating transcription factor 3 (ATF3) in T2DOP and explored its specific regulatory mechanism, providing a new treatment target for T2DOP. We cultured hFob1.19 cells in high glucose (HG, 35 mM) and knocked down ATF3 using short hairpin RNA (shRNA). We then measured cell viability, assessed morphology, quantified the expression of ATF3 and glutathione peroxidase 4 (GPX4), detected the levels of reactive oxygen species (ROS) and lipid peroxides, and determined the osteogenic function of osteoblasts. Cystine/glutamate antiporter (system Xc-) activity was evaluated by determining the expression of SLC7A11 and the levels of glutathione (GSH) and extracellular glutamate. We constructed a T2DOP rat model and observed the effect of ATF3 on ferroptosis and T2DOP by knocking down ATF3 using small interfering RNA (siRNA). Then, we evaluated the levels of iron metabolism, lipid peroxidation, and bone turnover in serum, detected the expression of ATF3, SLC7A11, and GPX4 in bone tissues, and assessed bone microstructure using microcomputed tomography. ATF3 expression was increased in osteoblasts under HG condition and in T2DOP rats. Inhibiting the function of ATF3 increased GPX4 levels and reduced the accumulation of ROS and lipid peroxides. These changes inhibited the ferroptosis of osteoblasts and improved osteogenic function. In addition, HG induced ATF3 upregulation, resulting in decreased SLC7A11 expression and lower levels of intracellular GSH and extracellular glutamate. Osteoblast ferroptosis under HG conditions is induced by ATF3-mediated inhibition of system Xc- activity, and these events contribute to T2DOP pathogenesis.

Mitochondria-targeted photosensitizer based nanoplatform loading glutathione inhibitor for enhanced breast cancer photodynamic therapy

Photodynamic therapy (PDT) is an approved photoactivated technique that has been widely used for anti-tumor therapy through the generation of excessive ROS. However, the poor water solubility and targeting ability of photosensitizer and ROS depleting by high concentrations of glutathione (GSH) in the tumor microenvironment will limit the efficiency of PDT and synergistic therapy. Herein, we report a mitochondria targeted heterodimeric photosensitizer (TPP-TK-PPa) that can form co-delivery nanoplatform together with DSPE-PEG2000 and GSH-depleting agent DEM through the self-assembly. The TPP-TK-PPa/DEM NPs reveals excellent mitochondrial targeting ability and can efficiently deliver the therapeutic agents to tumor cells and release them into the mitochondria in an ROS-responsive manner. Additionally, The TPP-TK-PPa/DEM NPs can induce in situ production of ROS during PDT and reduce ROS consumption by decreasing intracellular GSH levels, which is attributed to the progressive release of DEM after cellular internalization. In addition, generation of ROS during PDT leads to distinct changes in mitochondrial membrane potential and morphology, which ultimately leads to apoptosis. More importantly, the in vitro antitumor activity results demonstrate that TPP-TK-PPa/DEM NPs showed remarkable phototoxicity and the IC50 values of TPP-TK-PPa/DEM NPs in MDA-MB-231 breast cancer cells was as low as 250 nM after PDT. This work provides a strategy for the development of photosensitizers with specific organelle targeting ability, which is of great importance for precise and effective PDT.

Antioxidant activity analysis of collagen peptide-magnesium chelate

Antioxidant peptides from natural polymer collagen are highly desirable and their ability to coordinate with minerals will provide new leads for the prevention and treatment of oxidative stress-related diseases. Here, we tested the changes of structures and functional properties of bovine bone collagen peptides (BBCP) after magnesium chelation. The Fourier transform infrared (FTIR) results revealed that Mg(II) chelated with BBCP mainly via amino nitrogen and carboxyl oxygen atoms. The fluorescence and circular dichroism (CD) results indicated that Mg(II) could induce and stabilize the conformational structures, and change the amino acid distribution on the surfaces of BBCP. The chelation of BBCP and Mg(II) led to the improvement of radical scavenging activities, and enhanced the intracellular antioxidant enzyme activities and GSH levels, thus demonstrating higher protective and reparative effects against H2O2-induced oxidative damage. This work provides mechanistic insights for further development of peptide-magnesium chelates for oxidative stress mitigation.

The microbiota catabolites of quercetin glycosides concertedly enhance the resistance against acetaldehyde-induced oxidative stress

3,4-Dihydroxyphenylacetic acid (DOPAC) and 3-hydroxyphenylacetic acid (OPAC) are the predominant catabolites of quercetin glycosides, such as quercetin 4′-O-β-glucoside from the onion, produced by intestinal microbiota. Although each catabolite has been reported to protect the cells from acetaldehyde-induced cytotoxicity, the effect of their combination remains to be clarified. The purpose of this study was to determine whether the combination of DOPAC and OPAC enhances the resistance against the acetaldehyde-induced oxidative stress in the cultured hepatocytes. The pretreatment of the combination of DOPAC (5 μM) and OPAC (5 μM) showed significant protection against the acetaldehyde- and hydrogen peroxide-induced cytotoxicity, even though each compound at the same concentration did not. This combination also significantly inhibited the intracellular dichlorofluorescin diacetate-detectable reactive oxygen species (ROS) level, whereas the solo treatment did slightly, suggesting that reducing mechanisms of ROS or compounds that enhance ROS production are involved in the cytoprotective effect. The combinatory treatment significantly enhanced the gene expression of not only the aldehyde dehydrogenases (ALDHs), but also glutamate-cysteine ligase, catalytic subunit, the first rate-limiting enzyme of glutathione (GSH) synthesis. Accordingly, both the intracellular GSH level and the total ALDH activity were enhanced by DOPAC plus OPAC. Involvement of GSH in the cytoprotection as well as ALDH up-regulation by the combination was confirmed by the experiments using a GSH biosynthesis inhibitor, buthionine sulfoximine. Taken together, the present results suggested that the quercetin microbiota catabolites concertedly protect the cells from acetaldehyde through a pre-enhanced resistance against oxidative stress by the GSH-dependent up-regulation of ALDHs.

P042: Metabolic regulation of adaptive response to arginine deprivation in Hodgkin Lymphoma

Our group has previously shown an altered aminoacidic homeostasis in the microenvironment of classic Hodgkin`s Lymphoma (HL), due to arginine deprivation, which plays a major role in the immune-escape mechanisms and T-cell anergy induction. To study the metabolic adaptive response to arginine deprivation in vitro, we cultured three HL human cell lines (L428, L540, KMH2) with customized complete media or lacking Arg (R0), supplemented with 10% dialyzed fetal bovine serum, in six independent experiments to collect their global metabolomic analysis by gas chromatography-mass spectrometry (GC/MS) and liquid chromatography-tandem mass spectrometry (LC/MS/MS) platforms by Metabolon Inc and transcriptome profiling by RNA-seq. Apotpostosis and mitochondrial depolarization were measured by FACS. Findings were validated by qRT-PCR and Western Blot analysis. Gene set enrichment analysis (GSEA) showed deep transcriptome rearrangements in KMH2 and HDMYZ cell lines, involving upregulation of genes required for the unfolded protein response, p53 pathway and networks and proteosome degradation, with a minimal effect on metabolism features, except for the genes involved in lactate metabolism. Arg deprivation caused mitochondrial distress and transcriptional reprogramming, via induction of oxidative stress, affecting the mitochondrial activity, switching from glucose-based metabolism to mitochondrial oxidation of fatty acids, requiring the transfer of fatty acids from lipid droplets to mitochondria. RNA-seq analysis showed that induction of ferroptosis key genes GPX4, TXNRD1 and ACSL4 together with reduced intracellular GSH levels and increased amount of cystine and methionine sulfoxide occurred upon arginine deprivation. The low-energy metabolic state induced by the adaptive response to arg-deprivation posed KMH2-HL cells into a quiescence state, with elevated HMOX1 and HMOX-2 to scavenge the excess ROS with subsequent genome instability as shown by increased γHA2X+/ATM+ cells. In HDMYZ, the increased oxidative stress due to arginine deprivation induced the engagement of the UFMylation pathway. These findings were confirmed in vivo, since in peripheral blood of HL patients we found reduced amount of GSH, glutamine and S-adenosylhomocysteine, citrate, methionine (p<0.001), as detected by HPLC. Taken together, our data suggest how arginine deprivation can regulate lipid trafficking, ferroptosis and UFMylation, novel potential targets to overcome drug resistance in cHL.

Exogenous Glutathione Promotes the Proliferation of Pinus koraiensis Embryonic Cells and the Synthesis of Glutathione and Ascorbic Acid.

Somatic embryogenesis (SE), which leads to the formation of embryonic callus (EC) tissue, is the most promising method for large-scale production and selective breeding of woody plants. However, in many species, SE suffers from low proliferation rates, hindering the production of improved plant materials. One way of improving proliferation rates is achieved by improving the redox status of the culture medium. In this study, we investigated the effects of exogenous glutathione (GSH) and L-buthionine sulfoximine (BSO, the inhibitor of glutathione synthase) on the EC proliferation rate in Korean pine (Pinus koraiensis), using cell lines with both high (F: 001#-001) and low (S: 001#-010) proliferation potential. We found that exogenous GSH promoted cell proliferation in both cell lines, while exogenous BSO inhibited proliferation in both cell lines. At 35 d with exogenous GSH treatment, the fresh weight of F and S cell lines increased by 35.48% and 48.39%, respectively, compared with the control. The exogenous application of GSH increased the intracellular levels of GSH, total GSH (T-GSH), oxidized glutathione (GSSG), ascorbic acid (ASA), total ASA (T-ASA), and the ratios of GSH:T-GSH and ASA:T-ASA in both F and S cell lines. Furthermore, exogenous GSH increased the activity of both glutathione reductase (GR) and dehydroascorbate reductase (DHAR) while decreasing the activity of ascorbate peroxidase (APX) in both cell lines. It appears that the application of exogenous GSH promotes a reducing cultural environment, which is conducive to EC proliferation in Korean pine. By helping to reveal the mechanism whereby GSH regulates redox homeostasis in Korean pine EC cells, we have laid the foundation for a large-scale breeding of Korean pine somatic embryogenesis technology system.

Chitosan derivatives as nanocarriers for hLDHA inhibitors delivery to hepatic cells: A selective strategy for targeting primary hyperoxaluria diseases

Primary hyperoxalurias (PHs) are a group of inherited alterations of the hepatic glyoxylate metabolism that result in an excess of oxalate production by the oxidation of glyoxylate by the human lactate dehydrogenase A enzyme (hLDHA). The selective liver inhibition of this enzyme is one of the therapeutic strategies followed in the treatment of this disease. Even though several efforts have been recently performed using gene silencing by the RNA interference approach, small-molecule inhibitors that selectively reach hepatocytes are preferred since they present the advantages of a lower production cost and better pharmacological properties. In that sense, the design, synthesis, and physicochemical characterization by NMR, FTIR, DLS and TEM of two nanocarriers based on chitosan conjugates (1, non-redox-sensitive; 2, redox-sensitive) have been performed to (i) achieve the selective transport of hLDHA inhibitors into hepatocytes and (ii) their disruption once they reach the hepatocytes cytosol. Polymer 2 self-assembled into micelles in water and showed high drug loadings (19.8–24.5 %) and encapsulation efficiencies (31.9–40.8%) for the hLDHA inhibitors (I-III) tested. The non-redox-sensitive micelle 1 remained stable under different glutathione (GSH) concentrations (10 μM and 10 mM), and just a residual release of the inhibitor encapsulated was observed (less than 10 %). On the other hand, micelle 2 was sufficiently stable under in vitro physiological conditions (10 μM, GSH) but it quickly disassembled under the simulated reducing conditions present inside hepatocytes (10 mM GSH), achieving a 60 % release of the hLDHA inhibitor encapsulated after 24 h, confirming the responsiveness of the developed carrier to the high levels of intracellular GSH.

Improved Tolerance of Lactiplantibacillus plantarum in the Presence of Acid by the Heterologous Expression of trxA from Oenococcus oeni

Oenococcus oeni is the main microorganism that undergoes malolactic fermentation (MLF) in the winemaking industry due to its excellent adaptability to harsh wine environments. The start of MLF is often delayed or even fails, and low pH appears to be a crucial parameter. To study the function of the trxA gene in acid stress, a plasmid containing the trxA gene of O. oeni SD-2a was heterologously expressed in Lactiplantibacillus plantarum WCFS1. The recombinant strain (WCFS1-trxA) grew better than the control strain (WCFS1-Vector) under acid stress. The expression of thioredoxin system genes was much higher in the recombinant strain compared with the control strain under acid stress. In addition, a series of physiological and biochemical assays were conducted. The ATP content was lower in the recombinant strain, while the cell membrane fluidity and integrity improved in the recombinant strain. Moreover, reactive oxygen species (ROS) accumulation, intracellular GSH level, and superoxide dismutase (SOD) activity assays showed that the recombinant strain decreased the intracellular reactive oxygen species (ROS) accumulation by improving the SOD activity. In conclusion, heterologous expression of trxA improves the SOD activity of L. plantarum WCFS1, reducing bacterial ROS and increasing cell membrane fluidity and integrity, enhancing the tolerance of Lactiplantibacillus plantarum WCFS1 under acid stress.

Different Inhibition of Nrf2 by Two Keap1 Isoforms α and β to Shape Malignant Behaviour of Human Hepatocellular Carcinoma.

Nrf2 (nuclear factor E2-related factor 2, encoded by Nfe2l2) acts as a master transcriptional regulator in mediating antioxidant, detoxification, and cytoprotective responses against oxidative, electrophilic, and metabolic stress, but also plays a crucial role in cancer metabolism and multiple oncogenic pathways, whereas the redox sensor Keap1 functions as a predominant inhibitor of Nrf2 and, hence, changes in its expression abundance directly affect the Nrf2 stability and transcriptional activity. However, nuanced functional isoforms of Keap1 α and β have rarely been identified to date. Herein, we have established four distinct cell models stably expressing Keap1−/−, Keap1β(Keap1Δ1–31), Keap1-Restored, and Keap1α-Restored aiming to gain a better understanding of similarities and differences of two Keap1 isoforms between their distinct regulatory profiles. Our experimental evidence revealed that although Keap1 and its isoforms are still localized in the cytoplasmic compartments, they elicited differential inhibitory effects on Nrf2 and its target HO-1. Furthermore, transcriptome sequencing unraveled that they possess similar but different functions. Such functions were further determined by multiple experiments in vivo (i.e., subcutaneous tumour formation in nude mice) and in vitro (e.g., cell cloning, infection, migration, wound healing, cell cycle, apoptosis, CAT enzymatic activity, and intracellular GSH levels). Of note, the results obtained from tumourigenesis experiments in xenograft model mice were verified based on the prominent changes in the PTEN signaling to the PI3K-AKT-mTOR pathways, in addition to substantially aberrant expression patterns of those typical genes involved in the EMT (epithelial–mesenchymal transition), cell cycle, and apoptosis.