Tumor-Specific Catalysis-Mediated Enhanced Chemodynamic Therapy in Synergy with Mitophagy Inhibition Improves Therapeutic Efficacy in Endometrial Cancer

BackgroundChemodynamic therapy (CDT) relies on tumor microenvironment (e.g. high H2O2 level) responsive Fenton-like reactions to produce hydroxyl radicals (·OH) against tumors. However, endogenous H2O2 is insufficient for effective chemodynamic reactions.ResultsAn NAD(P)H: quinone oxidoreductase 1 (NQO1)highCatalase (CAT)low therapeutic window for the use of NQO1 bioactive drug β-lapachone (β-Lap) was firstly identified in endometrial cancer (EC). Accompanied by NADH depletion, β-Lap was catalyzed by NQO1 to produce excess H2O2 initiating oxidative stress, which selectively suppressed NQO1high EC cell proliferation, induced DNA double-strand breaks and promoted apoptosis. SiRNA-mediated NQO1 knockdown or dicoumarol rescued NQO1high EC cells from β-Lap-induced cytotoxicity. Arginine-glycine-aspartic acid (RGD)-functionalized iron-based metal organic frameworks-MOF(Fe) further promoted the conversion of accumulated H2O2 into highly oxidative ·OH, and in turn exacerbated the oxidative damage to RGD-positive target cells. Mitophagy inhibition by Mdivi-1 blocked a powerful antioxidant defense approach, ultimately ensuring the antitumor efficacy of stepwise amplified ROS signals. The tumor growth inhibition rate was about 85.92%.ConclusionsTumor specific chemotherapy in combination with CDT-triggered therapeutic modality presented unprecedented therapeutic advantages for the treatment of NQO1+ advanced type I or type II EC.

Impact of Hydrogen Sulfide on Mitochondrial and Bacterial Bioenergetics

This review focuses on the effects of hydrogen sulfide (H2S) on the unique bioenergetic molecular machines in mitochondria and bacteria—the protein complexes of electron transport chains and associated enzymes. H2S, along with nitric oxide and carbon monoxide, belongs to the class of endogenous gaseous signaling molecules. This compound plays critical roles in physiology and pathophysiology. Enzymes implicated in H2S metabolism and physiological actions are promising targets for novel pharmaceutical agents. The biological effects of H2S are biphasic, changing from cytoprotection to cytotoxicity through increasing the compound concentration. In mammals, H2S enhances the activity of FoF1-ATP (adenosine triphosphate) synthase and lactate dehydrogenase via their S-sulfhydration, thereby stimulating mitochondrial electron transport. H2S serves as an electron donor for the mitochondrial respiratory chain via sulfide quinone oxidoreductase and cytochrome c oxidase at low H2S levels. The latter enzyme is inhibited by high H2S concentrations, resulting in the reversible inhibition of electron transport and ATP production in mitochondria. In the branched respiratory chain of Escherichia coli, H2S inhibits the bo3 terminal oxidase but does not affect the alternative bd-type oxidases. Thus, in E. coli and presumably other bacteria, cytochrome bd permits respiration and cell growth in H2S-rich environments. A complete picture of the impact of H2S on bioenergetics is lacking, but this field is fast-moving, and active ongoing research on this topic will likely shed light on additional, yet unknown biological effects.

Activation of The P62-Keap1-NRF2 Pathway Protects Against Ferroptosis in Radiation-Induced Lung Injury

Background Radiation-induced lung injury (RILI) is one of the most common, serious and dose-limiting complications of thoracic radiotherapy. A primary reason for this is the radiation-induced cell death. Ferroptosis is a recently recognized form of regulated cell death, characterized by the accumulation of lipid peroxidation products and lethal reactive oxygen species (ROS). The ROS induced by irradiation might be the original trigger of ferroptosis in RILI. Furthermore, activation of the P62-Kelch-like ECH-associated protein 1 (Keap1)-nuclear factor erythroid 2-related factor 2 (NRF2) pathway has been shown to exert a protective effect, blunting ferroptosis. Therefore, this study aims to explore the protective effect of the P62-Keap1-NRF2 pathway against radiation-induced ferroptosis in alveolar epithelial cells. Results Firstly, our results demonstrated that radiation induced ferroptosis in vitro RILI cell model, which could be significantly reduced by Ferrostatin-1 (Fer-1), a specific inhibitor of ferroptosis. Then, we found that overexpression of P62 interacted with Keap1 to promote NRF2 translocation into the nucleus and upregulation its target proteins quinone oxidoreductase 1 (NQO1), heme oxygenase 1 (HO1) and ferritin heavy chain 1 (FTH1). Conclusion Collectively, the activation of the P62-Keap1-NRF2 pathway prevents radiation-induced ferroptosis in RILI cells, providing a theoretical basis for further research to find a potential approach for RILI therapy.

In Vitro and in Vivo Characterisation of P. Aeruginosa Oxidoreductase Enzymes in Pathogenesis and Therapy

<p>Pseudomonas aeruginosa, an increasingly multi-drug resistant human pathogen, is now one of the top three causes of opportunistic infection and there is much interest in identifying novel therapeutic targets for treatment. As a bacterial pathogen, P. aeruginosa encounters innate immune system defences and must continue to adapt to its defence strategies to accommodate the ever-changing environment. Though P. aeruginosa virulence determinants have been heavily characterised over the last several decades, most recent work acknowledges the complex interaction between the human host and the pathogen as an on-going dialogue of virulence factors adapting to the continuum that is the immune response. A major challenge that P. aeruginosa must overcome are reactive oxygen species (ROS) that are released at all stages of infection. Based on previous work which demonstrated a role for soluble nitro- and quinone oxidoreductase (NQOR) enzymes in protecting a related bacterium (Pseudomonas putida) from oxidative stress, we hypothesized that P. aeruginosa would similarly utilize NQORs to withstand ROS. This thesis seeks to understand the role of ROS-protecting enzymes in pathogenesis as well as their potential applications in a therapeutic context. Several NQORs of P. aeruginosa were identified to possess biochemical characteristics consistent with the enzymatic capacity to indirectly reduce reactive species like H₂O₂. However, when individual genes encoding NQORs were deleted from P. aeruginosa, no apparent H₂O₂ sensitivity was seen. In contrast, when candidate genes were over-expressed, certain NQOR enzymes conferred the ability to tolerate H₂O₂ challenge at low concentrations; indicating that these NQORs may play a protective role whose effects are masked in vitro by genetic redundancy as well as a highly active endogenous catalase. By developing a novel in vivo cell culture infection model, the survival of P. aeruginosa post exposure to immunocompetent murine macrophages was also assessed. This not only demonstrated that several putative NQORs were activated in the presence of macrophages but also that an in vivo modelling system is likely to be more appropriate for discovering virulence determinants. In a different aspect of this study it was investigated whether the reductive capacity of the P. aeruginosa-derived NQORs might hold potential for gene-directed enzyme-prodrug therapy (GDEPT). Prodrugs, such as 5-(aziridin-1-yl)-2,4-dinitrobenzamide (CB1954) or the nitro-chloromethyl benzindoline SN 26438, are nontoxic in their native form, but become highly toxic upon reduction of their nitro functional groups. The P. aeruginosa NQORs, were tested to identify enzymes capable of efficient activation of CB1954 or SN 26438. Although none of these enzymes exhibited greater activity with CB1954 than the “best in class” Eschericha coli enzymes NfsA or NfsB, the P. aeruginosa NfsB orthologue (PA5190) demonstrated greater than 20-fold improved activity over NfsB from Escherichia coli in its ability to sensitise human cells to SN 26438. This finding offers promise for development of PA5190 and SN 26438 as a novel enzyme-prodrug paradigm for GDEPT.</p>

In Vitro and in Vivo Characterisation of P. Aeruginosa Oxidoreductase Enzymes in Pathogenesis and Therapy

<p>Pseudomonas aeruginosa, an increasingly multi-drug resistant human pathogen, is now one of the top three causes of opportunistic infection and there is much interest in identifying novel therapeutic targets for treatment. As a bacterial pathogen, P. aeruginosa encounters innate immune system defences and must continue to adapt to its defence strategies to accommodate the ever-changing environment. Though P. aeruginosa virulence determinants have been heavily characterised over the last several decades, most recent work acknowledges the complex interaction between the human host and the pathogen as an on-going dialogue of virulence factors adapting to the continuum that is the immune response. A major challenge that P. aeruginosa must overcome are reactive oxygen species (ROS) that are released at all stages of infection. Based on previous work which demonstrated a role for soluble nitro- and quinone oxidoreductase (NQOR) enzymes in protecting a related bacterium (Pseudomonas putida) from oxidative stress, we hypothesized that P. aeruginosa would similarly utilize NQORs to withstand ROS. This thesis seeks to understand the role of ROS-protecting enzymes in pathogenesis as well as their potential applications in a therapeutic context. Several NQORs of P. aeruginosa were identified to possess biochemical characteristics consistent with the enzymatic capacity to indirectly reduce reactive species like H₂O₂. However, when individual genes encoding NQORs were deleted from P. aeruginosa, no apparent H₂O₂ sensitivity was seen. In contrast, when candidate genes were over-expressed, certain NQOR enzymes conferred the ability to tolerate H₂O₂ challenge at low concentrations; indicating that these NQORs may play a protective role whose effects are masked in vitro by genetic redundancy as well as a highly active endogenous catalase. By developing a novel in vivo cell culture infection model, the survival of P. aeruginosa post exposure to immunocompetent murine macrophages was also assessed. This not only demonstrated that several putative NQORs were activated in the presence of macrophages but also that an in vivo modelling system is likely to be more appropriate for discovering virulence determinants. In a different aspect of this study it was investigated whether the reductive capacity of the P. aeruginosa-derived NQORs might hold potential for gene-directed enzyme-prodrug therapy (GDEPT). Prodrugs, such as 5-(aziridin-1-yl)-2,4-dinitrobenzamide (CB1954) or the nitro-chloromethyl benzindoline SN 26438, are nontoxic in their native form, but become highly toxic upon reduction of their nitro functional groups. The P. aeruginosa NQORs, were tested to identify enzymes capable of efficient activation of CB1954 or SN 26438. Although none of these enzymes exhibited greater activity with CB1954 than the “best in class” Eschericha coli enzymes NfsA or NfsB, the P. aeruginosa NfsB orthologue (PA5190) demonstrated greater than 20-fold improved activity over NfsB from Escherichia coli in its ability to sensitise human cells to SN 26438. This finding offers promise for development of PA5190 and SN 26438 as a novel enzyme-prodrug paradigm for GDEPT.</p>

Neuroprotection of Kolaviron by Regulation of Nuclear Factor Erythroid 2-related Factor 2 in 1-Methyl-4-phenyl-1,2,3,6-tetrahydropyridine Mice Model of Parkinson Disease

Objectives: Parkinson’s disease (PD) is the most prevalent movement disorder. Available therapies are palliative with no effect on disease progression. We have previously demonstrated that kolaviron (KV), a natural anti-inflammatory and antioxidant agent, suppressed behavioral defect, redo-inflammation, and nigrostriatal pathology in rotenone PD model. The present study investigates the neuroprotective effect of KV focusing on DJ-1/nuclear factor erythroid 2-related factor 2 (Nrf2) signaling pathway. Material and Methods: All-trans retinoic acid (ATRA, 10 mg/kg/day) was used to inhibit Nrf2. PD was established with four doses of 1-methyl-4-phenyl-1,2,3,6-tetrahydropyridine (MPTP) (20 mg/kg) at 2 h interval. MPTP mice were pre-treated with either KV (200 mg/kg/day), ATRA or both for 7 days before MPTP. Mice were evaluated for locomotor defects and indices of oxidative stress, neuroinflammation and neurotransmission as well as pathological tyrosine hydroxylase expression PD were evaluated in the striatum. Results: ATRA alone in mice did not exhibit neurobehavioral defect but caused striatal toxicity, mild nigrostriatal pathology, significant nitrosative stress, and Nrf2 cascade inhibition. KV+ATRA mice were slow in movement with frequent short-lived interruptions and oxidative striatal pathology. ATRA aggravated MPTP-associated locomotor incompetence and could not prevent nigrostriatal toxicity with evident vacuolated striosome and pyknotic/degenerating dopaminergic neurons. MPTP induced acute locomotor, exploratory, and motor incompetence, which was prevented by KV treatment. In addition, KV treatment restored MPTP-mediated depletion of endogenous antioxidant, striatal nitrosative stress, and oxidative damage with elevated DJ-1 level, potentiated Nrf2/NAD(P)H; quinone oxidoreductase-1 cytoprotective capacity, reduced Kelch-like ECH-associated protein 1 expression, and limited striatal pathology. However, ATRA treatment attenuated all the protective effects of KV on MPTP-challenged mice. Meanwhile, other ATRA-combinations elicited significant DJ-1 and Nrf2 induction but are associated striatal toxicity/pathology. Conclusion: This suggests that KV may be conferring protection through a yet-undetermined DJ-1 downstream cytoprotective effect dependent on the KV-mediated attenuation of oxidative environment.

Modulation of Cellular NAD+ Attenuates Cancer-Associated Hypercoagulability and Thrombosis via the Inhibition of Tissue Factor and Formation of Neutrophil Extracellular Traps

Cancer-associated thrombosis is the second-leading cause of mortality in patients with cancer and presents a poor prognosis, with a lack of effective treatment strategies. NAD(P)H quinone oxidoreductase 1 (NQO1) increases the cellular nicotinamide adenine dinucleotide (NAD+) levels by accelerating the oxidation of NADH to NAD+, thus playing important roles in cellular homeostasis, energy metabolism, and inflammatory responses. Using a murine orthotopic 4T1 breast cancer model, in which multiple thrombi are generated in the lungs at the late stage of cancer development, we investigated the effects of regulating the cellular NAD+ levels on cancer-associated thrombosis. In this study, we show that dunnione (a strong substrate of NQO1) attenuates the prothrombotic state and lung thrombosis in tumor-bearing mice by inhibiting the expression of tissue factor and formation of neutrophil extracellular traps (NETs). Dunnione increases the cellular NAD+ levels in lung tissues of tumor-bearing mice to restore the declining sirtuin 1 (SIRT1) activity, thus deacetylating nuclear factor-kappa B (NF-κB) and preventing the overexpression of tissue factor in bronchial epithelial and vascular endothelial cells. In addition, we demonstrated that dunnione abolishes the ability of neutrophils to generate NETs by suppressing histone acetylation and NADPH oxidase (NOX) activity. Overall, our results reveal that the regulation of cellular NAD+ levels by pharmacological agents may inhibit pulmonary embolism in tumor-bearing mice, which may potentially be used as a viable therapeutic approach for the treatment of cancer-associated thrombosis.

Development of whole-cell catalyst system for sulfide biotreatment based on the engineered haloalkaliphilic bacterium

Microorganisms play an essential role in sulfide removal. Alkaline absorption solution facilitates the sulfide’s dissolution and oxidative degradation, so haloalkaliphile is a prospective source for environmental-friendly and cost-effective biodesulfurization. In this research, 484 sulfide oxidation genes were identified from the metagenomes of the soda-saline lakes and a haloalkaliphilic heterotrophic bacterium Halomonas salifodinae IM328 (=CGMCC 22183) was isolated from the same habitat as the host for expression of a representative sequence. The genetic manipulation was successfully achieved through the conjugation transformation method, and sulfide: quinone oxidoreductase gene (sqr) was expressed via pBBR1MCS derivative plasmid. Furthermore, a whole-cell catalyst system was developed by using the engineered strain that exhibited a higher rate of sulfide oxidation under the optimal alkaline pH of 9.0. The whole-cell catalyst could be recycled six times to maintain the sulfide oxidation rates from 41.451 to 80.216 µmol·min−1·g−1 dry cell mass. To summarize, a whole-cell catalyst system based on the engineered haloalkaliphilic bacterium is potentiated to be applied in the sulfide treatment at a reduced cost.

Role of Human NADPH Quinone Oxidoreductase (NQO1) in Oxygen-Mediated Cellular Injury and Oxidative DNA Damage in Human Pulmonary Cells

Supplemental oxygen administration is frequently used in premature infants and adults with pulmonary insufficiency. NADPH quinone oxidoreductase (NQO1) protects cells from oxidative injury by decreasing reactive oxygen species (ROS). In this investigation, we tested the hypothesis that overexpression of NQO1 in BEAS-2B cells will mitigate cell injury and oxidative DNA damage caused by hyperoxia and that A-1221C single nucleotide polymorphism (SNP) in the NQO1 promoter would display altered susceptibility to hyperoxia-mediated toxicity. Using stable transfected BEAS-2B cells, we demonstrated that hyperoxia decreased cell viability in control cells (Ctr), but this effect was differentially mitigated in cells overexpressing NQO1 under the regulation of the CMV viral promoter, the wild-type NQO1 promoter (NQO1-NQO1), or the NQO1 promoter carrying the SNP. Interestingly, hyperoxia decreased the formation of bulky oxidative DNA adducts or 8-hydroxy-2 ′ -deoxyguanosine (8-OHdG) in Ctr cells. qPCR studies showed that mRNA levels of CYP1A1 and NQO1 were inversely related to DNA adduct formation, suggesting the protective role of these enzymes against oxidative DNA injury. In SiRNA experiments entailing the NQO1-NQO1 promoter, hyperoxia caused decreased cell viability, and this effect was potentiated in cells treated with CYP1A1 siRNA. We also found that hyperoxia caused a marked induction of DNA repair genes DDB2 and XPC in Ctr cells, supporting the idea that hyperoxia in part caused attenuation of bulky oxidative DNA lesions by enhancing nucleotide excision repair (NER) pathways. In summary, our data support a protective role for human NQO1 against oxygen-mediated toxicity and oxidative DNA lesions in human pulmonary cells, and protection against toxicity was partially lost in SNP cells. Moreover, we also demonstrate a novel protective role for CYP1A1 in the attenuation of oxidative cells and DNA injury. Future studies on the mechanisms of attenuation of oxidative injury by NQO1 should help in developing novel approaches for the prevention/treatment of ARDS in humans.