High-Dose IV Hydroxocobalamin (Vitamin B12) in Septic Shock: A Double-Blind, Allocation-Concealed, Placebo-Controlled Single-Center Pilot Randomized Controlled Trial (The Intravenous Hydroxocobalamin in Septic Shock Trial).

BACKGROUND: Elevated hydrogen sulfide (H2S) contributes to vasodilatation and hypotension in septic shock, and traditional therapies do not target this pathophysiologic mechanism. High-dose IV hydroxocobalamin scavenges and prevents H2S formation, which may restore vascular tone and may accentuate recovery. No experimental human studies have tested high-dose IV hydroxocobalamin in adults with septic shock. RESEARCH QUESTION: In adults with septic shock, is comparing high-dose IV hydroxocobalamin with placebo feasible? STUDY DESIGN AND METHODS: We conducted a phase 2 single-center, double-blind, allocation-concealed, placebo-controlled, parallel-group pilot randomized controlled trial comparing high-dose IV hydroxocobalamin with placebo in critically ill adults with septic shock. Patients meeting Sepsis 3 criteria were randomized 1:1 to receive a single 5-g dose of high-dose IV hydroxocobalamin or equivalent volume 0.9% saline solution as placebo. The primary outcome was study feasibility (enrollment rate, clinical and laboratory compliance rate, and contamination rate). Secondary outcomes included between-group differences in plasma H2S concentrations and vasopressor dose before and after infusion. RESULTS: Twenty patients were enrolled over 19 months, establishing an enrollment rate of 1.05 patients per month. Protocol adherence rates were 100% with zero contamination. In the high-dose IV hydroxocobalamin group, compared to placebo, there was a greater reduction in vasopressor dose between randomization and postinfusion (-36% vs 4%, P < .001) and randomization and 3-h postinfusion (-28% vs 10%, P = .019). In the high-dose IV hydroxocobalamin group, the plasma H2S level was reduced over 45 mins by -0.80 ± 1.73 µM, as compared with -0.21 ± 0.64 µM in the placebo group (P = .3). INTERPRETATION: This pilot trial established favorable feasibility metrics. Consistent with the proposed mechanism of benefit, high-dose IV hydroxocobalamin compared with placebo was associated with reduced vasopressor dose and H2S levels at all time points and without serious adverse events. These data provide the first proof of concept for feasibility of delivering high-dose IV hydroxocobalamin in septic shock. TRIAL REGISTRY: ClinicalTrials.gov; No.: NCT03783091; URL: www. CLINICALTRIALS: gov.

Magnolia extract is effective for the chemoprevention of oral cancer through its ability to inhibit mitochondrial respiration at complex I.

BACKGROUND: Magnolia extract (ME) is known to inhibit cancer growth and metastasis in several cell types in vitro and in animal models. However, there is no detailed study on the preventive efficacy of ME for oral cancer, and the key components in ME and their exact mechanisms of action are not clear. The overall goal of this study is to characterize ME preclinically as a potent oral cancer chemopreventive agent and to determine the key components and their molecular mechanism(s) that underlie its chemopreventive efficacy. METHODS: The antitumor efficacy of ME in oral cancer was investigated in a 4-nitroquinoline-1-oxide (4NQO)-induced mouse model and in two oral cancer orthotopic models. The effects of ME on mitochondrial electron transport chain activity and ROS production in mouse oral tumors was also investigated. RESULTS: ME did not cause detectable side effects indicating that it is a promising and safe chemopreventive agent for oral cancer. Three major key active compounds in ME (honokiol, magnolol and 4-O-methylhonokiol) contribute to its chemopreventive effects. ME inhibits mitochondrial respiration at complex I of the electron transport chain, oxidizes peroxiredoxins, activates AMPK, and inhibits STAT3 phosphorylation, resulting in inhibition of the growth and proliferation of oral cancer cells. CONCLUSION: Our data using highly relevant preclinical oral cancer models, which share histopathological features seen in human oral carcinogenesis, suggest a novel signaling and regulatory role for mitochondria-generated superoxide and hydrogen peroxide in suppressing oral cancer cell proliferation, progression, and metastasis. Video abstract.

High-Throughput Screening of NOX Inhibitors.

Development of new, selective inhibitors of nicotinamide adenine dinucleotide phosphate oxidase (NOX) isoforms is important both for basic studies on the role of these enzymes in cellular redox signaling, cell physiology, and proliferation and for development of new drugs for diseases carrying a component of increased NOX activity, such as several types of cancer and cardiovascular and neurodegenerative diseases. High-throughput screening (HTS) of large libraries of compounds remains the major approach for development of new NOX inhibitors. Here, we describe the protocol for the HTS campaign for NOX inhibitors using rigorous assays for superoxide radical anion and hydrogen peroxide, based on oxidation of hydropropidine, coumarin boronic acid, and Amplex Red. We propose using these three probes to screen for and identify new inhibitors, by selecting positive hits that show inhibitory effects in all three assays. Protocols for the synthesis of hydropropidine and for confirmatory assays, including oxygen consumption measurements, electron paramagnetic resonance spin trapping of superoxide, and simultaneous monitoring of superoxide and hydrogen peroxide, are also provided.

Detection of mitochondria-generated reactive oxygen species in cells using multiple probes and methods: Potentials, pitfalls, and the future.

Reactive oxygen and nitrogen species (ROS/RNS) such as superoxide (O2̇̄), hydrogen peroxide, lipid hydroperoxides, peroxynitrite, and hypochlorous and hypobromous acids play a key role in many pathophysiological processes. Recent studies have focused on mitochondrial ROS as redox signaling species responsible for promoting cell division, modulating and regulating kinases and phosphatases, and activating transcription factors. Many ROS also stimulate cell death and senescence. The extent to which these processes occur is attributed to ROS levels (low or high) in cells. However, the exact nature of ROS remains unknown. Investigators have used redox-active probes that, upon oxidation by ROS, yield products exhibiting fluorescence, chemiluminescence, or bioluminescence. Mitochondria-targeted probes can be used to detect ROS generated in mitochondria. However, because most of these redox-active probes (untargeted and mitochondria-targeted) are oxidized by several ROS species, attributing redox probe oxidation to specific ROS species is difficult. It is conceivable that redox-active probes are oxidized in common one-electron oxidation pathways, resulting in a radical intermediate that either reacts with another oxidant (including oxygen to produce O2̇̄) and forms a stable fluorescent product or reacts with O2̇̄ to form a fluorescent marker product. Here, we propose the use of multiple probes and complementary techniques (HPLC, LC-MS, redox blotting, and EPR) and the measurement of intracellular probe uptake and specific marker products to identify specific ROS generated in cells. The low-temperature EPR technique developed to investigate cellular/mitochondrial oxidants can easily be extended to animal and human tissues.

A review of the basics of mitochondrial bioenergetics, metabolism, and related signaling pathways in cancer cells: Therapeutic targeting of tumor mitochondria with lipophilic cationic compounds.

The present review is a sequel to the previous review on cancer metabolism published in this journal. This review focuses on the selective antiproliferative and cytotoxic effects of mitochondria-targeted therapeutics (MTTs) in cancer cells. Emerging research reveals a key role of mitochondrial respiration on tumor proliferation. Previously, a mitochondria-targeted nitroxide was shown to selectively inhibit colon cancer cell proliferation at submicromolar levels. This review is centered on the therapeutic use of MTTs and their bioenergetic profiling in cancer cells. Triphenylphosphonium cation conjugated to a parent molecule (e.g., vitamin-E or chromanol, ubiquinone, and metformin) via a linker alkyl chain is considered an MTT. MTTs selectively and potently inhibit proliferation of cancer cells and, in some cases, induce cytotoxicity. MTTs inhibit mitochondrial complex I activity and induce mitochondrial stress in cancer cells through generation of reactive oxygen species. MTTs in combination with glycolytic inhibitors synergistically inhibit tumor cell proliferation. This review discusses how signaling molecules traditionally linked to tumor cell proliferation affect tumor metabolism and bioenergetics (glycolysis, TCA cycle, and glutaminolysis).

Antiproliferative effects of mitochondria-targeted cationic antioxidants and analogs: Role of mitochondrial bioenergetics and energy-sensing mechanism.

One of the proposed mechanisms for tumor proliferation involves redox signaling mediated by reactive oxygen species such as superoxide and hydrogen peroxide generated at moderate levels. Thus, the antiproliferative and anti-tumor effects of certain antioxidants were attributed to their ability to mitigate intracellular reactive oxygen species (ROS). Recent reports support a role for mitochondrial ROS in stimulating tumor cell proliferation. In this study, we compared the antiproliferative effects and the effects on mitochondrial bioenergetic functions of a mitochondria-targeted cationic carboxyproxyl nitroxide (Mito-CP), exhibiting superoxide dismutase (SOD)-like activity and a synthetic cationic acetamide analog (Mito-CP-Ac) lacking the nitroxide moiety responsible for the SOD activity. Results indicate that both Mito-CP and Mito-CP-Ac potently inhibited tumor cell proliferation. Both compounds altered mitochondrial and glycolytic functions, and intracellular citrate levels. Both Mito-CP and Mito-CP-Ac synergized with 2-deoxy-glucose (2-DG) to deplete intracellular ATP, inhibit cell proliferation and induce apoptosis in pancreatic cancer cells. We conclude that mitochondria-targeted cationic agents inhibit tumor proliferation via modification of mitochondrial bioenergetics pathways rather than by dismutating and detoxifying mitochondrial superoxide.

Alterations in bioenergetic function induced by Parkinson's disease mimetic compounds: lack of correlation with superoxide generation.

In vitro and in vivo models of Parkinson's disease (PD) suggest that increased oxidant production leads to mitochondrial dysfunction in dopaminergic neurons and subsequent cell death. However, it remains unclear if cell death in these models is caused by inhibition of mitochondrial function or oxidant production. The objective of this study was to determine the relationship between mitochondrial dysfunction and oxidant production in response to multiple PD neurotoxicant mimetics. MPP(+) caused a dose-dependent decrease in the basal oxygen consumption rate in dopaminergic N27 cells, indicating a loss of mitochondrial function. In parallel, we found that MPP(+) only modestly increased oxidation of hydroethidine as a diagnostic marker of superoxide production in these cells. Similar results were found using rotenone as a mitochondrial inhibitor, or 6-hydroxydopamine (6-OHDA) as a mechanistically distinct PD neurotoxicant, but not with exposure to paraquat. In addition, the extracellular acidification rate, used as a marker of glycolysis, was stimulated to compensate for oxygen consumption rate inhibition after exposure to MPP(+), rotenone, or 6-OHDA, but not paraquat. Together these data indicate that MPP(+), rotenone, and 6-OHDA dramatically shift bioenergetic function away from the mitochondria and towards glycolysis in N27 cells.