Hydrogen Peroxide Mediates Artemisinin-Derived C-16 Carba-Dimer-Induced Toxicity of Human Cancer Cells.

This study used a nitroaliphatic chemistry approach to synthesize a novel artemisinin-derived carba-dimer (AG-1) and determined its anti-proliferative effects in human normal and cancer cells. AG-1 treatments selectively inhibit proliferation of cancer cells compared to normal human fibroblasts. Compared to artemisinin, AG-1 is more toxic to human breast, prostate, head-neck, pancreas and skin cancer cells; 50% inhibition (IC50) 123 µM in AG-1 vs. 290 µM in artemisinin-treated breast cancer cells. AG-1 treatment decreased (~ 5 folds) cyclin D1 protein expression that correlated with an increase in the percentage of cells in the G1-phase, suggesting a G1 delay. AG-1-induced toxicity was independent of the DNA damage at 72 h post-treatment, as measured by micronuclei frequency and H2AX protein levels. Results from electron paramagnetic resonance spectroscopy showed Fe-catalyzed formation of AG-1 carbon-centered radicals in a cell-free system. Flow cytometry analysis of H2DCF-DA oxidation showed a significant increase in the steady-state levels of reactive oxygen species (ROS) in AG-1-treated cells. Pre-treatment with N-acetyl-l-cysteine and antioxidant enzymes (superoxide dismutase and catalase) significantly suppressed AG-1-induced toxicity, suggesting that superoxide and hydrogen peroxide contribute to AG-1-induced toxicity in human cancer cells. AG-1 represents a novel class of anti-cancer drug that is more potent than its parent compound, artemisinin.

Altered Glycolysis and Mitochondrial Respiration in a Zebrafish Model of Dravet Syndrome.

Altered metabolism is an important feature of many epileptic syndromes but has not been reported in Dravet syndrome (DS), a catastrophic childhood epilepsy associated with mutations in a voltage-activated sodium channel, Nav1.1 (SCN1A). To address this, we developed novel methodology to assess real-time changes in bioenergetics in zebrafish larvae between 4 and 6 d postfertilization (dpf). Baseline and 4-aminopyridine (4-AP) stimulated glycolytic flux and mitochondrial respiration were simultaneously assessed using a Seahorse Biosciences extracellular flux analyzer. Scn1Lab mutant zebrafish showed a decrease in baseline glycolytic rate and oxygen consumption rate (OCR) compared to controls. A ketogenic diet formulation rescued mutant zebrafish metabolism to control levels. Increasing neuronal excitability with 4-AP resulted in an immediate increase in glycolytic rates in wild-type zebrafish, whereas mitochondrial OCR increased slightly and quickly recovered to baseline values. In contrast, scn1Lab mutant zebrafish showed a significantly slower and exaggerated increase of both glycolytic rates and OCR after 4-AP. The underlying mechanism of decreased baseline OCR in scn1Lab mutants was not because of altered mitochondrial DNA content or dysfunction of enzymes in the electron transport chain or tricarboxylic acid cycle. Examination of glucose metabolism using a PCR array identified five glycolytic genes that were downregulated in scn1Lab mutant zebrafish. Our findings in scn1Lab mutant zebrafish suggest that glucose and mitochondrial hypometabolism contribute to the pathophysiology of DS.

Epilepsy, Behavioral Abnormalities, and Physiological Comorbidities in Syntaxin-Binding Protein 1 (STXBP1) Mutant Zebrafish.

Mutations in the synaptic machinery gene syntaxin-binding protein 1, STXBP1 (also known as MUNC18-1), are linked to childhood epilepsies and other neurodevelopmental disorders. Zebrafish STXBP1 homologs (stxbp1a and stxbp1b) have highly conserved sequence and are prominently expressed in the larval zebrafish brain. To understand the functions of stxbp1a and stxbp1b, we generated loss-of-function mutations using CRISPR/Cas9 gene editing and studied brain electrical activity, behavior, development, heart physiology, metabolism, and survival in larval zebrafish. Homozygous stxbp1a mutants exhibited a profound lack of movement, low electrical brain activity, low heart rate, decreased glucose and mitochondrial metabolism, and early fatality compared to controls. On the other hand, homozygous stxbp1b mutants had spontaneous electrographic seizures, and reduced locomotor activity response to a movement-inducing "dark-flash" visual stimulus, despite showing normal metabolism, heart rate, survival, and baseline locomotor activity. Our findings in these newly generated mutant lines of zebrafish suggest that zebrafish recapitulate clinical phenotypes associated with human syntaxin-binding protein 1 mutations.

Reactive oxygen species mediate microRNA-302 regulation of AT-rich interacting domain 4a and C-C motif ligand 5 expression during transitions between quiescence and proliferation.

Normal cell growth consists of two distinct phases, quiescence and proliferation. Quiescence, or G(0), is a reversible growth arrest in which cells retain the ability to reenter the proliferative cycle (G(1), S, G(2), and M). Although not actively dividing, quiescent cells are metabolically active and quiescence is actively maintained. Our results from microRNA PCR arrays and Taqman PCR assays showed a significant decrease (4-fold) in miR-302 levels during quiescence compared to proliferating normal human fibroblasts, suggesting that miR-302 could regulate cellular proliferation. Results from a Q-RT-PCR and dual-luciferase-3'-UTR reporter assays identified ARID4a (AT-rich interacting domain 4a, also known as RBP1) and CCL5 (C-C motif ligand 5) as targets for miR-302. Ionizing radiation decreased miR-302 levels, which was associated with an increase in its target mRNA levels, ARID4a and CCL5. Such an inverse correlation was also observed in cells treated with hydrogen peroxide as well as SOD2-overexpressing cells. Overexpression of miR-302 suppresses ARID4a and CCL5 mRNA levels, and increased the percentage of S-phase cells. These results identified miR-302 as an ROS-sensitive regulator of ARID4a and CCL5 mRNAs as well as demonstrate a regulatory role of miR-302 during quiescence and proliferation.

MnSOD activity regulates hydroxytyrosol-induced extension of chronological lifespan.

Chronological lifespan (CLS) is defined as the duration of quiescence in which normal cells retain the capacity to reenter the proliferative cycle. This study investigates whether hydroxytyrosol (HT), a naturally occurring polyphenol found in olives, extends CLS in normal human fibroblasts (NHFs). Quiescent NHFs cultured for a long duration (30-60 days) lose their capacity to repopulate. Approximately 60% of these cells exit the cell cycle permanently; a significant increase in the doubling time of the cell population was observed. CLS was extended in quiescent NHFs that were cultured in the presence of HT for 30-60 days. HT-induced extension of CLS was associated with an approximately 3-fold increase in manganese superoxide dismutase (MnSOD) activity while there was no change in copper-zinc superoxide dismutase, catalase, or glutathione peroxidase protein levels. Quiescent NHFs overexpressing a dominant-negative mutant form of MnSOD failed to extend CLS. HT suppressed age-associated increase in mitochondrial ROS levels. Results from spectroscopy assays indicate that HT in the presence of peroxidases can undergo catechol-semiquinone-quinone redox cycling generating superoxide, which in a cellular context can activate the antioxidant system, e.g., MnSOD expression. These results demonstrate that HT extends CLS by increasing MnSOD activity and decreasing age-associated mitochondrial reactive oxygen species accumulation.

Redox control of the cell cycle in health and disease.

The cellular oxidation and reduction (redox) environment is influenced by the production and removal of reactive oxygen species (ROS). In recent years, several reports support the hypothesis that cellular ROS levels could function as ''second messengers'' regulating numerous cellular processes, including proliferation. Periodic oscillations in the cellular redox environment, a redox cycle, regulate cell-cycle progression from quiescence (G(0)) to proliferation (G(1), S, G(2), and M) and back to quiescence. A loss in the redox control of the cell cycle could lead to aberrant proliferation, a hallmark of various human pathologies. This review discusses the literature that supports the concept of a redox cycle controlling the mammalian cell cycle, with an emphasis on how this control relates to proliferative disorders including cancer, wound healing, fibrosis, cardiovascular diseases, diabetes, and neurodegenerative diseases. We hypothesize that reestablishing the redox control of the cell cycle by manipulating the cellular redox environment could improve many aspects of the proliferative disorders.

Late ROS accumulation and radiosensitivity in SOD1-overexpressing human glioma cells.

This study investigates the hypothesis that CuZn superoxide dismutase (SOD1) overexpression confers radioresistance to human glioma cells by regulating the late accumulation of reactive oxygen species (ROS) and the G(2)/M-checkpoint pathway. U118-9 human glioma cells (wild type, neo vector control, and stably overexpressing SOD1) were irradiated (0-10 Gy) and assayed for cell survival, cellular ROS levels, cell-cycle-phase distributions, and cyclin B1 expression. SOD1-overexpressing cells were radioresistant compared to wild-type (wt) and neo vector control (neo) cells. Irradiated wt and neo cells showed a significant increase (approximately twofold) in DHE fluorescence beginning at 2 days postirradiation, which remained elevated at 8 days postirradiation. Interestingly, the late accumulation of ROS was suppressed in irradiated SOD1-overexpressing cells. The increase in ROS levels was followed by a decrease in cell growth and viability and an increase in the percentage of cells with sub-G(1) DNA content. SOD1 overexpression enhanced radiation-induced G(2) accumulation within 24 h postirradiation, which was accompanied by a decrease in cyclin B1 mRNA and protein levels. These results support the hypothesis that long after radiation exposure a "metabolic redox response" regulates radiosensitivity of human glioma cells.