CellROX Green Research Articles

Thymoquinone Induces Mitochondrial Damage and Death of Cerebellar Granule Neurons

Thymoquinone (TQ) exhibits a wide spectrum of biological activities. Most studies on the neurotoxic action of TQ have been carried out in cancer cell lines. Here, we studied the toxic effect of TQ in primary neuronal cultures in vitro. Incubation with 0.04-0.05 mM TQ for 24 h induced the death of cultured cerebellar granule neurons (CGNs) in a dose-dependent manner. Neuronal death was preceded by an increase in the reactive oxygen species (ROS) generation, as demonstrated using CellROX Green and MitoSOX Red. Confocal and electron microscopy showed that incubation with 0.05 mM TQ for 5 h induced changes in the intracellular localization of mitochondria and mitochondria hypertrophy and cell swelling. The antioxidant N-acetyl-L-cysteine (2 mM) protected CGNs from the toxic action of TQ. Taken together, these facts suggest that TQ is toxic for normal neurons, while ROS-induced changes in the mitochondria can be one of the major causes of the TQ-induced neuronal damage and death.

Pathogenic Pathways in Early-Onset Autosomal Recessive Parkinson's Disease Discovered Using Isogenic Human Dopaminergic Neurons.

SummaryParkinson's disease (PD) is a complex and highly variable neurodegenerative disease. Familial PD is caused by mutations in several genes with diverse and mostly unknown functions. It is unclear how dysregulation of these genes results in the relatively selective death of nigral dopaminergic neurons (DNs). To address this question, we modeled PD by knocking out the PD genes PARKIN (PRKN), DJ-1 (PARK7), and ATP13A2 (PARK9) in independent isogenic human pluripotent stem cell (hPSC) lines. We found increased levels of oxidative stress in all PD lines. Increased death of DNs upon differentiation was found only in the PARKIN knockout line. Using quantitative proteomics, we observed dysregulation of mitochondrial and lysosomal function in all of the lines, as well as common and distinct molecular defects caused by the different PD genes. Our results suggest that precise delineation of PD subtypes will require evaluation of molecular and clinical data.

Reactive oxygen species (ROS) generation is stimulated by κ opioid receptor activation through phosphorylated c-Jun N-terminal kinase and inhibited by p38 mitogen-activated protein kinase (MAPK) activation

Activation of the mitogen-activated protein kinase (MAPK) c-Jun N-terminal kinase (JNK) by the Gi/o protein-coupled κ opioid receptor (KOR), μ opioid, and D2 dopamine receptors stimulates peroxiredoxin 6 (PRDX6)-mediated production of reactive oxygen species (ROS). ROS production by KOR-inactivating antagonists norbinaltorphimine (norBNI) and JDTic blocks Gαi protein activation, but the signaling mechanisms and consequences of JNK activation by KOR agonists remain uncharacterized. Binding of arrestins to KOR causes desensitization of G protein signaling and acts as a scaffold to initiate MAPK activation. Here, we found that the KOR agonists U50,488 and dynorphin B stimulated biphasic JNK activation with an early arrestin-independent phase, requiring the small G protein RAC family small GTPase 1 (RAC1) and protein kinase C (PKC), and a later arrestin-scaffolded phase, requiring RAC1 and Ras homolog family member (RHO) kinase. JNK activation by U50,488 and dynorphin B also stimulated PRDX6-dependent ROS production but with an inverted U-shaped dose-response relationship. KOR agonist-induced ROS generation resulted from the early arrestin-independent phase of JNK activation, and this ROS response was suppressed by arrestin-dependent activation of the MAPK p38. The apparent balance between p38 MAPK and JNK/ROS signaling has important physiological implications for understanding of dynorphin activities during the stress response. To visualize these activities, we monitored KOR agonist-mediated activation of ROS in transfected live cells by two fluorescent sensors, CellROX Green and HyPerRed. These findings establish an important aspect of opioid receptor signaling and suggest that ROS induction may be part of the physiological response to KOR activation.

Novel Endoperoxides Increase Apoptosis in Cancer, but not Normal Cells, and May Involve Differences in Transferrin Receptor (TfR) Expression

Artemisinin (ART), a common anti‐malarial drug, and its analogs are also useful as repurposed anti‐cancer drugs, but their properties are not fully elucidated. We previously synthesized novel trioxane (DMR) and dioxazanine (HSM) ART analogs and showed that: a. at low doses (5–10 μM), HSM > than DMR in inducing apoptosis in human colon and lung cancer cells, but not in normal lung cells; b. The actions involved reactive oxygen species (ROS) (Faseb J, ′18:32, 616.2). TfR expression is increased in cancer vs. normal cells. Thus, we hypothesize that, in cancer cells, TfR increases [Fe2+]i which acts on the endoperoxide to produce ROS and induce apoptosis. We examined the mechanism of action of DMR/HSM in inducing apoptosis in human breast, lung, and colon cancer cell lines as compared to normal cell lines. To increase the efficacy of the drug, we also designed and synthesized a 3rd novel analog with dual dioxazanine pharmacophores, PMW.Confluent normal (Breast: MCF10A, Lung: A549) and cancer (Breast: MCF7, Colon: T84, Lung: A549) cells were treated (18 H) with 1–50μM of DMR, HSM or PMW±Deferoxamine (DFO, iron chelator; 1–25 μM). Cells were stained with FITC‐Annexin V (apoptosis), propidium iodide (cell death) or CellRox Green (mitochondrial/nuclear ROS), imaged and quantified by flow cytometry and/or microscopy (Image J). Normal and cancer cell lysates (30 μg) were subjected to SDS‐PAGE and immunoblotting with polyclonal TfR antibody or anti‐GAPDH (control).As in other cancer cells, both DMR and HSM (5 – 50μM) induced apoptosis in breast cancer, but not normal, cells. In contrast to A549 lung cells, where HSM was more effective than DMR, in MCF7 breast cells, 50μM DMR was more efficient (~40%) in inducing apoptosis than HSM (10%). This dictates the development of novel tissue‐specific analogs. The new analog, PMW (1 – 50μM, 18H) did not induce apoptosis in the cancer cells. The larger size of PMW may have slowed entry, suggesting longer incubations.Image J analysis (mean pixel intensity; 18 H) of CellRox+ lung cells (n=3) showed that HSM dose‐dependently increased ROS in cancer A549: (DMSO: 1.5±0.2; HSM, 5 μM: 19±2; 10 μM: 48±4; 50 μM: 59±2); but not normal BEAS2B: (DMSO: 1.0±0.3; HSM, 5 μM: 1.8±1; 10 μM: 2.2±0.4; 50 μM: 2.9±2). Similarly, in breast cancer cells (n=3), DMR dose‐dependently increased ROS in MCF7 (DMSO: 1.0±0.3; DMR, 5 μM: 12±2; 10 μM: 51±5; 50 μM: 42±3); but not in MCF10A (DMSO: 1.0±0.2; DMR, 5 μM: 1.6±1; 10 μM: 3.1±1; 50 μM: 2.5±1).Immunoblotting detected a distinct 94 kDa TfR protein in colon, lung, and breast cancer, but not normal cells. The effect of DMR/HSM±DFO on apoptosis was examined to study the role of TfR. Albeit showing an inhibitory trend, DFO did not significantly alter ART analogs actions (% Annexin V+ cells,18 H: A549: HSM,10μM: 46±4; HSM+5μM DFO: 38±6; HSM +10μM DFO: 33±9; MCF7: DMR,10μM: 36±4; DMR+5μM DFO: 31±7; DMR+10μM DFO: 29±5; n≥3).Synthesizing novel ART‐analogs with improved pharmacokinetics to specifically target cancer cells and elucidating their mechanism of action will help develop new alternatives to treat cancer.Support or Funding InformationNSF ‐ MRI: DBI‐1427937 to JS; Ben U Funds to JS and DMR; UIC Funds to MCR; APS‐STRIDE to UD; APS‐UGSRF to MHThis abstract is from the Experimental Biology 2019 Meeting. There is no full text article associated with this abstract published in The FASEB Journal.

Single-Cell, Time-Lapse Reactive Oxygen Species Detection in E. coli.

Detection of reactive oxygen species (ROS) in bacteria has been limited to bulk biochemical assays. Although they are powerful and quantitative tools to understand the overall production of ROS in E. coli, such assays provide limited spatial and temporal information when correlating cellular phenotype with perturbations such as antibiotics or other treatments. We have developed single-cell, time-lapse assays to detect ROS in live E. coli. The assays utilize flow systems on a fluorescence microscope to correlate symptoms aroused from biological or chemical perturbations with the in situ detection of ROS. ROS is detected by fluorogenic dyes that accumulate inside the cell, allowing detection of ROS in single cells in both homogeneous and heterogeneous samples using CellROX Green and Amplex® Red/APEX2. © 2018 by John Wiley & Sons, Inc.

Abstract 3530: Coenzyme Q10 (BPM31510-IV in clinical trials) increases mitochondrial Q-pool and modulates electron transport chain function to elicit cell death in pancreatic cancer cells

Abstract Mitochondria play a multifaceted role in tumorigenesis through regulation of energy production, biomass, redox state, and engagement of cell death pathways. The mitochondrial Q (coenzyme Q10)-pool facilitates electron transport from Complexes I and II to III and is essential for regulating these activities. Therefore, altering mitochondrial Q-pool homeostasis represents a potential therapeutic strategy in cancer. BPM 31510 is a Coenzyme Q10 containing lipid nanodispersion currently in clinical trials for pancreatic cancer. Here, we used BPM 31510 to assess how modulation of mitochondrial Q-pool homeostasis impacts mitochondrial electron transport chain function to activate regulated cell death. Pancreatic cancer cell lines (MIA PaCa-2 and Panc-1) represent models with sensitivity to BPM 31510 both in vitro and in vivo. Treatment with BPM 31510 (EC50 dose, 24 h) resulted in significant mitochondrial enrichment of CoQ10 compared to other subcellular compartments, and quantitatively, CoQ10 levels were 100 times higher in mitochondria isolated from BPM 31510 treated cells over untreated controls (MIA PaCa-2 untreated, 0.31 nmol/mg; treated, 39.4 nmol/mg; Panc1 untreated, 0.21 nmol/mg; treated, 29.3 nmol/mg). Alterations in mitochondrial respiration characterized by dose-dependent decreases in succinate- or glycerol-3-phosphate-fueled respiration were observed in cells treated with BPM 31510, while pyruvate or TMPD/ascorbate-fueled respiration was only modestly affected, suggesting that BPM31510 specifically impairs respiratory responses dependent on Complexes II/III. Moreover, in the presence of multiple mitochondrial substrates, total respiratory capacity was decreased and reliance on pyruvate (Complex I)-fueled respiration was increased with BPM 31510 treatment, indicative of bioenergetic remodeling. Concomitantly to BPM 31510-dependent changes in mitochondrial respiratory responses, BPM 31510 exposure increased oxidation of the reactive oxygen species (ROS) probes, CellROX Green and DCF-DA, increased oxidized glutathione, and decreased levels of the cellular reducing equivalent NADPH. Importantly, BPM 31510-induced death could be partially rescued by agents which alleviate electron transport chain impairment linking respiratory function to the anti-cancer mechanism of action of BPM 31510. Together, these data indicate that BPM31510 directly impairs the mitochondrial Q-pool and respiratory function resulting in oxidative stress and consequential cell death and thus provide mechanistic understanding of the anti-cancer activity of BPM31510. Citation Format: Pallavi Awate, Tulin Dadali, Ryan Ng, Saie Mogre, Anne R. Diers, Hannah Rockwell, Justice McDaniel, Emily Chen, Fei Gao, Michael Kiebish, Stephane Gesta, Vivek Vishnudas, Niven R. Narain, Rangaprasad Sarangarajan. Coenzyme Q10 (BPM31510-IV in clinical trials) increases mitochondrial Q-pool and modulates electron transport chain function to elicit cell death in pancreatic cancer cells [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2018; 2018 Apr 14-18; Chicago, IL. Philadelphia (PA): AACR; Cancer Res 2018;78(13 Suppl):Abstract nr 3530.

N-Acetyl-L-cysteine in the Presence of Cu2+ Induces Oxidative Stress and Death of Granule Neurons in Dissociated Cultures of Rat Cerebellum.

Addition into the culture medium of the antioxidant N-acetylcysteine (NAC, 1mM) in the presence of Cu2+ (0.0005-0.001mM) induced intensive death of cultured rat cerebellar granule neurons, which was significantly decreased by the zinc ion chelator TPEN (N,N,N',N'-tetrakis(2-pyridylmethyl)ethylenediamine). However, the combined action of NAC and Zn2+ did not induce destruction of the neurons. Measurement of the relative intracellular concentration of Zn2+ with the fluorescent probe FluoZin-3 AM or of free radical production using a CellROX Green showed that incubation of the culture for 4h with Cu2+ and NAC induced an intensive increase in the fluorescence of CellROX Green but not of FluoZin-3. Probably, the protective effect of TPEN in this case could be mediated by its ability to chelate Cu2+. Incubation of cultures in a balanced salt solution in the presence of 0.01mM Cu2+ caused neuronal death already after 1h if the NAC concentration in the solution was within 0.005-0.05mM. NAC at higher concentrations (0.1-1mM) together with 0.01mM Cu2+ did not cause the death of neurons. These data imply that the antioxidant NAC can be potentially harmful to neurons even in the presence of nanomolar concentrations of variable valence metals.

Edaravone Prevents Retinal Degeneration in Adult Mice Following Optic Nerve Injury.

To assess the therapeutic potential of edaravone, a free radical scavenger that is used for the treatment of acute brain infarction and amyotrophic lateral sclerosis, in a mouse model of optic nerve injury (ONI). Two microliters of edaravone (7.2 mM) or vehicle were injected intraocularly 3 minutes after ONI. Optical coherence tomography, retrograde labeling of retinal ganglion cells (RGCs), histopathology, and immunohistochemical analyses of phosphorylated apoptosis signal-regulating kinase-1 (ASK1) and p38 mitogen-activated protein kinase (MAPK) in the retina were performed after ONI. Reactive oxygen species (ROS) levels were assessed with a CellROX Green Reagent. Edaravone ameliorated ONI-induced ROS production, RGC death, and inner retinal degeneration. Also, activation of the ASK1-p38 MAPK pathway that induces RGC death following ONI was suppressed with edaravone treatment. The results of this study suggest that intraocular administration of edaravone may be a useful treatment for posttraumatic complications.

Oxidative stress induced in E. coli by the human antimicrobial peptide LL-37.

Antimicrobial peptides (AMPs) are thought to kill bacterial cells by permeabilizing their membranes. However, some antimicrobial peptides inhibit E. coli growth more efficiently in aerobic than in anaerobic conditions. In the attack of the human cathelicidin LL-37 on E. coli, real-time, single-cell fluorescence imaging reveals the timing of membrane permeabilization and the onset of oxidative stress. For cells growing aerobically, a CellROX Green assay indicates that LL-37 induces rapid formation of oxidative species after entry into the periplasm, but before permeabilization of the cytoplasmic membrane (CM). A cytoplasmic Amplex Red assay signals a subsequent burst of oxidative species, most likely hydrogen peroxide, shortly after permeabilization of the CM. These signals are much stronger in the presence of oxygen, a functional electron transport chain, and a large proton motive force (PMF). They are much weaker in cells growing anaerobically, by either fermentation or anaerobic respiration. In aerobic growth, the oxidative signals are attenuated in a cytochrome oxidase–bd deletion mutant, but not in a –bo3 deletion mutant, suggesting a specific effect of LL-37 on the electron transport chain. The AMPs melittin and LL-37 induce strong oxidative signals and exhibit O2-sensitive MICs, while the AMPs indolicidin and cecropin A do not. These results suggest that AMP activity in different tissues may be tuned according to the local oxygen level. This may be significant for control of opportunistic pathogens while enabling growth of commensal bacteria.

Production of Superoxide in Bacteria Is Stress- and Cell State-Dependent: A Gating-Optimized Flow Cytometry Method that Minimizes ROS Measurement Artifacts with Fluorescent Dyes.

The role of reactive oxygen species (ROS) in microbial metabolism and stress response has emerged as a major theme in microbiology and infectious disease. Reactive fluorescent dyes have the potential to advance the study of ROS in the complex intracellular environment, especially for high-content and high-throughput analyses. However, current dye-based approaches to measuring intracellular ROS have the potential for significant artifacts. Here, we describe a robust platform for flow cytometric quantification of ROS in bacteria using fluorescent dyes, with ROS measurements in 10s-of-1000s of individual cells under a variety of conditions. False positives and variability among sample types (e.g., bacterial species, stress conditions) are reduced with a flexible four-step gating scheme that accounts for side- and forward-scattered light (morphological changes), background fluorescence, DNA content, and dye uptake to identify cells producing ROS. Using CellROX Green dye with Escherichia coli, Mycobacterium smegmatis, and Mycobacterium bovis BCG as diverse model bacteria, we show that (1) the generation of a quantifiable CellROX Green signal for superoxide, but not hydrogen peroxide-induced hydroxyl radicals, validates this dye as a superoxide detector; (2) the level of dye-detectable superoxide does not correlate with cytotoxicity or antibiotic sensitivity; (3) the non-replicating, antibiotic tolerant state of nutrient-deprived mycobacteria is associated with high levels of superoxide; and (4) antibiotic-induced production of superoxide is idiosyncratic with regard to both the species and the physiological state of the bacteria. We also show that the gating method is applicable to other fluorescent indicator dyes, such as the 5-carboxyfluorescein diacetate acetoxymethyl ester and 5-cyano-2,3-ditolyl tetrazolium chloride for cellular esterase and reductive respiratory activities, respectively. These results demonstrate that properly controlled flow cytometry coupled with fluorescent probes provides precise and accurate quantitative analysis of ROS generation and metabolic changes in stressed bacteria.

ROS Detection in Botryococcus braunii Colonies with CellROX Green Reagent.

We analyzed the reactive oxygen species (ROS) accumulation in the colony-forming green microalga Botryococcus braunii in response to several stress inducers such as NaCl, NaHCO3, salicylic acid (SA), methyl jasmonate, and acetic acid. A staining assay using the fluorescent dye CellROX Green was used. CellROX Green is a fluorogenic probe used for measuring oxidative stress in live cells. The dye is weakly fluorescent inside cells in a reduced state but exhibits bright green photostable fluorescence upon oxidation by ROS and subsequent binding to DNA. The large amount of liquid hydrocarbons produced and excreted by B. braunii, creates a highly hydrophobic extracellular environment that makes difficult to study short times defense responses on this microalga. The procedure developed here allowed us to detect ROS in this microalga even within a short period of time (in minutes) after treatment of cells with different stress inducers.

Glucose deprivation stimulates Cu(2+) toxicity in cultured cerebellar granule neurons and Cu(2+)-dependent zinc release.

Copper chloride (0.01mM, 2h) did not have significant influence on the survival of cerebellar granule neurons (CGNs) incubated in balanced salt solution. However, CuCl2 caused severe neuronal damage by glucose deprivation (GD). The glutamate NMDA-receptors blocker MK-801 partially and antioxidant N-acetyl-l-cysteine (NAC) or Zn(2+) chelator, N,N,N',N'-tetrakis(2-pyridylmethyl)ethylenediamine (TPEN) almost entirely protected CGNs from this toxic effect. Measurements of intracellular calcium ions using Fluo-4 AM, or zinc ions with FluoZin-3 AM demonstrated that 1 h-exposure to GD induced intensive increase of Fluo-4 but not FluoZin-3 fluorescence in neurons. The supplementation of solution with CuCl2 caused an increase of FluoZin-3, Fluo-4 and CellROX Green (reactive oxygen species probe) fluorescence by GD. The stimulation of Fluo-4 but not FluoZin-3 fluorescence by copper could be prevented partially by MK-801 and as well as CellROX Green fluorescence by NAC at GD. This data imply that during GD copper ions induce intense displacement zinc ions from intracellular stores, in addition free radical production, glutamate release and Ca(2+) overload of CGNs, that causes death of neurons as a result.

Tetrahydroanthraquinone Derivative (±)-4-Deoxyaustrocortilutein Induces Cell Cycle Arrest and Apoptosis in Melanoma Cells via Upregulation of p21 and p53 and Downregulation of NF-kappaB.

Background: Malignant melanoma is an aggressive type of skin cancer with high risk for metastasis and chemoresistance. Disruption of tightly regulated processes such as cell cycle, cell adhesion, cell differentiation and cell death are predominant in melanoma development. So far, conventional treatment options have been insufficient to treat metastatic melanoma and survival rates are poor. Anthraquinone compounds have been reported to have anti-tumorigenic potential by DNA-interaction, promotion of apoptosis and suppression of proliferation in various cancer cells.Methods: In the current study, the racemic tetrahydroanthraquinone derivative (±)-4-deoxyaustrocortilutein (4-DACL) was synthesized and the cytotoxic activity against melanoma cells and melanoma spheroids determined by CellTiter-Blue viability Assay and phase contrast microscopy. Generation of reactive oxygen species (ROS) was determined with CellROX Green and Deep Red Reagent kit and microplate-based fluorometry. Luciferase reporter gene assays for nuclear factor kappa B (NF-κB) and p53 activities and western blotting analysis were carried out to detect the expression of anti-proliferative or pro-apoptotic (p53, p21, p27, MDM2, and GADD45M) and anti-apoptotic (p65, IκB-α, IKK) proteins. Cell cycle distribution and apoptosis rate were detected by flow cytometry, the morphological changes visualized by fluorescence microscopy and the activation of different caspase cascades distinguished by Caspase Glo 3/7, 8 and 9 Assays.Results: We demonstrated that 4-DACL displayed high activity against different malignant melanoma cells and melanoma spheroids and only low toxicity to melanocytes and other primary cells. In particular, 4-DACL treatment induced mitochondrial ROS, reduced NF-κB signaling activity and increased up-regulation of the cell cycle inhibitors cyclin-dependent kinase inhibitor p21 (p21WAF1/Cip1) and the tumor suppressor protein p53 in a dose-dependent manner, which was accompanied by decreased cell proliferation and apoptosis via the intrinsic pathway.Conclusion: According to these results, we suggest that 4-DACL may be a promising therapeutic agent for the treatment of malignant melanoma.

Detection of Reactive Oxygen Species Using MitoSOX and CellROX in Zebrafish

The production of free radicals is the result of normal cellular metabolism. Free radicals are involved in innumerable different cellular and biological functions such as signaling, proliferation, cell death, aging, inflammation, etc. Under physiological conditions, the levels of reactive oxygen species (ROS) are strictly regulated by the cells. However, during stressful conditions such as oxidative stress, ROS levels increase causing damages to different molecules like DNA, lipids and proteins. Increased levels of ROS have been associated with a growing list of different diseases. In this protocol, we used MitoSOX and CellROX Green oxidative stress probes to label the intracellular ROS and detect the fluorescence using cell sorting and confocal analyses.

Hyperbaric Oxygen Increases Sensitivity to Chemotherapy in Acute Leukemia

Introduction: Chemotherapy resistance remains a significant challenge in the treatment of adults with Acute Myeloid Leukemia (AML). Although new targets and novel agents are under clinical investigation, the majority of AML patients are treated with conventional induction chemotherapy consisting of an anthracycline and cytarabine, with little modification in over 40 years. Insights into the biology of resistance to cytarabine and anthracyclines may suggest novel strategies in the treatment of AML. Hyperbaric oxygen (HBO) treatment is commonly used in the treatment of decompression sickness from scuba diving, wound healing and infections. 100% oxygen is administered at increased pressure. Limited published data suggest a role for HBO to cause in vitro apoptosis of leukemia cell lines, but no mechanism has been described. We examined the role of HBO in AML sensitivity to chemotherapy, reactive oxygen species (ROS) generation, glycolytic pathway activation and gene expression.Methods: Human AML cell lines HL-60 and MV-411 were treated with escalating doses of cytarabine or daunorubicin as single agents followed by HBO treatment for 2 hours or normobaric conditions. HBO treatment consisted of exposure to 100% oxygen at 2.5 atmosphere absolutes for a total of 2 hours in a specifically designed in vitro HBO chamber. At 24 hours after chemotherapy dosing, leukemia cells were analyzed for proliferation using Cell Titer 96 Aqueous MTS assay (Promega). Apoptosis was measured using Annexin V-FITC and analyzed by flow cytometry. In order to understand potential mechanisms of HBO activity on leukemia cells, studies of HBO-treated cells without use of chemotherapy were also performed. CellROX Oxidative Stress assay (Life Technologies) was performed at 24 hours. CellROX Green and Deep Red reagents measured ROS level in the DNA and cytoplasm. RNA was isolated and real-time PCR was performed using CFX384 Real-time PCR system using primer sets specific for human hexokinase2 and phosphofructokinase to examine the effects of HBO on glycolysis. Gene expression profiling was performed using the Human Transcriptome Array 2.0 (Affymetrix) and examined differences in gene expression following HBO treatment of HL-60 cells and bone marrow from an AML patient.Results: HBO-treated leukemia cells had increased sensitivity to cytarabine (Table 1, Figure 1) (MV411 response to cytarabine: IC50 2649nM without HBO treatment, 1921nM with HBO, p=0.02; HL60 response to cytarabine: linear response without HBO, IC50 1625nM with HBO, p<0.035). Decreased proliferation was also seen with HBO-treated cells in response to daunorubicin but was not statistically significant (Table 1). Similarly, apoptosis from cytarabine in HL-60 cells was increased with HBO treatment (p<0.02), but the results with daunorubicin were not significant. Significant increases in ROS levels were observed at 24 hours post-HBO for both cell lines when compared to non-treated cells (HL60 DNA p<0.0001, HL60 cytoplasm p<0.01, MV411 DNA p<0.01, MV411 cytoplasm p<0.01). Glycolysis pathway components human hexokinase 2 and phosphofructokinase were upregulated in HL60 and MV411 cells treated with HBO compared to controls. Gene expression profiling showed that approximately 200 genes in coding regions were upregulated in HBO-treated HL-60 cells; specific piRNA clusters were downregulated in both AML cell line HL60 and a bone marrow sample from a patient with relapsed AML when treated with HBO.Conclusions: HBO treatment significantly increases the sensitivity to cytarabine AML cell lines in vitro. Increased ROS, upregulation of glycolysis-related enzymes and changes in gene expression profiling provide insight into the potential mechanisms of HBO enhanced chemotherapy sensitivity. Given the safety and commonplace use of HBO for other indications, and the significant challenge of chemotherapy resistance, it is important to pursue further studies to understand the biology of HBO treatment in AML.Table 1IC50 (nM) of chemotherapy agents in AML cell lines with and without co-treatment with HBO.CytarabineDaunorubicinno HBOHBOp-valueno HBOHBOp-valueMV411264919210.0214709.1641.1nsHL60*16250.0346639406.5ns* linear response, no IC50 available [Display omitted] DisclosuresNo relevant conflicts of interest to declare.

Melissa officinalis extract induces apoptosis and inhibits proliferation in colon cancer cells through formation of reactive oxygen species

PurposeEfficient strategies for the prevention of colon cancer are extensively being explored, including dietary intervention and the development of novel phytopharmaceuticals. Safe extracts of edible plants contain structurally diverse molecules that can effectively interfere with multi-factorial diseases such as colon cancer. In this study, we describe the antiproliferative and proapoptotic effects of ethanolic lemon balm (Melissa officinalis) leaves extract in human colon carcinoma cells. We further investigated the role of extra- and intracellular reactive oxygen species (ROS). MethodsAntitumor effects of lemon balm extract (LBE) were investigated in HT-29 and T84 human colon carcinoma cells. Inhibition of proliferation was analyzed by DNA quantification. The causal cell cycle arrest was determined by flow cytometry of propidium iodide-stained cells and by immunoblotting of cell cycle regulator proteins. To investigate apoptosis, cleavage of caspases 3 and 7 was detected by immunoblotting and fluorescence microscopy. Phosphatidylserine externalization was measured by Annexin V assays. Mechanistic insights were gained by measurement of ROS using the indicator dyes CM-H2DCFDA and Cell ROX Green. ResultsAfter 3 and 4 days of treatment, LBE inhibited the proliferation of HT-29 and T84 colon carcinoma cells with an inhibitory concentration (IC50) of 346 and 120 µg/ml, respectively. Antiproliferative effects were associated with a G2/M cell cycle arrest and reduced protein expression of cyclin dependent kinases (CDK) 2, 4, 6, cyclin D3, and induced expression of cyclin-dependent kinase inhibitor 2C (p18) and 1A (p21). LBE (600 µg/ml) induced cleavage of caspases 3 and 7 and phosphatidylserine externalization. LBE-induced apoptosis was further associated with formation of ROS, whereas quenching of ROS by antioxidants completely rescued the colon carcinoma cells from LBE-induced apoptosis. ConclusionsLemon balm (Melissa officinalis) extract inhibits the proliferation of colon carcinoma cells and induces apoptosis through formation of ROS. Taken together, LBE or subfractions thereof could be used for the prevention of colon cancer.

Monitoring Cr Intermediates and Reactive Oxygen Species with Fluorescent Probes during Chromate Reduction

Cr(VI) genotoxicity is caused by products of its reductive metabolism inside the cells. Reactive oxygen species (ROS) and Cr(V,IV) intermediates are potential sources of oxidative damage by Cr(VI). Here, we investigated seven fluorescent probes for the detection of ROS and non-ROS oxidants in Cr(VI) reactions with its main reducers. We found that Cr(V)-skipping metabolism of Cr(VI) by ascorbate in vitro gave no responses with all tested dyes, indicating nonreactivity of Cr(IV) and absence of ROS. Cr(VI) reduction with glutathione (GSH) or Cys strongly enhanced the fluorescence of dichlorofluorescein (DCF) and dihydrorhodamine 123 (DHR123) but produced minimal fluorescence with dihydroethidium and no increases with aminophenylfluorescein and CellRox Green, Orange, and Red. Several tests showed that Cr(VI)-thiol reactions lacked ROS and that Cr(V) caused oxidation of DCF and DHR123. DCF reacted only with free Cr(V), whereas DHR123 detected both the free Cr(V) and Cr(V)-GSH complex. We estimated that Cr(VI)-GSH reactions generated approximately 75% Cr(V)-GSH and 25% free Cr(V), whereas Cys reactions appeared to produce only free Cr(V). DHR123 measurements in H460 cells showed that reduction of Cr(VI) was complete within 20 min postexposure, but it lasted at least 1 h without GSH. Cells with restored ascorbate levels exhibited no DCF or DHR123 oxidation by Cr(VI). Overall, our results demonstrated that Cr(VI) metabolism with its biological reducers lacked ROS and that DHR123 and DCF responses were indicators of total and free Cr(V), respectively. CellRox dyes, dihydroethidium and aminophenylfluorescein, are insensitive to Cr(V,IV) and can be used for monitoring ROS during coexposure to Cr(VI) and oxidants.