N-alkyl triphenylvinylpyridinium conjugated dihydroartemisinin perturbs mitochondrial functions resulting in enhanced cancer versus normal cell toxicity.

Dihydroartemisinin (DHA) is an FDA-approved antimalarial drug that has been repurposed for cancer therapy because of its preferential antiproliferative effects on cancer versus normal cells. Mitochondria represent an attractive target for cancer therapy based on their regulatory role in proliferation and cell death. This study investigates whether DHA conjugated to innately fluorescent N-alkyl triphenylvinylpyridinium (TPVP) perturbs mitochondrial functions resulting in a differential toxicity of cancer versus normal cells. TPVP-DHA treatments resulted in a dose-dependent toxicity of human melanoma and pancreatic cancer cells, whereas normal human fibroblasts were resistant to this treatment. TPVP-DHA treatments resulted in a G1-delay of the cancer cell cycle, which was also associated with a significant inhibition of the mTOR-metabolic and ERK1/2-proliferative signaling pathways. TPVP-DHA treatments perturbed mitochondrial functions, which correlated with increases in mitochondrial fission. In summary, TPVP mediated mitochondrial targeting of DHA enhanced cancer cell toxicity by perturbing mitochondrial functions and morphology.

Arachidonate 12-lipoxygenase and 12-hydroxyeicosatetraenoic acid contribute to stromal aging-induced progression of pancreatic cancer.

The incidence of pancreatic cancer increases with age, suggesting that chronological aging is a significant risk factor for this disease. Fibroblasts are the major nonmalignant cell type in the stroma of human pancreatic ductal adenocarcinoma (PDAC). In this study, we investigated whether the chronological aging of normal human fibroblasts (NHFs), a previously underappreciated area in pancreatic cancer research, influences the progression and therapeutic outcomes of PDAC. Results from experiments with murine xenografts and 2D and 3D co-cultures of NHFs and PDAC cells revealed that older NHFs stimulate proliferation of and confer resistance to radiation therapy of PDAC. MS-based metabolite analysis indicated that older NHFs have significantly increased arachidonic acid 12-lipoxygenase (ALOX12) expression and elevated levels of its mitogenic metabolite, 12-(S)-hydroxy-5,8,10,14-eicosatetraenoic acid (12-(S)-HETE) compared with their younger counterparts. In co-cultures with older rather than with younger NHFs, PDAC cells exhibited increases in mitogen-activated protein kinase signaling and cellular metabolism, as well as a lower oxidation state that correlated with their enhanced proliferation and resistance to radiation therapy. Expression of ALOX12 was found to be significantly lower in PDAC cell lines and tumor biopsies, suggesting that PDAC cells rely on a stromal supply of mitogens for their proliferative needs. Pharmacological (hydroxytyrosol) and molecular (siRNA) interventions of ALOX12 in older NHFs suppressed their ability to stimulate proliferation of PDAC cells. We conclude that chronological aging of NHFs contributes to PDAC progression and that ALOX12 and 12-(S)-HETE may be potential stromal targets for interventions that seek to halt progression and improve therapy outcomes.

Dual Oxidase-Induced Sustained Generation of Hydrogen Peroxide Contributes to Pharmacologic Ascorbate-Induced Cytotoxicity.

Pharmacologic ascorbate treatment (P-AscH-, high-dose, intravenous vitamin C) results in a transient short-term increase in the flux of hydrogen peroxide that is preferentially cytotoxic to cancer cells versus normal cells. This study examines whether an increase in hydrogen peroxide is sustained posttreatment and potential mechanisms involved in this process. Cellular bioenergetic profiling following treatment with P-AscH- was examined in tumorigenic and nontumorigenic cells. P-AscH- resulted in sustained increases in the rate of cellular oxygen consumption (OCR) and reactive oxygen species (ROS) in tumor cells, with no changes in nontumorigenic cells. Sources for this increase in ROS and OCR were DUOX 1 and 2, which are silenced in pancreatic ductal adenocarcinoma, but upregulated with P-AscH- treatment. An inducible catalase system, to test causality for the role of hydrogen peroxide, reversed the P-AscH--induced increases in DUOX, whereas DUOX inhibition partially rescued P-AscH--induced toxicity. In addition, DUOX was significantly downregulated in pancreatic cancer specimens compared with normal pancreas tissues. Together, these results suggest that P-AscH--induced toxicity may be enhanced by late metabolic shifts in tumor cells, resulting in a feed-forward mechanism for generation of hydrogen peroxide and induction of metabolic stress through enhanced DUOX expression and rate of oxygen consumption. SIGNIFICANCE: A high dose of vitamin C, in addition to delivering an acute exposure of H2O2 to tumor cells, activates DUOX in pancreatic cancer cells, which provide sustained production of H2O2.

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.

Methylseleninic Acid Induces Lipid Peroxidation and Radiation Sensitivity in Head and Neck Cancer Cells.

Combination radiation and chemotherapy are commonly used to treat locoregionally advanced head and neck squamous cell carcinoma (HNSCC). Aggressive dosing of these therapies is significantly hampered by side effects due to normal tissue toxicity. Selenium represents an adjuvant that selectively sensitizes cancer cells to these treatments modalities, potentially by inducing lipid peroxidation (LPO). This study investigated whether one such selenium compound, methylseleninic acid (MSA), induces LPO and radiation sensitivity in HNSCC cells. Results from 4,4-difluoro-4-bora-3a,4a-diaza-S-indacene (BODIPY) C11 oxidation and ferric thiocyanate assays revealed that MSA induced LPO in cells rapidly and persistently. Propidium iodide (PI) exclusion assay found that MSA was more toxic to cancer cells than other related selenium compounds; this toxicity was abrogated by treatment with α-tocopherol, an LPO inhibitor. MSA exhibited no toxicity to normal fibroblasts at similar doses. MSA also sensitized HNSCC cells to radiation as determined by clonogenic assay. Intracellular glutathione in cancer cells was depleted following MSA treatment, and supplementation of the intracellular glutathione pool with N-acetylcysteine sensitized cells to MSA. The addition of MSA to a cell-free solution of glutathione resulted in an increase in oxygen consumption, which was abrogated by catalase, suggesting the formation of H2O2. Results from this study identify MSA as an inducer of LPO, and reveal its capability to sensitize HNSCC to radiation. MSA may represent a potent adjuvant to radiation therapy in HNSCC.

Enhanced Pharmacological Ascorbate Oxidation Radiosensitizes Pancreatic Cancer.

Pharmacologic ascorbate (P-AscH-) is emerging as a promising adjuvant for advanced pancreatic cancer. P-AscH- generates hydrogen peroxide (H2O2), leading to selective cancer cell cytotoxicity. Catalytic manganoporphyrins, such as MnT4MPyP, can increase the rate of oxidation of P-AscH-, thereby increasing the flux of H2O2, resulting in increased cytotoxicity. We hypothesized that a multimodal treatment approach, utilizing a combination of P-AscH-, ionizing radiation and MnT4MPyP, would result in significant flux of H2O2 and pancreatic cancer cytotoxicity. P-AscH- with MnT4MPyP increased the rate of oxidation of P-AscH- and produced radiosensitization in all pancreatic cancer cell lines tested. Three-dimensional (3D) cell cultures demonstrated resistance to P-AscH-, radiation or MnT4MPyP treatments alone; however, combined treatment with P-AscH- and MnT4MPyP resulted in the inhibition of tumor growth, particularly when also combined with radiation. In vivo experiments using a murine model demonstrated an increased rate of ascorbate oxidation when combinations of P-AscH- with MnT4MPyP were given, thus acting as a radiosensitizer. The translational potential was demonstrated by measuring increased ascorbate oxidation ex vivo, whereby MnT4MPyP was added exogenously to plasma samples from patients treated with P-AscH- and radiation. Combination treatment utilizing P-AscH-, manganoporphyrin and radiation results in significant cytotoxicity secondary to enhanced ascorbate oxidation and an increased flux of H2O2. This multimodal approach has the potential to be an effective treatment for pancreatic ductal adenocarcinoma.

MnSOD and Cyclin B1 Coordinate a Mito-Checkpoint during Cell Cycle Response to Oxidative Stress.

Communication between the nucleus and mitochondrion could coordinate many cellular processes. While the mechanisms regulating this communication are not completely understood, we hypothesize that cell cycle checkpoint proteins coordinate the cross-talk between nuclear and mitochondrial functions following oxidative stress. Human normal skin fibroblasts, representative of the G2-phase, were irradiated with 6 Gy of ionizing radiation and assayed for cyclin B1 translocation, mitochondrial function, reactive oxygen species (ROS) levels, and cytotoxicity. In un-irradiated controls, cyclin B1 was found primarily in the nucleus of G2-cells. However, following irradiation, cyclin B1 was excluded from the nucleus and translocated to the cytoplasm and mitochondria. These observations were confirmed further by performing transmission electron microscopy and cell fractionation assays. Cyclin B1 was absent in mitochondria isolated from un-irradiated G2-cells and present in irradiated G2-cells. Radiation-induced translocation of cyclin B1 from the nucleus to the mitochondrion preceded changes in the activities of mitochondrial proteins, that included decreases in the activities of aconitase and the mitochondrial antioxidant enzyme, manganese superoxide dismutase (MnSOD), and increases in complex II activity. Changes in the activities of mito-proteins were followed by an increase in dihydroethidium (DHE) oxidation (indicative of increased superoxide levels) and loss of the mitochondrial membrane potential, events that preceded the restart of the stalled cell cycle and subsequently the loss in cell viability. Comparable results were also observed in un-irradiated control cells overexpressing mitochondria-targeted cyclin B1. These results indicate that MnSOD and cyclin B1 coordinate a cross-talk between nuclear and mitochondrial functions, to regulate a mito-checkpoint during the cell cycle response to oxidative stress.

Mitofusin 1 and optic atrophy 1 shift metabolism to mitochondrial respiration during aging.

Replicative and chronological lifespan are two different modes of cellular aging. Chronological lifespan is defined as the duration during which quiescent normal cells retain their capacity to re-enter the proliferative cycle. This study investigated whether changes in metabolism occur during aging of quiescent normal human fibroblasts (NHFs) and the mechanisms that regulate these changes. Bioenergetics measurements were taken in quiescent NHFs from younger (newborn, 3-day, 5-month, and 1-year) and older (58-, 61-, 63-, 68-, and 70-year) healthy donors as well as NHFs from the same individual at different ages (29, 36, and 46 years). Results show significant changes in cellular metabolism during aging of quiescent NHFs: Old NHFs exhibit a significant decrease in glycolytic flux and lactate levels, and increase in oxygen consumption rate (OCR) and ATP levels compared to young NHFs. Results from the Seahorse XF Cell Mito Stress Test show that old NHFs with a lower Bioenergetic Health Index (BHI) are more prone to oxidative stress compared to young NHFs with a higher BHI. The increase in OCR in old NHFs is associated with a shift in mitochondrial dynamics more toward fusion. Genetic knockdown of mitofusin 1 (MFN1) and optic atrophy 1 (OPA1) in old NHFs decreased OCR and shifted metabolism more toward glycolysis. Downregulation of MFN1 and OPA1 also suppressed the radiation-induced increase in doubling time of NHFs. In summary, results show that a metabolic shift from glycolysis in young to mitochondrial respiration in old NHFs occurs during chronological lifespan, and MFN1 and OPA1 regulate this process.

N-acetyl-L-cysteine increases MnSOD activity and enhances the recruitment of quiescent human fibroblasts to the proliferation cycle during wound healing.

The rebuilding of the connective tissue during wound healing requires the recruitment of fibroblasts to the wound area as well as reentry of quiescent fibroblasts to the proliferative cycle. Whether this process can be modulated by a small molecular weight thiol antioxidant N-acetyl-L-cysteine (NAC) was tested in normal human skin fibroblasts (NHFs) using a uni-directional wound healing assay. NAC treated cells demonstrated a decreased migration rate but increased number of proliferating cells recruited into the wound area post wounding. Fifteen day quiescent control and NAC treated NHFs were re-plated at a lower density and cell numbers counted at different days post-plating. Interestingly, NAC treated cells exhibited increased cellular proliferation indicated by both decreased cell population doubling time and increased S phase cells. NAC treated cells demonstrated decreased steady state levels of reactive oxygen species as well as increased protein and activity levels of manganese superoxide dismutase (MnSOD). NAC treatment failed to induce proliferation in quiescent cells lacking MnSOD expression. These results demonstrate that NAC enhanced the recruitment of quiescent NHFs into proliferation cycle during wound healing. Our results also suggest that the wound healing properties of NAC might be due to its ability to induce and enhance MnSOD expression and activity. Altogether, these findings suggest NAC might be potentially developed as a dietary intervention to improve tissue injury in animals and humans.

Succinate dehydrogenase activity regulates PCB3-quinone-induced metabolic oxidative stress and toxicity in HaCaT human keratinocytes.

Polychlorinated biphenyls (PCBs) and their metabolites are environmental pollutants that are known to have adverse health effects. 1-(4-Chlorophenyl)-benzo-2,5-quinone (4-ClBQ), a quinone metabolite of 4-monochlorobiphenyl (PCB3, present in the environment and human blood) is toxic to human skin keratinocytes, and breast and prostate epithelial cells. This study investigates the hypothesis that 4-ClBQ-induced metabolic oxidative stress regulates toxicity in human keratinocytes. Results from Seahorse XF96 Analyzer showed that the 4-ClBQ treatment increased extracellular acidification rate, proton production rate, oxygen consumption rate and ATP content, indicative of metabolic oxidative stress. Results from a q-RT-PCR assay showed significant increases in the mRNA levels of hexokinase 2 (hk2), pyruvate kinase M2 (pkm2) and glucose-6-phosphate dehydrogenase (g6pd), and decreases in the mRNA levels of succinate dehydrogenase (complex II) subunit C and D (sdhc and sdhd). Pharmacological inhibition of G6PD-activity enhanced the toxicity of 4-ClBQ, suggesting that the protective function of the pentose phosphate pathway is functional in 4-ClBQ-treated cells. The decrease in sdhc and sdhd expression was associated with a significant decrease in complex II activity and increase in mitochondrial levels of ROS. Overexpression of sdhc and sdhd suppressed 4-ClBQ-induced inhibition of complex II activity, increase in mitochondrial levels of ROS, and toxicity. These results suggest that the 4-ClBQ treatment induces metabolic oxidative stress in HaCaT cells, and while the protective function of the pentose phosphate pathway is active, inhibition of complex II activity sensitizes HaCaT cells to 4-ClBQ-induced toxicity.

Forkhead box M1 regulates quiescence-associated radioresistance of human head and neck squamous carcinoma cells.

Cellular quiescence is a reversible growth arrest in which cells retain their ability to enter into and exit from the proliferative cycle. This study investigates the hypothesis that cell growth-state specific oxidative stress response regulates radiosensitivity of cancer cells. Results showed that quiescent (low proliferative index; >75% G1 phase and lower RNA content) Cal27 and FaDu human head and neck squamous cell carcinoma (HNSCC) are radioresistant compared to proliferating cells. Quiescent cells exhibited a three to tenfold increase in mRNA levels of Mn-superoxide dismutase (MnSOD), dual oxidase 2 (DUOX2) and dual-specificity phosphatase 1 (DUSP1), while mRNA levels of catalase (CAT), peroxiredoxin 3 (PRDX3) and C-C motif ligand 5 (CCL5) were approximately two to threefold lower compared to proliferating cells. mRNA levels of forkhead box M1 (FOXM1) showed the largest decrease in quiescent cells at approximately 18-fold. Surprisingly, radiation treatment resulted in a distinct gene expression pattern that is specific to proliferating and quiescent cells. Specifically, FOXM1 expression increased two to threefold in irradiated quiescent cells, while the same treatment had no net effect on FOXM1 mRNA expression in proliferating cells. RNA interference and pharmacological-based downregulation of FOXM1 abrogated radioresistance of quiescent cells. Furthermore, radioresistance of quiescent cells was associated with an increase in glucose consumption and expression of glucose-6-phosphate dehydrogenase (G6PD). Knockdown of FOXM1 resulted in a significant decrease in G6PD expression, and pharmacological-inhibition of G6PD sensitized quiescent cells to radiation. Taken together, these results suggest that targeting FOXM1 may overcome radioresistance of quiescent HNSCC.

PCB 126 perturbs hypoxia-induced HIF-1α activity and glucose consumption in human HepG2 cells.

Aerobic organisms strongly depend on the availability of oxygen for respiration and countless other metabolic processes to maintain cellular homeostasis. Under certain conditions, the amount of available oxygen can be limited. To support survival in environments with limited oxygen supply, hypoxia-inducible factors (HIFs) reprogram vital components of cellular metabolism. HIF-1α is an important mediator of acute and adaptive responses to hypoxic stress. Interestingly, the heterodimeric partner required by HIF-1α to function as transcription factor, known as ARNT, is also an essential part of the aryl hydrocarbon receptor (AhR) transcription factor complex. Thus, via ARNT a crosstalk exists between these two pathways that might affect HIF-1α-mediated processes. In this study we sought to assess the effect of the AhR agonist PCB 126 on HIF-1α activity as well as on HIF-1α-regulated targets involved in cellular metabolism in human HepG2 cells. Our results show that PCB 126 reduced HIF-1α localization to the nucleus. Furthermore, in an in vivo setting, rats exposed to parenteral PCB 126 also displayed reduced hepatocyte nuclear localization of HIF-1α. Additionally, HepG2 cells exposed to PCB 126 displayed reduced hypoxia-regulated HRE-luciferase reporter gene expression as well as a reduction in glucose consumption in conditions of hypoxia. In summary, this study reveals that HIF-1α-regulated cellular metabolic processes are negatively affected by PCB 126 which might ultimately affect adaptive responses and cell survival in hypoxic environments.

Hydroxytyrosol inhibits chemokine C-C motif ligand 5 mediated aged quiescent fibroblast-induced stimulation of breast cancer cell proliferation.

Cancer is an age-associated disease. Although the mechanisms of age-associated increase in cancer incidence are not completely understood, it is believed that the tumor stromal environment significantly influences epithelial malignancy. Fibroblasts are a major cell type in the stroma and, under normal conditions, fibroblasts reside in the quiescent state. Cellular quiescence is a reversible process where cells enter into the proliferative cycle and then exit back to quiescence. We have shown previously that quiescent fibroblasts lose their proliferative capacity as they age, and we defined this mode of cellular aging as chronological life span. Using conditioned media and co-culture experiments, results from this study show that normal human fibroblasts (NHFs) nearing the end of their chronological life span stimulate the proliferation of MB231 and MCF7 human breast epithelial cancer cells. Chemokine C-C motif ligand 5 (CCL5) expression was found to be approximately 8-fold higher in old compared to that in young quiescent NHFs, which correlated with an increase in the ERK1/2-cyclin D1 pro-proliferative pathway in MB231 cells. Conditioned media treated with anti-CCL5 antibody suppressed the activation of the ERK1/2-cyclin D1 pathway and proliferation of MB231 cells. Hydroxytyrosol, a dietary polyphenol and an active ingredient of olive, inhibited CCL5 expression in aging quiescent NHFs. This inhibition was associated with NHFs inability to activate the ERK1/2-cyclin D1 pathway and enhance proliferation of MB231 cells. These results show that fibroblasts nearing the end of their chronological life span promote proliferation of human breast epithelial cancer cells and dietary polyphenols inhibit this process.

Manganese superoxide dismutase regulates a redox cycle within the cell cycle.

SIGNIFICANCE: Manganese superoxide dismutase (MnSOD) is a nuclear-encoded and mitochondria-matrix-localized oxidation-reduction (redox) enzyme that regulates cellular redox homeostasis. Cellular redox processes are known to regulate proliferative and quiescent growth states. Therefore, MnSOD and mitochondria-generated reactive oxygen species (ROS) are believed to be critical regulators of quiescent cells' entry into the cell cycle and exit from the proliferative cycle back to the quiescent state. RECENT ADVANCES/CRITICAL ISSUES: Recent evidence suggests that the intracellular redox environment fluctuates during the cell cycle, shifting toward a more oxidized status during mitosis. MnSOD activity is higher in G0/G1 cells compared with S, G2 and M phases. After cell division, MnSOD activity increases in the G1 phase of the daughter generation. The periodic fluctuation in MnSOD activity during the cell cycle inversely correlates with cellular superoxide levels as well as glucose and oxygen consumption. Based on an inverse correlation between MnSOD activity and glucose consumption during the cell cycle, it is proposed that MnSOD is a central molecular player for the "Warburg effect." FUTURE DIRECTIONS: In general, loss of MnSOD activity results in aberrant proliferation. A better understanding of the MnSOD and mitochondrial ROS-dependent cell cycle processes may lead to novel approaches to overcome aberrant proliferation. Since ROS have both deleterious (pathological) and beneficial (physiological) effects, it is proposed that "eustress" should be used when discussing ROS processes that regulate normal physiological functions, while "oxidative stress" should be used to discuss the deleterious effects of ROS.

Selenoprotein P inhibits radiation-induced late reactive oxygen species accumulation and normal cell injury.

PURPOSE: Radiation is a common mode of cancer therapy whose outcome is often limited because of normal tissue toxicity. We have shown previously that the accumulation of radiation-induced late reactive oxygen species (ROS) precedes cell death, suggesting that metabolic oxidative stress could regulate cellular radiation response. The purpose of this study was to investigate whether selenoprotein P (SEPP1), a major supplier of selenium to tissues and an antioxidant, regulates late ROS accumulation and toxicity in irradiated normal human fibroblasts (NHFs). METHODS AND MATERIALS: Flow cytometry analysis of cell viability, cell cycle phase distribution, and dihydroethidium oxidation, along with clonogenic assays, were used to measure oxidative stress and toxicity. Human antioxidant mechanisms array and quantitative real-time polymerase chain reaction assays were used to measure gene expression during late ROS accumulation in irradiated NHFs. Sodium selenite addition and SEPP1 overexpression were used to determine the causality of SEPP1 regulating late ROS accumulation and toxicity in irradiated NHFs. RESULTS: Irradiated NHFs showed late ROS accumulation (4.5-fold increase from control; P<.05) that occurs after activation of the cell cycle checkpoint pathways and precedes cell death. The mRNA levels of CuZn- and Mn-superoxide dismutase, catalase, peroxiredoxin 3, and thioredoxin reductase 1 increased approximately 2- to 3-fold, whereas mRNA levels of cold shock domain containing E1 and SEPP1 increased more than 6-fold (P<.05). The addition of sodium selenite before the radiation treatment suppressed toxicity (45%; P<.05). SEPP1 overexpression suppressed radiation-induced late ROS accumulation (35%; P<.05) and protected NHFs from radiation-induced toxicity (58%; P<.05). CONCLUSION: SEPP1 mitigates radiation-induced late ROS accumulation and normal cell injury.

Selenoprotein P regulates 1-(4-Chlorophenyl)-benzo-2,5-quinone-induced oxidative stress and toxicity in human keratinocytes.

Polychlorinated biphenyls and their metabolites are environmental pollutants that are believed to have adverse health effects presumably by inducing oxidative stress. To determine if 1-(4-Chlorophenyl)-benzo-2,5-quinone (4-ClBQ; metabolite of 4-monochlorobiphenyl, PCB3)-induced oxidative stress is associated with changes in the expression of specific antioxidant genes, mRNA levels of 92 oxidative stress-response genes were analyzed using TaqMan Array Human Antioxidant Mechanisms (Life Technologies), and results were verified by performing quantitative RT-PCR assays. The expression of selenoprotein P (sepp1) was significantly downregulated (8- to 10-fold) in 4-ClBQ-treated HaCaT human skin keratinocytes, which correlated with a significant increase in MitoSOX oxidation. Overexpression of Mn-superoxide dismutase or catalase or treatment with N-acetyl-l-cysteine suppressed 4-ClBQ-induced toxicity. Sodium selenite supplementation also suppressed 4-ClBQ-induced decrease in sepp1 expression, which was associated with a significant inhibition in cell death. Furthermore, HaCaT cells overexpressing sepp1 were resistant to 4-ClBQ-induced oxidative stress and toxicity. These results demonstrate that SEPP1 represents a previously unrecognized regulator of PCB-induced biological effects. These results support the speculation that selenoproteins can be an attractive countermeasure for PCB-induced adverse biological effects.

Manganese superoxide dismutase regulates a metabolic switch during the mammalian cell cycle.

Proliferating cells consume more glucose to cope with the bioenergetics and biosynthetic demands of rapidly dividing cells as well as to counter a shift in cellular redox environment. This study investigates the hypothesis that manganese superoxide dismutase (MnSOD) regulates cellular redox flux and glucose consumption during the cell cycle. A direct correlation was observed between glucose consumption and percentage of S-phase cells in MnSOD wild-type fibroblasts, which was absent in MnSOD homozygous knockout fibroblasts. Results from electron paramagnetic resonance spectroscopy and flow cytometric assays showed a significant increase in cellular superoxide levels in S-phase cells, which was associated with an increase in glucose and oxygen consumption, and a decrease in MnSOD activity. Mass spectrometry results showed a complex pattern of MnSOD-methylation at both lysine (68, 89, 122, and 202) and arginine (197 and 216) residues. MnSOD protein carrying a K89A mutation had significantly lower activity compared with wild-type MnSOD. Computational-based simulations indicate that lysine and arginine methylation of MnSOD during quiescence would allow greater accessibility to the enzyme active site as well as increase the positive electrostatic potential around and within the active site. Methylation-dependent changes in the MnSOD conformation and subsequent changes in the electrostatic potential around the active site during quiescence versus proliferation could increase the accessibility of superoxide, a negatively charged substrate. These results support the hypothesis that MnSOD regulates a "metabolic switch" during progression from quiescent through the proliferative cycle. We propose MnSOD as a new molecular player contributing to the Warburg effect.

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.

PCB-153 exposure coordinates cell cycle progression and cellular metabolism in human mammary epithelial cells.

2,2',4,4',5,5'-Hexachlorobiphenyl (PCB-153) is a non-metabolizable environmental chemical contaminant commonly found in breast milk of PCB exposed individuals, suggesting that chronic exposure to PCB-153 could have adverse health effects. We have shown previously that PCB-153 increased reactive oxygen species levels in non-tumorigenic MCF-10A human mammary epithelial cells, which were associated with DNA damage, growth inhibition, and cytotoxicity. This study investigates the hypothesis that PCB-153 exposure coordinates cell cycle progression and cellular metabolism by inhibiting cyclin D1 accumulation. PCB-153 treated MCF-10A cells exhibited a dose and time dependent decrease in cyclin D1 protein levels. The decrease in cyclin D1 protein levels was associated with an inhibition in AKT and GSK-3beta phosphorylation, which correlated with an increase in cyclin D1-T286 phosphorylation. Fibroblasts carrying a mutant form of cyclin D1 (T286A) were resistant to PCB-153 induced degradation of cyclin D1. Pre-treatment of cells with a proteasome inhibitor (MG132) suppressed PCB-153 induced decrease in cyclin D1 protein levels. Interestingly, suppression in cyclin D1 accumulation was associated with an increase in cellular glucose consumption, and hexokinase II and pyruvate kinase protein levels. These results suggest that cyclin D1 coordinates cell cycle progression and cellular metabolism in PCB-153 treated non-tumorigenic human mammary epithelial cells.