UVA Radiation Enhances Lomefloxacin-Mediated Cytotoxic,Growth-Inhibitory and Pro-Apoptotic Effect in Human Melanoma Cells through Excessive Reactive Oxygen Species Generation

Melanoma, the most dangerous type of cutaneous neoplasia, contributes to about 75% of all skin cancer-related deaths. Thus, searching for new melanoma treatment options is an important field of study. The current study was designed to assess whether the condition of mild and low-dose UVA radiation augments the lomefloxacin-mediated cytotoxic, growth-inhibitory and pro-apoptotic effect of the drug in melanoma cancer cells through excessive oxidative stress generation. C32 amelanotic and COLO829 melanotic (BRAF-mutant) melanoma cell lines were used as an experimental model system. The combined exposure of cells to both lomefloxacin and UVA irradiation caused higher alterations of redox signalling pathways, as shown by intracellular reactive oxygen species overproduction and endogenous glutathione depletion when compared to non-irradiated but lomefloxacin-treated melanoma cells. The obtained results also showed that lomefloxacin decreased both C32 and COLO829 cells’ viability in a concentration-dependent manner. This effect significantly intensified when melanoma cells were exposed to UVA irradiation and the drug. For melanoma cells exposed to lomefloxacin or lomefloxacin co-treatment with UVA irradiation, the concentrations of the drug that decreased the cells’ viability by 50% (EC₅₀) were found to be 0.97, 0.17, 1.01, 0.18 mM, respectively. Moreover, we found that the redox imbalance, mitochondrial membrane potential breakdown, induction of DNA fragmentation, and changes in the melanoma cells’ cell cycle distribution (including G₂/M, S as well as Sub-G₁-phase blockade) were lomefloxacin in a dose-dependent manner and were significantly augmented by UVA radiation. This is the first experimental work that assesses the impact of excessive reactive oxygen species generation upon UVA radiation exposure on lomefloxacin-mediated cytotoxic, growth-inhibitory and pro-apoptotic effects towards human melanoma cells, indicating the possibility of the usage of this drug in the photochemotherapy of malignant melanoma as an innovative medical treatment option which could improve the effectiveness of therapy. The obtained results also revealed that the redox imbalance intensification mediated by the phototoxic potential of fluoroquinolones may be considered as a more efficient treatment model of malignant melanoma and may constitute the basis for the development of new compounds with a high ability to excessive oxidative stress generation upon UVA radiation in cancer cells.     

Hyperthermia Induces Apoptosis through Endoplasmic Reticulum and Reactive Oxygen Species in Human Osteosarcoma Cells

Osteosarcoma (OS) is a relatively rare form of cancer, but OS is the most commonly diagnosed bone cancer in children and adolescents. Chemotherapy has side effects and induces drug resistance in OS. Since an effective adjuvant therapy was insufficient for treating OS, researching novel and adequate remedies is critical. Hyperthermia can induce cell death in various cancer cells, and thus, in this study, we investigated the anticancer method of hyperthermia in human OS (U-2 OS) cells. Treatment at 43 °C for 60 min induced apoptosis in human OS cell lines, but not in primary bone cells. Furthermore, hyperthermia was associated with increases of intracellular reactive oxygen species (ROS) and caspase-3 activation in U-2 OS cells. Mitochondrial dysfunction was followed by the release of cytochrome c from the mitochondria, and was accompanied by decreased anti-apoptotic Bcl-2 and Bcl-xL, and increased pro-apoptotic proteins Bak and Bax. Hyperthermia triggered endoplasmic reticulum (ER) stress, which was characterized by changes in cytosolic calcium levels, as well as increased calpain expression and activity. In addition, cells treated with calcium chelator (BAPTA-AM) blocked hyperthermia-induced cell apoptosis in U-2 OS cells. In conclusion, hyperthermia induced cell apoptosis substantially via the ROS, ER stress, mitochondria, and caspase pathways. Thus, hyperthermia may be a novel anticancer method for treating OS.     

Hypomagnetic Field Induces the Production of Reactive Oxygen Species and Cognitive Deficits in Mice Hippocampus

Previous studies have found that hypomagnetic field (HMF) exposure impairs cognition behaviors in animals; however, the underlying neural mechanisms of cognitive dysfunction are unclear. The hippocampus plays important roles in magnetoreception, memory, and spatial navigation in mammals. Therefore, the hippocampus may be the key region in the brain to reveal its neural mechanisms. We recently reported that long-term HMF exposure impairs adult hippocampal neurogenesis and cognition through reducing endogenous reactive oxygen species (ROS) levels in adult neural stem cells that are confined in the subgranular zone (SGZ) of the hippocampus. In addition to adult neural stem cells, the redox state of other cells in the hippocampus is also an important factor affecting the functions of the hippocampus. However, it is unclear whether and how long-term HMF exposure affects ROS levels in the entire hippocampus (i.e., the dentate gyrus (DG) and ammonia horn (CA) regions). Here, we demonstrate that male C57BL/6J mice exposed to 8-week HMF exhibit cognitive impairments. We then found that the ROS levels of the hippocampus were significantly higher in these HMF-exposed mice than in the geomagnetic field (GMF) group. PCR array analysis revealed that the elevated ROS levels were due to HMF-regulating genes that maintain the redox balance in vivo, such as Nox4, Gpx3. Since high levels of ROS may cause hippocampal oxidative stress, we suggest that this is another reason why HMF exposure induces cognitive impairment, besides the hippocampal neurogenesis impairments. Our study further demonstrates that GMF plays an important role in maintaining hippocampal function by regulating the appropriate endogenous ROS levels.     

The Differential Reactive Oxygen Species Production of Tear Neutrophils in Response to Various Stimuli In Vitro

A large number of polymorphonuclear neutrophils (PMNs) invade the ocular surface during prolonged eye closure (sleep); these leukocytes are commonly referred as tear PMNs. PMNs contribute to homeostasis and possess an arsenal of inflammatory mediators to protect against pathogens and foreign materials. This study examined the ability of tear PMNs to generate reactive oxygen species (ROS), an essential killing mechanism for PMNs which can lead to oxidative stress and imbalance. Cells were collected after sleep from healthy participants using a gentle eye wash. ROS production in stimulated (phorbol-12-myristate-13-acetate (PMA), lipopolysaccharides (LPS) or N-Formylmethionyl-leucyl-phenylalanine (fMLP)) and unstimulated tear PMNs was measured using luminol-enhanced chemiluminescence for 60 min. A high level of constitutive/spontaneous ROS production was observed in tear PMNs in the absence of any stimulus. While tear PMNs were able to produce ROS in response to PMA, they failed to appropriately respond to LPS and fMLP, although fMLP-stimulated tear PMNs generated ROS extracellularly in the first three minutes. Higher ROS generation was observed in isolated tear PMNs which may be due to priming from the magnetic bead cell separation system. The differential responses of tear PMNs in ROS generation provide further evidence of their potential inflammatory roles in ocular complications involving oxidative stress.     

Cellular Metabolism and Bioenergetic Function in Human Fibroblasts and Preadipocytes of Type 2 Familial Partial Lipodystrophy

LMNA mutation is associated with type-2 familial partial lipodystrophy (FPLD2). The disease causes a disorder characterized by anomalous accumulation of body fat in humans. The dysfunction at the molecular level is triggered by a lamin A/C mutation, impairing the cell metabolism. In human fibroblasts and preadipocytes, a trend for ATP production, mainly supported by mitochondrial oxidative metabolism, is detected. Moreover, primary cell lines with FPLD2 mutation decrease the mitochondrial ATP production if compared with the control, even if no differences are observed in the oxygen consumption rate of bioenergetic parameters (i.e., basal and maximal respiration, spare respiratory capacity, and ATP turnover). Conversely, glycolysis is only inhibited in FPLD2 fibroblast cell lines. We notice that the amount of ATP produced in the fibroblasts is higher than in the preadipocytes, and likewise in the control, with respect to FPLD2, due to a more active oxidative phosphorylation (OXPHOS) and glycolysis. Moreover, the proton leak parameter, which characterizes the transformation of white adipose tissue to brown/beige adipose tissue, is unaffected by FPLD2 mutation. The metabolic profile of fibroblasts and preadipocytes is confirmed by the ability of these cell lines to increase the metabolic potential of both OXPHOS and glycolysis under energy required independently by the FPLD2 mutation.     

Anticonvulsant Effects of Topiramate and Lacosamide on Pilocarpine-Induced Status Epilepticus in Rats: A Role of Reactive Oxygen Species and Inflammation

Background: Status epilepticus (SE) is a neurological disorder characterized by a prolonged epileptic activity followed by subsequent epileptogenic processes. The aim of the present study was to evaluate the early effects of topiramate (TPM) and lacosamide (LCM) treatment on oxidative stress and inflammatory damage in a model of pilocarpine-induced SE. Methods: Male Wistar rats were randomly divided into six groups and the two antiepileptic drugs (AEDs), TPM (40 and 80 mg/kg, i.p.) and LCM (10 and 30 mg/kg, i.p.), were injected three times repeatedly after pilocarpine administration. Rats were sacrificed 24 h post-SE and several parameters of oxidative stress and inflammatory response have been explored in the hippocampus. Results: The two drugs TPM and LCM, in both doses used, succeeded in attenuating the number of motor seizures compared to the SE-veh group 30 min after administration. Pilocarpine-induced SE decreased the superoxide dismutase (SOD) activity and reduced glutathione (GSH) levels while increasing the catalase (CAT) activity, malondialdehyde (MDA), and IL-1β levels compared to the control group. Groups with SE did not affect the TNF-α levels. The treatment with a higher dose of 30 mg/kg LCM restored to control level the SOD activity in the SE group. The two AEDs, in both doses applied, also normalized the CAT activity and MDA levels to control values. In conclusion, we suggest that the antioxidant effect of TPM and LCM might contribute to their anticonvulsant effect against pilocarpine-induced SE, whereas their weak anti-inflammatory effect in the hippocampus is a consequence of reduced SE severity.     

Genetically Encoded Biosensors to Monitor Intracellular Reactive Oxygen and Nitrogen Species and Glutathione Redox Potential in Skeletal Muscle Cells

Reactive oxygen and nitrogen species (RONS) play an important role in the pathophysiology of skeletal muscle and are involved in the regulation of intracellular signaling pathways, which drive metabolism, regeneration, and adaptation in skeletal muscle. However, the molecular mechanisms underlying these processes are unknown or partially uncovered. We implemented a combination of methodological approaches that are funded for the use of genetically encoded biosensors associated with quantitative fluorescence microscopy imaging to study redox biology in skeletal muscle. Therefore, it was possible to detect and monitor RONS and glutathione redox potential with high specificity and spatio-temporal resolution in two models, isolated skeletal muscle fibers and C2C12 myoblasts/myotubes. Biosensors HyPer3 and roGFP2-Orp1 were examined for the detection of cytosolic hydrogen peroxide; HyPer-mito and HyPer-nuc for the detection of mitochondrial and nuclear hydrogen peroxide; Mito-Grx1-roGFP2 and cyto-Grx1-roGFP2 were used for registration of the glutathione redox potential in mitochondria and cytosol. G-geNOp was proven to detect cytosolic nitric oxide. The fluorescence emitted by the biosensors is affected by pH, and this might have masked the results; therefore, environmental CO₂ must be controlled to avoid pH fluctuations. In conclusion, genetically encoded biosensors and quantitative fluorescence microscopy provide a robust methodology to investigate the pathophysiological processes associated with the redox biology of skeletal muscle.     

The Chemistry of Reactive Oxygen Species (ROS) Revisited: Outlining Their Role in Biological Macromolecules (DNA,Lipids and Proteins) and Induced Pathologies

Living species are continuously subjected to all extrinsic forms of reactive oxidants and others that are produced endogenously. There is extensive literature on the generation and effects of reactive oxygen species (ROS) in biological processes, both in terms of alteration and their role in cellular signaling and regulatory pathways. Cells produce ROS as a controlled physiological process, but increasing ROS becomes pathological and leads to oxidative stress and disease. The induction of oxidative stress is an imbalance between the production of radical species and the antioxidant defense systems, which can cause damage to cellular biomolecules, including lipids, proteins and DNA. Cellular and biochemical experiments have been complemented in various ways to explain the biological chemistry of ROS oxidants. However, it is often unclear how this translates into chemical reactions involving redox changes. This review addresses this question and includes a robust mechanistic explanation of the chemical reactions of ROS and oxidative stress.     

Modulatory Effect of Myokines on Reactive Oxygen Species in Ischemia/Reperfusion

There is a growing body of evidence showing the importance of physical activity against acute ischemic events in various organs. Ischemia/reperfusion injury (I/R) is characterized by tissue damage as a result of restriction and subsequent restoration of blood supply to an organ. Oxidative stress due to increased reactive oxygen species formation and/or insufficient antioxidant defense is considered to play an important role in I/R. Physical activity not only decreases the general risk factors for ischemia but also confers direct anti-ischemic protection via myokine production. Myokines are skeletal muscle-derived cytokines, representing multifunctional communication channels between the contracting skeletal muscle and other organs through an endocrine manner. In this review, we discuss the most prominent members of the myokines (i.e., brain-derived neurotrophic factor (BDNF), cathepsin B, decorin, fibroblast growth factors-2 and -21, follistatin, follistatin-like, insulin-like growth factor-1; interleukin-6, interleukin-7, interleukin-15, irisin, leukemia inhibitory factor, meteorin-like, myonectin, musclin, myostatin, and osteoglycin) with a particular interest in their potential influence on reactive oxygen and nitrogen species formation or antioxidant capacity. A better understanding of the mechanism of action of myokines and particularly their participation in the regulation of oxidative stress may widen their possible therapeutic use and, thereby, may support the fight against I/R.     

Streptozotocin-Induced Cytotoxicity, Oxidative Stress and Mitochondrial Dysfunction in Human Hepatoma HepG2 Cells

Streptozotocin (STZ) is an antibiotic often used in the treatment of different types of cancers. It is also highly cytotoxic to the pancreatic beta-cells and therefore is commonly used to induce experimental type 1 diabetes in rodents. Resistance towards STZ-induced cytotoxicity in cancer cells has also been reported. Our previous studies have reported organ-specific toxicity and metabolic alterations in STZ-induced diabetic rats. STZ induces oxidative stress and metabolic complications. The precise molecular mechanism of STZ-induced toxicity in different tissues and carcinomas is, however, unclear. We have, therefore, investigated the mechanism of cytotoxicity of STZ in HepG2 hepatoma cells in culture. Cells were treated with different doses of STZ for various time intervals and the cytotoxicity was studied by observing the alterations in oxidative stress, mitochondrial redox and metabolic functions. STZ induced ROS and RNS formation and oxidative stress as measured by an increase in the lipid peroxidation as well as alterations in the GSH-dependent antioxidant metabolism. The mitochondria appear to be a highly sensitive target for STZ toxicity. The mitochondrial membrane potential and enzyme activities were altered in STZ treated cells resulting in the inhibition of ATP synthesis. ROS-sensitive mitochondrial aconitase activity was markedly inhibited suggesting increased oxidative stress in STZ-induced mitochondrial toxicity. These results suggest that STZ-induced cytotoxicity in HepG2 cells is mediated, at least in part, by the increase in ROS/RNS production, oxidative stress and mitochondrial dysfunction. Our study may be significant for better understanding the mechanisms of STZ action in chemotherapy and drug induced toxicity.     

Protective Effects of a Lutein Ester Prodrug,Lutein Diglutaric Acid,against H2O2-Induced Oxidative Stress in Human Retinal Pigment Epithelial Cells

Oxidative stress-induced cell damage and death of the retinal pigmented epithelium (RPE), a polarized monolayer that maintains retinal health and homeostasis, lead to the development of age-related macular degeneration (AMD). Several studies show that the naturally occurring antioxidant Lutein (Lut) can protect RPE cells from oxidative stress. However, the poor solubility and low oral bioavailability limit the potential of Lut as a therapeutic agent. In this study, lutein diglutaric acid (Lut-DG), a prodrug of Lut, was synthesized and its ability to protect human ARPE-19 cells from oxidative stress was tested compared to Lut. Both Lut and Lut-DG significantly decreased H₂O₂-induced reactive oxygen species (ROS) production and protected RPE cells from oxidative stress-induced death. Moreover, the immunoblotting analysis indicated that both drugs exerted their protective effects by modulating phosphorylated MAPKs (p38, ERK1/2 and SAPK/JNK) and downstream molecules Bax, Bcl-2 and Cytochrome c. In addition, the enzymatic antioxidants glutathione peroxidase (GPx) and catalase (CAT) and non-enzymatic antioxidant glutathione (GSH) were enhanced in cells treated with Lut and Lut-DG. In all cases, Lut-DG was more effective than its parent drug against oxidative stress-induced damage to RPE cells. These findings highlight Lut-DG as a more potent compound than Lut with the protective effects against oxidative stress in RPE cells through the modulation of key MAPKs, apoptotic and antioxidant molecular pathways.     

Elevated Production of Mitochondrial Reactive Oxygen Species via Hyperthermia Enhanced Cytotoxic Effect of Doxorubicin in Human Breast Cancer Cell Lines MDA-MB-453 and MCF-7

Hyperthermia (HT) treatment is a noninvasive cancer therapy, often used with radiation therapy and chemotherapy. Compared with 37 °C, 42 °C is mild heat stress for cells and produces reactive oxygen species (ROS) from mitochondria. To involve subsequent intracellular accumulation of DOX, we have previously reported that the expression of ATP-binding cassette sub-family G member 2 (ABCG2), an exporter of doxorubicin (DOX), was suppressed by a larger amount of intracellular mitochondrial ROS. We then hypothesized that the additive effect of HT and chemotherapy would be induced by the downregulation of ABCG2 expression via intracellular ROS increase. We used human breast cancer cell lines, MCF-7 and MDA-MB-453, incubated at 37 °C or 42 °C for 1 h to clarify this hypothesis. Intracellular ROS production after HT was detected via electron spin resonance (ESR), and DOX cytotoxicity was calculated. Additionally, ABCG2 expression in whole cells was analyzed using Western blotting. We confirmed that the ESR signal peak with HT became higher than that without HT, indicating that the intracellular ROS level was increased by HT. ABCG2 expression was downregulated by HT, and cells were injured after DOX treatment. DOX cytotoxicity enhancement with HT was considered a result of ABCG2 expression downregulation via the increase of ROS production. HT increased intracellular ROS production and downregulated ABCG2 protein expression, leading to cell damage enhancement via DOX.     

Acrylamide and Potential Risk of Diabetes Mellitus: Effects on Human Population,Glucose Metabolism and Beta-Cell Toxicity

Diabetes mellitus is a frequent endocrine disorder characterized by hyperglycemia. Acrylamide (AA) is food contaminant formed during the high-temperature processing of food rich in carbohydrates and low in proteins. Recent human epidemiological studies have shown a potential association between AA exposure and the prevalence of diabetes in the general population. In male rats, AA treatment promoted pancreatic islet remodeling, which was determined by alpha-cell expansion and beta-cell reduction, while in female rats AA caused hyperglycemia and histopathological changes in pancreatic islets. In vitro and in vivo rodent model systems have revealed that AA induces oxidative stress in beta cells and that AA impairs glucose metabolism and the insulin signaling pathway. Animal studies have shown that diabetic rodents are more sensitive to acrylamide and that AA aggravates the diabetic state. In this review, we provide an overview of human epidemiological studies that examined the relation between AA exposure and glucose disorders. In addition, the effects of AA treatment on pancreatic islet structure, beta-cell function and glucose metabolism in animal models are comprehensively analyzed with an emphasis on sex-related responses. Furthermore, oxidative stress as a putative mechanism of AA-induced toxicity in beta cells is explored. Finally, we discuss the effects of AA on diabetics in a rodent model system.     

Protective Effects of Mitochondrial Uncoupling Protein 2 against Aristolochic Acid I-Induced Toxicity in HK-2 Cells

Aristolochic acid I (AA I) is one of the most abundant and toxic aristolochic acids that is reported to cause Aristolochic acid nephropathy (AAN). This paper was designed to assess whether mitochondrial Uncoupling Protein 2 (UCP2), which plays an antioxidative and antiapoptotic role, could protect human renal proximal tubular epithelial (HK-2) cells from toxicity induced by AA I. In this study, HK-2 cells were treated with different concentrations of AA I with or without UCP2 inhibitor (genipin). To upregulate the expression of UCP2 in HK-2 cells, UCP2-DNA transfection was performed. The cell viability was evaluated by colorimetric method using MTT. A series of related biological events such as Reactive Oxygen Species (ROS), Glutathione peroxidase (GSH-Px), and Malondialdehyde (MDA) were evaluated. The results showed that the cytotoxicity of AA I with genipin group was much higher than that of AA I alone. Genipin dramatically boosted oxidative stress and exacerbated AA I-induced apoptosis. Furthermore, the increased expression of UCP2 can reduce the toxicity of AA I on HK-2 cells and upregulation of UCP2 expression can reduce AA I-induced oxidative stress and apoptosis. In conclusion, UCP2 might be a potential target for alleviating AA I-induced nephrotoxicity.     

Damaged DNA Binding Protein 2 in Reactive Oxygen Species (ROS) Regulation and Premature Senescence

Premature senescence induced by DNA damage or oncogene is a critical mechanism of tumor suppression. Reactive oxygen species (ROS) have been implicated in the induction of premature senescence response. Several pathological disorders such as cancer, aging and age related neurological abnormalities have been linked to ROS deregulation. Here, we discuss how Damaged DNA binding Protein-2 (DDB2), a nucleotide excision repair protein, plays an important role in ROS regulation by epigenetically repressing the antioxidant genes MnSOD and Catalase. We further revisit a model in which DDB2 plays an instrumental role in DNA damage induced ROS accumulation, ROS induced premature senescence and inhibition of skin tumorigenesis.     

Effects of Oxygen Supply Modes on the Production of Human Growth Hormone in Different Scale Bioreactors

Recombinant Escherichia coli has been studied as a main host for recombinant protein productions, but it is still difficult to cultivate E. coli in a large industrial‐scale process due to the oxygen supply limitation. In this study, E. coli BL(21) harboring a new constructed plasmid (pEHUb‐hGH) was used for producing recombinant human growth hormone (r‐hGH) in 5‐L and 30‐L scale fermentors by supplying air and high purity oxygen, respectively, where the high purity oxygen was produced from a vacuum pressure swing adsorption (PSA). The impact of oxygen supply modes, i.e., air and high purity oxygen, on cell growth and r‐hGH production was investigated in different scale fermentors. In the case of high purity oxygen supply, the final cell density and r‐hGH concentrations were 63.0 and 4.8 g/L in the 5‐L fermentor, 51.6 and 4.0 g/L in the 30‐L fermentor, respectively. In addition, the productivity of r‐hGH was doubled in the 5‐L fermentor, and increased 4‐fold in the 30‐L fermentor, compared to the results obtained in the case of the air supply. The supply of high purity oxygen eliminated the oxygen limitation and acetate formation effectively, and apparently, did not affect the degradation of r‐hGH. This shows that the recombinant E. coli cultivation with high purity oxygen produced from PSA may provide an effective method for large‐scale industrial production of recombinant proteins.     

The Effects of Silencing PTX3 on the Proteome of Human Endothelial Cells

The human long pentraxin PTX3 has complex regulatory roles at the crossroad of innate immunity, inflammation, and tissue repair. PTX3 can be produced by various cell types, including vascular endothelial cells (ECs), in response to pro-inflammatory cytokines or bacterial molecules. PTX3 has also been involved in the regulation of cardiovascular biology, even if ambiguous results have been so far provided in both preclinical and clinical research. In this study, we compared the proteomic profiles of human ECs (human umbilical vein ECs, HUVECs), focusing on differentially expressed proteins between the control and PTX3-silenced ECs. We identified 19 proteins that were more abundant in the proteome of control ECs and 23 proteins that were more expressed in PTX3-silenced cells. Among the latter, proteins with multifunctional roles in angiogenesis, oxidative stress, and inflammation were found, and were further validated by assessing their mRNAs with RT-qPCR. Nevertheless, the knock down of PTX3 did not affect in vitro angiogenesis. On the contrary, the lack of the protein induced an increase in pro-inflammatory markers and a shift to the more oxidative profile of PTX3-deficient ECs. Altogether, our results support the idea of a protective function for PTX3 in the control of endothelial homeostasis, and more generally, in cardiovascular biology.