Modification of Proliferation and Apoptosis in Breast Cancer Cells by Exposure of Antioxidant Nanoparticles Due to Modulation of the Cellular Redox State Induced by Doxorubicin Exposure

In this report, we investigated whether the use of chitosan-carrying-glutathione nanoparticles (CH-GSH NPs) can modify proliferation and apoptosis, and reduce cell damage induced by doxorubicin on breast cancer cells. Doxorubicin is a widely used antineoplasic agent for the treatment of various types of cancer. However, it is also a highly toxic drug because it induces oxidative stress. Thus, the use of antioxidant molecules has been considered to reduce the toxicity of doxorubicin. CH-GSH NPs were characterized in size, zeta potential, concentration, and shape. When breast cancer cells were treated with CH-GSH nanoparticles, they were localized in the cellular cytoplasm. Combined doxorubicin exposure with nanoparticles increased intracellular GSH levels. At the same time, decreasing levels of reactive oxygen species and malondialdehyde were observed and modified antioxidant enzyme activity. Levels of the Ki67 protein were evaluated as a marker of cell proliferation and the activity of the Casp-3 protein related to cell apoptosis was measured. Our data suggests that CH-GSH NPs can modify cell proliferation by decreasing Ki67 levels, induce apoptosis by increasing caspase-3 activity, and reduce the oxidative stress induced by doxorubicin in breast cancer cells by modulating molecules associated with the cellular redox state. CH-GSH NPs could be used to reduce the toxic effects of this antineoplastic. Considering these results, CH-GSH NPs represent a novel delivery system offering new opportunities in pharmacy, material science, and biomedicine.

Role of insulin/glucagon ratio and cell redox state in the hyperglycaemia induced by exposure to a 60-Hz magnetic field in rats

The exposure to extremely low-frequency electromagnetic fields (EMFs) could adversely affect the endocrine system and cellular proliferative response. Nonetheless, the use of 60-Hz EMFs in the form of magneto-therapy exerts beneficial actions on human health but can also induce hyperglycaemia. Therefore, the present study was aimed to search for metabolic responses of fed or fasted male rats to a single EMF exposure. We performed a 15 min-single exposure to 60-Hz (3.8 mT, intensity) EMF, and determined serum levels of glucose, lipids, and indicators of cellular redox state and energy parameters. A single exposure to a 60-Hz EMF induced hyperglycaemia in both animal groups, and an attenuated second serum insulin peak. The 60-Hz EMF also decreased free fatty acids and lactate serum levels, oppositely increasing pyruvate and acetoacetate levels. Significant increases in blood glucose level and rat’s glucose metabolism were related to a more oxidized cellular redox state and variations in insulin and glucagon secretion. The 60-Hz EMF’s effects were not modified in animals previously subjected to chronic EMFs exposure (14 days). In conclusion, increased serum glucose levels and glucose metabolism induced by a single 60-Hz EMF exposure were closely related to the cellular redox state and the insulin/glucagon ratio.

Redox Metabolism Measurement in Mammalian Cells and Tissues by LC-MS

Cellular redox state is highly dynamic and delicately balanced between constant production of reactive oxygen species (ROS), and neutralization by endogenous antioxidants, such as glutathione. Physiologic ROS levels can function as signal transduction messengers, while high levels of ROS can react with and damage various molecules eliciting cellular toxicity. The redox state is reflective of the cell’s metabolic status and can inform on regulated cell-state transitions or various pathologies including aging and cancer. Therefore, methods that enable reliable, quantitative readout of the cellular redox state are imperative for scientists from multiple fields. Liquid-chromatography mass-spectrometry (LC-MS) based methods to detect small molecules that reflect the redox balance in the cell such as glutathione, NADH, and NADPH, have been developed and applied successfully, but because redox metabolites are very labile, these methods are not easily standardized or consolidated. Here, we report a robust LC-MS method for the simultaneous detection of several redox-reactive metabolites that is compatible with parallel global metabolic profiling in mammalian cells. We performed a comprehensive comparison between three commercial hydrophilic interaction chromatography (HILIC) columns, and we describe the application of our method in mammalian cells and tissues. The presented method is easily applicable and will enable the study of ROS function and oxidative stress in mammalian cells by researchers from various fields.

Redox Metabolism Measurement in Mammalian Cells and Tissues by LC-MS

Cellular redox state is highly dynamic and delicately balanced between constant production of reactive oxygen species (ROS), and neutralization by endogenous antioxidants, such as glutathione. Physiologic ROS levels can function as signal transduction messengers, while high levels of ROS can react with and damage various molecules eliciting cellular toxicity. The redox state is reflective of the cell’s metabolic status and can inform on regulated cell-state transitions or various pathologies including aging and cancer. Therefore, methods that enable reliable, quantitative readout of the cellular redox state are imperative for scientists from multiple fields. Liquid-chromatography mass-spectrometry (LC-MS) based methods to detect small molecules that reflect the redox balance in the cell such as glutathione, NADH and NADPH, have been developed and applied successfully, but because redox metabolites are very labile, these methods are not easily standardized or consolidated. Here we report a robust LC-MS method for the simultaneous detection of several redox-reactive metabolites that is compatible with parallel global metabolic profiling in mammalian cells. We performed a comprehensive comparison between three commercial hydrophilic interaction chromatography (HILIC) columns, and we describe the application of our method in mammalian cells and tissues. The presented method is easily applicable and will enable the study of ROS function and oxidative stress in mammalian cells by researchers from various fields.

Role of Insulin/Glucagon Ratio and Cell Redox State in the Hyperglycaemia Induced by Exposure to Electromagnetic Fields in Rats.

The exposure to extremely low-frequency electromagnetic fields (EMFs) with a might adversely affect the endocrine system and reproductive functions. Nonetheless, the use of EMFs in the form of magneto-therapy exerts beneficial actions in human medicine, but also affecting glucose and lipids metabolism, inducing hyperglycaemia and a “diabetic” response. Therefore, we used fed or fasted male rats for a single exposure to 60-Hz EMFs, determining serum levels of glucose, lipids, and indicators of cellular redox state and energy parameters. A single exposure to EMFs induced hyperglycaemia in both, fed and fasted rats, accompanied by an attenuated second serum insulin peak. In addition, EMFs also decreased serum levels of free fatty acids and lactate, while they increased those of pyruvate and acetoacetate. Changes in blood glucose level were related with a more oxidized cellular redox state and with the insulin/glucagon ratio. Moreover, rat’s glucose metabolism also coincided with variations in insulin and glucagon secretion, probably depending on cell redox state. The EMFs effects were not modified in animals previously subjected to a chronic exposure (14 days). In conclusion, increased serum glucose levels and glucose metabolism induced by EMFs exposure were closely related with the cellular redox state and with the insulin/glucagon ratio.

Cellular Redox State Acts as Switch to Determine the Direction of NNT-Catalyzed Reaction in Cystic Fibrosis Cells

The redox states of NAD and NADP are linked to each other in the mitochondria thanks to the enzyme nicotinamide nucleotide transhydrogenase (NNT) which, by utilizing the mitochondrial membrane potential (mΔΨ), catalyzes the transfer of redox potential between these two coenzymes, reducing one at the expense of the oxidation of the other. In order to define NNT reaction direction in CF cells, NNT activity under different redox states of cell has been investigated. Using spectrophotometric and western blotting techniques, the presence, abundance and activity level of NNT were determined. In parallel, the levels of NADPH and NADH as well as of mitochondrial and cellular ROS were also quantified. CF cells showed a 70% increase in protein expression compared to the Wt sample; however, regarding NNT activity, it was surprisingly lower in CF cells than healthy cells (about 30%). The cellular redox state, together with the low mΔΨ, pushes to drive NNT reverse reaction, at the expense of its antioxidant potential, thus consuming NADPH to support NADH production. At the same time, the reduced NNT activity prevents the NADH, produced by the reaction, from causing an explosion of ROS by the damaged respiratory chain, in accordance with the reduced level of mitochondrial ROS in NNT-loss cells. This new information on cellular bioenergetics represents an important building block for further understanding the molecular mechanisms responsible for cellular dysfunction in cystic fibrosis.