Involvement of necroptosis in the selective toxicity of the natural compound (±) gossypol on squamous skin cancer cells in vitro.

Cutaneous basal and squamous cell carcinoma reflect the first and second most common type of non-melanoma skin cancer, respectively. Especially cutaneous squamous cell carcinoma has the tendency to metastasize, finally resulting in a rather poor prognosis. Therapeutic options comprise surgery, radiation therapy, and a systemic or targeted chemotherapy. There are some good treatment results, but overall, the response rate of newly developed drugs is still modest. Drug repurposing represents an alternative approach where already available and clinically approved substances are used, which originally intended for other clinical benefits. In this context, we tested the effect of the naturally occurring polyphenolic aldehyde (±) gossypol with concentrations between 1 and 5 µM on the invasive squamous cell carcinoma cell line SCL-1 and normal human epidermal keratinocytes. Gossypol treatment up to 96 h resulted in a selective cytotoxicity of SCL-1 cells (IC50: 1.7 µM, 96 h) compared with normal keratinocytes (IC50: ≥ 5.4 µM, 96 h) which is mediated by mitochondrial dysfunction and finally leading to necroptotic cell death. Taken together, gossypol shows a high potential as an alternative anticancer drug for the treatment of cutaneous squamous cell carcinoma.

Carbon monoxide exposure activates ULK1 via AMPK phosphorylation in murine embryonic fibroblasts.

Carbon monoxide (CO) is endogenously produced upon degradation of heme by heme oxygenases (HOs) and is suggested to act as a gaseous signaling molecule. The expression of HO-1 is triggered by the Nrf2-Keap1 signaling pathway which responds to exogenous stress signals and dietary constituents such as flavonoids and glucosinolates or reactive metabolic intermediates like 4-hydroxynonenal. Endogenous CO affects energy metabolism, regulates the utilization of glucose and addresses CYP450 enzymes. Using the CO releasing molecule-401 (CORM-401), we studied the effect of endogenous CO on ATP synthesis, AMP-signaling and activation of the AMPK pathway in cell culture. Upon exposure of cells to CORM-401, the mitochondrial ATP production rate was significantly decreased (P=0.007) to about 50%, while glycolytic ATP synthesis was unchanged (P=0.489). Total ATP levels were less affected as determined by mass spectrometry. Instead, levels of ADP and AMP were elevated following CORM-401 exposure by about two- (P=0.022) and four-fold (P=0.012) compared to control, respectively. Increased concentrations of AMP activate AMPK which was demonstrated by a 10 to 15-fold increased phosphorylation of Thr172 of the α-subunit of AMPK (P=0.025). A downstream target of AMPK is the kinase ULK1 which triggers autophagic and mitophagic processes. Activation of ULK1 after CO exposure was proven by a 3 to 5-fold elevated phosphorylation of ULK1 at Ser555 (P=0.004). The present data suggest that production of endogenous CO leads to increasing amounts of AMP which mediates AMPK-dependent downstream effects and likely triggers autophagic processes. Since dietary constituents and their metabolites induce the expression of the CO producing enzyme HO-1, CO signaling may also be involved in the cellular response to nutritional factors.

The BH3 mimetic (±) gossypol induces ROS-independent apoptosis and mitochondrial dysfunction in human A375 melanoma cells in vitro.

A major challenge in current cancer therapy is still the treatment of metastatic melanomas of the skin. BH3 mimetics represent a novel group of substances inducing apoptosis. In this study, we investigated the cytotoxic effect of (±) gossypol (GP), a natural compound from cotton seed, on A375 melanoma cells and the underlying biochemical mechanisms. To prevent undesired side effects due to toxicity on normal (healthy) cells, concentrations only toxic for tumor cells have been elaborated. Viability assays were performed to determine the cytotoxicity of GP in A375 melanoma and normal (healthy) cells. For the majority of experiments, a concentration of 2.5 µM GP was used resulting in a ROS-independent but caspase-dependent cell death of A375 melanoma cells. At this level, GP was non-toxic for normal human epidermal melanocytes. GP has a very short half-life, however, it was demonstrated that only the "parent" compound and not decomposition products are responsible for the cytotoxic effect in A375 melanoma cells. GP significantly decreased mitochondrial membrane potential accompanied by a Drp1-dependent loss of mitochondrial integrity (fragmentation) in tumor cells. Taken together, GP induced a ROS-independent intrinsic apoptosis leading to the conclusion that within a specific concentration range, GP may work as effective anticancer drug without harmful side effects.

Effects of frequently applied carbon monoxide releasing molecules (CORMs) in typical CO-sensitive model systems - A comparative in vitro study.

Intracellular carbon monoxide (CO) is a gaseous signaling molecule and is generated enzymatically by heme oxygenases upon degradation of heme to billiverdin. Target structures for intracellular produced CO are heme proteins including cytochrome c oxidase of the respiratory chain, cytochrome P450-dependent monooxygenases, or myoglobin. For studies on CO signaling, CO-releasing molecules (CORMs) of different structure are available. Here, three frequently used CORMs (CORM-2, CORM-3 and CORM-401) were studied for their properties to provide CO in biological test systems and address susceptible heme proteins. CO release was investigated in the myoglobin binding assay and found to be rapid (<5 min) with CORM-2- and CORM-3, whereas CORM-401 continuously provided CO (>50 min). Storage stability of CORM stock solutions was also assessed with the myoglobin assay. Only CORM-401 stock solutions were stable over a period of 7 days. Incubation of CORMs with recombinant cytochrome P450 led to an inhibition of enzyme activity. However, only CORM-3 and CORM-401 proved to be suitable in this test system because controls with the inactivated CORM-2 (iCORM-2) also led to a loss of enzyme activity. The impact of CORMs on the respiratory chain was investigated with high resolution respirometry and extracellular flux technology. In the first approach interferences of CORM-2 and CORM-3 with oxygen measurement occurred, since a rapid depletion of oxygen was detected in the medium even when no cells were present. However, CORM-401 did not interfere with oxygen measurement and the expected inhibition of cellular respiration was observed. CORM-2 was not suitable for use in oxygen measurements with the extracellular flux technology and CORM-3 application did not show any effect in this system. However, CO-dependent inhibition of cellular respiration was observed with CORM-401. Based on the present experiments it is concluded, that CORM-401 produced most reliable CO-specific results for the modulation of typical CO targets. For studies on CO-dependent biological effects on intracellular heme groups, CORM-2 and CORM-3 were less suitable. Depending on the experimental setting, data achieved with these compounds should be evaluated with caution.

UVA-1 exposure in vivo leads to an IL-6 surge within the skin.

UVA-1 is a known promotor of skin ageing. Cytokines like IL-1α, Il-1ß or TNF-α, VEGF and IL-6 orchestrate UV effects, and IL-6 is furthermore an effector of UVA-induced photoageing. We investigated how fractionated UVA-1 doses influence the cytokine milieu and especially the IL-6 levels in the skin in vivo. In a study with 35 participants, we exposed previously unirradiated human skin to three UVA-1 irradiation regimes. Cytokine levels in interstitial skin fluid were measured up to 48 hours postexposure and compared to unirradiated control skin fluid. Our results show that IL-6 levels increased significantly after UVA-1 exposure at selected time points. The other candidates IL-1α, Il-1ß or TNF-α and VEGF show no significant response after UVA-1 exposure in vivo. UVA-1 thus raises selectively IL-6 levels in vivo, a fact that underlines its role in photoageing and has potential implications for its modulatory effect on photoageing pathology.

A three-dimensional skin equivalent reflecting some aspects of in vivo aged skin.

Human skin undergoes morphological, biochemical and functional modifications during the ageing process. This study was designed to produce a 3-dimensional (3D) skin equivalent in vitro reflecting some aspects of in vivo aged skin. Reconstructed skin was generated by co-culturing skin fibroblasts and keratinocytes on a collagen-glycosaminoglycan-chitosan scaffold, and ageing was induced by the exposition of fibroblasts to Mitomycin-C (MMC). Recently published data showed that MMC treatment resulted in a drug-induced accelerated senescence (DIAS) in human dermal fibroblast cultures. Next to established ageing markers, histological changes were analysed in comparison with in vivo aged skin. In aged epidermis, the filaggrin expression is reduced in vivo and in vitro. Furthermore, in dermal tissue, the amount of elastin and collagen is lowered in aged skin in vivo as well as after the treatment of 3D skin equivalents with MMC in vitro. Our results show histological signs and some aspects of ageing in a 3D skin equivalent in vitro, which mimics aged skin in vivo.

The role of GAPDH in the selective toxicity of CNP in melanoma cells.

BACKGROUND: Malignant melanoma is the most aggressive form of skin cancer with a rather poor prognosis. Standard chemotherapy often results in severe side effects on normal (healthy) cells finally being difficult to tolerate for the patients. Shown by us earlier, cerium oxide nanoparticles (CNP, nanoceria) selectively killed A375 melanoma cells while not being cytotoxic at identical concentrations on non-cancerous cells. In conclusion, the redox-active CNP exhibited both prooxidative as well as antioxidative properties. In that context, CNP induced mitochondrial dysfunction in the studied melanoma cells via generation of reactive oxygene species (primarily hydrogen peroxide (H2O2)), but that does not account for 100% of the toxicity. AIM: Cancer cells often show an increased glycolytic rate (Warburg effect), therefore we focused on CNP mediated changes of the glucose metabolism. RESULTS: It has been shown before that glyceraldehyde 3-phosphate dehydrogenase (GAPDH) activity is regulated via oxidation of a cysteine in the active center of the enzyme with a subsequent loss of activity. Upon CNP treatment, formation of cellular lactate and GAPDH activity were significantly lowered. The treatment of melanoma cells and melanocytes with the GAPDH inhibitor heptelidic acid (HA) decreased viability to a much higher extent in the cancer cells than in the studied normal (healthy) cells, highlighting and supporting the important role of GAPDH in cancer cells. CONCLUSION: We identified glyceraldehyde 3-phosphate dehydrogenase (GAPDH) as a target protein for CNP mediated thiol oxidation.

Investigating the surfactant antagonism with the Open Source Reconstructed Epidermis (OS-Rep) model.

Under the current EU chemicals legislation, in vitro test methods became the preferred methods to identify and classify the skin irritation potential of chemicals and mixtures. Among these, especially in vitro skin models are widely used. For surfactants, a well-known group of typically irritating chemicals, it is a long-standing experience that the irritation potential of a mixture of surfactants is typically lower than the irritation potential of the single surfactants, an effect usually described as surfactant antagonism. In order to evaluate if this effect can be observed in skin model systems as well, the irritation potential of the surfactants and of their mixtures was determined in the Open Source Reconstructed Epidermis (OS-REp) models. Combinations of sodium dodecyl sulfate or linear alkylbenzene sulfonate with cocoamidopropyl betain and alkyl polyglycosid, respectively, resulted in a clear decrease of the irritation potential compared to the irritation exerted by the single surfactants. The effect appeared to be primarily driven by the mixture's lower ability to damage the skin model's barrier, as shown by a reduced fluorescein permeation.

The natural chalcone cardamonin selectively induces apoptosis in human neuroblastoma cells.

Neuroblastoma is the most common extracranial malignant tumor in childhood. Approximately 60% of all patients are classified as high-risk and require intensive treatment including non-selective chemotherapeutic agents leading to severe side effects. Recently, phytochemicals like the natural chalcone cardamonin (CD) have gained attention in cancer research. For the first time, we investigated the selective anti-cancer effects of CD in SH-SY5Y human neuroblastoma cells compared to healthy (normal) fibroblasts (NHDF). Our study revealed selective and dose-dependent cytotoxicity of CD in SH-SY5Y. The natural chalcone CD specifically altered the mitochondrial membrane potential (ΔΨm), as an early marker of apoptosis, in human neuroblastoma cells. Caspase activity was also selectively induced and the amount of cleaved caspase substrates such as PARP was thus increased in human neuroblastoma cells. CD-mediated apoptotic cell death was rescued by pan caspase inhibitor Z-VAD-FMK. The natural chalcone CD selectively induced apoptosis, the programmed cell death, in SH-SY5Y human neuroblastoma cells whereas NHDF being a model for normal (healthy) cells were unaffected. Our data indicates a clinical potential of CD in the more selective and less harmful treatment of neuroblastoma.

The Antimalarial Drug Artesunate Mediates Selective Cytotoxicity by Upregulating HO-1 in Melanoma Cells.

Despite great efforts to develop new therapeutic strategies to combat melanoma, the prognosis remains rather poor. Artesunate (ART) is an antimalarial drug displaying anti-cancer effects in vitro and in vivo. In this in vitro study, we investigated the selectivity of ART on melanoma cells. Furthermore, we aimed to further elucidate the mechanism of the drug with a focus on the role of iron, the induction of oxidative stress and the implication of the enzyme heme oxygenase 1 (HO-1). ART treatment decreased the cell viability of A375 melanoma cells while it did not affect the viability of normal human dermal fibroblasts, used as a model for normal (healthy) cells. ART's toxicity was shown to be dependent on intracellular iron and the drug induced high levels of oxidative stress as well as upregulation of HO-1. Melanoma cells deficient in HO-1 or treated with a HO-1 inhibitor were less sensitive towards ART. Taken together, our study demonstrates that ART induces oxidative stress resulting in the upregulation of HO-1 in melanoma cells, which subsequently triggers the effect of ART's own toxicity. This new finding that HO-1 is involved in ART-mediated toxicity may open up new perspectives in cancer therapy.

CNP mediated selective toxicity on melanoma cells is accompanied by mitochondrial dysfunction.

Cerium (Ce) oxide nanoparticles (CNP; nanoceria) are reported to have cytotoxic effects on certain cancerous cell lines, while at the same concentration they show no cytotoxicity on normal (healthy) cells. Redox-active CNP exhibit both selective prooxidative as well as antioxidative properties. The former is proposed to be responsible for impairment of tumor growth and invasion and the latter for rescuing normal cells from reactive oxygen species (ROS)-induced damage. Here we address possible underlying mechanisms of prooxidative effects of CNP in a metastatic human melanoma cell line. Malignant melanoma is the most aggressive form of skin cancer, and once it becomes metastatic the prognosis is very poor. We have shown earlier that CNP selectively kill A375 melanoma cells by increasing intracellular ROS levels, whose basic amount is significantly higher than in the normal (healthy) counterpart, the melanocytes. Here we show that CNP initiate a mitochondrial increase of ROS levels accompanied by an increase in mitochondrial thiol oxidation. Furthermore, we observed CNP-induced changes in mitochondrial bioenergetics, dynamics, and cristae morphology demonstrating mitochondrial dysfunction which finally led to tumor cell death. CNP-induced cell death is abolished by administration of PEG-conjugated catalase. Overall, we propose that cerium oxide nanoparticles mediate cell death via hydrogen peroxide production linked to mitochondrial dysfunction.

Ammonia inhibits energy metabolism in astrocytes in a rapid and glutamate dehydrogenase 2-dependent manner.

Astrocyte dysfunction is a primary factor in hepatic encephalopathy (HE) impairing neuronal activity under hyperammonemia. In particular, the early events causing ammonia-induced toxicity to astrocytes are not well understood. Using established cellular HE models, we show that mitochondria rapidly undergo fragmentation in a reversible manner upon hyperammonemia. Further, in our analyses, within a timescale of minutes, mitochondrial respiration and glycolysis were hampered, which occurred in a pH-independent manner. Using metabolomics, an accumulation of glucose and numerous amino acids, including branched chain amino acids, was observed. Metabolomic tracking of 15N-labeled ammonia showed rapid incorporation of 15N into glutamate and glutamate-derived amino acids. Downregulating human GLUD2 [encoding mitochondrial glutamate dehydrogenase 2 (GDH2)], inhibiting GDH2 activity by SIRT4 overexpression, and supplementing cells with glutamate or glutamine alleviated ammonia-induced inhibition of mitochondrial respiration. Metabolomic tracking of 13C-glutamine showed that hyperammonemia can inhibit anaplerosis of tricarboxylic acid (TCA) cycle intermediates. Contrary to its classical anaplerotic role, we show that, under hyperammonemia, GDH2 catalyzes the removal of ammonia by reductive amination of α-ketoglutarate, which efficiently and rapidly inhibits the TCA cycle. Overall, we propose a critical GDH2-dependent mechanism in HE models that helps to remove ammonia, but also impairs energy metabolism in mitochondria rapidly.

Endogenous Carbon Monoxide Signaling Modulates Mitochondrial Function and Intracellular Glucose Utilization: Impact of the Heme Oxygenase Substrate Hemin.

Stress-inducible heme oxygenase-1 (HO-1) catalyzes the oxidative cleavage of heme yielding biliverdin, ferrous iron, and carbon monoxide (CO). Heme oxygenase activity has been attributed to antioxidant defense via the redox cycling system of biliverdin and bilirubin. There is increasing evidence that CO is a gaseous signaling molecule and plays a role in the regulation of energy metabolism. Inhibitory effects of CO on the respiratory chain are well established, but the implication of such a process on the cellular stress response is not well understood. By means of extracellular flux analyses and isotopic tracing, we studied the effects of CO, either released from the CO donor CORM-401 or endogenously produced by heme oxygenases, on the respiratory chain and glucose metabolism. CORM-401 was thereby used as a tool to mimic endogenous CO production by heme oxygenases. In the long term (>60 min), CORM-401-derived CO exposure inhibited mitochondrial respiration, which was compensated by increased glycolysis accompanied by a loss of the ATP production rate and an increase in proton leakage. This effect pattern was likewise observed after endogenous CO production by heme oxygenases. However, in the present setting, these effects were only observed when sufficient substrate for heme oxygenases (hemin) was provided. Modulation of the HO-1 protein level was less important. The long-term influence of CO on glucose metabolism via glycolysis was preceded by a short-term response (<30 min) of the cells to CO. Stable isotope-labeling experiments and metabolic flux analysis revealed a short-term shift of glucose consumption from glycolysis to the pentose phosphate pathway (PPP) along with an increase in reactive oxygen species (ROS) generation. Overall, we suggest that signaling by endogenous CO stimulates the rapid formation of reduction equivalents (NADPH) via the PPP, and plays an additional role in antioxidant defense, e.g., via feed-forward stimulation of the bilirubin/biliverdin redox cycling system.

Carbon monoxide releasing molecule 401 (CORM-401) modulates phase I metabolism of xenobiotics.

Next to its well-studied toxicity, carbon monoxide (CO) is recognized as a signalling molecule in various cellular processes. Thus, CO-releasing molecules (CORMs) are of considerable interest for basic research and drug development. Aim of the present study was to investigate if CO, released from CORMs, inhibits cytochrome P450-dependent monooxygenase (CYP) activity and modulates xenobiotic metabolism. CORM-401 was used as a model CO delivering compound; inactive CORM-401 (iCORM-401), unable to release CO, served as control compound. CO release from CORM-401, but not from iCORM-401, was validated using the cell free myoglobin assay. CO-dependent inhibition of CYP activity was shown by 7-ethoxyresorufin-O-deethylation (EROD) with recombinant CYP and HepG2 cells. Upon CORM-401 exposure EROD activity of recombinant CYP decreased concentration dependently, while iCORM-401 had no effect. Treatment with CORM-401 decreased EROD activity in HepG2 cells at concentrations higher than 50 µM CORM-401, while iCORM-401 showed no effect. At the given concentrations cell viability was not affected. Amitriptyline was selected as a model xenobiotic and formation of its metabolite nortriptyline by recombinant CYP was determined by HPLC. CORM-401 treatment inhibited the formation of nortriptyline whereas iCORM-401 treatment did not. Overall, we demonstrate CO-mediated inhibitory effects on CYP activity when applying CORMs. Since CORMs are currently under drug development, the findings emphasize the importance to take into account that this class of compounds may interfere with xenobiotic metabolism.

In vitro selective cytotoxicity of the dietary chalcone cardamonin (CD) on melanoma compared to healthy cells is mediated by apoptosis.

Malignant melanoma is an aggressive type of cancer and the deadliest form of skin cancer. Even though enormous efforts have been undertaken, in particular the treatment options against the metastasizing form are challenging and the prognosis is generally poor. A novel therapeutical approach is the application of secondary plant constituents occurring in food and food products. Herein, the effect of the dietary chalcone cardamonin, inter alia found in Alpinia species, was tested using human malignant melanoma cells. These data were compared to cardamonin treated normal melanocytes and dermal fibroblasts representing healthy cells. To investigate the impact of cardamonin on tumor and normal cells, it was added to monolayer cell cultures and cytotoxicity, proliferation, tumor invasion, and apoptosis were studied with appropriate cell biological and biochemical methods. Cardamonin treatment resulted in an apoptosis-mediated increase in cytotoxicity towards tumor cells, a decrease in their proliferation rate, and a lowered invasive capacity, whereas the viability of melanocytes and fibroblasts was hardly affected at such concentrations. A selective cytotoxic effect of cardamonin on melanoma cells compared to normal (healthy) cells was shown in vitro. This study along with others highlights that dietary chalcones may be a valuable tool in anticancer therapies which has to be proven in the future in vivo.

Stromal resistance of fibroblasts against oxidative damage: involvement of tumor cell-secreted platelet-derived growth factor (PDGF) and phosphoinositide 3-kinase (PI3K) activation.

A critical step in tumor progression is the interaction of malignant and stromal cells via paracrine mechanisms. Stromal cells, particularly fibroblasts, support cancer cells in invasion of the surrounding tissue for access to the vascular system. Here, the question is addressed of whether tumor cells induce 'stromal resistance', i.e. protect the microenvironment from oxidative damage. The supernatant of cultured skin-derived tumor cells was added to fibroblasts and was shown to protect the fibroblasts from hydrogen peroxide-mediated cell toxicity. The platelet-derived growth factor secreted from the cancer cells was identified as trigger of this protection in fibroblasts via the phosphoinositide 3-kinase pathway. These data suggest that prosurvival signals in stromal fibroblasts as initiated by tumor cells constitute a strategy of 'stromal resistance', illustrating a novel biological role of fibroblasts for the tumor microenvironment.

Nanotherapy and Reactive Oxygen Species (ROS) in Cancer: A Novel Perspective.

The incidence of numerous types of cancer has been increasing over recent years, representing the second-most frequent cause of death after cardiovascular diseases. Even though, the number of effective anticancer drugs is increasing as well, a large number of patients suffer from severe side effects (e.g., cardiomyopathies) caused by these drugs. This adversely affects the patients' well-being and quality of life. On the molecular level, tumor cells that survive treatment modalities can become chemotherapy-resistant. In addition, adverse impacts on normal (healthy, stromal) cells occur concomitantly. Strategies that minimize these negative impacts on normal cells and which at the same time target tumor cells efficiently are needed. Recent studies suggest that redox-based combinational nanotherapies may represent one option in this direction. Here, we discuss recent advances in the application of nanoparticles, alone or in combination with other drugs, as a promising anticancer tool. Such novel strategies could well minimize harmful side effects and improve patients' health prognoses.

The BH3 mimetic compound BH3I-1 impairs mitochondrial dynamics and promotes stress response in addition to its pro-apoptotic key function.

BH3 mimetics, such as BH3I-1, act as Bcl-2 antagonists, promote apoptosis and are used in basic research studies on apoptotic signaling and are currently tested as experimental anti-tumor agents. The present study addresses time- and dose-dependent responses of BH3I-1 on apoptosis, cellular stress defense mediated by heme oxygenase-1 (HO-1), and mitochondrial morphology. As expected, treatment of normal human dermal fibroblasts with BH3I-1 induced apoptosis as determined by typical markers including cytochrome c release, loss of procaspase-3, and PARP cleavage. Induction of the cellular stress response marker HO-1 precedes apoptosis induction whereas fragmentation of the mitochondrial network was triggered even more rapidly. No difference in apoptosis induction was found upon depletion of HO-1 by siRNA compared to controls suggesting that apoptosis induction by BH3I-1 is not affected by HO-1. To evaluate the functional interplay between mitochondrial fragmentation and HO-1 induction, murine embryonic fibroblasts lacking the fission factor Drp1 were used. In Drp1 knock out cells, HO-1 levels were low compared to wild type cells, both in untreated controls as well as after BH3I-1 exposure, demonstrating that Drp1 is at least in part required for determining basal and inducible HO-1 levels. Considering the sequence of events, it was shown here that BH3I-1 dependent apoptosis is a rather late event, while effects on mitochondrial morphology and cellular stress response (HO-1 induction) are observed rapidly after exposure of cells to the compound. We propose that BH3I-1 is a valuable tool for studying cellular stress responses as well as mitochondrial dynamics in future studies. Since BH3 mimetics are promising experimental anticancer drugs, our data further imply that additional biological effects such as upregulation of detoxifying systems or changes in mitochondrial dynamics could interfere, in combination therapy, with selective drug toxicity and thus need to be taken into account for drug development.

Efficacy of Different Compositions of Cerium Oxide Nanoparticles in Tumor-Stroma Interaction.

The biomedical application of cerium oxide nanoparticles (nanoceria) is a focal point of research for a few years. The biochemical effects of nanoceria depend on various factors including particle size, oxidation state of cerium, oxygen vacancies on the surface, use of dispersants or coatings, pH and cell type. Due to their autocatalytic redox-activity, these particles are considered to act as a specific anti-cancer tool with less side effects on healthy cells and tissues, as the particles kill tumor cells, while protecting healthy cells from oxidative damage. In the present study, four different types of nanoceria were investigated with regard to their impact on biochemical parameters in vitro, which play a pivotal role in tumor-stroma interaction. The obtained data and presented in vitro test parameters will be helpful in designing nanoceria compositions, which are ideally suited for anticancer therapy, either as a 'stand alone drug' or in combination with other chemotherapeutics.

Involvement of selenoprotein P in protection of human astrocytes from oxidative damage.

Selenoprotein P (SeP) is a highly glycosylated, selenium-rich plasma protein. Aside from its role as selenium carrier protein, an antioxidative function of SeP has been suggested. Astrocytes, which detoxify reactive oxygen species in the brain, were described as potential target cells of SeP. We investigated the expression of SeP in human astrocytes and its involvement in the protection of these cells against tert-butyl hydroperoxide (t-BHP)-induced oxidative damage. We show that primary human astrocytes and the human astrocytoma cell line MOG-G-CCM express SeP as an unglycosylated protein, which is not secreted. SeP expression in astrocytes is constitutive. Preincubation of astrocytes with hepatocyte-derived SeP mimicks the protective effect of low-molecular-weight selenocompounds such as sodium selenite or selenomethionine against oxidative damage, shielding astrocytes from t-BHP-induced cytotoxicity. Selenium supplementation of astrocytes counteracts oxidative stress via an increase in expression and activity of the selenoenzyme cytosolic glutathione peroxidase (cGPx). Furthermore, specific downregulation of SeP expression by small interfering RNA decreases cell viability of human astrocytes and makes them more susceptible to t-BHP-induced cytotoxicity. Our results implicate an antioxidant activity of constitutively expressed SeP in selenium-deficient astrocytes, while during adequate selenium supply the enhanced protection against oxidative stress is exerted by cGPx.