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Retarded growth and neurodegeneration are hallmarks of the premature aging disease Cockayne syndrome (CS). Cockayne syndrome proteins take part in the key step of ribosomal biogenesis, transcription of RNA polymerase I. Here, we identify a mechanism originating from a disturbed RNA polymerase I transcription that impacts translational fidelity of the ribosomes and consequently produces misfolded proteins. In cells from CS patients, the misfolded proteins are oxidized by the elevated reactive oxygen species (ROS) and provoke an unfolded protein response that represses RNA polymerase I transcription. This pathomechanism can be disrupted by the addition of pharmacological chaperones, suggesting a treatment strategy for CS. Additionally, this loss of proteostasis was not observed in mouse models of CS., (Copyright © 2018 The Author(s). Published by Elsevier Inc. All rights reserved.)

Quality by design principles (QbD) were used to assist the formulation of prednisolone-loaded long-circulating liposomes (LCL-PLP) in order to gain a more comprehensive understanding of the preparation process. This approach enables us to improve the final product quality in terms of liposomal drug concentration, encapsulation efficiency and size, and to minimize preparation variability. A 19-run D-optimal experimental design was used to study the impact of the highest risk factors on PLP liposomal concentration (Y 1 - μg/ml), encapsulation efficiency (Y 2 -%) and size (Y 3 -nm). Out of six investigated factors, four of them were identified as critical parameters affecting the studied responses. PLP molar concentration and the molar ratio of DPPC to MPEG-2000-DSPE had a positive impact on both Y 1 and Y 2 , while the rotation speed at the formation of the lipid film had a negative impact. Y 3 was highly influenced by prednisolone molar concentration and extrusion temperature. The accuracy and robustness of the model was further on confirmed. The developed model was used to optimize the formulation of LCL-PLP for efficient accumulation of the drug to tumor tissue. The cytotoxicity of the optimized LCL-PLP on C26 murine colon carcinoma cells was assessed. LCL-PLP exerted significant anti-angiogenic and anti-inflammatory effects on M2 macrophages, affecting indirectly the C26 colon carcinoma cell proliferation and development.

PGC-1α is a versatile inducer of mitochondrial biogenesis and responsive to the changing energy demands of the cell. As mitochondrial ATP production requires proteins that derive from translation products of cytosolic ribosomes, we asked whether PGC-1α directly takes part in ribosomal biogenesis. Here, we show that a fraction of cellular PGC-1α localizes to the nucleolus, the site of ribosomal transcription by RNA polymerase I. Upon activation PGC-1α associates with the ribosomal DNA and boosts recruitment of RNA polymerase I and UBF to the rDNA promoter. This induces RNA polymerase I transcription under different stress conditions in cell culture and mouse models as well as in healthy humans and is impaired already in early stages of human Huntington's disease. This novel molecular link between ribosomal and mitochondrial biogenesis helps to explain sarcopenia and cachexia in diseases of neurodegenerative origin.

The nucleolus has long been considered to be a pure ribosome factory. However, over the last two decades it became clear that the nucleolus is involved in numerous other functions besides ribosome biogenesis. Our experiments indicate that the activity of RNA polymerase I (Pol I) transcription monitors the integrity of the DNA and influences the response to nucleolar stress as well as the rate of survival. Cells with a repressed ribosomal DNA (rDNA) transcription activity showed an increased and prolonged p53 stabilisation after UVC-irradiation. Furthermore, p53 stabilisation after inhibition and especially after UVC-irradiation might be due to abrogation of the HDM2-p53 degradation pathway by ribosomal proteins (RPs). Apoptosis mediated by highly activated p53 is a typical hallmark of Cockayne syndrome cells and transcriptional abnormalities and the following activation of the RP-HDM2-p53 pathway would be a possible explanation.

The role of tumor-associated macrophages (TAMs) in the development of colon carcinoma is still controversial. Therefore, the present study aimed to investigate the TAM‑driven processes that may affect colon cancer cell proliferation. To achieve this purpose, murine macrophages were co-cultured with C26 murine colon carcinoma cells at a cell density ratio that approximates physiological conditions for colon carcinoma development in vivo. In this respect, the effects of TAM-mediated angiogenesis, inflammation and oxidative stress on the proliferative capacity of C26 murine colon carcinoma cells were studied. To gain insight into the TAM-driven oxidative stress, NADPH oxidase, the main pro-oxidant enzyme in macrophages, was inhibited. Our data revealed that the stimulatory effects of TAMs on C26 cell proliferation may be related mainly to their pro-oxidant actions exerted by NADPH oxidase activity, which maintains the redox status and the angiogenic capacity of the tumor microenvironment. Additionally, the anti-inflammatory and pro-angiogenic effects of TAMs on tumor cells were found to create a favorable microenvironment for C26 colon carcinoma development and progression. In conclusion, our data confirmed the protumor role of TAMs in the development of colon carcinoma in an oxidative stress-dependent manner that potentiates the angiogenic capacity of the tumor microenvironment. These data may offer valuable information for future tumor-targeted therapies based on TAM 're-education' strategies.

Previous studies have demonstrated that tumor-associated macrophages (TAMs) are pivotal players in tumor progression via modulation of tumor angiogenesis, inflammation, metastasis and oxidative stress, as well as of the response of cancer cells to cytotoxic drugs. Nevertheless, the role of TAMs in the prognosis of colorectal cancer remains controversial. Therefore, the present study aimed to investigate how TAMs mediate the response of C26 colon carcinoma cells to the cytotoxic drug 5-fluorouracil (5-FU), upon TAM co-cultivation with these cancer cells in vitro . In this respect, 5-FU cytotoxicity was assessed in C26 cells in standard culture and in a co-culture with peritoneal macrophages, the production of NF-κB was determined by western blot analysis, and the production of angiogenic/inflammatory proteins in each experimental model was evaluated by protein array analysis. To gain further evidence of the effect of TAMs on oxidative stress, malondialdehyde was measured through high-performance liquid chromatography, and the total nonenzymatic antioxidant levels and the production of nitrites were measured through colorimetric assays. The results demonstrated that TAMs exerted a dual role in the response of C26 cells to 5-FU administration in the co-culture model. Thus, on one side, TAMs sensitized C26 cells to 5-FU administration through inhibition of the production of inflammatory and angiogenic proteins in these cancer cells; however, they also protected cancer cells against 5-FU-induced oxidative stress. Collectively, the present findings suggest that the combined administration of 5-FU with pharmacological agents that prevent TAMs to maintain the physiological range of tumor cell oxidative stress may highly improve the therapeutic potential of this drug.

Statins possess antitumor actions at doses 100- to 500-fold higher than those needed to lower cholesterol levels. Thus, the antitumor efficacy of statins could be improved greatly by using tumor-targeted delivery systems. Therefore the present work aims to investigate the antitumor activity of long-circulating liposome-encapsulated simvastatin (LCL-SIM) versus free SIM in B16.F10 murine melanoma-bearing mice. Our results showed that LCL-SIM inhibits strongly the B16.F10 melanoma growth (by 85%) whereas free SIM was ineffective. Moreover, the antitumor activity of LCL-SIM depends on the presence of functional tumor-associated macrophages (TAM) in tumor tissue and is mainly based on the reduction of the TAM-mediated oxidative stress as well as of the production of the hypoxia-inducible factor 1 α (HIF-1 α) in tumors. In conclusion, our findings suggest that the antitumor activity of LCL-SIM on B16.F10 melanoma growth is a result of the tumor-targeting property of the liposome formulation and is tightly dependent on the presence of TAM in tumor tissue., (Copyright © 2014. Published by Elsevier Ireland Ltd.)

Simvastatin (SIM) is a lipophilic statin that has potential benefits for prevention and treatment of several types of malignancies. However, its low water solubility and the toxicity associated with administration of high doses recommend it for encapsulation in carriers able to deliver the therapeutic dose in the tumor. In this work, liposomes with long-circulating properties were proposed as delivery systems for SIM. The objective of this study was to optimize the formulation of SIM-loaded long-circulating liposomes (LCL-SIM) by using D-optimal experimental design. The influence of phospholipids concentration, phospholipids to cholesterol molar ratio and SIM concentration was studied on SIM liposomal concentration, encapsulation efficiency and liposomal size. The optimized formulation had liposomal SIM concentration 6238 µg/ml, EE % of 83.4% and vesicle size of 190.5 nm. Additionally we evaluated the in vitro cytotoxicity of the optimized liposomal SIM (LCL-SIM-OPT) on C26 murine colon carcinoma cells cultivated in monoculture as well as in co-culture with murine peritoneal macrophages at a cell density ratio that provides an approximation of physiological conditions of colon carcinoma development in vivo. Our preliminary studies suggested that LCL-SIM-OPT exerted cytotoxicity on C26 cells probably via enhancement of oxidative stress in co-culture environment.

Statins, as inhibitors of de-novo synthesis of cholesterol, exert cytotoxic actions on tumor cells. Despite the increasing data on the antitumoral activities of statins, their complete mechanisms of action still remain obscure. Therefore, the present study aims to investigate the mechanisms of lipophilic statin-induced cytotoxicity on B16.F10 murine melanoma cells in vitro. In-vitro effects of two lipophilic statins, simvastatin and lovastatin, and a hydrophilic statin, pravastatin, were investigated with respect to B16.F10 murine melanoma cell proliferation and viability. Our results show that only lipophilic statins exerted strong cytotoxic effects on B16.F10 melanoma cells. To gain further evidence on the pleiotropic effects of statins responsible for their cytotoxicity in B16.F10 cells, we have assessed their proapoptotic effects by Annexin V-fluorescein isothiocyanate/propidium iodide staining and measured tumor cell production of the hypoxia-inducible factor 1α by western blot analysis, nonenzymatic antioxidant levels by an antioxidant colorimetric assay, and superoxide dismutase activity through an indirect method on the basis of inhibition of xanthine oxidase activity. Protein array was also used to assess angiogenic/inflammatory protein production in B16.F10 cells. Our results pointed out that the cytotoxic actions exerted by lipophilic statins were mainly based on the suppressive actions of these drugs on hypoxia-inducible factor 1α expression and nonenzymatic antioxidant levels, as well as because of the inhibition of superoxide dismutase activity in B16.F10 melanoma cells. In addition, the reduction in the angiogenic/inflammatory capacity of tumor cells induced by lipophilic statins can strengthen and support their cytotoxicity.

Because polycystic ovarian syndrome (PCOS) is a risk factor for type 2 diabetes, the affected women can present frequently prediabetic states such as impaired fasting glycaemia and/or impaired glucose tolerance. The purpose of our study is to explore the effect of antiandrogenic spironolactone on glucose metabolism and oxidative stress (OS) parameters in oestradiol valerate (OV) induced PCOS rat model.72 female Wistar rats were distributed either to PCOS group (n=65, OV dissolved in sesame oil, 5 mg/0.4 ml), or to non-PCOS control group (n=7, sesame oil, 0.4 ml). After a month, ultrasound was performed to assess the ovarian morphology, and the results of an initial oral glucose tolerance test (OGTT) were used to identify the animals with altered glucose metabolism (AGM). Glucose transporter 4 (GLUT4) was evaluated from muscle biopsies, OS parameters were assessed from blood and muscle samples, and ovaries of 3 rats were removed for histopathological examination. Afterwards, the AGM group was divided in a treated PCOS group denoted as Sp+D (per os spironolactone dissolved in DMSO, 2 mg/0.2 ml), and a PCOS control treated with DMSO (0.2 ml). After one month of daily treatment, a final OGTT was performed. GLUT4 and OS parameters were again evaluated and ovaries were removed for histopathological examination.As compared to the values prior to the treatment, Sp+D reversed fasting hyperglycaemia (p<0.001), increased GLUT4 immunoreactivity in the perinuclear compartment (p<0.05) and translocation to plasmalemma (p<0.001) and improved superoxide dismutase (0.001

There is a need for new strategies for noninvasive imaging of pathological conditions within the human body. The approach of combining the unique physical properties of noble-metal nanoparticles with their chemical specificity and an easy way of conjugation open up new routes toward building bio-nano-objects for biomedical tracking and imaging. This work reports the design and assessment of a novel class of biocompatible, highly sensitive SERS nanotags based on chitosan-coated silver nanotriangles (Chit-AgNTs) labeled with para-aminothiophenol (p-ATP). The triangular nanoparticles are used as Raman scattering enhancers and have proved to yield a reproducible and strong SERS signal. When tested inside lung cancer cells (A549) this class of SERS nanotags presents low in vitro toxicity, without interfering with cell proliferation. Easily internalized by the cells, as demonstrated by imaging using both reflected bright-light optical microscopy and SERS spectroscopy, the particles are proved to be detectable inside cells under a wide window of excitation wavelengths, ranging from visible to near infrared (NIR). Their high sensitivity and NIR availability make this class of SERS nanotags a promising candidate for noninvasive imaging of cancer cells.