High-Dose Fenofibrate Stimulates Multiple Cellular Stress Pathways in the Kidney of Old Rats.

We investigated the age-related effects of the lipid-lowering drug fenofibrate on renal stress-associated effectors. Young and old rats were fed standard chow with 0.1% or 0.5% fenofibrate. The kidney cortex tissue structure showed typical aging-related changes. In old rats, 0.1% fenofibrate reduced the thickening of basement membranes, but 0.5% fenofibrate exacerbated interstitial fibrosis. The PCR array for stress and toxicity-related targets showed that 0.1% fenofibrate mildly downregulated, whereas 0.5% upregulated multiple genes. In young rats, 0.1% fenofibrate increased some antioxidant genes' expression and decreased the immunoreactivity of oxidative stress marker 4-HNE. However, the activation of cellular antioxidant defenses was impaired in old rats. Fenofibrate modulated the expression of factors involved in hypoxia and osmotic stress signaling similarly in both age groups. Inflammatory response genes were variably modulated in the young rats, whereas old animals presented elevated expression of proinflammatory genes and TNFα immunoreactivity after 0.5% fenofibrate. In old rats, 0.1% fenofibrate more prominently than in young animals induced phospho-AMPK and PGC1α levels, and upregulated fatty acid oxidation genes. Our results show divergent effects of fenofibrate in young and old rat kidneys. The activation of multiple stress-associated effectors by high-dose fenofibrate in the aged kidney warrants caution when applying fenofibrate therapy to the elderly.

Aging and short-term calorie restriction differently affect the cardiac and skeletal muscle expression of genes regulating energy substrate utilization in male rats.

Aging affects the energy metabolism differently in the cardiac and skeletal muscles. The study aim was to assess the effects of short-term calorie restriction (SCR) and refeeding on the expression of genes involved in the control of cardiac and skeletal muscle energy metabolism in old vs. young male rats. Young (4 mo) and old (24 mo) rats were subjected to 60% SCR for 30 days, and refed ad libitum for 2 or 4 days. In the cardiac (CM) and skeletal muscles (SM) we compared the gene expression (qPCR) of carnitine palmitoyltransferase-I (Cpt-I), peroxisome proliferator-activated receptor beta/delta (Ppar-ß/δ), glucose transporter 4 (Glut4), peroxisome proliferator-activated receptor-γ coactivator-1α (Pgc-1α), and sirtuin 3 (Sirt3). In CM, aging increased Cpt-I expression but did not affect the other genes. In SM, Cpt-I, Glut4, Pgc-1α, and Sirt3 mRNA levels were lower in old than young rats. In CM of only young rats SCR increased Cpt-I expression which remained elevated after refeeding. Upon SCR, the expression of Ppar-ß/δ, Glut4, Pgc-1α, and Sirt3 in CM increased in young but not old rats, and refeeding re-established control levels. In SM of young rats SCR increased Ppar-ß/δ and Pgc-1α, and decreased Sirt3 expression, whereas refeeding generally decreased these mRNA levels. In SM of old rats SCR decreased only Pgc-1α expression. The adaptive response to SCR and subsequent refeeding is muscle tissue-specific and differs in young and old male rats. SCR appears to increase the efficiency of glucose and fatty acid utilization in the cardiac muscle of young, but not old male rats.

Sestrins' Expression in the Liver Is Not Altered by Short-Term Calorie Restriction in Young and Old Rats.

INTRODUCTION: Short-term calorie restriction (SCR) may have a positive impact on health. We hypothesized that sestrins, a family of stress-inducible proteins (Sesn1, Sesn2, Sesn3) are involved in the response to SCR in the liver. METHODS: Young-adult (4-month) and old (24-month) male Wistar rats were either fed ad libitum (control groups) or received 60% of food intake on a daily basis for 30 days (SCR groups). In blood sera, biochemical parameters and fibroblast growth factor 21 (FGF21) concentration were measured (ELISA). Liver samples were collected for analyses of genes' expression (real-time PCR) and protein levels (Western blotting). RESULTS: SCR caused improvements in blood glucose and lipids and parameters of liver function but did not affect the serum FGF21 concentration. SCR caused changes typically associated with calorie restriction in the gene expression of fatty acid synthase (fasn), ATP-citrate lyase (acly), and sirtuin 1 (sirt1). In the liver of young SCR rats, protein level of Sesn2 tended to increase, while Sesn3 tended to decrease, accompanied by reduced sesn3 expression. In old SCR rats, reduced sesn1 expression was reflected by decreasing trend for Sesn1 content. Activation of AMP-activated protein kinase (phospho-Thr172) and protein content of peroxisome proliferator-activated receptor gamma coactivator 1-alpha were not affected by SCR. CONCLUSION: Sestrins' hepatic expression is only minimally affected by SCR in young and old rats. We propose that sestrins may not be a major effector of mild SCR in the liver of young or old rats.

Homocysteine-induced decrease in HUVEC cells' resistance to oxidative stress is mediated by Akt-dependent changes in iron metabolism.

PURPOSE: Hyperhomocysteinemia is an independent risk factor for cardiovascular diseases and also promotes neuronal death in various neurodegenerative diseases. There is evidence that iron can mediate homocysteine (Hcy) toxicity. Thus, the aim of this study was to investigate the effect of Hcy on iron metabolism in HUVEC and SH-SY5Y cells. METHODS: HUVEC and SH-SY5Y cells were treated with 3 mM Hcy for a defined time. RESULTS: We demonstrate that Hcy induced the upregulation of ferritins type L and H in HUVEC cells in a time-dependent manner and had no effect on the ferritins in SH-SY5Y cells. The change in ferritin expression was preceded by a significant decrease in the cellular level of the active form of Akt kinase in HUVEC but not in SH-SY5Y cells. An increase in ferritin L and H protein levels was observed in the Akt1, Akt2, Akt3 siRNA transfected cells, while in the cells transfected with FOXO3a siRNA, a decrease in both ferritins levels was noticed. Moreover, in the HUVEC cells treated with Hcy for 6 days, the active form of kinase Akt returned to the control level and it was accompanied by a drop in ferritin L and H protein levels. Cytotoxicity of hydrogen peroxide significantly increased in HUVEC cells pre-treated with Hcy for 24 h. CONCLUSIONS: These data indicate that Hcy induces an increase in cellular ferritin level, and the process is mediated by alterations in Akt-FOXO3a signaling pathway.

Piceatannol, a Structural Analog of Resveratrol, Is an Apoptosis Inducer and a Multidrug Resistance Modulator in HL-60 Human Acute Myeloid Leukemia Cells.

Acute myeloid leukemia is characterized by uncontrolled clonal proliferation of abnormal myeloid progenitor cells. Despite recent advances in the treatment of this disease, the prognosis and overall long-term survival for patients remain poor, which drives the search for new chemotherapeutics and treatment strategies. Piceatannol, a polyphenolic compound present in grapes and wine, appears to be a promising chemotherapeutic agent in the treatment of leukemia. The aim of the present study was to examine whether piceatannol induces autophagy and/or apoptosis in HL-60 human acute myeloid leukemia cells and whether HL-60 cells are able to acquire resistance to piceatannol toxicity. We found that piceatannol at the IC90 concentration of 14 µM did not induce autophagy in HL-60 cells. However, it induced caspase-dependent apoptosis characterized by phosphatidylserine externalization, disruption of the mitochondrial membrane potential, caspase-3 activation, internucleosomal DNA fragmentation, PARP1 cleavage, chromatin condensation, and fragmentation of cell nuclei. Our findings also imply that HL-60 cells are able to acquire resistance to piceatannol toxicity via mechanisms related to MRP1 activity. Our results suggest that the use of piceatannol as a potential chemotherapeutic agent may be associated with the risk of multidrug resistance, warranting its use in combination with other chemotherapeutic agents.

Age-Related Changes in Sirtuin 7 Expression in Calorie-Restricted and Refed Rats.

BACKGROUND: Sirtuins (SIRT1-7) have been implicated to mediate the beneficial effects of calorie restriction for healthy aging. While the physiological functions of SIRT7 are still poorly understood, SIRT7 has recently been shown to affect ribosome biogenesis, mitochondrial gene expression, and hepatic lipid metabolism. OBJECTIVE: To analyze the effects of age and short-term calorie restriction (SCR) and subsequent refeeding on SIRT7 expression in key metabolic tissues. METHODS: Four- and 24-month-old male Wistar rats were subjected to 40% SCR for 30 days, followed by ad libitum feeding for 2 or 4 days. Liver, white adipose tissue (WAT), heart and skeletal muscle samples were analyzed by real-time PCR and Western blotting for SIRT7 mRNA and protein expression, respectively. RESULTS: Aging had diverse effects on SIRT7 levels in lipogenic tissues: both the mRNA and protein levels increased in the retroperitoneal depot (rWAT), did not change in the epididymal depot (eWAT), and decreased in the subcutaneous depot (sWAT) and the liver of old as compared to young animals. In the heart, extensor digitorum longus muscle (EDL) and soleus muscle (SOL), Sirt7 gene but not protein expression was lower in old than in young control rats. SCR did not affect SIRT7 expression in WAT and the liver in both age groups. In the heart of young animals, SCR did not affect SIRT7 mRNA or protein level. In EDL, SIRT7 protein but not mRNA levels decreased after SCR and remained reduced upon refeeding. In SOL, both SIRT7 mRNA and protein expression were inhibited by refeeding. In old rats, cardiac Sirt7 expression increased after SCR and refeeding. In old rats' EDL and SOL muscles, SIRT7 protein expression was inhibited by refeeding. CONCLUSION: Age-related changes of SIRT7 gene expression in key organs of energy homeostasis are tissue dependent.

White Adipose Tissue Depot-Specific Activity of Lipogenic Enzymes in Response to Fasting and Refeeding in Young and Old Rats.

BACKGROUND: Although the heterogeneity of white adipose tissue (WAT) in different anatomical sites is a well-known phenomenon, there are scarce data on aging-associated metabolic alterations in various WAT depots. OBJECTIVE: We used the model of fasting and refeeding to analyze the effect of aging on the activity of key lipogenic enzymes in retroperitoneal (rWAT), epididymal (eWAT), and subcutaneous (sWAT) adipose tissue depots. METHODS: 5- and 24-month-old male Wistar rats were fasted for 48 h or were fasted for 2 days and subsequently refed for 2 or 4 days. Control animals had ad libitum access to chow. Samples obtained from three WAT deposits were analyzed for the enzymatic activities of ATP citrate lyase (ACL), fatty acid synthase (FAS), and glucose-6-phosphate dehydrogenase (G6PD). Concentrations of lipids and proteins were measured in the blood serum. RESULTS: Fasting for 2 days decreased the concentration of free fatty acids only in the young rats. The basal activities of ACL and FAS were lower in eWAT than in rWAT and sWAT of the young rats. In the young rats, fasting did not change ACL and FAS activities in any of the studied depots. Refeeding increased these activities more quickly in rWAT than in eWAT, while in sWAT no induction was observed. ACL and FAS activities were manifold lower in all WAT depots of the old than in those of the young rats. In the old animals fasting had no effect on ACL activity in any depot and decreased FAS activity only in sWAT. After 4 days of refeeding, FAS activity increased in rWAT and sWAT, but no change in ACL activity occurred. G6PD activity in the young rats was lower by 40% in eWAT than in rWAT. The induction of the enzyme by refeeding occurred faster in rWAT than in eWAT, while in sWAT no change in G6PD activity was observed. G6PD activity did not change with aging. Fasting of the old rats decreased G6PD activity in rWAT and sWAT. Refeeding failed to induce the enzyme in these depots, whereas in eWAT G6PD activity increased by 76% after 4 days of refeeding. CONCLUSION: Fasting and refeeding revealed WAT depot-specific, age-related changes of the activities of lipogenic enzymes.

Identification of a suitable qPCR reference gene in metastatic clear cell renal cell carcinoma.

There is no data on reference gene (RG) selection in metastatic clear-cell renal cell carcinoma (mccRCC) for quantitative PCR (qPCR) data normalization. We aimed at selecting the most stable RG for further determination of new prognostic markers. Thirty-five nonmetastatic and 35 mccRCC patients undergoing radical nephrectomy were included. Paired primary tumor (T, n = 70) and normal (C, n = 70) kidney fragments were collected; from 12 out of 35 mccRCC cases, we also collected metastasized regional lymph nodes and adrenal gland tissues (M, n = 12). After RNA extraction, reverse transcription and qPCR were performed. Samples were divided into four analyzed groups. Fifteen candidate RGs were tested by RefFinder tool and manual statistics. To present the importance of RG selection, TP53 gene expression levels in samples were normalized with the use of RG data. RPL13 gene was the most stable RG in analysis of 35 primary tumor nonmetastatic versus 35 mccRCC samples and matched metastasized T/C/M samples (n = 12, each group). GUSB was the most suitable RG in total 152 samples and in paired T and C (n = 140) kidney samples. Expression of GUSB, RPL13, and the RPL13 + RPLP0 pair were independent of clinical/sample variables. Normalization of TP53 expression levels showed variability of GAPDH and ACTB assays. GUSB or RPL13 assays should be used in mccRCC for qPCR data normalization whereas GAPDH and ACTB assays should be avoided. Prior RG studies should precede each qPCR gene expression study since RG selection is associated with the origin and proportion of specimens.

Age-related effects of fenofibrate on the hepatic expression of sirtuin 1, sirtuin 3, and lipid metabolism-related genes.

BACKGROUND: Sirtuin 1 (Sirt1) and sirtuin 3 (Sirt3) participate in the regulation of lipid metabolism. Our aim was to investigate the effects of the hypolipemic drug fenofibrate (FN) on hepatic Sirt1 and Sirt3 expression, in relation to the expression of lipid metabolism-related genes and in the context of aging. METHODS AND RESULTS: Young and old male Wistar rats were fed standard chow or supplemented with 0.1% or 0.5% FN for 30 days (n=7-10 in each group). In young rats, 0.1% FN did not affect Sirt1 expression, however, 0.5% FN decreased Sirt1 and both doses reduced Sirt3 protein levels. In old rats, 0.5% FN decreased hepatic Sirt1 mRNA and both doses reduced Sirt1 protein levels, but not Sirt3 expression. Although hepatic Pparα protein levels did not change, FN treatment of young rats induced Cpt1b expression, whereas Lcad, Acox1, Pmp70, and Hmgcs2 expression increased only after 0.1% FN, and Fas2 expression decreased after 0.5% FN. In the liver of old rats, both doses increased Cpt1b and Lcad expression. Only 0.1% FN increased Pmp70 and Hmgcs2 expression, and only 0.5% FN increased Acox1 and Fas2 mRNA levels. CONCLUSIONS: Treatment with fenofibrate at low or high doses may downregulate the expression of Sirt1 and Sirt3 proteins in the rat liver. The dosage of FN affects molecular changes, and aging alters the response to 0.5% FN.

Short-term fenofibrate treatment improves ultrastructure of hepatocytes of old rats.

INTRODUCTION: Fenofibrate (FN) is a hypolipemic drug used for the treatment of mixed dyslipidemia. Since in our previous study FN administration to young and old rats adversely affected the serum activity of liver marker enzymes, we decided to examine the effects of FN on liver ultrastructure of young and old animals. MATERIAL AND METHODS: Young and old rats were fed standard rodent chow supplemented with 0.1% FN for 30 days. Liver samples obtained from animals under full anesthesia were processed by routine methods to obtain ultrathin and histological sections for the examination by light microscopy (LM) and transmission electron microscopy (TEM). Furthermore, liver lysates were analyzed by Western blotting for the expression of the autophagy-related proteins LC3A/B and beclin 1. RESULTS: The ultrastructure of hepatocytes in both age groups was well-preserved, with the presence of abundant mitochondria, numerous peroxisomes and lysosomes, glycogen stored in the form of rosettes, and occasionally autolysosomes. However, hepatocytes of old control rats contained less mitochondria and peroxisomes, and more lipid droplets than cells of young animals. The effects of FN on liver ultrastructure were age-depended. FN increased the relative number of mitochondria and peroxisomes in the hepatocytes of old, and did not affect their number in young rats. Moreover, FN decreased and increased the relative number of lipid droplets in the hepatocytes of old and young rats, respectively. At the LM level, Oil Red O staining revealed smaller and larger lipid droplets within hepatocytes and non-parenchymal liver cells. In the livers of young and old rats lipid droplets were distributed mainly in the periportal zones of hepatic lobules. Morphometric analysis confirmed that livers of control old rats contained more lipid-stainable areas than those of young ones; however, no effect of FN was observed either in young or old rats. Despite larger size of autolysosomes and autophagic vacuoles in hepatocytes of old rats, the expression of autophagy-related proteins did not differ in the livers of control and fenofibrate-treated young and old animals. CONCLUSIONS: The results of our study suggest that fenofibrate, apart from its hypolipemic action, may have beneficial effect on the energy metabolism in the liver of old individuals by increasing the number of mitochondria and peroxisomes in hepatocytes.

Effect of Corrosion Protection Method on Properties of RSW and RFSSW Lap Joints Applied in Production of Thin-Walled Aerostructures.

Aluminum structures, and in particular an element of aerostructures, are strongly exposed to the effects of weather conditions. In the case of using new techniques of joining these structural elements, the selection of proper corrosion protection without losing the required properties of the joint may determine its potential application. This paper presents the results of experimental research concerning the influence of corrosion protection on the microstructure and mechanical strength of resistance spot welded (RSW) and refill friction stir spot welded (RFSSW) joints. The tests were carried out on 2024 T3 aluminum alloy, both sides alcladed. For comparison purposes, the following joints were welded: without any protection, with the primer layer, with anodic oxide coating, and with anodic oxide coating plus sealant between the overlapping surface of the welded metal sheet. The samples were visually inspected, and metallographic and mechanical strength examination was conducted. The test results indicate that the application of the protective layers and its type have an impact on the strength of RSW and RFSSW joints. The use of an adhesive or sealant in welded joints provides an increase in the load capacity of the joint.

Fenofibrate impairs liver function and structure more pronounced in old than young rats.

INTRODUCTION: Since old animals are known to accumulate lipids in some organs, we compared effects of fenofibrate (FN) on systemic lipid metabolism, activity of liver marker enzymes and structure in young and old rats. MATERIAL AND METHODS: Young and old rats were fed chow supplemented with 0.1 % or 0.5 % FN. After 30 days, intraperitoneal glucose tolerance test (IPGTT) was performed, and blood and liver samples were collected. RESULTS: In young rats, 0.1 % FN, but not 0.5 % FN, decreased serum Chol by 74 %, and did not affect TG levels at either doses. In old rats, 0.5 % FN, but not 0.1 % FN, decreased Chol and TG level by 56 % and 49 %, respectively. In young rats, 0.1 % and 0.5 % FN increased serum activity of ALP by 227 % and 260 %, respectively, and did not affect AST and ALT activities. In old rats, only 0.5 % FN increased serum ALP activity by 150 %, respectively. In old rats, neither dose of FN affected serum AST activity, and only 0.5 % FN increased serum ALT activity by 200 %. The histological examination of liver structure revealed that both doses of FN impaired lobular architecture, expansion of bile canaliculi, and degeneration of parenchymal cells with the presence of cells containing fat droplets; administration of FN increased area occupied by collagen fibers. CONCLUSIONS: Although 0.5 % FN decreased serum Chol concentration, it increased serum ALP activity and impaired liver structure in both in both age groups of rats. Thus, FN treatment should be under the control of liver function, especially in older patients.

In vitro generation of pancreatic ß-cells for diabetes treatment. I. ß-like cells derived from human pluripotent stem cells.

Diabetes mellitus is a chronic disease that affects hundreds of millions of people worldwide. Type 1 diabetes (T1D) is characterized by the lack of pancreatic ß-cells that had been destroyed as a result of an autoimmune response. Therefore, in patients with T1D, the replacement therapy with functional ß-cells derived from extrinsic sources could be a preferable option as compared to insulin treatment. Unfortunately, successful transplantation of whole pancreata or pancreatic islets into patients with diabetes is available only to a fraction of them due to the scarcity of donors. The rapid development of cell reprogramming methods made it possible to generate large numbers of human ß-like cells derived from human embryonic stem cells (hESCs) or human induced pluripotent stem cells (hiPSCs). This review describes the basis of in vitro differentiaton protocols of ß-like cells that mimic changes of the main signaling pathways during the key stages of human and murine pancreas development, which are described first. During the last 15 years it was found that there are no important differences between hESCs and hiPSCs in their differentiation capacities into ß-like cells and the expression profiles of the key transcription factors. The in vitro produced ß-like cells are immature as demonstrated by functional tests in rodents and single-cell transcriptomic and proteomic analyses. After the transplantation of the ß cell progenitors into immunocompromised diabetic mice, a few weeks have to pass before the increased insulin levels in response to glucose load appear. There is a continuous progress in the development of open-type encapsulation devices which allow the vascularization of the transplanted cells and protect them against host's immune cells. The results of the first clinical trial of human partially differentiated endocrine progenitors of ß cells transplanted into patients with T1D will be published in the year 2019. It is hoped that further improvements in the techniques of large-scale generation of the ß-like cells derived from human pluripotent stem cells will bring us closer to their clinical application as a form of cause-directed therapy for people with diabetes.

Effects of 5-FU and anti-EGFR antibody in combination with ASA on the spherical culture system of HCT116 and HT29 colorectal cancer cell lines.

The aim of this study was to examine the effects of 5­fluorouracil (5­FU), anti­epidermal growth factor receptor (EGFR) antibody and aspirin (ASA) on the characteristics of two CRC cell lines, HCT116 and HT29, maintained in a spherical culture system. We observed that the morphology of both the HCT116 and HT29 cell­derived spheres was significantly impaired and the size of the colonospheres was markedly reduced following treatment with the aforementioned three drugs. In contrast to adherent cultures, the spherical cultures were more resistant to the tested drugs, as was reflected by their capacity to re­create the colonospheres when sustained in serum­free medium. Flow cytometric analysis of the drug­treated HCT116 cell­derived spheres revealed changes in the fraction of cells expressing markers of cancer stem cells (CSCs), whereas the CSC phenotype of HT29 cell­derived colonospheres was affected to a lesser extent. All reagents enhanced the percentage of non­viable cells in the colonospheres despite the diminished fraction of active caspase­3­positive cells following treatment of the HT29 cell­derived spheres with anti­EGFR antibody. Increased autophagy, assessed by acridine orange staining, was noted following the incubation of the HT29­colonospheres with ASA and 5­FU in comparison to the control. Notably, the percentage of cyclooxygenase (COX)­2­positive cells was not affected by ASA, although its activity was markedly elevated in the colonospheres incubated with anti­EGFR antibody. On the whole, the findings of this study indicate that all the tested drugs were involved in different cellular processes, which suggests that they should be considered for the combined therapeutic treatment of CRC, particularly for targeting the population of CSC­like cells. Thus, cancer cell­derived spheres may be used as a preferable model for in vitro anticancer drug testing.

Prognostic significance of VHL, HIF1A, HIF2A, VEGFA and p53 expression in patients with clear­cell renal cell carcinoma treated with sunitinib as first­line treatment.

Clear cell renal cell carcinoma (ccRCC) is the most common subtype of renal cell cancer, characterized by the highest mortality rate among other RCC subtypes due to the occurrence of metastasis and drug resistance following surgery. The Von Hippel­Lindau tumor suppressor (VHL)­hypoxia­inducible factor 1 subunit α (HIF1A)/hypoxia­inducible factor 2α (HIF2A)­vascular endothelial growth factor A (VEGFA) protein axis is involved in the development and progression of ccRCC, whereas sunitinib, a tyrosine kinase inhibitor, blocks the binding of VEGFA to its receptor. The aim of the present study was to examine the possible association of the gene expression of VHL, HIF1A, HIF2A, VEGFA and tumor protein P53 (P53) in cancer tissue with the outcome of ccRCC patients who were treated with sunitinib as first­line therapy following nephrectomy. A total of 36 ccRCC patients were enrolled, 11 of whom were administered sunitinib post­operatively. Tumor and control samples were collected, and mRNA and protein levels were assessed by reverse transcription­quantitative polymerase chain reaction and western blot analysis, respectively. High mRNA and protein expression levels of HIF2A and VEGFA were found to be associated with shorter overall survival (OS) and progression­free survival (PFS) rates, as well as with unfavorable risk factors of cancer recurrence and mortality. Resistance to sunitinib was also observed; the OS and PFS rates were shorter (median OS and PFS: 12 and 6 months, respectively, vs. undetermined). Sunitinib resistance was associated with high HIF2A and VEGFA protein levels (b=0.57 and b=0.69 for OS and PFS, respectively; P<0.001). Taken together, the findings of this study suggest that the protein levels of HIF2A and VEGFA in tumor tissue may serve as independent prognostic factors in ccRCC. ccRCC patients with increased intratumoral HIF2A and VEGFA protein levels, and unaltered VHL protein levels, are not likely to benefit from sunitinib treatment following nephrectomy; however, this hypothesis requires verification by large­scale replication studies.

The Designer Drug 3-Fluoromethcathinone Induces Oxidative Stress and Activates Autophagy in HT22 Neuronal Cells.

Synthetic cathinones are psychoactive substances, derivatives of a natural psychostimulant cathinone. Although many synthetic cathinones have lost their legal status in many countries, their abuse still continues worldwide. Recently, they have been reported to exert neurotoxic effects in vitro and in vivo. The molecular mechanisms of their action have not been fully elucidated. Recently, they have been linked to the induction of oxidative stress, autophagy, and apoptosis. The aim of this study was to investigate whether 3-fluoromethcathinone (3-FMC), a synthetic cathinone, is able to induce oxidative stress, autophagy, and apoptosis in HT22 immortalized mouse hippocampal cells. We found that treatment of HT22 cells with this compound results in a concentration-dependent increase in the intracellular production of reactive oxygen species. Moreover, 3-FMC induced concentration-dependent conversion of cytosolic LC3-I to membrane-bound LC3-II and formation of autophagic vacuoles. Additionally, the level of p62/SQSTM1 protein decreased after 3-FMC treatment, suggesting that accumulation of autophagic vacuoles resulted from activation rather than inhibition of autophagy. Our results also showed that 3-FMC at millimolar concentration is able to induce caspase-dependent apoptotic cell death in HT22 cells. Our findings suggest that abuse of 3-FMC may disturb neuronal homeostasis and impair functioning of the central nervous system.

Overexpression of the YAP1 oncogene in clear cell renal cell carcinoma is associated with poor outcome.

Clear cell renal cell carcinoma (ccRCC) is the most common subtype of RCC (70-80%). Yes-associated protein 1 (YAP1) protein is a nuclear effector of the Hippo pathway and acts as a transcriptional co-activator of genes involved in the processes of growth and development of tissues. Hippo signaling, with its key kinases, MST2 and large tumor suppressor kinase 1 (LATS1), plays a significant role in the negative regulation of the amount and activity of YAP1 protein. Components of the Hippo pathway and YAP1 levels are frequently dysregulated in a variety of tumors, suggestive of their possible involvement in carcinogenesis. Our aim was to evaluate gene and protein expression profiles of YAP1, MST2 and LATS1 and the methylation status of MST2 and LATS1 promoters in ccRCC. mRNA levels of MST2, LATS1 and YAP1 genes were assessed in the tumor and matched normal kidney tissues of 86 patients, and in 12 samples of local metastases by quantitative PCR (qPCR). Proteins were semi-quantified in 58 patient samples by western blotting. Hypermethylation of LATS1 and MST2 promoters was measured by methylation­specific high­resolution-melting qPCR. We found that LATS1 promoter hypermethylation, decreased LATS1 mRNA/protein and increased YAP1 mRNA/protein levels in tumor samples were associated with higher TNM and Fuhrman's stages and patient survival. Higher YAP1 mRNA levels were associated with poor outcome (HR=4.03, p=0.036). No changes in MST2 (promoter/mRNA/protein) were found. We propose that deregulation of LATS1 and YAP1 expression is associated with ccRCC progression and poor patient survival. Measurement of YAP1 mRNA levels in paired tumor-normal kidney tissue samples may serve as a new prognostic factor in ccRCC.

Decreased expression of p73 in colorectal cancer.

INTRODUCTION: The colorectal cancer (CRC) is one of the most frequent cancer in Poland and worldwide. This disease is characterized by distinct genetic alterations. p73 belongs to the p53 gene family; however, its role in the pathogenesis of CRC has not been completely understood. p73 gene encodes several mRNA variants and protein isoforms with its longest and fully functional p73a (mRNA) and TAp73a (protein) isoform. The aim of the study was to investigate p73 gene expression at the mRNA (p73a) and protein (TAp73a) levels in CRC. MATERIAL AND METHODS: Small sections of the crc tumor tissue and macroscopically unchanged colon mucosa and submucosa from the dissection margin were collected from 23 patients diagnosed with CRC. p73 mRNA levels were measured by Real-time PCR (QPCR) method and the expression level of TAp73a protein was assessed by Western blotting (WB) and immunohistochemical (IHC) staining. RESULTS: We found a 37% decrease in the level of p73a mRNA in neoplastically changed (tumor) compared with unchanged normal colon tissue from the surgical margin (p = 0.041). No correlations were found between mRNA levels in cancer tissue and clinical-pathological parameters. The semi-quantification of TAp73a protein revealed lower and higher TAp73a protein contents in 11/23 and 12/23 of tumor samples, respectively, when compared with the median value of TAp73a protein in normal colon tissue (p = 0.61). The level of TAp73a protein level was 5 times lower in poorly differentiated cancer cells (G3) in comparison to moderately differentiated ones (G2; p = 0.02). No statistically significant correlations were observed between the level of the TAp73a protein and clinical-pathological patients' characteristics. The IHC analysis of TAp73a protein presence in CRC samples showed decreased immunoreactivity when compared with matched sections of the unchanged colon wall in 4/9 patients, similar intensity of the IHC reaction in 4/9 patients and increased immunoreactivity in 1/9 patients. The TAp73a protein was localized mainly in the cytoplasm of the cancer cells. No statistically significant correlations between IHC results and clinical-pathological features of the patients were found. CONCLUSIONS: The obtained results suggest that the p73 gene may play a role as a tumor suppressor in the CRC progression.

Decreased expression of RASSF1A tumor suppressor gene is associated with worse prognosis in clear cell renal cell carcinoma.

Clear-cell renal cell carcinoma (ccRCC) is the most common subtype of RCC (70-80%) and is associated with poor prognosis in 40% of cases mainly due to metastasis in the course of the disease. RASSF1, with its isoforms RASSF1A and RASSF1C, is a tumor suppressor gene which has not been fully analyzed in ccRCC yet. The epigenetic downregulation of RASSF1A is commonly associated with promoter hypermethylation. The aim of the present study was to compare the ccRCC outcomes with the expression of RASSF1A and RASSF1C. Tissues were obtained from 86 ccRCC patients. RASSF1A and RASSF1C mRNA levels were assessed in tumor and matched normal kidney tissue, and in 12 samples of local metastases by quantitative PCR (qPCR). RASSF1A and RASSF1C proteins levels were semi-quantified in 58 samples by western blot analysis and their tissue localization was assessed by immunohistochemistry. Hypermethylation of RASSF1A promoter was measured by high-resolution-melting methylation-specific qPCR. RASSF1A mRNA levels were 4 and 5 times lower in 66% of tumor and 75% metastasized samples. RASSF1A hypermethylation was found in 40% of analyzed T cases. RASSF1A protein expression was 5 or 20 times decreased in 70% tumor and 75% metastatic samples, respectively. RASSF1A hypermethylation, mRNA and protein levels were associated with TNM progression and higher Fuhrman's grading. Decreased RASSF1A expression, hypermethylation, TNM and Fuhrman's grading were associated with poorer overall survival (OS). Cox hazard ratio (HR) analysis revealed predictor role of RASSF1A mRNA levels on OS and progression-free survival (PFS) in relation to Fuhrman's grading (OS HR=2.25, PFS HR=2.93). RASSF1C levels were increased in ccRCC; no correlations with clinicopathological variables were found. We conclude that RASSF1C gene is not involved in ccRCC progression and we propose that the measurements of RASSF1A mRNA levels in paired tumor-normal kidney tissue could serve as a new prognostic factor in ccRCC.