Thioredoxin-interacting protein (TXNIP) inhibits the activity of thioredoxin (TXN), leading to increased oxidative stress. Expression of the TXNIP gene is regulated by DNA methylation. However, no study has reported the influence of lifestyle factors on TXNIP DNA methylation. Our goal was to determine the association between smoking habits and TXNIP DNA methylation levels in a Japanese population. We conducted a cross-sectional study of 417 subjects (180 males and 237 females) participating in a health examination. We used a pyrosequencing assay to determine TXNIP DNA methylation levels in leukocytes. The mean TXNIP DNA methylation level in current smokers (75.3%) was significantly lower than that in never and ex-smokers (never: 78.1%, p < 0.001; ex: 76.9%, p = 0.013). Multivariable logistic regression analyses showed that the OR for TXNIP DNA hypomethylation was significantly higher in current smokers than that in never smokers, and significantly higher in current smokers with years of smoking ≥ 35 and Brinkman Index ≥ 600 compared to that in non-smokers. In conclusion, we found that current smokers had TXNIP DNA hypomethylation compared to never and ex-smokers. Moreover, long-term smoking and high smoking exposure also were associated with TXNIP DNA hypomethylation.

Inflammation is associated with production of reactive oxygen species (ROS) and results in the induction of thioredoxin (TXN) and peroxiredoxins (PRDXs) and activation of nuclear factor-like 2 (Nrf2). In this study we have used the mouse RAW 264.7 macrophage and the human THP-1 monocyte cell line to investigate the pattern of expression of three Nrf2 target genes, PRDX1, TXN reductase (TXNRD1) and heme oxygenase (HMOX1), by activation of different Toll-like receptors (TLRs). We found that, while the TLR4 agonist lipopolysaccharide (LPS) induces all three genes, the pattern of induction with agonists for TLR1/2, TLR3, TLR2/6 and TLR7/8 differs depending on the gene and the cell line. In all cases, the extent of induction was HMOX1>TXNRD1>PRDX1. Since LPS was a good inducer of all genes in both cell lines, we studied the mechanisms mediating LPS induction of the three genes using mouse RAW 264.7 cells. To assess the role of ROS we used the antioxidant N-acetylcysteine (NAC). Only LPS induction of HMOX1 was inhibited by NAC while that of TXNRD1 and PRDX1 was unaffected. These three genes were also induced by phorbol myristate acetate (PMA), a ROS-inducer acting by activation of protein kinase C (PKC). The protein kinase inhibitor staurosporine inhibited the induction of all three genes by PMA but only that of HMOX1 by LPS. This indicates that activation of these genes by inflammatory agents is regulated by different mechanisms involving either ROS or protein kinases, or both.

Scope: Sphingolipids including ceramides are implicated in the pathogenesis of obesity and insulin resistance. Correspondingly, inhibition of pro-inflammatory and neurotoxic ceramide accumulation prevents obesity-mediated insulin resistance and cognitive impairment. Increasing evidence suggests the farnesoid X receptor (FXR) is involved in ceramide metabolism, as bile acid-FXR crosstalk controls ceramide levels along the gut-liver axis. The authors previously reported that FXR agonist xanthohumol (XN), the principal prenylated flavonoid in hops (Humulus lupulus), and its hydrogenated derivatives, α,β-dihydroxanthohumol (DXN), and tetrahydroxanthohumol (TXN), ameliorated obesity-mediated insulin resistance, and cognitive impairment in mice fed a high-fat diet.

Methods and results: To better understand how the flavonoids improve both, lipid and bile acid profiles in the liver are analyzed, sphingolipid relative abundance in the hippocampus is measured, and linked them to metabolic and neurocognitive performance. XN, DXN, and TXN (30 mg kg^-1 BW per day) decrease ceramide content in liver and hippocampus; the latter is linked to improvements in spatial learning and memory. In addition, XN, DXN, and TXN decrease hepatic cholesterol content by enhancing de novo synthesis of bile acids.

Conclusion: These observations suggest that XN, DXN, and TXN may alleviate obesity-induced metabolic and neurocognitive impairments by targeting the liver-brain axis.

Keywords: bile acids; ceramide; cholesterol; obesity.

This study aimed to evaluate the effects of priming on seed germination under salt stress and gene expression in seeds and seedlings of P. angulata L. After priming for 10 days, seed germination was tested in plastic trays containing 15 ml of water (0 dS m-1 - control) or 15 ml of NaCl solution (2, 4, 6, 8, 10, 12, 14 and 16 dS m-1). Fresh and dry weight of shoots and roots of seedlings were evaluated at 0, 2, 4, 6, 8 dS m-1. Total RNA was extracted from whole seeds and seedlings followed by RT-qPCR. The target genes selected for this study were: ascorbate peroxidase (APX), glutathione-S-transferase (GST), thioredoxin (TXN), high affinity potassium transporter protein 1 (HAK1) and salt overly sensitive 1 (SOS1). At an electroconductivity of 14 dS m-1 the primed seeds still germinated to 72%, in contrast with the non-primed seeds which did not germinate. The relative expression of APX was higher in primed seeds and this may have contributed to the maintenance of high germination in primed seeds at high salt concentrations. GST and TXN displayed increased transcript levels in shoots and roots of seedlings from primed seeds. Priming improved seed germination as well as salt tolerance and this is correlated with increased expression of APX in seeds and SOS1, GST and TXN in seedlings.

The genes of top athletes are a valuable genetic resource for the human race, and could be exploited to identify novel genes related to sports ability, as well as other functions. We analyzed the expressed sequence tags from top half-pipe snowboarding athletes using the SMART complementary DNA (cDNA) library construction method to elucidate the characteristics of the athlete genome and the differential expression of the genes it contains. Overall, we established a full-length cDNA library from the lymphocytes of half-pipe snowboarding athletes and analyzed the inserted gene fragments. We also classified those genes according to molecular function, biological characteristics, cellular composition, protein types, and signal paths. A total of 201 functional genes were noted, which were distributed in 27 pathways. TXN, MDH1, ARL1, ARPC3, ACTG1, and other genes measured in sequence may be associated with physical ability. This suggests that the SMART cDNA library constructed from the genetic material from top athletes is an effective tool for preserving genetic sports resources and providing genetic markers of physical ability for athlete selection.

To serve as an initial step in developing an ideal genetic marker map for the house musk shrew, Suncus murinus, 318 comparative anchor tagged sequence (CATS) primer pairs were assessed for polymorphism ascertainment and linkage mapping. Of the 112 (35.2%) CATS primer pairs that were successfully amplified by PCR in the shrew, 18 (16.1%) showed polymorphism between two mutant strains, BAN-kc, oeb and WZ. Linkage analysis of the polymorphic CATS markers and three visible mutant genes, kc, oeb and wz, genotyped in a 77 F2 mapping panel from a cross of the two mutant strains, assigned wz and five CATS markers into three linkage groups. Sequence analysis revealed that two (ADA and TXN) out of nine CATS amplified sequences had a total of six deletions of varying sizes and 17 single nucleotide polymorphisms (SNPs). BLAST search identified three CATS (ADA, CYP1A2, and TXN) products matching the genes from which they were originally designed, while the remaining six markers could not be identified. Together with the use of the detected SNPs as genetic markers, the five CATS markers linkage mapped in this species will serve as anchors in establishing the first framework map for locating loci affecting all heritable qualitative and quantitative traits in the musk shrew.

Expression of thioredoxin-1 (TXN) and CXCL9 is not restricted to THRLBCL macrophages, but may be observed in histiocytes and neoplastic (HRS) cells of EBV + mixed cellularity (MC) classical Hodgkin lymphoma (cHL) and nodular lymphocyte predominant HL. We aimed to validate and extend the above observations in 174 cHL patients evaluating the immunohistochemical expression of TXN, CXCL9 and IFN-γ. HRS-cell CXCL9 expression was higher in latent membrane protein-1 (LMP1)+, MC and Stage IV. TXN and CXCL9 expression by cHL histiocytes was more frequent in LMP1+, MC and older patients (only for CXCL9). TXN expression by HRS cells (≥80%) was independently associated with better failure-free survival. In conclusion, markers of TCHRLBCL histiocytes (TXN, CXCL9), as well as IFN-γ are also expressed by histiocyte subsets and neoplastic cells of cHL. The expression of some of them is more prominent in EBV + MC, but not restricted to this subtype. The prognostic implication of TXN needs further evaluation.

OBJECTIVE

Distant metastasis is a major cause of mortality for patients with lung adenocarcinoma. So far, the mechanism of tumor metastasis is unknown. This study was to screen the gene expression signature in relation to lymphatic metastasis of human lung adenocarcinoma.

METHODS

Primary lung adenocarcinoma tissues and regional lymph nodes were obtained from 22 patients underwent radical resection. The samples were classified into three groups: 11 cases of primary lung adenocarcinoma without lymphatic metastasis (TxN-), 11 cases of primary lung adenocarcinoma with lymphatic metastasis (TxN+), and 11 cases of the corresponding tumor cells from metastatic lymph nodes(N+). Total RNA was extracted from laser microdissected tumor samples. Adequate RNA starting materials from the primary tumors or metastatic nodes were labeled and then hybridized into the same microarray containing 6000 known human genes or expressed sequence tags (ESTs). After scanning, data analyses were performed using GeneSpring 6.2.

RESULTS

Among 17 differentially expressed genes between the TxN+ and TxN-groups, 12 genes were significantly elevated and five genes were significantly downregulated in the TxN+ group compared with the TxN-group. There were 53 differentially regulated genes between the N+ and TxN+ groups, among which 25 genes were overexpressed and 28 genes were suppressed in the N+ group.

CONCLUSIONS

The combination of early oncogenic alterations and later acquisition of a set of genetic alterations may determine the metastatic potential of lung adenocarcinoma.

Nineteen patients with stage D1 (TXN/MO) prostatic adenocarcinomas were treated with radical prostatectomy and adjuvant radiation therapy. The latter was aortoiliac for stage B tumors and aortoiliac plus pelvic for stage C tumors. No hormonal therapy was associated to this. Lymph node invasion was multifocal in 10 of 19 cases, bilateral in 3 cases, and massive with rupture of the capsule and invasion of the fat surrounding the lymph nodes in 3. The maximum follow-up was 17 years, with a minimum follow-up of 1 year. The dosage of the prostatic specific antigen (PSA) has been an essential element of surveillance since 1987. Eight patients died of their cancer, 11 are alive, including 9 alive and healthy (47%). Among the latter, 5 (26%) are alive without any treatment, with a prostatic antigen level around zero at a follow-up of 6 to 12 years. The review of the literature shows that nonsurgical treatments have a palliative effect for stage D1 tumors, and that surgery alone is not always sufficient. Surgery must be followed by radiation therapy, the only modality likely to complete the effect of radical surgery with the same curative purpose.

To define the molecular mechanisms that mediate hyperthermia-induced apoptosis, we performed microarray and computational gene expression analyses. U937 cells, a human myelomonocytic lymphoma cell line, were treated with hyperthermia at 42 degrees C for 90 min and cultured at 37 degrees C. Apoptotic cells ( approximately 15%) were seen 6 h after hyperthermic treatment, and elevated expression of heat shock proteins (HSPs) including Hsp27, Hsp40, and Hsp70 was detected, following the activation of heat shock factor-1. Of the 54,675 probe sets analyzed, 1334 were upregulated and 4214 were downregulated by >2.0-fold in the cells treated with hyperthermia. A non-hierarchical gene clustering algorithm, K-means clustering, demonstrated 10 gene clusters. The gene network U1 or U2 that was obtained from up-regulated genes in cluster I or IX contained HSPA1B, DNAJB1, HSPH1, and TXN or PML, LYN, and DUSP1, and were mainly associated with cellular compromise, and cellular function and maintenance or death, and cancer, respectively. In the decreased gene cluster II, the gene network D1 including CCNE1 and CEBPE was associated with the cell cycle and cellular growth and proliferation. These findings will provide a basis for understanding the detailed molecular mechanisms of apoptosis induced by hyperthermia at 42 degrees C in cells.

In our previous study, five different secretory proteins, including GSN, ADAMTSL4, CALR, PPIA and TXN, have been identified to be associated with the nasopharyngeal carcinoma (NPC) metastasis.In this work, the 5 proteins were further investigated. Bioinformatics analysis suggested that they might play an important role in the process of NPC development.Western blotting analysis showed that all of these 5 targets could be secreted into extracellular by both high metastatic NPC 5-8F cells and non-metastatic NPC 6-10B cells. Except for GSN, the expressions of ADAMTSL4, CALR, PPIA and TXN proteins in extracts of the 5-8F and 6-10B cells were significantly different (P < 0.05). Thus, the expressions of these 4 differentially expressed proteins were further tested in a cohort of NPC tissue specimens. The results indicated that the expression levels of ADAMTSL4 and TXN were highly correlated with the lymph node and distant metastasis (P<0.05) in NPC patients. Moreover, Enzyme-linked immunosorbent assay (ELISA) was used to investigate the concentrations of the ADAMTSL4 and TXN in serum specimens of NPC patients. The results revealed that serum ADAMTSL4 expression level was closely correlated with lymph node metastasis and clinical stage (P<0.05) in NPC patients, and it was able to discriminate metastasis NPC from non-metastasis NPC with a sensitivity of 75.6% and a specificity of 64.7%. The present data show for the first time that the ADAMTSL4 and TXN may be novel and potential biomarkers for predicting the NPC metastasis.Furthermore, the serum ADAMTSL4 could be a potential serum tumor biomarker for prognosis of NPC.

Comparison of protein expression in intestinal and diffuse stomach tumors by 2D gel electrophoresis led to identification of three proteins (SOD2, S100A6, and TXN), which are overexpressed in tumors as compared to normal controls. It was shown, that overexpression of proteins SOD2 and TXN occurs much more frequently in diffuse tumors than in intestinal ones. A control panel of eleven proteins overexpressed in stomach tumors has been selected based on the data of comparative 2D analysis described in the literature. Bioinformatics search for mRNAs encoding proteins from the control panel in Oncomine database (which contains the results of determination of mRNA transcription level in tumor vs. normal samples) demonstrated the coincidence of proteomic and transcriptomic data for seven out of 11 proteins.

Context: HemoHIM is an herbal preparation containing Angelica gigas Nakai (Apiaceae), Cnidium officinale Makino (Umbelliferae), and Paeonia lactiflora Pallas (Paeoniaceae) developed for immune regulation. To date, studies on the antifatigue effects of HemoHIM have not been conducted.

Objective: The antifatigue effects of HemoHIM using models of citrinin and exercise-induced chronic fatigue syndrome were investigated.

Materials and methods: Citrinin-induced L6 skeletal muscle cells were treated with HemoHIM (125, 250, and 500 μg/mL). The antioxidant factors were analysed. ICR mice were divided into four groups (n = 10): control, HemoHIM 250, 500 mg/kg, and creatine 300 mg/kg, respectively. Mice were orally administered HemoHIM or creatine for three weeks; during this time, both rotarod test and forced swimming test (FST) were conducted. The latency time was investigated and antioxidant, antifatigue factors were analysed.

Results: HemoHIM significantly restored reduced antioxidant enzymes (SOD, CAT, Txn, GPx, GSr, and GCLC in HemoHIM 500 μg/mL) compared to the citrinin group in L6 cells. In vivo, HemoHIM significantly improved the latency time (FST; 279.88 ± 50.32 sec, rotarod test; 552.35 ± 23.50 sec in HemoHIM 500 mg/kg). Moreover, the FST-induced reduction in glucose and glutathione significantly increased by 3-fold (HemoHIM 500 mg/kg) and increase in LDH and MDA were significantly inhibited by 1.6, 2.1-fold in the HemoHIM 500 mg/kg compared to the control group.

Keywords: Fatigue; forced swimming test; rotarod test.

Careful analysis and comparison of optical and electrochemical data available in recent literature for multi-thiophene molecular assemblies suggested a few basic rules for the design of structurally simple and easily accessible oligothiophenes endowed with properties not far from those exhibited by much more complex and synthetically demanding architectures. The synthesis and computational investigation of three examples of a class of oligothiophenes (spider-like) tailored according to these indications are reported together with their exhaustive optical and electrochemical characterization. The new compounds (T9 5, T14 6, T19 7) are characterized by a thiophene, a 2,2'-bithiophene and a 2,2',5',2''-terthiophene unit (the spider body) fully substituted with 5-(2,2'-bithiophen)yl pendants (the spider legs). Absorption and electrochemical data are in good agreement and point to a high pi-conjugation level, comparable to those displayed by much larger assemblies. Electrode potential cycling in proximity of the first oxidation peak affords fast and reproducible formation of conducting, highly stable [TXn]m films, mainly consisting of dimers (m=2). Electrooxidation kinetic experiments on deuterium-labelled T9 5, coupled to laser-desorption-ionization mass spectroscopy on the resulting dimer demonstrated that the coupling process is extremely regioselective in the alpha positions of the more conjugated pentathiophene chain. The optical and the electrochemical properties of the films are reported and discussed. A peculiar feature is their impressive charge-trapping ability. Spider-like oligothiophenes are promising materials for applications as active layers in multifunctional organic devices.

Background: Oxidative stress induced by nutritional overload has been linked to the pathogenesis of insulin resistance, which is associated with metabolic syndrome, obesity, type 2 diabetes and diabetic vascular complications. Postprandial changes in expression of oxidative stress pathway genes in obese vs. lean individuals, following intake of different types of meals varying in macronutrient composition have not been characterized to date. Here we aimed to test whether/how oxidative stress responses in obese vs. lean individuals are modulated by meal composition. Methods: High-carbohydrate (HC), high-fat (HF), or high-protein (HP) liquid mixed meals were administered to study subjects (lean insulin-sensitive, n = 9 and obese insulin-resistant, n = 9). Plasma levels of glucose and insulin, lipid profile, urinary F₂-isoprostanes (F₂-IsoP), and expression levels of genes of oxidative stress pathways were assessed in mononuclear cells (MNC) derived from fresh peripheral blood, at baseline and up to 6-h postprandial states. Differences in these parameters were compared between insulin-sensitive/resistant groups undergoing aforementioned meal challenges. Results: Obese individuals exhibited increased pro-oxidant (i.e., CYBB and CYBA) and anti-oxidant (i.e., TXN RD1) gene expression in the postprandial state, compared with lean subjects, regardless of meal type (P interaction for group × time < 0.05). By contrast, lean subjects had higher expression of NCF-4 gene (pro-oxidant) after HC meal and SOD1 gene (anti-oxidant) after HC and HF meals (P interaction for group × meal < 0.05). There was an increase in postprandial level of urinary F₂-IsoP in the obese (P < 0.05) but not lean group. Conclusions: These findings may represent an adaptive oxidative response to mitigate increased stress induced by acute nutritional excess. Further, the results suggest an increased predisposition of obese subjects to oxidative stress. Chronic nutritional excess resulting in increases in body weight and adiposity might lead to decompensation leading to worsening insulin resistance and its sequel. Insights from this study could impact on nutritional recommendations for obese subjects at high-risk of cardiovascular diseases.

The analgesic effects of alcohol infusion of the pituitary, high-dose progesterone and their association on chronic intractable pain in TxN+M+ breast cancer patients no longer responsive to combination chemotherapy, radiotherapy and hormone therapy are assessed. The antalgic effect of medroxyprogesterone proved to be more lasting than that of alcohol infusion of the pituitary. Moreover, 6 of the 20 patients treated with the former presented regression of the disease whereas none of those treated with alcohol did so.

Late allograft failure (LAF) is a common cause of end stage renal disease. These patients face interrelated challenges regarding immunosuppression management, risk of graft intolerance syndrome (GIS), and sensitization. This retrospective study analyzes sensitization, pathology, imaging, and transfusion requirements in 33 LAFs presenting either with GIS (22) or grafts remaining quiescent (11). All patients underwent immunosuppression weaning to discontinuation at LAF. Profound increases in sensitization were noted for all groups and occurred in the GIS group prior to transplant nephrectomy (TxN). Patients with GIS experienced a major upswing in sensitization at, or before the time of their symptomatic presentation. For both GIS and quiescent grafts, sensitization appeared to be closely linked to immunosuppression withdrawal. Most transfusion naïve patients became highly sensitized. Fourteen patients in the GIS group underwent TxN which revealed grade II acute cellular rejection or worse, with grade 3 chronic active T-cell-mediated rejection. Blinded comparisons of computed tomography scan of GIS group revealed swollen allografts with fluid collections compared with the quiescent allografts (QAs), which were shrunken and atrophic. The renal volume on imaging and weight of explants nearly matched. Future studies should focus on interventions to avoid sensitization and GIS.

Polysaccharides including β-glucans are important bioactive components of mushroom. Xylaria nigripes is a popular medicinal fungus that has been used for treating trauma, insomnia and mental illness. This study examined the physicochemical characteristics and anti-inflammatory activities of water soluble non-digestible polysaccharides (TXNP and CXNP) from fruiting bodies of two cultivated X. nigripes strains (TXN and CXN). Results showed that both TXNP and CXNP possessed relatively similar FT-IR spectra. TXNP had a triple helix conformation and molecular weight of 853.8 kDa, whereas the molecular weight of CXNP was 14.7 kDa. The monosaccharide composition of TXNP was predominantly glucose, whereas CXNP contained xylose, mannose and glucose. Although both TXNP and CXNP dose-dependently suppressed the production of NO, IL-1β, TNF-α and PGE2, as well as the expression of iNOS, COX-2 and NF-κB in the lipopolysaccharide-induced RAW264.7 macrophages, the potency of TXNP was stronger. This study reveals that under similar conditions of cultivation and extraction procedures, the different physicochemical characteristics of polysaccharides from TXN and CXN may have contributed to the differences in their anti-inflammatory potency.

Elevated birth weight is linked to glucose intolerance and obesity health-related complications later in life. No studies have examined if infant birth weight is associated with gene expression markers of obesity and inflammation in a tissue that comes directly from the infant following birth. We evaluated the association between birth weight and gene expression on fetal programming of obesity. Foreskin samples were collected following circumcision, and gene expression analyzed comparing the 15% greatest birth weight infants (n=7) v. the remainder of the cohort (n=40). Multivariate linear regression models were fit to relate expression levels on differentially expressed genes to birth weight group with adjustment for variables selected from a list of maternal and infant characteristics. Glucose transporter type 4 (GLUT4), insulin receptor substrate 2 (IRS2), leptin receptor (LEPR), lipoprotein lipase (LPL), low-density lipoprotein receptor-related protein 1 (LRP1), matrix metalloproteinase 2 (MMP2), plasminogen activator inhibitor-1 (PAI-1) and transcription factor 7-like 2 (TCF7L2) were significantly upregulated and histone deacetylase 1 (HDAC1) and thioredoxin (TXN) downregulated in the larger birth weight neonates v.

METHODS

Multivariate modeling revealed that the estimated adjusted birth weight group difference exceeded one standard deviation of the expression level for eight of the 10 genes. Between 25 and 50% of variation in expression level was explained by multivariate modeling for eight of the 10 genes. Gene expression related to glycemic control, appetite/energy balance, obesity and inflammation were altered in tissue from babies with elevated birth weight, and these genes may provide important information regarding fetal programming in macrosomic babies.

Traumatic optic neuropathy is an important cause of severe vision loss. So, many attempts were performed to transplant stem cells systemically or locally to regenerate the injured retina. In this study, we investigated the effect of human umbilical cord blood mesenchymal stem cells (hUBMSCs) on histological structure, apoptotic, antiapoptotic, oxidant and antioxidant markers in an experimental model of cryo-induced retinal damage in mice. Forty-eight mice were included with 4 major groups; group I contained 18 mice as controls. The others included 30 mice exposed to cryo-induced retinal injury and were subdivided into three equal groups: group II received no treatment after injury. Group III was intravenously injected with hUCBMSCs after injury and group IV received an intravitreal injection with hUCBMSCs into both eyes. Retinal tissues were used for histopathological, immunological and gene expression studies. Real time-PCR was performed to assess B-cell lymphoma 2 (bcl2), Bcl2-associated X protein (bax), heme oxygenase-1 (hmox-1) and thioredoxin-2 (tnx-2) expression and to assess the differentiation of the stem cells into the retinal tissue. Immunohistochemical analysis was performed to assess caspase-3, 3-nitrotyrosine (3-NT) and basic fibroblast growth factor (bFGF). Disturbed retinal structure was seen in cryo-injured mice while hUCBMSCs treated groups showed nearly normal structure. By real time-PCR, significantly reduced mRNA expressions of Bax and notably enhanced mRNA expression of Bcl-2, hmox-1 and txn-2 were demonstrated in retinal injured mice with hUCBMSCs treatment compared to those without. In addition, immunohistochemical analysis confirmed downregulation of 3-NT and caspase-3 and upregulation of bFGF after hUCBMSCs injection in injured retina. Furthermore, there was no differentiation of transplanted stem cells into the retinal tissue. In conclusions, hUCBMSCs could improve the morphological retinal structure in cryo-induced retinal damage model by modulation of the oxidant-apoptotic status and by increased the expression of bFGF. © 2017 IUBMB Life, 69(3):188-201, 2017.

Withaferin A (WFA), the Indian ginseng bioactive compound, exhibits an antiproliferation effect on several kinds of cancer, but it was rarely reported in bladder cancer cells. This study aims to assess the anticancer effect and mechanism of WFA in bladder cancer cells. WFA shows antiproliferation to bladder cancer J82 cells based on the finding of the MTS assay. WFA disturbs cell cycle progression associated with subG1 accumulation in J82 cells. Furthermore, WFA triggers apoptosis as determined by flow cytometry assays using annexin V/7-aminoactinomycin D and pancaspase detection. Western blotting also supports WFA-induced apoptosis by increasing cleavage of caspases 3, 8, and 9 and poly ADP-ribose polymerase. Mechanistically, WFA triggers oxidative stress-association changes, such as the generation of reactive oxygen species and mitochondrial superoxide and diminishment of the mitochondrial membrane potential, in J82 cells. In response to oxidative stresses, mRNA for antioxidant signaling, such as nuclear factor erythroid 2-like 2 (NFE2L2), catalase (CAT), superoxide dismutase 1 (SOD1), thioredoxin (TXN), glutathione-disulfide reductase (GSR), quinone dehydrogenase 1 (NQO1), and heme oxygenase 1 (HMOX1), are overexpressed in J82 cells. In addition, WFA causes DNA strand breaks and oxidative DNA damages. Moreover, the ROS scavenger N-acetylcysteine reverts all tested WFA-modulating effects. In conclusion, WFA possesses anti-bladder cancer effects by inducing antiproliferation, apoptosis, and DNA damage in an oxidative stress-dependent manner.

Keywords: DNA damage; Withaferin A; apoptosis; bladder cancer; oxidative stress.

Each cell contains many large DNA polymers packed in a nucleus of approx. 10 μm in diameter. With histones, these DNA polymers are known to form chromatins. How chromatins further compact in the nucleus is unclear but it inevitably depends on an extensive non-chromatin nuclear scaffold. Imaging of endogenous chromatin network and the complementary scaffold that support this network has not been achieved but biochemical and proteomic investigations of the scaffold can still provide important insights into this chromatin-organizing network. However, this demands highly inclusive and reproducible extraction of the nuclear scaffold. We have recently developed a simple protocol for releasing the scaffold components from chromatins. The inclusiveness of the extract was testified by the observation that, upon its extraction from the nuclei, the remaining nuclear chromatins were liberated into extended and often parallel chromatin fibers. Basically, this protocol includes the generation of pure nuclei, treatment of the nuclei with Triton X-100 to generate envelope-depleted nuclei (TxN), and extraction of the nuclei at 500 mM NaCl in a sucrose-containing buffer. This combined extract of TxN is known as TxNE.

Keywords: Chromatin; Extract; Nuclei; Nucleophosmin-1; Scaffold.