Pediatric acute lymphoblastic leukemia (ALL) is the most common childhood cancer and the leading cause of cancer-related mortality in children. T cell ALL (T-ALL) represents about 15% of pediatric ALL cases and is considered a high-risk disease. T-ALL is often associated with resistance to treatment, including steroids, which are currently the cornerstone for treating ALL; moreover, initial steroid response strongly predicts survival and cure. However, the cellular mechanisms underlying steroid resistance in T-ALL patients are poorly understood. In this study, we combined various genomic datasets in order to identify candidate genetic mechanisms underlying steroid resistance in children undergoing T-ALL treatment.

Extracellular matrix (ECM) increase in diabetic nephropathy (DN) is closely related to mitochondrial dysfunction. The mechanism of protective function of mitofusin 2 (Mfn2) for mitochondria remains largely unknown. In this study, the molecular mechanisms for the effect of Mfn2 on mitochondria and subsequent collagen IV expression in DN were investigated. Ras-binding-deficient mitofusin 2 (Mfn2-Ras(Δ)) were overexpressed in rat glomerular mesangial cells, and then the cells were detected for mitochondrial morphology, cellular reactive oxygen species (ROS), mRNA and protein expression of collagen IV with advanced glycation end-product (AGE) stimulation. Preliminary results reveal that the mitochondrial dysfunction and the increased synthesis of collagen IV after AGE stimulation were reverted by Mfn2-Ras(Δ) overexpression. Bioinformatical computations were performed to search transcriptional factor motifs in the promoter region of collagen IV. Three specific regions for TFAP2A binding were identified, followed by validation with chromatin immunoprecipitation experiments. Knocking down TFAP2A significantly decreased the TF binding in the first two regions and the gene expression of collagen IV. Furthermore, results reveal that Mfn2-Ras(Δ) overexpression significantly mitigated TFAP2A binding and also reverted the histone acetylation at Regions 1 and 2 after AGE stimulation. In streptozotocin-induced diabetic rats, Mfn2-Ras(Δ) overexpression also ameliorated glomerular mesangial lesions with decreased collagen IV expression, accompanied by decreased acetylation and TFAP2A binding at Region 1. In conclusion, this study highlights the pathway by which mitochondria affect the histone acetylation of gene promoter and provides a new potential therapy approach for DN.

A new targeting chemotherapeutic agent, Pt-Mal-LHRH, was synthesized by linking activated cisplatin to luteinizing hormone releasing hormone (LHRH). The compound's efficacy and selectivity toward 4T1 breast cancer cells were evaluated. Carboplatin was selected as the comparative platinum complex, since the Pt-Mal-LHRH malonate linker chelates platinum in a similar manner to carboplatin. Breast cancer and normal cell viability were analyzed by an MTT assay comparing Pt-Mal-LHRH with carboplatin. Cells were also treated with either Pt-Mal-LHRH or carboplatin to evaluate platinum uptake by ICP-MS and cell migration using an in vitro scratch-migration assay. Tumor volume and metastasis were evaluated using an in vivo 4T1 mouse tumor model. Mice were administered Pt-Mal-LHRH (carboplatin molar equivalent dosage) through ip injection and compared to those treated with carboplatin (5 (mg/kg)/week), no treatment, and LHRH plus carboplatin (unbound) controls. An MTT assay showed a reduction in cell viability (p < 0.01) in 4T1 and MDA-MB-231 breast cancer cells treated with Pt-Mal-LHRH compared to carboplatin. Pt-Mal-LHRH was confirmed to be cytotoxic by flow cytometry using a propidium iodide stain. Pt-Mal-LHRH displayed a 20-fold increase in 4T1 cellular uptake compared to carboplatin. There was a decrease (p < 0.0001) in 4T1 cell viability compared to 3T3 normal fibroblast cells. Treatment with Pt-Mal-LHRH also resulted in a significant decrease in cell-migration compared to carboplatin. In vivo testing found a significant reduction in tumor volume (p < 0.05) and metastatic tumor colonization in the lungs with Pt-Mal-LHRH compared to carboplatin. There was a slight decrease in lung weight and no difference in liver weight between treatment groups. Together, our data indicate that Pt-Mal-LHRH is a more potent and selective chemotherapeutic agent than untargeted carboplatin.

It has been shown that calcitriol and all-trans retinoic acid (ATRA) have modulatory effects on the immune system. The present study investigates the synergistic effects of combination treatment of calcitriol and ATRA in experimental autoimmune encephalomyelitis (EAE), an animal model of multiple sclerosis (MS).

Curcumin has been shown to have a wide variety of therapeutic effects, ranging from anti-inflammatory, chemopreventive, anti-proliferative, and anti-metastatic. This review provides an overview of the recent research conducted to overcome the problems with the bioavailability of curcumin, and of the preclinical and clinical studies that have reported success in combinatorial strategies coupling curcumin with other treatments. Research on the signaling pathways that curcumin treatment targets shows that it potently acts on major intracellular components involved in key processes such as genomic modulations, cell invasion and cell death pathways. Curcumin is a promising molecule for the prevention and treatment of cancer.

Although chronic lymphocytic leukemia (CLL) is the most common adult leukemia in Western world, it remains incurable with conventional chemotherapeutic agents. Tumor necrosis factor (TNF)-related apoptosis-inducing ligand (TRAIL) is an antitumor candidate in cancer therapy. This study examines the proapoptotic effects of poly(propylene imine) (PPI) glycodendrimers modified with the maltotriose residues (PPI-G4-OS-Mal-III and PPI-G4-DS-Mal-III) on the TNF family in CLL cells. The combination of an understanding of the signaling pathways associated with CLL and the development of a molecular profiling is a key issue for the design of personalized approaches to therapy. Gene expression is determined with two-color microarray 8 × 60K. The findings indicate that PPI-G4-OS/DS-Mal-III affect gene expression from the TRAIL apoptotic pathway and exert a strong effect on CLL cells comparable with fludarabine. Dendrimer-targeted technology may well prove to bridge the gap between the ineffective treatment of today and the effective personalized therapy of the future.

Hemorrhagic cystitis is an inflammatory and ulcerative bladder condition associated with systemic chemotherapeutics, like cyclophosphomide. Earlier, we reported reactive oxygen species resulting from cyclophosphamide metabolite, acrolein, causes global methylation followed by silencing of DNA damage repair genes. Ogg1 (8-oxoguanine DNA glycosylase) is one such silenced base excision repair enzyme that can restore DNA integrity. The accumulation of DNA damage results in subsequent inflammation associated with pyroptotic death of bladder smooth muscle cells. We hypothesized that reversing inflammasome-induced imprinting in the bladder smooth muscle could prevent the inflammatory phenotype. Elevated recruitment of Dnmt1 and Dnmt3b to the Ogg1 promoter in acrolein treated bladder muscle cells was validated by the pattern of CpG methylation revealed by bisulfite sequencing. Knockout of Ogg1 in detrusor cells resulted in accumulation of reactive oxygen mediated 8-Oxo-dG and spontaneous pyroptotic signaling. Histone deacetylase (HDAC) inhibitor, suberoylanilide hydroxamic acid (SAHA), restored Ogg1 expression in cells treated with acrolein and mice treated with cyclophosphamide superior to the standard of care, mesna or nicotinamide-induced DNA demethylation. SAHA restored cyclophosphamide-induced bladder pathology to that of untreated control mice. The observed epigenetic imprinting induced by inflammation suggests a new therapeutic target for the treatment of hemorrhagic cystitis.

Alzheimer's disease is most common neurodegenerative disorder and is characterized by increased production of soluble amyloid-β oligomers, the main toxic species predominantly formed from aggregation of monomeric amyloid-β (Aβ). Increased production of Aβ invokes a cascade of oxidative damages to neurons and eventually leads to neuronal death. This study was aimed to investigate the neuroprotective effects of a β-sheet breaker α/β-hybrid peptide (BSBHp) and the underlying mechanisms against Aβ40 -induced neurotoxicity in human neuroblastoma SH-SY5Y cells. Cells were pretreated with the peptide Aβ40 to induce neurotoxicity. Assays for cell viability, cell membrane damage, cellular apoptosis, generation of reactive oxygen species (ROS), intracellular free Ca2+ , and key apoptotic protein levels were performed in vitro. Our results showed that pretreatment with BSBHp significantly attenuates Aβ40 -induced toxicity by retaining cell viability, suppressing generation of ROS, Ca2+ levels, and effectively protects neuronal apoptosis by suppressing pro-apoptotic protein Bax and up-regulating antiapoptotic protein Bcl-2. These results suggest that α/β-hybrid peptide has neuroprotective effects against Aβ40 -induced oxidative stress, which might be a potential therapeutic agent for treating or preventing neurodegenerative diseases.

Abnormal expression and dysfunction of methyl-CpG binding protein 2 (MeCP2) cause Rett syndrome (RTT). The diverse phosphorylation modifications modulate MeCP2 function in neural cells. Using western blot and immunohistochemistry, we examined the expression patterns of MeCP2 and three phospho-MeCP2s (pMeCP2s) in the developing rat brain. The expression of MeCP2 and phospho-S80 (pS80) MeCP2 increased while pS421 MeCP2 and pS292 MeCP2 decreased with brain maturation. In contrast to the nuclear localization of MeCP2 and pS80 MeCP2, pS421 MeCP2 and pS292 MeCP2 were mainly expressed in the cytoplasmic compartment. Apart from their distribution in neurons, they were also detected at a low level in astrocytes. Postnatally-initiated MeCP2 deficiency affected cerebellar neural cell development, as determined by the abnormal expression of GFAP, DCX, Tuj1, MAP-2, and calbindin-D28k. Together, these results demonstrate that MeCP2 and diverse pMeCP2s have distinct features of spatio-temporal expression in the rat brain, and that the precise levels of MeCP2 in the postnatal period are vital to cerebellar neural cell development.

Oxytocin is a neuropeptide widely expressed in the brain. Oxytocin plays a role in both proliferation and differentiation of various cells. Previous studies have suggested that oxytocin could affect the morphology of neuronal cells, therefore the objective of the present study was to test whether (1) oxytocin receptor stimulation/inhibition by specific ligands may change cell morphology and gene expression of selected cytoskeletal proteins (2) oxytocin receptor silencing/knockdown may decrease the length of cell projections (3) oxytocin receptor knockdown may affect human glioblastoma U-87MG cell survival. We confirmed the stimulatory effect of retinoic acid (10 µM) and oxytocin (1 µM) on projection growth. The combination of retinoic acid (10 µM) and oxytocin receptor antagonist (L-371,257, 1 µM) decreased projections length. Contrary to our assumptions, oxytocin receptor silencing did not prevent stimulation of length of projection by retinoic acid. Retinoic acid's and oxytocin's stimulation of projections length was significantly blunted in U-87MG cells with oxytocin receptor knockdown. Cell viability was significantly decreased in U-87MG cells with oxytocin receptor knockdown. Significantly higher levels of mRNA for cytoskeletal proteins drebrin and vimentin were observed in response to oxytocin incubation for 48 h. The data obtained in the present study clearly show that oxytocin induces formation and elongation of cell projections in astrocyte-like U-87MG cells. The effect is mediated by oxytocin receptors and it is accompanied by an increase in gene expression of drebrin and vimentin. Thus, oxytocin receptor signaling, particularly in the glial cells, may play an important role in native cell life, differentiation processes, and tumor progression, as well.

We investigated the effects of sildenafil citrate (SC) on podocyturia in N ω-nitro-L-arginine methyl ester hydrochloride (L-NAME) model of pre-eclampsia (PE). One hundred and twenty Sprague-Dawley rats (SDR) were divided into five groups like pregnant control (PC), early-onset PE (EOPE), late-onset PE(LOPE), early and late-onset PE with SC-treated groups [EOPE (SC); LOPE (SC)]. PE was induced in SDR by oral administration of L-NAME in drinking water for 4-8 days for EOPE and 8-14 day for LOPE. The blood pressure, urine volume and total urine protein were increased in EOPE and LOPE groups when compared to PC, and all the above parameters decreased in EOPE (SC) and LOPE (SC) groups when compared to EOPE and LOPE groups, respectively. The EOPE and LOPE groups showed an increase in urinary nephrin mRNA and podocin mRNA levels compared to PC group. Increases in serum and renal soluble fms-like tyrosine kinase-1 (sFlt-1) expression levels and decreases in renal vascular endothelial growth factor (VEGF) expression and serum placenta growth factor (PlGF) levels were observed in EOPE and LOPE groups when compared to PC group. In addition, decreases in serum and renal sFlt-1 expression levels and increases in renal VEGF expression and serum PlGF levels were observed in EOPE (SC) and LOPE (SC) groups when compared to EOPE and LOPE groups, respectively. The light microscopy showed that the renal tissue of L-NAME-treated rats had extensive glomerular damage, tubular damage and infiltration by mononuclear cells when compared to PC group. Therefore, SC ameliorated podocyturia through its effects on the antiangiogenic/angiogenic status in this animal model.

Although high-density lipoprotein is atheroprotective, it can become dysfunctional in chronic inflammatory conditions and increase cardiovascular risk. We previously demonstrated that HDL from subjects with documented coronary artery disease is dysfunctional and is pro-oxidant/proinflammatory in macrophages. Here we examined the influence of dysfunctional/proinflammatory HDL (piHDL) on lipid accumulation in human macrophages, in comparison to functional HDL (nHDL). Exposure of macrophages to piHDL, in contrast to nHDL, resulted in oxidative stress and marked uptake of lipids from piHDL, leading to the formation of foam cell phenotype as noted by oil red O staining with concomitant increase in total cellular cholesterol content. Using western blotting, we identified that piHDL profoundly upregulated the expression of scavenger receptor CD36 and suppressed the expression of ABCG1 and SRB1 in macrophages, thereby facilitating cholesterol influx capacity of macrophages. We then identified that CD36 did not act alone, indeed it was activated in macrophages along with ERK/MAPK, in response to piHDL, which in turn led to lipid accumulation as well as proinflammatory response via activation of NFkB and subsequent release of proinflammatory markers-TNF-ά and MMP-9. These effects were confirmed using pharmacological inhibitors for either CD36 or ERK/MAPK. Furthermore, piHDL treatment moderately activated PPAR-γ and Nrf2, the known regulators of CD36 in macrophages, suggesting that the two forms of HDL differentially regulate CD36 expression. Taken together, the results demonstrate that a novel CD36-ERK/MAPK-dependent mechanism is involved in macrophage lipid accumulation by piHDL, there by revealing the importance of functional deficiency in HDL and its potential link to atherogenesis.

This study is to investigate the role of adenovirus 36 (Ad36) in regulating expression of peroxisome proliferator-activated receptor γ (PPARγ) and cell death-inducing DFFA-like effector c (CIDEC) in Ad36-induced adipocyte differentiation. Human adipose-derived mesenchymal stem cells (hAMSCs) were isolated and cultured, and then infected with Ad36. Ad36-induced adipocytes were identified using quantitative real-time PCR and Oil red O staining. The expression levels of PPARγ and CIDEC in Ad36-induced adipocytes were determined by quantitative real-time PCR and Western blot analysis. Glucose uptake and intracellular triglyceride content were also determined in these induced cells. Our results from the Oil red O staining showed that Ad36 induced the differentiation of hAMSCs into human adipocytes in vitro. Moreover, the medium glucose concentration was significantly decreased, while the intracellular triglyceride content was significantly increased, in the Ad36-induced adipocytes, compared with the control group. Furthermore, our results showed that, the mRNA and protein expression levels of PPARγ and CIDEC were significantly upregulated in Ad36-induced adipocytes, in a time-dependent manner. On the other hand, compared with the control group, the CIDEC expression was downregulated when the Ad36-induced adipocytes were treated with the PPARγ inhibitor, GW9662. Ad36 could upregulate the expression level of CIDEC through increasing PPARγ expression during the adipocyte differentiation process.

Many studies of the toxic effects of zinc oxide nanoparticles (ZnO NPs) in aquatic organisms have been performed because of increasing ZnO NP use. However, the toxicological pathways are not understood. In this study, ZnO NPs were found to be more toxic than ZnSO4 to zebrafish larvae, but ZnO NP toxicity did not involve transcript alterations. Biological processes affected by ZnO NPs and ZnSO4 were investigated by performing ingenuity pathway analysis on differently expressed genes in larvae exposed to sub-lethal ZnO NP and ZnSO4 concentrations. We identified upregulated and downregulated differently expressed genes in fish exposed to ZnO NPs and ZnSO4, and found that ZnO NPs slightly induced cell differentiation and pathways associated with the immune system and activated several key genes involved in cancer cell signaling. The results may be key to predicting and elucidating the mechanisms involved in ZnO NP and ZnSO4 toxicity in zebrafish larvae.

Purpose: To identify the molecular signature associated with abiraterone acetate (AA) response and mechanisms underlying AA resistance in castration-resistant prostate cancer patient-derived xenografts (PDXs).Experimental Design: SCID mice bearing LuCaP 136CR, 77CR, 96CR, and 35CR PDXs were treated with AA. Tumor volume and prostate-specific antigen were monitored, and tumors were harvested 7 days after treatment or at end of study for gene expression and immunohistochemical studies.Results: Three phenotypic groups were observed based on AA response. An ultraresponsive phenotype was identified in LuCaP 136CR with significant inhibition of tumor progression and increased survival, intermediate responders LuCaP 77CR and LuCaP 96CR with a modest tumor inhibition and survival benefit, and LuCaP 35CR with minimal tumor inhibition and no survival benefit upon AA treatment. We identified a molecular signature of secreted proteins associated with the AA ultraresponsive phenotype. Upon resistance, AA ultraresponder LuCaP 136CR displayed reduced androgen receptor (AR) signaling and sustainably low nuclear glucocorticoid receptor (nGR) localization, accompanied by steroid metabolism alteration and epithelial-mesenchymal transition phenotype enrichment with increased expression of NF-κB-regulated genes; intermediate and minimal responders maintained sustained AR signaling and increased tumoral nGR localization.Conclusions: We identified a molecular signature of secreted proteins associated with AA ultraresponsiveness and sustained AR/GR signaling upon AA resistance in intermediate or minimal responders. These data will inform development of noninvasive biomarkers predicting AA response and suggest that further inhibition along the AR/GR signaling axis may be effective only in AA-resistant patients who are intermediate or minimal responders. These findings require verification in prospective clinical trials. Clin Cancer Res; 23(9); 2301-12. ©2016 AACR.

Although proteasome inhibitors (PIs) are used as anticancer drugs to treat various cancers, their relative therapeutic efficacy on stem cells vs. bulk cancers remains unknown. Here, we show that stem cells derived from gliomas, GSCs, are up to 1,000-fold more sensitive to PIs (IC50, 27-70 nM) compared with their differentiated controls (IC50, 47 to »100 μM). The stemness of GSCs correlates to increased ubiquitination, whose misregulation readily triggers apoptosis. PI-induced apoptosis of GSCs is independent of NF-κB but involves the phosphorylation of c-Jun N-terminal kinase as well as the transcriptional activation of endoplasmic reticulum (ER) stress-associated proapoptotic mediators. In contrast to the general notion that ER stress-associated apoptosis is signaled by prolonged unfolded protein response (UPR), GSC-selective apoptosis is instead counteracted by the UPR ATF3 is a key mediator in GSC-selective apoptosis. Pharmaceutical uncoupling of the UPR from its downstream apoptosis sensitizes GSCs to PIs in vitro and during tumorigenesis in mice. Thus, a combinational treatment of a PI with an inhibitor of UPR-coupled apoptosis may enhance targeting of stem cells in gliomas.

Alcohol dependence is associated with deficits in glutamate uptake and impairment of glutamate homeostasis in different brain reward regions. Glutamate transporter subtype 1 (GLT-1), cystine-glutamate exchanger (xCT) and glutamate/aspartate transporter (GLAST) are one of the key players in regulating extracellular glutamate concentration in the brain. Parenteral treatment with ceftriaxone, β-lactam antibiotic, has been reported to attenuate ethanol consumption and reinstatement to cocaine-seeking behavior, in part, by restoring the expression of GLT-1 and xCT in mesocorticolimbic brain regions in rats. In this study, we focused to test Augmentin (amoxicillin/clavulanate), which can be administered orally to subjects. Therefore, we examined the effects of orally administered Augmentin on ethanol intake as well as GLT-1, xCT and GLAST expression in male alcohol-preferring (P) rats. We found that orally administered Augmentin significantly attenuated ethanol consumption in P rats as compared to the vehicle-treated group. Importantly, the attenuation in ethanol consumption was associated with a significant upregulation of GLT-1 and xCT expression in nucleus accumbens (NAc) and prefrontal cortex (PFC). There was no effect of orally administered Augmentin on GLAST expression in either NAc or PFC. These findings present strong evidence that oral administration of Augmentin can be used as an alternative to parenteral treatment.

The present study reports the evaluation of immunomodulatory and therapeutic potential of a purified Aspergillus panamensis lectin. The immunomodulatory potential of the purified lectin was determined in swiss albino mice by studying its effect on anaphylaxis reaction, arthus reaction, respiratory burst activity, nitric oxide production and quantification of cytokine levels. The therapeutic potential of the lectin was evaluated in male wistar rat models by studying its curative effect on ulcerative colitis. The purified lectin inhibited systemic anaphylaxis and arthus reaction. It enhanced the functional ability of macrophages which was evident from increase in reduction of nitroblue tetrazolium dye and nitric oxide production. It also stimulated the production of Th-1 cytokine IFN-γ and Th-2 cytokine IL-6. Maximum immunomodulatory effect was seen at lectin concentration of 1.5mg/kg body weight. The lectin also showed curative effect against trinitrobenzene sulphonic acid induced ulcerative colitis. The results of this study adequately reflect the role of purified A. panamensis lectin in improving the immune status of mice models. They also show the effect of lectin in reducing the severity of incidence and decrease in clinical symptoms of ulcerative colitis.

The purpose of this study was to investigate the short-term effects of a single oral dose of T-2 and HT-2 toxin at 0.15, 0.33 and 1.82 mg kg-1 body weight, or deoxynivalenol (DON) and 15-acetyl-DON at 0.13, 0.31 and 1.75 mg kg-1 body weight in common carp. Conjugated dienes and trienes (the early markers of lipid peroxidation) were elevated in all DON-treated groups at the 16th hour, while thiobarbituric acid reactive substances (TBARS; termination marker) were increased at the highest dose of DON at the 16th and 24th hours. T-2 toxin did not cause changes in these parameters. Glutathione content and glutathione peroxidase activity showed higher levels at the 16th hour as the effect of both mycotoxins. The expression of glutathione peroxidase (GPx4) genes (gpx4a and gpx4b) revealed a dual response. Downregulation was observed at the 8th hour, followed by an induction at the 16th hour, at the lowest dose of both mycotoxins. Higher doses revealed long-drawn emergence and an elevation was observed only at the 24th hour. However, at the lowest and highest doses of DON or T-2 toxin the changes in gene expression were delayed, which may be related to the low oxidative stress response, as suggested by the expression profiles of the nrf2, keap1, gpx4a and gpx4b genes.

The aim of the present study was to investigate the effects of butyrate as a feed supplement on the expression of insulin signalling proteins as potent regulators of metabolism and growth in Ross 308 broiler chickens fed maize- or wheat-based diets. Both diets were supplemented with non-protected butyrate (1.5 and 3.0 g/kg of diet, respectively) or with protected butyrate (0.2 g/kg of diet); the diet of the control groups was prepared without any additives (control). On day 42 of life, systemic blood samples were drawn for analyses of glucose and insulin concentrations, and tissue samples (liver, gastrocnemius muscle and subcutaneous adipose tissue) were taken for Western blotting examinations. The expression of key insulin signalling proteins (IRβ, PKCζ and mTOR) was assessed by semiquantitative Western blotting from the tissues mentioned. The type of diet had a remarkable influence on the insulin homeostasis of chickens. The wheat-based diet significantly increased IRβ and mTOR expression in the liver as well as mTOR and PKCζ expression in the adipose tissue when compared to animals kept on a maize-based diet. IRβ expression in the liver was stimulated by the lower dose of non-protected butyrate as well, suggesting the potential of butyrate as a feed additive to affect insulin sensitivity. Based on the results obtained, the present study shows new aspects of nutritional factors by comparing the special effects of butyrate as a feed additive and those of the cereal type, presumably in association with dietary non-starch polysaccharide- (NSP-) driven enteric shortchain fatty acid release including butyrate, influencing insulin homeostasis in chickens. As the tissues of chickens have physiologically lower insulin sensitivity compared to mammals, diet-associated induction of the insulin signalling pathway can be of special importance in improving growth and metabolic health.