Cell Signaling Proteins Research Articles

The role of cholesterol and cholesterol-driven membrane raft domains in prostate cancer.

Membrane rafts are heterogeneous and dynamic domains that are characterized by tight packing of lipids. They are enriched in cholesterol, sphingolipids, and certain types of proteins. Among these are various cell signaling proteins, which indicate that rafts play an important role in cell signal transduction pathways, including some involved in cancer development, progression, and invasiveness. Due to their increased cholesterol content, raft domains exhibit lower fluidity than the surrounding membrane. The cell membranes of some solid tumors, such as breast and prostate cancer, contain higher levels of cholesterol, which means larger raft domain can form in those membranes. This may stimulate signaling pathways to promote tumor growth and progression. This review focuses on the known raft-dependent regulatory mechanisms that promote prostate cancer progression.Impact statementProstate cancer remains the most common malignancy and second most frequent cause of cancer-related death in men. Cholesterol levels are usually higher in prostate cancer cells. This affects the cell membrane composition, with cholesterol and sphingolipid-containing raft membrane domains becoming a greater component. In addition to polar lipids, these domains recruit and regulate certain types of protein, including various cell signaling proteins that are critical to cancer cell survival and invasiveness. This suggests that membrane rafts have a regulatory role in tumor progression, making them a potential target in prostate cancer treatment.

Catalytically inactive receptor tyrosine kinase PTK7 activates FGFR1 independent of FGF.

Protein tyrosine kinase 7 (PTK7), a catalytically defective receptor protein tyrosine kinase (RPTK), plays an oncogenic role by activating an unidentified TKI-258 (dovitinib)-sensitive RPTK in esophageal squamous cell carcinoma (ESCC) cells. Here, we demonstrate that among TKI-258–sensitive RPTKs, fibroblast growth factor receptor (FGFR) 1 is significantly up-regulated in ESCC tissues and cell lines. We show that PTK7 colocalizes with FGFR1 and binds it via its extracellular domain in human embryonic kidney 293 and ESCC TE-10 cells. PTK7 knockdown not only reduced ligand-free and fibroblast growth factor (FGF)-induced phosphorylation of FGFR1 but also the interaction of signaling adaptor proteins with FGFR1 and activation of downstream signaling proteins in TE-10 cells. In addition, PTK7 knockdown reduced FGF-induced oncogenic phenotypes including proliferation, anchorage-independent colony formation, wound healing, and invasion in ESCC cells. Taken together, our data demonstrate that PTK7 binds and activates FGFR1 independent of FGF and thus increases oncogenicity of PTK7- and FGFR1-positive cancers such as ESCC.—Shin, W.-S., Lee, H. W., Lee, S.-T. Catalytically inactive receptor tyrosine kinase PTK7 activates FGFR1 independent of FGF.

How Phosphorylation by PINK1 Remodels the Ubiquitin System: A Perspective from Structure and Dynamics.

Ubiquitin is an important signaling protein in cells. It functions by covalent attachment to substrate proteins and by noncovalent interactions with target proteins. Ubiquitins are also concatenated, and the resulting polyubiquitins recognize target proteins multivalently with enhanced specificity. The function of ubiquitin is enabled by the conformational dynamics of ubiquitin and polyubiquitins, which spans over 12 orders of magnitude in a time scale. Recently, it was found that ubiquitin can be phosphorylated by PINK1 at residues S65 and T66. Only sparsely populated for the unmodified ubiquitin, a C-terminally retracted conformation is stabilized for phosphorylated ubiquitin and is further enriched at an increasing pH. The modulation of tertiary structure further impacts the quaternary arrangements of ubiquitin subunits in polyubiquitins. Additionally, ubiquitin phosphorylation inhibits the activities of many enzymes responsible for attaching and removing polyubiquitins, thus remodeling the composition and length of polyubiquitins. The phosphorylation-remolded polyubiquitins can then recognize different target proteins. As PINK1 and ubiquitin phosphorylation levels are up-regulated under certain pathophysiological conditions, the remodeled ubiquitin system may be involved in the divergence of cell fate.

Maladaptive remodeling of pulmonary artery root autografts after Ross procedure: A proteomic study

ObjectivePulmonary autograft root dilatation is the major long-term complication after Ross procedure and the leading cause for reoperation. However, the mechanisms underlying dilatation remain to be elucidated. This study analyzed the proteomic changes seen in the dilated pulmonary autograft compared with normal pulmonary artery and aorta tissues. MethodsPulmonary autograft surgical samples were taken from 9 consecutive patients (mean age 37 ± 14; 15-51 years) with mean diameters of 5.2 ± 0.5 cm (4.6-5.8 cm) reoperated 8 to 16 years after Ross procedure. Control pulmonary artery and aorta samples were from 7 age- and sex-matched cardiac donors. Tunicae mediae from all samples were processed for proteomic analysis via 2-dimensional electrophoresis, matrix-assisted-laser-desorption-ionization-time of flight/mass spectrometry, and bioinformatics. The thus-identified putatively relevant proteins were validated via Western immunoblotting. ResultsPulmonary autograft proteome features differed markedly from control pulmonary arteries, since proteins related to focal adhesions (eg, paxillin), cytoskeleton (eg, vimentin), and metalloprotease-regulating proteoglycans (eg, testican-2) were significantly up-regulated, whereas significant decreases occurred in microfibril-associated glycoprotein1, which controls elastic fiber buildup. Profound changes also occurred in cell-signaling proteins, ie, increases in soluble Jagged-1 fragment and ectodysplasin-2 receptor, and decreases in Notch-1 intracellular domain fragment. Moreover, pulmonary autograft expression levels of Paxillin, Vimentin, Jagged-1 fragment, and Notch1 intracellular domain fragment also differed from those of control aorta. ConclusionsThis study provides the first description of the specific proteomic features of dilated pulmonary autograft tunica media, which separate them sharply not only from those of control pulmonary artery and aorta but also of aortic aneurysms. These findings suggest that dilated pulmonary autografts undergo a unique maladaptive remodeling process deserving further investigation.

Can Allosteric Receptor-Protein Interactions in Receptor Complexes Be a Molecular Mechanism Involved in Cancer Immune Therapy?

Based on the work in the Central Nervous System with discoveries of allosteric receptor-receptor interactions in homo- and heteroreceptor complexes representing a major integrative mechanism in synapses and extrasynaptic regions, it is proposed that a similar mechanism may exist in the immunological synapses. We discuss a putative additional molecular mechanism for the ability of the inhibitory T cell signaling proteins CTLA-4 and PD-1 and the adenosine A2AR to diminish T cell activation leading to enhancement of cancer development. We suggest that in the same immunological synapse involving T cells and antigen presenting cells multiple heteroreceptor complexes may participate and be in balance with each other. Their composition can vary between functional states and among different types of T cells. The T cell receptor (TCR) and its accelerators, strongly enhancing T cell activation, can be under inhibitory control by T cell signaling proteins CTLA4 and PD-1 and also the adenosine A2AR through inhibitory allosteric receptor-receptor interactions in different types of heteroreceptor complexes. As a result, inhibitory tumor induced immunosuppression can develop due to a dominance of the inhibitory signaling causing a brake on the TCR and/or its accelerator and the cancer immunotherapy becomes blocked.

Liquid Chromatography-Multiple Reaction Monitoring Mass Spectrometry Quantification of Extracellular Vesicles Signaling Proteins in placental cells under different physiological conditions

Perinatal sodium overload induces endothelial dysfunction in adult offspring, but the underlying mechanisms are not fully known. The involvement of tissue renin-angiotensin system on high sodium-programmed endothelial dysfunction was examined.Acetylcholine and angiotensin I and II responses were analyzed in aorta and mesenteric resistance arteries from 24-week-old male offspring of normal-salt (O-NS, 1.3% NaCl) and high-salt (O-HS, 8% NaCl) fed dams. COX-2 expression, O2– production and angiotensin converting enzyme (ACE) activity were determined. A separated O-HS was treated with losartan (15 mg kg−1/day) for eight weeks.Compared to O-NS, O-HS were normotensive. Acetylcholine-induced relaxation was impaired in O-HS arteries, which was improved by tempol, apocynin or indomethacin. The angiotensin I-induced contraction was greater in O-HS arteries, whereas the angiotensin II responses were unchanged. ACE activity, O2– production and COX-2 expression were increased in O-HS arteries. In this group, the increased O2– production was inhibited by apocynin or losartan. Chronic losartan decreased COX-2 expression and restored the endothelium-dependent vasodilation in O-HS.Our findings reiterate that perinatal sodium overload programs endothelial dysfunction in adult offspring through a blood pressure-independent mechanism. Our results also suggest that vascular angiotensin II is the main mediator of high sodium-programmed endothelial dysfunction, promoting COX-2 expression and oxidative stress.

Long-chain n-3 fatty acids as an essential link between musculoskeletal and cardio-metabolic health in older adults.

This narrative review aims to critically evaluate scientific evidence exploring the therapeutic role(s) of long-chain n-3 PUFA in the context of ageing, and specifically, sarcopenia. We highlight that beyond impairments in physical function and a lack of independence, the age-related decline in muscle mass has ramifications for cardio-metabolic health. Specifically, skeletal muscle is crucial in regulating blood glucose homeostasis (and by extension reducing type 2 diabetes mellitus risk) and providing gluconeogenic precursors that are critical for survival during muscle wasting conditions (i.e. AIDS). Recent interest in the potential anabolic action of n-3 PUFA is based on findings from experimental studies that measured acute changes in the stimulation of muscle protein synthesis (MPS) and/or chronic changes in muscle mass and strength in response to fish oil-derived n-3 PUFA supplementation. Key findings include a potentiated response of MPS to amino acid provision or resistance-based exercise with n-3 PUFA in healthy older adults that extrapolated to longer-term changes in muscle mass and strength. The key mechanism(s) underpinning this enhanced response of MPS remains to be fully elucidated, but is likely driven by the incorporation of exogenous n-3 PUFA into the muscle phospholipid membrane and subsequent up-regulation of cell signalling proteins known to control MPS. In conclusion, multiple lines of evidence suggest that dietary n-3 PUFA provide an essential link between musculoskeletal and cardio-metabolic health in older adults. Given that western diets are typically meagre in n-3 PUFA content, nutritional recommendations for maintaining muscle health with advancing age should place greater emphasis on dietary n-3 PUFA intake.

Abstract 1861: Aviscumine (ME-503) suppresses the growth of melanoma by potentially targeting c-Myc pathway

Abstract Aviscumine, a recombinant mistletoe lectin I, is produced in E. coli and has been evaluated for its antitumor activities in various experimental in-vitro and in-vivo tumor models as well as in clinical trials. A phase II trial demonstrated the safety and efficacy of Aviscumine in pretreated patients with metastatic melanoma (stage IV). However, the mechanism(s) underlying the effect of Aviscumine in melanoma is inconclusive. Here, the antitumor activities and relevant mode of actions of Aviscumine were investigated in human melanoma A375, mouse melanoma Yummer and B16 cells as well as their relevant xenograft animal models. Cells were treated with Aviscumine (0-20 ng/mL) for 48 and 72 hrs, and cell proliferation was measured by MTT assay. Alteration of cell growth regulatory proteins and cell signaling proteins were determined by Reverse Phase Proteomic Array (RPPA) and validated with western blotting. Aviscumine exerted much stronger anti-proliferative activity in both A375 and Yummer cells with IC50 of 0.18 ± 0.03 ng/ml and 0.41 ± 0.06 ng/ml, respectively than that of B16 cells (IC50 15.93 ± 3.48 ng/ml). In A375 cells, Aviscumine significantly increased subG0/G1 population suggesting Aviscumine treatment led to apoptotic and necrotic cell death. Additionally, downregulation of various proteins associated with apoptotic cell death (pCDK1, pRB, MCl-1) was also observed by RPPA. Furthermore, Aviscumine significantly decreased c-Myc protein expression in a concentration-dependent manner measured by both RPPA and western blot. Interestingly, baseline c-Myc protein expression was much higher in the Aviscumine-sensitive A375 and Yummer cells than that in the Aviscumine-resistant B16 cells. Finally, the effects of Aviscumine on tumor growth were tested via subcutaneous injection (30 ng/kg, twice per week for three weeks) in mice bearing A375 or B16 melanoma. At 3 weeks, A375 tumors were markedly smaller in Aviscumune treated mice (383.8 ± 102.2 mg) than in the control-treated group (922.4 ± 296.1 mg). c-Myc protein expression was also significantly reduced (62% vs. control) in Aviscumine treated A375 tumors, as were levels of multiple downstream metabolites (pyruvate, malate, and glutamate) (p< 005). In contrast, similar to the in vitro data, Aviscumine treatment showed no tumor growth inhibitory effects in B16 tumor-bearing mice evidenced by minimum reduction on terminal tumor weight between vehicle control (1455 ± 430 mg) and Aviscumine treated group (1323 ± 270 mg). Together, these preliminary data implicate c-Myc as both a target and predictive biomarker for Aviscumine elicited antitumor activity. Citation Format: Peiying Yang, Tara Conway, Patrea Rhea, Dongmei Chen, Bo Wei, Jibin Ding, Hans Lentzen, Jennifer McQuade. Aviscumine (ME-503) suppresses the growth of melanoma by potentially targeting c-Myc pathway [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2019; 2019 Mar 29-Apr 3; Atlanta, GA. Philadelphia (PA): AACR; Cancer Res 2019;79(13 Suppl):Abstract nr 1861.

Abstract 3879: Inhibition of CXCR4 driven colorectal cancer progression by Nef-M1 peptide

Abstract Introduction: The Nef-M1 peptide (Nef-M1) competes effectively with the natural ligand of CXCR4, SDF-1α, and exhibits anti-tumorigenic activity in human colorectal cancer (CRC). However, the molecular mechanisms of action of Nef-M1 on CRC remain unclear. In this study, we evaluated the inhibition of CXCR4 driven molecular signaling mechanisms that is involved in CRC progression. Experimental Design: Cell line derived xenografts (CDX), patient derived explant (PDE) and in vitro cell based models were used to examine the mechanisms of action of Nef-M1. The severe combined immunodeficient (SCID) mice with tumor were treated intraperitoneally with either Nef-M1 or scrambled amino acids sequence Nef-M1 (sNef-M1) that is the control. Preparation of CRC tissue, explant complete media, and cultures and treatment for 48 hours were performed for the PDE study. Sections from tumors were evaluated by immunostaining (IHC) for signaling proteins thus, implicating CRC progression. Western blot (WB) analyses were also performed on lysates of both cell lines and tumors to assess the effect of Nef-M1 on the signaling pathways that promote CRC progression. Results: The present study revealed that PDE recapitulate multiple biological features of the disease and these were found to be very similar to the corresponding original CRC. CXCR4 overexpressing CRC cells displayed activation of CXCR4/CREB signaling as demonstrated by an increased activation of CREB, and expression of B-Myb and APOBEC3B (A3B). IHC analysis for Ub-H2B, a stem cell signaling protein that correlates with advanced disease and metastasis indicated that Nef-M1 treated CDX or PDE tumors had low expression of Ub-H2B. However, sNef-M1 treated tumors had high expression of Ub-H2B. WB analyses indicated that Nef-M1 not only suppressed the expression of Ub-H2B, but also significantly suppressed the expression of A3B. Cells expressing CXCR4 became susceptible to Nef-M1-induced inhibition of Akt, mTOR activation and Ub-H2B expression. Conclusions: These results indicate that Nef-M1 suppresses CXCR4 driven activation of CXCR4/CREB signaling and the expression of Ub-H2B. Therefore, Nef-M1 inhibition of these signaling pathways may be a promising therapeutic strategy for CRC. This work was supported by NIH/NCI Workforce Diversity Grant R21-CA171251. Citation Format: Venkat R. Katkoori, Zeynoire Anderson, Upender Manne, Harvey Bumpers. Inhibition of CXCR4 driven colorectal cancer progression by Nef-M1 peptide [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2019; 2019 Mar 29-Apr 3; Atlanta, GA. Philadelphia (PA): AACR; Cancer Res 2019;79(13 Suppl):Abstract nr 3879.

To See or Not to See: A Systematic Review of the Importance of Human Ocular Surface Cytokine Biosignatures in Ocular Allergy

Cytokines are key cell signalling proteins in a number of immune and homeostatic pathways of the human body. In particular, they mediate intracellular mechanisms of allergy on the ocular surface by triggering cellular responses that result in typical physiological ocular allergy symptoms, such as itchiness, watery eyes, irritation, and swelling. Given the recent research focus in optometry on the aetiology of corneal ectasia subtypes like keratoconus, there is an increasing need for the development of new clinical diagnostic methods. An increasing trend is evident among recent publications in cytokine studies, whereby the concentrations of cytokines in healthy and disease states are compared to derive a specific cytokine profile for that disease referred to as ‘biosignatures’. Biosignatures have diagnostic applications in ocular allergy as a cheap, non-invasive alternative to current techniques like IgE antibody testing and skin prick tests. Cytokine detection from tear samples collected via microcapillary flow can be analysed either by enzyme-linked immunosorbent assays (ELISA), multiplex magnetic bead assays, or immunoblot assays. Characterising patient hypersensitivities through diagnostic tests is the first step to managing exposure to triggers. Investigating cytokine biosignatures in ocular allergy and their links to physiology are imperative and will be the focus of this systematic review article.

Proteomic changes across a natural temperature gradient in a marine gastropod

Responses of marine ectotherms to variable environmental temperature often entails maintanence of cellular homeostasis and physiological function through temperature compensation and physiological changes. We investigated the physiological response to thermal stress by examining proteomic changes in the marine kelp forest gastropod and emerging fisheries species Kellet's whelk (Kelletia kelletii) across a naturally-existing thermal gradient that ranges from a warmer-water site inside the species' native range and extends to the northern, cold-water edge of the range. We hypothesized that abundance of cellular stress response and energy metabolism proteins would increase with decreasing temperature in support of cold-compensation. Our exploratory proteomic analysis of whelk gill tissue (N = 6 whelks) from each of the four California Channel Island sites revealed protein abundance changes related to the cytoskeleton, energy metabolism/oxidative stress, and cell signaling. The changes did not correlate consistently with temperature. Nonetheless, whelks from the coldest island site showed increased abundance of energy metabolism and oxidative stress proteins, possibly suggesting oxidative damage of lipid membranes that is ameliorated by antioxidants and may aid in their cold stress response. Similarly, our exploratory analysis revealed abundances of cell signaling proteins that were higher at the coldest site compared to the warmest site, possibly indicating an importance for cell signaling regulation in relatively cooler environments. This study provides protein targets for future studies related to thermal effects in marine animals and may contribute to understanding the physiological response of marine organisms to future ocean conditions.

Low-Dose Rapamycin Facilitates Recovery from Exercise-Induced Muscle Injury

Rapamycin has been shown to have a dose-dependent effect on multiple signaling proteins in skeletal muscle cells that influence protein synthesis and calcium handling. However, it has yet to be determined if a low-dose of rapamycin impacts skeletal muscle during recovery from an exercise-induced injury. PURPOSE: To determine if low-dose rapamycin affects the rate of isometric strength recovery, muscle ubiquitination levels and markers of autophagy compared to saline control 14 days after exercise induced injury. METHODS: Mice were injected with either saline (SAL; 0.9%) or low-dose rapamycin (RAP; 10 μg/kg body weight) every other day for 2 weeks before and after a single bout of 150 eccentric contractions of the left anterior crural muscles. The recovery of strength of the anterior crural muscles was measured in vivo immediately, 7 days, and 14 days after injury induction. The magnitude of expression of beclin-1, ubiquitin, and ubiquitinated protein in injured and contralateral control TA muscles (i.e., primary anterior crural muscle) were analyzed via Western blot at 14 days after injury. RESULTS: Isometric twitch torque values did not differ between groups at any time point. No group differences in peak isometric tetanic torque were observed pre-injury, post-injury or 7 days following injury. However at 14 days, RAP mice recovered to pre-injury peak isometric torque values (Pre= 2.30 ± .07 Nmm; 14d= 2.25 ± 0.08 Nmm) while SAL group was significantly lower than pre-injury. At 14 days, RAP mice generated 15.4% higher maximal torque than SAL group (p = 0.04). Beclin-1 and free ubiquitin expression in TA muscles were significantly increased in both SAL (1.4-fold and 2.3-fold, respectively) and RAP (2.2-fold and 8.0-fold, respectively) mice at 14 days after injury compared to the uninjured muscle. The increase in the free ubiquitin in the injured muscle was 3.3-fold greater in the RAP treatment compared to SAL (p = 0.001). There were no significant changes in the ubiquitination of proteins among the groups at 14 days post-injury. CONCLUSION: Chronic low-dose rapamycin treatment in mice enhances recovery of skeletal muscle from eccentric contraction-induced injury at the 14th day and accentuates the upregulation of free ubiquitin.

PB1769 INTERLEUKIN‐10 (1082G/A) GENE POLYMORPHISM IS ASSOCIATED WITH RISK OF ACUTE MYELOID LEUKEMIA IN SUDANESE POPULATION

Background:Acute myeloid leukemia (AML) is a cytogenetically and molecularly heterogeneous diseases, and description of transforming genetic events is becoming increasingly significant. Interleukins (ILs) are a diverse set of small cell signaling protein molecules. Single nucleotide polymorphisms (SNPs) of ILs alter their function, increasing predisposition to different illnesses.Aims:We investigated the association between a single nucleotide polymorphism (SNP) in IL‐10 ‐1082G/A (rs1800896) in Sudanese acute myeloid leukemia patientsMethods:A total of 30 patients with Acute Myeloid Leukemia and 30 control subjects were enrolled in this study. Blood samples were collected from all participants in EDTA containing tubes. Genomic DNA was extracted from all blood samples using salting out method. The genotypic variants of IL‐10(‐1082G/A) polymorphism were detected by allele specific‐PCR.Results:We found that (36.7%) of patients have homogenous GG genotype, (43.3%) have heterogeneous GA genotype and (20.0%) have AA genotype. GA genotype was significantly higher risk of AML compared with the homozygous, there is no association between IL‐10(‐1082G/A) polymorphism and AML sub‐type, gender, age group, mean of hematological parameter (TWBCs, Blast%, Hb and platelet count).Summary/Conclusion:In summary we conclude that GA genotype of IL‐10 ‐1082G/A (rs1800896) polymorphism is a risk factor for AML and G allele is insignificantly higher than A allele in AML patient. No association between IL‐10(‐1082G/A) polymorphism and AML sub‐type, gender, age group, mean of hematological parameter (TWBCs, Blast%, Hb and platelet count).

Study on the metabolism toxicity, susceptibility and mechanism of di-(2-ethylhexyl) phthalate on rat liver BRL cells with insulin resistance in vitro

Di-(2-ethylhexyl) phthalate (DEHP) is the most commonly used plasticizer which could be easily absorbed by humans and animals through various channels. It has been found that exposure to DEHP could increase the incidence of insulin resistance. In this study, therefore, the metabolism toxicity, susceptibility and mechanism of DEHP (5500 and 50,000 nM exposure for 24 h) on normal BRL cells (Buffalo Rat Liver cells) and BRL cells with insulin resistance induced by insulin were investigated. The results showed that DEHP could cause cell damage with ALT and AST activities and MDA levels increased, cell apoptosis with Caspase-3 levels increased and insulin resistance with IR-β levels decreased in BRL cells with resistance and normal BRL cells. Western-blot analysis and Q-PCR showed that the levels and gene expressions of insulin signaling proteins (IRS-1, GLUT4, GSK-3α, GSK-3β, PI3K, AKT, mTOR), cell signaling proteins (RAS, ERK1/2, MEK1/2, BAD, BAX, BCL-2) and immediate early genes in insulin resistance cells and normal cells were significantly altered by DEHP. DEHP significantly promoted serine phosphorylation of IRS-1. The insulin resistance cells in metabolism toxicity were more sensitive to DEHP than normal cells. Intervention with insulin could improve the metabolism toxicity and insulin resistance. The results indicated that DEHP exerted metabolic toxic effects and increased insulin resistance through interfering with glucose metabolism and insulin signaling transduction pathway. Moreover, the risks of DEHP-induced metabolic toxicity and insulin resistance in BRL cells with insulin resistance were higher than that of normal BRL cells.

Dihydrotestosterone increases the risk of bladder cancer in men

Men are at a higher risk of developing bladder cancer than women. Although the urinary bladder is not regarded as an sex organ, it has the potential to respond to androgen signals. The mechanisms responsible for the gender differences remain unexplained. Androgen receptor (AR) after binding with 5α-dihydrotestosteron (DHT) undergoes a conformational change and translocates to nucleus to induce transcriptional regulation of target genes. However androgen/AR signaling can also be activated by interacting with several signaling molecules and exert its non-genomic function. The aim of present study was to explain whether the progression of bladder cancer in men is dependent on androgen/AR signaling. Studies were carried out on human bladder cancer cell lines: HCV29, T24, HT1376 and HTB9. Bladder cancer cells were treated for 48 h with 10 nM DHT or not, with replacement after 24 h. Expression of cell signaling proteins, was analyzed using Western Blot and RT-PCR. Subcellular localization of protein was studied using the ProteoExtract Subcellular Proteome Extraction Kit and Western blot analysis. We showed that DHT treatment significantly increased AR expression in bladder cell line HCV29. We also observed DHT-mediated activation of Akt/GSK-3β signaling pathway which plays a central role in cancer progression. Presented results also show that androgen/AR signaling is implicated in phosphorylation of eIF4E which can promote epithelial–mesenchymal transition (EMT). We indicate that AR plays an essential role in bladder cancer progression in male patients. Therefore, androgen-activated AR signaling is an attractive regulatory target for the inhibition or prevention of bladder cancer incidence in men.

Electrophilic additions of nitrated fatty acids with biological thiols: comparison with type-2 alkenes

We investigated the adduct formations of cysteine and glutathione with electrophilic substrates. Density functional theory with a continuum model was applied to explore the reactions that serve as models for cell signaling proteins and electrophiles. Biological electrophilic species including nitrated fatty acids (NO2-FAs), 15-deoxy-delta12,14-prostaglandin J2 (15d-PGJ2) and neurotoxic type-2 alkenes were included in our investigation. The reaction sequence begins with a thiol proton transfer with the base, followed by nucleophilic attack, and ends with a second proton transfer to accomplish thiol additions to the substrates. It was observed that adduct formations between the thiols and type-2 alkenes are comparatively more exergonic than those with NO2-FAs and 15d-PGJ2. The type-2 alkenes that were most exergonic in adduct formations were experimentally identified to be the most toxic. The computational approach presented in this work may serve as a convenient method to predict the degree of neurotoxicity.

Expansion of IL-23 receptor bearing TNFR2+ T cells is associated with molecular resistance to anti-TNF therapy in Crohn’s disease

ObjectiveAnti-tumour necrosis factor (TNF) antibodies are successfully used for treatment of Crohn’s disease. Nevertheless, approximately 40% of patients display failure to anti-TNF therapy. Here, we characterised molecular mechanisms that are...

Atg14 protects the intestinal epithelium from TNF-triggered villus atrophy

ABSTRACTRegulation of intestinal epithelial turnover is a key component of villus maintenance in the intestine. The balance of cell turnover can be perturbed by various extrinsic factors including the cytokine TNF, a cell signaling protein that mediates both proliferative and cytotoxic outcomes. Under conditions of infection and damage, defects in autophagy are associated with TNF-mediated cell death and tissue damage in the intestinal epithelium. However, a direct role of autophagy within the context of enterocyte cell death during homeostasis is lacking. Here, we generated mice lacking ATG14 (autophagy related 14) within the intestinal epithelium [Atg14f/f Vil1-Cre (VC)+]. These mice developed spontaneous villus loss and intestinal epithelial cell death within the small intestine. Based on marker studies, the increased cell death in these mice was due to apoptosis. Atg14f/f VC+ intestinal epithelial cells demonstrated sensitivity to TNF-triggered apoptosis. Correspondingly, both TNF blocking antibody and genetic deletion of Tnfrsf1a/Tnfr1 rescued villus loss and cell death phenotype in Atg14f/f VC+ mice. Lastly, we identified a similar pattern of spontaneous villus atrophy and cell death when Rb1cc1/Fip200 was conditionally deleted from the intestinal epithelium (Rb1cc1f/f VC+). Overall, these findings are consistent with the hypothesis that factors that control entry into the autophagy pathway are also required during homeostasis to prevent TNF triggered death in the intestine.Abbreviations: ANOVA: analysis of variance; Atg14: autophagy related 14; Atg16l1: autophagy related 16-like 1 (S. cerevisiae); Atg5: autophagy related 5; cCASP3: cleaved CASP3/caspase-3; cCASP8: cleaved CASP8/caspase-8; CHX: cycloheximide; EdU: 5-ethynyl-2´-deoxyuridine thymidine; f/f: flox/flox; H&E: hematoxylin and eosin; MTT: 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide; Nec-1: necrostatin-1; Rb1cc1/Fip200: RB1-inducible coiled-coil 1; Ripk1: receptor (TNFRSF)-interacting serine-threonine kinase 1; Ripk3: receptor (TNFRSF)-interacting serine-threonine kinase 3; Tnfrsf1a/Tnfr1: tumor necrosis factor receptor superfamily, member 1a; Tnf/ Tnfsf1a: tumor necrosis factor; VC: Vil1/villin 1-Cre

In Vitro and In Vivo Antimetastatic Effects of ZSTK474 on Prostate Cancer DU145 Cells.

The lethality of prostate cancer is mainly due to metastasis. Inhibition of metastasis is expected to be a promising approach for prostate cancer therapy. Phosphatidylinositol 3-kinase (PI3K)/Akt pathway is reported to be closely involved in cell growth, migration, etc. Objective: The study investigated the antimetastatic activities of pan-PI3K inhibitor ZSTK474 on DU145 cells. 1. The In vitro effect of ZSTK474 on the migration, invasion and adhesion of DU145 cells was determined with Transwell migration assay and wound healing assay, Tranwell invasion assay and adhesion assay, respectively. 2. In vitro effect of ZSTK474 on the signal proteins in DU145 cells was determined with Western blot analysis and ELISA. 3. Moreover, the In vivo antimetastatic effect of ZSTK474 was evaluated with MicroCT and histology analysis. ZSTK474 potently attenuated the capability of migration, invasion and adhesion of DU145 cells, negatively regulated Girdin, Integrinβ1 and matrix metalloproteinases (MMPs). In addition, the expression of hypoxia-inducible factor-1α (HIF-1α) and vascular endothelial growth factor (VEGF), which are known to be related to angiogenesis and metastasis, was also inhibited. Oral administration of ZSTK474 (200 mg/kg) ameliorated in vivo bone metastasis of DU145 cells, with improved bone structure and bone mineral density (BMD). Tissue staining indicated a reduction in metastatic DU145 cells and osteoclasts in the bones of ZSTK474-treated mice, compared with the non-treated group. Our result demonstrated the antimetastatic activity of ZSTK474 on prostate cancer DU145 cells, suggesting the potential application in prostate cancer patients.

Iron transitions during activation of allosteric heme proteins in cell signaling.

Allosteric heme proteins can fulfill a very large number of different functions thanks to the remarkable chemical versatility of heme through the entire living kingdom. Their efficacy resides in the ability of heme to transmit both iron coordination changes and iron redox state changes to the protein structure. Besides the properties of iron, proteins may impose a particular heme geometry leading to distortion, which allows selection or modulation of the electronic properties of heme. This review focusses on the mechanisms of allosteric protein activation triggered by heme coordination changes following diatomic binding to proteins as diverse as the human NO-receptor, cytochromes, NO-transporters and sensors, and a heme-activated potassium channel. It describes at the molecular level the chemical capabilities of heme to achieve very different tasks and emphasizes how the properties of heme are determined by the protein structure. Particularly, this reviews aims at giving an overview of the exquisite adaptability of heme, from bacteria to mammals.