Total Protein Expression Research Articles

Destabilizing NF1 variants act in a dominant negative manner through neurofibromin dimerization

The majority of pathogenic mutations in the neurofibromatosis type I (NF1) gene reduce total neurofibromin protein expression through premature truncation or microdeletion, but it is less well understood how loss-of-function missense variants drive NF1 disease. We have found that patient variants in codons 844 to 848, which correlate with a severe phenotype, cause protein instability and exert an additional dominant-negative action whereby wild-type neurofibromin also becomes destabilized through protein dimerization. We have used our neurofibromin cryogenic electron microscopy structure to predict and validate other patient variants that act through a similar mechanism. This provides a foundation for understanding genotype-phenotype correlations and has important implications for patient counseling, disease management, and therapeutics.

The role of CSE1L silencing in the regulation of proliferation and apoptosis via the AMPK/mTOR signaling pathway in chronic myeloid leukemia

ABSTRACTObjectivesChromosome segregation 1-like (CSE1L) is abundant and strongly expressed in solid tumors. However, the expression and role of CSE1L in chronic myeloid leukemia(CML) remain largely unknown.Materials and methodsThe relative expression levels of CSE1L in bone marrow granulocytes from patients with primary CML and non-hematologic controls were measured by flow cytometry. Cell counting kit-8 analysis, DNA Content Quantitation Assay, and Annexin V-PE/7-AAD staining were applied to assess the effects of CSE1L knockdown on cell proliferation, cell cycle progression, and apoptosis.ResultsElevated expression of CSE1L was detected in bone marrow granulocytes of patients with primary CML. In the CML cell line K562 cells, CSE1L knockdown impaired cell proliferation blocked the cell cycle shift from G0/G1 phase to the S phase, and promoted apoptosis. Knockdown of CSE1L reduced Bcl-2 protein expression and increased Bax protein expression. Meanwhile, knockdown of CSE1L enhanced the expression of phospho-AMPK protein and decreased the expression of phospho-mTOR protein. The expression of total AMPK and mTOR proteins was not affected. In addition, CSE1L expression levels were decreased in imatinib-treated K562 cells.ConclusionsCSE1L plays a pivotal role in K562 cell survival and growth. These functions may be partially dependent on the AMPK/mTOR signaling pathway to achieve. In addition, CSE1L may have had a future impact on the treatment of CML patients.

Alkaloid from Alstonia yunnanensis diels root against gastrointestinal cancer: Acetoxytabernosine inhibits apoptosis in hepatocellular carcinoma cells.

Liver cancer belongs to Gastrointestinal (GI) malignancies which is a common clinical disease, a thorny public health problem, and one of the major diseases that endanger human health. Molecules from natural products (NPs) or their derivatives play an increasingly important role in various chronic diseases such as GI cancers. The chemical composition of the Alstonia yunnanensis Diels roots was studied using silica column chromatography, gel chromatography, recrystallization, and HPLC, and the compounds were structurally identified by modern spectral analysis using mass spectrometry (MS) and nuclear magnetic resonance (1H-, 13C-, HMQC-, HMBC-, and 1H-1HCOSY-NMR), ultraviolet and visible spectrum (UV), and electronic Circular Dichroism (ECD). Acetoxytabernosine (AC), an indole alkaloid with antitumor activity, was isolated from Alstonia yunnanensis Diels root. The current study aimed to investigate the influence of AC on the cell proliferation of BEL-7402 and SMMC7721 and to elucidate the underlying mechanism. The absolute configuration of AC was calculated by ECD (electronic circular dichroism). The effects of AC on the viability of different tumor cell lines were studied by the SRB method. The death mode of human hepatoma cells caused by AC was studied by TUNEL cell apoptosis detection and AnnexinV-FITC/PI double staining image. Mitochondrial membrane potential was detected by JC-1. The effects of AC on the expression of apoptosis-related proteins (Caspase9, Caspase3, and Parp-1) in SMMC7721 and BEL-7402 cells were detected by western blot. It was found that the absolute configuration of AC is 19(s), 20(s)-Acetoxytabernosine. AC could induce apoptosis of SMMC7721 and BEL-7402, and block the replication of DNA in the G1 phase. Under the treatment of AC, the total protein expression of apoptosis-related proteins (Caspase9, Caspase3, and Parp-1) significantly decreased in SMMC7721 and BEL-7402. The results suggested that AC induced apoptosis through a caspase-dependent intrinsic pathway in SMMC7721 and BEL-7402, and natural product-based drug development is an important direction in antitumor drug discovery and research.

Protective mechanism of gold nanoparticles on human neural stem cells injured by β-amyloid protein through miR-21–5p/SOCS6 pathway

Alzheimer’s disease (AD) is a neurodegenerative disorder with progressive memory loss in dementia. Gold nanoparticles (AuNPs) were reported beneficial for human neural stem cells (hNSCs) treated with Amyloid-beta (Aβ), but the neuroprotective mechanisms still are unknown. First, the hNSCs induced by Aβ to construct AD cell model in vitro and AuNPs was performed to assess the therapeutic effect of Aβ-targeted AD treatment. Then, we investigated the effects of AuNPs on hNSCs viability and proinflammatory factors (interleukin 6 and tumor necrosis factor-alpha) by Cell Counting Kit-8 (CCK-8) and enzyme-linked immunosorbent (ELISA). FACS was carried out to determinate Tuj-1 and glial fibrillary acidic protein (GFAP). Reactive oxygen species (ROS) generation and mitochondrial membrane potential was evaluated by ROS and JC-1 assay kit. In addition, miRNA array was used to systematically detect the differential miRNAs. Dual-luciferase reporter assay was applied to verify the targeting relationship between miR-21–5p and the suppressor of cytokine signalling 6(SOCS6). Quantitative PCR (qPCR) and Western blot assessments were also used to detect related gene expression intracellularly or in the supernatant. The results demonstrate that AuNPs co-treatment repressed the high expression of total tau (T-tau), phosphorylated tau (P-tau), and Aβ protein, and reduced apoptosis rate of hNSCs. Aβ-induced decreased mitochondrial membrane potential and mitochondria in the hNSCs were damaged, while AuNPs co-treatment showed a protective effect on mitochondrial membrane potential. Co-treatment with AuNPs significantly increased dynamin-related protein 1 (DRP1), nuclear respiratory factor 1 (NRF1), and mitochondrial transcription factor A (TFAM) mRNA levels. AuNPs may improve mitochondrial function impairment due to Aβ by elevating mitochondrial membrane potential, upregulating regulators of mitochondrial biogenesis, and inhibiting ROS production. hNSCs transfected with miR-21–5p inhibitor reversed AuNPs mediated cytoprotection induced by Aβ. AuNPs upregulation of miR-21–5p expression and exert a mitochondrial protective function. Overexpression of miR-21–5p contributes to enhancing the effect of cytoprotection of AuNPs. MiR-21–5p direct targeting SOCS6 and overexpression SOCS6 exerted opposite effects on hNSCs compared with miR-21–5p mimic group. In conclusion, AuNPs can protect hNSCs from Aβ injury and decrease mitochondrial damage by regulating the miR-21–5p/SOCS6 pathway.

Design, synthesis, and anti-hepatocellular carcinoma of thiopyrimidine/chalcone hybrids as dual STAT3/STAT5 inhibitors.

Among the promising therapeutic targets for treating cancer are the continuously active STAT proteins, which are important in the progression of many malignancies. Here, we detail the STAT3/5 inhibitory action and thiopyrimidine/chalcone hybrid design, production, and anti-hepatocellular carcinoma activity. The prepared hybrids were assessed for their cytotoxic effect on HepG2 and Huh7 liver cancer cells. The most active compounds 5e and 5h (IC50 range from 0.55 to 2.58 μM) were further evaluated against normal THLE cells to examine their safety profiles. The hybrids 5e and 5h were additionally tested for their potential to inhibit STAT3 and STAT5a. They showed dual inhibitory action, with a decrease in the level of STAT3 by 65 and 87 times, respectively, and a decrease in the level of STAT5 by 60 and 79.5 times, respectively, compared to the control. Additionally, western blot analysis of compound 5h revealed inhibition of STAT3 and STAT5 phosphorylation at Tyr705 and Tyr694, respectively, with only a slight decrease in the total expression of STAT3 and STAT5 proteins. And lastly, molecular docking research provided additional insight on the 5h binding mechanism in the STAT3 and STAT5 SH2 domains.

PS-BPB10-6: THE RELATION BETWEEN PERICYTE DETACHMENT AND INTERSTITIAL FIBROSIS IN DAHL SALT-SENSITIVE HYPERTENSIVE (DAHLS) RATS

Background: Congestive heart failure produces the state of fluid volume overload, central venous pressure (CVP) elevation, and consequently renal venous pressure (RVP) elevation. We have recently developed a novel rat unilateral renal venous congestion model, in which ligation of the inferior vena cava between both renal veins artificially induced renal congestion in the left kidney only. In this model, pericyte detachment and pericyte-myofibroblast transition (PMT) around the vasa recta were involved in interstitial injury and fibrosis. Dahl salt-sensitive hypertensive (DahlS) rats on high salt diets exhibit hypertension, congestive heart failure, fluid volume overload, CVP and RVP elevation, and renal damage, considering to be a non-artificial renal congestion model. The relation, however, between renal interstitial injury and fibrosis, elevated RVP, and pericyte detachment in DahlS rats has not been reported. In the present study, we assessed the relation of pericyte detachment around the vasa recta and PMT with interstitial fibrosis in DahlS rats. Methods: Eight-week-old DahlS rats (Dahl/Mcwi) were divided into three groups: normal diet, 4% NaCl diet, and 8% NaCl diet groups, and their kidneys were analyzed after 2 weeks. Total mRNA and protein expression levels were quantified by real-time PCR and western blot, respectively. Tubulointerstitial damage and fibrosis were identified by histological analysis. Furthermore, we evaluated pericyte detachment around the vasa recta by low-vacuum scanning electron microscopy (LV-SEM). Results: The expression of KIM1, a tubulointerstitial damage marker; CNN1 and αSMA, interstitial fibrosis markers; and TAGLN, a smooth muscle protein22 and PMT marker; was significantly increased in a salt-dependent manner in DahlS rats. In histological analysis, hyperplasia of the extracellular matrix and renal tubular injury were detected in 4% and 8% NaCl diet groups. The positive area of PMT markers in the interstitial space around the vasa recta was also increased in the NaCl diet groups. Pericyte detachment from the vasa recta was observed in the NaCl diet groups by LV-SEM. Conclusions: Pericyte detachment and PMT are involved in tubulointerstitial injury and fibrosis of DahlS rats. Renal venous congestion and subsequent physiological changes could have a role in renal damage and fibrosis. Arterial hypertension has been emphasized for a long time in renal injury, and venous hypertension must also be focused on with the concept of renal congestion. Further understanding of this concept may lead to new therapeutic strategies for renal injury.

Low-Dose Metformin Treatment Reduces In Vitro Growth of the LL/2 Non-small Cell Lung Cancer Cell Line.

Lung cancer maintains a relatively small survival rate (~19%) over a 5-year period and up to 80-85% of all lung cancer diagnoses are Non-Small Cell Lung Cancer (NSCLC). To determine whether metformin reduces non-small cell lung cancer (NSCLC) LL/2 cell growth, cells were grown in vitro and treated with metformin for 48 h. qPCR was used to assess genes related to cell cycle regulation and pro-apoptotic markers, namely Cyclin D, CDK4, p27, p21, and HES1. Treatment with 10 mM metformin significantly reduced HES1 expression (p = 0.011). Furthermore, 10 mM metformin treatment significantly decreased REDD1 (p = 0.0082) and increased p-mTOR Ser2448 (p = 0.003) protein expression. Control cells showed significant reductions in phosphorylated p53 protein expression (p = 0.0367), whereas metformin treated cells exhibited reduced total p53 protein expression (p = 0.0078). There were no significant reductions in AMPK, PKB/AKT, or STAT3. In addition, NSCLC cells were treated for 48 h. with 10 mM metformin, 4 µM gamma-secretase inhibitor (GSI), or the combination of metformin (10 mM) and GSI (4 µM) to determine the contribution of respective signaling pathways. Metformin treatment significantly reduced total nucleus expression of the proliferation maker Ki-67 with an above 65% reduction in Ki-67 expression between control and metformin-treated cells (p = 0.0021). GSI (4 µM) treatment significantly reduced Ki-67 expression by ~20% over 48 h (p = 0.0028). Combination treatment (10 mM metformin and 4 µM GSI) significantly reduced Ki-67 expression by more than 50% over 48 h (p = 0.0245). As such, direct administration of metformin (10 mM for 48 h) proved to be an effective pharmaceutical agent in reducing the proliferation of cultured non-small cell cancer cells. These intriguing in vitro results, therefore, support the further study of metformin in appropriate in vivo models as an anti-oncogenic agent and/or an adjunctive therapy.

2-Aryl Propionic Acid Amide Modification of Naproxen and Ibuprofen Dimers for Anti-neuroinflammatory Activity in BV2 mouse Microglial Cells

Inflammation is a common link in the pathophysiology of many neurological illnesses, including Alzheimer’s disease. Activated glial cells contribute to neuroinflammation by producing pro-inflammatory mediators. Naproxen and ibuprofen are nonsteroidal anti-inflammatory drugs with 2-aryl(s) propionic acid as a common pharmacophore. Here we designed a small series of naproxen and ibuprofen amide dimers and tested their effects on the expression of inducible nitric oxide synthase (iNOS), a neuroinflammatory enzyme in lipopolysaccharide (LPS)-stimulated BV2 mouse microglial cells. Of note, treatment with CNU 019, 020, 021, 023, 024, and 027 at 10 M markedly inhibited the LPS-induced iNOS expression in BV2 cells. CNU 024 was tested further at different concentrations to regulate the LPS-induced iNOS expression in BV2 cells. Treatment with CNU 024 at 5, 10, or 20 M dose-dependently suppressed the LPS-induced iNOS protein and mRNA expression levels in BV2 cells, in which maximal inhibition was seen at 20 M. CNU 024 treatment at doses tested further led to a concentration-dependent inhibition of the LPS-induced phosphorylation (activation) of p38 mitogen-activated protein kinase (MAPK) without influencing its total protein expression in BV2 cells, but it did not affect the LPS-induced activation of c-jun N-terminal kinase-1/2 and extracellular signal-regulated kinases-1/2 in these cells. In summary, our results demonstrate that CNU 024 inhibits the LPS-induced iNOS expression in BV2 cells, partly mediated by the inhibition of p38 MAPK. This work shows that CNU 024 could be a valuable ligand for further development as a potential drug candidate for treating neuroinflammatory pathologies.

Dickkopf-1 drives tumor immune evasion by inducing PD-L1 expression in hepatocellular carcinoma

Understanding the mechanisms regulating PD-L1 expression in hepatocellular carcinoma (HCC) is important to improve the response rate to PD-1/PD-L1 blockade therapy. Here, we show that DKK1 expression is positively associated with PD-L1 expression and inversely correlated with CD8+ T cell infiltration in human HCC tumor specimens. In a subcutaneous xenograft tumor model, overexpression of DKK1 significantly promotes tumor growth, tumoral PD-L1 expression, but reduces tumoral CD8+ T cell infiltration; whereas knockdown of DKK1 has opposite effects. Moreover, enforced expression of DKK1 dramatically promotes PD-L1 expression, Akt activation, β-catenin phosphorylation and total protein expression in HCC cells. By contrast, knockdown of DKK1 inhibits all, relative to controls. In addition, CKAP4 depletion, Akt inhibition, or β-catenin depletion remarkably abrogates DKK1 overexpression-induced transcriptional expression of PD-L1 in HCC cells. Reconstituted expression of the active Akt1 largely increased PD-L1 transcriptional expression in HCC cells. Similarly, expression of WT β-catenin, but not the phosphorylation-defective β-catenin S552A mutant, significantly promotes PD-L1 expression. Correlation analysis of human HCC tumor specimens further revealed that DKK1 and PD-L1 expression were positively correlated with p-β-catenin expression. Together, our findings revealed that DKK1 promotes PD-L1 expression through the activation of Akt/β-catenin signaling, providing a potential strategy to enhance the clinical efficacy of PD-1/PD-L1 blockade therapy in HCC patients.

Estradiol regulates the expression of plasma membrane Ca2+-ATPase isoform 2 in inner ear of rats

Objective: To observe the effects of estradiol on expression of plasma membrane Ca2+-ATPase isoform 2 in inner ear of rats. Methods: Twenty-five Three-months-old female Sprague-Dawley rats were randomly and equally divided into five groups by random number table mathod,with five rats in each group. Animals in Sham group were sham-operated while others were bilateral ovariactmized. One month after modeling, the OVX groups were supplemented with estradiol (E2 group), progesterone (P group), estradiol and progesterone (E2+P group)and vehicle sesame oil (Veh group), while the Sham operation group (Sham group) was supplemented with vehicle sesame oil.All rats were sacrificed and otocysts were obtained immediately. Enzyme-linked immunosorbent assay was used to detect the changes in serum estradiol and progesterone levels of each group of rats before operation, before treatment and before sacrifice. Western blot and quantitative real-time polymerase chain reaction were used to detect the expression of total PMCA2 protein and mRNA in the inner ear of each group. Results: There was no significant difference in serum estradiol and progesterone levels among the five groups before operated(P>0.05). Before treatment, the serum estradiol and progesterone levels of rats in each group were significantly lower than those in Sham group (P<0.05). The serum estradiol level in E2 group and E2+P group was not significantly different from that in Sham group (P>0.05), while the serum estradiol level in P group and Veh group was significantly different from that in Sham group (P<0.05). The level of progesterone in P group and E2+P group was higher than that in Sham group (P<0.05), while the level of progesterone in Veh group and E2 group was lower than that in Sham group (P<0.05). Protein and mRNA expression of PMCA2 in P and Veh groups were significantly decreased compared with that of Sham group (P<0.05) while the expression levels underwent no significantly change in E2 and E2+P groups (P<0.05). Conclusion: The decrease of serum estradiol level can reduce the expression of otolith regulatory protein PMCA2 in rats, and then affect otolith metabolism, which may be an important link of estrogen affecting otolith metabolism.

Tetrahydroxy Stilbene Glucoside Alleviates Ischemic Stroke by Regulating Conformation-Dependent Intracellular Distribution of PKM2 for M2 Macrophage Polarization.

Tetrahydroxy stilbene glucoside (TSG) is a bioactive ingredient with powerful anti-inflammatory and neuroprotective properties. However, the detailed mechanisms concerning the neuroprotective effect of TSG are not fully understood. This study aims to address the molecular mechanism involved in the protective effects of TSG on murine ischemic stroke. We found that TSG meliorated the phenotypes of ischemic stroke in vivo, which was correlated with the increased percentage of infiltrated M2 macrophages in brain after stroke. Mechanistically, TSG regulated macrophage polarization by significantly downregulating the transcriptional levels of M1 marker genes (iNOS and IL-1β) but upregulating that of the M2 marker genes (arg-1 and IL-4) following lipopolysaccharide/interferon-γ stimulation. Consistently, TSG reversed the metabolic profiling of M1 macrophage toward the M2 status at intracellular energy levels. Surprisingly, the knockdown of an established metabolic enzyme pyruvate kinase M2 (PKM2) that is important for M1 switch in macrophages abolished the promotive effect of TSG on the M2 polarization. Further investigation revealed that TSG markedly downregulated the intracellular ratio of dimer/monomer to the tetramer of PKM2 without affecting its total protein expression, leading to a suppressed nuclear translocation of functioning PKM2 in macrophages for M1 differentiation. Taken together, we identified a novel mechanism for macrophage M2 polarization regulation by a small-molecule chemical that controls the quality (conformation) rather than the quantity (expression) of an intracellular M1-promoting metabolic enzyme, which offers a better understanding of the mechanisms of macrophage plasticity and has serious implication in translational strategies for the treatment of macrophage-mediated neurological diseases with natural bioactive products.

Psychosocial Stress‐Accelerates Alzheimer’s Disease Progression via the Xanthine Oxidase Pathway

AbstractBackgroundVascular contributions to cognitive impairment and dementia are a key pathology associated with Alzheimer’s disease (AD). We have shown that chronic stress induces cerebrovascular dysfunction due to a pro‐oxidative environment, a phenotype similar to that noted with AD. Epidemiological studies also suggest a link between chronic stress and dementia. We tested the hypothesis that chronic stress accelerates the AD pathology via the xanthine oxidase (XO) pathway.MethodsWild type (WT) and Triple transgenic (AD) mice were separated into 1) WT control, 2) WT+UCMS, 3) AD control, 4) AD control+Feb, 5) AD+UCMS, 6) AD+UCMS+Feb. At 4 months‐of‐age, dependent upon group, the unpredicted chronic mild stress (UCMS) model with or without Febuxostat (Feb) treated water (50mg/L) began for 8 weeks. Mice were either euthanized at 6 or 9 months‐of‐age. The middle cerebral artery (MCA) was removed for pressure myography. Brains were homogenized to extract soluble and insoluble fractions of tau proteins using Western blot normalized to GAPDH or B‐actin.ResultsAt 6 months‐of‐age, AD mice displayed a 72% impaired MCA dilation to acetylcholine compared to WT control mice (p&lt;0.05). WT and AD mice that underwent UCMS displayed similar MCA dysfunction compared to AD control mice. UCMS accelerated total and phosphorylated tau protein expression by 83% (p&lt;0.05) and 350% (p&lt;0.001), respectively, compared to AD controls. Treatment with Feb prevented MCA dysfunction in AD control mice, WT UCMS mice, and AD UCMS mice. Feb prevented the increased expression of phosphorylated tau and lowered the total tau protein expression in the AD‐UCMS group. However, total tau protein expression remained higher compared to AD control levels. At 9 months‐of‐age, the majority of these trends persisted with the one exception being a significant impairment in the AD UCMS group compared to both AD control or WT UCMS mice. Tau levels have yet to be processed for 9‐months.ConclusionOverall, these data suggest that chronic stress accelerates cerebrovascular pathology with AD and via a pro‐oxidative state driven by activation of the XO pathway. We will further explore Aβ1‐42 and XO in 3xTg brain homogenates at 6 and 9 months‐of‐age, to better understand how stress accelerates AD pathology.

MODL-08. ESTABLISHING IN VITRO MODELS TO RECAPITULATE PROTEIN-LEVEL PROGNOSTIC CELL PHENOTYPES FROM PRIMARY HUMAN GLIOBLASTOMA

Abstract Glioblastoma (GBM) is a highly aggressive primary central nervous system tumor for which median survival continues to be low, despite multimodal treatment and surgical resection. Heterogeneity at many levels (genomic, cytoarchitectural, and metabolic) likely contributes to these poor outcomes, and single-cell approaches can help to identify prognostically meaningful cells and features within heterogeneous tumors at each of these levels. Recent studies using mass cytometry (CyTOF) and the accompanying analysis pipeline Risk Assessment Population Identification (RAPID) measured per-cell protein levels in &amp;gt; 2 million cells from 28 IDH1/2-wildtype primary glioblastoma tumors and identified 2 phenotypes within these tumors that are present in different abundances across patients and have contrasting prognostic value. Increased abundance of Glioblastoma Negative Prognostic (GNP) cells within these tumors is predictive of shorter overall survival (OS HR = 1.07 [95% CI 1.02–1.12], p = 0.003), while the converse is true for Glioblastoma Positive Prognostic (GPP) cells (OS HR = 0.93 [0.87–1.0], p = 0.05). To effectively study these phenotypes, reproducible in vitro models are needed that recapitulate the total protein expression and phosphorylation events that distinguish GNP and GPP cells. Using available RNA transcript data, a candidate subset of glioma stem cell models were identified and are now being profiled using standardized mass-tagged antibody panels. These culture models will be quantitatively compared against established GNP and GPP populations in patient tissue samples using the Marker Enrichment Modeling statistic as well as other machine learning tools. Development of these models will be critical to future experiments aimed at identifying possible mechanistic therapies that effectively eliminate each of these identified populations, as well as aligning these protein-level phenotypes to other genomic and functional data in glioblastoma samples and models.

Bone Metastases of Diverse Primary Origin Frequently Express the VDR (Vitamin D Receptor) and CYP24A1.

Active vitamin D (1,25(OH)2D3) is known to exert direct anti-cancer actions on various malignant tissues through binding to the vitamin D receptor (VDR). These effects have been demonstrated in breast, prostate, renal and thyroid cancers, which all have a high propensity to metastasise to bone. In addition, there is evidence that vitamin D catabolism via 24-hydroxylase (CYP24A1) is altered in tumour cells, thus, reducing local active vitamin D levels in cancer cells. The aim of this study was to assess VDR and CYP24A1 expression in various types of bone metastases by using immunohistochemistry. Overall, a high total VDR protein expression was detected in 59% of cases (39/66). There was a non-significant trend of high-grade tumours towards the low nuclear VDR expression (p = 0.07). Notably, patients with further distant metastases had a reduced nuclear VDR expression (p = 0.03). Furthermore, a high CYP24A1 expression was detected in 59% (39/66) of bone metastases. There was a significant positive correlation between nuclear VDR and CYP24A1 expression (p = 0.001). Collectively, the VDR and CYP24A1 were widely expressed in a multitude of bone metastases, pointing to a potential role of vitamin D signalling in cancer progression. This is of high clinical relevance, as vitamin D deficiency is frequent in patients with bone metastases.

Predictors of muscle hypertrophy responsiveness to electrically evoked resistance training after spinal cord injury.

The purpose of the study was to identify potential predictors of muscle hypertrophy responsiveness following neuromuscular electrical stimulation resistance training (NMES-RT) in persons with chronic spinal cord injury (SCI). Data for twenty individuals with motor complete SCI who completed twice weekly NMES-RT lasting 12-16weeks as part of their participation in one of two separate clinical trials were pooled and retrospectively analyzed. Magnetic resonance imaging (MRI) was used to measure muscle cross-sectional area (CSA) of the whole thigh and knee extensor muscle before and after NMES-RT. Muscle biopsies and fasting biomarkers were also measured. Following the completion of the respective NMES-RT trials, participants were classified into either high-responders (n = 8; muscle CSA > 20%) or low-responders (n = 12; muscle CSA < 20%) based on whole thigh muscle CSA hypertrophy. Whole thigh muscle and knee extensors CSAs were significantly greater (P < 0.0001) in high-responders (29 ± 7% and 47 ± 15%, respectively) compared to low-responders (12 ± 3% and 19 ± 6%, respectively). There were no differences in total caloric intake or macronutrient intake between groups. Extensor spasticity was lower in the high-responders compared to the low-responders as was the dosage of baclofen. Prior to the intervention, the high-responders had greater body mass compared to the low-responders with SCI (87.8 ± 13.7 vs. 70.4 ± 15.8kg; P = 0.012), body mass index (BMI: 27.6 ± 2.7 vs. 22.9 ± 6.0kg/m2; P = 0.04), as well as greater percentage in whole body and regional fat mass (P < 0.05). Furthermore, high-responders had a 69% greater increase (P = 0.086) in total Akt protein expression than low-responders. High-responders also exhibited reduced circulating IGF-1 with a concomitant increase in IGFBP-3. Exploratory analyses revealed upregulation of mRNAs for muscle hypertrophy markers [IRS-1, Akt, mTOR] and downregulation of protein degradation markers [myostatin, MurF-1, and PDK4] in the high-responders compared to low-responders. The findings indicate that body composition, spasticity, baclofen usage, and multiple signaling pathways (anabolic and catabolic) are involved in the differential muscle hypertrophy response to NMES-RT in persons with chronic SCI.

Inhibition of Glypican-3 Cleavage Results in Reduced Cell Proliferation in a Liver Cancer Cell Line

IntroductionGlypican-3 (GPC3) is a surface-bound proteoglycan overexpressed in pediatric liver cancer and utilized clinically as an immunohistochemical tumor marker. Furin is a proprotein convertase that is ubiquitously expressed and shown to modify GPC3 post-translationally. In experimental models of epithelial-based cancers, furin inhibition decreased tumor cell migration and proliferation representing a potential therapeutic target. MethodsUsing a synthetic furin inhibitor, we evaluated proliferation, migration, protein, and RNA expression in two liver cancer cell lines, HepG2 (GPC3-positive) and SKHep1 cells (GPC3-negative). Total furin protein and GPC3 protein expression were assessed to evaluate functional levels of furin. ResultsThere was a reduction in HepG2 proliferation with addition of furin inhibitor at the 48-h timepoint, however there was an increase in HepG2 migration. ConclusionsGPC3 cleavage in hepatoblastoma (HB) has a role in cell proliferation with therapeutic potential, however furin inhibition is not an appropriate target for GPC3-expressing HB due to increased migration which may enhance metastatic potential.

Response Prediction Using 18F-FAPI-04 PET/CT in Patients with Esophageal Squamous Cell Carcinoma Treated with Concurrent Chemoradiotherapy.

This prospective study examined whether imaging results obtained using the tracer 18F-AlF-NOTA-fibroblast activation protein inhibitor (FAPI)-04 (denoted as 18F-FAPI-04) in PET/CT can predict the short-term outcome in patients with locally advanced esophageal squamous cell carcinoma (LA-ESCC) treated with concurrent chemoradiotherapy (CCRT). Methods: The 18 enrolled LA-ESCC patients underwent 18F-FAPI-04 PET/CT scanning before CCRT. The SUVmax, SUVmean, SUVpeak, metabolic tumor volume, and total lesion fibroblast activation protein expression of the primary tumor were recorded. Additionally, the SUVmax of the primary tumor and SUVmean of normal tissue (muscle and blood) were measured, and their ratios were denoted as target-to-background ratios (TBRmuscle and TBRblood). Patients were classified as responders or nonresponders according to RECIST (version 1.1), and variables were compared between the 2 groups. Results: The TBRblood, TBRmuscle, and SUVmean were significantly higher in nonresponders than in responders (all P < 0.05). Receiver-operating-characteristic curve analysis identified TBRblood (area under the curve [AUC], 0.883; P = 0.008), TBRmuscle (AUC, 0.896; P = 0.006) and SUVmean (AUC, 0.870; P = 0.010) as significant predictors of the response to CCRT, with cutoffs of 10.68, 10.95, and 6.88, respectively. The sensitivity, specificity, positive predictive value, negative predictive value, and accuracy were also determined for TBRblood (100.0%, 72.7%, 66.7%, 88.9%, and 77.8%, respectively), TBRmuscle (100.0%, 72.7%, 66.7%, 88.9%, and 77.8%, respectively), and SUVmean (85.7%, 81.8%, 75.0%, 90.0%, and 83.3%, respectively). On univariate logistic regression analysis, TBRblood (P = 0.026), TBRmuscle (P = 0.036), SUVmean (P = 0.045), and tumor site (P = 0.032) were significantly correlated with the short-term outcome. On multivariable logistic regression analysis, TBRblood (P = 0.046) was an independent prognostic factor for short-term outcome. Conclusion: A higher baseline TBRblood on 18F-FAPI-04 PET/CT scans was associated with a poor response to CCRT in LA-ESCC patients, and thus, TBRblood may be useful for screening LA-ESCC patients before CCRT treatment.

Transcriptional Responses of Copper-Transport-Related Genes ctr1, ctr2 and atox1 and Their Roles in the Regulation of Cu Homeostasis in Yellow Catfish Pelteobagrus fulvidraco.

Here, we characterized the function of ctr1, ctr2 and atox1 promoters in yellow catfish Pelteobagrus fulvidraco, a common freshwater teleost in Asian countries. We obtained 1359 bp, 1842 bp and 1825 bp sequences of ctr1, ctr2 and atox1 promoters, and predicted key transcription factor binding sites on their promoters, including MRE, SREBP1, NRF2, KLF4 and STAT3. Cu differentially influenced the activities of ctr1, ctr2 and atox1 promoters from different regions. We found that the −326/−334 bp and −1232/−1240 bp locus in the atox1 promoter were functional NRF2 binding sites, which negatively controlled the activity of the atox1 promoter. The −91/−100 bp locus in the ctr1 promoter and −232/−241 bp and −699/−708 bp locus in the atox1 promoter were functional SREBP1 binding sites, which positively controlled the activities of ctr1 and atox1 promoters. Cu inhibited the NRF2 binding ability to the atox1 promoter, but promoted the SREBP1 binding ability to the ctr1 and atox1 promoters. Dietary Cu excess significantly down-regulated hepatic mRNA and total protein expression of CTR1, CTR2 and ATOX1 of yellow catfish, compared to the adequate dietary Cu group. The subcellular localization showed that CTR1 was mainly localized on the cell membrane, CTR2 in the cell membrane and the lysosome, and ATOX1 in the cytoplasm. In conclusion, we demonstrated the regulatory mechanism of three Cu transporters at the transcription levels, and found the functional NRF2 and SREBP1 response elements in ctr1, ctr2 and atox1 promoters, which provided new insights into their roles in the regulation of Cu homeostasis in fish.

Equine insulin dysregulation causes tissue specific alterations of proinflammatory cytokines and acute phase proteins in a NF-kB independent manner

Similar to human diabetes, equine metabolic syndrome (EMS) causes insulin dysregulation leading to debilitating sequela including laminitis. The pathophysiological mechanisms underlying EMS and laminitis are not well known. Therefore, using an insulin-resistant equine model, we hypothesized that insulin dysregulation induces an increased expression of inflammatory proteins in a tissue specific manner. Two groups of horses (n = -5/group) were categorized as insulin-resistant (IR) or insulin-sensitive (IS), using a frequently sampled intra-venous glucose tolerance test. Biopsies from skeletal muscle, and visceral and subcutaneous adipose tissues were collected in both groups. Protein expression was quantified via Western blotting in order to investigate HSP90, α 2 macroglobulin (A2M), Fibrinogen α, β, γ isoforms as well as cytokines, including interleukin-1β (IL-1β) and interleukin-6 (IL-6), in muscle and adipose tissues. Protein expression of HSP90, A2M and IL1-β was significantly greater in visceral adipose tissue of IR horses compared to IS horses. Fibrinogen (α and γ) expression was only significantly increased in subcutaneous adipose tissue of IR group compared to IS group. In contrast, no statistically significant difference in protein expression of proinflammatory cytokines and acute phase proteins was reported in skeletal muscle of IR vs. IS horses. Relative protein expression of total and phospho-NFκB protein expression was not statistically significantly changed in adipose tissues of IR horses compared to IS horses. In conclusion, proinflammatory cytokines and acute phase proteins were upregulated in adipose tissue, but not in skeletal muscle, through an NF-kB independent pathway. Insights from this study could reveal novel biomarkers and potential therapeutic targets for EMS.

Cisplatin-induced HSF1-HSP90 axis enhances the expression of functional PD-L1 in oral squamous cell carcinoma.

Immune checkpoint inhibitor-based cancer immunotherapy has provided an additional therapeutic option for oral squamous cell carcinoma (OSCC) with recurrence or distant metastases. However, further improvement of OSCC treatment is required to develop the optimal combination or order for chemoradiotherapy and immunotherapy. Along with the accumulation of clinical knowledge and evidence, it is also essential to clarify the biological impact of chemo-radiotherapeutic agents on the cancer immune microenvironment. In this study, we investigated the effects of cisplatin (CDDP), a key therapeutic agent for OSCC, on programmed death-ligand 1 (PD-L1) expression in OSCC lines. Although CDDP treatment increased the surface levels of PD-L1 on OSCC cell lines, the gene and total protein expression levels of PD-L1 were not altered. We also demonstrated that the phosphorylation of heat shock factor 1 and heat shock protein 90 was involved in this process. In addition, CDDP-induced PD-L1 attenuated the target-specific cytotoxic T lymphocyte reaction to OSCC. These results provide an immunobiological basis for the response of OSCC to CDDP and will contribute to our biological understanding of the action of novel combination therapy including immunotherapy together with platinum-based chemotherapy for OSCC.