Vitamin C Suppresses Pancreatic Carcinogenesis through the Inhibition of Both Glucose Metabolism and Wnt Signaling

Cumulative studies have indicated that high-dose vitamin C has antitumor effects against a variety of cancers. However, the molecular mechanisms underlying these inhibitory effects against tumorigenesis and metastasis, particularly in relation to pancreatic cancer, are unclear. Here, we report that vitamin C at high concentrations impairs the growth and survival of pancreatic ductal adenocarcinoma (PDAC) cells by inhibiting glucose metabolism. Vitamin C was also found to trigger apoptosis in a caspase-independent manner. We further demonstrate that it suppresses the invasion and metastasis of PDAC cells by inhibiting the Wnt/β-catenin-mediated epithelial-mesenchymal transition (EMT). Taken together, our results suggest that vitamin C has therapeutic effects against pancreatic cancer.     

Cytotoxic T Cell Expression of Leukocyte-Associated Immunoglobulin-Like Receptor-1 (LAIR-1) in Viral Hepatitis C-Mediated Hepatocellular Carcinoma

Virus-related hepatocellular carcinoma (HCC) pathogenesis involves liver inflammation, therefore, despite successful treatment, hepatitis C virus (HCV) may progress to HCC from initiated liver cirrhosis. Cytotoxic T cells (Tcs) are known to be involved in HCV-related cirrhotic complications and HCC pathogenesis. The inhibitory checkpoint leukocyte-associated immunoglobulin-like receptor-1 (LAIR-1) is expressed on Tcs. Therefore, we aimed to determine whether the Tc expression level of LAIR-1 is associated with HCC progression and to evaluate LAIR-1 expression as a noninvasive biomarker for HCC progression in the context of liver cirrhosis related to HCV genotype 4 (G4) in Egyptian patients’ peripheral venous blood liquid biopsy. A total of 64 patients with HCC and 37 patients with liver cirrhosis were enrolled in this case-controlled study, and their LAIR-1 expression on Tc related to the progression of liver cirrhosis was examined and compared to that of the apparently healthy control group (n = 20). LAIR-1 expression was analyzed using flow cytometry. Results: The HCC group had significantly higher LAIR-1 expression on Tc and percentage of Tc positive for LAIR-1 (LAIR-1+Tc%) than the HCV G4-related liver cirrhosis group. LAIR-1+Tc% was correlated with the HCC surrogate tumor marker AFP (r = 0.367, p = 0.001) and insulin resistance and inflammation prognostic ratios/indices. A receiver operating characteristic (ROC) curve revealed that adding LAIR-1+Tc% to AFP can distinguish HCC transformation in the Egyptian patients’ cohort. Upregulated LAIR-1 expression on Tc could be a potential screening noninvasive molecular marker for chronic inflammatory HCV G4 related liver cirrhosis. Moreover, LAIR-1 expression on Tc may be one of the players involved in the progression of liver cirrhosis to HCC.     

Oligomerization of Human Cystatin C—An Amyloidogenic Protein: An Analysis of Small Oligomeric Subspecies

Human cystatin C (HCC), an amyloidogenic protein, forms dimers and higher oligomers (trimers, tetramers and donut like large oligomers) via a domain-swapping mechanism. The aim of this study was the characterization of the HCC oligomeric states observed within the pH range from 2.2 to 10.0 and also in conditions promoting oligomerization. The HCC oligomeric forms obtained in different conditions were characterized using size exclusion chromatography, dynamic light scattering and small-angle X-ray scattering. The marked ability of HCC to form tetramers at low pH (2.3 or 3.0) and dimers at pH 4.0–5.0 was observed. HCC remains monomeric at pH levels above 6.0. Based on the SAXS data, the structure of the HCC tetramer was proposed. Changes in the environment (from acid to neutral) induced a breakdown of the HCC tetramers to dimers. The tetrameric forms of human cystatin C are formed by the association of the dimers without a domain-swapping mechanism. These observations were confirmed by their dissociation to dimers at pH 7.4.     

The PINK1 p.Asn521Thr Variant Is Associated with Earlier Disease Onset in GRN/C9orf72 Frontotemporal Lobar Degeneration

Genetic frontotemporal lobar degeneration (FTLD) is characterized by heterogeneous phenotypic expression, with a disease onset highly variable even in patients carrying the same mutation. Herein we investigated if variants in lysosomal genes modulate the age of onset both in FTLD due to GRN null mutations and C9orf72 expansion. In a total of 127 subjects (n = 74 GRN mutations and n = 53 C9orf72 expansion carriers), we performed targeted sequencing of the top 98 genes belonging to the lysosomal pathway, selected based on their high expression in multiple brain regions. We described an earlier disease onset in GRN/C9orf72 pedigrees in subjects carrying the p.Asn521Thr variant (rs1043424) in PTEN-induced kinase 1 (PINK1), a gene that is already known to be involved in neurodegenerative diseases. We found that: (i) the PINK1 rs1043424 C allele is significantly associated with the age of onset; (ii) every risk C allele increases hazard by 2.11%; (iii) the estimated median age of onset in homozygous risk allele carriers is 10–12 years earlier than heterozygous/wild type homozygous subjects. A replication study in GRN/C9orf72 negative FTLD patients confirmed that the rs1043424 C allele was associated with earlier disease onset (−5.5 years in CC versus A carriers). Understanding the potential mechanisms behind the observed modulating effect of the PINK1 gene in FTLD might prove critical for identifying biomarkers and/or designing drugs to modify the age of onset, especially in GRN/C9orf72-driven disease.     

Curcumin Sensitises Cancerous Kidney Cells to TRAIL Induced Apoptosis via Let-7C Mediated Deregulation of Cell Cycle Proteins and Cellular Metabolism

Targeted therapies are the most attractive options in the treatment of different tumours, including kidney cancers. Such therapies have entered a golden era due to advancements in research, breakthroughs in scientific knowledge, and a better understanding of cancer therapy mechanisms, which significantly improve the survival rates and life expectancy of patients. The use of tumour necrosis factor (TNF)-related apoptosis inducing ligand (TRAIL) as an anticancer therapy has attracted the attention of the scientific community and created great excitement due to its selectivity in targeting cancerous cells with no toxic impacts on normal tissues. However, clinical studies disappointingly showed the emergence of resistance against TRAIL. This study aimed to employ curcumin to sensitise TRAIL-resistant kidney cancerous ACHN cells, as well as to gain insight into the molecular mechanisms of TRAIL sensitization. Curcumin deregulated the expression of apoptosis-regulating micro Ribonucleic Acid (miRNAs), most notably, let-7C. Transfecting ACHN cells with a let-7C antagomir significantly increased the expression of several cell cycle protein, namely beta (β)-catenin, cyclin dependent kinase (CDK)1/2/4/6 and cyclin B/D. Further, it overexpressed the expression of the two key glycolysis regulating proteins including hypoxia-inducible factor 1-alpha (HIF-1α) and pyruvate dehydrogenase kinase 1 (PDK1). Curcumin also suppressed the expression of the overexpressed proteins when added to the antagomir transfected cells. Overall, curcumin targeted ACHN cell cycle and cellular metabolism by promoting the differential expression of let-7C. To the best of our knowledge, this is the first study to mechanistically report the cancer chemosensitisation potential of curcumin in kidney cancer cells via induction of let-7C.     

Compound C Inhibits Renca Renal Epithelial Carcinoma Growth in Syngeneic Mouse Models by Blocking Cell Cycle Progression,Adhesion and Invasion

Compound C (CompC), an inhibitor of AMP-activated protein kinase, reduces the viability of various renal carcinoma cells. The molecular mechanism underlying anti-proliferative effect was investigated by flow cytometry and western blot analysis in Renca cells. Its effect on the growth of Renca xenografts was also examined in a syngeneic BALB/c mouse model. Subsequent results demonstrated that CompC reduced platelet-derived growth factor receptor signaling pathways and increased ERK1/2 activation as well as reactive oxygen species (ROS) production. CompC also increased the level of active Wee1 tyrosine kinase (P-Ser⁶⁴²-Wee1) and the inactive form of Cdk1 (P-Tyr¹⁵-Cdk1) while reducing the level of active histone H3 (P-Ser¹⁰-H3). ROS-dependent ERK1/2 activation and sequential alterations in Wee1, Cdk1, and histone H3 might be responsible for the CompC-induced G2/M cell cycle arrest and cell viability reduction. In addition, CompC reduced the adhesion, migration, and invasion of Renca cells in the in vitro cell systems, and growth of Renca xenografts in the BALB/c mouse model. Taken together, the inhibition of in vivo tumor growth by CompC may be attributed to the blockage of cell cycle progression, adhesion, migration, and invasion of tumor cells. These findings suggest the therapeutic potential of CompC against tumor development and progression.     

Quantitative Plasma Proteomics to Identify Candidate Biomarkers of Relapse in Pediatric/Adolescent Hodgkin Lymphoma

Classical pediatric Hodgkin Lymphoma (HL) is a rare malignancy. Therapeutic regimens for its management may be optimized by establishing treatment response early on. The aim of this study was to identify plasma protein biomarkers enabling the prediction of relapse in pediatric/adolescent HL patients treated under the pediatric EuroNet-PHL-C2 trial. We used untargeted liquid chromatography-tandem mass spectrometry (LC-MS/MS)-based proteomics at the time of diagnosis—before any therapy—as semiquantitative method to profile plasma proteins specifically associated with relapse in 42 children with nodular sclerosing HL. In both the exploratory and the validation cohorts, six proteins (apolipoprotein E, C4b-binding protein α chain, clusterin, fibrinogen γ chain, prothrombin, and vitronectin) were more abundant in the plasma of patients whose HL relapsed (|fold change| ≥ 1.2, p < 0.05, Student’s t-test). Predicting protein function with the Gene Ontology classification model, the proteins were included in four biological processes (p < 0.01). Using immunoblotting and Luminex assays, we validated two of these candidate biomarkers—C4b-binding protein α chain and clusterin—linked to innate immune response function (GO:0045087). This study identified C4b-binding protein α chain and clusterin as candidate early plasma biomarkers of HL relapse, and important for the purpose of shedding light on the molecular scenario associated with immune response in patients treated under the EuroNet-PHL-C2 trial.     

Clostridiumnovyi’s Alpha-Toxin Changes Proteome and Phosphoproteome of HEp-2 Cells

C. novyi type A produces the alpha-toxin (TcnA) that belongs to the large clostridial glucosylating toxins (LCGTs) and is able to modify small GTPases by N-acetylglucosamination on conserved threonine residues. In contrast, other LCGTs including Clostridioides difficile toxin A and toxin B (TcdA; TcdB) modify small GTPases by mono-o-glucosylation. Both modifications inactivate the GTPases and cause strong effects on GTPase-dependent signal transduction pathways and the consequent reorganization of the actin cytoskeleton leading to cell rounding and finally cell death. However, the effect of TcnA on target cells is largely unexplored. Therefore, we performed a comprehensive screening approach of TcnA treated HEp-2 cells and analyzed their proteome and their phosphoproteome using LC-MS-based methods. With this data-dependent acquisition (DDA) approach, 5086 proteins and 9427 phosphosites could be identified and quantified. Of these, 35 proteins were found to be significantly altered after toxin treatment, and 1832 phosphosites were responsive to TcnA treatment. By analyzing the TcnA-induced proteomic effects of HEp-2 cells, 23 common signaling pathways were identified to be altered, including Actin Cytoskeleton Signaling, Epithelial Adherens Junction Signaling, and Signaling by Rho Family GTPases. All these pathways are also regulated after application of TcdA or TcdB of C. difficile. After TcnA treatment the regulation on phosphorylation level was much stronger compared to the proteome level, in terms of both strength of regulation and the number of regulated phosphosites. Interestingly, various signaling pathways such as Signaling by Rho Family GTPases or Integrin Signaling were activated on proteome level while being inhibited on phosphorylation level or vice versa as observed for the Role of BRCA1 in DNA Damage Response. ZIP kinase, as well as Calmodulin-dependent protein kinases IV & II, were observed as activated while Aurora-A kinase and CDK kinases tended to be inhibited in cells treated with TcnA based on their substrate regulation pattern.     

Coagulation Factor XIIIa and Activated Protein C Activate Platelets via GPVI and PAR1

Platelet and coagulation activation are highly reciprocal processes driven by multi-molecular interactions. Activated platelets secrete several coagulation factors and expose phosphatidylserine, which supports the activation of coagulation factor proteins. On the other hand, the coagulation cascade generates known ligands for platelet receptors, such as thrombin and fibrin. Coagulation factor (F)Xa, (F)XIIIa and activated protein C (APC) can also bind to platelets, but the functional consequences are unclear. Here, we investigated the effects of the activated (anti)coagulation factors on platelets, other than thrombin. Multicolor flow cytometry and aggregation experiments revealed that the ‘supernatant of (hirudin-treated) coagulated plasma’ (SCP) enhanced CRP-XL-induced platelet responses, i.e., integrin α_IIbβ₃ activation, P-selectin exposure and aggregate formation. We demonstrated that FXIIIa in combination with APC enhanced platelet activation in solution, and separately immobilized FXIIIa and APC resulted in platelet spreading. Platelet activation by FXIIIa was inhibited by molecular blockade of glycoprotein VI (GPVI) or Syk kinase. In contrast, platelet spreading on immobilized APC was inhibited by PAR1 blockade. Immobilized, but not soluble, FXIIIa and APC also enhanced in vitro adhesion and aggregation under flow. In conclusion, in coagulation, factors other than thrombin or fibrin can induce platelet activation via GPVI and PAR receptors.     

基于智能型电动机保护器的监控系统设计与实现

针对目前电动机保护器的在使用过程中的特点,介绍了一种基于智能型的可通信电动机保护器的监控系统的设计方案。上位机与可通信电动机保护器依靠现场总线技术组成监控网络。简单介绍了现场总线技术、MODBUS通信协议,用Visualc＋＋设计了一种上位机监控软件,并对监控软件的关键技术做了介绍。