Enhanced Cell Survival Research Articles

Conducting Polymer-ECM Scaffolds for Human Neuronal Cell Differentiation.

3D cell culture formats more closely resemble tissue architecture complexity than 2D systems, which are lacking most of the cell-cell and cell-microenvironment interactions of the in vivo milieu. Scaffold-based systems integrating natural biomaterials are extensively employed in tissue engineering to improve cell survival and outgrowth, by providing the chemical and physical cues of the natural extracellular matrix (ECM). Using the freeze-drying technique, porous 3D composite scaffolds consisting of poly(3,4-ethylene-dioxythiophene) doped with polystyrene sulfonate (PEDOT:PSS), containing ECM components (i.e., collagen, hyaluronic acid, and laminin) are engineered for hosting neuronal cells. The resulting scaffolds exhibit a highly porous microstructure and good conductivity, determined by scanning electron microscopy and electrochemical impedance spectroscopy, respectively. These supports boast excellent mechanical stability and water uptake capacity, making them ideal candidates for cell infiltration. SH-SY5Y human neuroblastoma cells show enhanced cell survival and proliferation in the presence of ECM compared to PEDOT:PSS alone. Whole-cell patch-clamp recordings acquired from differentiated SHSY5Y cells in the scaffolds demonstrate that ECM constituents promote neuronal differentiation in situ. These findings reinforce the usability of 3D conducting supports as engineered highly biomimetic and functional in vitro tissue-like platforms for drug or disease modeling.

Recent Advances in Cell and Functional Biomaterial Treatment for Spinal Cord Injury.

Spinal cord injury (SCI) is a devastating central nervous system disease caused by accidental events, resulting in loss of sensory and motor function. Considering the multiple effects of primary and secondary injuries after spinal cord injury, including oxidative stress, tissue apoptosis, inflammatory response, and neuronal autophagy, it is crucial to understand the underlying pathophysiological mechanisms, local microenvironment changes, and neural tissue functional recovery for preparing novel treatment strategies. Treatment based on cell transplantation has become the forefront of spinal cord injury therapy. The transplanted cells provide physical and nutritional support for the damaged tissue. At the same time, the implantation of biomaterials with specific biological functions at the site of the SCI has also been proved to improve the local inhibitory microenvironment and promote axonal regeneration, etc. The combined transplantation of cells and functional biomaterials for SCI treatment can result in greater neuroprotective and regenerative effects by regulating cell differentiation, enhancing cell survival, and providing physical and directional support for axon regeneration and neural circuit remodeling. This article reviews the pathophysiology of the spinal cord, changes in the microenvironment after injury, and the mechanisms and strategies for spinal cord regeneration and repair. The article will focus on summarizing and discussing the latest intervention models based on cell and functional biomaterial transplantation and the latest progress in combinational therapies in SCI repair. Finally, we propose the future prospects and challenges of current treatment regimens for SCI repair, to provide references for scientists and clinicians to seek better SCI repair strategies in the future.

Transcript analysis and expression of the glbO gene, encoding truncated hemoglobin,O, of M. Smegmatis implicate its role under hypoxia and oxidative stress

Although truncated hemoglobin O, (trHbO), is ubiquitous among mycobacteria, its physiological function is not very obvious and may be diverse. In an attempt to understand role of trHbO in cellular metabolism of a non-pathogenic mycobacterium, we analysed expression profile of the glbO gene, encoding trHbO, in M. smegmatis and studied implications of its overexpression on physiology of its host under different environmental conditions. Quantitative RT-PCR indicated that transcript level of the glbO gene remains low at a basal level under aerobic growth cycle of M. smegmatis but its level gets induced significantly during low oxygen, oxidative stress and macrophage infection. Overexpression of the glbO gene enhanced growth of M. smegmatis under hypoxia, promoted pellicle biofilm formation and provided resistance towards oxidative stress. Additionally, glbO gene overexpressing M. smegmatis exhibited enhanced cell survival over isogenic control cells and altered the level of pro- and anti- inflammatory cytokines during intracellular infection. These results suggested important role of trHbO, in supporting the cellular metabolism and survival of M, smegmatis both under low oxygen and oxidative stress.

HIF-1α Stabilization Boosts Pulp Regeneration by Modulating Cell Metabolism

Stem cell–based therapeutics is a promising strategy in dental pulp regeneration. However, low cell viability after transplantation in vivo due to the ischemic microenvironment is still a critical challenge for future clinical application. With the aim of improving postimplantation cell survival and pulp tissue regeneration, stem cells from human exfoliated deciduous teeth (SHED) were preconditioned to a hypoxic condition by hypoxia-inducible factor 1α (HIF-1α) stabilization via knockdown of prolyl hydroxylase domain-containing protein 2 (PHD2) using lentiviral short hairpin RNA. HIF-1α–stabilized SHED were encapsulated in PuraMatrix hydrogel, injected into root canals of human tooth fragments, and implanted in the subcutaneous space of immunodeficient mice. After 28 d, enhanced dental pulp–like tissue formation was observed with a significantly higher level of vascularization, which could be attributed to both endothelial differentiation of SHED and recruitment of host blood vessels. Furthermore, dentin-like tissue formation in vivo and accelerated odontogenic/osteogenic differentiation both in vivo and in vitro were observed. At 7 d postimplantation, significantly less DNA damage and higher Ki67 expression were detected in the HIF-1α–stabilized SHED group compared with the control SHED. Accordingly, cell viability assay and staining for Ki67 and apoptotic cells in vitro showed that HIF-1α stabilization could decrease cell apoptosis and enhance cell survival significantly. We demonstrated that PI3K/AKT pathway activation had resulted in low caspase 3 expression in HIF-1α–stabilized SHED in hypoxic conditions. Furthermore, we found that HIF-1α–induced cell survival could also be attributed to the upregulated expression of PDK1, HK2, and Glut1, which contributes to the maintenance of reactive oxygen species homeostasis and metabolic adaptation in hypoxia. In addition, we identified Smad7 as 1 of the top 3 upregulated genes through RNA sequencing in HIF-1α–stabilized SHED and demonstrated its essential role in HK2 and Glut1 upregulation. Taken together, HIF-1α stabilization enhances cell survival of SHED through modulating various target genes and potential signaling pathways, as well as odontogenic tissue formation during dental pulp regeneration, which could benefit stem cell–based therapy in general.

Bioinformatics Analysis Reveals FOXM1/BUB1B Signaling Pathway as a Key Target of Neosetophomone B in Human Leukemic Cells: A Gene Network-Based Microarray Analysis.

Abnormal expression of Forkhead box protein M1 (FOXM1) and serine/threonine kinase Budding uninhibited by benzimidazoles 1 (BUB1B) contributes to the development and progression of several cancers, including chronic myelogenous leukemia (CML). However, the molecular mechanism of the FOXM1/BUB1B regulatory network and the role of Neosetophomone-B (NSP-B) in leukemia remains unclear. NSP-B, a meroterpenoid fungal secondary metabolite, possesses anticancer potential in human leukemic cells lines; however, the underlying mechanism has not been elucidated. The present study aimed to explore the role of NSP-B on FOXM1/BUB1B signaling and the underlying molecular mechanism of apoptosis induction in leukemic cells. We performed gene expression profiling of NSP-B-treated and untreated leukemic cells to search for differentially expressed genes (DEGs). Interestingly BUB1B was found to be significantly downregulated (logFC -2.60, adjusted p = 0.001) in the treated cell line with the highest connectivity score among cancer genes. Analysis of TCGA data revealed overexpression of BUB1B compared to normal in most cancers and overexpression was associated with poor prognosis. BUB1B also showed a highly significant positive correlation with FOXM1 in all the TCGA cancer types. We used human leukemic cell lines (K562 and U937) as an in vitro study model to validate our findings. We found that NSP-B treatment of leukemic cells suppressed the expression of FOXM1 and BUB1B in a dose-dependent manner. In addition, NSP-B also resulted in the downregulation of FOXM1-regulated genes such as Aurora kinase A, Aurora kinase B, CDK4, and CDK6. Suppression of FOXM1 either by siRNA or NSP-B reduced BUB1B expression and enhanced cell survival inhibition and induction of apoptosis. Interestingly combination treatment of thiostrepton and NSP-B suppressed of cell viability and inducted apoptosis in leukemic cells via enhancing the activation of caspase-3 and caspase-8 compared with single-agent treatment. These results demonstrate the important role of the FOXM1/BUB1B pathway in leukemia and thus a potential therapeutic target.

Upregulating microRNA‐210 to Inhibit Apoptosis of Neural Stem Cells with an MRI–Visible Nanomedicine for Stroke Therapy

Transplantation of neural stem cells (NSCs) is a promising paradigm for treating stroke. However, the poor survival of transplanted NSCs greatly limits the therapeutic potential. microRNA‐210 (miR‐210), a key hypoxia‐regulated miRNA, can enhance cell survival by targeting the expression of multiple apoptosis‐related genes, such as caspase‐8‐associated protein‐2 (casp8ap2), Bax, and Bcl‐2. Meanwhile, a noninvasive cell‐tracking method is also indispensable for monitoring the in vivo cell‐based therapy. Herein, an MRI–visible nanomedicine is developed to codeliver superparamagnetic iron oxide (SPIO) nanoparticles and miR‐210 into NSCs. This therapeutic nanomedicine not only promotes the survival of NSCs via upregulating miR‐210 to inhibit NSCs apoptosis but also allows an in vivo tracking of transplanted NSCs with MRI. The enhanced NSCs survivability significantly promotes the structural and functional recovery after stroke onset, which highlights the great potential of the multifunctional nanomedicine to improve the therapeutic efficacy of NSCs for stroke treatment.

Abstract 408: Development of a first-in-class notch transcription factor inhibitor CB-103 to overcome resistance to chemotherapy and targeted therapies

Abstract Human cancers possess striking ability to develop resistance to Standard of Care (SoC) designed to target specific hallmarks of cancer. This capability to develop resistance is in part due to cancer cell’s ability to activate alternative pathways and modulate tumor microenvironment to enhance cell survival, evade cell death and immunosurveillance. One such mechanism of developing resistance to SoC is the activation of the NOTCH pathway. NOTCH signaling is a developmental pathway which is known to be activated in several human cancers as a key oncogenic hit. In addition to its role as an oncogenic driver, activation of NOTCH is also known to induce resistance to several anti-cancer therapeutics in breast cancer and colorectal cancer. NOTCH signaling is known to cooperate with parallel oncogenic signals to impede activity of several drugs as a single agent. Previous agents targeting the NOTCH pathway upstream of the transcription complex were halted early in clinical development due to dose-limiting toxicities.Here we report activity of a novel first-in-class clinical stage CSL-NICD inhibitor in a panel of cell lines, xenograft and PDX models of human cancers in combination with several chemo and targeted therapies. Guided by in vitro and in vivo studies pre-clinical studies, CB-103 is currently being investigated in clinical trials as a single agent and in combination with SoC. Preliminary pre-clinical and clinical data shows desirable safety and efficacy profile of CB-103 enabling clinical development in combination with several therapeutic agents. Citation Format: Michele Vigolo, Charlotte Urech, Sebastien Lamy, Samarpan Majumder, Lucio Miele, Rajwinder Lehal. Development of a first-in-class notch transcription factor inhibitor CB-103 to overcome resistance to chemotherapy and targeted therapies [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2022; 2022 Apr 8-13. Philadelphia (PA): AACR; Cancer Res 2022;82(12_Suppl):Abstract nr 408.

Abstract 3017: The poly-aneuploid cancer cell state utilizes lipid droplets as a mechanism of survival

Abstract Cancer is the second leading cause of all death in the world, killing over 10 million people in 2020. Metastatic cancer is responsible for 90% of all cancer related deaths. Metastatic disease remains incurable as a population of cancer cells within a tumor are or become resistant to all known natural and synthetic therapies. Despite decades of research, the source of this therapeutic resistance still remains largely undefined. We have identified that the polyaneuploid cancer cell (PACC) transition state is associated with therapeutic resistance. This state of cells are large in size, have high genomic content, and are the only cell remaining in tissue culture conditions after high dose chemotherapy. The PACC transition state has been documented in many different cancer types but their survival mechanisms have remained largely unexplored. We have discovered that the PACC transition state contains a higher number of lipid droplets (LDs) than the parental cells they are derived from. LDs are central anti-lipotoxic organelles that control fatty acid, diacylglycerol, cholesterol, and ceramide lipotoxicity by sequestering toxic lipids into inert triglycerides, cholesterol esters, and acylceramides. LDs are important in cancer as higher levels of LDs are associated with higher tumor aggressiveness and chemotherapy resistance. Utilizing TCGA data we discovered that PLIN1, an exclusive LD surface protein, is higher in prostate cancer patients with lower disease-free survival, higher Gleason score, and higher tumor stage. This indicates that more aggressive prostate cancer, which has been shown to have a higher number of PACCs in patient samples, is associated with higher levels of LDs. We hypothesize that the PACC state is surviving chemotherapeutic stress by sequestering toxic lipids into LDs to enhance cell survival. To determine when and how LDs form in the PACC transition state we used live cell imaging, western blotting, flow cytometry, and staining of key lipid droplet proteins. We utilized four different cancer cell lines (PC3, DU145, LNCaP, MDA-MB-231) with three different classes of chemotherapies (cisplatin, docetaxel, etoposide). We established that LDs form in the PACC transition state as early as three hours post chemotherapy treatment. This suggests that the PACC state utilizes a key mechanism of sequestering toxic lipids into LDs as soon as therapy is applied to avoid lipotoxicity and cell death. We also discovered that hormone-sensitive lipase (HSL), an enzyme that mobilizes fats stored in LDs, was down in the PACC state compared to parental cells, indicating that these LDs are not readily being broken down after formation. Our future work will focus on inhibiting LD formation to increase cellular lipotoxicity in the PACC transition state leading to PACC death and an increase in overall cancer cells killed. Citation Format: Laurie Gayle Kostecka, Sarah Amend, Kenneth Pienta. The poly-aneuploid cancer cell state utilizes lipid droplets as a mechanism of survival [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2022; 2022 Apr 8-13. Philadelphia (PA): AACR; Cancer Res 2022;82(12_Suppl):Abstract nr 3017.

Abstract 6034: Characterization of a novel syngeneic cSCC mouse model to advance immunotherapy treatments

Abstract Cutaneous Squamous Cell Carcinoma (cSCC) is the second most common malignancy diagnosed in Caucasians with an incidence of about one million cases per year. TP53 and p16 are the most common mutated genes in cSCC. Previous studies of cSCC have shown that patients with both p53 and p16 mutations have lower survival rates. There are currently no syngeneic mouse preclinical models to accelerate the efficacy of immunotherapy combinations to treat cSCC. Our laboratory has generated several genetically engineered mouse models (GEMMs) containing tissue-specific p53 and p16 gene mutations. Recently, we have immortalized three murine cSCC cell lines (cSCC-5690, cSCC-8006, and cSCC-8271) derived from spontaneous skin tumors from GEMMs. Therefore, we hypothesized that the double mutant cell line cSCC-5690 enhances cell survival, tumor aggressiveness, and has lower sensitivity to antibody immunotherapy. Protein and RNA expression of the cell lines were validated by western blotting and qRT-PCR assays. Sphere and soft agar assays were used to evaluate the stem cell population and tumorigenicity of the cell lines. Then, we examined the chemosensitivity with cisplatin through clonogenic assays. To further study the tumorigenicity of cells, we evaluated in vivo tumor growth on C57BL/6J immunocompetent mice by injecting subcutaneously the tumor cell lines. The three cell lines expressed different levels of keratin 14 (K14), but cSCC 5690 and 8006 expressed high levels of genes related to oncogenic pathways as well as mesenchymal markers. The cell line cSCC-5690 expressing mutant p53 R172H with p16 gene deletion, showed an enhanced sphere, and colony tumor formation and both tumorigenic cell lines (cSCC-5690, 8006) showed different sensitivities to cisplatin treatment. A higher tumor growth rate was observed with cSCC-5690 compared to cSCC-8006, while cSCC-8271 showed no tumor growth. Immunohistochemistry studies demonstrated that both cSCC-5690 and 8006 tumors expressed vimentin but only cSCC-5690 expressed K14. The most aggressive syngeneic tumor model (cSCC-5690) was used to evaluate the anti-tumor efficiency of anti-PD1, anti-CD47 antibodies, and STING-agonist. Preliminary results revealed a significant tumor response only to STING agonist (c-di-GMP) which activates the innate and adaptive immune response. Therefore, we were able to characterize all three cell lines and established a new mouse model of cSCC as a relevant platform for immunotherapy studies. Citation Format: Alanis Enid Rodriguez Rosario, Roberto Rangel, Jeffrey Myers. Characterization of a novel syngeneic cSCC mouse model to advance immunotherapy treatments [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2022; 2022 Apr 8-13. Philadelphia (PA): AACR; Cancer Res 2022;82(12_Suppl):Abstract nr 6034.

Abstract 3991: Ts65Dn Down syndrome mice reveal a potential therapeutic vulnerability in a BBN bladder cancer model and subset of bladder cancer cell lines

Abstract Trisomy 21, Down Syndrome, results in a constellation of symptoms including increased risk for leukemia. In contrast, mounting evidence indicates that people with Down Syndrome have lower rates of numerous solid tumors, particularly carcinogen driven cancers, even when adjusted for age. The mechanisms that dictate why this occurs are not well understood but could potentially yield actionable targets for treating or preventing cancer formation. We hypothesized Ts65Dn mice, a murine model of Down Syndrome, would be protected against BCa formation and could be evaluated as a genetic basis for the discovery of novel therapeutic targets in bladder cancer. Using the well characterized N-butyl-N-(4-hydroxybutyl) nitrosamine (BBN) chemical carcinogen bladder cancer model we initiated tumors in Ts65Dn and WT mice. After 16 weeks BBN exposure, TS65Dn mice were found to have smaller bladder tumors by weight with a trend toward lower stages as compared to WT animals. RNA-sequencing was used to assess broad genomic changes in the tumors. Reactome analysis of the RNA-sequencing data indicated pathways associated with mitosis and the cell cycle were most affected in Ts65Dn mice, including downregulation of pathways associated with Aurora Kinase B (AURKB). AURKB is a pro-tumorigenic kinase known to enhance cell survival and proliferation. Bioinformatics analysis indicates that while AURKB is broadly expressed in most tissues, it is potently upregulated in bladder cancers, offering a potential therapeutic target. Using the AURKB specific inhibitor barasertib-HQPA we evaluated drug sensitivity in a set of BCa cell lines. Cell lines associated with a basal subtype gene signature (5637, HT-1376) were largely susceptible to barasertib with IC50 values in nM concentrations, whereas other BCa cell lines (HTB-5, T24) had IC50 values nearly 1,000X higher. We next assessed histone H3 phosphorylation, a known substrate of AURKB, to validate drug efficacy. Interestingly, inhibition of histone H3 phosphorylation was noted in all cell lines at nM concentrations, indicating that while barasertib is functionally active in both sets of cells, the anti-proliferative activity is not dependent upon phosphorylation of histone H3, but rather other factors that we have yet to determine. In conclusion, we have found Ts65Dn mice have reduced tumor formation when exposed to the BCa carcinogen BBN and furthermore, have identified AURKB as a potential therapeutic target. Use of Ts65Dn mice may be a relevant model for identifying potentially undefined tumorigenic agents in multiple cancers including bladder. Citation Format: Binh Vo, Erika Abbott, Carolyn J. Vivian, Xena Moore, Ganeshkumar Rajendran, Katie Dennis, Benjamin L. Woolbright, Scott J. Weir, John A. Taylor. Ts65Dn Down syndrome mice reveal a potential therapeutic vulnerability in a BBN bladder cancer model and subset of bladder cancer cell lines [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2022; 2022 Apr 8-13. Philadelphia (PA): AACR; Cancer Res 2022;82(12_Suppl):Abstract nr 3991.

Neural stem cell-derived exosome as a nano-sized carrier for BDNF delivery to a rat model of ischemic stroke

Our previous study demonstrated the potential therapeutic role of human neural stem cell-derived exosomes (hNSC-Exo) in ischemic stroke. Here, we loaded brain-derived neurotrophic factor (BDNF) into exosomes derived from NSCs to construct engineered exosomes (BDNF-hNSC-Exo) and compared their effects with those of hNSC-Exo on ischemic stroke both in vitro and in vivo. In a model of H2O2-induced oxidative stress in NSCs, BDNF-hNSC-Exo markedly enhanced cell survival. In a rat middle cerebral artery occlusion model, BDNF-hNSC-Exo not only inhibited the activation of microglia, but also promoted the differentiation of endogenous NSCs into neurons. These results suggest that BDNF can improve the function of NSC-derived exosomes in the treatment of ischemic stroke. Our research may support the clinical use of other neurotrophic factors for central nervous system diseases.

Mitochondrial matrix-localized Src kinase regulates mitochondrial morphology

The architecture of mitochondria adapts to physiological contexts: while mitochondrial fragmentation is usually associated to quality control and cell death, mitochondrial elongation often enhances cell survival during stress. Understanding how these events are regulated is important to elucidate how mitochondrial dynamics control cell fate. Here, we show that the tyrosine kinase Src regulates mitochondrial morphology. Deletion of Src increased mitochondrial size and reduced cellular respiration independently of mitochondrial mass, mitochondrial membrane potential or ATP levels. Re-expression of Src targeted to the mitochondrial matrix, but not of Src targeted to the plasma membrane, rescued mitochondrial morphology in a kinase activity-dependent manner. These findings highlight a novel function for Src in the control of mitochondrial dynamics.

IL-27 improves adoptive CD8+ T cells' antitumor activity via enhancing cell survival and memory T cell differentiation.

IL‐27 is an anti‐inflammatory cytokine that triggers enhanced antitumor immunity, particularly cytotoxic T lymphocyte responses. In the present study, we sought to develop IL‐27 into a therapeutic adjutant for adoptive T cell therapy using our well‐established models. We have found that IL‐27 directly improved the survival status and cytotoxicity of adoptive OT‐1 CD8+ T cells in vitro and in vivo. Meanwhile, IL‐27 treatment programs memory T cell differentiation in CD8+ T cells, characterized by upregulation of genes associated with T cell memory differentiation (T‐bet, Eomes, Blimp1, and Ly6C). Additionally, we engineered the adoptive OT‐1 CD8+ T cells to deliver IL‐27. In mice, the established tumors treated with OT‐1 CD8+ T‐IL‐27 were completely rejected, which demonstrated that IL‐27 delivered via tumor antigen–specific T cells enhances adoptive T cells’ cancer immunity. To our knowledge, this is the first application of CD8+ T cells as a vehicle to deliver IL‐27 to treat tumors. Thus, this study demonstrates IL‐27 is a feasible approach for enhancing CD8+ T cells’ antitumor immunity and can be used as a therapeutic adjutant for T cell adoptive transfer to treat cancer.

New insights into the molecular mechanisms of ROR1, ROR2, and PTK7 signaling from the proteomics and pharmacological modulation of ROR1 interactome

ROR1, ROR2, and PTK7 are Wnt ligand-binding members of the receptor tyrosine kinase family. Despite their lack of catalytic activity, these receptors regulate skeletal, cardiorespiratory, and neurological development during embryonic and fetal stages. However, their overexpression in adult tissue is strongly connected to tumor development and metastasis, suggesting a strong pharmacological potential for these molecules. Wnt5a ligand can activate these receptors, but lead to divergent signaling and functional outcomes through mechanisms that remain largely unknown. Here, we developed a cellular model by stably expressing ROR1, ROR2, and PTK7 in BaF3 cells that allowed us to readily investigate side-by-side their signaling capability and functional outcome. We applied proteomic profiling to BaF3 clones and identified distinctive roles for ROR1, ROR2, and PTK7 pseudokinases in modulating the expression of proteins involved in cytoskeleton dynamics, apoptotic, and metabolic signaling. Functionally, we show that ROR1 expression enhances cell survival and Wnt-mediated cell proliferation, while ROR2 and PTK7 expression is linked to cell migration. We also demonstrate that the distal C-terminal regions of ROR1 and ROR2 are required for receptors stability and downstream signaling. To probe the pharmacological modulation of ROR1 oncogenic signaling, we used affinity purification coupled to mass spectrometry (AP-MS) and proximity-dependent biotin identification (BioID) to map its interactome before and after binding of GZD824, a small molecule inhibitor previously shown to bind to the ROR1 pseudokinase domain. Our findings bring new insight into the molecular mechanisms of ROR1, ROR2, and PTK7, and highlight the therapeutic potential of targeting ROR1 with small molecule inhibitors binding to its vestigial ATP-binding site.

Cancer Relevance of Human Genes.

We hypothesize that genes that directly or indirectly interact with core cancer genes (CCGs) in a comprehensive gene-gene interaction network may have functional importance in cancer. We categorized 12 767 human genes into CCGs (n = 468), 1 (n = 5467), 2 (n = 5573), 3 (n = 915), and more than 3 steps (n = 416) removed from the nearest CCG in the Search Tool for the Retrieval of Interacting Genes/Proteins network. We estimated cancer-relevant functional importance in these neighborhood categories using 1) gene dependency score, which reflects the effect of a gene on cell viability after knockdown; 2) somatic mutation frequency in The Cancer Genome Atlas; 3) effect size that estimates to what extent a mutation in a gene enhances cell survival; and 4) negative selection pressure of germline protein-truncating variants in healthy populations. Cancer biology-related functional importance of genes decreases as their distance from the CCGs increases. Genes closer to cancer genes show greater connectedness in the network, have greater importance in maintaining cancer cell viability, are under greater negative germline selection pressure, and have higher somatic mutation frequency in cancer. Based on these 4 metrics, we provide cancer relevance annotation to known human genes. A large number of human genes are connected to CCGs and could influence cancer biology to various extent when dysregulated; any given mutation may be functionally important in one but not in another individual depending on genomic context.

Selective cleavage of ncRNA and antiviral activity by RNase2/EDN in THP1-induced macrophages

RNase2 is the member of the RNaseA family most abundant in macrophages. Here, we knocked out RNase2 in THP-1 cells and analysed the response to Respiratory Syncytial Virus (RSV). RSV induced RNase2 expression, which significantly enhanced cell survival. Next, by cP-RNAseq sequencing, which amplifies the cyclic-phosphate endonuclease products, we analysed the ncRNA population. Among the ncRNAs accumulated in WT vs KO cells, we found mostly tRNA-derived fragments (tRFs) and second miRNAs. Differential sequence coverage identified tRFs from only few parental tRNAs, revealing a predominant cleavage at anticodon and d-loops at U/C (B1) and A (B2) sites. Selective tRNA cleavage was confirmed in vitro using the recombinant protein. Likewise, only few miRNAs were significantly more abundant in WT vs RNase2-KO cells. Complementarily, by screening of a tRF & tiRNA array, we identified an enriched population associated to RNase2 expression and RSV exposure. The results confirm the protein antiviral action and provide the first evidence of its cleavage selectivity on ncRNAs.Graphical abstract

The Role of NEDD4 E3 Ubiquitin-Protein Ligases in Parkinson's Disease.

Parkinson’s disease (PD) is a debilitating neurodegenerative disease that causes a great clinical burden. However, its exact molecular pathologies are not fully understood. Whilst there are a number of avenues for research into slowing, halting, or reversing PD, one central idea is to enhance the clearance of the proposed aetiological protein, oligomeric α-synuclein. Oligomeric α-synuclein is the main constituent protein in Lewy bodies and neurites and is considered neurotoxic. Multiple E3 ubiquitin-protein ligases, including the NEDD4 (neural precursor cell expressed developmentally downregulated protein 4) family, parkin, SIAH (mammalian homologues of Drosophila seven in absentia), CHIP (carboxy-terminus of Hsc70 interacting protein), and SCFFXBL5 SCF ubiquitin ligase assembled by the S-phase kinase-associated protein (SKP1), cullin-1 (Cul1), a zinc-binding RING finger protein, and the F-box domain/Leucine-rich repeat protein 5-containing protein FBXL5), have been shown to be able to ubiquitinate α-synuclein, influencing its subsequent degradation via the proteasome or lysosome. Here, we explore the link between NEDD4 ligases and PD, which is not only via α-synuclein but further strengthened by several additional substrates and interaction partners. Some members of the NEDD4 family of ligases are thought to crosstalk even with PD-related genes and proteins found to be mutated in familial forms of PD. Mutations in NEDD4 family genes have not been observed in PD patients, most likely because of their essential survival function during development. Following further in vivo studies, it has been thought that NEDD4 ligases may be viable therapeutic targets in PD. NEDD4 family members could clear toxic proteins, enhancing cell survival and slowing disease progression, or might diminish beneficial proteins, reducing cell survival and accelerating disease progression. Here, we review studies to date on the expression and function of NEDD4 ubiquitin ligases in the brain and their possible impact on PD pathology.

Saikosaponin d alleviates high-fat-diet induced hepatic steatosis in hybrid grouper (Epinephelus lanceolatus♂ × Epinephelus fuscoguttatus♀) by targeting AMPK/PPARα pathway

High-fat diets generally cause the accumulation of hepatic steatosis in farmed fish tissues, which results in metabolic disorders, abnormal oxidative status and immune system suppression, seriously affecting sustainable aquaculture development. The main bioactive components of Bupleurum are saikosaponins, saikosaponins d (SSd) exhibiting the strongest pharmacological activity. The aim of this research was to examine the preventive and therapeutic benefits of SSd on hepatic steatosis, in addition to the underlying processes of hybrid groupers in vitro and in vivo. SSd was found to enhance cell survival in vitro by lowering apoptosis and decrease lipid accumulation in hepatocytes by reducing oxidative stress, restricting lipogenesis and enhancing lipid oxidation. In vivo, 300 hybrid groupers were fed SSd at dosages of 0, 100, 200, 400, and 800 mg/kg that was supplemented with a high-lipid basal diet for eight weeks. Pretreatment with SSd caused a raised expression in lipolysis-related genes (AMPKα, PPARα, CPT1, LPL) while decreasing the expression of adipogenesis-related genes in the liver of hybrid groupers (G6PD, ME1, FAS and DGAT2). Furthermore, kinsenoside was found to increase ATGL-mediated lipolysis, amplified by AMP-activated protein kinase (AMPK) activation, in addition to the hydrolysis of triglycerides to glycerol and fatty acids, which require transit into mitochondria for further oxidation. AMPK activation was found to increase ATGL expression for lipolysis, while also increasing CPT1 expression for fatty acid transport to the mitochondria. The findings of this study add to a growing body of evidence suggesting SSd therapy can be used for the prevention hepatic steatosis in hybrid groupers via the AMPK/PPAR pathway.

Telmisartan is the most effective ARB to increase adiponectin via PPARα in adipocytes.

Telmisartan and irbesartan are angiotensin II receptor blockers (ARBs) and reportedly stimulate adiponectin secretion from adipocytes via partial peroxisome proliferator-activated receptor γ (PPARγ) activation. However, quantitative evaluation among different ARBs has not been performed. Adiponectin exerts strong protection against a number of pathological events by suppressing cell death, inhibiting inflammation, and enhancing cell survival, while leptin promotes inflammation, oxidative stress, atherogenesis, and thrombosis. The aim of this study was to identify the most effective ARB enhancing adiponectin secretion without raising leptin secretion from human white adipocytes (HWAs). Among seven ARBs (azilsartan, candesartan, irbesartan, losartan, olmesartan, telmisartan, and valsartan), telmisartan was the most effective ARB for the increase of adiponectin secretion and irbesartan was the second, whereas the other ARBs at 1 µM had no effect on adiponectin secretion. GW9662, a PPARγ antagonist, completely blocked pioglitazone (PPARγ agonist)-induced adiponectin secretion and mRNA expression, whereas it unexpectedly blocked neither telmisartan- nor irbesartan-induced adiponectin secretion and mRNA expression but rather increased them. GW6471, PPARα antagonist, and siRNA for PPARα suppressed telmisartan- and irbesartan-induced adiponectin secretion, suggesting that PPARα is the main target of these ARBs to increase adiponectin secretion in HWAs. Leptin secretion was not affected by any ARBs at 1 µM and GW9662 significantly decreased the basal secretion of leptin, suggesting that basal leptin secretion is regulated in a PPARγ-dependent manner. We conclude that telmisartan is the most effective ARB to increase adiponectin secretion via PPARα without raising leptin secretion from HWAs.

Effect of pulsed electromagnetic field on in vitro and in vivo neural differentiation, cell survival of human mesenchymal stem cells

Low frequency-pulsed electromagnetic fields (LF-PEMFs) affect many biological processes such as differentiation, regulation of transcription factor and cell proliferation. However, the fundamental mechanisms responsible for these effects remain unclear. The aim of this study is to understand the mechanisms underlying PEMF-mediated suppression of apoptosis and promotion of survival, including PEMF-induced neuronal differentiation on in vivo and in vitro. Treatment of induced human BM-MSCs with PEMF increased the expression of neural markers such as NF-L, NeuroD1 and Tau. Moreover, treatment of induced human BMMSCs with PEMF greatly decreased cell death in a dose- and time-dependent manner. There is evidence that Akt and Ras are involved in neuronal survival and protection. Activation of Akt and Ras results in the regulation of survival proteins such as Bad and Bcl-xL. Thus, the Akt/Ras signaling pathway may be a desirable target for enhancing cell survival and treatment of neurological disease. C57B6 mice were exposed to LF-PEMF (F = 60Hz, Bm = 10mT) after photothrombotic occlusion. We measured the BDNF/TrkB/Akt signaling pathway, pro-apoptotic and pro-survival protein and gene expressions, and the expression of inflammatory mediators and performed behavioral tests in both LF-PEMF-treated and untreated ischemic stroke mice. Our analyses showed that PEMF exposure dramatically increased the activity of Akt, Rsk, Creb, Erk, Bcl-xL and Bad via phosphorylation. PEMFdependent cell protection was reversed by pretreatment with LY294002, a specific inhibitor of phosphatidylinositol 3-kinase (PI3K). Our data suggest that the PI3K/Akt/Bad signaling pathway may be a possible mechanism for the cell-protective effects of PEMF. Also results indicated that LF-PEMF treatment promotes activation of the BDNF/TrkB/Akt signaling pathway. Subsequently, pro-survival proteins were significantly increased, while proapoptotic proteins and inflammatory mediators were decreased in ischemic stroke mice after LF-PEMF treatment. The results demonstrated that LF-PEMF exposure has a neuroprotective effect after ischemic stroke in mice during the recovery process.