Leucyl-tRNA Synthetase Regulates Lactation and Cell Proliferation via mTOR Signaling in Dairy Cow Mammary Epithelial Cells

The role of LeuRS, an aminoacyl-tRNA synthetase, as an intracellular l-leucine sensor for the mTORC1 pathway has been the subject of much research recently. Despite this, the association between LeuRS and lactation in dairy cow mammary epithelial cells (DCMECs) remains unknown. In this study, we found that LeuRS expression in mammary gland tissue was significantly higher during lactation than pregnancy. Moreover, our data demonstrates that LeuRS is localized in the cytoplasm. Treatment with leucine increased DCMECs viability and proliferation, as well as mammalian target of rapamycin (mTOR), p-mTOR, ribosomal protein S6 kinase 1 (S6K1), p-S6K1, β-Casein, sterol regulatory element binding protein 1c (SREBP-1c), glucose transporter 1 (GLUT1), and Cyclin D1 mRNA and protein expression. Secretion of lactose and triglyceride were also increased. siRNA-mediated knockdown of LeuRS led to reduction in all of these processes. Based on these data, LeuRS up-regulates the mTOR pathway to promote proliferation and lactation of DCMECs in response to changes in the intracellular leucine concentration.     

CRISPR-Cas9–Mediated TIM3 Knockout in Human Natural Killer Cells Enhances Growth Inhibitory Effects on Human Glioma Cells

Glioblastoma (GBM) is the most common and aggressive primary malignant brain tumor in adults. Natural Killer (NK) cells are potent cytotoxic effector cells against tumor cells inducing GBM cells; therefore, NK cell based- immunotherapy might be a promising target in GBM. T cell immunoglobulin mucin family member 3 (TIM3), a receptor expressed on NK cells, has been suggested as a marker of dysfunctional NK cells. We established TIM3 knockout in NK cells, using the clustered regularly interspaced short palindromic repeats (CRISPR)-CRISPR-associated protein 9 (Cas9). Electroporating of TIM3 exon 2- or exon 5-targeting guide RNA- Cas9 protein complexes (RNPs) inhibited TIM3 expression on NK cells with varying efficacy. T7 endonuclease I mutation detection assays showed that both RNPs disrupted the intended genome sites. The expression of other checkpoint receptors, i.e., programmed cell death 1 (PD1), Lymphocyte-activation gene 3 (LAG3), T cell immunoreceptor with Ig and ITIM domains (TIGIT), and TACTILE (CD96) were unchanged on the TIM3 knockout NK cells. Real time cell growth assays revealed that TIM3 knockout enhanced NK cell–mediated growth inhibition of GBM cells. These results demonstrated that TIM3 knockout enhanced human NK cell mediated cytotoxicity on GBM cells. Future, CRISPR-Cas9 mediated TIM3 knockout in NK cells may prove to be a promising immunotherapeutic alternative in patient with GBM.     

Insulin-like Growth Factor 1 Promotes Cell Proliferation by Downregulation of G-Protein-Coupled Receptor 17 Expression via PI3K/Akt/FoxO1 Signaling in SK-N-SH Cells

Insulin-like growth factor 1 (IGF-1) not only regulates neuronal function and development but also is neuroprotective in the setting of acute ischemic stroke. G-protein-coupled receptor 17 (GPR17) expression in brain tissue serves as an indicator of brain damage. As whether IGF-1 regulates GPR17 expression remains unknown, the aim of this study is to investigate how IGF-1 regulates GPR17 expression in vitro. Human neuroblastoma SK-N-SH cells were used. Lentivirus-mediated short hairpin RNA (shRNA) was constructed to mediate the silencing of FoxO1, while adenoviral vectors were used for its overexpression. Verification of the relevant signaling cascade was performed using a FoxO1 inhibitor (AS1842856), a phosphatidylinositol 3-kinase (PI3K) inhibitor (LY294002), and a GPR17 antagonist (cangrelor). Cell proliferation was analyzed using EdU staining; immunofluorescence staining was used to detect the expression and subcellular localization of FoxO1. Chromatin immunoprecipitation was used to analyze the binding of FoxO1 to the GPR17 promoter in SK-N-SH cells. The expression of FoxO1, GPR17, and protein kinase B (also known as Akt) mRNA and protein as well as the levels of FoxO1 and Akt phosphorylation were investigated in this study. IGF-1 was found to downregulate FoxO1 and GPR17 expression in SK-N-SH cells while promoting cell viability and proliferation. Inhibition of FoxO1 and antagonism of GPR17 were found to play a role similar to that of IGF-1. Silencing of FoxO1 by lentivirus-mediated shRNA resulted in the downregulation of FoxO1 and GPR17 expression. The overexpression of FoxO1 via adenoviral vectors resulted in the upregulation of FoxO1 and GPR17 expression. Blocking of PI3K signaling by LY294002 inhibited the effect of IGF-1 on GPR17 suppression. Results from chromatin immunoprecipitation revealed that IGF-1 promotes FoxO1 nuclear export and reduces FoxO1 binding to the GPR17 promoter in SK-N-SH cells. Here, we conclude that IGF-1 enhances cell viability and proliferation in SK-N-SH cells via the promotion of FoxO1 nuclear export and reduction of FoxO1 binding to the GPR17 promoter via PI3K/Akt signaling. Our findings suggest that the enhancement of IGF-1 signaling to antagonize GPR17 serves as a potential therapeutic strategy in the management of acute ischemic stroke.     

GCN5 Potentiates Glioma Proliferation and Invasion via STAT3 and AKT Signaling Pathways

The general control of nucleotide synthesis 5 (GCN5), which is one kind of lysine acetyltransferases, regulates a number of cellular processes, such as cell proliferation, differentiation, cell cycle and DNA damage repair. However, its biological role in human glioma development remains elusive. In the present study, we firstly reported that GCN5 was frequently overexpressed in human glioma tissues and GCN5 was positively correlated with proliferation of cell nuclear antigen PCNA and matrix metallopeptidase MMP9. Meanwhile, down-regulation of GCN5 by siRNA interfering inhibited glioma cell proliferation and invasion. In addition, GCN5 knockdown reduced expression of p-STAT3, p-AKT, PCNA and MMP9 and increased the expression of p21 in glioma cells. In conclusion, GCN5 exhibited critical roles in glioma development by regulating cell proliferation and invasion, which suggested that GCN5 might be a potential molecular target for glioma treatment.     

Suppression of Proliferation of Human Glioblastoma Cells by Combined Phosphodiesterase and Multidrug Resistance-Associated Protein 1 Inhibition

The paucity of currently available therapies for glioblastoma multiforme requires novel approaches to the treatment of this brain tumour. Disrupting cyclic nucleotide-signalling through phosphodiesterase (PDE) inhibition may be a promising way of suppressing glioblastoma growth. Here, we examined the effects of 28 PDE inhibitors, covering all the major PDE classes, on the proliferation of the human U87MG, A172 and T98G glioblastoma cells. The PDE10A inhibitors PF-2545920, PQ10 and papaverine, the PDE3/4 inhibitor trequinsin and the putative PDE5 inhibitor MY-5445 potently decreased glioblastoma cell proliferation. The synergistic suppression of glioblastoma cell proliferation was achieved by combining PF-2545920 and MY-5445. Furthermore, a co-incubation with drugs that block the activity of the multidrug resistance-associated protein 1 (MRP1) augmented these effects. In particular, a combination comprising the MRP1 inhibitor reversan, PF-2545920 and MY-5445, all at low micromolar concentrations, afforded nearly complete inhibition of glioblastoma cell growth. Thus, the potent suppression of glioblastoma cell viability may be achieved by combining MRP1 inhibitors with PDE inhibitors at a lower toxicity than that of the standard chemotherapeutic agents, thereby providing a new combination therapy for this challenging malignancy.     

Effects of the Anti-Tumorigenic Agent AT101 on Human Glioblastoma Cells in the Microenvironmental Glioma Stem Cell Niche

Glioblastoma (GBM) is a barely treatable disease due to its profound chemoresistance. A distinct inter- and intratumoral heterogeneity reflected by specialized microenvironmental niches and different tumor cell subpopulations allows GBMs to evade therapy regimens. Thus, there is an urgent need to develop alternative treatment strategies. A promising candidate for the treatment of GBMs is AT101, the R(-) enantiomer of gossypol. The present study evaluates the effects of AT101, alone or in combination with temozolomide (TMZ), in a microenvironmental glioma stem cell niche model of two GBM cell lines (U251MG and U87MG). AT101 was found to induce strong cytotoxic effects on U251MG and U87MG stem-like cells in comparison to the respective native cells. Moreover, a higher sensitivity against treatment with AT101 was observed upon incubation of native cells with a stem-like cell-conditioned medium. This higher sensitivity was reflected by a specific inhibitory influence on the p-p42/44 signaling pathway. Further, the expression of CXCR7 and the interleukin-6 receptor was significantly regulated upon these stimulatory conditions. Since tumor stem-like cells are known to mediate the development of tumor recurrences and were observed to strongly respond to the AT101 treatment, this might represent a promising approach to prevent the development of GBM recurrences.     

The Role of Sonic Hedgehog in Human Holoprosencephaly and Short-Rib Polydactyly Syndromes

The Hedgehog (HH) signalling pathway is one of the major pathways controlling cell differentiation and proliferation during human development. This pathway is complex, with HH function influenced by inhibitors, promotors, interactions with other signalling pathways, and non-genetic and cellular factors. Many aspects of this pathway are not yet clarified. The main features of Sonic Hedgehog (SHH) signalling are discussed in relation to its function in human development. The possible role of SHH will be considered using examples of holoprosencephaly and short-rib polydactyly (SRP) syndromes. In these syndromes, there is wide variability in phenotype even with the same genetic mutation, so that other factors must influence the outcome. SHH mutations were the first identified genetic causes of holoprosencephaly, but many other genes and environmental factors can cause malformations in the holoprosencephaly spectrum. Many patients with SRP have genetic defects affecting primary cilia, structures found on most mammalian cells which are thought to be necessary for canonical HH signal transduction. Although SHH signalling is affected in both these genetic conditions, there is little overlap in phenotype. Possible explanations will be canvassed, using data from published human and animal studies. Implications for the understanding of SHH signalling in humans will be discussed.     

Inhibition of Citrinin-Induced Apoptotic Biochemical Signaling in Human Hepatoma G2 Cells by Resveratrol

The mycotoxin citrinin (CTN), a natural contaminant in foodstuffs and animal feeds, exerts cytotoxic and genotoxic effects on various mammalian cells. CTN causes cell injury, including apoptosis, but its precise regulatory mechanisms of action are currently unclear. Resveratrol, a member of the phytoalexin family found in grapes and other dietary plants, possesses antioxidant and anti-tumor properties. In the present study, we examined the effects of resveratrol on apoptotic biochemical events in Hep G2 cells induced by CTN. Resveratrol inhibited CTN-induced ROS generation, activation of JNK, loss of mitochondrial membrane potential (MMP), as well as activation of caspase-9, caspase-3 and PAK2. Moreover, resveratrol and the ROS scavengers, NAC and α-tocopherol, abolished CTN-stimulated intracellular oxidative stress and apoptosis. Active JNK was required for CTN-induced mitochondria-dependent apoptotic biochemical changes, including loss of MMP, and activation of caspases and PAK2. Activation of PAK2 was essential for apoptosis triggered by CTN. These results collectively demonstrate that CTN stimulates ROS generation and JNK activation for mitochondria-dependent apoptotic signaling in Hep G2 cells, and these apoptotic biochemical events are blocked by pretreatment with resveratrol, which exerts antioxidant effects.     

Lactoferrin Deficiency Impairs Proliferation of Satellite Cells via Downregulating the ERK1/2 Signaling Pathway

Lactoferrin (Ltf), a naturally active glycoprotein, possesses anti-inflammatory, anti-microbial, anti-tumor, and immunomodulatory activities. Many published studies have indicated that Ltf modulates the proliferation of stem cells. However, the role of Ltf in the proliferation of satellite cells, an important cell type in muscle regeneration, has not yet been reported. Here, by using Ltf systemic knockout mice, we illustrate the role of Ltf in skeletal muscle. Results shows that Ltf deficiency impaired proliferation of satellite cells (SCs) and the regenerative capability of skeletal muscle. Mechanistic studies showed that ERK1/2 phosphorylation was significantly downregulated after Ltf deletion in SCs. Simultaneously, the cell cycle-related proteins cyclin D and CDK4 were significantly downregulated. Intervention with exogenous recombinant lactoferrin (R-Ltf) at a concentration of 1000 μg/mL promoted proliferation of SCs. In addition, intraperitoneal injection of Ltf effectively ameliorated the skeletal muscle of mice injured by 1.2% BaCl₂ solution. Our results suggest a protective effect of Ltf in the repair of skeletal muscle damage. Ltf holds promise as a novel therapeutic agent for skeletal muscle injuries.     

Hypoxia-Induced FAM13A Regulates the Proliferation and Metastasis of Non-Small Cell Lung Cancer Cells

Hypoxia in non-small cell lung cancer (NSCLC) affects cancer progression, metastasis and metabolism. We previously showed that FAM13A was induced by hypoxia in NSCLC but the biological function of this gene has not been fully elucidated. This study aimed to investigate the role of hypoxia-induced FAM13A in NSCLC progression and metastasis. Lentiviral shRNAs were used for FAM13A gene silencing in NSCLC cell lines (A549, CORL-105). MTS assay, cell tracking VPD540 dye, wound healing assay, invasion assay, BrdU assay and APC Annexin V staining assays were performed to examine cell proliferation ability, migration, invasion and apoptosis rate in NSCLC cells. The results of VPD540 dye and MTS assays showed a significant reduction in cell proliferation after FAM13A knockdown in A549 cells cultured under normal and hypoxia (1% O₂) conditions (p < 0.05), while the effect of FAM13A downregulation on CORL-105 cells was observed after 96 h exposition to hypoxia. Moreover, FAM13A inhibition induced S phase cell cycle arrest in A549 cells under hypoxia conditions. Silencing of FAM13A significantly suppressed migration of A549 and CORL-105 cells in both oxygen conditions, especially after 72 and 96 h (p < 0.001 in normoxia, p < 0.01 after hypoxia). It was showed that FAM13A reduction resulted in disruption of the F-actin cytoskeleton altering A549 cell migration. Cell invasion rates were significantly decreased in A549 FAM13A depleted cells compared to controls (p < 0.05), mostly under hypoxia. FAM13A silencing had no effect on apoptosis induction in NSCLC cells. In the present study, we found that FAM13A silencing has a negative effect on proliferation, migration and invasion activity in NSCLC cells in normal and hypoxic conditions. Our data demonstrated that FAM13A depleted post-hypoxic cells have a decreased cell proliferation ability and metastatic potential, which indicates FAM13A as a potential therapeutic target in lung cancer.     

Caffeic Acid Phenethyl Ester Suppresses Proliferation and Survival of TW2.6 Human Oral Cancer Cells via Inhibition of Akt Signaling

Caffeic acid phenethyl ester (CAPE) is a bioactive component extracted from honeybee hive propolis. Our observations indicated that CAPE treatment suppressed cell proliferation and colony formation of TW2.6 human oral squamous cell carcinoma (OSCC) cells dose-dependently. CAPE treatment decreased G1 phase cell population, increased G2/M phase cell population, and induced apoptosis in TW2.6 cells. Treatment with CAPE decreased protein abundance of Akt, Akt1, Akt2, Akt3, phospho-Akt Ser473, phospho-Akt Thr 308, GSK3β, FOXO1, FOXO3a, phospho-FOXO1 Thr24, phospho-FoxO3a Thr32, NF-κB, phospho-NF-κB Ser536, Rb, phospho-Rb Ser807/811, Skp2, and cyclin D1, but increased cell cycle inhibitor p27^Kip. Overexpression of Akt1 or Akt2 in TW2.6 cells rescued growth inhibition caused by CAPE treatment. Co-treating TW2.6 cells with CAPE and 5-fluorouracil, a commonly used chemotherapeutic drug for oral cancers, exhibited additive cell proliferation inhibition. Our study suggested that administration of CAPE is a potential adjuvant therapy for patients with OSCC oral cancer.     

Costimulatory CD226 Signaling Regulates Proliferation of Memory-like NK Cells in Healthy Individuals with Latent Mycobacterium tuberculosis Infection

It is now widely accepted that NK cells can acquire memory, and this makes them more effective to protect against some pathogens. Prior reports indicate memory-like NK cells (mlNKs) in murine model of Mycobacterium tuberculosis (Mtb) as well as in healthy individuals with latent TB infection (LTBI). The increased expression of CD226 was evident in mlNKs from LTBI+ people after stimulation with γ-irradiated Mtb (γ-Mtb). We thus evaluated the contribution of costimulatory CD226 signaling in the functionality of mlNKs in LTBI+ people. We found that blockade of CD226 signaling using the antibody- or CRISPR/Cas9-mediated deletion of the CD226 gene in NK cells diminished the proliferation of mlNKs from LTBI+ people. Blocking CD226 signaling also reduced the phosphorylation of FOXO1 and cMyc expression. Additionally, cMyc inhibition using a chemical inhibitor reduced proliferation by mlNKs from LTBI+ people. Moreover, blocking CD226 signaling reduced glycolysis in NK cells, and the inhibition of glycolysis led to reduced effector function of mlNKs from LTBI+ people. Overall, our results provide a role for CD226 signaling in mlNK responses to Mtb.     

An Easy‐to‐Fabricate Hydrogel Platform with Tunable Stiffness and Cell Anchorage: Validation of Its Feasibility in Modulating Sonic Hedgehog Signaling Pathway Physically

The biophysical factors of microenvironments have increasingly been recognized as playing key roles in driving cell functions along with biochemical cues. However, current synthetic extracellular matrix materials with tunable biophysical properties are both costly and technically complicated to prepare, therefore limiting their application in the molecular biology field. Herein, an interpenetration hydrogel group with gradient stiffness and cell anchor points is developed, and the matrix stiffness and adhesion property functions as key regulators to determine the final output of Sonic Hedgehog (Shh) signaling is reported. Specifically, it is shown that the spreading areas of mouse embryonic fibroblasts are mainly regulated by stiffness of the matrix. The Shh‐induced Gli1 expression also increases with the cell spreading area. Finally, this work shows that b1‐integrin and focal adhesion kinase, respectively, act as sensors of matrix stiffness and adhesion properties, demonstrating the significance of the precise regulation of Shh signaling by cellular microenvironments. These results demonstrate the feasibility of using this hydrogel platform to study the impact of biophysical changes of the microenvironment on particular cellular signal pathways, and can potentially be applied in low‐cost, high throughput screening of signal pathways which are responsive to the biophysical changes of microenvironments.