Presence Of Epidermal Growth Factor Research Articles

The evaluation of melatonin and EGF interaction on breast cancer metastasis.

Metastasis in breast cancer is the first cause of death in patients. The epidermal growth factor (EGF) increases cancer cells' invasion, and migration. Melatonin's inhibitory effects on various types of cancer were confirmed. This study aimed to investigate whether melatonin could apply its impact through the EGF-related pathways or not. First, MDA-MB-231 and MCF7 cells were cultured, and then melatonin effects on cell viability were determined by MTT assay. Transwell invasion assay was applied to identify the invasiveness of these breast cancer cell lines under treatment of EGF and melatonin. Real-time RT-PCR then investigated the expression of MMP9 and MMP2 in determined groups. Cell proliferation was also assayed under EGF and melatonin treatment using Ki67 assessment by flow cytometry. The rate of invasion and migration of EGF-treated cells increased in both groups, in which melatonin caused increased invasion by EGF just in MCF7 cells. MMP9 and MMP2 expression increased significantly in both cell lines under EGF treatment, and melatonin increased these genes' expression in both cell lines (p<0.05). EGF increased the MMP9 and MMP2 gene expression, and melatonin increased EGF-induced expression (p<0.05). The EGF reduced the expression of the Ki67 protein in the MCF7 cell line, which was negatively affected by melatonin and EGF. In contrast, along with melatonin, EGF did not affect the proliferation of the MDA-MB-231 cell line. The results of this study show that melatonin in the presence of EGF does not show the anti-cancer properties previously described for this substance.

Abstract PO4-14-01: Synergistic Activity of CDK8/19 Inhibitor RVU120 and MEK Inhibitors in Hormone-Negative Breast Cancer: Implications for Targeted Therapy

Abstract Breast cancer (BC) is a complex disease with distinct subtypes, including triple-negative breast cancer (TNBC), which lacks targeted therapies. CDK8 and CDK19, components of the mediator complex involved in transcriptional regulation, have emerged as potential therapeutic targets in BC. Transcriptomic analysis revealed that elevated CDK8 expression was associated with poor prognosis across all BC subtypes. Furthermore, previous studies demonstrated that sensitivity to CDK8/CDK19 inhibition correlated with high STAT3 phosphorylation and enrichment of TNF/NF-kB and STAT target genes in responder cells. In this study, we evaluated the responsiveness of TNBC cell lines to RVU120, a highly selective and potent CDK8/CDK19 inhibitor currently undergoing clinical trials for acute myeloid leukemia (AML), high-risk myelodysplastic syndrome (HR-MDS), and solid tumors. Notably, the sensitivity of TNBC cell lines to RVU120 varied depending on the culture conditions, with the presence of epidermal growth factor (EGF) identified as a major determinant of response variability. Considering the role of EGFR in activating downstream signaling pathways, including MAPK and STAT, and previous observations of synergy between small molecule CDK8 inhibitors and MEK inhibitors in neuroblastoma, we investigated MEK inhibitors as potential combination partners in hormone-negative BC. Among the 15 tested BC cell lines, a synergy between CDK8/CDK19 inhibitors and MEK inhibitors was observed in 5 cell lines, antagonism in another 5 cell lines, and no synergy in the remaining 5 cell lines, based on the analysis with Combenefit software (Loewe additivity model). Importantly, cell lines where synergy of the combination therapy has been observed, exhibited EGFR amplification and markers of activated RAS pathway. This pattern of response was consistent across different MEK and MEK/RAF inhibitors, including selumetinib, trametinib, and avutometinib. These findings highlight the synergistic potential of combining RVU120 with MEK inhibitors in hormone-negative BC, particularly in TNBC with EGFR amplification and an active RAS pathway. Ongoing studies involving RNAseq analysis and organoid screening aim to further elucidate the underlying mechanisms and confirm the efficacy of this combination therapy. Furthermore, in vivo efficacy experiments are necessary to validate in vitro activity of both compounds. Successful development of this therapeutic concept relies on the validation and implementation of selected partner drugs targeting RAS pathway, as well as the identification of suitable biomarkers such as EGFRamp or RAS-score. Promising findings of this study provide a rationale for exploring the combination of CDK8/CDK19 inhibitor RVU120 and MEK inhibitors as a potential targeted therapy strategy for hormone-negative BC. Citation Format: Urszula Pakulska, Adrianna Moszynska, Justyna Martyka, Jerzy Woznicki, Elzbieta Adamczyk, Marta Obacz, Kinga Keska Izworska, Katarzyna Wiklik, Agata Stachowicz, Hendrik Nogai, Milena Mazan, Tomasz Rzymski. Synergistic Activity of CDK8/19 Inhibitor RVU120 and MEK Inhibitors in Hormone-Negative Breast Cancer: Implications for Targeted Therapy [abstract]. In: Proceedings of the 2023 San Antonio Breast Cancer Symposium; 2023 Dec 5-9; San Antonio, TX. Philadelphia (PA): AACR; Cancer Res 2024;84(9 Suppl):Abstract nr PO4-14-01.

Nuclear Factor I A and Nuclear Factor I B Are Jointly Required for Mouse Postnatal Neural Stem Cell Self-Renewal.

Mouse postnatal neural stem cells (pNSCs) can be expanded in vitro in the presence of epidermal growth factor and fibroblast growth factor 2 and upon removal of these factors cease proliferation and generate neurons, astrocytes, and oligodendrocytes. The genetic requirements for self-renewal and lineage-commitment of pNSCs are incompletely understood. In this study, we show that the transcription factors NFIA and NFIB, previously shown individually, to be essential for the normal commitment of pNSCs to the astrocytic lineage in vivo, are jointly required for normal self-renewal of pNSCs in vitro and in vivo. Using conditional knockout alleles of Nfia and Nfib, we show that the simultaneous loss of these two genes under self-renewal conditions in vitro reduces the expression of the proliferation markers PCNA and Ki67, eliminates clonogenicity of the cells, reduces the number of cells in S phase, and induces aberrant differentiation primarily into the neuroblast lineage. This phenotype requires the loss of both genes and is not seen upon loss of Nfia or Nfib alone, nor with combined loss of Nfia and Nfix or Nfib and Nfix. These data demonstrate a unique combined requirement for both Nfia and Nfib for pNSC self-renewal.

Epidermal growth factor alters silica nanoparticle uptake and improves gold-nanoparticle-mediated gene silencing in A549 cells.

Delivery of therapeutic nanoparticles (NPs) to cancer cells represents a promising approach for biomedical applications. A key challenge for nanotechnology translation from the bench to the bedside is the low amount of administered NPs dose that effectively enters target cells. To improve NPs delivery, several studies proposed NPs conjugation with ligands, which specifically deliver NPs to target cells via receptor binding. One such example is epidermal growth factor (EGF), a peptide involved in cell signaling pathways that control cell division by binding to epidermal growth factor receptor (EGFR). However, very few studies assessed the influence of EGF present in the cell environment, on the cellular uptake of NPs. We tested if the stimulation of EGFR-expressing lung carcinomacells A549 with EGF affects the uptake of 59 nm and 422 nm silica (SiO2) NPs. Additionally, we investigated whether the uptake enhancement can be achieved with gold NPs, suitable to downregulate the expression of cancer oncogene c-MYC. Our findings show that EGF binding to its receptor results in receptor autophosphorylation and initiate signaling pathways, leading to enhanced endocytosis of 59 nm SiO2 NPs, but not 422 nm SiO2 NPs. Additionally, we demonstrated an enhanced gold (Au) NPs endocytosis and subsequently a higher downregulation of c-MYC. These findings contribute to a better understanding of NPs uptake in the presence of EGF and that is a promising approach for improved NPs delivery.

Oocyte-secreted factor TGFB2 enables mouse cumulus cell expansion in vitro.

Cumulus expansion is necessary for the release of a fertilizable oocyte from the ovary, which is critical for the normal fertilization of mammals. Cumulus expansion requires cooperation between epidermal growth factor (EGF)-like growth factors and oocyte paracrine factors. Growth differentiation factor 9 (GDF9) and bone morphogenetic protein 15 (BMP15) are well-known paracrine factors secreted by oocytes. In addition, transforming growth factor-β2 (TGFB2) was primarily expressed in oocytes and its membrane receptors type 1 receptor (TGFBR1) and type 2 receptor (TGFBR2) were located in cumulus cells. In our present study, TGFB2 induced expansion of oocytectomized (OOX) complexes and increased the expression of expansion-related genes in the presence of EGF, suggesting that TGFB2 enables cumulus expansion. Inhibition of TGF-β signaling with SD208 blocked TGFB2-promoted cumulus expansion. Furthermore, in the culture of OOX complexes from mice of Tgfbr2-specific depletion in granulosa cells, TGFB2-promoted cumulus expansion and the expression of expansion-related genes were impaired. These results suggest that TGFB2 could induce cumulus expansion through TGFBR-SMAD2/3 signaling. Tgfb2-specific depletion in oocytes using Zp3-Cre mice had no effect on cumulus expansion in vivo, possibly due to the compensatory effect of other cumulus expansion-enabling factors. Taken together, TGFB2 is involved in expansion-related gene expression and consequent cumulus expansion.

Endothelin A (ETA) and Endothelin B (ETB) Receptor Subtypes Potentiate Epidermal Growth Factor (EGF)-Mediated Proliferation in Human Asthmatic Bronchial Airway Smooth Muscle.

BackgroundAsthma is a chronic disease characterized by chronic inflammation, reversible airway obstruction, airway hyperresponsiveness (AHR), and airway remodeling. One of the important features of asthma is airway remodeling, which plays a central role in airflow limitation. Airway remodeling involves numerous changes in the bronchial walls, including airway smooth muscle (ASM) cell hypertrophy and hyperplasia. Studies have shown that ASM hyperplasia in asthma is mediated by the increased production of mitogens. Endothelin-1 (ET-1) has been shown to induce proliferation and function as a co-mitogen in vascular and ASM. In patients with asthma, plasma and bronchoalveolar lavage fluid have been shown to have elevated ET-1 levels, which have been linked to airway remodeling and airflow obstruction in severe asthma. This study investigates the role of ET-1 in proliferation, the receptor subtype mediating its effect, and the signaling pathway.MethodologyNormal and asthmatic bronchial airway smooth muscle (BASM) cells were seeded into 5 × 103 cells/well. Cell proliferation was assayed using 5-bromo-2’-deoxyuridine (BrdU) incorporation. Confluent cells were treated with different concentrations of ET-1 in the presence or absence of the epidermal growth factor (EGF). Signaling pathways were explored using pretreatment of BASM with antagonists 15 minutes before ET-1/EGF stimulation.ResultsIn asthmatic BASM, ET-1 (0.1 nM) functions as a co-mitogen in the presence of EGF (10 nM), showing a significantly greater effect on asthmatic BASM proliferation compared with normal BASM. The ETA receptor antagonist BQ-123 (10-1,000 nM) significantly reduced the proliferative effect of ET-1/EGF on asthmatic BASM more than normal BASM. Moreover, the effect of ETB antagonist BQ-788 (1,000 nM) or pretreatment with the ETB agonist S6C (1-10 nM) followed by co-treatment with EGF in asthmatic BASM showed a small but significant decrease when pretreated with the inhibitor and increased with the agonist, thereby suggesting that the co-mitogenic effect of ET-1 is mainly via the activation of ETA receptors, with a small contribution by the ETB receptors in asthmatic BASM. Finally, pertussis toxin (PTX) pretreatment (25 and 50 ng/mL) showed that EGF and ET-1/EGF mitogenic and co-mitogenic signaling utilizes Gi/0-mediated transactivation by EGF and ET receptors, especially in asthmatic BASM, leading to the activation of Ras-ERK-PI3K pathways. Enhanced ERK and PI3K effects on proliferation suggested that these kinases modulate the co-mitogenic effect of ET-1 in asthmatic BASM. Enhanced cross-talk between ET and EGF receptors may be a potential mechanism contributing to airway remodeling in asthmatic BASM.ConclusionsET-1 enhances the mitogenic effect of EGF predominantly via the ETA receptor in asthmatic BASM with the activation of Ras, ERK, and PI3K. The cross-talk mechanism between ET and EGF receptors may be a potential therapeutic target to prevent the progression of airway remodeling in ASM in patients with asthma.

MYO10 promotes transzonal projection-dependent germ line-somatic contact during mammalian folliculogenesis†.

Granulosa cells of growing ovarian follicles elaborate filopodia-like structures termed transzonal projections (TZPs) that supply the enclosed oocyte with factors essential for its development. Little is known, however, of the mechanisms underlying the generation of TZPs. We show in mouse and human that filopodia, defined by an actin backbone, emerge from granulosa cells in early stage primary follicles and that actin-rich TZPs become detectable as soon as a space corresponding to the zona pellucida appears. mRNA encoding Myosin10 (MYO10), a motor protein that accumulates at the base and tips of filopodia and has been implicated in their initiation and elongation, is present in granulosa cells and oocytes of growing follicles. MYO10 protein accumulates in foci located mainly between the oocyte and innermost layer of granulosa cells, where it colocalizes with actin. In both mouse and human, the number of MYO10 foci increases as oocytes grow, corresponding to the increase in the number of actin-TZPs. RNAi-mediated depletion of MYO10 in cultured mouse granulosa cell-oocyte complexes is associated with a 52% reduction in the number of MYO10 foci and a 28% reduction in the number of actin-TZPs. Moreover, incubation of cumulus-oocyte complexes in the presence of epidermal growth factor, which triggers a 93% reduction in the number of actin-TZPs, is associated with a 55% reduction in the number of MYO10 foci. These results suggest that granulosa cells possess an ability to elaborate filopodia, which when directed toward the oocyte become actin-TZPs, and that MYO10 increases the efficiency of formation or maintenance of actin-TZPs.

Subcellular regulation of glucose metabolism through multienzyme glucosome assemblies by EGF–ERK1/2 signaling pathways

A multienzyme metabolic assembly for human glucose metabolism, namely the glucosome, has been previously demonstrated to partition glucose flux between glycolysis and building block biosynthesis in an assembly size-dependent manner. Among three different sizes of glucosome assemblies, we have shown that large-sized glucosomes are functionally associated with the promotion of serine biosynthesis in the presence of epidermal growth factor (EGF). However, due to multifunctional roles of EGF in signaling pathways, it is unclear which EGF-mediated signaling pathways promote these large glucosome assemblies in cancer cells. In this study, we used Luminex multiplexing assays and high-content single-cell imaging to demonstrate that EGF triggers temporal activation of extracellular signal-regulated kinases 1/2 (ERK1/2) in Hs578T cells. Subsequently, we found that treatments with a pharmacological inhibitor of ERK1/2, SCH772984, or short-hairpin RNAs targeting ERK1/2 promote the dissociation of large-sized assemblies to medium-sized assemblies in Hs578T cells. In addition, our Western blot analyses revealed that EGF treatment does not increase the expression levels of enzymes that are involved in both glucose metabolism and serine biosynthesis. The observed spatial transition of glucosome assemblies between large and medium sizes appears to be mediated by the degree of dynamic partitioning of glucosome enzymes without changing their expression levels. Collectively, our study demonstrates that EGF–ERK1/2 signaling pathways play an important role in the upregulation of large-sized glucosomes in cancer cells, thus functionally governing the promotion of glycolysis-derived serine biosynthesis.

Вплив нейротрофічних факторів на популяцію серотонінергічних нейронів n.raphe в умовах культивування

Актуальність. Молекулярні механізми спеціалізації серотонінергічних нейронів зі стовбурових і прогеніторних нервових клітин вивчені далеко не повністю. Мета дослідження — визначення найбільш ефективних нейроіндукторів, що підтримують життєздатність та функціональну активність серотонінергічних нейронів та їх детермінованих попередників. Як критерії оцінки були обрані: експресія відповідних генів, загальний вміст серотоніну та чисельність серотонінергічних нейронів, візуалізованих за допомогою гістохімічної реакції. Матеріали та методи. Робота була виконана на суспензійній культурі клітин зони n.raphe з використанням гістохімічних методів, імуноферментного аналізу та полімеразної ланцюгової реакції. Результати. Установлено, що BDNF та NGF стимулюють збільшення чисельності серотонінергічних нейронів у дисоційованій культурі в 1,6 та 2 рази порівняно з контрольними зразками. Додавання в культуральне середовище BDNF та ретиноєвої кислоти призводить до зростання вмісту серотоніну в культуральному матеріалі. Експресія гена Nkx 2.2, що є маркером мітотично-активних попередників серотонінергічних нейронів, підтримується за наявності BDNF і збільшується під впливом EGF та FGFb. Експресія гена Pet1, що свідчить про наявність детермінованих прогеніторів серотонінергічних нейронів, підтримується всіма досліджуваними факторами, за виключенням екзогенного серотоніну. Експресія гена Tph1, маркера початку синтезу серотоніну, спостерігається під впливом FGFb, EGF та NGF. Висновки. Таким чином, наші дані показали, що вищевказані нейроіндуктори впливають на різні фази серотонінгенезу і можуть бути використані для його регуляції.

PSX-B-13 Effect of epidermal growth factor and prolactin on the quality of bovine oocytes aging in vitro

Abstract The evaluation of factors responsible for the protection of the oocytes attained the metaphase-II stage from aging is importance for successful in vitro embryo reproduction. The aim of the present research was to study dose-dependent effects of epidermal growth factor (EGF) and prolactin (PRL) on the quality of bovine oocytes after their aging in vitro. Bovine cumulus-enclosed oocytes (CEOs) were matured in vitro for 20 h in TCM 199 containing 0.2 mM sodium pyruvate, 10% fetal calf serum (FCS), 10 μg/ml FSH and LH. At the end of in vitro maturation, oocytes were transferred to TCM 199 supplemented with 10% FCS (aging medium) and cultured for additional 24 h in the absence (Control) and in presence of EGF (10 and 50 ng/ml) and PRL (20 and 50 ng/ml). After prolonged culture oocytes were used for apoptosis detection (TUNEL staining, n=251) and the state of chromosomes evaluation (Tarkowski’s cytogenetic method, n=359). The data from 3–4 replicates were analyzed by ANOVA. At the end of prolonged culture (24 h) the rate of apoptotic oocytes in the Control group was 47.4±8.5%. EGF at concentration of 10 ng/ml and PRL at both doses decreased this rate to 15.0–22.1% (p &amp;lt; 0.05). Furthermore, PRL (not EGF) reduced the frequency of abnormal chromosome modifications (decondensation, adherence, clumping) at concentrations of 20–50 ng/ml from 58.7±2.1% (Control) to 41.2±1.9 and 45.6±2.7% respectively (p &amp;lt; 0.01). Thus, EGF and PRL is able to maintain the apoptosis resistance of bovine oocytes during their prolonged in vitro culture as well as PRL have the decelerating effect on abnormal modifications of M-II chromosomes. The research was supported by RFBR (17-29-08035) and the Ministry of Science and Higher Education of Russia.

The phospholipase D inhibitor FIPI potently blocks EGF-induced calcium signaling in human breast cancer cells

BackgroundPhosphotyrosine kinase (PTK)-mediated phospholipase C-γ1 (PLC-γ1) signaling plays a crucial role in the release of the universal second messenger calcium from intracellular stores, which is mandatory for several cellular processes, including cell migration. However, PLC-γ1 could also be activated in a PTK-independent manner by phospholipase D (PLD)-derived phosphatidic acid (PA). Because both higher PLD expression levels and PLD activity have also been associated with breast cancer cell invasion and migration, we wondered whether there might be a link between PLD and PLC-γ1, which was investigated in this study.MaterialsMDA-MB-468-NEO (EGFR positive) and MDA-MB-468-HER2 (EGFR and HER2 positive) human breast cancer cells were used in this study. The migratory behavior of the cells in the presence of epidermal growth factor (EGF) and the PLD inhibitor 5-fluoro-2-indolyl-des-chlorohalopemide (FIPI) was analyzed using the 3D collagen matrix migration assay. Changes in cytosolic calcium levels in the presence of EGF, FIPI and Sig-1R agonists and antagonists as well as in PLD1 siRNA knockdown cells were determined by flow cytometry. Western blot analyses were performed to determine the basal expression levels and phosphorylation patterns of EGFR, HER2, AKT, MAPKp42/44, PLC-γ1 and Sig-1R.ResultsThe EGF-induced migration of MDA-MB-468-NEO and MDA-MB-468-HER2 cells was significantly impaired by FIPI. Likewise, FIPI also significantly abolished EGF-induced calcium release in both cell lines. However, neither the expression levels nor the phosphorylation patterns of EGFR, HER2, AKT, MAPKp42/44 and PLC-γ1 were markedly changed by FIPI. Knockdown of PLD1 expression by siRNA also significantly impaired EGF-induced calcium release in both cell lines. Targeting Sig-1R, which interacts with IP3R, with the antagonist BD1047 also abrogated EGF-induced calcium release. However, EGF-induced calcium release was also impaired if cells were treated with the Sig-1R agonists PRE084 and PPBP maleate.ConclusionIn summary, blocking PLD activity with the specific inhibitor FIPI or knocking down PDL1 expression by siRNA significantly impaired EGF-induced calcium release in MDA-MB-468-NEO and MDA-MB-468-HER2 cells, likely indicating a connection between PLD activity and PLC-γ1-mediated calcium signaling. However, how PLD activity interferes with the release of calcium from intracellular stores remains unclear.5hw_Xw1J4QoujjE82ULAZvVideo

Directional Migration of Breast Cancer Cells Hindered by Induced Electric Fields May Be Due to Accompanying Alteration of Metabolic Activity

Background: Induced electric fields (iEFs) control directional breast cancer cell migration. While the connection between migration and metabolism is appreciated in the context of cancer and metastasis, effects of iEFs on metabolic pathways especially as they relate to migration, remain unexplored. Materials and Methods: Quantitative cell migration data in the presence and absence of an epidermal growth factor (EGF) gradient in the microfluidic bidirectional microtrack assay was retrospectively analyzed for additional effects of iEFs on cell motility and directionality. Surrogate markers of oxidative phosphorylation (succinate dehydrogenase [SDH] activity) and glycolysis (lactate dehydrogenase activity) were assessed in MDA-MB-231 breast cancer cells and normal MCF10A mammary epithelial cells exposed to iEFs and EGF. Results: Retrospective analysis of migration results suggests that iEFs increase forward cell migration speeds while extending the time cells spend migrating slowly in the reverse direction or remaining stationary. Furthermore, in the presence of EGF, iEFs differentially altered flux through oxidative phosphorylation in MDA-MB-231 cells and glycolysis in MCF10A cells. Conclusions: iEFs interfere with MDA-MB-231 cell migration, potentially, by altering mitochondrial metabolism, observed as an inhibition of SDH activity in the presence of EGF. The energy intensive process of migration in these highly metastatic breast cancer cells may be hindered by iEFs, thus, through hampering of oxidative phosphorylation.

Hexameric procyanidins inhibit colorectal cancer cell growth through both redox and non-redox regulation of the epidermal growth factor signaling pathway

Dietary proanthocyanidins (PAC) consumption is associated with a decreased risk for colorectal cancer (CRC). Dysregulation of the epidermal growth factor (EGF) receptor (EGFR) signaling pathway is frequent in human cancers, including CRC. We previously showed that hexameric PAC (Hex) exert anti-proliferative and pro-apoptotic actions in human CRC cells. This work investigated if Hex could exert anti-CRC effects through its capacity to regulate the EGFR pathway. In proliferating Caco-2 cells, Hex acted attenuating EGF-induced EGFR dimerization and NADPH oxidase-dependent phosphorylation at Tyr 1068, decreasing EGFR location at lipid rafts, and inhibiting the downstream activation of pro-proliferative and anti-apoptotic pathways, i.e. Raf/MEK/ERK1/2 and PI3K/Akt. Hex also promoted EGFR internalization both in the absence and presence of EGF. While Hex decreased EGFR phosphorylation at Tyr 1068, it increased EGFR Tyr 1045 phosphorylation. The latter provides a docking site for the ubiquitin ligase c-Cbl and promotes EGFR degradation by lysosomes. Importantly, Hex acted synergistically with the EGFR-targeted chemotherapeutic drug Erlotinib, both in their capacity to decrease EGFR phosphorylation and inhibit cell growth. Thus, dietary PAC could exert anti-CRC actions by modulating, through both redox- and non-redox-regulated mechanisms, the EGFR pro-oncogenic signaling pathway. Additionally, Hex could also potentiate the actions of EGFR-targeted drugs.

Epidermal growth factor promotes glioblastoma cell death under glucose deprivation via upregulation of xCT (SLC7A11)

The cystine/glutamate antiporter xCT (SLC7A11) is frequently overexpressed in many cancers, including glioblastoma. Cystine taken up by the cells via xCT is reduced to cysteine, which is used to synthesize glutathione for antioxidant cellular defense. However, overexpression of xCT causes cell death under glucose-limited conditions. We found that stimulation of glioblastoma cells with epidermal growth factor (EGF) induces the upregulation of xCT and promotes cell death under glucose deprivation. Treatment with the mTOR inhibitor Torin 1 suppressed the EGF-induced upregulation of xCT and cell death. EGF increased xCT mRNA levels, which was suppressed by Torin 1. The lysosome inhibitor bafilomycin A1 increased xCT protein levels in the absence of EGF or in the presence of EGF and Torin 1. Taken together, our study suggests that EGF promotes glioblastoma cell death under glucose-limited conditions via the upregulation of xCT at transcriptional and protein levels in an mTOR-dependent manner.

Calmodulin downregulation in conditional knockout HeLa cells inhibits cell migration

The study of calmodulin (CaM) functions in living cells has been tackled up to date using cell-permeant CaM inhibitors or interference-RNA methods. CaM inhibitors may lack specificity and the siRNA interference approach is challenging, as all three CaM genes expressing an identical protein in mammals have to be blocked. Therefore, we recently introduced a novel genetic system using CRISPR/Cas9-mediated gene deletion and conditional CaM expression to study the function of CaM in HeLa cells. Here, we describe the effect of CaM downregulation on the basal and epidermal growth factor (EGF)-dependent 2D- and 3D-migration in HeLa cells. CaM downregulation inhibited cell migration on a 2D-surface in the absence but not in the presence of EGF. In contrast, CaM downregulation led to inhibition of 3D-migration across a porous membrane both in the absence and presence of EGF. CaM downregulation decreased the expression of Rac1, Cdc42 and RhoA, all known to play crucial roles in cell migration. These results show that EGF-dependent 2D- and 3D-migration utilize distinct CaM-regulated systems and identify several essential migratory proteins directly or indirectly regulated by CaM.

Epidermal Growth Factor Stimulates Fatty Acid Synthesis Mainly via PLC-γ1/Akt Signaling Pathway in Dairy Goat Mammary Epithelial Cells.

Simple SummaryGoat milk contains an abundance of fatty acids which are benefit to human health. Epidermal growth factor (EGF) is a small peptide which could positively regulate the growth, development and differentiation of the mammary gland during lactation. However, little information is available about EGF in regulating lipid metabolism in the mammary gland. This study investigated the effects of EGF on the triglyceride (TG) synthesis, lipogenic genes expression and the downstream signal protein levels in goat mammary epithelial cells (GMECs). Our findings indicated EGF might be beneficial to improve milk fat synthesis of dairy goats.EGF acts as a ligand of the EGF receptor (EGFR) to activate the EGFR-mediated signaling pathways and is involved in the regulation of cell physiology. However, the roles of EGFR mediated signaling pathways in the regulation of lipid metabolism in goat mammary epithelial cells (GMECs) are poorly understood. To evaluate the impact of EGF on GMECs, the triglyceride (TG) content and lipid droplet were detected, using TG assay and immunofluorescence. Further, expression of lipogenic genes, the protein kinase B (Akt), phospholipase C-γ1 (PLC-γ1) and extracellular signal-regulated kinases (ERK)1/2 signaling pathways were measured by real-time polymerase chain reaction and Western blot, respectively. The results showed that the mRNA expression of EGFR gene was significantly upregulated in lactating goat mammary gland tissues compared to non-lactation period (p < 0.05). TG contents in EGF-treated GMECs were significantly increased (p < 0.05), and an increase of lipid droplets was also detected. In vitro studies demonstrated that the mRNA levels of lipogenesis-related FASN, ACC, SCD1, LXRa, LXRb and SP1 genes were positively correlated to the mRNA level of EGFR gene shown by gene overexpression and silencing (p < 0.05). The phosphorylations of Akt, ERK1/2 and PLC-γ1 in GMECs were greatly upregulated in the presence of EGF, and specific inhibitors were capable of blocking the phosphorylation of Akt, ERK1/2 and PLC-γ1. Compared with EGF-treated GMECs, the mRNA levels of FASN, ACC and SCD1 were significantly decreased in GMECs co-treated with PLC-γ1 and Akt inhibitor and EGF (p < 0.05), and TG content was also dropped significantly. These observations implied that EGFR plays an important role in regulating de novo fatty acid synthesis in GMECs, mainly mediated by Akt and PLC-γ1 signaling pathways.

Gene expression dynamic analysis reveals co-activation of Sonic Hedgehog and epidermal growth factor followed by dynamic silencing.

Aberrant activation of the Sonic Hedgehog (SHH) gene is observed in various cancers. Previous studies have shown a “cross-talk” effect between the canonical Hedgehog signaling pathway and the Epidermal Growth Factor (EGF) pathway when SHH is active in the presence of EGF. However, the precise mechanism of the cross-talk effect on the entire gene population has not been investigated. Here, we re-analyzed publicly available data to study how SHH and EGF cooperate to affect the dynamic activity of the gene population. We used genome dynamic analysis to explore the expression profiles under different conditions in a human medulloblastoma cell line. Ordinary differential equations, equipped with solid statistical and computational tools, were exploited to extract the information hidden in the dynamic behavior of the gene population. Our results revealed that EGF stimulation plays a dominant role, overshadowing most of the SHH effects. We also identified cross-talk genes that exhibited expression profiles dissimilar to that seen under SHH or EGF stimulation alone. These unique cross-talk patterns were validated in a cell culture model. These cross-talk genes identified here may serve as valuable markers to study or test for EGF co-stimulatory effects in an SHH+ environment. Furthermore, these cross-talk genes may play roles in cancer progression, thus they may be further explored as cancer treatment targets.

The inhibitory effect of ECG and EGCG dimeric procyanidins on colorectal cancer cells growth is associated with their actions at lipid rafts and the inhibition of the epidermal growth factor receptor signaling

Colorectal cancer (CRC) is one of the most common cancers worldwide. Epidemiological studies indicate that consumption of fruits and vegetables containing procyanidins is associated with lower CRC risk. This study investigated the capacity of two dimeric procyanidins composed of epicatechin gallate (ECG) or epigallocatechin gallate (EGCG) isolated from persimmons, to inhibit CRC cell growth and promote apoptosis, characterizing the underlying mechanisms. ECG and EGCG dimers reduced the growth of five human CRC cell lines in a concentration (10–60 μM)- and time (24–72 h)-dependent manner, with a 72 h-IC50 value in Caco-2 cells of 10 and 30 μM, respectively. ECG and EGCG dimers inhibited Caco-2 cell proliferation by arresting the cell cycle in G2/M phase and by inducing apoptosis via the mitochondrial pathway. In addition, ECG and EGCG dimers inhibited cell migration, invasion, and adhesion, decreasing the activity of matrix metalloproteinases (MMP-2/9). Mechanistically, ECG and EGCG dimers inhibited the activation of lipid raft-associated epidermal growth factor (EGF) receptor (EGFR), without affecting its localization at lipid rafts. In particular, ECG and EGCG dimers reduced EGFR phosphorylation at Tyr1068 residue, prevented EGFR dimerization and activation upon stimulation, and induced EGFR internalization both in the absence and presence of EGF. Furthermore, ECG and EGCG dimers increased EGFR phosphorylation at Tyr1045 residue, providing a docking site for ubiquitin ligase c-Cbl and induced EGFR degradation by the proteasome. Downstream of EGFR, ECG and EGCG dimers inhibited the activation of the MEK/ERK1/2 and PI3K/AKT signaling pathways, downregulating proteins involved in the modulation of cell survival. In conclusion, ECG and EGCG dimers reduced CRC cell growth by inhibiting EGFR activation at multiple steps, including the disruption of lipid rafts integrity and promoting EGFR degradation. These results shed light on a potential molecular mechanism on how procyanidins-rich diets may lower CRC risk.

Identification of neurospheres generated from human dental pulp stem cells in xeno-/serum-free conditions

IntroductionCell-based therapies require an emerging alternative treatment using easily harvested cell sources. Neural stem cells derived from various tissues, including brain, bone marrow, skin and retina can give rise to both neurons and glial cells. Recently, human dental pulp stem cells (DPSCs) and stem cells from human exfoliated deciduous teeth (SHED) were demonstrated to have mesenchymal stem cell-like abilities such as self-renewal and multi-lineage differentiation, including neuron and glial cells. Moreover, DPSCs and SHED show a higher proliferation rate and a higher number of population doublings compared with adult bone marrow stromal stem cells. Therefore, DPSCs are a useful source that can be applied in cell replacement therapy for various neurological disorders. Generally, the conventional culture methods for DPSCs have used serum, therefore the undefined components in culture medium may complicate investigations of the molecular mechanisms that control the self-renewal and differentiation of DPSCs. However, neural stem cells proliferate to form ‘neurospheres’ in suspension in vitro in the presence of epidermal growth factor (EGF) and basic fibroblast growth factor (bFGF). No study to date has obtained neurospheres from DPSCs in serum-free conditions in primary culture. Thus, the aim of this study was to establish a method for the proliferation and neural differentiation of DPSCs in xeno- and serum-free conditions in primary culture.MethodsDPSCs were obtained from the dental pulp of wisdom teeth from healthy individuals (18–41 years old) and cultured in conventional medium containing 15% fetal bovine serum and xeno-/serum-free medium. We evaluated the proliferation of DPSCs, neurosphere generation, and neural differentiation under xeno-/serum-free conditions by flow cytometry, immunohistochemistry, and real-time polymerase chain reaction.ResultsIn proliferation medium without xeno/serum, DPSCs can proliferate and generate neurospheres, however, the neurospheres had limited self-renewal ability. Under differentiation conditions, class III β-tubulin (TUBB3) and microtubule-associated protein (MAP2) were more significantly expressed in neurospheres derived from DPSCs in xeno-/serum-free culture conditions than in DPSCs in conventional culture conditions.ConclusionsOur result demonstrated that neurosphere generation from DPSCs in xeno-/serum-free culture may be an accessible source for clinical cell replacement therapies for neuronal degenerative diseases.

189 Effects of epidermal growth factor and progesterone on oocyte meiotic resumption and expression of maturation-related transcripts in bovine oocytes from medium-size antral follicles

The aims of this study were to evaluate the effects of epidermal growth factor (EGF) and progesterone (P4) on maturation and expression transcripts for GDF9, CCNB1, H1FOO, cMOS, PARN, and eIF4E after prematuration of cumulus-oocyte complexes (COCs) from antral follicles. Bovine COCs (3-6mm) were aspirated and pre-matured for 20h in control medium [TCM-199 containing 5.0mgmL−1 LH, 0.5mgmL−1 FSH, 0.4% bovine serum albumin, cilostamide (10μM) and follicular hemisections] alone or supplemented with EGF (10ngmL−1), P4 (100 µM), or both EGF (10ngmL−1) and P4 (100 µM). After that, COCs were matured for 24h in the same medium, without EGF, P4, cilostamide, and follicular hemisections. Oocyte diameters were evaluated with the software Nis Elements (Nikon Instruments Inc.). To evaluate meiotic progression, the oocytes were fixed in 4% paraformaldehyde and transferred to 0.5% Triton X-100. The chromatin configuration during meiosis was assessed by 10μgmL−1 bisbenzimide (Hoechst 33342) and analysed under an epi-fluorescent inverted microscope (DMI4000B; Leica). Oocytes were classified according to the nuclear maturation stage as germinal vesicle, metaphase I, anaphase I, telophase I, and metaphase II. To evaluate mRNA expression, oocytes were stored in micr-centrifuge tubes at −80°C until RNA extraction. RNA was extracted using Trizol according to the manufacturer's instructions (Invitrogen). After reverse transcription, mRNA for GDF9, cyclin B1, H1FOO, cMOS, PARN, eIF4E, and GAPDH (housekeeping gene) was quantified by real-time PCR and analysed by Kruskal-Wallis test. The percentages of oocytes in each stage of maturation were compared by Mann-Whitney test (P&amp;lt;0.05). The results showed that prematuration of COCs in the presence of P4 and both EGF and P4 promoted an increase in oocyte diameter compared with the control or EGF treatment alone. The presence of cilostamide inhibited early meiotic resumption, benefiting oocyte capacitation, but the presence of EGF, P4, or EGF and P4 together in the prematuration medium did not influence meiosis resumption rates. The presence of EGF or P4 in prematuration medium increased the mRNA levels for cMOS in oocytes (P&amp;lt;0.05). The H1FOO mRNA levels in oocytes cultured with EGF and P4 increased significantly compared with oocytes cultured in EGF alone (P&amp;lt;0.05). In contrast, mRNA levels for cyclin B1 in oocytes cultured with P4 were higher than those cultured in the presence of EGF alone (P&amp;lt;0.05). In addition, levels of mRNA for eIF4E showed a significant reduction in oocytes cultured with P4 compared with those pre-matured with EGF or both EGF and P4. The EGF treatment reduced the levels of mRNA for GDF9 compared with control medium. The mRNA levels of PARN did not differ significantly between treatments. In conclusion, EGF, P4, and EGF and P4 combined did not influence oocyte growth and meiotic resumption. However, EGF or P4 increased the mRNA expression of cMOS, whereas EGF reduced the levels of transcripts for GDF9.