Action Of Epidermal Growth Factor Research Articles

Plumbagin as a preferential lead molecule to combat EGFR-driven matrix abundance and migration of cervical carcinoma cells.

The handshake between the complex networks of matrix components in the tumor micro-environment (TME) is considered as a crucial event in the progression of several cancers including cervical carcinoma (CC). A number of studies report a connection between epidermal growth factor (EGF) and matrix component production. Studies demonstrate that the mechano-transduction trigger by collagen, influences the tumor cells to undergo epithelial-mesenchymal transition (EMT) and block the entryof drugs. We hypothesize that the intervention to prevent EGF triggered deposition of matrix components could sensitize several therapies for CC cells. We utilized morphological assessment, MTT assay, mitored tracking, acridine orange (AO)/ ethidium bromide (EtBr) staining and bromodeoxyuridine (BrdU) assay to measure the cell viability, mitochondrial activity, cellular apoptosis, and DNA synthesis. Clonogenic assay and scratch healing assay were executed to address the stemness and migratory potential. Detection of glycosaminoglycan's (GAGs), collagen, matrix metalloproteinase (MMP)-2/9 secretion and calcium (Ca2+) ions were performed to assess the production of matrix components. Finally, the interaction between EGFR and plumbagin was evaluated by employing molecular dynamics (MD) simulation. Pre-treating the cells with plumbagin inhibited the EGF-induced EMT along with reduction in cell proliferation, migration, clonogenesis and depletion of matrix components. The actions of EGF and plumbagin were more pronounced in HPV-positive CC cells than HPV-negative CC cells. This study identified that increased matrix production triggered by EGF-rich milieu is inhibited by plumbagin in human papilloma viral (HPV) 68 positive ME180, HPV 16 positive SiHa and HPV-negative C33A cell lines. Delivery of plumbagin directly to TME would effectively accelerate the clearance of CC cells, reduce metastasis and matrix abundance by employing targeted delivery to minimize the undesired effects of plumbagin.

Ellagic acid protected the gingival tissue via fibroblast and epidermal growth factors in rats.

To investigate the effect of ellagic acid (EA) in gingival tissues injury in rats. Twenty rats were categorized into two groups. In burn group, an excisional wound area was created by removing a 4-mm diameter flap from the left molar region in the mucoperiosteal region of the gingiva. In burn + ellagic acid group, 1.2mg/mL EA was administered as irrigation for one week. Animals was sacrificed under anesthesia at the end of experiment. Malondialdehyde (MDA), myeloperoxidase (MPO) and glutathione (GSH) level were measured. Hematoxylin and eosin, fibroblast growth factor (FGF) and epidermal growth factor (EGF) immunostainings were applied to tissues. MDA, MPO, inflammation and leukocyte infiltration were high in burn group. Degeneration epithelium, edema and inflammatory cell infiltration in connective tissue areas, and dilatation and congestion in blood vessels were observed in burn group. In burn + EA group, the gingival epithelium improved, collagen fiber production increased and organized dermis were observed. After burn injury, FGF and EGF activity was increased in EA treated groups. We suggest that EA have the potential for better healing outcomes in oral wounds. EA seems to have promising therapeutic efficacy to enhance oral wound healing.

EGCG attenuate EGF triggered matrix abundance and migration in HPV positive and HPV negative cervical cancer cells.

Our previous laboratory findings suggested the beneficial effects of epigallocatechin gallate (EGCG) against cervical cancer (CC) cells survival. The present study is aimed at identifying the effects of EGCG in preventing the actions of epidermal growth factor (EGF) in human papilloma virus (HPV) 68 positive ME180 and HPV negative C33A CC cells. An elevated level of EGF in tumor micro-environment (TME) is linked to the metastasis of several cancers including CC. We hypothesized that EGCG has the ability to block the actions of EGF. To test this, survival assay was performed in cells treated with or without EGF and EGCG. The mitochondrial activity of cells was ascertained using MTT assay and mitored staining. Protein and non-protein components in the extracellular matrix such as collagen and sulphated glycosaminoglycans (GAGs) were evaluated using sirius red and alcian blue staining, respectively. Matrix metalloproteinase-2 (MMP-2) gene expression and enzymatic activity were assessed using real time-reverse transcriptase-polymerase chain reaction (RT-PCR) and gelatin zymography. Wound healing assay was performed to assess the EGF induced migratory ability and its inhibition by EGCG pre-treatment. Clonogenic assay showed thatEGCG pre-treatment blocked the EGF driven colony formation. In silico analysis performed identified the efficacy of EGCG in binding with different domains of EGF receptor (EGFR). EGCG pre-treatment prevented the epithelial-mesenchymal transition (EMT) and metabolic activity induced by EGF, this is associated with concomitant reduction in the gene expression and enzyme activity of MMP-2. Further, reduced migration and ability to form colonies were observed in EGCG pre-treated cells when stimulated with EGF. HPV positive ME180 cells showed increased migratory and clonogenic ability upon EGF stimulation, whose effects were not much significant in HPV negative C33A cells. EGCG effectively blocked the actions of EGF in both HPV positive and HPV negative conditions and can be advocated as supplementary therapy for the management of EGF driven CC. However, further studies using cell line-derived xenograft(CDX)/patient-derived xenograft(PDX) model system is warranted to validate the therapeutic utility of EGCG.

Plumbagin binds to epidermal growth factor receptor and mitigate the effects of epidermal growth factor micro-environment in PANC-1 cells.

A sustained increase in the mortality of pancreatic cancer (PC) and sudden metastasis-related mortality is a cause for concern. Aberrant expression of epidermal growth factor (EGF) receptor (EGFR) is noted in several cases of PC metastasis. The present study is aimed at analyzing the expression of EGFR in PC and its relevance to the progression of PC. Despite the number of studies that have shown the benefits of plumbagin on PC cells, its role on cancer stem cells remains largely unknown. To this end, the study used an EGF micro-environment to make cancer stem cells in vitro and ascertained the role of plumbagin in mitigating the actions of EGF. The kaplan-meier (KM) plot indicated reduced overall survival (OS) analysis in PC patients with high EGFR than low EGFR expression. Plumbagin pre-treatment significantly prevented EGF-induced survival, epithelial-to-mesenchymal transition (EMT), clonogenesis, migration, matrix metalloproteinase -2 (MMP-2) gene expression and its secretion, and matrix protein hyaluron production in PANC-1 cells. The computational studies indicate the greater affinity of plumbagin with different domains of EGFR than gefitinib. Several hallmarks of resistance and migration due to EGF are effectively attenuated by plumbagin. Collectively, these results warrant investigating the actions of plumbagin in a pre-clinical study to substantiate these findings.

Effects of gallic acid on gingival wounds.

Our aim in this study was to investigate the effect of Gallic acid (GA) on gingival tissue injury. Twenty rats were categorized into two groups. In the burn group, an excisional wound area was created by removing a 4 mm diameter flap from the left molar region in the mucoperiosteal region of the gingiva. In the Burn+gallic acid group, 1.2 mg/ml GA was administered as irrigation for 1 week. Animals were sacrificed under anesthesia at the end of experiment. Malondialdehyde (MDA), myeloperoxidase (MPO) and glutathione (GSH) levels were measured. Hematoxylin Eosin, fibroblast growth factor (FGF) and epidermal growth factor (EGF) immunostaining was applied to tissues. MDA and MPO levels increased, and GSH, epithelization, FGF and EGF expression levels were decreased. Gallic acid treatment improved these scores. Degenerated gingival epithelium, disintegrity in epithelial and connective tissue fibers, edema and inflammatory cells were observed in the burn group. Gallic acid treatment after burn improved the pathologies. After burn injury, FGF and EGF activity was increased in Gallic acid-treated groups. We suggest that GA has the potential for better healing outcomes in oral wounds. GA seems to have promising therapeutic efficacy in enhancing oral wound healing.

Overexpressed cold inducible RNA-binding protein improves cell viability and EGF expression in glial cells

BackgroundCold inducible RNA-binding protein (CIRP) is a key protein in the hypothermic therapy. Highly expressed CIRP exerts a neuroprotective effect on neurons. The aim of this study is to provide the evidence of the protective effects of CIRP on the glial cells and explore the downstream pathway of CIRP.ResultsThe results of this study demonstrated that the cell viability of the glial cells with CIRP overexpression was increased significantly compared to the control. With CIRP overexpression, the epidermal growth factor (EGF) mRNA expression was found increasing significantly and the mRNA expressions of derived neurotrophic factor (BDNF), bcl-2, vascular endothelial growth factor (VEGF) and nerve growth factor (NGF) were not upregulated compared to the control. EGF and CIRP co-expression was demonstrated on the glial cells. With CIRP expression, EGF expression on the glial cells was increased statistically compared to the control.ConclusionCIRP overexpression increases the cell viability of the glial cells, exerting a neuroprotective effect. EGF expression is activated on the glial cells with CIRP overexpression, implying a pathway of CIRP neuroprotection via EGF activation.

Beta-carotene regulates the biological activity of EGF in IEC6 cells by alleviating the inflammatory process

ABSTRACT Epidermal growth factor (EGF) has many important biological functions. It plays an important role in regulating the growth, survival, migration, apoptosis, proliferation, and differentiation of intestinal tissues and cells. However, until now, the effect of inflammation on the biological activity of EGF in intestinal cells or tissues is still unclear. For this reason, in the current research, we have conducted a detailed study on this issue. Using the rat small intestinal crypt epithelial cell line (IEC6) was used as an in vitro model, and Confocal laser scanning microscope (CLSM), Flow cytometry (FCM), Indirect immunofluorescence assay (IFA), Western-blotting (WB), and Quantitative real-time RT-PCR (QRT-PCR) methods were used to explore the effects of inflammation on EGF/EGFR biological activity and signal transduction profiles. We found that the EGF/EGFR nuclear signal almost disappeared in the inflammatory state, and the phosphorylation levels of EGFR, AKT, and STAT3 were all significantly down-regulated. In addition, we also studied the effect of β-carotene on the biological activity of EGF, and found that when cells were pretreated with β-carotene, the cellular behavior, biological activity, and nuclear signal of EGF/EGFR under inflammation stimulation were partially restored. In summary, the current study shows that inflammation can disrupt EGF/EGFR-mediated signaling in IEC6 cells, suggesting that inflammation negatively regulates the biological activity of EGF/EGFR. Furthermore, we found that β-carotene not only attenuated lipopolysaccharide (LPS)-induced inflammation but also partially restored the biological activity of EGF in IEC6 cells, laying a solid foundation for studying the biological functions of EGF and β-carotene.

Uric acid, as a double-edged sword, affects the activity of epidermal growth factor (EGF) on human umbilical vein endothelial cells by regulating aging process

ABSTRACT Uric acid (UA) is the main metabolite of the human body. Although UA is only a product of metabolism, it is important biological regulator. Epidermal growth factor (EGF) has important biological functions. However, so far, the effect of UA on EGF’s activity has not been revealed. For this, in the current study, we systematically studied the effect of OA on the biological activity of EGF. Human Umbilical Vein Endothelial Cells (HUVECs) were used as an in vitro model, and Western-blot, RT-PCR, laser scanning confocal microscopy (CLSM) and co-localization analyses were carried out. The results showed that high concentration of UA (10 mg/dl) severely affected the biological activity of EGF. High concentration of UA suppressed the activity of EGF, and inhibited the biological effect of EGF on the HUVECs. However, it is interesting that EGF-mediated intracellular signaling was significantly down-regulated in the H2O2-induced senescent HUVEC, and physiological concentration of UA could at least partially restore the EGF-mediated signaling. Further work showed that physiological concentration of UA (5 mg/dl) shows the anti-aging effect. Taken together, current research indicates that UA may be a ‘double-edged sword’, physiological concentration of UA may be beneficial, but high concentrations of uric acid (UA) are harmful.

Temporally Controlled Photouncaged Epidermal Growth Factor Influences Cell Fate in Hydrogels.

Hydrogels are powerful materials that more accurately mimic the cellular microenvironment over static two-dimensional culture. Photochemical strategies enable dynamic complexity to be achieved within hydrogels to better mimic the extracellular matrix; however, many photochemical systems to pattern proteins within hydrogels are complicated by long reaction times to immobilize these proteins wherein the protein can lose activity. As proof-of-concept, we demonstrate an elegant method where photocaged proteins are immobilized in hydrogels and then directly photoactivated. Specifically, we immobilized streptavidin-ortho-nitrobenzyl-modified epidermal growth factor (EGF) to cross-linked hyaluronan hydrogels and cultured two EGF-responsive cancer cells of breast and lung therein. We used light to temporally uncage and control EGF activation, thereby inducing cell death in breast cancer cells and proliferation in lung cancer cells. These results show how temporal, photochemical, protein activation influences cellular response and lays the foundation for further advances in manipulating the in vitro environment to control cell fate.

Isoprocurcumenol Supports Keratinocyte Growth and Survival through Epidermal Growth Factor Receptor Activation.

Although proliferation of keratinocytes, a major type of skin cells, is a key factor in maintaining the function of skin, their ability to proliferate tends to diminish with age. To solve such a problem, researchers in medical and skin cosmetic fields have tried to utilize epidermal growth factor (EGF), but achieved limited success. Therefore, a small natural compound that can mimic the activity of EGF is highly desired in both medical and cosmetic fields. Here, using the modified biosensor system, we observed that natural small-compound isoprocurcumenol, which is a terpenoid molecule derived from turmeric, can activate EGFR signaling. It increased the phosphorylation of ERK and AKT, and upregulated the expression of genes related to cell growth and proliferation, such as c-myc, c-jun, c-fos, and egr-1. In addition, isoprocurcumenol induced the proliferation of keratinocytes in both physical and UVB-induced cellular damage, indicative of its function in skin regeneration. These findings reveal that EGF-like isoprocurcumenol promotes the proliferation of keratinocytes and further suggest its potential as an ingredient for medical and cosmetics use.

Protease Inhibitors Protect Bovine Colostrum or Chicken Egg Growth Factors from Pancreatic Enzyme Digestion in AGS Cells or Colitic Rats

BackgroundBovine colostrum (BC) and chicken egg contain proteins possessing growth factor activity. Epidermal growth factor (EGF) provides much of the pro-reparative activity within BC. Clinical use of orally administered peptide growth factors is hampered by digestion from pancreatic proteases. ObjectivesWe examined whether adding a protease inhibitor [soybean trypsin inhibitor (SBTI) or ovomucoid] protected bioactivity of BC ± egg or EGF alone against pancreatic digestion using in vitro and in vivo models. MethodsBC, egg, or EGF alone or in combination with trypsin inhibitors were tested for proliferative (Alamar blue) activity using human gastric adenocarcinoma (AGS) cells, prior to and after incubation with HCl/pepsin and trypsin/chymotrypsin. Data were analyzed using 2-factor ANOVA. Eight groups (n = 10) of adult female Sprague-Dawley rats (mean: 188.3 ± 0.8 g) received 20 mg/kg/d of BC + egg, 100 μg/d of EGF, 5 mg/d ovomucoid, or 10.8 mg/d SBTI, alone or in combination (in 1 mL 3% NaHCO3) by gavage for 9 d and dextran sodium sulfate (DSS; 5% in drinking water) for the final 7 d. Histology, microscopic damage score, and myeloperoxidase (MPO) were assessed and analyzed using 1-factor ANOVA. ResultsProliferative activities of BC, egg, or EGF were reduced 40–57% by HCl/pepsin exposure and further reduced 14–24% by chymotrypsin/trypsin. Co-addition of SBTI or ovomucoid truncated the decrease in proliferative bioactivity caused by chymotrypsin/trypsin by 54–100% (P < 0.01). In vivo study showed oral EGF alone or protease inhibitors given alone were ineffective in reducing DSS damage, whereas SBTI with EGF or ovomucoid with BC + egg improved protective effects on weight gain, disease activity score, colonic MPO, and histology damage by 3–4-fold (P < 0.01). ConclusionsStudies using AGS, cells, and Sprague-Dawley rats showed the protease inhibitors ovomucoid and SBTI protected BC, egg, and EGF against loss of bioactivity due to pancreatic enzymes and, when given with NaHCO3, enhanced colonic protection against DSS damage.

Propofol Affects EGF's Activity in Intestinal Cell by Down-Regulating EGFR-Mediated Intracellular Signaling.

Propofol is a commonly used anesthetic drug in clinic. In recent years, a series of non-anesthetic effects of propofol have been discovered. Studies have shown that propofol has many effects on the intestine. Epidermal growth factor (EGF) is one of the most important growth factors that could regulate intestinal growth and development. In the current study, we studied the effect of protocol on the biological activity of EGF on intestinal tissue and cell models. Through flow cytometry, indirect immunofluorescence and Western-blot and other technologies, it was found that propofol reduced the activity of EGF on intestinal cells, which inhibited EGF-induced intestinal cell proliferation and changed the cell behavior of EGF. To further explore the potential mechanism by which propofol down-regulated epidermal growth factor receptor (EGFR)-induced signaling, we carried out a series of related experiments, and found that propofol may inhibit the proliferation of intestinal cells by inhibiting the EGFR-mediated intracellular signaling pathway. The current research will lay the theoretical and experimental basis for further study of the effect of propofol on the intestine.

EGFR Inhibitors and Apoptosis Inducers : Design, Docking, Synthesis, and Anticancer Activity of Novel Tri-Chalcone Derivatives

Estrogen receptors (ERs) are primarily expressed in cancer cells and interfere with the epidermal growth factor (EGFR) signal pathway. However, data are limited regarding the interaction between cytoplasmic ER expression and EGFR-tyrosine kinase inhibitor response. The objective of this study was to design multi-target molecules for enhancing anti-cancer activity and decreasing drug resistance. Two distinct series of tri-chalcone derivatives S1(1-7), S2(1-7) with promising anti-cancer activity was incorporated into the current study. Compounds S1(1-2) and S2(1-2) demonstrated the highest binding affinity for interactions with the active EGFR binding site. Interestingly, compounds S1-1 and S1-2 have shown a potent cytotoxicity of MCF-7 (2.23 ± 0.11 and 2.04 ± 0.71 µM) and MDA-MB-231 (6.44 ± 0.01 and 3.75 ± 0.26 µM), respectively, relative to tamoxifen IC50 of 9.3 ± 0.44 and 18.92 ± 1.43 µM. Thus, in-vitro EGFR activity was evaluated for compounds S1-1 and S1-2. The results indicated that compounds S1-1 and S1-2 showed a significant improvement in the anti-cancer resistant treatment due to their potential effects on the EGFR and the possible anti-ER effect.

Therapeutic effects of EGF-modified curcumin/chitosan nano-spray on wound healing.

Dermal injury, including trauma, surgical incisions, and burns, remain the most prevalent socio-economical health care issue in the clinic. Nanomedicine represents a reliable administration strategy that can promote the healing of skin lesions, but the lack of effective drug delivery methods can limit its effectiveness. In this study, we developed a novel nano-drug delivery system to treat skin defects through spraying. We prepared curcumin-loaded chitosan nanoparticles modified with epidermal growth factor (EGF) to develop an aqueous EGF-modified spray (EGF@CCN) for the treatment of dermal wounds. In vitro assays showed that the EGF@CCN displayed low cytotoxicity, and that curcumin was continuously and slowly released from the EGF@CCN. In vivo efficacy on wound healing was then evaluated using full-thickness dermal defect models in Wistar rats, showing that the EGF@CCN had significant advantages in promoting wound healing. On day 12 post-operation, skin defects in the rats of the EGF@CCN group were almost completely restored. These effects were related to the activity of curcumin and EGF on skin healing, and the high compatibility of the nano formulation. We therefore conclude that the prepared nano-scaled EGF@CCN spray represents a promising strategy for the treatment of dermal wounds.

Recombinant human epidermal growth factor combined with vacuum sealing drainage for wound healing in Bama pigs

BackgroundVacuum sealing drainage (VSD) and epidermal growth factor (EGF) both play an important role in the treatment of wounds. This study aims to explore the effects of the combination of VSD and EGF on wound healing and the optimal concentration and time of EGF.MethodsWe tested the proliferation and migration capacity of HaCaT and L929 cells at different EGF concentrations (0, 1, 5, 10, and 100 ng/ml) and different EGF action times (2, 10, and 30 min). A full-thickness skin defect model was established using male, 30-week-old Bama pigs. The experiment included groups as follows: routine dressing change after covering with sterile auxiliary material (Control), continuous negative pressure drainage of the wound (VSD), continuous negative pressure drainage of the wound and injection of EGF 10 min followed by removal by continuous lavage (V + E 10 min), and continuous negative pressure drainage of the wound and injection of EGF 30 min followed by removal by continuous lavage (V + E 30 min). The wound healing rate, histological repair effect and collagen deposition were compared among the four groups.ResultsAn EGF concentration of 10 ng/ml and an action time of 10 min had optimal effects on the proliferation and migration capacities of HaCaT and L929 cells. The drug dispersion effect was better than drug infusion after bolus injection effect, and the contact surface was wider. Compared with other groups, the V + E 10 min group promoted wound healing to the greatest extent and obtained the best histological score.ConclusionsA recombinant human epidermal growth factor (rhEGF) concentration of 10 ng/ml can promote the proliferation and migration of epithelial cells and fibroblasts to the greatest extent in vitro. VSD combined with rhEGF kept in place for 10 min and then washed, can promote wound healing better than the other treatments in vivo.

Acetogenin Extracted from Annona muricata Prevented the Actions of EGF in PA-1 Ovarian Cancer Cells.

In individuals with ovarian cancer, an increase in the circulating level of the epidermal growth factor (EGF) is readily apparent. Ovarian cancer cells exhibit signaling pathway of the epidermal growth factor (EGFR) and respond to the EGF. Annona muricata (AM) has been shown to decrease ovarian cell proliferation however, role of AM in regulating EGF actions is not yet to be reported. In this study, we proposed that the fractionated compound acetogenin can inhibit the activation of EGFR-regulated signaling cascades such as MAPK7 / PI3K-Akt / mTOR / STAT upon EGF stimulation. Ethanolic extract was prepared for the whole AM plant and Thin Layer Chromatography (TLC) was performed to characterize the secondary metabolites and each fraction was assessed using kedde reagent for the presence of acetogenin. The effects of acetogenins were then tested on the survival of PA-1 ovarian cancer cells under basal and EGF stimulated conditions. To delineate the role of acetogenin in EGFR signaling cascades, the in silico docking studies were conducted. The fraction of acetogenin decreased the viability of EGF induced PA-1 ovarian cancer cells that indicating the EGF inhibitory effects of acetogenin. The docking studies specifically illustrated that when the acetogenin binding with tyrosine kinase (TK) and regulatory unit (RU) which subsequently resulted in a reduction in EGF induced the survival of PA-1 ovarian cancer cells. The vital regulatory role of acetogenin reported in this study indicate significant anticancer activities of acetogenin from AM. The in silico study of the acetogenin function predicted that it binds specifically to Asp837 (phosphor-acceptor site) of EGFR, essential for phosphorylation of substrates in the TK domain and RU which promote downstream signaling. Acetogenin isolated from AM effectively inhibited the survival of PA-1 ovarian cancer cells through impaired EGF signaling.

Pituitary Actions of EGF on Gonadotropins, Growth Hormone, Prolactin and Somatolactins in Grass Carp

Simple SummaryIn mammals, the functions of epidermal growth factor (EGF) have been widely studied. However, little is known about the pituitary actions of EGF in teleost. Using primary cultured grass carp pituitary cells as model, we found that EGF could reduce pituitary luteinizing hormone β (LHβ) mRNA expression, but induce pituitary growth hormone (GH), prolactin (PRL) and somatolactins (SL) mRNA expression. Furthermore, we also found that NKB could suppress EGF-induced PRL mRNA expression in grass carp pituitary cells. These results suggested that EGF could directly regulate pituitary hormones expression in teleost.In mammals, epidermal growth factor (EGF) plays a vital role in both pituitary physiology and pathology. However, the functional role of EGF in the regulation of pituitary hormones has rarely reported in teleost. In our study, using primary cultured grass carp pituitary cells as an in vitro model, we examined the effects of EGF on pituitary hormone secretion and gene expression as well as the post-receptor signaling mechanisms involved. Firstly, we found that EGF significantly reduced luteinizing hormone (LHβ) mRNA expression via ErbB1 coupled to ERK1/2 pathway, but had no effect on LH release in grass carp pituitary cells. Secondly, the results showed that EGF was effective in up-regulating mRNA expression of growth hormone (GH), somatolactin α (SLα) and somatolactin β (SLβ) via ErbB1 and ErbB2 and subsequently coupled to MEK1/2/ERK1/2 and PI3K/Akt/mTOR pathways, respectively. However, EGF was not effective in GH release in pituitary cells. Thirdly, we found that EGF strongly induced pituitary prolactin (PRL) release and mRNA expression, which was mediated by ErbB1 and subsequent stimulation of MEK1/2/ERK1/2 and PI3K/Akt/mTOR pathways. Interestingly, subsequent study further found that neurokinin B (NKB) significantly suppressed EGF-induced PRL mRNA expression, which was mediated by neurokinin receptor (NK2R) and coupled to AC/cAMP/PKA signal pathway. These results suggested that EGF could differently regulate the pituitary hormones expression in grass carp pituitary cells.

Abstract 1430: CaMKK2 regulates EGF-dependent activation of oncogenic Akt in ovarian cancer cells

Abstract Background-The canonical PI3K/Akt pathway is hyperactive in a variety of cancers including ovarian cancer (OVCa). Using baculovirus-expressed, FPLC-purified enzymes, we previously demonstrated that Calcium/Calmodulin-dependent CaM Kinase Kinase 2 (CaMKK2) activates the oncogene Akt by direct phosphorylation of its primary activation site T308. In OVCAR-3 and other OVCa cells, RNAi-mediated knockdown of CaMKK2 decreased basal Akt phosphorylation at T308 in a Phosphoinositide-Dependent Kinase-1 (PDK1)-independent manner. CaMKK2 knockdown also decreased Akt phosphorylation at its secondary activation site, S473 by an apparently indirect mechanism. Results- In this study we show that CaMKK2 mediates downstream effects of epidermal growth factor (EGF) on the Akt cascade in OVCa cells. Treatment of OVCAR3 cells with EGF produced a prominent enhancement of p-Akt T308 which was reduced ~2-fold, by either CaMKK2 or PDK1 knockdowns. Knockdown of CaMKK2 but not PDK1 reduced p-Akt S473 after EGF treatment. Similar results were obtained with Insulin-like growth factor 1 (IGF1). To discern how CaMKK2 is engaged by EGF action we examined whether, in vitro, CaMKK2 phosphorylation of WT Akt or a synthetic peptide encompassing T308 is stimulated by Calcium/CaM and/or Phosphatidylinositol (3,4,5)-trisphosphate (PIP3). CaMKK2 activity was enhanced by Calcium/CaM but not by soluble PIP3. CaMKK2 did not bind to a dot blot of various phosphoinositides, although strong binding to PIP3 was observed with PDK1. Alternatively, Akt regulation by CaMKK2 may occur via a Calcium/CaM-dependent pathway. Increased p-Akt T308 and S473 upon treatment of cells with the calcium-ionophore, ionomycin, was significantly reduced by CaMKK2 knockdown. EGF stimulation of Akt phosphorylation at both T308 and S473 was significantly reduced by the intracellular calcium chelator, BAPTA-AM and by the CaM inhibitor, W7. We also probed the mechanism by which optimal cellular Akt phosphorylation at S473 requires CaMKK2. mTOR complex 2 (mTORC2) and DNA-dependent protein kinase (DNA-PKcs) are considered candidate kinases for p-Akt S473. CaMKK2 knockdown regulated p-Akt S473 in M059J cells, a DNA-PKcs null cell line similarly to that observed with OVCAR3 cells, suggesting that mTORC2 may be regulated by CaMKK2. Using an MS/MS Protein-protein interaction (PPI) screen we observed CaMMK2 interaction with multiple ribosomal proteins an observation consistent with suggested mTORC2 ribosomal regulation of Akt S473 phosphorylation. Conclusions-This study documents Akt activating phosphorylation by CaMKK2 in response to EGF in a Calcium/CaM-dependent, PI3K/PIP3 independent, manner. Besides directly phosphorylating Akt at T308, CaMKK2 regulates p-Akt S473 potentially through the regulation of mTORC2 activity. Our results highlight the importance of CaMKK2 activated by growth factor signals as a parallel system to the oncogenic PI3K/PDK1 pathway. Citation Format: Shuhang Dai, Angela M. Gocher, Arthur M. Edelman. CaMKK2 regulates EGF-dependent activation of oncogenic Akt in ovarian cancer cells [abstract]. In: Proceedings of the Annual Meeting of the American Association for Cancer Research 2020; 2020 Apr 27-28 and Jun 22-24. Philadelphia (PA): AACR; Cancer Res 2020;80(16 Suppl):Abstract nr 1430.

Epidermal Growth Factor (EGF) Augments the Invasive Potential of Human Glioblastoma Multiforme Cells via the Activation of Collaborative EGFR/ROS-Dependent Signaling.

Abnormal secretion of epidermal growth factor (EGF) by non-neuronal cells (e.g., glioma-associated microglia) establishes a feedback loop between glioblastoma multiforme (GBM) invasion and a functional disruption of brain tissue. Considering the postulated significance of this vicious circle for GBM progression, we scrutinized mechanisms of EGF-dependent pro-invasive signaling in terms of its interrelations with energy metabolism and reactive oxygen species (ROS) production. The effects of EGF on the invasiveness of human glioblastoma T98G cells were estimated using time-lapse video microscopy, immunocytochemistry, cell cycle assay, immunoblot analyses, and Transwell® assay. These techniques were followed by quantification of the effect of EGFR (Epidermal Growth Factor Receptor) and ROS inhibitors on the EGF-induced T98G invasiveness and intracellular ROS, ATP, and lactate levels and mitochondrial metabolism. The EGF remarkably augmented the proliferation and motility of the T98G cells. Responses of these cells were accompanied by cellular rear–front polarization, translocation of vinculin to the leading lamellae, and increased promptness of penetration of micropore barriers. Erlotinib (the EGFR inhibitor) significantly attenuated the EGF-induced T98G invasiveness and metabolic reprogramming of the T98G cells, otherwise illustrated by the increased mitochondrial activity, glycolysis, and ROS production in the EGF-treated cells. In turn, ROS inhibition by N-acetyl-L-cysteine (NAC) had no effect on T98G morphology, but considerably attenuated EGF-induced cell motility. Our data confirmed the EGFR/ROS-dependent pro-neoplastic and pro-invasive activity of EGF in human GBM. These EGF effects may depend on metabolic reprogramming of GBM cells and are executed by alternative ROS-dependent/-independent pathways. The EGF may thus preserve bioenergetic homeostasis of GBM cells in hypoxic regions of brain tissue.

EGF released from human placental mesenchymal stem cells improves premature ovarian insufficiency via NRF2/HO-1 activation.

Human placental mesenchymal stem cells (hPMSCs) have the ability to release cytokines and to differentiate into the three germ layers. To date, the relevance of hPMSCs for the treatment of premature ovarian insufficiency (POI) disease through the regulation of oxidative stress is still unclear. Therefore, to evaluate the therapeutic efficiency and investigate the mechanism of hPMSCs, we generated a mouse model of POI and collected human ovarian granule cells (hGCs) from patients with POI. hPMSCs displayed therapeutic effects on POI ovarian function, including recovered follicular numbers and increased expression of oocyte markers. Furthermore, secretion of the cytokine EGF (epidermal growth factor) was higher from hPMSCs than it was from other cells. FACS and Western blot analyses showed that EGF elevated the proliferation and reduced the apoptosis in hGCs. hPMSCs and EGF inhibited oxidative stress levels. Protein assays demonstrated that EGF suppressed oxidative stress by dose-dependently upregulating the expression of the NRF2/HO-1 pathway, and it inhibited the apoptosis by regulating the PTEN/PI3K/AKT pathway. These findings provide an experimental foundation for hPMSCs in improving ovarian function through the secretion of EGF. The mechanism of action of EGF is related to protection from oxidative stress by activation of the NRF2/HO-1.