HB-EGF Research Articles

Reverse of the resistance to paclitaxel of the heparin binding-epidermal growth factor-like growth factor inhibitor in ovarian cancer

Objective: To investigate the effect and mechanism of CRM197, the heparin binding-epidermal growth factor-like growth factor (HB-EGF) inhibitor, on the reverse of the resistance of ovarian cancer to paclitaxel. Methods: (1)The effect of CRM197 on the 50% inhibitory concentrations (IC(50)) of human ovarian carcinoma cell line A2780 and paclitaxel-resistant ovarian carcinoma cell line A2780/Taxol was tested by methyl thiazolyl tetrazolium (MTT) assay. Western blot was used to detect the effect of CRM197 on the expression of HB-EGF, epidermal growth factor receptor (EGFR) and plasma membrane glycoprotein (P-gp) protein in A2780 and A2780/Taxol cells. Real-time PCR was used to examine the MDR1 mRNA expression in these cells. (2) A2780/Taxol cells were divided into 4 groups, including the cells transfected with empty vector and saline treatment (empty vector group), MDR1 small interference RNA (siRNA) vector and saline treatment (MDR1 siRNA group), empty vector and CRM197 treatment (empty vector+CRM197 group) and MDR1 siRNA vector and CRM197 treatment (MDR1 siRNA+CRM197 group), respectively. Flow cytometry was used to detecte the effect of intracellular rhodomine 123 (Rh123) accumulation, and caspase-3 activity assay was used to test the effect of apoptosis in four groups of A2780/Taxol cells. (3) In experiments in vivo, A2780/Taxol cells were inoculated to nude mouse subcutaneously to determine the EGFR and P-gp protein expression following CRM197 treatment by immunohistochemistry. Results: (1) In vitro, MTT examination showed that the IC(50) of A2780/Taxol cells to paclitaxel in A2780/Taxol+CRM197 group [(6.4±0.3) μmol/L] was significantly lower than the IC(50) in A2780/Taxol group [ (34.1±0.5) μmol/L, P<0.01], and the reveral fold of CRM197 was 5.3. The expression level of HB-EGF protein in A2780/Taxol+CRM197 group (1.44±0.29) was significantly lower than HB-EGF protein in A2780/Taxol group (2.72±0.32), respectively (P<0.05). The expression level of EGFR protein (0.71±0.25) and P-gp protein (0.82±0.19) in A2780/Taxol+CRM197 group was significantly lower than EGFR protein (1.87±0.31) and P-gp protein (1.84±0.27) of A2780/Taxol group (P<0.05). Compared with A2780/Taxol group (1.78±0.27) , MDR1 mRNA was significantly down-regulated in A2780/Taxol+CRM197 group (0.79±0.13, P<0.05). (2) The fluorescence intensity of Rh123 of the A2780/Taxol cells in empty vector group, MDR1 siRNA group,empty vector+CRM197 group, MDR1 siRNA+CRM197 group was 33.4±1.6, 56.3±3.3, 43.5±3.1,100.4±7.4, and the pNA of the A2780/Taxol cells was (11.4±1.2) , (52.8±0.9) , (71.2±3.6) , (82.7±3.8) μmol/L. The expression levels in MDR1 siRNA+CRM197 group were both higher than the expression levels in empty vector+CRM197 group, and the expression levels in empty vector+CRM197 group, MDR1 siRNA group were both higher than the expression levels in empty vector group (P<0.05). (3) In vivo, the expression scores of EGFR protein in A2780/Taxol+CRM197 tumors (4.4±1.4) were lower than that in A2780/Taxol tumors (10.2±3.1, P<0.05). The expression scores of P-gp protein in A2780/Taxol+CRM197 tumors (3.8±1.1) were lower than that in A2780/Taxol tumors (8.8±2.7, P<0.05). Conclusion: CRM197 reverses the resistance of ovarian cancer to paclitaxel by increasing caspase-3 activity to advance apoptosis via EGFR/MDR1/P-gp pathway.

HB-EGF expression as a potential biomarker of acquired middle ear cholesteatoma

Conclusions: The heparin-binding epidermal growth factor-like growth factor (HB-EGF) plays an essential role in the development and invasiveness of cholesteatoma. This study may help to realize the molecular mechanisms underlying the pathogenesis of cholesteatoma and make HB-EGF a promising target for drug intervention of cholesteatoma.Objective: To detect HB-EGF expression in human surgical specimens of acquired middle ear cholesteatoma and analyze its functional role as a regulator of epithelial keratinocytes hyperproliferation.Methods: A total of 34 patients who underwent surgical treatment for middle ear cholesteatoma were recruited in the study. The mRNA and protein expression of HB-EGF in middle ear cholesteatoma tissues and normal postauricular skin tissues was investigated by real-time quantitative reverse-transcription-polymerase chain reaction (RT-qPCR), immunohistochemical staining, and western blot. The correlation between bone resorption degree and HB-EGF expression was also analyzed.Results: On average, compared with normal postauricular skin, expression of HB-EGF mRNA in the cholesteatoma epithelium was significantly elevated 2.41-fold by RT-qPCR, and HB-EGF protein significantly upregulated 2.32-fold by western blot. Positive HB-EGF immunostaining observed in the basal and suprabasal layers of cholesteatoma epithelium was significantly stronger than in normal postauricular skin. Meanwhile, an obviously positive correlation between HB-EGF protein expression and bone resorption degree was discovered.

Heparin-binding epidermal growth factor-like growth factor promotes neuroblastoma differentiation.

High-risk neuroblastoma is characterized by undifferentiated neuroblasts and low schwannian stroma content. The tumor stroma contributes to the suppression of tumor growth by releasing soluble factors that promote neuroblast differentiation. Here we identify heparin-binding epidermal growth factor-like growth factor (HBEGF) as a potent prodifferentiating factor in neuroblastoma. HBEGF mRNA expression is decreased in human neuroblastoma tumors compared with benign tumors, with loss correlating with decreased survival. HBEGF protein is expressed only in stromal compartments of human neuroblastoma specimens, with tissue from high-stage disease containing very little stroma or HBEGF expression. In 3 human neuroblastoma cell lines (SK-N-AS, SK-N-BE2, and SH-SY5Y), soluble HBEGF is sufficient to promote neuroblast differentiation and decrease proliferation. Heparan sulfate proteoglycans and heparin derivatives further enhance HBEGF-induced differentiation by forming a complex with the epidermal growth factor receptor, leading to activation of the ERK1/2 and STAT3 pathways and up-regulation of the inhibitor of DNA binding transcription factor. These data support a role for loss of HBEGF in the neuroblastoma tumor microenvironment in neuroblastoma pathogenesis.-Gaviglio, A. L., Knelson, E. H., Blobe, G. C. Heparin-binding epidermal growth factor-like growth factor promotes neuroblastoma differentiation.

BK-UM in patients with recurrent ovarian cancer or peritoneal cancer: a first-in-human phase-I study

BackgroundBK-UM (CRM197) is a mutant form of diphtheria toxin and a specific inhibitor of heparin-binding epidermal growth factor-like growth factor (HB-EGF). We assessed the safety, pharmacokinetics, recommended dose, and efficacy of BK-UM in patients with recurrent ovarian cancer (OC) or peritoneal cancer (PC), and measured HB-EGF levels in serum and abdominal fluid after BK-UM administration.MethodsEleven patients with advanced or recurrent OC or PC were enrolled and treated with BK-UM via the intraperitoneal route. The dose was escalated (1.0, 2.0, 3.3, and 5.0 mg/m2) using a 3 + 3 design.ResultsEight of 11 patients completed treatment. No dose-limiting toxicity (DLT) was experienced at dose levels 1 (1.0 mg/m2) and 2 (2.0 mg/m2). Grade 3 transient hypotension as an adverse event (defined as a DLT in the present study) was observed in two of four patients at dose level 3 (3.3 mg/m2). Treatment with BK-UM was associated with decreases in HB-EGF levels in serum and abdominal fluid in seven of 11 patients and five of eight patients, respectively. Clinical outcomes included a partial response in one patient, stable disease in five patients, and progressive disease in five patients.ConclusionsBK-UM was well tolerated at doses of 1.0 and 2.0 mg/m2, with evidence for clinical efficacy in patients with recurrent OC or PC. A dose of 2.0 mg/m2 BK-UM is recommended for subsequent clinical trials.Trial registrationThis trial was prospectively performed as an investigator-initiated clinical trial. The trial numbers are UMIN000001002 and UMIN000001001, with registration dates of 1/30/2008 and 2/4/2008, respectively. UMIN000001001 was registered as a trial for the continuous administration of BK-UM after UMIN000001002.

Functional Outcomes of Heparin-Binding Epidermal Growth Factor-Like Growth Factor for Regeneration of Chronic Tympanic Membrane Perforations in Mice.

We aim to demonstrate that regeneration of chronic tympanic perforations with heparin-binding epidermal growth factor-like growth factor (HB-EGF) delivered by an injectable hydrogel restored hearing to levels similar to that of nonperforated tympanic membranes. Chronic tympanic membrane perforation is currently managed as an outpatient surgery with tympanoplasty. Due to the costs of this procedure in the developed world and a lack of accessibility and resources in developing countries, there is a great need for a new treatment that does not require surgery. In this study, we show in a mouse model through measurement of auditory brainstem response and distortion product otoacoustic emissions that tympanic perforations lead to hearing loss and this can be predominantly recovered with HB-EGF treatment (5 μg/mL). Our animal model suggests a return to function between 2 and 6 months after treatment. Auditory brainstem response thresholds had returned to the control levels at 2 months, but the distortion product otoacoustic emissions returned between 2 and 6 months. We also show how the vibration characteristics of the regenerated tympanic membrane, as measured by laser Doppler vibrometry, can be similar to that of an unperforated tympanic membrane. Using the best available methods for preclinical evaluation in animal models, it is likely that HB-EGF-like growth factor treatment leads to regeneration of chronic tympanic membrane perforations and restoration of the tympanic membrane to normal function, suggesting a potential route for nonsurgical treatment.

P2.01-059 Regulation of Glycodelin Expression - An Immunomodulatory and Pregnancy Associated Protein in NSCLC

Glycodelin (gene name: progesterone-associated endometrial protein, PAEP) is a protein initially described as an immune system modulator during the establishment of pregnancy. Former studies determined an atypical expression and secretion of glycodelin in non-small cell lung cancer (NSCLC), the most common type of lung cancer. To date, there is not much known about the signaling pathway which regulates PAEP/glycodelin expression in cancer. However, initial experiments already revealed an inducing effect of epidermal growth factor (EGF), heparin-binding epidermal growth factor-like growth factor (HB-EGF), lysophosphatidic acid (LPA) and Phorbol 12-myristate 13 acetate (PMA) on PAEP/glycodelin expression in two NSCLC cell lines (H1975 and 2106T). In this study, we analyzed an extended number of possible regulatory candidates to acquire a more detailed view on the regulatory pathway of PAEP/glycodelin in NSCLC. A lung adenocarcinoma (H1975) and a lung squamous cell carcinoma cell line (2106T) were transfected with siRNA targeting nuclear factor κB1 (NFκB1) or treated with human chorionic gonadotropin (hCG), transforming growth factor-β (TGF-β) 1, -2, -3, protein kinase C (PKC) activator bryostatin 1 and PKC inhibitor GF109203x, respectively. Additionally, combined treatments with GF109203x and TGF-β 1,-2, EGF or HB-EGF were performed. PAEP expression in the manipulated cells was determined by quantitative polymerase chain reaction (qPCR), while glycodelin expression or secretion was detected by western blot analysis. NFκB1 siRNA transfection resulted in decreased PAEP and glycodelin amounts (H1975 and 2106T), whereas hCG (H1975 and 2106T) and TGF-β 1, -2, -3 (2106T) treatment led to higher levels. In bryostatin treated cells (H1975 and 2106T), PAEP/glycodelin expression was upregulated. The contradictory effect could be demonstrated for cells treated with the PKC inhibitor GF109203x alone and in combination with TGF-β 1,-2, EGF or HB-EGF (H1975 and 2106T). This study revealed that there are different regulation mechanisms of PAEP/glycodelin induction in NSCLC. Especially, PKC seems to be involved as a key molecule. The investigated candidates which play a crucial role in driving this signaling pathway are all known to promote the development of cancer. Elucidating the regulatory pathway of the immune system modulating protein glycodelin might reveal a potential strategy to weaken the immune system defense of lung tumors.

Arachidonate 12-Lipoxygenase Inhibitors Promote S100A3 Citrullination in Cultured SW480 Cells and Isolated Hair Follicles.

The human hair shaft is covered with multiple scale-like cuticular layers. During the terminal differentiation stage of immature cuticular cells within the hair follicle, cysteine-rich calcium binding S100A3 protein is predominantly translated, and its arginine residues are converted to citrullines by peptidylarginine deiminases (PADI). In this study, we found several naturally occurring compounds (e.g., hinokitiol, escletin, and quercetin) elevate S100A3 citrullination in a human colorectal adenocarcinoma cell line (SW480). Selected compounds similarly promoted cuticular differentiation within isolated human hair follicles. Their promotive activities correlated with the previously reported inhibitory activities of arachidonate 12-lipoxygenase (ALOX12) in vitro. Microarray analysis revealed that ALOX12 inhibitor remarkably up-regulated heparin-binding epidermal growth factor-like growth factor (HBEGF). ALOX12 inhibitor and recombinant HBEGF similarly regulated expression of PADI genes in SW480 cells. In isolated hair follicles, arachidonic acid strongly promoted S100A3 citrullination along with elevation of HBEGF. These results suggest that ALOX12 inhibition efficiently triggers hair cuticle maturation by modulating arachidonate metabolism in concert with HBEGF.

Localisation of stem cell factor, stanniocalcin-1, connective tissue growth factor and heparin-binding epidermal growth factor in the bovine uterus at the time of blastocyst formation.

Early embryonic losses before implantation account for the highest rates of reproductive failure in mammals, in particular when in vitro-produced embryos are transferred. In the present study, we used molecular biology techniques (real-time quantitative polymerase chain reaction), classical immunohistochemical staining coupled with confocal microscopy and proteomic analysis (multiple reaction monitoring and western blot analysis) to investigate the role of four growth factors in embryo-uterine interactions during blastocyst development. Supported by a validated embryo transfer model, the study investigated: (1) the expression of stem cell factor (SCF), stanniocalcin-1 (STC1), connective tissue growth factor (CTGF) and heparin-binding epidermal growth factor-like growth factor (HB-EGF) in bovine uterine fluid; (2) the presence of SCF, STC1, CTGF and HB-EGF mRNA and protein in the bovine endometrium and embryos; and (3) the existence of reciprocal regulation between endometrial and embryonic expression of SCF, STC1, CTGF and HB-EGF. The results suggest that these growth factors most likely play an important role during preimplantation embryo development in cattle. The information obtained from the present study can contribute to improving the performance of in vitro culture technology in cattle and other species.

Expression of PGR, HBEGF, ITGAV, ITGB3 and SPP1 genes in eutopic endometrium of infertile women with endometriosis during the implantation window: a pilot study.

Alterations in endometrial receptivity may be involved in the etiopathogenesis of endometriosis-related infertility. The literature has suggested that patients with endometriosis present progestin resistance, which could affect embryo implantation. We question the presence of alterations in the expression of the progesterone receptor gene (PGR) and the genes related to endometrium-embryo interaction regulated by progesterone. This pilot study compared the expression of PGR, HBEGF, ITGAV, ITGB3, and SPP1 genes in eutopic endometrium during the implantation window (IW) in infertile women with endometriosis with that observed in the endometrium of fertile and infertile controls. In this prospective case-control study, endometrial biopsies were performed during the IW in patients aged between 18 and 45 years old, with regular cycles and without endocrine/systemic dysfunctions, divided into endometriosis (END), infertile control (IC) and fertile control (FC) groups. Total RNA extraction, cDNA synthesis, and gene expression analysis by Real-Time PCR were performed. We assessed the size of the difference that our series was powered to detect. From the 687 patients who underwent diagnostic videolaparoscopy or tubal ligation at the University Hospital, 130 were eligible. Of these, 32 had endometrial samples collected, with 17 confirmed in the IW. Fifteen samples (5 END, 5 IC and 5 FC) were analyzed. There was no significant difference in the expression of any studied gene. Our sample size allowed us to identify or discard large differences (two standard deviations) among the groups. Endometriosis doesn't cause large changes in the endometrial expression of PGR, HBEGF, ITGAV, ITGB3 and SPP1 during the IW.

Matrix Metalloproteinase 14 promotes lung cancer by cleavage of Heparin-Binding EGF-like Growth Factor

Molecularly targeted therapies benefit approximately 15–20% of non-small cell lung cancer (NSCLC) patients carrying specific drug-sensitive mutations. Thus, there is a clinically unmet need for the identification of novel targets for drug development. Here, we performed RNA-deep sequencing to identify altered gene expression between malignant and non-malignant lung tissue. Matrix Metalloproteinase 14 (MMP14), a membrane-bound proteinase, was significantly up-regulated in the tumor epithelial cells and intratumoral myeloid compartments in both mouse and human NSCLC. Overexpression of a soluble dominant negative MMP14 (DN-MMP14) or pharmacological inhibition of MMP14 blocked invasion of lung cancer cells through a collagen I matrix in vitro and reduced tumor incidence in an orthotopic K-RasG12D/+p53−/− mouse model of lung cancer. Additionally, MMP14 activity mediated proteolytic processing and activation of Heparin-Binding EGF-like Growth Factor (HB-EGF), stimulating the EGFR signaling pathway to increase proliferation and tumor growth. This study highlights the potential for development of therapeutic strategies that target MMP14 in NSCLC with particular focus on MMP14-HB-EGF axis.

High early growth response 1 (EGR1) expression correlates with resistance to anti-EGFR treatment in vitro and with poorer outcome in metastatic colorectal cancer patients treated with cetuximab.

Biomarkers, such as mutant RAS, predict resistance to anti-EGFR therapy in only a proportion of patients, and hence, other predictive biomarkers are needed. The aims were to identify candidate genes upregulated in colorectal cancer cell lines resistant to anti-EGFR monoclonal antibody treatment, to knockdown (KD) these genes in the resistant cell lines to determine if sensitivity to anti-EGFR antibody was restored, and finally to perform a pilot correlative study of EGR1 expression and outcomes in a cohort of metastatic colorectal cancer (mCRC) patients given cetuximab therapy. Comparative expression array analysis of resistant cell lines (SW48, COLO-320DM, and SNU-C1) vs sensitive cell lines (LIM1215, CaCo2, and SW948) was performed. The highest up-regulated gene in each resistant cell line was knocked down (KD) using RNA interference, and effect on proliferation was assessed with and without anti-EGFR treatment. Expression of the candidate genes in patients' tumours treated with cetuximab was assessed by immunohistochemistry; survival analyses were performed comparing high vs low expression. Genes significantly upregulated in resistant cell lines were EGR1 (early growth response protein 1), HBEGF (heparin-binding epidermal growth factor-like growth factor), and AKT3 (AKT serine/threonine kinase 3). KD of each gene resulted in the respective cells being more sensitive to anti-EGFR treatment, suggesting that the resistant phenotype was reversed. In the pilot study of mCRC patients treated with cetuximab, both median PFS (1.38months vs 6.79months; HR 2.77 95%CI 1.2-19.4) and median OS (2.59months vs 9.82months; HR 3.0 95%CI 1.3-23.2) were significantly worse for those patients with high EGR1 expression. High EGR1 expression may be a candidate biomarker of resistance to anti-EGFR therapy.

Abstract A65: Initiation of pancreatic metaplasia requires ADAM17-mediated autocrine signaling, not paracrine signaling derived from macrophages

Abstract Acinar-to-ductal metaplasia (ADM) has been frequently seen in the damaged pancreatic tissues. It is also associated with PanIN lesions in PDAC samples derived from human and mouse and has been shown to act as a preneoplastic lesion in mice. Therefore, understanding the mechanisms of lesion formation at the earliest phase would provide needed information for studying pancreatic tumorigenesis. Previously, we have shown that EGFR activity is required for metaplastic duct formation and KRas-induced pancreatic tumorigenesis. In addition, a prominent macrophage infiltration has been shown to be important in the early phase of lesion formation. To investigate the origin of EGFR ligands for EGFR activation during ADM formation in vivo, we examined the metaplasia formation by administration of cerulein in the mice with the conditional knockout of a key EGFR ligand sheddase ADAM17 in the pancreas (ADAM17fl/fl;Ptf1aCre/+) or in the myeloid cells (ADAM17fl/fl;LysM-Cre). With 2 weeks cerulein treatment, wild type and ADAM17fl/fl;LysM-Cre mice showed a similar degree of metaplastic and fibrotic response. However, the mice with ADAM17-null pancreas displayed significant protection from chronic pancreatitis induced by cerulein, including a lack of significant macrophage infiltration, making the analysis of any additional contribution from macrophage derived EGFR ligands. To remedy this, we conditionally knocked out HB-EGF (Heparin-binding EGF-like growth factor) one of the most robustly expressed EGFR ligands in macrophages using LysM-Cre. We examined cerulein-induced metaplasia in HB-EGFfl/fl;LysM-Cre mice. Compared to wild type mice, HB-EGFfl/fl;LysM-Cre mice showed a similar degree of metaplastic and fibrotic response and a decrease in cell proliferation; moreover, myeloid cell-specific ablation of HB-EGF dramatically delayed the healing and the restoration of normal pancreatic epithelium after cessation of cerulein treatment. These results indicate that an ADAM17/EGFR ligand/EGFR autocrine loop derived from pancreatic parenchymal cells is required for metaplastic duct formation, and macrophage-derived HBEGF acting in a paracrine fashion contributes to cell survival and tissue remodeling. Citation Format: Hui-Ju Wen, Howard C. Crawford.{Authors}. Initiation of pancreatic metaplasia requires ADAM17-mediated autocrine signaling, not paracrine signaling derived from macrophages. [abstract]. In: Proceedings of the AACR Special Conference on Pancreatic Cancer: Advances in Science and Clinical Care; 2016 May 12-15; Orlando, FL. Philadelphia (PA): AACR; Cancer Res 2016;76(24 Suppl):Abstract nr A65.

Receptor-Mediated Enhanced Cellular Delivery of Nanoparticles Using Recombinant Receptor-Binding Domain of Diphtheria Toxin

Antibodies and peptides are often used to home nanoparticles (NPs) to specific cells. Here in this work, we have used recombinant receptor-binding domain of diphtheria toxin (RDT) as a homing molecule for NPs. Diphtheria toxin binds to heparin binding EGF-like growth factor (HB-EGF) through its receptor-binding domain. HB-EGF is often overexpressed as cell surface molecule in various types of cancer. We have prepared monodispersed, spherical PLGA NPs and coated these NPs with RDT. These NPs are characterized by FESEM and FT-IR spectroscopy. Using flow cytometry and fluorescence spectroscopy, we show that coating with RDT increases cellular uptake of PLGA NPs. We further show that RDT-coated nanoparticles are internalized through clathrin-dependent receptor-mediated endocytosis that can be reduced by specific inhibitor. These RDT-coated nanoparticles (RDT-NP) were further used for preferential delivery of Irinotecan, a chemotherapeutic agent, to cells overexpressing HB-EGF. We show that receptor-mediated enhanced uptake of RDT-NPs increases the potency of irinotecan in these cells.

Attenuation of the Type IV Pilus Retraction Motor Influences Neisseria gonorrhoeae Social and Infection Behavior.

ABSTRACTRetraction of the type IV pilus (Tfp) mediates DNA uptake, motility, and social and infection behavior in a wide variety of prokaryotes. To date, investigations into Tfp retraction-dependent activities have used a mutant deleted of PilT, the ATPase motor protein that causes the pilus fiber to retract. ΔpilT cells are nontransformable, nonmotile, and cannot aggregate into microcolonies. We tested the hypothesis that these retraction-dependent activities are sensitive to the strength of PilT enzymatic activity by using the pathogen Neisseria gonorrhoeae as a model. We constructed an N. gonorrhoeae mutant with an amino acid substitution in the PilT Walker B box (a substitution of cysteine for leucine at position 201, encoded by pilTL201C). Purified PilTL201C forms a native hexamer, but mutant hexamers hydrolyze ATP at half the maximal rate. N. gonorrhoeae pilTL201C cells produce Tfp fibers, crawl at the same speed as the wild-type (wt) parent, and are equally transformable. However, the social behavior of pilTL201C cells is intermediate between the behaviors of wt and ΔpilT cells. The infection behavior of pilTL201C is also defective, due to its failure to activate the epidermal growth factor receptor (EGFR)-heparin-binding EGF-like growth factor (HB-EGF) pathway. Our study indicates that pilus retraction, per se, is not sufficient for N. gonorrhoeae microcolony formation or infectivity; rather, these activities are sensitive to the strength of PilT enzymatic activity. We discuss the implications of these findings for Neisseria pathogenesis in the context of mechanobiology.

Highly Multiplexed Proteomic Analysis of Quantiferon Supernatants To Identify Biomarkers of Latent Tuberculosis Infection.

ABSTRACTThe tests for diagnosing latent tuberculosis infection (LTBI) are limited by a poor predictive value for identifying people at the highest risk for progressing to active tuberculosis (TB) and have various sensitivities and specificities in different populations. Identifying a more robust signature for LTBI is important for TB prevention and elimination. A pilot study was conducted with samples from immigrants to the United States that were screened for LTBI by the three commercially approved tests, namely, the tuberculin skin test (TST), the Quantiferon-TB Gold in-tube (QFT-GIT), and the T-SPOT.TB (T-SPOT). QFT-GIT supernatants from 13 people with concordant positive results and 26 people with concordant negative results were analyzed via the highly multiplexed SOMAscan proteomic assay. The proteins in the stimulated supernatants that distinguished LTBI from controls included interleukin-2 (IL-2), monocyte chemotactic protein 2 (MCP-2), interferon gamma inducible protein-10 (IP-10), interferon gamma (IFN-γ), tumor necrosis factor superfamily member 14 (TNFSF14, also known as LIGHT), monokine induced by gamma interferon (MIG), and granzyme B (P <0.00001). In addition, antigen stimulation increased the expression of heparin-binding EGF-like growth factor (HB-EGF) and activin AB in LTBI samples. In nil tubes, LIGHT was the most significant marker (P <0.0001) and was elevated in LTBI subjects. Other prominent markers in nonstimulated QFT-GIT supernatants were the complement-3 components C3b, iC3b, and C3d, which were upregulated in LTBI and markedly decreased upon stimulation. We found known and novel proteins that warrant further studies for developing improved tests for LTBI, for predicting progression to active disease, and for discriminating LTBI from active TB.

Heparin-binding EGF-like growth factor in diagnosis of malignant phyllodes tumor of the breast: a case report

Heparin-binding EGF-like growth factor in diagnosis of malignant phyllodes tumor of the breast: a case report

Heparin-binding epidermal growth factor-like growth factor and hepatocyte growth factor inhibit cholestatic liver injury in mice through different mechanisms.

In contrast to hepatocyte growth factor (HGF), the therapeutic potential and pathophysiologic roles of heparin-binding epidermal growth factor-like growth factor (HB-EGF) in liver diseases remain relatively unknown. To address the lack of effective pharmacologic treatments for cholestatic liver injuries, as well as to clarify the biologic features of these growth factors, we explored the effects of HB-EGF and HGF in mice with cholestatic liver injury induced by bile duct ligation (BDL). The mice were assessed 3, 5 and/or 14 days after BDL (acute, subacute and/or chronic phases, respectively) and intravenous injection of adenoviral vector expressing LacZ (control), HB-EGF, HGF, or HB-EGF and HGF. HB-EGF, HGF, or a combination of the growth factors exerted potent antioncotic (antinecrotic), antiapoptotic, anticholestatic, and regenerative effects on hepatocytes in vivo, whereas no robust antiapoptotic or regenerative effects were detected in interlobular bile ducts. Based on serum transaminase levels, the acute protective effects of HB-EGF on hepatocytes were greater than those of HGF. On the other hand, liver fibrosis and cholestasis during the chronic phase were more potently inhibited by HGF compared with HB-EGF. Compared with either growth factor alone, combining HB-EGF and HGF produced greater anticholestatic and regenerative effects during the chronic phase. Taken together, these findings suggest that HB-EGF and HGF inhibited BDL-induced cholestatic liver injury, predominantly by exerting acute cytoprotective and chronic antifibrotic effects, respectively; combining the growth factors enhanced the anticholestatic effects and liver regeneration during the chronic phase. Our results contribute to a better understanding of the pathophysiologic roles of HB-EGF and HGF, as well as to the development of novel effective therapies for cholestatic liver injuries.

A novel trichosanthin fusion protein with increased cytotoxicity to tumor cells.

To evaluate the anti-tumor effects of trichosanthin after fusion with a cell penetrating peptide, heparin-binding peptide (HBP), derived from human heparin-binding EGF-like growth factor (HB-EGF). The fusion protein of trichosanthin-HBP was expressed in Escherichia coli BL21 and purified by Ni-NTA affinity chromatography. The HBP domain had no influence on the topological inactivation activity and N-glycosidase activity of trichosanthin. Trichosanthin-HBP significantly inhibited the growth of tested cancer cells which are impervious to trichosanthin. Tumor cell apoptosis and both the mitochondrial- and death receptor-mediated apoptotic signaling pathways induced by trichosanthin-HBP were more significant than those induced by trichosanthin in HeLa cells. HBP is an efficient intracellular delivery vehicle for trichosanthin and makes trichosanthin-HBP become a promising agent for cancer therapy.

Epidermal growth factor (EGF) receptor-ligand based molecular staging predicts prognosis in head and neck squamous cell carcinoma partly due to deregulated EGF- induced amphiregulin expression.

BackgroundIncreased expression of epidermal growth factor receptor (EGFR) and its ligands is associated with poor prognosis and chemoresistance in many carcinoma types, but its role in head and neck squamous cell carcinoma (HNSCC) is unclear. Our aim was to clarify whether mRNA expression of EGFR-ligands was linked to prognosis and cisplatin resistance, and if so, which ligand was most important and how was the expression regulated.MethodsTo examine the prognostic effect of EGFR-ligand expression, we analyzed tumorous mRNA expression in 399 HNSCC patients. The intracellular signaling pathways controlling epidermal growth factor (EGF)-induced amphiregulin (AREG) expression were examined in three oral squamous cell carcinoma (OSCC) cell lines. Effect of AREG on cisplatin resistance was examined by viability assays in four-, and by association in 11 OSCC cell lines.ResultsThe patients were divided into five groups according to the median mRNA expression levels of four EGFR ligands, i.e. AREG, EGF, heparin-binding EGF-like growth factor (HBEGF) and beta-cellulin (BTC). The number of increased-expressed EGFR-ligands were progressively correlated to five-year survival, even in advanced TNM-stage IV patients, where five-year mortality increased from 26 % if tumor expressed none to one EGFR-ligand, to 45 % in three to four ligand expressing tumors. Thus, staging the tumor according to these EGFR-ligand mRNA expression pattern completely out performed TNM staging in predicting prognosis. Multivariate analysis identified AREG as the dominating predictor, and AREG was overexpressed in OSCC compared to tumors from other sites. Both EGF and HBEGF stimulation induced strong AREG increase in OSCC cell lines, which was partially mediated by the extracellular signal-regulated kinase 1/2 pathway, and negatively regulated by p38, c-Jun N-terminal kinase, and phosphoinositide-3 kinase. Although increased AREG mRNA expression predicted unfavorable prognosis in platinum treated HNSCC patients, AREG did not mediate cisplatin resistance in the OSCC cell lines.ConclusionsIncreased tumorous mRNA expression of four EGFR ligands was progressively associated with poor prognosis in HNSCC. Thus, EGFR-ligands mRNA expression pattern may be a new prognostic biomarker. The tightly regulated EGF-induced AREG mRNA expression was partly lost in the OSCC cell lines and restoring its regulation may be a new target in cancer treatment.Trial registrationNot applicable as the clinical data of the 498 HNSCC patients and their mRNA expression profiles were collected from the open TCGA database: http://cancergenome.nih.gov/cancersselected/headandneck.

EGF receptor ligands: recent advances.

Seven ligands bind to and activate the mammalian epidermal growth factor (EGF) receptor (EGFR/ERBB1/HER1): EGF, transforming growth factor-alpha (TGFA), heparin-binding EGF-like growth factor (HBEGF), betacellulin (BTC), amphiregulin (AREG), epiregulin (EREG), and epigen (EPGN). Of these, EGF, TGFA, HBEGF, and BTC are thought to be high-affinity ligands, whereas AREG, EREG, and EPGN constitute low-affinity ligands. This focused review is meant to highlight recent studies related to actions of the individual EGFR ligands, the interesting biology that has been uncovered, and relevant advances related to ligand interactions with the EGFR.