In this study,mouse models of benign prostatic hyperplasia induced by subcutaneous injection of testosterone propionate was used to investigate the therapeutic effect and mechanism of Urtica hyperborean( UW) extracts on prostate hyperplasia in mice. The effects of UW extracts on prostate index,serum epidermal growth factor( EGF) and dihydrotestosterone( DHT) in model mice were observed,and the EGF and anti-apoptotic factor( Bcl-2) mRNA expression levels were detected as well as pathological changes in prostate tissue. The results showed that the ethyl acetate extraction and alcohol soluble fraction of the UW could significantly reduce the prostate index,reduce the serum DHT and EGF levels( P<0. 01),and significantly decrease the EGF and Bcl-2 mRNA expression( P<0. 01),significantly improved the morphological structure of prostate tissue. The above results confirmed that ethyl acetate extract and alcohol-soluble parts of UW have a good preventive effect on mice prostatic hyperplasia model,and its mechanism may be to reduce androgen levels by regulating polypeptide growth factors and/or inhibiting cell hyperproliferation and promoting apoptosis. This study laid the foundation for the further research on UW.
Animals ; Dihydrotestosterone ; blood ; Epidermal Growth Factor ; blood ; Male ; Medicine, Tibetan Traditional ; Mice ; Plant Extracts ; pharmacology ; Prostatic Hyperplasia ; chemically induced ; drug therapy ; Proto-Oncogene Proteins c-bcl-2 ; metabolism ; Testosterone Propionate ; Urticaceae ; chemistry

Angiogenesis is a crucial process in the development of inflammatory diseases, including cancer, psoriasis and rheumatoid arthritis. Recently, several alkaloids from Picrasma quassioides had been screened for angiogenic activity in the zebrafish model, and the results indicated that 1-methoxycarbony-β-carboline (MCC) could effectively inhibit blood vessel formation. In this study, we further confirmed that MCC can inhibit, in a concentration-dependent manner, the viability, migration, invasion, and tube formation of human umbilical vein endothelial cells (HUVECs) in vitro, as well as the regenerative vascular outgrowth of zebrafish caudal fin in vivo. In the zebrafish xenograft assay, MCC inhibited the growth of tumor masses and the metastatic transplanted DU145 tumor cells. The proteome profile array of the MCC-treated HUVECs showed that MCC could down-regulate several angiogenesis-related self-secreted proteins, including ANG, EGF, bFGF, GRO, IGF-1, PLG and MMP-1. In addition, the expression of two key membrane receptor proteins in angiogenesis, TIE-2 and uPAR, were also down-regulated after MCC treatment. Taken together, these results shed light on the potential therapeutic application of MCC as a potent natural angiogenesis inhibitor via multiple molecular targets.
Angiogenesis Inhibitors ; chemistry ; pharmacology ; Animals ; Carbolines ; chemistry ; pharmacology ; Cell Movement ; drug effects ; Cell Proliferation ; drug effects ; Epidermal Growth Factor ; genetics ; metabolism ; Fibroblast Growth Factors ; genetics ; metabolism ; Human Umbilical Vein Endothelial Cells ; cytology ; drug effects ; metabolism ; Humans ; Insulin-Like Growth Factor I ; genetics ; metabolism ; Neovascularization, Physiologic ; drug effects ; Picrasma ; chemistry ; Plant Extracts ; chemistry ; pharmacology ; Receptor, TIE-2 ; genetics ; metabolism ; Zebrafish ; embryology

PURPOSE: Rearrangement of the proto-oncogene rearranged during transfection (RET) has been newly identified potential driver mutation in lung adenocarcinoma. Clinically available tyrosine kinase inhibitors (TKIs) target RET kinase activity, which suggests that patients with RET fusion genes may be treatable with a kinase inhibitor. Nevertheless, the mechanisms of resistance to these agents remain largely unknown. Thus, the present study aimed to determine whether epidermal growth factor (EGF) and hepatocyte growth factor (HGF) trigger RET inhibitor resistance in LC-2/ad cells with CCDC6-RET fusion genes. MATERIALS AND METHODS: The effects of EGF and HGF on the susceptibility of a CCDC6-RET lung cancer cell line to RET inhibitors (sunitinib, E7080, vandetanib, and sorafenib) were examined. RESULTS: CCDC6-RET lung cancer cells were highly sensitive to RET inhibitors. EGF activated epidermal growth factor receptor (EGFR) and triggered resistance to sunitinib, E7080, vandetanib, and sorafenib by transducing bypass survival signaling through ERK and AKT. Reversible EGFR-TKI (gefitinib) resensitized cancer cells to RET inhibitors, even in the presence of EGF. Endothelial cells, which are known to produce EGF, decreased the sensitivity of CCDC6-RET lung cancer cells to RET inhibitors, an effect that was inhibited by EGFR small interfering RNA (siRNA), anti-EGFR antibody (cetuximab), and EGFR-TKI (Iressa). HGF had relatively little effect on the sensitivity to RET inhibitors. CONCLUSION: EGF could trigger resistance to RET inhibition in CCDC6-RET lung cancer cells, and endothelial cells may confer resistance to RET inhibitors by EGF. E7080 and other RET inhibitors may provide therapeutic benefits in the treatment of RET-positive lung cancer patients.
Adenocarcinoma/drug therapy/*genetics ; Cell Line, Tumor ; Cetuximab/pharmacology ; Drug Resistance, Neoplasm/drug effects/*genetics ; Epidermal Growth Factor/metabolism/*pharmacology ; *Gene Rearrangement ; Hepatocyte Growth Factor/*pharmacology ; Humans ; Indoles/pharmacology ; Lung Neoplasms/drug therapy/*genetics ; MAP Kinase Signaling System ; *Mutation ; Niacinamide/analogs & derivatives/pharmacology ; Phenylurea Compounds/pharmacology ; Piperidines/pharmacology ; Protein Kinase Inhibitors/therapeutic use ; Proto-Oncogene Proteins c-ret/*antagonists & inhibitors/genetics ; Pyrroles/pharmacology ; Quinazolines/pharmacology ; RNA, Small Interfering/pharmacology ; Receptor, Epidermal Growth Factor/genetics/metabolism ; Signal Transduction/drug effects ; fms-Like Tyrosine Kinase 3/metabolism

BACKGROUNDTwo recent whole-exome sequencing researches identifying somatic mutations in the ubiquitin-specific protease 8 (USP8) gene in pituitary corticotroph adenomas provide exciting advances in this field. These mutations drive increased epidermal growth factor receptor (EGFR) signaling and promote adrenocorticotropic hormone (ACTH) production. This study was to investigate whether the inhibition of USP8 activity could be a strategy for the treatment of Cushing's disease (CD).

METHODSThe anticancer effect of USP8 inhibitor was determined by testing cell viability, colony formation, apoptosis, and ACTH secretion. The immunoblotting and quantitative reverse transcription polymerase chain reaction were conducted to explore the signaling pathway by USP8 inhibition.

RESULTSInhibition of USP8-induced degradation of receptor tyrosine kinases including EGFR, EGFR-2 (ERBB2), and Met leading to a suppression of AtT20 cell growth and ACTH secretion. Moreover, treatment with USP8 inhibitor markedly induced AtT20 cells apoptosis.

CONCLUSIONSInhibition of USP8 activity could be an effective strategy for CD. It might provide a novel pharmacological approach for the treatment of CD.

Adrenocorticotropic Hormone ; metabolism ; Animals ; Apoptosis ; drug effects ; Cell Proliferation ; drug effects ; physiology ; Cell Survival ; drug effects ; physiology ; Endopeptidases ; metabolism ; Endosomal Sorting Complexes Required for Transport ; antagonists & inhibitors ; metabolism ; Enzyme Inhibitors ; pharmacology ; Humans ; Indenes ; pharmacology ; Mice ; Pyrazines ; pharmacology ; Receptor, Epidermal Growth Factor ; metabolism ; Ubiquitin Thiolesterase ; antagonists & inhibitors ; metabolism

BACKGROUND/AIMS: Silibinin, the main component of silymarin, is used as a hepatoprotectant and exhibits anticancer effects against various cancer cells. This study evaluated the effects of a combination of silibinin with either gefitinib or sorafenib on hepatocellular carcinoma (HCC) cells. METHODS: Several different human HCC cell lines were used to test the growth-inhibiting effects and cell toxicity of silibinin both alone and in combination with either gefitinib or sorafenib. The cell viability and growth inhibition were assessed using the 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide assay, trypan blue staining, and a colony-forming assay. Furthermore, changes in epidermal growth factor receptor (EGFR)-related signals were evaluated by Western blot analysis. RESULTS: Gefitinib, sorafenib, and silibinin individually exhibited dose-dependent antiproliferative effects on HCC cells. Combined treatment with silibinin enhanced the gefitinib-induced growth-inhibiting effects in some HCC cell lines. The combination effect of gefitinib and silibinin was synergistic in the SNU761 cell line, but was only additive in the Huh-BAT cell line. The combination effect may be attributable to inhibition of EGFR-dependent Akt signaling. Enhanced growth-inhibiting effects were also observed in HCC cells treated with a combination of sorafenib and silibinin. CONCLUSIONS: Combined treatment with silibinin enhanced the growth-inhibiting effects of both gefitinib and sorafenib. Therefore, the combination of silibinin with either sorafenib or gefitinib could be a useful treatment approach for HCC in the future.
Antineoplastic Agents/*pharmacology ; Carcinoma, Hepatocellular/metabolism/pathology ; Cell Line, Tumor ; Cell Proliferation/*drug effects ; Cell Survival/drug effects ; Down-Regulation/drug effects ; Drug Screening Assays, Antitumor ; Drug Synergism ; Humans ; Liver Neoplasms/metabolism/pathology ; Niacinamide/*analogs & derivatives/pharmacology ; Phenylurea Compounds/*pharmacology ; Proto-Oncogene Proteins c-akt/metabolism ; Quinazolines/*pharmacology ; Receptor, Epidermal Growth Factor/metabolism ; Signal Transduction/drug effects ; Silymarin/*pharmacology

OBJECTIVETo study the effect of aqueous extract of Taxus chinensis var. mairei (AETC) combined Erlotnib on the growth of A549 xenograft in nude mice and its mechanism.

METHODSThe xenograft model in nude mice was established by inoculating A549 cells subcutaneously. BALB/c nude mice bearing A549 xenograft were randomly divided into six groups, i.e., the low dose Erlotinib group (A) , the standard dose Erlotnib group (B) , the low dose Erlotinib combined AETC group (C), the standard dose Erlotnib combined AETC group (D), the AETC group (E), the control group (F), 12 in each group. Different medication was performed for 7 successive weeks after 24 h. One mL blood was withdrawn and tumor tissues taken. The tumor inhibition rate was calculated. The combined effect was analyzed by Jin's Formula [Q = Ea + b/(Ea + Eb-Ea x Eb) ]. mRNA and protein expression levels of epidermal growth factor receptor (EGFR), cyclooxygenase-2 (COX-2), and B cell lymphoma-2 (Bcl-2) in xenografts were detected using real-time RT-PCR and ELISA.

RESULTSCompared with Group F, the xenograft weight was obviously lowered in Group B-E (P < 0.05, P < 0.01). The q value was 0.92 in Group C and 0.96 in Group D, which was obtained by simple adding of the two drugs. Compared with Group F, EG- FR mRNA expression in Group D and E, COX-2 mRNA expression in Group A-E; Bcl-2 mRNA expression in Group B-D; COX-2 protein expression in Group B-E; Bcl-2 protein expression in Group C and D were obviously lowered with statistical difference (P < 0.05, P < 0.01).

CONCLUSIONSAETC combined low dose and standard dose Erlotinib had synergistic effect on tumor inhibition. Its mechanism might be associated with down-regulating mRNA and protein expression levels of COX-2 and Bcl-2.

Animals ; Antineoplastic Agents ; pharmacology ; Antineoplastic Combined Chemotherapy Protocols ; pharmacology ; Cell Line, Tumor ; Cyclooxygenase 2 ; metabolism ; Drugs, Chinese Herbal ; pharmacology ; Enzyme Inhibitors ; pharmacology ; Erlotinib Hydrochloride ; pharmacology ; Heterografts ; Lung Neoplasms ; Mice ; Mice, Inbred BALB C ; Mice, Nude ; Receptor, Epidermal Growth Factor ; metabolism ; Taxus ; Transplantation, Heterologous

OBJECTIVETo investigate the effects of bufalin in reversing hepatocyte growth factor (HGF)-induced resistance to afatinib in H1975 lung cancer cells, and explore its possible mechanism.

METHODSThe afatinib-resistant H1975 lung cancer cells (H1975AR) were induced by exogenous HGF and transfected with recombinant adenoviral vector Ad-HGF-GFP. The cytostatic effects of bufalin, afatinib and bufalin plus afatinib on H1975AR cells were evaluated by MTT assay. The impact of combined therapy with bufalin and afatinib on invasion of H1975AR cells was determined by transwell migration assay. The concentrations of HGF in the culture supernatants of H1975/Vec and H1975/HGF cells were determined by ELISA. The expression of EGFR, cMET and EMT signal pathway-related proteins in H1975AR cells treated with bufalin, afatinib and bufalin plus afatinib were detected by Western blot.

RESULTSThe results of MTT assay showed that afatinib did not inhibit the growth of H1975 cells, but after 72 h of the combined treatment with bufalin and afatinib and in the presence of HGF, the growth rate of H1975 cells was (38.67 ± 8.76)%, significantly lower than the growth rate of (63.45 ± 12.65)% in the H1975 cells treated with HGF alone (P < 0.05). The results of transwell migration assay showed that in the presence of HGF, afatinib plus bufalin combination therapy markedly decreased the number of invaded H1975 cells through the Matrigel chamber (48.98 ± 11.43), significantly lower than the 118.92 ± 37.29 of afatinib-treated or the 88.84 ± 19.53 of bufalin-treated cells (P < 0.05 for all). The result of ELISA showed that H1975/HGF cells secreted high levels of HGF, and afatinib and bufalin had no effect on the HGF secretion in H1975/HGF cells. The results of Western blot analysis showed that the expression of p-EGFR, p-cMet, p-AKT, p-ERK, vimentin and snail in H1975AR cells treated with bufalin puls afatinb was down-regulated markedly, and the expression of E-cadherin was up-regulated markedly.

CONCLUSIONSCombination of bufalin and afatinib strongly inhibits the growth of H1975AR lung cancer cells and decreases their invasion ability. The possible mechanism of combined treatment with bufalin and afatinib may be related to the blocking of cMet/PI3K/AKT and cMet/MAPK/ERK pathways and inhibiting of epithelial-mesenchymal transition.

Antineoplastic Agents ; pharmacology ; Antineoplastic Combined Chemotherapy Protocols ; pharmacology ; Bufanolides ; pharmacology ; Cadherins ; metabolism ; Cell Line, Tumor ; Cell Proliferation ; drug effects ; Coloring Agents ; Drug Resistance, Neoplasm ; drug effects ; Epithelial-Mesenchymal Transition ; drug effects ; Hepatocyte Growth Factor ; pharmacology ; Humans ; Lung Neoplasms ; drug therapy ; metabolism ; pathology ; MAP Kinase Signaling System ; Neoplasm Proteins ; metabolism ; Phosphatidylinositol 3-Kinases ; Quinazolines ; pharmacology ; Receptor, Epidermal Growth Factor ; Signal Transduction ; Tetrazolium Salts ; Thiazoles

OBJECTIVETo investigate the effect of silicon dioxide (SiO₂) on the expression of E-cadherin, α-smooth muscle actin (α-SMA), and transforming growth factor β₁(TGF-β₁) in human pulmonary epithelial cells (A549) with epithelial-mesenchymal transition (EMT), and to study the roles of epidermal growth factor receptor (EGFR) signaling pathway in SiO₂-induced EMT in A549 cells in vitro.

METHODSAlveolar macrophages (AMs) were stimulated with 50 µg/ml SiO₂for 3, 6, 12, 18, 24, or 36 h, and the supernatants were collected to measure the expression of TGF-β₁protein by ELISA. The AM supernatant in which TGF-β₁reached the highest expression (T=18 h) was used as AM-conditioned supernatant. A549 cells were cultured in AM-conditioned supernatant and stimulated with indicated doses of SiO₂(0, 50, 100, and 200 µg/ml) for 48 h. The cell morphological changes were observed using an inverted microscope. The cells were collected at different times, and the mRNA and protein expression levels of E-cadherin, α-SMA, and EGFR were measured by RT-PCR and immunocytofluorescence, respectively.

RESULTSAfter stimulation by SiO₂, the expression level of TGF-β₁protein at each time point was significantly higher in the presence of AM supernatants than in the absence of AM supernatants (P<0.05). With the action time, the expression level of TGF-β₁protein increased at first and then decreased, and the highest level was reached at 18 h. After exposure to SiO₂, A549 cells exhibited mesenchymal characteristics, such as a spindle shape, pseudopodia change, and fibroblast-like morphology, as observed by inverted microscope, especially in the 200 µg/ml group. With increased concentration of SiO₂, the mRNA and protein expression of E-cadherin was down-regulated gradually, especially in the 200 µg/ml group, whereas the mRNA and protein expression of α-SMA and EGFR was up-regulated gradually, especially in the 200 µg/m1 group. There were significant differences between the SiO₂-treated groups (50, 100, and 200 µg/ml SiO₂) and the control group (P<0.05).

CONCLUSIONAfter being stimulated by SiO₂in vitro, AMs have significantly increased expression level of TGF-β₁protein. The AM supernatant together with SiO₂can induce the transition of pulmonary epithelial cells to mesenchymal cells, and its mechanism may be related to the EGFR signaling pathway.

Actins ; metabolism ; Cadherins ; metabolism ; Cell Line, Tumor ; Epithelial Cells ; cytology ; metabolism ; Epithelial-Mesenchymal Transition ; drug effects ; Humans ; Lung ; cytology ; Macrophages, Alveolar ; metabolism ; Receptor, Epidermal Growth Factor ; metabolism ; Signal Transduction ; Silicon Dioxide ; pharmacology ; Transforming Growth Factor beta1 ; metabolism

To explore the feasibility of mesenchymal stem cells (MSCs) acting as seed cells in tissue engineering, we isolated human bone marrow MSCs and differentiated them into vascular endothelial-like cells (ELCs) in vitro. Bone marrow mononuclear cells (BMSCs) were isolated by the method of percoll density centrifugation, and seeded in Dulbecco Modified Eagle Medium supplemented with 10% fetal bovine serum. MSCs were purified through multiple adherent cultures, and differentiated into ELCs induced by endothelial cell growth medium-2 (EBM-2) medium containing vascular endothelial growth factor (VEGF), human fibroblast growth factor (hFGF), insulin like growth factors 1 (IGF-1), and human epidermal growth factor (hEGF). The relative biologic characteristics of ELCs including cell morphology and phenotype were studied by inverted microscope and flow cytometry. The induced cells were identified by immunofluorescence with CD31 and Von Willebrand factor (vWF). The results showed that the morphology of MSCs was long-spindle and vortex-like growth. After induction of differentiation, the cells were round, and similar to vascular endothelial cells (ECs). Flow cytometric analysis revealed that ELCs expressed ECs specific surface markers of CD31 and vascular endothelial cadherin (VE-cadherin), but not CD133. Immunofluorescence results also confirmed that ELCs expressed CD31 and vWF. The results suggested that ELCs possed similar cell biological characteristics with ECs. In one word, human MSCs derived from bone marrow have the potential to differentiate into ECs in vitro, and show clinical feasibility acting as ideal donor cells of vascular tissue engineering.
Antigens, CD ; metabolism ; Bone Marrow Cells ; Cadherins ; metabolism ; Cell Culture Techniques ; Cell Differentiation ; Cells, Cultured ; Culture Media ; chemistry ; Endothelial Cells ; cytology ; Epidermal Growth Factor ; pharmacology ; Fibroblast Growth Factors ; pharmacology ; Flow Cytometry ; Humans ; Insulin-Like Growth Factor I ; pharmacology ; Mesenchymal Stromal Cells ; cytology ; Platelet Endothelial Cell Adhesion Molecule-1 ; metabolism ; Tissue Engineering ; Vascular Endothelial Growth Factor A ; pharmacology ; von Willebrand Factor ; metabolism

BACKGROUNDReplacement of spiral ganglion neurons would be one prioritized step in an attempt to restore sensory neuronal hearing loss. However, the possibility that transplanted neurons could regenerate new synaptic connections to hair cells has not been explored. The objective of this study was to test whether neural stem cell (NSC)-derived neurons can form synaptic connections with hair cells in vitro.

METHODSNSCs were mechanically separated from the hippocampus in SD rat embryos (E12-E14) and cultured in a serum-free medium containing basic fibroblast growth factor and epidermal growth factor. Rat NSCs were co-cultured with explants of cochlea sensory epithelia obtained from postnatal Day 3 rats under transway filter membrane.

RESULTSAt Day 3, the NSCs began to show chemotactic differentiation and grew toward cochlea sensory epithelia. After 9-day co-culture, neurites of NSC-derived neurons predominantly elongated toward hair cells. Immunohistochemical analyses revealed the fibers overlapped with synapsin and hair cells, indicating the formation of new synaptic connections. After 14-day culture, triple staining revealed the fibers overlapped with PSD95 (postsynaptic density) which is juxtaposed with CtBP2 (presynaptic vesicle), indicating the formation of new ribbon synapse.

CONCLUSIONSNSC-derived neurons can make synaptic connections with hair cells and provide a model for studying synaptic plasticity and regeneration. Whether the newly forming synapse is functional merits further electrophysiological study.

Animals ; Cell Differentiation ; drug effects ; Cells, Cultured ; Coculture Techniques ; Epidermal Growth Factor ; pharmacology ; Fibroblast Growth Factor 2 ; pharmacology ; Hair Cells, Auditory ; cytology ; drug effects ; ultrastructure ; Neural Stem Cells ; cytology ; drug effects ; ultrastructure ; Neurons ; cytology ; drug effects ; ultrastructure ; Rats ; Rats, Sprague-Dawley ; Synapses ; drug effects ; metabolism