Journal: Scientific Reports

Article Title: Acetylcholine acts through M3 muscarinic receptor to activate the EGFR signaling and promotes gastric cancer cell proliferation

doi: 10.1038/srep40802

Figure Lengend Snippet: ( a ) Cells were treated with indicated concentrations of ACh for 2 h, EGFR and phosphorylated EGFR were analyzed by western blot. ( b ) Cells were treated with 200 μM ACh for 15 min to 120 min, EGFR and phosphorylated EGFR were analyzed by western blot. ( c ) After knockdown of M3 AChR expression by transfecting small interfering RNA, p-EGFR expression stimulated by ACh were analyzed by western blot in MKN45 and BGC823 cells. ( d ) After adding 10 μM EGFR specific inhibitor AG1478 2 h prior to ACh addition, protein changes were analyzed by western blot. ( e ) AG1478 was added 2 h before ACh addition, CCK-8 assay was used to study the role of EGFR in ACh induced cell proliferation. (**P < 0.01).

Article Snippet: ACh, neostigmine and AFDX-116 was obtained from Sigma (St Louis, MO, USA), AG1478, trihexyphenidyl and darifenacin were obtained from MCE (Monmouth Junction, NJ, USA).

Techniques: Western Blot, Knockdown, Expressing, Small Interfering RNA, CCK-8 Assay

Journal: Scientific Reports

Article Title: Acetylcholine acts through M3 muscarinic receptor to activate the EGFR signaling and promotes gastric cancer cell proliferation

doi: 10.1038/srep40802

Figure Lengend Snippet: ( a ) In gastric cancer cells, cytoplasm ChAT protein catalyzes ACh synthesis, then ACh is secreted and released into the extracellular space, the extracellular ACh could combine and activate M3 receptor located in cytomembrane of cells, following activating EGFR signaling, and then leading to the increasement of p-ERK1/2 and p-AKT, thus, promote gastric cancer cell proliferation and survival. ( b ) When the function of M3R was blocked by specific antagonists (such as 4-DAMP), or its downstream effector EGFR, inhibited by specific inhibitor AG1478, the auto-stimulating role of ACh in cell proliferation will be weaken in some degree. The M3R/EGFR signaling inhibitors can enhance the chemosensitivity of 5-Fu and reduce cell viability in gastric cancer. The combined strategy may provide a feasible strategy for gastric cancer therapy.

Article Snippet: ACh, neostigmine and AFDX-116 was obtained from Sigma (St Louis, MO, USA), AG1478, trihexyphenidyl and darifenacin were obtained from MCE (Monmouth Junction, NJ, USA).

Techniques:

Journal: Molecular cancer

Article Title: Disruption of ETV6 leads to TWIST1-dependent progression and resistance to epidermal growth factor receptor tyrosine kinase inhibitors in prostate cancer.

doi: 10.1186/s12943-018-0785-1

Figure Lengend Snippet: Fig. 3 Epidermal growth factor receptor (EGFR) signaling triggers ETV6-mediated suppression of TWIST1. (a) Monitoring ETV6 mRNA following treatments with EGF and CI1033. PBS and DMSO, vehicle control of EGF and CI1033, respectively. (b) Monitoring TWIST1 mRNA following treatments with CI1033 and stable ETV6 expression. EV, control vector. (c) Western blot assay with cellular lysates from RasB1 cells. Cells were treated with EGFR modulators (EGF, CI1033, left panel) or a stable ETV6-expressing vector (EV vs. ETV6, right panel). (d, e) Two androgen receptor (AR)-positive cell lines transiently transfected with ETV6-specific siRNA (scr. vs. siETV6) were analyzed for TWIST1 mRNA (d) and for the Western blot assay (e). (f) Monitoring TWIST1 mRNA in 22RV1 cells transiently transfected with ETV6-specific siRNA (scr. vs. siETV6). TWIST1 mRNA was measured in response to EGFR activation (PBS vs. EGF) or inactivation (DMSO vs. CI1033). Quantification of mRNA is presented as the mean ± SEM, n = 3. *p < 0.05, **p < 0.01, ***p < 0.001

Article Snippet: The EGF was from R&D Systems (Minneapolis, MN, USA), and the EGFR inhibitors (CI1033 and AG1478) were from Selleck (Houston, TX, USA).

Techniques: Control, Expressing, Plasmid Preparation, Western Blot, Transfection, Activation Assay

Journal: Molecular cancer

Article Title: Disruption of ETV6 leads to TWIST1-dependent progression and resistance to epidermal growth factor receptor tyrosine kinase inhibitors in prostate cancer.

doi: 10.1186/s12943-018-0785-1

Figure Lengend Snippet: Fig. 5 ETV6-TWIST1 signaling is involved in the molecular mechanism of drug resistance. (a) Proliferation assay in three DU145 derived cells treated with a tyrosine kinase inhibitor (TKI: AG1478, 0.1~ 10 μM), n = 8. shLacZ, control; shETV6, ETV6-knockdown; shETV6 + siTWIST1, both ETV6- and TWIST1-knockdown. (b) Proliferation assay in three stable RasB1 derived cells treated with a TKI (CI1033, 0.1~ 10 nM), n = 8. EV, control vector; ETV6, ETV6-expressing vector; ETV6 + TWIST1, both ETV6- and TWIST1-expressing vectors. (c) Tumor growth analysis of stable RasB1 cell lines (EV vs. ETV6) subcutaneously inoculated in male nude mice followed by treatment with CI1033. Tumor sizes were monitored weekly (left, n = 5). At the end, tumor weights were also measured (right, n = 5). (d) Selected images of mice from panel C, containing tumors (arrows) derived from stable RasB1 cell lines (EV vs. ETV6). (e) Western blot analysis of human prostate cancer cells. RasB1 cells were introduced with exogenous ETV6 or under ETV6-knockdown in 22RV1 and LNCaP cells by an siRNA approach (siETV6). EV, empty vector; scr., siRNA control. (f) Working model, activation of the epidermal growth factor receptor (EGFR) promotes tumor progression and drug resistance through RAS signaling and suppression of ETV6, which lead to TWIST1-dependent malignant phenotypes. A mutual inhibitory circuit exists between EGFR-RAS signaling and ETV6

Article Snippet: The EGF was from R&D Systems (Minneapolis, MN, USA), and the EGFR inhibitors (CI1033 and AG1478) were from Selleck (Houston, TX, USA).

Techniques: Proliferation Assay, Derivative Assay, Control, Knockdown, Plasmid Preparation, Expressing, Western Blot, Activation Assay

Journal: JCI Insight

Article Title: Mevastatin promotes healing by targeting caveolin-1 to restore EGFR signaling

doi: 10.1172/jci.insight.129320

Figure Lengend Snippet: (A) G-LISA Rac1-GTP activation assay in HEKs treated with 5 μM mevastatin for 48 hours (n = 6). Treatment with 12.5 ng/mL EGF for 5 minutes served as positive control. Mevastatin induced Rac1-GTP activation. (B) ITGB5 and phalloidin staining of human keratinocytes (HEKs) treated with mevastatin or EGF in the presence or absence of 150 nM tyrphostin AG 1478. Scale bar: 10 μm. LP, lamellipodia. (C) Percentage of ITGB5+ lamellipodia+ cells (n = 3). Mevastatin induced lamellipodia formation, whereas tyrphostin AG 1478 inhibited mevastatin-induced lamellipodia. Data are represented as mean ± SD and were analyzed by a 1-way ANOVA followed by Holm-Sidak’s post hoc test; ****P < 0.0001.

Article Snippet: HEKs were grown to approximately 30% confluency and switched to basal media for 24 hours and treated with 5 μM mevastatin, 150 nM tyrphostin AG 1478 (Cell Signaling Technology), or both in combination, and 12.5 ng/ml EGF treatment for 5 minutes served as a positive control.

Techniques: Activation Assay, Positive Control, Staining

Journal: Slas Discovery

Article Title: Development of a 3D Tissue Culture–Based High-Content Screening Platform That Uses Phenotypic Profiling to Discriminate Selective Inhibitors of Receptor Tyrosine Kinases

doi: 10.1177/1087057116657269

Figure Lengend Snippet: Identification of selective c-Met/EGFR inhibitors in a Vichem epidermal growth factor (EGF) receptor (EGFR)/c-Met inhibiting compound library. ( A ) Compound screen of 80 Vichem compounds tested at 10, 3.16, and 1µM. Compounds were divided over triplicate plates and coexposed with either hepatocyte growth factor (HGF) (20 ng/mL) or EGF (20 ng/mL). Principal components analysis (PCA) was trained on unstimulated and stimulated controls separately for plates exposed to HGF or EGF, respectively, to obtain a principal component that could separate c-Met and EGFR responses. Data points represent mean determinations from three wells. Controls are color-coded as indicated in the legend. For stimulated (top middle) and unstimulated controls (bottom left), a 2D-density estimation (contour lines) is shown. ( B ) Representative 2D projected images derived from the F-actin staining after 96 h of compound treatment. Except for AG1478, which was 3.16 µM, shown treatment concentrations were 10 µM. Pictures were obtained using a BD Pathway 855 microscope (images trimmed to 300 × 300 pixels for presentation purposes). ( C ) Cell count (viability) is a poor criterion for selecting c-Met and EGFR inhibitors. Number of nuclei per well was percent normalized to stimulated control (100%) and lowest detected cell count and 0%, top panel. The top ranking 10% of compounds affecting cell count are color-coded in red. Middle and lower panels show the same compounds ranked on efficacy on c-Met and EGFR profiles (distance to unstimulated control). Mean values shown; each chart contains compounds at three different concentrations.

Article Snippet: EGFR inhibitor AG1478 was obtained from Santa Cruz (SC-200613; Bio-Connect B.V. Huissen).

Techniques: Drug discovery, Derivative Assay, Staining, Microscopy, Cell Counting, Control

Journal: PLoS ONE

Article Title: LIV-1 Promotes Prostate Cancer Epithelial-to-Mesenchymal Transition and Metastasis through HB-EGF Shedding and EGFR-Mediated ERK Signaling

doi: 10.1371/journal.pone.0027720

Figure Lengend Snippet: LIV-1 overexpressing cells (LIV#8 and LIV#14) showed increased phosphorylated EGFR (p-EGFR) and ERK (p-ERK). B, EGFR inhibitor (AG1478) treatment reduced phosphorylated EGFR and ERK in LIV#8 clone. C, inhibition of EGFR suppressed migratory ability and invasive ability of LIV-1 overexpressing clones in transwell assays 24 hours after treatment. D, inhibition of ERK signaling resulted in similar suppression of cellular motility as EGFR inhibition. * indicates statistical significance compared to the control of the same group ( P <0.05).

Article Snippet: Tyrphostin AG1478, U0126 and MMP2/9 inhibitor III were obtained from Alomone labs (Jerusalem, Israel), Cell Signaling Technology (Danvers, MA), and Calbiochem (Darmstadt, Germany), respectively.

Techniques: Inhibition, Clone Assay

Journal: PLoS ONE

Article Title: LIV-1 Promotes Prostate Cancer Epithelial-to-Mesenchymal Transition and Metastasis through HB-EGF Shedding and EGFR-Mediated ERK Signaling

doi: 10.1371/journal.pone.0027720

Figure Lengend Snippet: A, LIV-1 overexpressing cells (LIV#8) and control cells (con1) were treated with EGF and AG1478 for 2 hours. Western blotting showed that the EGF-elicited EGFR and ERK phosphorylation was blocked by the AG1478 inhibitor. B, RT-PCR showed increased HB-EGF, MMP2 and MMP9 expression in LIV-1 overexpressing cells. C, LIV-1 overexpressing cells (LIV#8 and LIV#14) were cultured in serum-free medium for 24 hours and the culture media were used to determine the MMP2 and MMP9 enzymatic activity by zymogram assay. D, the effect of MMP2/9 enzymatic activity on HB-EGF shedding was evaluated by ELISA. LIV-1 overexpressing clones (LIV#8 and LIV#14) secreted more HB-EGF than control clones (con1 and con2), and the secretion was reduced by MMP2/9 inhibition. ∗ indicates statistical significance compared to the control of the group ( P <0.05). E, Western blotting showed that inhibition of MMP2/9 activity suppressed EGFR and the downstream ERK phosphorylation. F, LIV-1 overexpressing cells were treated with MMP 2/9 inhibitor III for 24 hours in Transwell motility assays. Both migration and invasion of the treated cells were decreased. * indicates statistical significance compared to the control group ( P <0.05). G, diagram depicts the proposed role of LIV-1 in prostate cancer cell EMT and metastasis.

Article Snippet: Tyrphostin AG1478, U0126 and MMP2/9 inhibitor III were obtained from Alomone labs (Jerusalem, Israel), Cell Signaling Technology (Danvers, MA), and Calbiochem (Darmstadt, Germany), respectively.

Techniques: Western Blot, Reverse Transcription Polymerase Chain Reaction, Expressing, Cell Culture, Activity Assay, Enzyme-linked Immunosorbent Assay, Clone Assay, Inhibition, Migration