Journal: The Journal of Biological Chemistry

Article Title: Inhibition of Oncogenic Epidermal Growth Factor Receptor Kinase Triggers Release of Exosome-like Extracellular Vesicles and Impacts Their Phosphoprotein and DNA Content *

doi: 10.1074/jbc.M115.679217

Figure Lengend Snippet: Responses of EGFR-driven cancer cells to EGFR inhibitors. A–C, A431 cells harboring genomic amplification of the EGFR gene were grown in monolayer cultures and exposed for 24 h to the indicated irreversible EGFR kinase inhibitors (EKIs: CI-1033 and PF-00299804) or neutralizing anti-EGFR antibody (Cetuximab) at increasing concentrations. The cells were tested for metabolic activity using the MTS assay. EKIs, but not Cetuximab, triggered marked and dose-dependent reduction in metabolic activity. D, effects of drug treatment on EGFR phosphorylation (P-EGFR; Western blotting); numerical values represent mean ± S.D. of several independent experiments; *, p > 0.05.

Article Snippet: The clonal A431-CD63/GFP cell line was prepared by transfecting A431 parental cells with the plasmid pCMV6-AC-GFP (OriGene, Rockville, MD), clone RG 201733, encoding the CD63-GFP fusion protein.

Techniques: Amplification, Activity Assay, MTS Assay, Western Blot

Journal: The Journal of Biological Chemistry

Article Title: Inhibition of Oncogenic Epidermal Growth Factor Receptor Kinase Triggers Release of Exosome-like Extracellular Vesicles and Impacts Their Phosphoprotein and DNA Content *

doi: 10.1074/jbc.M115.679217

Figure Lengend Snippet: Marked increase in the EV-mediated EGFR emission from EKI-treated cancer cells. A, unchanged levels of EGFR protein in lysates of A431 cells cultured for 24 h in the presence of control medium (control), CI-1033 (5 μm), PF-00299804 (5 μm), or Cetuximab (Cetux., 50 μg/ml), as measured by ELISA. B, unchanged levels of EGFR protein in lysates of A431 cells cultured in the presence of CI-1033 (5 μm), TGFα (50 ng/ml), or both (EGFR ELISA). C, dramatic increase in EGFR signal in the EV (P4) fraction of the conditioned medium corresponding to CI-1033 and PF-00299804 treatment; pooled data were from two independent experiments. D, increase in EV-mediated emission of EGFR upon treatment with CI-1033 and TGFα/CI-1033. The combined treatment highlights the ability of EGFR kinase inhibitor to trigger EV release; p values were as indicated. The results were independently reproduced using an antibody array (data not shown). NS, not significant.

Article Snippet: The clonal A431-CD63/GFP cell line was prepared by transfecting A431 parental cells with the plasmid pCMV6-AC-GFP (OriGene, Rockville, MD), clone RG 201733, encoding the CD63-GFP fusion protein.

Techniques: Cell Culture, Enzyme-linked Immunosorbent Assay, Ab Array

Journal: The Journal of Biological Chemistry

Article Title: Inhibition of Oncogenic Epidermal Growth Factor Receptor Kinase Triggers Release of Exosome-like Extracellular Vesicles and Impacts Their Phosphoprotein and DNA Content *

doi: 10.1074/jbc.M115.679217

Figure Lengend Snippet: Differential EGFR phosphorylation profiles of cancer cells and their EVs in the presence or absence of PF-00299804 treatment. A431 cells and corresponding EV preparations were subjected to Western blotting for EGFR and for three different EGFR phosphosites (Tyr-845, Tyr-1068, and Tyr-1173). EVs differ in their EGFR phosphorylation profiles from their parental cells, including preponderance of 2 out of 3 phosphoisoforms of EGFR following drug treatment.

Article Snippet: The clonal A431-CD63/GFP cell line was prepared by transfecting A431 parental cells with the plasmid pCMV6-AC-GFP (OriGene, Rockville, MD), clone RG 201733, encoding the CD63-GFP fusion protein.

Techniques: Western Blot

Journal: The Journal of Biological Chemistry

Article Title: Inhibition of Oncogenic Epidermal Growth Factor Receptor Kinase Triggers Release of Exosome-like Extracellular Vesicles and Impacts Their Phosphoprotein and DNA Content *

doi: 10.1074/jbc.M115.679217

Figure Lengend Snippet: Differential phosphorylation profiles of EGFR, AKT, and ERK in cancer cells and their EVs following treatment with pan-Erb kinase inhibitor CI-1033. A431 cells were treated with the EGFR agonist (TGFα, 50 ng/ml), irreversible ErbB kinase inhibitor (CI-1033, 5 μm), or both. The cells and EVs were collected 24 h later and immunoblotted for total EGFR, three different EGFR phosphosites (P-EGFR Tyr-845, Tyr-1068, and Tyr-1173), AKT, P-AKT (Ser-473), ERK, and P-ERK (Thr-202/Tyr-Y204). As loading controls, β-actin and Ponceau red were used for cell and EV lysates, respectively. EVs differ in their EGFR phosphorylation profile from their parental cells. P-EGFR is less abundant in EVs than in corresponding cells. EVs accumulate Tyr-845 and Tyr-1068 P-EGFR isoforms, but not Tyr-1173, following treatment with CI-1033. EV-associated Tyr-1173 is increased following stimulation with TGFα. EVs contain no detectable P-AKT but are enriched for ERK and P-ERK following CI-1033 treatment (see text for details).

Article Snippet: The clonal A431-CD63/GFP cell line was prepared by transfecting A431 parental cells with the plasmid pCMV6-AC-GFP (OriGene, Rockville, MD), clone RG 201733, encoding the CD63-GFP fusion protein.

Techniques:

Journal: The Journal of Biological Chemistry

Article Title: Inhibition of Oncogenic Epidermal Growth Factor Receptor Kinase Triggers Release of Exosome-like Extracellular Vesicles and Impacts Their Phosphoprotein and DNA Content *

doi: 10.1074/jbc.M115.679217

Figure Lengend Snippet: Pharmacological blockade of the oncogenic EGFR triggers emission of EVs with exosomal characteristics. A431-CD63/GFP cells were cultured for 24 h in the presence of control media or EGFR blocking concentrations of CI-1033 (5 μm). A, EVs were collected by ultracentrifugation at either 2500 × g (EV-P2) or 110,000 × g (EV-P4) and immunoblotted for EGFR or GFP, the latter to reveal the exogenous GFP/CD63 chimeric protein marker of exosomes. P4 fraction containing exosome-sized EVs was enriched in EGFR and GFP, especially after the CI-1033 treatment. B and C, A431-derived EVs were floated on the sucrose gradient, and the respective fractions were profiled for size and numerical EV distribution using nanoparticle tracking analysis system (NTA, Nanosight). Of note is the fact that CI-1033 treatment stimulated production of mainly small EVs (exosome-like), with sizes ranging between 51 and 150 nm and density between 1.11 and 1.21 g/ml, which corresponded to sucrose density fractions 3–8. NTA of individual fractions containing cancer cell-derived EVs suggests a highly heterogeneous size distribution. D and E, exosomal fractions (1.12–1.17 g/ml) of EVs purified from conditioned medium of control A431-CD63/GFP cells contain both EGFR and GFP (CD63) markers (immunoblotting). CI-1033 treatment triggered the increase in the EGFR and exosomal marker content (GFP-CD63 and CD9) across exosomal fractions 1.10–1.21 g/ml of A431-GFP/CD63-derived EVs. Data are representative of three independent experiments (see text for details).

Article Snippet: The clonal A431-CD63/GFP cell line was prepared by transfecting A431 parental cells with the plasmid pCMV6-AC-GFP (OriGene, Rockville, MD), clone RG 201733, encoding the CD63-GFP fusion protein.

Techniques: Cell Culture, Blocking Assay, Marker, Derivative Assay, Purification, Western Blot

Journal: The Journal of Biological Chemistry

Article Title: Inhibition of Oncogenic Epidermal Growth Factor Receptor Kinase Triggers Release of Exosome-like Extracellular Vesicles and Impacts Their Phosphoprotein and DNA Content *

doi: 10.1074/jbc.M115.679217

Figure Lengend Snippet: EKI-induced cellular vesiculation depends on the neutral sphingomyelinase pathway of exosomal biogenesis. A, NTA analysis of EVs emitted from A431 cells treated with EKI (CI-1033 (CI)), inhibitor of neutral sphingomyelinase (GW4869), inhibitor of acidic sphingomyelinase (FTY720), or their indicated combinations. Of note is a shift to the right in the median size peak of EVs in presence of GW4869 indicative of reduced numbers of exosome-seized vesicles and the preponderance of larger EV sizes. B, EV output into the A431 conditioned medium as measured within the median exosome size (range 150–200 nm) of vesicles. CI-1033-induced increase in EV numbers is attenuated by GW4869 pretreatment but not by FTY720 pretreatment, which increases the EV production; compilation is of three independent experiments, and p value is as indicated. NS, nonsignificant.

Article Snippet: The clonal A431-CD63/GFP cell line was prepared by transfecting A431 parental cells with the plasmid pCMV6-AC-GFP (OriGene, Rockville, MD), clone RG 201733, encoding the CD63-GFP fusion protein.

Techniques:

Journal: The Journal of Biological Chemistry

Article Title: Inhibition of Oncogenic Epidermal Growth Factor Receptor Kinase Triggers Release of Exosome-like Extracellular Vesicles and Impacts Their Phosphoprotein and DNA Content *

doi: 10.1074/jbc.M115.679217

Figure Lengend Snippet: EKI-triggered vesiculation involves caspase activity. A, activation of caspase 3 in A431 cells treated with CI-1033 and etoposide. Floating (P1) cells reveal the cleaved caspase 3 (lower) band. Cell cultures were treated with growth inhibitory (5 μm) and subthreshold (1 μm) concentrations of CI-1033, and with pro-apoptotic concentrations of etoposide. In all cases caspase 3 cleavage was inhibited by ZVAD peptide. No cleaved caspase was detectable in adherent cells. Caspase 3 was undetectable in microvesicle-like (P2) and exosome-like (P4) EV fractions. B–E, NTA profile reveals that growth inhibitory dosing of CI-1033 (5 μm), but not of etoposide, evoked vesicular emissions from A431 cells, despite caspase 3 activation in both cases. CI-1033-induced EV release was selectively inhibited by ZVAD pretreatment, as demonstrated by NTA of total (C), and exosome-like (150–200 nm (D)) vesicle populations, but this effect was not observed in larger microvesicle-like EV subsets (350–400 nm; E); data are representative of three independent experiments, p value is as indicated. NS, nonsignificant.

Article Snippet: The clonal A431-CD63/GFP cell line was prepared by transfecting A431 parental cells with the plasmid pCMV6-AC-GFP (OriGene, Rockville, MD), clone RG 201733, encoding the CD63-GFP fusion protein.

Techniques: Activity Assay, Activation Assay

Journal: The Journal of Biological Chemistry

Article Title: Inhibition of Oncogenic Epidermal Growth Factor Receptor Kinase Triggers Release of Exosome-like Extracellular Vesicles and Impacts Their Phosphoprotein and DNA Content *

doi: 10.1074/jbc.M115.679217

Figure Lengend Snippet: EKI treatment leads to cellular emission of genomic DNA within exosome-like fraction of EVs. A, A431 cells were cultured and treated with CI-1033 (5 μm), and sucrose fractions of their EVs were tested for extracellular genomic DNA using QC Analyzer (A) or PCR (B) assays. EV fractions are as follows: 3 (below exosomal density), 6 (exosomal like), and 9 (above exosomal density) were analyzed for DNA content. DNA was detected (arrowhead) only in fractions 6 and 9, and upon treatment with CI-1033. B, PCR amplification of the EGFR (exon 18) genomic sequence in A431-derived EV fractions obtained with and without CI-1033 treatment. These profiles suggest the emission of DNA within exosome-like fractions of EVs following the EGFR blockade. EVs at a higher density contain gDNA regardless of treatment (see text for details).

Article Snippet: The clonal A431-CD63/GFP cell line was prepared by transfecting A431 parental cells with the plasmid pCMV6-AC-GFP (OriGene, Rockville, MD), clone RG 201733, encoding the CD63-GFP fusion protein.

Techniques: Cell Culture, Amplification, Sequencing, Derivative Assay

Journal: The Journal of Biological Chemistry

Article Title: Inhibition of Oncogenic Epidermal Growth Factor Receptor Kinase Triggers Release of Exosome-like Extracellular Vesicles and Impacts Their Phosphoprotein and DNA Content *

doi: 10.1074/jbc.M115.679217

Figure Lengend Snippet: EV-mediated extracellular co-emission of EGFR and DNA following cancer cell exposure to EKIs. A, experimental design: ImmunoCapture of pre-filtered, EGFR-containing EVs produced by A431 cells either in the absence of in the presence of EKIs, followed by detection of extracellular genomic DNA (exo-gDNA; PCR). B, analysis of EGFR genomic sequences in EV fractions captured on plates coated with anti-EGFR antibody. This assay reveals that the same population of small EGFR-positive exosome-like EVs, which are able to pass through both 0.8- and 0.45-μm filters, is enriched in exo-gDNA, but only if the cells were pretreated with growth inhibitory doses of EKIs, CI-1033 or PF-00299804 (see text for details). UC, ultracentrifugation; -, possible but not obligatory step in the procedure.

Article Snippet: The clonal A431-CD63/GFP cell line was prepared by transfecting A431 parental cells with the plasmid pCMV6-AC-GFP (OriGene, Rockville, MD), clone RG 201733, encoding the CD63-GFP fusion protein.

Techniques: Produced, Genomic Sequencing

Journal: Oncology Letters

Article Title: Antitumor effects of dioscin in A431 cells via adjusting ATM/p53-mediated cell apoptosis, DNA damage and migration

doi: 10.3892/ol.2020.12321

Figure Lengend Snippet: Inhibitory effects of dioscin on the viability, colony formation and migration of A431 cells. (A) Chemical structure of dioscin. (B) Effects of dioscin on the viability of A431 cells detected via MTT assay. (C) Representative morphological images of cells treated with different concentrations of dioscin (2.9, 5.8 and 11.6 µM) for 24 h. (D) Effects of dioscin treatment (2.9, 5.8 and 11.6 µM) for 24 h on colony formation in A431 cells. (E) Effects of dioscin treatment (2.9, 5.8 and 11.6 µM) for 24 h on migratory and invasive properties of A431. Scale bar, 100 µm. Data are presented as the mean ± SD (n=5). *P<0.05 and **P<0.01 vs. control. Dio, dioscin.

Article Snippet: The human skin carcinoma A431 cell line was purchased from Wuhan Boster Biological Technology, Ltd.

Techniques: Migration, MTT Assay, Control

Journal: Oncology Letters

Article Title: Antitumor effects of dioscin in A431 cells via adjusting ATM/p53-mediated cell apoptosis, DNA damage and migration

doi: 10.3892/ol.2020.12321

Figure Lengend Snippet: Dioscin induces apoptosis and DNA damage, and suppresses invasion and migration of A431 cells. (A) Representative figures of dioscin on apoptosis and DNA damage via TUNEL and comet assays, respectively. (B) Expression levels of p-ATM after dioscin treatment via immunofluorescence and western blotting. (C) Effects of dioscin on the expression levels of MMP2/9, RHO and cdc42. Data are presented as the mean ± SD (n=3). *P<0.05 and **P<0.01 vs. control. Dio, dioscin; p-ATM, phosphorylated ataxia telangiectasia-mutated; MMP, matrix metalloproteinase; TUNEL, terminal deoxynucleotidyl transferase dUTP nick-end labeling.

Article Snippet: The human skin carcinoma A431 cell line was purchased from Wuhan Boster Biological Technology, Ltd.

Techniques: Migration, TUNEL Assay, Expressing, Immunofluorescence, Western Blot, Control

Journal: Oncology Letters

Article Title: Antitumor effects of dioscin in A431 cells via adjusting ATM/p53-mediated cell apoptosis, DNA damage and migration

doi: 10.3892/ol.2020.12321

Figure Lengend Snippet: p53-siRNA abrogates the inhibitory effects of dioscin on A431 cells. (A) A431 cells were transfected with p53-siRNA and the viability of cells was measured. Effects of dioscin and p53-siRNA on (B) cell colony formation ability, (C) cell migration, apoptosis and DNA damage, (D) p53 expression via immunofluorescence and (E) protein expression levels of cleaved-PARP, cleaved caspase-3/9, Bax, Bcl-2, RHO and cdc42. Scale bar, 100 µm. Data are presented as the mean ± SD (n=3). **P<0.01 vs. dioscin group. PARP, poly (ADP-ribose) polymerase; ns, not significant; siRNA, small interfering RNA.

Article Snippet: The human skin carcinoma A431 cell line was purchased from Wuhan Boster Biological Technology, Ltd.

Techniques: Transfection, Migration, Expressing, Immunofluorescence, Small Interfering RNA