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  • 99
    Millipore afatinib
    Regression of ear tumors by EGFR inhibitors (A-D) Assessment of ear tumor regression by MRI scan before and after treatment with EGFR inhibitors. SP-C/mEGFR L+T mice with head tilt were randomized to 4 groups (3 mice/group) and then treated with cetuximab for 7 days (A), cetuximab combined with erlotinib or <t>afatinib</t> for 7 days (B and C), and WZ4002 for 5 days (D) as described dosing and schedule in Materials and Methods. See also Table S1 . White ovals indicate dense areas of ear tumors. L, left side. Columns , mean of relative ear tumor volume from 3 mice in before and after treatment with each regimen. bars , standard deviation. Statistical analysis was performed with unpaired t test. P values of less than 0.05 were considered significant. (E) Change of tumor volume from baseline by EGFR inhibitors for individual SP-C/mEGFR L+T mice bearing ear tumors in Table S1 . CR, complete response; PR, partial response; SD, stable disease; PD, progressive disease. See also the criteria to classify tumor responses to drug treatment as described in Supplementary Materials and Methods .
    Afatinib, supplied by Millipore, used in various techniques. Bioz Stars score: 99/100, based on 46 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
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    99
    Selleck Chemicals afatinib
    Increased expression of FGF2 and FGFR1 upon acquisition of <t>afatinib</t> resistance (A) Both resistant sublclones produced more than 30-fold higher levels of FGF2 than PC9 and Rev1. (B) Phosphorylation of EGFR, Akt and Erk in Rev1 was similarly susceptible to the inhibitory effect of afatinib (6 h) in PC9 when phosphorylation of Akt and Erk was resistant to the inhibitory effect of the drug in both resistant subclones. (C) Time kinetics for treatment with FGF showed enhanced phosphorylation of FGFR in both B19 and B20, accompanying by enhanced activation of Akt and Erk. (D) Increasing dose-dependent activation of FGFR, Akt and Erk in B19 and B20 upon treatment with various doses of FGF. All experiments were performed under serum free condition.(E) Autocrine stimulation of B19 by secreted endogenous growth factor was responsible for activation of FGFR phosphorylation
    Afatinib, supplied by Selleck Chemicals, used in various techniques. Bioz Stars score: 99/100, based on 397 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
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    93
    Boehringer Ingelheim afatinib
    ERBB2 extracellular domain mutant cervical cancers are sensitive to ERBB2 inhibition by <t>afatinib</t> or neratinib in vitro and in vivo. ( A and B ) Mean IC 50 values of ERBB2-mutated cervical carcinoma (CC) compared to ERBB2 wild-type CC cell lines ( P
    Afatinib, supplied by Boehringer Ingelheim, used in various techniques. Bioz Stars score: 93/100, based on 361 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
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    92
    LC Laboratories afatinib
    Targeted inhibition of EGFR and CDK4/6. A and B, cell titer blue viability assays for lapatinib (Lap), <t>afatinib</t> (Afa), palbociclib (Pal), and combination treatment (Combo). Coefficient of interaction values at different effective doses for the combination
    Afatinib, supplied by LC Laboratories, used in various techniques. Bioz Stars score: 92/100, based on 108 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
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    93
    Santa Cruz Biotechnology afatinib
    <t>Afatinib</t> induced cell apoptosis and proliferation arrest in ICC cell lines. Cell viability in JCK and OZ cells treated with (A) different afatinib doses and (B) for different durations, showing that afatinib blocked the cell growth in a time- and dose-dependent manner. (C) Flow cytometry using TUNEL-staining, and (D) cell apoptosis results, demonstrating that afatinib notably induced ICC cell apoptosis. (E) Western blots and (F) quantified protein levels, revealing that afatinib significantly inhibited pEGFR and pSTAT3 (n=3). *P
    Afatinib, supplied by Santa Cruz Biotechnology, used in various techniques. Bioz Stars score: 93/100, based on 29 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
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    92
    Sequoia Research afatinib
    ( A ) HCC1419 cells were treated with varying concentrations of either neratinib or <t>afatinib</t> for 1 week. Cells were then fixed and stained for SA-β-gal activity and compared to untreated control and lapatinib treated cells. ( B ) HCC1419 cells were treated with 10 µg/mL trastuzumab alone or in combination with 250 nM lapatinib for 1 week, prior to SA-β-gal activity testing. ( C ) SKBR3 cells were treated with 10 µg/mL trastuzumab alone or in combination with 250 nM lapatinib for 2 weeks, prior to SA-β-gal activity testing. Images taken at 400× magnification.
    Afatinib, supplied by Sequoia Research, used in various techniques. Bioz Stars score: 92/100, based on 13 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
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    93
    Cayman Chemical afatinib
    Effects of <t>Afatinib</t> on apoptotic response and DNA damage repair in irradiated PC-9-GR cells (A). Apoptosis analysis. Cells were treated with afatinib (350.0 nM), IR (6Gy) or afatinib for two hours followed by IR. Apoptosis was determined 36 hours later after IR with flow cytometry analysis. (B). Effect of pan-caspase inhibitor z-VAD-fmk on afatinib-enhanced apoptotic response in irradiated PC-9-GR cells. Cells were pretreated with 20 M z-VAD-fmk, followed by indicated treatments. Apoptosis was determined with flow cytometry analysis. Diagram showing the changes of early, late and total apoptosis in these experiments. (C). Immunofluorescence analysis. Immunofluorescence analyses were performed to examine the formation and changes of nuclear γ-H2AX foci. Representative images of nuclear γ-H2AX foci in PC-9-GR cells treated with IR or the combination are shown in top panel, and diagram showing the change in the cell fractions with positive γ-H2AX foci are present in bottom panel. Data represent the average of three experiments. Error bars indicate standard deviation. * indicates statistical significance. (D). Effect of afatinib on DNA-pKcs expression. Cell lysates were collected from PC-9-GR cells treated with afatinib, IR or the combination for twenty-four hours. Expression of DNA-pKcs protein was examined with western blot assay. Anti-GAPDH antibody was included as a loading control. (E). Clonogenic survival assay. PC-9-GR cells were transiently transfected with DNA-pKcs siRNA, or scramble-siRNA as control. Clonogenic survival assay was performed as described above. Western blot results showing the inhibitory effect of siRNAs on DNA-pKcs protein in cells collected 72 hours after transfections.
    Afatinib, supplied by Cayman Chemical, used in various techniques. Bioz Stars score: 93/100, based on 11 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
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    90
    Euromedex afatinib
    L861Q TaqMan assay (droplet digital PCR). 2D plots and amounts of L861Q-positive droplets (indicated in red) obtained via ddPCR with FFPE DNA ( a ) or cfDNA ( b to h ). a ) the primary tumour, b ) on the day of surgery (D0), c ) before concomitant chemotherapy (CT) and radiotherapy (RT) (pre-RT), d ) 1 month after RT (post-RT), e ) 1 month after the first cycle of CT (CT-1), f ) 1 month after four cycles of CT (CT-4), g ) after 9 weeks of <t>afatinib</t> treatment (post-TKI), and h ) after two cycles of lomustine with bevacizumab (Lom + Bev-2). The plasma DNA concentration is expressed as the amount per ml of plasma. ddPCR, droplet digital PCR; FFPE, formalin-fixed, paraffin-embedded; pDNA, plasma DNA; AU, arbitrary units; WT, wild type; CT, chemotherapy; RT, radiotherapy
    Afatinib, supplied by Euromedex, used in various techniques. Bioz Stars score: 90/100, based on 9 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
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    92
    SYNkinase afatinib
    Effects of <t>afatinib</t> treatment on apoptosis and the IL-6/JAK1/STAT3 signaling pathway in H1975 cells. (A) Cell viability was measured with the CCK-8 assay and shown by the absorbance at 450 nm following treatment with various concentrations of afatinib for 24 h. (B) Protein levels of IL-6 from the supernatant of H1975 cells treated with or without 1 µM afatinib were detected by ELISA. (C) Activation of the JAK1/STAT3 signaling pathway was detected in H1975 cells in the presence or absence of 1 µM afatinib by western blot. β-actin was included as an mRNA and protein loading control. Data are representative of three independent experiments and expressed as means ± SEM. **P
    Afatinib, supplied by SYNkinase, used in various techniques. Bioz Stars score: 92/100, based on 12 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
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    85
    Boehringer Ingelheim irreversible erbb family blocker afatinib
    Effects of <t>afatinib</t> treatment on apoptosis and the IL-6/JAK1/STAT3 signaling pathway in H1975 cells. (A) Cell viability was measured with the CCK-8 assay and shown by the absorbance at 450 nm following treatment with various concentrations of afatinib for 24 h. (B) Protein levels of IL-6 from the supernatant of H1975 cells treated with or without 1 µM afatinib were detected by ELISA. (C) Activation of the JAK1/STAT3 signaling pathway was detected in H1975 cells in the presence or absence of 1 µM afatinib by western blot. β-actin was included as an mRNA and protein loading control. Data are representative of three independent experiments and expressed as means ± SEM. **P
    Irreversible Erbb Family Blocker Afatinib, supplied by Boehringer Ingelheim, used in various techniques. Bioz Stars score: 85/100, based on 8 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
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    88
    Boehringer Ingelheim afatinib bibw
    Effects of <t>afatinib</t> treatment on apoptosis and the IL-6/JAK1/STAT3 signaling pathway in H1975 cells. (A) Cell viability was measured with the CCK-8 assay and shown by the absorbance at 450 nm following treatment with various concentrations of afatinib for 24 h. (B) Protein levels of IL-6 from the supernatant of H1975 cells treated with or without 1 µM afatinib were detected by ELISA. (C) Activation of the JAK1/STAT3 signaling pathway was detected in H1975 cells in the presence or absence of 1 µM afatinib by western blot. β-actin was included as an mRNA and protein loading control. Data are representative of three independent experiments and expressed as means ± SEM. **P
    Afatinib Bibw, supplied by Boehringer Ingelheim, used in various techniques. Bioz Stars score: 88/100, based on 6 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
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    91
    AstaTech Inc afatinib
    Inhibition of TNF induces sensitivity of EGFRwt-expressing NSCLC cells to EGFR inhibition. ( A and B ) AlamarBlue cell viability assay in H441 or A549 cells. TNFR1 was silenced by siRNA transfection for 48 hours, and cells were exposed to erlotinib (1 μM) for 72 hours in RPMI-1640 with 5% FBS. ( C ) Silencing of TNFR1 was confirmed by Western blot. Western blot results are representative of at least 3 independent replicates. ( D and E ) Thalidomide sensitizes H441 and A549 cells to EGFR inhibition with erlotinib. Thalidomide (5 μg/ml) and erlotinib were added to H441 and A549 cells concurrently, and AlamarBlue assay was done after 72 hours. ( F and G ) A similar experiment was done using etanercept (100 μg/ml) and erlotinib in H441 and A549 cells. ( H ) H441 cells were treated with <t>afatinib</t> (1 μM) in the presence or absence of etanercept. AlamarBlue assay was conducted after 72 hours. ( I ) H441 cells were treated with afatinib and thalidomide for 72 hours, followed by AlamarBlue assay. ( J and K ) Similar experiments were done as described in H and I in A549 cells. ( L and M ) EGFRwt cells were seeded in 6-well plates at 1,000 cells per well, and incubated with 20 μg/ml thalidomide and/or 1 μM erlotinib. Fourteen days later, cell colonies were fixed by 100% methanol and then stained by 0.5% crystal violet in 25% methanol. Images were captured by a scanner, and colony counts were processed by ImageJ. Data represent the mean percentage of control ± SEM. n = 3 biologically independent experimental replicates ( A , B , and D – M ). * P
    Afatinib, supplied by AstaTech Inc, used in various techniques. Bioz Stars score: 91/100, based on 7 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
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    92
    Sequoia Sciences afatinib
    A) CENPE protein expression in response to a 12 hour treatment with 150nM <t>Afatinib,</t> 150nM Trastuzumab (Her), 150nM Trastuzumab + 1μM Lapatinib (Lap) and 1μM Lapatinib + 20μM Capecitabine (Cap) with densitometric measurement of fold change (control vs. drug treated) in B) the SKBR3 cell line and C) the BT474 cell line. * represents significance at p
    Afatinib, supplied by Sequoia Sciences, used in various techniques. Bioz Stars score: 92/100, based on 6 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
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    86
    Boehringer Ingelheim fda approved afatinib
    A) CENPE protein expression in response to a 12 hour treatment with 150nM <t>Afatinib,</t> 150nM Trastuzumab (Her), 150nM Trastuzumab + 1μM Lapatinib (Lap) and 1μM Lapatinib + 20μM Capecitabine (Cap) with densitometric measurement of fold change (control vs. drug treated) in B) the SKBR3 cell line and C) the BT474 cell line. * represents significance at p
    Fda Approved Afatinib, supplied by Boehringer Ingelheim, used in various techniques. Bioz Stars score: 86/100, based on 5 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
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    92
    Boehringer Ingelheim giotrif afatinib tablets
    A) CENPE protein expression in response to a 12 hour treatment with 150nM <t>Afatinib,</t> 150nM Trastuzumab (Her), 150nM Trastuzumab + 1μM Lapatinib (Lap) and 1μM Lapatinib + 20μM Capecitabine (Cap) with densitometric measurement of fold change (control vs. drug treated) in B) the SKBR3 cell line and C) the BT474 cell line. * represents significance at p
    Giotrif Afatinib Tablets, supplied by Boehringer Ingelheim, used in various techniques. Bioz Stars score: 92/100, based on 2 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
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    88
    Boehringer Ingelheim o afatinibe
    A) CENPE protein expression in response to a 12 hour treatment with 150nM <t>Afatinib,</t> 150nM Trastuzumab (Her), 150nM Trastuzumab + 1μM Lapatinib (Lap) and 1μM Lapatinib + 20μM Capecitabine (Cap) with densitometric measurement of fold change (control vs. drug treated) in B) the SKBR3 cell line and C) the BT474 cell line. * represents significance at p
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    91
    Boehringer Ingelheim tki afatinib
    A) CENPE protein expression in response to a 12 hour treatment with 150nM <t>Afatinib,</t> 150nM Trastuzumab (Her), 150nM Trastuzumab + 1μM Lapatinib (Lap) and 1μM Lapatinib + 20μM Capecitabine (Cap) with densitometric measurement of fold change (control vs. drug treated) in B) the SKBR3 cell line and C) the BT474 cell line. * represents significance at p
    Tki Afatinib, supplied by Boehringer Ingelheim, used in various techniques. Bioz Stars score: 91/100, based on 3 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
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    Image Search Results


    Regression of ear tumors by EGFR inhibitors (A-D) Assessment of ear tumor regression by MRI scan before and after treatment with EGFR inhibitors. SP-C/mEGFR L+T mice with head tilt were randomized to 4 groups (3 mice/group) and then treated with cetuximab for 7 days (A), cetuximab combined with erlotinib or afatinib for 7 days (B and C), and WZ4002 for 5 days (D) as described dosing and schedule in Materials and Methods. See also Table S1 . White ovals indicate dense areas of ear tumors. L, left side. Columns , mean of relative ear tumor volume from 3 mice in before and after treatment with each regimen. bars , standard deviation. Statistical analysis was performed with unpaired t test. P values of less than 0.05 were considered significant. (E) Change of tumor volume from baseline by EGFR inhibitors for individual SP-C/mEGFR L+T mice bearing ear tumors in Table S1 . CR, complete response; PR, partial response; SD, stable disease; PD, progressive disease. See also the criteria to classify tumor responses to drug treatment as described in Supplementary Materials and Methods .

    Journal: Oncotarget

    Article Title: Epidermal growth factor receptor as a novel molecular target for aggressive papillary tumors in the middle ear and temporal bone

    doi:

    Figure Lengend Snippet: Regression of ear tumors by EGFR inhibitors (A-D) Assessment of ear tumor regression by MRI scan before and after treatment with EGFR inhibitors. SP-C/mEGFR L+T mice with head tilt were randomized to 4 groups (3 mice/group) and then treated with cetuximab for 7 days (A), cetuximab combined with erlotinib or afatinib for 7 days (B and C), and WZ4002 for 5 days (D) as described dosing and schedule in Materials and Methods. See also Table S1 . White ovals indicate dense areas of ear tumors. L, left side. Columns , mean of relative ear tumor volume from 3 mice in before and after treatment with each regimen. bars , standard deviation. Statistical analysis was performed with unpaired t test. P values of less than 0.05 were considered significant. (E) Change of tumor volume from baseline by EGFR inhibitors for individual SP-C/mEGFR L+T mice bearing ear tumors in Table S1 . CR, complete response; PR, partial response; SD, stable disease; PD, progressive disease. See also the criteria to classify tumor responses to drug treatment as described in Supplementary Materials and Methods .

    Article Snippet: Afatinib and erlotinib were suspended in vehicle (0.5% Carboxymethylcellulose: Sigma C4888) and administrated by gavage at 20 mg/kg and 50 mg/kg once a day, daily for 7 days, respectively.

    Techniques: Magnetic Resonance Imaging, Mouse Assay, Standard Deviation

    Increased expression of FGF2 and FGFR1 upon acquisition of afatinib resistance (A) Both resistant sublclones produced more than 30-fold higher levels of FGF2 than PC9 and Rev1. (B) Phosphorylation of EGFR, Akt and Erk in Rev1 was similarly susceptible to the inhibitory effect of afatinib (6 h) in PC9 when phosphorylation of Akt and Erk was resistant to the inhibitory effect of the drug in both resistant subclones. (C) Time kinetics for treatment with FGF showed enhanced phosphorylation of FGFR in both B19 and B20, accompanying by enhanced activation of Akt and Erk. (D) Increasing dose-dependent activation of FGFR, Akt and Erk in B19 and B20 upon treatment with various doses of FGF. All experiments were performed under serum free condition.(E) Autocrine stimulation of B19 by secreted endogenous growth factor was responsible for activation of FGFR phosphorylation

    Journal: Oncotarget

    Article Title: FGFR1 activation is an escape mechanism in human lung cancer cells resistant to afatinib, a pan-EGFR family kinase inhibitor

    doi:

    Figure Lengend Snippet: Increased expression of FGF2 and FGFR1 upon acquisition of afatinib resistance (A) Both resistant sublclones produced more than 30-fold higher levels of FGF2 than PC9 and Rev1. (B) Phosphorylation of EGFR, Akt and Erk in Rev1 was similarly susceptible to the inhibitory effect of afatinib (6 h) in PC9 when phosphorylation of Akt and Erk was resistant to the inhibitory effect of the drug in both resistant subclones. (C) Time kinetics for treatment with FGF showed enhanced phosphorylation of FGFR in both B19 and B20, accompanying by enhanced activation of Akt and Erk. (D) Increasing dose-dependent activation of FGFR, Akt and Erk in B19 and B20 upon treatment with various doses of FGF. All experiments were performed under serum free condition.(E) Autocrine stimulation of B19 by secreted endogenous growth factor was responsible for activation of FGFR phosphorylation

    Article Snippet: Afatinib, lapatinib, foretinib, gefitinib, and dasatinib were purchased from Selleck (Houston, USA).

    Techniques: Expressing, Produced, Activation Assay

    Our hypothetic model shows how afatinib resistance is acquired in lung cancer cells In drug sensitive cell line, the cell survival and growth of human lung cancer cells harboring activating EGFR depends upon the EGFR/EGFR family driven PI3K/Akt and Erk pathways, and this cell survival and growth is highly susceptible to afatinib and other EGFR-TKIs. By contrast, afatinib-resistant subclones express elevated levels of FGFR1 together with FGF2, resulting in activation of Akt and Erk, when EGFR/EGFR family-driven cell growth/survival signaling pathways are mostly attenuated. Of EMT-related transcription factors, Twist seems to specifically responsible for elevated expression of FGFR1 in afatinib resistant cell lines.

    Journal: Oncotarget

    Article Title: FGFR1 activation is an escape mechanism in human lung cancer cells resistant to afatinib, a pan-EGFR family kinase inhibitor

    doi:

    Figure Lengend Snippet: Our hypothetic model shows how afatinib resistance is acquired in lung cancer cells In drug sensitive cell line, the cell survival and growth of human lung cancer cells harboring activating EGFR depends upon the EGFR/EGFR family driven PI3K/Akt and Erk pathways, and this cell survival and growth is highly susceptible to afatinib and other EGFR-TKIs. By contrast, afatinib-resistant subclones express elevated levels of FGFR1 together with FGF2, resulting in activation of Akt and Erk, when EGFR/EGFR family-driven cell growth/survival signaling pathways are mostly attenuated. Of EMT-related transcription factors, Twist seems to specifically responsible for elevated expression of FGFR1 in afatinib resistant cell lines.

    Article Snippet: Afatinib, lapatinib, foretinib, gefitinib, and dasatinib were purchased from Selleck (Houston, USA).

    Techniques: Activation Assay, Expressing

    Establishment of afatinib-resistant lung cancer cells (A) Dose response curves for PC9, and drug-resistant PC9BR, PC9BR (3Mo), (10Mo), (11Mo), and (21Mo) cells to various doses of afatinib were determined by WST assay. (B) Western blotting analysis was performed for biochemical profiling of these cells in the absence or presence of afatinib for 6 h. Expression of pEGFR, HER2/pHER2, and HER3/pHER3 were markedly downregulated by resistance to afatinib, and activation of downstream regulating molecules for cell growth and survival was found to be highly resistant to the drugs. Downregulation of EGFR family proteins and upregulation of FGFR1 by selecting for afatinib resistance. (C) Dose response curves for afatinib were acquired for PC9 and its drug-resistant subclones, B3, B19, B20 and Rev1, with various doses of afatinib. (D) B19 and B20 showed 2- to 5-fold higher collateral sensitivity to PD173074.(E) Increasing expression of FGFR1 and pFGFR in resistant sublclones relative to their parental cells

    Journal: Oncotarget

    Article Title: FGFR1 activation is an escape mechanism in human lung cancer cells resistant to afatinib, a pan-EGFR family kinase inhibitor

    doi:

    Figure Lengend Snippet: Establishment of afatinib-resistant lung cancer cells (A) Dose response curves for PC9, and drug-resistant PC9BR, PC9BR (3Mo), (10Mo), (11Mo), and (21Mo) cells to various doses of afatinib were determined by WST assay. (B) Western blotting analysis was performed for biochemical profiling of these cells in the absence or presence of afatinib for 6 h. Expression of pEGFR, HER2/pHER2, and HER3/pHER3 were markedly downregulated by resistance to afatinib, and activation of downstream regulating molecules for cell growth and survival was found to be highly resistant to the drugs. Downregulation of EGFR family proteins and upregulation of FGFR1 by selecting for afatinib resistance. (C) Dose response curves for afatinib were acquired for PC9 and its drug-resistant subclones, B3, B19, B20 and Rev1, with various doses of afatinib. (D) B19 and B20 showed 2- to 5-fold higher collateral sensitivity to PD173074.(E) Increasing expression of FGFR1 and pFGFR in resistant sublclones relative to their parental cells

    Article Snippet: Afatinib, lapatinib, foretinib, gefitinib, and dasatinib were purchased from Selleck (Houston, USA).

    Techniques: WST Assay, Western Blot, Expressing, Activation Assay

    The close association of FGFR activation with acquired resistance to afatinib (A) Effect of FGFR-TKI against afatinib-resistant cells. The phosphorylation of FGFR was blocked upon treatment with either PD173074 (1 μM) alone or with both PD173074 (1 μM) and afatinib (1 μM) for 24 h. (B) Growth of both resistant sublclones was blocked upon treatent with PD173074 (1 μM) alone or with PD173074 (1 μM) and afatinib (1 μM). (C) Treatment with FGFR1 siRNA reduced the expression of FGFR1, accompanied by inhibition of both Akt and Erk phosphorylation in B19 and B20 cells. Cleaved PARP was also induced when resistant sublclones were treated with siRNA FGFR1 in the absence or presence of afatinib (1 μM) for 24 h.

    Journal: Oncotarget

    Article Title: FGFR1 activation is an escape mechanism in human lung cancer cells resistant to afatinib, a pan-EGFR family kinase inhibitor

    doi:

    Figure Lengend Snippet: The close association of FGFR activation with acquired resistance to afatinib (A) Effect of FGFR-TKI against afatinib-resistant cells. The phosphorylation of FGFR was blocked upon treatment with either PD173074 (1 μM) alone or with both PD173074 (1 μM) and afatinib (1 μM) for 24 h. (B) Growth of both resistant sublclones was blocked upon treatent with PD173074 (1 μM) alone or with PD173074 (1 μM) and afatinib (1 μM). (C) Treatment with FGFR1 siRNA reduced the expression of FGFR1, accompanied by inhibition of both Akt and Erk phosphorylation in B19 and B20 cells. Cleaved PARP was also induced when resistant sublclones were treated with siRNA FGFR1 in the absence or presence of afatinib (1 μM) for 24 h.

    Article Snippet: Afatinib, lapatinib, foretinib, gefitinib, and dasatinib were purchased from Selleck (Houston, USA).

    Techniques: Activation Assay, Expressing, Inhibition

    Twist knockdown specifically blocked FGFR1 expression and Akt phospholylation in afatinib resistant cell lines (A) Microarray analysis showed that the resistant subclones B19 acquired typical EMT characteristics relative to their drug-sensitive parental PC9. (B) Expression of Twist, Snail, Slug, and ZEB1 was increased in resistant cells, accompanied by a decrease in the expression of E-cadherin and an increase in that of vimentin. (C) Western blot analyses showed that expression of all three transcription factors was downregulated by their cognate siRNA. Phosphorylation of Akt and Erk was decreased when expression of twist was knockedowned. (D) Real-time PCR analysis revealed that expression of Twist mRNA was downregulated by its cognate siRNA by RT-PCR (E) Expression of FGFR1 was almost completely blocked accompanying by decreased phosphorylation of Akt and ERK when B19 or B20 cells were treated with Twist siRNA for 24hr and 48hr. (F) Cell growth inhibition of B19 and B20 when treated with afatinib and Twist siRNA. (G) FGFR1 mRNA levels in PC9cells were also increased to 2.5-4 folds of the control when twist was overexpressed.

    Journal: Oncotarget

    Article Title: FGFR1 activation is an escape mechanism in human lung cancer cells resistant to afatinib, a pan-EGFR family kinase inhibitor

    doi:

    Figure Lengend Snippet: Twist knockdown specifically blocked FGFR1 expression and Akt phospholylation in afatinib resistant cell lines (A) Microarray analysis showed that the resistant subclones B19 acquired typical EMT characteristics relative to their drug-sensitive parental PC9. (B) Expression of Twist, Snail, Slug, and ZEB1 was increased in resistant cells, accompanied by a decrease in the expression of E-cadherin and an increase in that of vimentin. (C) Western blot analyses showed that expression of all three transcription factors was downregulated by their cognate siRNA. Phosphorylation of Akt and Erk was decreased when expression of twist was knockedowned. (D) Real-time PCR analysis revealed that expression of Twist mRNA was downregulated by its cognate siRNA by RT-PCR (E) Expression of FGFR1 was almost completely blocked accompanying by decreased phosphorylation of Akt and ERK when B19 or B20 cells were treated with Twist siRNA for 24hr and 48hr. (F) Cell growth inhibition of B19 and B20 when treated with afatinib and Twist siRNA. (G) FGFR1 mRNA levels in PC9cells were also increased to 2.5-4 folds of the control when twist was overexpressed.

    Article Snippet: Afatinib, lapatinib, foretinib, gefitinib, and dasatinib were purchased from Selleck (Houston, USA).

    Techniques: Expressing, Microarray, Western Blot, Real-time Polymerase Chain Reaction, Reverse Transcription Polymerase Chain Reaction, Inhibition

    Effect of Afatinib on the phosphorylation of JAK kinases in ARPE-19 cells invaded by T. gondii RH tachyzoites. The challenge of T. gondii induced phosphorylation of the Tyr1022/Tyr1023 site of JAK1 and the Tyr980 site of JAK3 in the ARPE-19 cells only at 2 hr post infection. The time of Afatinib challenge was the same as compared with T. gondii (RH) infection. Afatinib challenge was at 1 hr before infection and at 1 hr post infection. The concentration of Afatinib was 10 μM, and DMSO was used as the vehicle.

    Journal: The Korean Journal of Parasitology

    Article Title: Afatinib Reduces STAT6 Signaling of Host ARPE-19 Cells Infected with Toxoplasma gondii

    doi: 10.3347/kjp.2016.54.1.31

    Figure Lengend Snippet: Effect of Afatinib on the phosphorylation of JAK kinases in ARPE-19 cells invaded by T. gondii RH tachyzoites. The challenge of T. gondii induced phosphorylation of the Tyr1022/Tyr1023 site of JAK1 and the Tyr980 site of JAK3 in the ARPE-19 cells only at 2 hr post infection. The time of Afatinib challenge was the same as compared with T. gondii (RH) infection. Afatinib challenge was at 1 hr before infection and at 1 hr post infection. The concentration of Afatinib was 10 μM, and DMSO was used as the vehicle.

    Article Snippet: Drugs and antibodies Afatinib (BIBW2992) and Erlotinib (OSI-420) were purchased from Selleck Chemicals (Houston, Texas, USA).

    Techniques: Infection, Concentration Assay

    Afatinib efficiently inhibits the intracellular multiplication of T. gondii RH tachyzoites in ARPE-19 cells. (A) The number of parasites per parasitophorous vacuolar membrane (PVM) after Giemsa staining. DMSO was used as the negative vehicle control, whereas pyrimethamine 5 μM was used as the positive control. The highest concentrations of 3 tyrosine inhibitors were applied in this experiment. The treating concentrations of Afatinib, Erlotinib, and Sunitinib were 10, 50, and 10 μM, respectively. Data are means±SD from duplicate wells. (B) A representative result of Giemsa stain. The host cell is ARPE-19 cells, and parasites are tachyzoites of T. gondii RH strain.

    Journal: The Korean Journal of Parasitology

    Article Title: Afatinib Reduces STAT6 Signaling of Host ARPE-19 Cells Infected with Toxoplasma gondii

    doi: 10.3347/kjp.2016.54.1.31

    Figure Lengend Snippet: Afatinib efficiently inhibits the intracellular multiplication of T. gondii RH tachyzoites in ARPE-19 cells. (A) The number of parasites per parasitophorous vacuolar membrane (PVM) after Giemsa staining. DMSO was used as the negative vehicle control, whereas pyrimethamine 5 μM was used as the positive control. The highest concentrations of 3 tyrosine inhibitors were applied in this experiment. The treating concentrations of Afatinib, Erlotinib, and Sunitinib were 10, 50, and 10 μM, respectively. Data are means±SD from duplicate wells. (B) A representative result of Giemsa stain. The host cell is ARPE-19 cells, and parasites are tachyzoites of T. gondii RH strain.

    Article Snippet: Drugs and antibodies Afatinib (BIBW2992) and Erlotinib (OSI-420) were purchased from Selleck Chemicals (Houston, Texas, USA).

    Techniques: Staining, Positive Control, Giemsa Stain

    Afatinib affects the phosphorylation of Stat6 activated by T. gondii (RH) in ARPE-19 cells. (A) The phosphorylation of the Tyr641 site of STAT6, but not the Tyr701 site of STAT1 in the ARPE-19 cells after challenge with T. gondii RH tachyzoites by western blot. The time of Afatinib challenge was the same as T. gondii (RH) infection. Afatinib challenge was at 1 hr before infection and at 1 hr post infection. The concentration of Afatinib was 10 μM, and DMSO was used as the vehicle. (B) Immunofluorescence stain at 2 hr and 8 hr post infection. The corresponding results of the western blot in Fig. 4A are marked in red frame.

    Journal: The Korean Journal of Parasitology

    Article Title: Afatinib Reduces STAT6 Signaling of Host ARPE-19 Cells Infected with Toxoplasma gondii

    doi: 10.3347/kjp.2016.54.1.31

    Figure Lengend Snippet: Afatinib affects the phosphorylation of Stat6 activated by T. gondii (RH) in ARPE-19 cells. (A) The phosphorylation of the Tyr641 site of STAT6, but not the Tyr701 site of STAT1 in the ARPE-19 cells after challenge with T. gondii RH tachyzoites by western blot. The time of Afatinib challenge was the same as T. gondii (RH) infection. Afatinib challenge was at 1 hr before infection and at 1 hr post infection. The concentration of Afatinib was 10 μM, and DMSO was used as the vehicle. (B) Immunofluorescence stain at 2 hr and 8 hr post infection. The corresponding results of the western blot in Fig. 4A are marked in red frame.

    Article Snippet: Drugs and antibodies Afatinib (BIBW2992) and Erlotinib (OSI-420) were purchased from Selleck Chemicals (Houston, Texas, USA).

    Techniques: Western Blot, Infection, Concentration Assay, Immunofluorescence, Staining

    Afatinib halts the growth of T. gondii RH tachyzoites in ARPE-19 cells from the time of treatment. (A) The number of parasites per PVM after treatment with pyrimethamine 5.0 μM and Afatinib 10.0 μM at 24 hr and 48 hr post infection, respectively. DMSO was used as the negative vehicle control. (B) A representative result of Giemsa stain. The host cell is ARPE-19 cells, and parasites are tachyzoites of T. gondii RH strain.

    Journal: The Korean Journal of Parasitology

    Article Title: Afatinib Reduces STAT6 Signaling of Host ARPE-19 Cells Infected with Toxoplasma gondii

    doi: 10.3347/kjp.2016.54.1.31

    Figure Lengend Snippet: Afatinib halts the growth of T. gondii RH tachyzoites in ARPE-19 cells from the time of treatment. (A) The number of parasites per PVM after treatment with pyrimethamine 5.0 μM and Afatinib 10.0 μM at 24 hr and 48 hr post infection, respectively. DMSO was used as the negative vehicle control. (B) A representative result of Giemsa stain. The host cell is ARPE-19 cells, and parasites are tachyzoites of T. gondii RH strain.

    Article Snippet: Drugs and antibodies Afatinib (BIBW2992) and Erlotinib (OSI-420) were purchased from Selleck Chemicals (Houston, Texas, USA).

    Techniques: Infection, Giemsa Stain

    Afatinib inhibits the intracellular growth of the T. gondii RH strain in a dose-dependent manner. The number of parasites was counted per PVM after Giemsa staining after treatment with Afatinib 1, 5, and 10.0 μM concentrations. DMSO was used as the negative vehicle control, whereas pyrimethamine 5.0 μM was used as the positive control.

    Journal: The Korean Journal of Parasitology

    Article Title: Afatinib Reduces STAT6 Signaling of Host ARPE-19 Cells Infected with Toxoplasma gondii

    doi: 10.3347/kjp.2016.54.1.31

    Figure Lengend Snippet: Afatinib inhibits the intracellular growth of the T. gondii RH strain in a dose-dependent manner. The number of parasites was counted per PVM after Giemsa staining after treatment with Afatinib 1, 5, and 10.0 μM concentrations. DMSO was used as the negative vehicle control, whereas pyrimethamine 5.0 μM was used as the positive control.

    Article Snippet: Drugs and antibodies Afatinib (BIBW2992) and Erlotinib (OSI-420) were purchased from Selleck Chemicals (Houston, Texas, USA).

    Techniques: Staining, Positive Control

    CX3CL1/CX3CR1 activates the Src/FAK pathway. (A) VCaP cells were treated either with or without CX3CL1 (100 nM) for 5, 15, 30, 45 or 60 min, and the expression of Src, p-Src (Tyr416), FAK and p-FAK (Tyr576/577) was determined by western blotting. (B) VCaP cells were treated either with or without CX3CL1 (100 nM) for 5, 15, 30, 45 or 60 min, and the expression of EGFP, p-EGFR (Tyr992), PTK2B and p-PTK2B (Tyr402) was measured by western blotting. The expression of EGFR, p-EGFR (Tyr992), Src, p-Src (Tyr416), FAK and p-FAK (Tyr576/577) was measured by western blotting in CX3CL1-treated (100 nM), stable CX3CR1-overexpressing or siRNA-induced CX3CR1-knockdown cells: (C) VCaP; (D) PC-3. PC-3 cells were pretreated with (E) bosutinib (0.5 nM for 3 h) or with (F) PF-562271 (0.2 nM for 0.5 h), following which CX3CL1 was added to cells (100 nM for 5, 15, 30, 45 or 60 min), and the expression of p-Src (Tyr416) and p-FAK (Tyr576/577) was examined. VCaP cells were pretreated with either afatinib (1 or 10 µ M for 4 h) or bosutinib (0.25 or 2 µ M for 1 h), following which CX3CL1 (100 nM) was added to cells (100 nM) for 30 min, and the expression of (G) Src, p-Src (Tyr416), (H) EGFR and p-EGFR (Tyr992) were detected by western blotting. (I and J) The expression of MMP-9 and ROCK1 was measured by western blotting CX3CL1-treated (100 nM), stable CX3CR1-overexpressing or siRNA-induced CX3CR1-knockdown cells: (I) VCaP; (J) PC-3. Src, proto-oncogene tyrosine-protein kinase Src; FAK, focal adhesion kinase; EGFR, epidermal growth factor receptor; p, phosphorylated; OE, overexpression; NC, negative control; KD, knockdown; CON, control; CX3CL1, C-X3-C motif chemokine ligand 1; PTK2B, protein-tyrosine kinase 2-β; CX3CR1, C-X3-C motif chemokine receptor 1; MMP-9, matrix metal-loproteinase-9; ROCK1, Rho-associated protein kinase 1.

    Journal: International Journal of Oncology

    Article Title: CX3CL1/fractalkine enhances prostate cancer spinal metastasis by activating the Src/FAK pathway

    doi: 10.3892/ijo.2018.4487

    Figure Lengend Snippet: CX3CL1/CX3CR1 activates the Src/FAK pathway. (A) VCaP cells were treated either with or without CX3CL1 (100 nM) for 5, 15, 30, 45 or 60 min, and the expression of Src, p-Src (Tyr416), FAK and p-FAK (Tyr576/577) was determined by western blotting. (B) VCaP cells were treated either with or without CX3CL1 (100 nM) for 5, 15, 30, 45 or 60 min, and the expression of EGFP, p-EGFR (Tyr992), PTK2B and p-PTK2B (Tyr402) was measured by western blotting. The expression of EGFR, p-EGFR (Tyr992), Src, p-Src (Tyr416), FAK and p-FAK (Tyr576/577) was measured by western blotting in CX3CL1-treated (100 nM), stable CX3CR1-overexpressing or siRNA-induced CX3CR1-knockdown cells: (C) VCaP; (D) PC-3. PC-3 cells were pretreated with (E) bosutinib (0.5 nM for 3 h) or with (F) PF-562271 (0.2 nM for 0.5 h), following which CX3CL1 was added to cells (100 nM for 5, 15, 30, 45 or 60 min), and the expression of p-Src (Tyr416) and p-FAK (Tyr576/577) was examined. VCaP cells were pretreated with either afatinib (1 or 10 µ M for 4 h) or bosutinib (0.25 or 2 µ M for 1 h), following which CX3CL1 (100 nM) was added to cells (100 nM) for 30 min, and the expression of (G) Src, p-Src (Tyr416), (H) EGFR and p-EGFR (Tyr992) were detected by western blotting. (I and J) The expression of MMP-9 and ROCK1 was measured by western blotting CX3CL1-treated (100 nM), stable CX3CR1-overexpressing or siRNA-induced CX3CR1-knockdown cells: (I) VCaP; (J) PC-3. Src, proto-oncogene tyrosine-protein kinase Src; FAK, focal adhesion kinase; EGFR, epidermal growth factor receptor; p, phosphorylated; OE, overexpression; NC, negative control; KD, knockdown; CON, control; CX3CL1, C-X3-C motif chemokine ligand 1; PTK2B, protein-tyrosine kinase 2-β; CX3CR1, C-X3-C motif chemokine receptor 1; MMP-9, matrix metal-loproteinase-9; ROCK1, Rho-associated protein kinase 1.

    Article Snippet: Additionally, when EGFR inhibitor afatinib, Src inhibitor bosutinib, and FAK inhibitor PF-562271 (Selleck Chemicals, Houston, TX, USA) were used, the cells were pretreated in accordance with previous studies ( – ).

    Techniques: Expressing, Western Blot, Over Expression, Negative Control

    Quantifying life and death during real-time imaging of drug response. ( A ) Time-lapse XYZT acquisitions were visualized using color-coded depth projections and mitotic and apoptotic analysis were manually marked. ( B ) Increases (mitoses) and decreases (apoptosis) in cell numbers were chronologically ranked, thus reconstructing organoid size evolution in time. ( C ) Separate contributions of mitoses and apoptosis to development of P18T and P18T-KRAS G12D while incubated with vehicle (−) or afatinib + selumetinib (a+s). Whereas in P18T drug treatment (a+s) results in both proliferation block and apoptosis induction, P18T-KRAS G12D only show reduced proliferation but unchanged apoptosis rates. *p

    Journal: eLife

    Article Title: Targeting mutant RAS in patient-derived colorectal cancer organoids by combinatorial drug screening

    doi: 10.7554/eLife.18489

    Figure Lengend Snippet: Quantifying life and death during real-time imaging of drug response. ( A ) Time-lapse XYZT acquisitions were visualized using color-coded depth projections and mitotic and apoptotic analysis were manually marked. ( B ) Increases (mitoses) and decreases (apoptosis) in cell numbers were chronologically ranked, thus reconstructing organoid size evolution in time. ( C ) Separate contributions of mitoses and apoptosis to development of P18T and P18T-KRAS G12D while incubated with vehicle (−) or afatinib + selumetinib (a+s). Whereas in P18T drug treatment (a+s) results in both proliferation block and apoptosis induction, P18T-KRAS G12D only show reduced proliferation but unchanged apoptosis rates. *p

    Article Snippet: Targeted inhibitors Afatinib, Dacomitinib, Lapatinib, Selumetinib, Trametinib, BYL719, MK2206 and GDC-0994, navitoclax and venetoclax were purchased from Selleck Chemicals.

    Techniques: Imaging, Incubation, Blocking Assay

    Dose-response curves of patient-derived CRC organoids P18T-KRAS treated with different combination therapies against the EGFR-RAS-ERK pathway with addition of BCL2/BCLXL inhibitor navitoclax and the corresponding mono and dual therapy controls. Top panel: EGFR-RAS-ERK pathway inhibition with dual EGFR/HER2 inhibitor afatinib and MEK inhibitor trametinib. Lower panel: EGFR-RAS-ERK pathway inhibition with MEK inhibitor selumetinib and ERK inhibitor SCH772984. Cell viability was measured by an ATP-based assay after 72 hr of drug treatment. Inhibition of the EGFR-RAS-ERK pathway using fixed high concentrations of inhibitors against EGFR-RAS-MEK pathway effectors strongly sensitizes for navitoclax-induced reduction of cellular viability (black line). Horizontal colored-dashed lines represent the level of cellular viability of the respective anchor treatments without navitoclax. The representative images of the combined anchor treatments with and without a low dose of navitoclax are depicted on the right. Note that organoids remain viable with only robust EGFR-RAS-ERK inhibition. Dose-response curves are averages of n = 2. DOI: http://dx.doi.org/10.7554/eLife.18489.037

    Journal: eLife

    Article Title: Targeting mutant RAS in patient-derived colorectal cancer organoids by combinatorial drug screening

    doi: 10.7554/eLife.18489

    Figure Lengend Snippet: Dose-response curves of patient-derived CRC organoids P18T-KRAS treated with different combination therapies against the EGFR-RAS-ERK pathway with addition of BCL2/BCLXL inhibitor navitoclax and the corresponding mono and dual therapy controls. Top panel: EGFR-RAS-ERK pathway inhibition with dual EGFR/HER2 inhibitor afatinib and MEK inhibitor trametinib. Lower panel: EGFR-RAS-ERK pathway inhibition with MEK inhibitor selumetinib and ERK inhibitor SCH772984. Cell viability was measured by an ATP-based assay after 72 hr of drug treatment. Inhibition of the EGFR-RAS-ERK pathway using fixed high concentrations of inhibitors against EGFR-RAS-MEK pathway effectors strongly sensitizes for navitoclax-induced reduction of cellular viability (black line). Horizontal colored-dashed lines represent the level of cellular viability of the respective anchor treatments without navitoclax. The representative images of the combined anchor treatments with and without a low dose of navitoclax are depicted on the right. Note that organoids remain viable with only robust EGFR-RAS-ERK inhibition. Dose-response curves are averages of n = 2. DOI: http://dx.doi.org/10.7554/eLife.18489.037

    Article Snippet: Targeted inhibitors Afatinib, Dacomitinib, Lapatinib, Selumetinib, Trametinib, BYL719, MK2206 and GDC-0994, navitoclax and venetoclax were purchased from Selleck Chemicals.

    Techniques: Derivative Assay, Inhibition, ATP Assay

    Stills from representative time-lapse imaging (three days) of CRC organoids P18T and P18T-KRAS G12D treated with vehicle (DMSO) or a combination of targeted inhibitors afatinib (33 nM) and selumetinib (200 nM) (see also Video 2 ). In every panel, upper images show color-coded depth of maximum-projected z-stacks of H2B-mNeonGreen fluorescent organoids. Lower images: corresponding transmitted light images. Time interval: 15 min. Scale bars, 20 µm. Representative time-lapse of five organoids per condition. DOI: http://dx.doi.org/10.7554/eLife.18489.014

    Journal: eLife

    Article Title: Targeting mutant RAS in patient-derived colorectal cancer organoids by combinatorial drug screening

    doi: 10.7554/eLife.18489

    Figure Lengend Snippet: Stills from representative time-lapse imaging (three days) of CRC organoids P18T and P18T-KRAS G12D treated with vehicle (DMSO) or a combination of targeted inhibitors afatinib (33 nM) and selumetinib (200 nM) (see also Video 2 ). In every panel, upper images show color-coded depth of maximum-projected z-stacks of H2B-mNeonGreen fluorescent organoids. Lower images: corresponding transmitted light images. Time interval: 15 min. Scale bars, 20 µm. Representative time-lapse of five organoids per condition. DOI: http://dx.doi.org/10.7554/eLife.18489.014

    Article Snippet: Targeted inhibitors Afatinib, Dacomitinib, Lapatinib, Selumetinib, Trametinib, BYL719, MK2206 and GDC-0994, navitoclax and venetoclax were purchased from Selleck Chemicals.

    Techniques: Imaging

    Dose-response curves of patient-derived KRAS mutant CRC organoids P9T and P26T treated with the dual EGFR/HER2 inhibitor afatinib, MEK inhibitor selumetinib, BCL2/BCLXL inhibitor navitoclax or a combination thereof. Cell viability was measured by an ATP-based assay after 72 hr of drug treatment. Inhibition of the EGFR-RAS-ERK pathway using high concentrations for both afatinib and selumetinib (1 µM) strongly sensitizes for navitoclax-induced reduction of cellular viability (black line). Horizontal colored-dashed lines represent the level of cellular viability of the respective anchor treatments without navitoclax. Representative images of the combined anchor treatments with and without a low dose of navitoclax are depicted on the right. Note that organoids remain viable with only robust EGFR-RAS-ERK inhibition. Dose-response curves are averages of n = 2. DOI: http://dx.doi.org/10.7554/eLife.18489.036

    Journal: eLife

    Article Title: Targeting mutant RAS in patient-derived colorectal cancer organoids by combinatorial drug screening

    doi: 10.7554/eLife.18489

    Figure Lengend Snippet: Dose-response curves of patient-derived KRAS mutant CRC organoids P9T and P26T treated with the dual EGFR/HER2 inhibitor afatinib, MEK inhibitor selumetinib, BCL2/BCLXL inhibitor navitoclax or a combination thereof. Cell viability was measured by an ATP-based assay after 72 hr of drug treatment. Inhibition of the EGFR-RAS-ERK pathway using high concentrations for both afatinib and selumetinib (1 µM) strongly sensitizes for navitoclax-induced reduction of cellular viability (black line). Horizontal colored-dashed lines represent the level of cellular viability of the respective anchor treatments without navitoclax. Representative images of the combined anchor treatments with and without a low dose of navitoclax are depicted on the right. Note that organoids remain viable with only robust EGFR-RAS-ERK inhibition. Dose-response curves are averages of n = 2. DOI: http://dx.doi.org/10.7554/eLife.18489.036

    Article Snippet: Targeted inhibitors Afatinib, Dacomitinib, Lapatinib, Selumetinib, Trametinib, BYL719, MK2206 and GDC-0994, navitoclax and venetoclax were purchased from Selleck Chemicals.

    Techniques: Derivative Assay, Mutagenesis, ATP Assay, Inhibition

    Drug response of P18T-KRAS G12D and P26T CRC organoids examined by Western bot after 24 hr. Most effective reduction of p-ERK is detected when the organoids were treated with high concentrations of inhibitors afatinib (1 μM) and selumetinib (1 μM) and not at lower concentrations (65 nM of both drugs or 33 nM afatinib + 200 nM of selumetinib). DOI: http://dx.doi.org/10.7554/eLife.18489.038

    Journal: eLife

    Article Title: Targeting mutant RAS in patient-derived colorectal cancer organoids by combinatorial drug screening

    doi: 10.7554/eLife.18489

    Figure Lengend Snippet: Drug response of P18T-KRAS G12D and P26T CRC organoids examined by Western bot after 24 hr. Most effective reduction of p-ERK is detected when the organoids were treated with high concentrations of inhibitors afatinib (1 μM) and selumetinib (1 μM) and not at lower concentrations (65 nM of both drugs or 33 nM afatinib + 200 nM of selumetinib). DOI: http://dx.doi.org/10.7554/eLife.18489.038

    Article Snippet: Targeted inhibitors Afatinib, Dacomitinib, Lapatinib, Selumetinib, Trametinib, BYL719, MK2206 and GDC-0994, navitoclax and venetoclax were purchased from Selleck Chemicals.

    Techniques: Western Blot

    Drug response of CRC organoids as examined by Western blot. Combined Pan-HER/ MEK inhibition results in reduction of ERK phosphorylation in KRAS WT and KRAS G12D CRC organoids. Organoids were treated for 24 hr with MEK inhibitors selumetinib (1 μM), trametinib (10 nM), and the pan-HER inhibitor afatinib (1 μM) as indicated. WB is representative of four independent experiments. DOI: http://dx.doi.org/10.7554/eLife.18489.013

    Journal: eLife

    Article Title: Targeting mutant RAS in patient-derived colorectal cancer organoids by combinatorial drug screening

    doi: 10.7554/eLife.18489

    Figure Lengend Snippet: Drug response of CRC organoids as examined by Western blot. Combined Pan-HER/ MEK inhibition results in reduction of ERK phosphorylation in KRAS WT and KRAS G12D CRC organoids. Organoids were treated for 24 hr with MEK inhibitors selumetinib (1 μM), trametinib (10 nM), and the pan-HER inhibitor afatinib (1 μM) as indicated. WB is representative of four independent experiments. DOI: http://dx.doi.org/10.7554/eLife.18489.013

    Article Snippet: Targeted inhibitors Afatinib, Dacomitinib, Lapatinib, Selumetinib, Trametinib, BYL719, MK2206 and GDC-0994, navitoclax and venetoclax were purchased from Selleck Chemicals.

    Techniques: Western Blot, Inhibition

    Stills from representative time-lapse imaging (three days) of CRC organoids P8T and P26T treated with vehicle (DMSO) or a combination of targeted inhibitors afatinib (33 nM) and selumetinib (200 nM) (see also Video 2 ). In every panel, upper images show color-coded depth of maximum-projected z-stacks of H2B-mNeonGreen fluorescent organoids. Lower images: corresponding transmitted light images. Time interval: 15 min. Scale bars, 20 µm. Representative time-lapse of five organoids per condition. DOI: http://dx.doi.org/10.7554/eLife.18489.005

    Journal: eLife

    Article Title: Targeting mutant RAS in patient-derived colorectal cancer organoids by combinatorial drug screening

    doi: 10.7554/eLife.18489

    Figure Lengend Snippet: Stills from representative time-lapse imaging (three days) of CRC organoids P8T and P26T treated with vehicle (DMSO) or a combination of targeted inhibitors afatinib (33 nM) and selumetinib (200 nM) (see also Video 2 ). In every panel, upper images show color-coded depth of maximum-projected z-stacks of H2B-mNeonGreen fluorescent organoids. Lower images: corresponding transmitted light images. Time interval: 15 min. Scale bars, 20 µm. Representative time-lapse of five organoids per condition. DOI: http://dx.doi.org/10.7554/eLife.18489.005

    Article Snippet: Targeted inhibitors Afatinib, Dacomitinib, Lapatinib, Selumetinib, Trametinib, BYL719, MK2206 and GDC-0994, navitoclax and venetoclax were purchased from Selleck Chemicals.

    Techniques: Imaging

    Average growth speeds of the organoids were determined by linear fitting of the traces shown in Figure 8D . The two time frames roughly correspond to the first half and the second half of the experiment. Directly after drug removal, afatinib- and selumetinib-treated organoids show a significant reduction in growth speed as compared to vehicle-treated organoids. After 22–24 hr of recovery, growth rates return to the level of vehicle-treated organoids (possibly even slightly faster). *p

    Journal: eLife

    Article Title: Targeting mutant RAS in patient-derived colorectal cancer organoids by combinatorial drug screening

    doi: 10.7554/eLife.18489

    Figure Lengend Snippet: Average growth speeds of the organoids were determined by linear fitting of the traces shown in Figure 8D . The two time frames roughly correspond to the first half and the second half of the experiment. Directly after drug removal, afatinib- and selumetinib-treated organoids show a significant reduction in growth speed as compared to vehicle-treated organoids. After 22–24 hr of recovery, growth rates return to the level of vehicle-treated organoids (possibly even slightly faster). *p

    Article Snippet: Targeted inhibitors Afatinib, Dacomitinib, Lapatinib, Selumetinib, Trametinib, BYL719, MK2206 and GDC-0994, navitoclax and venetoclax were purchased from Selleck Chemicals.

    Techniques:

    Secondary organoid cultures were derived from xenografted P26T tumors (organoid-derived xenograft, ODX) of mice that have been treated with vehicle, afatinib (12,5 mg/kg; five days on, two days off), selumetinib (20 mg/kg; five days on, two days off) or both. Dose response curves were determined from these secondary post-xenograft cultures, as well as from the parental P26T organoid culture. Regardless of the in vivo applied drug treatment, the drug sensitivity phenotype of the organoids remained unaltered. DOI: http://dx.doi.org/10.7554/eLife.18489.009

    Journal: eLife

    Article Title: Targeting mutant RAS in patient-derived colorectal cancer organoids by combinatorial drug screening

    doi: 10.7554/eLife.18489

    Figure Lengend Snippet: Secondary organoid cultures were derived from xenografted P26T tumors (organoid-derived xenograft, ODX) of mice that have been treated with vehicle, afatinib (12,5 mg/kg; five days on, two days off), selumetinib (20 mg/kg; five days on, two days off) or both. Dose response curves were determined from these secondary post-xenograft cultures, as well as from the parental P26T organoid culture. Regardless of the in vivo applied drug treatment, the drug sensitivity phenotype of the organoids remained unaltered. DOI: http://dx.doi.org/10.7554/eLife.18489.009

    Article Snippet: Targeted inhibitors Afatinib, Dacomitinib, Lapatinib, Selumetinib, Trametinib, BYL719, MK2206 and GDC-0994, navitoclax and venetoclax were purchased from Selleck Chemicals.

    Techniques: Derivative Assay, Mouse Assay, In Vivo

    ERBB2 extracellular domain mutant cervical cancers are sensitive to ERBB2 inhibition by afatinib or neratinib in vitro and in vivo. ( A and B ) Mean IC 50 values of ERBB2-mutated cervical carcinoma (CC) compared to ERBB2 wild-type CC cell lines ( P

    Journal: Proceedings of the National Academy of Sciences of the United States of America

    Article Title: Whole-exome sequencing of cervical carcinomas identifies activating ERBB2 and PIK3CA mutations as targets for combination therapy

    doi: 10.1073/pnas.1911385116

    Figure Lengend Snippet: ERBB2 extracellular domain mutant cervical cancers are sensitive to ERBB2 inhibition by afatinib or neratinib in vitro and in vivo. ( A and B ) Mean IC 50 values of ERBB2-mutated cervical carcinoma (CC) compared to ERBB2 wild-type CC cell lines ( P

    Article Snippet: Since 5.8% of our cervical tumors (including 2 primary cell lines) ( ) harbored ERBB2 extracellular domain mutations, we evaluated the effect of afatinib (Boehringer Ingelheim GmbH, Ingelheim, Germany) and neratinib (Puma Biotechnologies, Los Angeles, CA) on cell growth, cell cycle distribution and signaling in 8 fully sequenced primary cell lines.

    Techniques: Mutagenesis, Inhibition, In Vitro, In Vivo

    Targeted inhibition of EGFR and CDK4/6. A and B, cell titer blue viability assays for lapatinib (Lap), afatinib (Afa), palbociclib (Pal), and combination treatment (Combo). Coefficient of interaction values at different effective doses for the combination

    Journal: Molecular cancer therapeutics

    Article Title: EGFR and RB1 as Dual Biomarkers in HPV-Negative Head and Neck Cancer

    doi: 10.1158/1535-7163.MCT-16-0243

    Figure Lengend Snippet: Targeted inhibition of EGFR and CDK4/6. A and B, cell titer blue viability assays for lapatinib (Lap), afatinib (Afa), palbociclib (Pal), and combination treatment (Combo). Coefficient of interaction values at different effective doses for the combination

    Article Snippet: After 24 hours, serially diluted concentrations of lapatinib or afatinib (A-8644, LC Laboratories), palbociclib, or combinations of palbociclib with lapatinib or afatinib (1:1 ratio) were added to cells.

    Techniques: Inhibition

    Afatinib induced cell apoptosis and proliferation arrest in ICC cell lines. Cell viability in JCK and OZ cells treated with (A) different afatinib doses and (B) for different durations, showing that afatinib blocked the cell growth in a time- and dose-dependent manner. (C) Flow cytometry using TUNEL-staining, and (D) cell apoptosis results, demonstrating that afatinib notably induced ICC cell apoptosis. (E) Western blots and (F) quantified protein levels, revealing that afatinib significantly inhibited pEGFR and pSTAT3 (n=3). *P

    Journal: Experimental and Therapeutic Medicine

    Article Title: Blocking of the EGFR-STAT3 signaling pathway through afatinib treatment inhibited the intrahepatic cholangiocarcinoma

    doi: 10.3892/etm.2018.6038

    Figure Lengend Snippet: Afatinib induced cell apoptosis and proliferation arrest in ICC cell lines. Cell viability in JCK and OZ cells treated with (A) different afatinib doses and (B) for different durations, showing that afatinib blocked the cell growth in a time- and dose-dependent manner. (C) Flow cytometry using TUNEL-staining, and (D) cell apoptosis results, demonstrating that afatinib notably induced ICC cell apoptosis. (E) Western blots and (F) quantified protein levels, revealing that afatinib significantly inhibited pEGFR and pSTAT3 (n=3). *P

    Article Snippet: Afatinib (cat. no. SC-364398) was obtained from Santa Cruz Biotechnology, Inc. (Santa Cruz, CA, USA).

    Techniques: Immunocytochemistry, Flow Cytometry, Cytometry, TUNEL Assay, Staining, Western Blot

    ( A ) HCC1419 cells were treated with varying concentrations of either neratinib or afatinib for 1 week. Cells were then fixed and stained for SA-β-gal activity and compared to untreated control and lapatinib treated cells. ( B ) HCC1419 cells were treated with 10 µg/mL trastuzumab alone or in combination with 250 nM lapatinib for 1 week, prior to SA-β-gal activity testing. ( C ) SKBR3 cells were treated with 10 µg/mL trastuzumab alone or in combination with 250 nM lapatinib for 2 weeks, prior to SA-β-gal activity testing. Images taken at 400× magnification.

    Journal: Cancers

    Article Title: HER2-Targeted Tyrosine Kinase Inhibitors Cause Therapy-Induced-Senescence in Breast Cancer Cells

    doi: 10.3390/cancers11020197

    Figure Lengend Snippet: ( A ) HCC1419 cells were treated with varying concentrations of either neratinib or afatinib for 1 week. Cells were then fixed and stained for SA-β-gal activity and compared to untreated control and lapatinib treated cells. ( B ) HCC1419 cells were treated with 10 µg/mL trastuzumab alone or in combination with 250 nM lapatinib for 1 week, prior to SA-β-gal activity testing. ( C ) SKBR3 cells were treated with 10 µg/mL trastuzumab alone or in combination with 250 nM lapatinib for 2 weeks, prior to SA-β-gal activity testing. Images taken at 400× magnification.

    Article Snippet: Solutions (10 mM) of lapatinib, neratinib and afatinib (Sequoia Research Products, Pangbourne, United Kingdom) and pifithrin (Sigma, St. Louis, MO, USA) were prepared in dimethyl sulfoxide (Sigma) and APR-246 (Tocris Bioscience, Bristol, United Kingdom) was prepared in sterile dH2 O. Trastuzumab was obtained from St. Vincent’s University Hospital, Dublin, Ireland.

    Techniques: Staining, Activity Assay

    ( A ) HCC1419 cells were treated with 250 nM for 1 week, EFM-192A and SKBR3 cells were treated with 250 nM for 2 weeks and MDA-MB-361 cells were treated with 500 nM lapatinib for 4 weeks. Senescent cells were then cultured in the absence of lapatinib for 1 week prior to SA-β-gal activity testing. ( B ) HCC1419 cells were treated with the indicated concentrations of lapatinib, afatinib or neratinib for 1 week; the cells were then cultured in the absence of drug for two weeks prior to SA-β-gal activity testing. Images taken at 400× magnification.

    Journal: Cancers

    Article Title: HER2-Targeted Tyrosine Kinase Inhibitors Cause Therapy-Induced-Senescence in Breast Cancer Cells

    doi: 10.3390/cancers11020197

    Figure Lengend Snippet: ( A ) HCC1419 cells were treated with 250 nM for 1 week, EFM-192A and SKBR3 cells were treated with 250 nM for 2 weeks and MDA-MB-361 cells were treated with 500 nM lapatinib for 4 weeks. Senescent cells were then cultured in the absence of lapatinib for 1 week prior to SA-β-gal activity testing. ( B ) HCC1419 cells were treated with the indicated concentrations of lapatinib, afatinib or neratinib for 1 week; the cells were then cultured in the absence of drug for two weeks prior to SA-β-gal activity testing. Images taken at 400× magnification.

    Article Snippet: Solutions (10 mM) of lapatinib, neratinib and afatinib (Sequoia Research Products, Pangbourne, United Kingdom) and pifithrin (Sigma, St. Louis, MO, USA) were prepared in dimethyl sulfoxide (Sigma) and APR-246 (Tocris Bioscience, Bristol, United Kingdom) was prepared in sterile dH2 O. Trastuzumab was obtained from St. Vincent’s University Hospital, Dublin, Ireland.

    Techniques: Multiple Displacement Amplification, Cell Culture, Activity Assay

    Effects of Afatinib on apoptotic response and DNA damage repair in irradiated PC-9-GR cells (A). Apoptosis analysis. Cells were treated with afatinib (350.0 nM), IR (6Gy) or afatinib for two hours followed by IR. Apoptosis was determined 36 hours later after IR with flow cytometry analysis. (B). Effect of pan-caspase inhibitor z-VAD-fmk on afatinib-enhanced apoptotic response in irradiated PC-9-GR cells. Cells were pretreated with 20 M z-VAD-fmk, followed by indicated treatments. Apoptosis was determined with flow cytometry analysis. Diagram showing the changes of early, late and total apoptosis in these experiments. (C). Immunofluorescence analysis. Immunofluorescence analyses were performed to examine the formation and changes of nuclear γ-H2AX foci. Representative images of nuclear γ-H2AX foci in PC-9-GR cells treated with IR or the combination are shown in top panel, and diagram showing the change in the cell fractions with positive γ-H2AX foci are present in bottom panel. Data represent the average of three experiments. Error bars indicate standard deviation. * indicates statistical significance. (D). Effect of afatinib on DNA-pKcs expression. Cell lysates were collected from PC-9-GR cells treated with afatinib, IR or the combination for twenty-four hours. Expression of DNA-pKcs protein was examined with western blot assay. Anti-GAPDH antibody was included as a loading control. (E). Clonogenic survival assay. PC-9-GR cells were transiently transfected with DNA-pKcs siRNA, or scramble-siRNA as control. Clonogenic survival assay was performed as described above. Western blot results showing the inhibitory effect of siRNAs on DNA-pKcs protein in cells collected 72 hours after transfections.

    Journal: Oncotarget

    Article Title: Afatinib increases sensitivity to radiation in non-small cell lung cancer cells with acquired EGFR T790M mutation

    doi:

    Figure Lengend Snippet: Effects of Afatinib on apoptotic response and DNA damage repair in irradiated PC-9-GR cells (A). Apoptosis analysis. Cells were treated with afatinib (350.0 nM), IR (6Gy) or afatinib for two hours followed by IR. Apoptosis was determined 36 hours later after IR with flow cytometry analysis. (B). Effect of pan-caspase inhibitor z-VAD-fmk on afatinib-enhanced apoptotic response in irradiated PC-9-GR cells. Cells were pretreated with 20 M z-VAD-fmk, followed by indicated treatments. Apoptosis was determined with flow cytometry analysis. Diagram showing the changes of early, late and total apoptosis in these experiments. (C). Immunofluorescence analysis. Immunofluorescence analyses were performed to examine the formation and changes of nuclear γ-H2AX foci. Representative images of nuclear γ-H2AX foci in PC-9-GR cells treated with IR or the combination are shown in top panel, and diagram showing the change in the cell fractions with positive γ-H2AX foci are present in bottom panel. Data represent the average of three experiments. Error bars indicate standard deviation. * indicates statistical significance. (D). Effect of afatinib on DNA-pKcs expression. Cell lysates were collected from PC-9-GR cells treated with afatinib, IR or the combination for twenty-four hours. Expression of DNA-pKcs protein was examined with western blot assay. Anti-GAPDH antibody was included as a loading control. (E). Clonogenic survival assay. PC-9-GR cells were transiently transfected with DNA-pKcs siRNA, or scramble-siRNA as control. Clonogenic survival assay was performed as described above. Western blot results showing the inhibitory effect of siRNAs on DNA-pKcs protein in cells collected 72 hours after transfections.

    Article Snippet: Afatinib (Cayman, San Diego, CA, USA) was dissolved in dimethyl sulfoxide (DMSO).

    Techniques: Irradiation, Flow Cytometry, Cytometry, Immunofluorescence, Standard Deviation, Expressing, Western Blot, Clonogenic Cell Survival Assay, Transfection

    Effect of afatinib on cell growth in NSCLC PC-9 (A), PC-9-GR (B), H1975 (C) and H460 (D). Cells were treated with afatinib at indicated concentrations, and cell viabilities were determined using MTS assay. Data represent the average of three experiments. Error bars indicate standard deviation.

    Journal: Oncotarget

    Article Title: Afatinib increases sensitivity to radiation in non-small cell lung cancer cells with acquired EGFR T790M mutation

    doi:

    Figure Lengend Snippet: Effect of afatinib on cell growth in NSCLC PC-9 (A), PC-9-GR (B), H1975 (C) and H460 (D). Cells were treated with afatinib at indicated concentrations, and cell viabilities were determined using MTS assay. Data represent the average of three experiments. Error bars indicate standard deviation.

    Article Snippet: Afatinib (Cayman, San Diego, CA, USA) was dissolved in dimethyl sulfoxide (DMSO).

    Techniques: MTS Assay, Standard Deviation

    Effect of afatinib on cell clonogenic survival in irradiated lung cancer cells PC-9 (A), PC-9-GR (B), H1975 (C) and H460 (D) cells were pre-treated with afatinib for 2 hours and then irradiated with indicated doses. Clonogenic survival assays were performed as described in Materials and Methods. Data represent the average of three experiments. Error bars indicate standard deviation.

    Journal: Oncotarget

    Article Title: Afatinib increases sensitivity to radiation in non-small cell lung cancer cells with acquired EGFR T790M mutation

    doi:

    Figure Lengend Snippet: Effect of afatinib on cell clonogenic survival in irradiated lung cancer cells PC-9 (A), PC-9-GR (B), H1975 (C) and H460 (D) cells were pre-treated with afatinib for 2 hours and then irradiated with indicated doses. Clonogenic survival assays were performed as described in Materials and Methods. Data represent the average of three experiments. Error bars indicate standard deviation.

    Article Snippet: Afatinib (Cayman, San Diego, CA, USA) was dissolved in dimethyl sulfoxide (DMSO).

    Techniques: Irradiation, Standard Deviation

    Effects of afatinib on protein phosphorylations (A-C). Western blot analysis. Cells were pretreated with afatinib for two hours followed by IR. Total cell lysates were collected from PC-9 (A), PC-9-GR (B) and H1975 (C) cells after indicated treatments, and analyzed for phosphorylations of EGFR, AKT and ERK proteins. Anti-GAPDH antibody was included as a loading control. (D). Quantitative analysis for the changes of protein phosphorylations. Densitometry for western blot signal for samples collected two hours post-treatments was conducted, and intensity for the targeted protein/modification was normalized to corresponding GAPDH. Data represent the average results from three independent experiments. (E). RTK array. Cell lysates were collected two hours after IR treatment or IR combined with afatinib, relative phosphorylation level of human receptor tyrosine kinases were determined with Phospho-RTK Array assay as described in Material and Methods.

    Journal: Oncotarget

    Article Title: Afatinib increases sensitivity to radiation in non-small cell lung cancer cells with acquired EGFR T790M mutation

    doi:

    Figure Lengend Snippet: Effects of afatinib on protein phosphorylations (A-C). Western blot analysis. Cells were pretreated with afatinib for two hours followed by IR. Total cell lysates were collected from PC-9 (A), PC-9-GR (B) and H1975 (C) cells after indicated treatments, and analyzed for phosphorylations of EGFR, AKT and ERK proteins. Anti-GAPDH antibody was included as a loading control. (D). Quantitative analysis for the changes of protein phosphorylations. Densitometry for western blot signal for samples collected two hours post-treatments was conducted, and intensity for the targeted protein/modification was normalized to corresponding GAPDH. Data represent the average results from three independent experiments. (E). RTK array. Cell lysates were collected two hours after IR treatment or IR combined with afatinib, relative phosphorylation level of human receptor tyrosine kinases were determined with Phospho-RTK Array assay as described in Material and Methods.

    Article Snippet: Afatinib (Cayman, San Diego, CA, USA) was dissolved in dimethyl sulfoxide (DMSO).

    Techniques: Western Blot, Modification

    (A). Afatinib enhances tumor growth inhibition in response to IR treatment in PC-9-GR xenograft (A). Athymic nude mice bearing isogenic PC-9-GR xenograft tumors were treated with afatinib, IR or the combination. Tumor growth was measured as described in Materials and Methods. The growth curves represent the average values of 8 mice in each group. Error bars indicate standard deviation. (B). IHC staining. xenograft tumors tissues were collected after 14 days of indicated treatments. Immunostaining was performed to test the changes of EGFR phosphorylation, expressions of Ki67 and DNA-pKcs proteins, and presence of γ-H2A.X and apoptotic markers CC3. Quantified H-scores were determined for each group (n=3 animals/group). The scale bar represents 100μm and all images are to the same scale.

    Journal: Oncotarget

    Article Title: Afatinib increases sensitivity to radiation in non-small cell lung cancer cells with acquired EGFR T790M mutation

    doi:

    Figure Lengend Snippet: (A). Afatinib enhances tumor growth inhibition in response to IR treatment in PC-9-GR xenograft (A). Athymic nude mice bearing isogenic PC-9-GR xenograft tumors were treated with afatinib, IR or the combination. Tumor growth was measured as described in Materials and Methods. The growth curves represent the average values of 8 mice in each group. Error bars indicate standard deviation. (B). IHC staining. xenograft tumors tissues were collected after 14 days of indicated treatments. Immunostaining was performed to test the changes of EGFR phosphorylation, expressions of Ki67 and DNA-pKcs proteins, and presence of γ-H2A.X and apoptotic markers CC3. Quantified H-scores were determined for each group (n=3 animals/group). The scale bar represents 100μm and all images are to the same scale.

    Article Snippet: Afatinib (Cayman, San Diego, CA, USA) was dissolved in dimethyl sulfoxide (DMSO).

    Techniques: Inhibition, Mouse Assay, Standard Deviation, Immunohistochemistry, Staining, Immunostaining

    L861Q TaqMan assay (droplet digital PCR). 2D plots and amounts of L861Q-positive droplets (indicated in red) obtained via ddPCR with FFPE DNA ( a ) or cfDNA ( b to h ). a ) the primary tumour, b ) on the day of surgery (D0), c ) before concomitant chemotherapy (CT) and radiotherapy (RT) (pre-RT), d ) 1 month after RT (post-RT), e ) 1 month after the first cycle of CT (CT-1), f ) 1 month after four cycles of CT (CT-4), g ) after 9 weeks of afatinib treatment (post-TKI), and h ) after two cycles of lomustine with bevacizumab (Lom + Bev-2). The plasma DNA concentration is expressed as the amount per ml of plasma. ddPCR, droplet digital PCR; FFPE, formalin-fixed, paraffin-embedded; pDNA, plasma DNA; AU, arbitrary units; WT, wild type; CT, chemotherapy; RT, radiotherapy

    Journal: BMC Cancer

    Article Title: Combining genomic analyses with tumour-derived slice cultures for the characterization of an EGFR-activating kinase mutation in a case of glioblastoma

    doi: 10.1186/s12885-018-4873-9

    Figure Lengend Snippet: L861Q TaqMan assay (droplet digital PCR). 2D plots and amounts of L861Q-positive droplets (indicated in red) obtained via ddPCR with FFPE DNA ( a ) or cfDNA ( b to h ). a ) the primary tumour, b ) on the day of surgery (D0), c ) before concomitant chemotherapy (CT) and radiotherapy (RT) (pre-RT), d ) 1 month after RT (post-RT), e ) 1 month after the first cycle of CT (CT-1), f ) 1 month after four cycles of CT (CT-4), g ) after 9 weeks of afatinib treatment (post-TKI), and h ) after two cycles of lomustine with bevacizumab (Lom + Bev-2). The plasma DNA concentration is expressed as the amount per ml of plasma. ddPCR, droplet digital PCR; FFPE, formalin-fixed, paraffin-embedded; pDNA, plasma DNA; AU, arbitrary units; WT, wild type; CT, chemotherapy; RT, radiotherapy

    Article Snippet: Cetuximab (Merck-Serono), erlotinib, and afatinib (both from Euromedex) were selected on the basis of their activity against the EGFR.

    Techniques: TaqMan Assay, Digital PCR, Formalin-fixed Paraffin-Embedded, Concentration Assay

    Inhibition of cell proliferation and the EGFR pathway in tumour explants. a ) Microscope images (× 40) of Ki67 immunostaining on tumour slices from the patient’s glioblastoma for each condition: control (CTL), cetuximab (CET), afatinib (AFA), and erlotinib (ERL). b ) Visual quantification of the proportion of Ki67-positive cells. There were no statistically significant differences between CTL on one hand and CET ( p = 0.45), AFA ( p = 0.6) and ERL ( p = 0.37). c ) Immunoblot of tumour slices treated for 48 h under four conditions: CTL, CET, AFA and ERL. To investigate the drugs’ efficacy, antibodies against phospho-ERK (P-ERK) and ERK (ERK) were applied, and actin was used as a loading control. d ) Quantification of the P-ERK/ERK ratio, as a percentage of the control P-ERK/ERK ratio. There were no statistically significant differences between the negative control on one hand and cetuximab ( p = 0.36), afatinib ( p = 0.99) and erlotinib ( p = 0.59) on the other. Values are expressed as the mean ( n = 4) and the standard error of the mean (represented by error bars) . The scale bar corresponds to 25 μm

    Journal: BMC Cancer

    Article Title: Combining genomic analyses with tumour-derived slice cultures for the characterization of an EGFR-activating kinase mutation in a case of glioblastoma

    doi: 10.1186/s12885-018-4873-9

    Figure Lengend Snippet: Inhibition of cell proliferation and the EGFR pathway in tumour explants. a ) Microscope images (× 40) of Ki67 immunostaining on tumour slices from the patient’s glioblastoma for each condition: control (CTL), cetuximab (CET), afatinib (AFA), and erlotinib (ERL). b ) Visual quantification of the proportion of Ki67-positive cells. There were no statistically significant differences between CTL on one hand and CET ( p = 0.45), AFA ( p = 0.6) and ERL ( p = 0.37). c ) Immunoblot of tumour slices treated for 48 h under four conditions: CTL, CET, AFA and ERL. To investigate the drugs’ efficacy, antibodies against phospho-ERK (P-ERK) and ERK (ERK) were applied, and actin was used as a loading control. d ) Quantification of the P-ERK/ERK ratio, as a percentage of the control P-ERK/ERK ratio. There were no statistically significant differences between the negative control on one hand and cetuximab ( p = 0.36), afatinib ( p = 0.99) and erlotinib ( p = 0.59) on the other. Values are expressed as the mean ( n = 4) and the standard error of the mean (represented by error bars) . The scale bar corresponds to 25 μm

    Article Snippet: Cetuximab (Merck-Serono), erlotinib, and afatinib (both from Euromedex) were selected on the basis of their activity against the EGFR.

    Techniques: Inhibition, Microscopy, Immunostaining, CTL Assay, Negative Control

    Magnetic resonance imaging. Gadolinium-contrast-enhanced T1-weighted 3D magnetic resonance imaging of the brain after radiotherapy ( a ), before afatinib treatment ( b ), and after 4 weeks ( c ) and 8 weeks ( d ) of afatinib treatment. The lesion is indicated by an arrow

    Journal: BMC Cancer

    Article Title: Combining genomic analyses with tumour-derived slice cultures for the characterization of an EGFR-activating kinase mutation in a case of glioblastoma

    doi: 10.1186/s12885-018-4873-9

    Figure Lengend Snippet: Magnetic resonance imaging. Gadolinium-contrast-enhanced T1-weighted 3D magnetic resonance imaging of the brain after radiotherapy ( a ), before afatinib treatment ( b ), and after 4 weeks ( c ) and 8 weeks ( d ) of afatinib treatment. The lesion is indicated by an arrow

    Article Snippet: Cetuximab (Merck-Serono), erlotinib, and afatinib (both from Euromedex) were selected on the basis of their activity against the EGFR.

    Techniques: Magnetic Resonance Imaging

    Effects of afatinib treatment on apoptosis and the IL-6/JAK1/STAT3 signaling pathway in H1975 cells. (A) Cell viability was measured with the CCK-8 assay and shown by the absorbance at 450 nm following treatment with various concentrations of afatinib for 24 h. (B) Protein levels of IL-6 from the supernatant of H1975 cells treated with or without 1 µM afatinib were detected by ELISA. (C) Activation of the JAK1/STAT3 signaling pathway was detected in H1975 cells in the presence or absence of 1 µM afatinib by western blot. β-actin was included as an mRNA and protein loading control. Data are representative of three independent experiments and expressed as means ± SEM. **P

    Journal: Molecular Medicine Reports

    Article Title: Matrine increases the inhibitory effects of afatinib on H1975 cells via the IL-6/JAK1/STAT3 signaling pathway

    doi: 10.3892/mmr.2017.6865

    Figure Lengend Snippet: Effects of afatinib treatment on apoptosis and the IL-6/JAK1/STAT3 signaling pathway in H1975 cells. (A) Cell viability was measured with the CCK-8 assay and shown by the absorbance at 450 nm following treatment with various concentrations of afatinib for 24 h. (B) Protein levels of IL-6 from the supernatant of H1975 cells treated with or without 1 µM afatinib were detected by ELISA. (C) Activation of the JAK1/STAT3 signaling pathway was detected in H1975 cells in the presence or absence of 1 µM afatinib by western blot. β-actin was included as an mRNA and protein loading control. Data are representative of three independent experiments and expressed as means ± SEM. **P

    Article Snippet: Afatinib was purchased from SYNkinase (San Diego, CA, USA).

    Techniques: CCK-8 Assay, Enzyme-linked Immunosorbent Assay, Activation Assay, Western Blot

    Effects of matrine and afatinib treatment on the growth of H1975 cells in vitro . (A) Protein levels of IL-6 from the supernatant of H1975 cells treated with or without 1 µM afatinib in the presence or absence of 2 mM matrine were detected by ELISA. (B) Protein levels of p-JAK1 and p-STAT3 were detected by western blot prepared from H1975 cells incubated with 1 µM afatinib combined with 2 mM matrine. (C) Effects of of afatinib and matrine treatment on the growth of H1975 cells were measured with the CCK-8 assay and shown by the absorbance at 450 nm. β-actin was included as an mRNA and protein loading control. Data are representative of three independent experiments and expressed as means ± SEM. *P

    Journal: Molecular Medicine Reports

    Article Title: Matrine increases the inhibitory effects of afatinib on H1975 cells via the IL-6/JAK1/STAT3 signaling pathway

    doi: 10.3892/mmr.2017.6865

    Figure Lengend Snippet: Effects of matrine and afatinib treatment on the growth of H1975 cells in vitro . (A) Protein levels of IL-6 from the supernatant of H1975 cells treated with or without 1 µM afatinib in the presence or absence of 2 mM matrine were detected by ELISA. (B) Protein levels of p-JAK1 and p-STAT3 were detected by western blot prepared from H1975 cells incubated with 1 µM afatinib combined with 2 mM matrine. (C) Effects of of afatinib and matrine treatment on the growth of H1975 cells were measured with the CCK-8 assay and shown by the absorbance at 450 nm. β-actin was included as an mRNA and protein loading control. Data are representative of three independent experiments and expressed as means ± SEM. *P

    Article Snippet: Afatinib was purchased from SYNkinase (San Diego, CA, USA).

    Techniques: In Vitro, Enzyme-linked Immunosorbent Assay, Western Blot, Incubation, CCK-8 Assay

    Effects of matrine and afatinib treatment on the growth of H1975 cells in vivo . After 5×10 6 cells were injected subcutaneously under the back skin of 5-to-6-week-old male BALB/c nude mice, mice were treated with or without matrine (5 g/kg) in the presence or absence of afatinib (5 mg/kg) formulated in saline by intraperitoneal injection once daily for 4 weeks and tumor volume was estimated once every three days with the formula: volume = l × w 2 × 0.536, where l and w are perpendicular measured diameters. Data are expressed as means ± SEM. *P

    Journal: Molecular Medicine Reports

    Article Title: Matrine increases the inhibitory effects of afatinib on H1975 cells via the IL-6/JAK1/STAT3 signaling pathway

    doi: 10.3892/mmr.2017.6865

    Figure Lengend Snippet: Effects of matrine and afatinib treatment on the growth of H1975 cells in vivo . After 5×10 6 cells were injected subcutaneously under the back skin of 5-to-6-week-old male BALB/c nude mice, mice were treated with or without matrine (5 g/kg) in the presence or absence of afatinib (5 mg/kg) formulated in saline by intraperitoneal injection once daily for 4 weeks and tumor volume was estimated once every three days with the formula: volume = l × w 2 × 0.536, where l and w are perpendicular measured diameters. Data are expressed as means ± SEM. *P

    Article Snippet: Afatinib was purchased from SYNkinase (San Diego, CA, USA).

    Techniques: In Vivo, Injection, Mouse Assay

    Inhibition of TNF induces sensitivity of EGFRwt-expressing NSCLC cells to EGFR inhibition. ( A and B ) AlamarBlue cell viability assay in H441 or A549 cells. TNFR1 was silenced by siRNA transfection for 48 hours, and cells were exposed to erlotinib (1 μM) for 72 hours in RPMI-1640 with 5% FBS. ( C ) Silencing of TNFR1 was confirmed by Western blot. Western blot results are representative of at least 3 independent replicates. ( D and E ) Thalidomide sensitizes H441 and A549 cells to EGFR inhibition with erlotinib. Thalidomide (5 μg/ml) and erlotinib were added to H441 and A549 cells concurrently, and AlamarBlue assay was done after 72 hours. ( F and G ) A similar experiment was done using etanercept (100 μg/ml) and erlotinib in H441 and A549 cells. ( H ) H441 cells were treated with afatinib (1 μM) in the presence or absence of etanercept. AlamarBlue assay was conducted after 72 hours. ( I ) H441 cells were treated with afatinib and thalidomide for 72 hours, followed by AlamarBlue assay. ( J and K ) Similar experiments were done as described in H and I in A549 cells. ( L and M ) EGFRwt cells were seeded in 6-well plates at 1,000 cells per well, and incubated with 20 μg/ml thalidomide and/or 1 μM erlotinib. Fourteen days later, cell colonies were fixed by 100% methanol and then stained by 0.5% crystal violet in 25% methanol. Images were captured by a scanner, and colony counts were processed by ImageJ. Data represent the mean percentage of control ± SEM. n = 3 biologically independent experimental replicates ( A , B , and D – M ). * P

    Journal: The Journal of Clinical Investigation

    Article Title: TNF-driven adaptive response mediates resistance to EGFR inhibition in lung cancer

    doi: 10.1172/JCI96148

    Figure Lengend Snippet: Inhibition of TNF induces sensitivity of EGFRwt-expressing NSCLC cells to EGFR inhibition. ( A and B ) AlamarBlue cell viability assay in H441 or A549 cells. TNFR1 was silenced by siRNA transfection for 48 hours, and cells were exposed to erlotinib (1 μM) for 72 hours in RPMI-1640 with 5% FBS. ( C ) Silencing of TNFR1 was confirmed by Western blot. Western blot results are representative of at least 3 independent replicates. ( D and E ) Thalidomide sensitizes H441 and A549 cells to EGFR inhibition with erlotinib. Thalidomide (5 μg/ml) and erlotinib were added to H441 and A549 cells concurrently, and AlamarBlue assay was done after 72 hours. ( F and G ) A similar experiment was done using etanercept (100 μg/ml) and erlotinib in H441 and A549 cells. ( H ) H441 cells were treated with afatinib (1 μM) in the presence or absence of etanercept. AlamarBlue assay was conducted after 72 hours. ( I ) H441 cells were treated with afatinib and thalidomide for 72 hours, followed by AlamarBlue assay. ( J and K ) Similar experiments were done as described in H and I in A549 cells. ( L and M ) EGFRwt cells were seeded in 6-well plates at 1,000 cells per well, and incubated with 20 μg/ml thalidomide and/or 1 μM erlotinib. Fourteen days later, cell colonies were fixed by 100% methanol and then stained by 0.5% crystal violet in 25% methanol. Images were captured by a scanner, and colony counts were processed by ImageJ. Data represent the mean percentage of control ± SEM. n = 3 biologically independent experimental replicates ( A , B , and D – M ). * P

    Article Snippet: Afatinib was bought from AstaTech Inc. Thalidomide, actinomycin D, and mithramycin A were from Cayman Chemical.

    Techniques: Inhibition, Expressing, Viability Assay, Transfection, Western Blot, Alamar Blue Assay, Incubation, Staining

    EGFR and TNF inhibition prevents the acquired resistance. ( A ) TetO-EGFR-L858R and CCSP-rtTA mice were exposed to doxycycline diets to induce tumors. Lung tumor formation was confirmed by MRI between weeks 4 and 5. Starting from week 5, mice were randomly divided into 4 groups and treated by 6.25 mg/kg erlotinib and/or 150 mg/kg thalidomide for 2 weeks. Tumor sizes were measured by 2 blinded researchers using ImageJ. Data were presented as each tumor size and the mean ± SEM. ( B ) MRI images from representative mice in each group. The tumors grow as diffuse lung opacities. ( C ) TNF mRNAs were detected in HCC827 parent and erlotinib-resistant cell lines by qPCR. ( D – F ) HCC827/ER3, HCC827/ER4A, and H1975 cells were treated with 5 μg/ml thalidomide and/or 100 nM erlotinib/afatinib for 72 hours followed by AlamarBlue assay. ( G ) 1 × 10 6 H1975 cells were injected s.c. into athymic mice. When tumors formed, mice were divided into 4 groups ( n = 6) and treated with 5 mg/kg afatinib by oral gavage or 150 mg/kg thalidomide i.p. for 24 days. ( H ) HCC827 cells were planted in a 96-well plate and treated with 100 nM erlotinib with or without 5 μg/ml thalidomide. The day when cells reached 100% confluence was considered the appearance of acquired resistance. ( I ) HCC827 cells were injected subcutaneously into athymic mice. When tumors reached 500 mm 3 , mice were divided into 4 groups ( n = 6) and treated with 6.25 mg/kg erlotinib by oral gavage or 150 mg/kg thalidomide by i.p. for 32 days. For ( C – F ) data represent mean ± SEM. n = 3 biologically independent experimental replicates. * P

    Journal: The Journal of Clinical Investigation

    Article Title: TNF-driven adaptive response mediates resistance to EGFR inhibition in lung cancer

    doi: 10.1172/JCI96148

    Figure Lengend Snippet: EGFR and TNF inhibition prevents the acquired resistance. ( A ) TetO-EGFR-L858R and CCSP-rtTA mice were exposed to doxycycline diets to induce tumors. Lung tumor formation was confirmed by MRI between weeks 4 and 5. Starting from week 5, mice were randomly divided into 4 groups and treated by 6.25 mg/kg erlotinib and/or 150 mg/kg thalidomide for 2 weeks. Tumor sizes were measured by 2 blinded researchers using ImageJ. Data were presented as each tumor size and the mean ± SEM. ( B ) MRI images from representative mice in each group. The tumors grow as diffuse lung opacities. ( C ) TNF mRNAs were detected in HCC827 parent and erlotinib-resistant cell lines by qPCR. ( D – F ) HCC827/ER3, HCC827/ER4A, and H1975 cells were treated with 5 μg/ml thalidomide and/or 100 nM erlotinib/afatinib for 72 hours followed by AlamarBlue assay. ( G ) 1 × 10 6 H1975 cells were injected s.c. into athymic mice. When tumors formed, mice were divided into 4 groups ( n = 6) and treated with 5 mg/kg afatinib by oral gavage or 150 mg/kg thalidomide i.p. for 24 days. ( H ) HCC827 cells were planted in a 96-well plate and treated with 100 nM erlotinib with or without 5 μg/ml thalidomide. The day when cells reached 100% confluence was considered the appearance of acquired resistance. ( I ) HCC827 cells were injected subcutaneously into athymic mice. When tumors reached 500 mm 3 , mice were divided into 4 groups ( n = 6) and treated with 6.25 mg/kg erlotinib by oral gavage or 150 mg/kg thalidomide by i.p. for 32 days. For ( C – F ) data represent mean ± SEM. n = 3 biologically independent experimental replicates. * P

    Article Snippet: Afatinib was bought from AstaTech Inc. Thalidomide, actinomycin D, and mithramycin A were from Cayman Chemical.

    Techniques: Inhibition, Mouse Assay, Magnetic Resonance Imaging, Real-time Polymerase Chain Reaction, Alamar Blue Assay, Injection

    Inhibition of TNF enhances sensitivity of NSCLC cells with EGFR activating mutations to EGFR inhibition. ( A and B ) AlamarBlue assay in HCC827 or H3255 cells. TNFR1 was silenced using siRNA, and 48 hours later cells were exposed to erlotinib for 72 hours. ( C ) Silencing of TNFR1 was confirmed by Western blot. Results are representative of 3 independent replicates. ( D and E ) Thalidomide sensitizes HCC827 and H3255 cells to EGFR inhibition. Thalidomide (5 μg/ml) and erlotinib were added concurrently, and AlamarBlue assay was done after 72 hours. ( F and G ) Similar experiments were done using etanercept (100 μg/ml) and erlotinib in HCC827 and H3255 cells. ( H and I ) HCC827 and H3255 cells were treated with afatinib with or without thalidomide for 72 hours, followed by AlamarBlue assay. ( J and K ) Similar experiments were performed in HCC827 and H3255 cells with afatinib and etanercept. The concentration of erlotinib or afatinib was 10 nM in A – K . ( L and M ) EGFR-mutant NSCLC cells were seeded in 6-well plates at 1,000 cells per well, and incubated with 5 μg/ml thalidomide and/or erlotinib 1 nM. Fourteen days later, cell colonies were fixed by 100% methanol and then stained by 0.5% crystal violet in 25% methanol. Images were captured by a scanner, and colony counting was processed by ImageJ. Data are presented as the average percentage of untreated colonies ± SEM from 3 experiments. ( N and O ) Exogenous TNF protects H3255 and HCC827 from all erlotinib-induced cell death. Cells were exposed to erlotinib (100 nM) with or without TNF (1 ng/ml). Cell viability was determined 72 hours later using AlamarBlue assay. Data represent the mean percentage of control ± SEM. n = 3 biologically independent experimental replicates ( A , B , and D – O ). * P

    Journal: The Journal of Clinical Investigation

    Article Title: TNF-driven adaptive response mediates resistance to EGFR inhibition in lung cancer

    doi: 10.1172/JCI96148

    Figure Lengend Snippet: Inhibition of TNF enhances sensitivity of NSCLC cells with EGFR activating mutations to EGFR inhibition. ( A and B ) AlamarBlue assay in HCC827 or H3255 cells. TNFR1 was silenced using siRNA, and 48 hours later cells were exposed to erlotinib for 72 hours. ( C ) Silencing of TNFR1 was confirmed by Western blot. Results are representative of 3 independent replicates. ( D and E ) Thalidomide sensitizes HCC827 and H3255 cells to EGFR inhibition. Thalidomide (5 μg/ml) and erlotinib were added concurrently, and AlamarBlue assay was done after 72 hours. ( F and G ) Similar experiments were done using etanercept (100 μg/ml) and erlotinib in HCC827 and H3255 cells. ( H and I ) HCC827 and H3255 cells were treated with afatinib with or without thalidomide for 72 hours, followed by AlamarBlue assay. ( J and K ) Similar experiments were performed in HCC827 and H3255 cells with afatinib and etanercept. The concentration of erlotinib or afatinib was 10 nM in A – K . ( L and M ) EGFR-mutant NSCLC cells were seeded in 6-well plates at 1,000 cells per well, and incubated with 5 μg/ml thalidomide and/or erlotinib 1 nM. Fourteen days later, cell colonies were fixed by 100% methanol and then stained by 0.5% crystal violet in 25% methanol. Images were captured by a scanner, and colony counting was processed by ImageJ. Data are presented as the average percentage of untreated colonies ± SEM from 3 experiments. ( N and O ) Exogenous TNF protects H3255 and HCC827 from all erlotinib-induced cell death. Cells were exposed to erlotinib (100 nM) with or without TNF (1 ng/ml). Cell viability was determined 72 hours later using AlamarBlue assay. Data represent the mean percentage of control ± SEM. n = 3 biologically independent experimental replicates ( A , B , and D – O ). * P

    Article Snippet: Afatinib was bought from AstaTech Inc. Thalidomide, actinomycin D, and mithramycin A were from Cayman Chemical.

    Techniques: Inhibition, Alamar Blue Assay, Western Blot, Concentration Assay, Mutagenesis, Incubation, Staining

    A) CENPE protein expression in response to a 12 hour treatment with 150nM Afatinib, 150nM Trastuzumab (Her), 150nM Trastuzumab + 1μM Lapatinib (Lap) and 1μM Lapatinib + 20μM Capecitabine (Cap) with densitometric measurement of fold change (control vs. drug treated) in B) the SKBR3 cell line and C) the BT474 cell line. * represents significance at p

    Journal: Journal of proteomics and genomics research

    Article Title: Determination of the Proteomic Response to Lapatinib Treatment using a comprehensive and reproducible ion-current-based proteomics strategy

    doi: 10.14302/issn.2326-0793.jpgr-13-257

    Figure Lengend Snippet: A) CENPE protein expression in response to a 12 hour treatment with 150nM Afatinib, 150nM Trastuzumab (Her), 150nM Trastuzumab + 1μM Lapatinib (Lap) and 1μM Lapatinib + 20μM Capecitabine (Cap) with densitometric measurement of fold change (control vs. drug treated) in B) the SKBR3 cell line and C) the BT474 cell line. * represents significance at p

    Article Snippet: Drug treatments were applied singly or in combinations as follows Laptinib 1μM (Sequoia Sciences, Saint Louis, MO, USA), 150 nM Herceptin (Roche IN, USA), 150 nM Afatinib (Sequoia Sciences) and 20 μM capecitabine (Sigma-Aldrich.

    Techniques: Expressing