Journal: Oncotarget

Article Title: Checkpoint kinase 1 inhibition sensitises transformed cells to dihydroorotate dehydrogenase inhibition

doi: 10.18632/oncotarget.19199

Figure Lengend Snippet: ( A ) Immunofluorescence of DNA (blue) and γH2AX (H2AX phosphorylation on serine 139) (green) in transformed MEFs that were exposed to either IC70 TFN (10 μM), IC10 PF477736 (0.7 μM), the combination of these compounds or 0.1 μM positive control camptothecin (CPT). Scale bar, 20 μm. ( B ) Western blotting analysis of Chk1 phosphorylation on serine 345 in cell lysates prepared 8 hours after the beginning of the exposure. Upper panel: transformed MEFs were exposed to vehicle, 0.1 μM camptothecin, IC10 PF477736, IC70 TFN ± IC10 PF477736 and IC70 IPP-A017-A04 (22 μM) ± IC10 PF477736. Lower panel: cells were exposed to vehicle, 0.1 μM camptothecin, IC70 TFN, IC10 PF477736, or increasing (IC50 = 1.45 μM, IC70 = 10 μM and IC90 = 25 μM) concentrations of teriflunomide ± IC10 PF477736.

Article Snippet: Transformed mouse embryonic fibroblasts were transfected with 700 pmoles of either a mix of 4 target-specific 19-25 nt small interfering RNA (siRNA) for mouse Chk1 (Santa Cruz Biotechnology sc-29270), or SASI_Mm01_00087610 and SASI_Mm01_00087610 MISSION ® siRNA for mouse DHODH (Sigma Aldrich), or 1400 pmoles of the combination of Chk1 and DHODH siRNA.

Techniques: Immunofluorescence, Phospho-proteomics, Transformation Assay, Positive Control, Western Blot

Journal: Lung cancer (Amsterdam, Netherlands)

Article Title: CHK1 levels correlate with sensitization to pemetrexed by CHK1 inhibitors in non-small cell lung cancer cells

doi: 10.1016/j.lungcan.2013.09.010

Figure Lengend Snippet: Relationship of CHK1 mRNA expression with patient survival, tumor differentiation and genomic complexity. (A) Analysis of gene expression of 442 lung adenocarcinomas using Kaplan–Meier survival curve shows that patients with high CHK1 mRNA expressing tumors demonstrate an overall poorer 5 year survival, comparing the higher 1/3 patients vs lower 2/3 patients based on CHK1 mRNA levels (log-rank test, p < 0.001). (B) This was verified in an independent cohort of patients with 101 lung adenocarcinomas with CHK1 mRNA verified by RT-PCR (p = 0.02). (C) Tumors with higher CHK1 mRNA expression are more likely to display a poorly-differentiated phenotype as compared to low CHK1 expressing tumors (p < 0.0001) in 442 lung adenocarcinomas. (D) This was verified in the same independent cohort of patients with 101 lung adenocarcinomas (p = 0.0003). CHK1 mRNA expression in lung adenocarcinomas also correlates with an increased number of genomic alterations, specifically gene mutations or amplifications. Tumors were classified into high, medium and low groups based on CHK1 mRNA expression (CHK1 mRNA >100, 100–50, and <50 as cutoff for these 3 groups). (E) Higher CHK1 mRNA expression correlated with an increased number of gene mutations in 54 lung adenocarcinomas (Pearson correlation, r = 0.993, p < 0.001, the mean mutation ±SD are 7.28 ±10.4, 4.23 ±3.7 and 1.14 ±0.99 for high, medium and low groups, respectively). (F) In a separate data-set of 93 lung adenocarcinomas, higher CHK1 mRNA correlated with DNA amplification/gain (Pearson correlation, r = 0.99, p < 0.001, mean amp/gain ±SD are 3.73 ±3.2, 2.29 ±2.1, and 1.0 ±0.76 for high, medium and low groups, respectively). (G) CHK1 mRNA levels do not correlate with gene deletions or LOH for the same 93 tumors.

Article Snippet: PathScan CHK1 Sandwich ELISA kit (Cell Signaling) was used according to manufacturer’s protocol.

Techniques: Expressing, Gene Expression, Reverse Transcription Polymerase Chain Reaction, Mutagenesis, DNA Amplification

Journal: Lung cancer (Amsterdam, Netherlands)

Article Title: CHK1 levels correlate with sensitization to pemetrexed by CHK1 inhibitors in non-small cell lung cancer cells

doi: 10.1016/j.lungcan.2013.09.010

Figure Lengend Snippet: (A) Gene expression analysis for CHK1 mRNA levels in representative sample of 15 cultured lung cancer cell lines and in nontransformed lung cells (NLC). Based on these results, we selected two apparent under-expressing cell lines, H1993 and H1437, and two over-expressing cell lines, H23 and H1299, for further analysis. (B) Relative expression of CHK1 to β-actin by qRT-PCR in selected NSCLC cell lines. Data shown is the aggregate of three independent assays, and suggests that H1437 is actually a CHK1 over-expressing cell line. CHK1 protein levels in selected NSCLC cell lines by Western Blot (C) and by ELISA (D) confirming the three higher CHK1 expressing cell lines (H23, H1437, and H1299) and the one lower expressing line (H1993). (E) CHK1 expression by immunohistochemistry in two representative cell lines (H1993 and H1299) showing increased nuclear CHK1 protein expression (brown stain) in the H1299 cells compared to the H1993 cells. (For interpretation of the references to color in this figure legend, the reader is referred to the web version of this article.)

Article Snippet: PathScan CHK1 Sandwich ELISA kit (Cell Signaling) was used according to manufacturer’s protocol.

Techniques: Gene Expression, Cell Culture, Expressing, Quantitative RT-PCR, Western Blot, Enzyme-linked Immunosorbent Assay, Immunohistochemistry, Staining

Journal: Lung cancer (Amsterdam, Netherlands)

Article Title: CHK1 levels correlate with sensitization to pemetrexed by CHK1 inhibitors in non-small cell lung cancer cells

doi: 10.1016/j.lungcan.2013.09.010

Figure Lengend Snippet: (A) Chemosensitization to PMX by the CHK1 inhibitor, MK-8776, as measured by WST-1 assay. Cells were treated with graded concentrations of PMX for 24 h (T0–T24 h) followed by 24 h treatment with MK-8776 at 500 nM (T24–T48 h). CHK1 inhibition preferentially sensitized cells expressing higher levels of CHK1 (H23, H1437 and H1299) as compared to low CHK1-expressing cells (H1993). At as low as 0.3 μM PMX concentration there was significant reduction in percent of proliferating cells in H23 (p = 0.0012), H1437 (p = 0.0006) and H1299 (p = 0.0006). No significant change in H1993 cells (p = 0.26). Each experiment was performed using 3 replicates for each drug concentration. All experiments were repeated a minimum of 3 times. (B) Chemosensitization by clonogenic survival assay shown in log scale. Low CHK1-expressing H1993 cells are not sensitized to PMX in combination with MK-8776, whereas over-expressing cell lines are sensitized.

Article Snippet: PathScan CHK1 Sandwich ELISA kit (Cell Signaling) was used according to manufacturer’s protocol.

Techniques: WST-1 Assay, Inhibition, Expressing, Concentration Assay, Clonogenic Cell Survival Assay

Journal: Lung cancer (Amsterdam, Netherlands)

Article Title: CHK1 levels correlate with sensitization to pemetrexed by CHK1 inhibitors in non-small cell lung cancer cells

doi: 10.1016/j.lungcan.2013.09.010

Figure Lengend Snippet: (A) Western Blot analyses of the MK-8776 plus PMX in low and high-expressing CHK1 cell lines. The higher CHK1 expressing line, H1299, displays a greater response in S345 autophosphorylation with combination treatment. Also of note, H1299 appears to have a higher baseline CDC25A expression which is reduced with PMX treatment alone. The addition of a CHK1 inhibitor increases CDC25A expression indicating mitotic entry. (B) Western Blot analysis of CHK1 siRNA treatment showing knockout of CHK1 protein expression at 24 and 48 h. In addition, cells were treated with non-target siRNAs (NT) and Lipofectamine RNAiMAX transfection reagent (Mock). (C) Effect of CHK1 siRNA on proliferation in H1993 and H1299 cell lines. H1299 cells show a greater reduction in proliferation than H1993 cells when treated with CHK1 siRNA vs NT siRNA. The combination of CHK1 siRNA and MK-8776 at 750 nM had no effect compared to siRNA use alone.

Article Snippet: PathScan CHK1 Sandwich ELISA kit (Cell Signaling) was used according to manufacturer’s protocol.

Techniques: Western Blot, Expressing, Knock-Out, Transfection

Journal: PLoS ONE

Article Title: NPRL2 Sensitizes Human Non-Small Cell Lung Cancer (NSCLC) Cells to Cisplatin Treatment by Regulating Key Components in the DNA Repair Pathway

doi: 10.1371/journal.pone.0011994

Figure Lengend Snippet: ( A ) H1229 cells treated with empty vector and IC 20 of cisplatin (3.0 µM), NPRL2, or NPRL2+ with IC 20 dose of cisplatin were harvested at 24, 48, and 72 h after treatment and analyzed for expression of Chk1, P-Chk1 (Ser345), Chk2, and P-Chk2 (Thr68). In cells treated with empty vector and IC 20 dose of cisplatin or NPRL2, P-Chk1 increased in a time-dependent manner. In contrast, in those treated with NPRL2 and IC 20 dose of cisplatin, P-Chk1 was dramatically enhanced. Although P-Chk2 was not detected in H1299 cells treated with empty vector and IC 20 dose of cisplatin, it was clearly increased in a time-dependent manner in those treated with NPRL2 or NPRL2+ IC 20 dose of cisplatin. ( B ) P-Chk2 expression was immunohistochemically analyzed in an orthotopic model of H322 pleural dissemination, as described in the section. P-Chk2 expression was slightly detected in pleural tumor cells from mice treated with NPRL2 nanoparticles, but not in those treated with LacZ or LacZ + cisplatin. In contrast, treatment by NPRL2 nanoparticles and cisplatin induced high phospho-Chk2 expression in tumor cells. Magnification: ×400. ( C ) The kinase activity of Chk1 and Chk2 in H1299 cells treated with empty vector + IC 20 dose of cisplatin, NPRL2 or NPRL2+ IC 20 dose of cisplatin was analyzed with use of a K-LISA Checkpoint Activity Kit, an enzyme-linked immunosorbent assay (ELISA)-based activity assay. Chk1 kinase activity was greater in cells treated with NPRL2+ cisplatin than in cells treated with empty vector or empty vector + cisplatin ( P <0.003). Chk2 kinase activity was more strongly enhanced in cells treated with NPRL2 or NPRL2+ cisplatin than in cells treated with empty vector or empty vector + cisplatin ( P <0.0001). Bars, SDs of the mean in four individual experiments.

Article Snippet: Anti-phospho-Chk1 (Ser345), anti-phospho-Chk2 (Thr68), anti-phospho-Chk1 (Ser345), anti-NBS1, anti-phospho-NBS1 (Ser343), anti-SMC1, anti-phospho-Cdc25C (Ser216), anti-phospho-Cdc2 (Tyr15), anti-caspase-3, and anti-caspase-9 were purchased from Cell Signaling Technology (Beverly, MA).

Techniques: Plasmid Preparation, Expressing, Activity Assay, Enzyme-linked Immunosorbent Assay

Journal: PLoS ONE

Article Title: NPRL2 Sensitizes Human Non-Small Cell Lung Cancer (NSCLC) Cells to Cisplatin Treatment by Regulating Key Components in the DNA Repair Pathway

doi: 10.1371/journal.pone.0011994

Figure Lengend Snippet: NPRL2+ cisplatin ( cis -diamminedichloroplatinum (II) [CDDP]) treatment effector caspase, caspase-3, is activated and PARP is cleaved. Therefore, our result that the combination treatment of NPRL2 and CDDP activates a caspase cascade and hyperphosphorylates H2AX suggests that this combination treatment can strongly enhance the apoptotic pathway. Adding NPRL2 treatment to CDDP significantly enhanced Chk2 and Chk1 kinase activity, and NPRL2+ CDDP treatment remarkably degraded the interaction of Cdc2 and cyclin B1, leading to the inactivation of the Cdc2/cyclin B1 complex and arrest in G2/M. The cells arrested at the G2/M phase ultimately promote apoptosis with characteristic nuclear fragmentation. This combination treatment strongly inactivates Cdc25A and Cdc25C, and phospho-Cdc2 (Tyr-15), an inactivated type of Cdc2, is clearly increased in NPRL2+ CDDP treatment.

Article Snippet: Anti-phospho-Chk1 (Ser345), anti-phospho-Chk2 (Thr68), anti-phospho-Chk1 (Ser345), anti-NBS1, anti-phospho-NBS1 (Ser343), anti-SMC1, anti-phospho-Cdc25C (Ser216), anti-phospho-Cdc2 (Tyr15), anti-caspase-3, and anti-caspase-9 were purchased from Cell Signaling Technology (Beverly, MA).

Techniques: Activity Assay

Journal: The American Journal of Pathology

Article Title: Conjugated Bile Acids Promote Invasive Growth of Esophageal Adenocarcinoma Cells and Cancer Stem Cell Expansion via Sphingosine 1-Phosphate Receptor 2–Mediated Yes-Associated Protein Activation

doi: 10.1016/j.ajpath.2018.05.015

Figure Lengend Snippet: List of Antibodies Used in This Study

Article Snippet: Antibodies used in this study are listed in . table ft1 table-wrap mode="anchored" t5 caption a7 Antibody Species Source Catalog number Application (dilution) S1PR1 Rabbit Abcam (Cambridge, UK) Ab23695 WB (1:500) S1PR2 Rabbit Santa Cruz Biotechnology (Dallas, TX) sc-25491 WB (1:200) S1PR3 Rabbit Santa Cruz Biotechnology sc-30024 WB (1:200) E-cadherin Rabbit Santa Cruz Biotechnology sc-7870 WB (1:200)/IF (1:50) Vimentin Rabbit Cell Signaling Technology (Danvers, MA) mAb 5741 WB (1:1000) CTGF Rabbit Sigma-Aldrich (St. Louis, MO) Sab1300909 WB (1:500) β-Catenin Mouse Santa Cruz Biotechnology sc-7963 WB (1:500) YAP Rabbit Sigma-Aldrich Y0771 WB (1:1000) p-YAP Rabbit Sigma-Aldrich Y4645 WB (1:1000) Lamin B Goat Santa Cruz Biotechnology sc-6216 WB (1:500) β-Actin (JLA20) Mouse DSHB at University of Iowa (Iowa City, IA) JLA20 WB (1:500) Open in a separate window CTGF, connective tissue growth factor; DSHB, Developmental Studies Hybridoma Bank; IF, immunofluorescence; mAb, monoclonal antibody; p-YAP, phosphorylated YAP; S1PR, sphingosine 1-phosphate receptor; WB, Western blotting.

Techniques:

Journal: The American Journal of Pathology

Article Title: Conjugated Bile Acids Promote Invasive Growth of Esophageal Adenocarcinoma Cells and Cancer Stem Cell Expansion via Sphingosine 1-Phosphate Receptor 2–Mediated Yes-Associated Protein Activation

doi: 10.1016/j.ajpath.2018.05.015

Figure Lengend Snippet: Sphingosine 1-phosphate receptor 2 (S1PR2) activation promotes transforming growth factor (TGF)-β–induced epithelial-to-mesenchymal transition in esophageal adenocarcinoma cells. A: Relative mRNA levels of E-cadherin in OE-19 and OE-33 cells. B: Representative images and relative protein levels of E-cadherin in OE-19 and OE-33 cells. C: Representative bright-field images and immunofluorescence images against E-cadherin of OE-19 and OE-33 cells. D: OE-19 and OE-33 cells were treated with 2.5 μg/mL TGF-β, 100 μmol/L taurocholate (TCA), or both for 72 hours. Representative bright-field images and immunofluorescence images of E-cadherin are shown. E: Protein levels of E-cadherin and vimentin in total lysate and β-catenin in nuclear and cytosolic fractions in OE-19 and OE-33 cells were determined by Western blots. Relative protein levels were analyzed using Lamin B as loading control for nuclear fraction and β-actin as loading control for cytosolic fraction and total lysate. Data are expressed as means ± SEM (A, B, and E). n = 3 (A, B, and E). ∗∗P < 0.01 versus OE-19; †P < 0.05, ††P < 0.01 versus control; ‡P < 0.05 versus no JTE-013 (S1PR2 antagonist); §P < 0.05 versus TGF-β. CTGF, connective tissue growth factor; E-Cad, E-cadherin.

Article Snippet: Antibodies used in this study are listed in . table ft1 table-wrap mode="anchored" t5 caption a7 Antibody Species Source Catalog number Application (dilution) S1PR1 Rabbit Abcam (Cambridge, UK) Ab23695 WB (1:500) S1PR2 Rabbit Santa Cruz Biotechnology (Dallas, TX) sc-25491 WB (1:200) S1PR3 Rabbit Santa Cruz Biotechnology sc-30024 WB (1:200) E-cadherin Rabbit Santa Cruz Biotechnology sc-7870 WB (1:200)/IF (1:50) Vimentin Rabbit Cell Signaling Technology (Danvers, MA) mAb 5741 WB (1:1000) CTGF Rabbit Sigma-Aldrich (St. Louis, MO) Sab1300909 WB (1:500) β-Catenin Mouse Santa Cruz Biotechnology sc-7963 WB (1:500) YAP Rabbit Sigma-Aldrich Y0771 WB (1:1000) p-YAP Rabbit Sigma-Aldrich Y4645 WB (1:1000) Lamin B Goat Santa Cruz Biotechnology sc-6216 WB (1:500) β-Actin (JLA20) Mouse DSHB at University of Iowa (Iowa City, IA) JLA20 WB (1:500) Open in a separate window CTGF, connective tissue growth factor; DSHB, Developmental Studies Hybridoma Bank; IF, immunofluorescence; mAb, monoclonal antibody; p-YAP, phosphorylated YAP; S1PR, sphingosine 1-phosphate receptor; WB, Western blotting.

Techniques: Activation Assay, Immunofluorescence, Western Blot, Control