Journal: Cancer

Article Title: Addition of an aminopeptidase N‐binding sequence to human endostatin improves inhibition of ovarian carcinoma growth

doi: 10.1002/cncr.21149

Figure Lengend Snippet: Characterization of NGR sequence‐modified endostatin (NGR‐endostatin). (A) Aminopeptidase N (APN)‐inhibitory activity. APN was extracted from human umbilical vein endothelial cell (HUVEC) cultures. Endostatin preparations, bestatin (positive control), and leupeptin (negative control) were used at a concentration of 5 μM. Endostatin‐RGD: endostatin containing an RGD sequence. (B) Cell attachment assay. HUVEC or WM35 cells that were prelabeled with 5‐(and‐6)‐carboxy fluorescein diacetate, succinimidyl ester, were added into triplicate wells coated with either endostatin or NGR‐endostatin at a concentration of 1 nmole per well. Wells coated with 0.2% gelatin were used as maximum attachment (100%). The attached cells were quantified by a fluorescence plate reader. Values represent the mean of two independent experiments. (C) The effect of endostatin and NGR‐endostatin on endothelial cell proliferation: Endostatin and NGR‐endostatin were used at a concentration of 2.5 μg/mL. Basic fibroblast growth factor (5 ng/mL) was used to induce proliferation of endothelial cells. The proliferation was determined by 5′‐bromo‐2′‐deoxyuridine uptake. (D) The effect of endostatin (open bar) and NGR‐endostatin (solid bar) on HUVEC migration. NGR‐containing peptide (SR‐1) plus endostatin (hatched bar) did not enhance the basal level of inhibition seen with endostatin alone (open bar). (E) Tumor localization: Human colon carcinoma cells (LS174T) were injected subcutaneously into female athymic nude mice. When the tumors reached a size of ≈ 500 mm 3 (10 days after inoculation), endostatin (open bars) or NGR‐endostatin (solid bars) was injected at a dose of 20 mg/kg subcutaneously. Tumor, liver, and lung tissues were resected and homogenized. Endostatin levels in the tissues and the sera were determined by enzyme‐linked immunoadsorbent assay in the soluble fraction. Endostatin levels are expressed as a relative concentration to serum levels of endostatin. Error bars indicate the standard error. Statistical significance was determined with a Student t test. An asterisk indicates P < 0.05.

Article Snippet: The human colon carcinoma cell line LS174T was obtained from the American Type Culture Collection (ATCC) (Rockville, MD).

Techniques: Sequencing, Modification, Activity Assay, Positive Control, Negative Control, Concentration Assay, Cell Attachment Assay, Fluorescence, Migration, Inhibition, Injection

Journal: The Journal of Experimental Medicine

Article Title: Targeting phospholipase D1 attenuates intestinal tumorigenesis by controlling β-catenin signaling in cancer-initiating cells

doi: 10.1084/jem.20141254

Figure Lengend Snippet: PLD1 is highly up-regulated in the CC-IC population and in vitro sphere-forming capacity. (A and B) Venn diagram for transcripts up- (A) and down-regulated (B) by PLD1 inhibition in DLD1 and HCT116 cells. Summary of the functional categories of genes significantly enriched upon treatment with PLD1 inhibitor. Gene ontology groups demonstrated enhanced statistical representation (P < 0.01). (C) Targeting PLD1 significantly represses the β-catenin signaling pathway but not other pathways in the CRC cells. In yellow: over-representation reaches statistical significance only for the β-catenin signatures. (D) q-RT-PCR analysis of β-catenin and C-IC markers under adherent and sphere culture conditions of xenografted DLD1 cells stably transfected with shPLD1 or treated with PLD1 inhibitor. Data are shown as mean ± SEM. ANOVA F-test was used. (E and F) In vitro LDAs of SFUs by PLD1 depletion (E) and PLD1 inhibition (F) in xenografted cells; error bars represent 95% confidence intervals. (G–I) IB (G) and (H) q-RT-PCR (H) analysis of PLD1 expression and cellular PA levels (I) in CD133 + CD44 + and CD133 – CD44 – cells sorted from xenografted CRC spheres. (E, F, H, and I) Data are shown as mean ± SEM. A Student’s t test was used. (J) After the indicated cells were sorted by flow cytometry using antibody to PLD1, the percentage of CD133 + CD44 + populations was analyzed by FACS. (K) Effect of PLD1 depletion on SP in CRC cells. (L and M) q-RT-PCR and IB analysis of the indicated C-IC markers in tumor tissues of the indicated mice. (N) IHC and q-RT-PCR analysis of the indicated C-IC markers in Apc Min/+ ( n = 7) and Apc Min/+ Pld1 Tg ( n = 6) mice. (O and P) After exposing the vehicle or PLD1-Inh group to 50 µg/ml 5-fluorouracil (5-Fu) and/or 100 µM oxaliplatin (Oxal), the percentage of apoptotic cells (O) and LDA analysis of SFUs (P) was measured. (L–P) Results are representative of at least three independent experiments and are shown as mean ± SEM. A Student’s t test was used. *, P < 0.05; **, P < 0.01; ***, P < 0.001. n.s., not significant; MW, molecular weight; a.u., arbitrary units. Bars, 100 µm.

Article Snippet: HEK293 and human CRC cells (HCT116, DLD1, SW480, and LS174T) were obtained from ATCC.

Techniques: In Vitro, Inhibition, Functional Assay, Reverse Transcription Polymerase Chain Reaction, Stable Transfection, Transfection, Expressing, Flow Cytometry, Molecular Weight

Journal: The Journal of Experimental Medicine

Article Title: Targeting phospholipase D1 attenuates intestinal tumorigenesis by controlling β-catenin signaling in cancer-initiating cells

doi: 10.1084/jem.20141254

Figure Lengend Snippet: Targeting PLD1 attenuates tumor-initiating capacity through the E2F1–miR-4496–β-catenin axis. (A) Effect of β-catenin depletion or premiR-4496 on the expression of the indicated genes. (B) Effect of anti–miR-4496 on the expression of the indicated proteins (left). The levels of anti-miR are shown as a control (right). (C) Effect of E2F1 depletion or miR-4496 on the binding of β-catenin/TCF to the promoter of C-IC marker genes. (D) Representative flow cytometric profiles of CD44 and CD133 expression under the indicated conditions (left). IB analysis of PLD1, E2F1, and β-catenin was shown as a control (right). (E) Frequency of secondary SFUs by transduction with anti–miR-4496 or shE2F1 in PLD1-depleted CRC cells, as determined by in vitro LDAs. (F) BrdU incorporation analysis in the indicated cells. (G) Photographs of excised tumors (left) and in vivo serial transplantation assays (right) from NOD/SCID mice ( n = 5 per group) injected with DLD1 cells derived from the indicated xenografts. Results are representative of at least three independent experiments and are shown as mean ± SEM. A Student’s t test was used. *, P < 0.05; **, P < 0.01; ***, P < 0.001. n.s., not significant; MW, molecular weight; a.u., arbitrary units. Bars: (E) 50 µm; (G) 1 cm.

Article Snippet: HEK293 and human CRC cells (HCT116, DLD1, SW480, and LS174T) were obtained from ATCC.

Techniques: Expressing, Control, Binding Assay, Marker, Transduction, In Vitro, BrdU Incorporation Assay, In Vivo, Transplantation Assay, Injection, Derivative Assay, Molecular Weight