Journal: Carcinogenesis

Article Title: Suppression of NF-kappaB activity by NDRG2 expression attenuates the invasive potential of highly malignant tumor cells.

doi: 10.1093/carcin/bgp072

Figure Lengend Snippet: Fig. 1. NDRG2 overexpression inhibits anchorage-independent growth of HT1080 human fibrosarcoma cells. (A) Expression levels of NDRG2 mRNA and protein in HT1080-mock and -NDRG2-transfected cells were examined by RT–PCR (upper lanes) and western blot (lower lanes) analysis, respectively. (B) Cell morphology was observed with phase-contrast microscopy and cell proliferation was determined with an 3-(4,5- dimethylthiazole-2-yl)-2,5-biphenyl tetrazolium bromide assay. (C) Cells (1 104) were suspended in 3 ml of the medium containing 0.3% agar and 10% FBS and were applied onto the pre-solidified 0.6% agar (3 ml) in 60 mm culture dishes. After 2 weeks of incubation, colonies on soft agar were observed under a phase-contrast microscope and the diameter of representative 20 colonies was measured; P , 0.01 (values are compared with HT1080-mock cells).

Article Snippet: Downregulation of NDRG2 gene expression by NDRG2 siRNA transfection NDRG2 siRNA (sc-40758) was purchased from Santa Cruz Biotechnology.

Techniques: Over Expression, Expressing, Transfection, Reverse Transcription Polymerase Chain Reaction, Western Blot, Microscopy, Incubation

Journal: Carcinogenesis

Article Title: Suppression of NF-kappaB activity by NDRG2 expression attenuates the invasive potential of highly malignant tumor cells.

doi: 10.1093/carcin/bgp072

Figure Lengend Snippet: Fig. 2. Effects of NDRG2 overexpression on the migration and invasion of HT1080 fibrosarcoma cells in an in vitro assay. (A) A fixed number of cells were plated onto the upper part of the transwell chamber. The media compartment of the lower chamber contained 10% FBS. After incubation for 20 h, cells migrating to the lower surface of the membrane were stained with a 0.2% crystal violet/20% methanol (vol/vol) solution and were observed with phase-contrast microscopy. Migrating cells were also examined using calcein-AM as a substrate for intracellular esterase. The fluorescent intensity of free calcein was measured with a fluorescence microplate reader set (PerkinElmer VictorTM). (B) A fixed number of cells were plated onto the upper part of the Matrigel-coated transwell chamber. After incubation for 20 h, invasive cells were characterized as described in Figure 2A. (C and D) The effect of NDRG2 overexpression on cell invasion in response to PMA stimulation was also investigated. P , 0.01 (values are compared with HT1080-mock cells).

Article Snippet: Downregulation of NDRG2 gene expression by NDRG2 siRNA transfection NDRG2 siRNA (sc-40758) was purchased from Santa Cruz Biotechnology.

Techniques: Over Expression, Migration, In Vitro, Incubation, Membrane, Staining, Microscopy

Journal: Carcinogenesis

Article Title: Suppression of NF-kappaB activity by NDRG2 expression attenuates the invasive potential of highly malignant tumor cells.

doi: 10.1093/carcin/bgp072

Figure Lengend Snippet: Fig. 3. NDRG2 overexpression in HT1080 fibrosarcoma cells reduces PMA-induced MMP expression, proteolytic activities and NF-jB activation. (A) mRNA expression of MMPs in a resting condition or PMA-simulated condition was measured by RT–PCR. Cells were treated with PMA for 24 h at concentrations of 2.5 and 5 nM. (B) Cells were incubated in serum-free media for 24–48 h, with or without 5 nM of PMA, and CM was collected. Concentrated CM by trichloroacetic acid precipitation was subjected to western blot for MMP-2 and -9. (C) CM was subjected to gelatin zymography for detecting MMP-2 and -9 activities. (D) Cells were transiently transfected with a luciferase reporter construct using Lipofectamine 2000 reagent. After incubation for 4 h, 5 nM of PMAwas added. After 18 h of additional incubation, cells were harvested in passive lysis buffer, and the luciferase activities were measured by a luminometer using the luciferase assay system. Changes in luciferase activity, with respect to the mock control, were calculated. (E) NF-jB and AP-1 luciferase activities after PMA or TNF-a stimulation were measured using the luciferase assay system. P , 0.01 (values are compared with HT1080-mock cells). (F) HT1080 wild-type cells were transfected with dominant negative forms of dnIKKa and dnIKKb and then exposed to PMA (5 nM) for 24 h. CM was subjected to gelatin zymography. (G and H) HT1080-mock and -NDRG2 cells were stimulated with 5 nM PMA for the indicated time, and cell lysates were subjected to western blot analysis. To examine the nuclear translocation of the NF-jB p65 subunit, cell lysates were fractionated into cytosolic (C) and nuclear (N) compartments. Results are from a single analysis, representative of two independent experiments.

Article Snippet: Downregulation of NDRG2 gene expression by NDRG2 siRNA transfection NDRG2 siRNA (sc-40758) was purchased from Santa Cruz Biotechnology.

Techniques: Over Expression, Expressing, Activation Assay, Reverse Transcription Polymerase Chain Reaction, Incubation, TCA Precipitation, Western Blot, Zymography, Transfection, Luciferase, Construct, Lysis, Activity Assay, Control, Dominant Negative Mutation, Translocation Assay

Journal: Carcinogenesis

Article Title: Suppression of NF-kappaB activity by NDRG2 expression attenuates the invasive potential of highly malignant tumor cells.

doi: 10.1093/carcin/bgp072

Figure Lengend Snippet: Fig. 4. NDRG2 overexpression in B16F10 murine melanoma cells also decreases their metastatic potential and suppresses NF-jB activation. (A) mRNA expression of MMP-2 and MMP-9 was measured by RT–PCR. (B) Concentrated CM was subjected to western blot analysis. (C) Gelatinolytic activities were analyzed by gelatin zymography using CM. TNF-a (10 ng/ml) was used to treat the cells for 24 h. (D) To examine anchorage-independent cell growth, a soft agar colony formation assay was performed. (E) A fixed number of cells were plated onto the upper part of the transwell chamber. After incubation for 20 h, cells migrating to the lower surface of the membrane were stained and observed by phase-contrast microscopy. Migrating cells were also determined using calcein-AM. A fixed number of cells were plated onto the upper part of the Matrigel-coated transwell chamber. After incubation for 20 h, invasive cells were characterized. (F) NF-jB and AP-1 luciferase activity, after treating cells with TNF-a (10 ng/ml) for 18 h, was measured using the luciferase assay system. P , 0.01 (values are compared with B16F10-mock cells). (G) After treating cells with TNF-a (10 ng/ml) for 15 min, cell lysates were subjected to western blot analysis.

Article Snippet: Downregulation of NDRG2 gene expression by NDRG2 siRNA transfection NDRG2 siRNA (sc-40758) was purchased from Santa Cruz Biotechnology.

Techniques: Over Expression, Activation Assay, Expressing, Reverse Transcription Polymerase Chain Reaction, Western Blot, Zymography, Soft Agar Assay, Incubation, Membrane, Staining, Microscopy, Luciferase, Activity Assay

Journal: Carcinogenesis

Article Title: Suppression of NF-kappaB activity by NDRG2 expression attenuates the invasive potential of highly malignant tumor cells.

doi: 10.1093/carcin/bgp072

Figure Lengend Snippet: Fig. 5. NDRG2 overexpression in B16F10 melanoma cells reduces pulmonary metastases and in vivo tumorigenesis. (A) Experimental metastatic potential was measured by a lung colonization assay. (B) Representative histological photographs of lung tissue sections stained with hematoxylin and eosin (200). NL, normal lung tissue; T, metastatic tumor lesion. (C) For the in vivo tumorigenicity assay, 6- to 8-week-old female C57BL/6J mice were shaved on the ventral side and challenged s.c. with 2 105 cells per 0.2 ml of PBS. Tumor diameters were measured every other day with a vernier caliper. (D) To examine lymph node metastasis, melanotic B16F10 cells were characterized in draining lymph nodes at day 20 after s.c. injection of tumor cells on the right buttocks. The pictures of lymph nodes are representative of inguinal lymph nodes (each group, n 5 3). (E) Cells were s.c. injected in the center of the abdominal region. After 20 days, inguinal lymph nodes on both sides were observed for melanotic tumor cells. (F) Lung colonization of tumor cells in the mice showing severe lymph node metastasis was observed macroscopically.

Article Snippet: Downregulation of NDRG2 gene expression by NDRG2 siRNA transfection NDRG2 siRNA (sc-40758) was purchased from Santa Cruz Biotechnology.

Techniques: Over Expression, In Vivo, Staining, Tumorigenicity Assay, Injection

Journal: Carcinogenesis

Article Title: Suppression of NF-kappaB activity by NDRG2 expression attenuates the invasive potential of highly malignant tumor cells.

doi: 10.1093/carcin/bgp072

Figure Lengend Snippet: Fig. 6. NDRG2 overexpression in B16F10 melanoma cells inhibits in vivo tumor angiogenesis. (A) Peritumoral vascularization was observed at day 12 or at day 18 after s.c. injection of B16F10-mock or -NDRG2 cells, respectively. Tumor volume of both mice was similar at the time of observation. After RNA extraction from a B16F10-mock and -NDRG2 tumor mass (at day 18 and 23 after s.c. injection), PECAM-1 mRNA expression was examined by RT–PCR. (B) The vascular density in solid tumors derived from s.c. injection of B1F10-mock or -NDRG2 cells was determined using immunohistochemical staining with an anti-CD31/ PECAM-1 antibody. Tissues were counterstained with hematoxylin. Brownish staining indicates endothelial cells (400). (C) B16F10-mock or -NDRG2 cells (3 106 cells per 0.2 ml of serum-free medium) with 0.4 ml of growth factor-reduced Matrigel were s.c. injected into the flank of C57BL/6J mice. After 7 days, Matrigel plugs were removed from the mice, and hemoglobin levels of Matrigel plugs were quantified with Drabkin’s reagent. Three independent experiments (n 5 2) were performed, and photographs and hemoglobin levels of representative Matrigel plugs were shown. (D) VEGF-a mRNA and protein secretion in the normoxia and hypoxia condition (10% CO2 and 1% O2) were measured by RT–PCR and enzyme-linked immunosorbent assay, respectively. P , 0.01 (values are compared with B16F10-mock cells).

Article Snippet: Downregulation of NDRG2 gene expression by NDRG2 siRNA transfection NDRG2 siRNA (sc-40758) was purchased from Santa Cruz Biotechnology.

Techniques: Over Expression, In Vivo, Injection, RNA Extraction, Expressing, Reverse Transcription Polymerase Chain Reaction, Derivative Assay, Immunohistochemical staining, Staining, Enzyme-linked Immunosorbent Assay

Journal: Carcinogenesis

Article Title: Suppression of NF-kappaB activity by NDRG2 expression attenuates the invasive potential of highly malignant tumor cells.

doi: 10.1093/carcin/bgp072

Figure Lengend Snippet: Fig. 7. siRNA-mediated knockdown of NDRG2-rescued NF-jB activity and increased migration as well as invasion. (A and B) The decrease of NDRG2 mRNA levels in B16F10-NDRG2 cells transfected with NDRG2-siRNA was confirmed by RT–PCR. Cell migration and invasion were analyzed after siRNA-mediated gene silencing of NDRG2. NF-jB luciferase activity was also examined. P , 0.01 (values are compared with control siRNA-transfected cells). (C) Increase in MMP-9 activity was observed in NDRG2-silenced SNU-620 cells. (D) NDRG2-silenced SNU-620 cells rescued the ability to proliferate in semisolid agar (upper) and to invade Matrigel barrier (lower).

Article Snippet: Downregulation of NDRG2 gene expression by NDRG2 siRNA transfection NDRG2 siRNA (sc-40758) was purchased from Santa Cruz Biotechnology.

Techniques: Knockdown, Activity Assay, Migration, Transfection, Reverse Transcription Polymerase Chain Reaction, Luciferase, Control