Journal: Asian Pacific Journal of Cancer Prevention : APJCP

Article Title: Epigenetic Downregulation and Growth Inhibition of IGFBP7 in Gastric Cancer

doi: 10.22034/APJCP.2018.19.3.667

Figure Lengend Snippet: Expression and Methylation of IGFBP7 in Gastric Cancer Cells and Tissues. (A) Expression of IGFBP7 mRNA was detected by qRT-PCR. (B) The methylation status of IGFBP7 exon 1 was examined by MSP. Distilled water (DW), negative loading control; UM, unmethylated DNA band; M, methylated DNA band. (C) By qRT-PCR, treatment with 5-aza-dc, TSA, or combination restored IGFBP7 mRNA expression. (D) IGFBP7 protein expression in gastric cancer (C) tissues compared to paired normal (N) gastric tissues was assessed by western blot. (E) A representative result of methylation was measured using MSP. (F) Immunohistochemical expression of IGFBP7. Left, negative staining for IGFBP7. Middle, weakly positive staining for IGFBP7. Right, strongly positive staining for IGFBP7. Original magnification, 100X. (G) Kaplan-Meier analysis of overall survival for different IGFBP7 expression in gastric cancer patients.

Article Snippet: IGFBP7 human cDNA clones were obtained from Origene Technologies, Inc (Rockville, MD, USA).

Techniques: Expressing, Methylation, Quantitative RT-PCR, Control, Western Blot, Immunohistochemical staining, Negative Staining, Staining

Journal: Asian Pacific Journal of Cancer Prevention : APJCP

Article Title: Epigenetic Downregulation and Growth Inhibition of IGFBP7 in Gastric Cancer

doi: 10.22034/APJCP.2018.19.3.667

Figure Lengend Snippet: Relationship Between IGFBP7 Expression and Methylation Frequency of IGFBP7 in Gastric Cancer Tissues

Article Snippet: IGFBP7 human cDNA clones were obtained from Origene Technologies, Inc (Rockville, MD, USA).

Techniques: Expressing, Methylation

Journal: Asian Pacific Journal of Cancer Prevention : APJCP

Article Title: Epigenetic Downregulation and Growth Inhibition of IGFBP7 in Gastric Cancer

doi: 10.22034/APJCP.2018.19.3.667

Figure Lengend Snippet: Correlation Between IGFBP7 Expression and Clinicopathologic Features

Article Snippet: IGFBP7 human cDNA clones were obtained from Origene Technologies, Inc (Rockville, MD, USA).

Techniques: Expressing

Journal: Asian Pacific Journal of Cancer Prevention : APJCP

Article Title: Epigenetic Downregulation and Growth Inhibition of IGFBP7 in Gastric Cancer

doi: 10.22034/APJCP.2018.19.3.667

Figure Lengend Snippet: Univariate and Multivariate Analysis of IGFBP7 Expression in Gastric Cancer

Article Snippet: IGFBP7 human cDNA clones were obtained from Origene Technologies, Inc (Rockville, MD, USA).

Techniques: Expressing

Journal: Asian Pacific Journal of Cancer Prevention : APJCP

Article Title: Epigenetic Downregulation and Growth Inhibition of IGFBP7 in Gastric Cancer

doi: 10.22034/APJCP.2018.19.3.667

Figure Lengend Snippet: Inhibition of IGFBP7 Expression Promotes Gastric Cancer Cell Growth. (A) Knock-down of IGFBP7 was determined using western blot and qRT-PCR. (B) Inhibition of IGFBP7 expression increased cell proliferation. (C) Colony formation was significantly promoted in IGFBP7 suppressed cells. (D) Western blot analysis was performed with the indicated antibodies on shRNA transduced cells. (E) Quantification of apoptosis by flow cytometry. Data presented mean ± SD and experiments were performed three times in triplicate. *p<0.05.

Article Snippet: IGFBP7 human cDNA clones were obtained from Origene Technologies, Inc (Rockville, MD, USA).

Techniques: Inhibition, Expressing, Knockdown, Western Blot, Quantitative RT-PCR, shRNA, Flow Cytometry

Journal: Asian Pacific Journal of Cancer Prevention : APJCP

Article Title: Epigenetic Downregulation and Growth Inhibition of IGFBP7 in Gastric Cancer

doi: 10.22034/APJCP.2018.19.3.667

Figure Lengend Snippet: Effect of IGFBP7 Overexpression on Gastric Cancer Cells. (A) Upregulation of IGFBP7 was proved by western blot and qRT-PCR. (B) Overexpression of IGFBP7 inhibited cell proliferation. (C) Colonies in transfected cells were visualized by staining and counted. (D) Western blot analysis was performed with the indicated antibodies on IGFBP7 overexpressed cells. (E) Apoptosis was detected by flow cytometry. (F) Detection of p-IGF-1R after IGF-1 ligand stimulation by western blot. (G) Cell growth rate was measured using proliferation assay. Data presented mean ± SD and experiments were performed three times in triplicate. *p<0.05.

Article Snippet: IGFBP7 human cDNA clones were obtained from Origene Technologies, Inc (Rockville, MD, USA).

Techniques: Over Expression, Western Blot, Quantitative RT-PCR, Transfection, Staining, Flow Cytometry, Proliferation Assay

Journal: Asian Pacific Journal of Cancer Prevention : APJCP

Article Title: Epigenetic Downregulation and Growth Inhibition of IGFBP7 in Gastric Cancer

doi: 10.22034/APJCP.2018.19.3.667

Figure Lengend Snippet: IGFBP7 Inhibits Gastric Cancer Cell Invasion and Migration. (A) The number of invading and migrating cells in IGFBP7 suppressed cells. (B) The number of invading and migrating cells in IGFBP7 overexpressed cells. (C) Cell migration was confirmed by a wound healing assay. Dotted lines indicate wound edges. Experiments were performed three times in triplicate, and data presented means ± SD. *p<0.05.

Article Snippet: IGFBP7 human cDNA clones were obtained from Origene Technologies, Inc (Rockville, MD, USA).

Techniques: Migration, Wound Healing Assay

Journal: International journal of cancer

Article Title: IGFBP7 downregulation is associated with tumor progression and clinical outcome in hepatocellular carcinoma.

doi: 10.1002/ijc.25994

Figure Lengend Snippet: Figure 1. Characteristics of PLC/PRF/5 transfected with shRNA against IGFBP7. (a) qRT-PCR (Left panel) and western blot analysis (Right

Article Snippet: Plasmid coding for short hairpin RNA (shRNA) against IGFBP7 and IGFBP7 expression plasmid were purchased from OriGene Technologies (Rockville, MD) and used to transfect HCC cells using Lipofectamine 2000 (Invitrogen, Carlsbad, CA) according to the instructions provided by the manufacturer.

Techniques: Transfection, shRNA, Quantitative RT-PCR, Western Blot

Journal: International journal of cancer

Article Title: IGFBP7 downregulation is associated with tumor progression and clinical outcome in hepatocellular carcinoma.

doi: 10.1002/ijc.25994

Figure Lengend Snippet: Figure 4. Characteristics of HuH7 transfected with IGFBP7 expression plasmid. (a) qRT-PCR (Left panel) and western blot

Article Snippet: Plasmid coding for short hairpin RNA (shRNA) against IGFBP7 and IGFBP7 expression plasmid were purchased from OriGene Technologies (Rockville, MD) and used to transfect HCC cells using Lipofectamine 2000 (Invitrogen, Carlsbad, CA) according to the instructions provided by the manufacturer.

Techniques: Transfection, Expressing, Plasmid Preparation, Quantitative RT-PCR, Western Blot

Journal: International journal of cancer

Article Title: IGFBP7 downregulation is associated with tumor progression and clinical outcome in hepatocellular carcinoma.

doi: 10.1002/ijc.25994

Figure Lengend Snippet: Figure 5. IGFBP7 expression and postoperative outcome in HCC patients. (a) Immunohistochemical findings in representative positive case (Left panel) and negative case (Right panel). T, tumoral lesion; N, non-tumoral lesion. Bar ¼ 200 lm. Disease-free survival (b) and overall

Article Snippet: Plasmid coding for short hairpin RNA (shRNA) against IGFBP7 and IGFBP7 expression plasmid were purchased from OriGene Technologies (Rockville, MD) and used to transfect HCC cells using Lipofectamine 2000 (Invitrogen, Carlsbad, CA) according to the instructions provided by the manufacturer.

Techniques: Expressing, Immunohistochemical staining

Journal: Cancer research

Article Title: IGFBP7 Deletion Promotes Hepatocellular Carcinoma

doi: 10.1158/0008-5472.CAN-16-2885

Figure Lengend Snippet: Igfbp7−/− mice develop spontaneous tumors. A. Igfbp7 mRNA levels in livers and spleens of Igfbp7+/+ and Igfbp7−/− mice. B. IGFBP7 protein levels in the indicated organs. β-actin: loading control. C. Immunohistochemistry for IGFBP7 in sections of liver and spleen of adult mice. D. Igfbp7 mRNA levels in total liver and in hepatocytes (Hep), stellate cells (Stell) and peritoneal macrophages (Mac). E. H&E staining of sections of the indicated organs in 24 months old mice. Arrows indicate lymphocytic infiltration. F. Afp and Il6 mRNA levels in livers and lungs of 24 months old Igfbp7+/+ and Igfbp7−/− mice. For graphs, data represent mean ± SD. *: p<0.01; #: p<0.05. A.U.: arbitrary unit. For all analyses at least 3 mice or more were used per group.

Article Snippet: Mouse Igfbp7 ( {"type":"entrez-nucleotide","attrs":{"text":"NM_001159518","term_id":"226958452"}} NM_001159518 ) expression plasmid was obtained from Origene Technologies, Inc. (MR222256).

Techniques: Control, Immunohistochemistry, Staining

Journal: Cancer research

Article Title: IGFBP7 Deletion Promotes Hepatocellular Carcinoma

doi: 10.1158/0008-5472.CAN-16-2885

Figure Lengend Snippet: Loss of Igfbp7 increases proliferation and inhibits senescence by activating IGF signaling pathway. A. Cell proliferation (left) and colony formation assays (right) in mouse embryonic fibroblasts (MEFs). B. Cell proliferation (left) and IGF-1-stimulated BrdU incorporation assays (right) in hepatocytes. C. Senescence-associated β-galactosidase (SA-β-Gal)-positive hepatocytes at day 8 of culture. D. γ-H2AX positive MEFs after 12 h of serum starvation. E. Cell cycle analysis of MEFs at 18 and 36 h after release from serum-free medium. F. Hepatocytes were cultured in insulin-free medium for 12 hours prior to treatment with IGF-1 (20ng/mL) for the indicated time points. Western blot analyses for the indicated proteins were performed. G. Western blot analysis for the indicated proteins in liver lysates of three independent Igfbp7+/+ and Igfbp7−/− mice. H. Western blot analysis for the indicated proteins in the indicated cells isolated from adult mice. For each cell type a representative EF1α blot is shown as loading control. I. Cell proliferation analysis in MEFs upon treatment with OSI-906 (4 µmol/L). For graphs, data represent mean ± SD. *: p<0.01.

Article Snippet: Mouse Igfbp7 ( {"type":"entrez-nucleotide","attrs":{"text":"NM_001159518","term_id":"226958452"}} NM_001159518 ) expression plasmid was obtained from Origene Technologies, Inc. (MR222256).

Techniques: BrdU Incorporation Assay, Cell Cycle Assay, Cell Culture, Western Blot, Isolation, Control

Journal: Cancer research

Article Title: IGFBP7 Deletion Promotes Hepatocellular Carcinoma

doi: 10.1158/0008-5472.CAN-16-2885

Figure Lengend Snippet: Genetic deletion of Igfbp7 accelerates DEN-induced HCC. Mice were treated with DEN (10 µg/gm) at 2 wks of age and were sacrificed at 32 wks. A. Photograph of DEN-treated mouse livers of the indicated genotypes. B. Number and sizes of liver nodules. C. Liver weight of DEN-injected mice. D–E. Total protein (D) and Aspartate aminotransferase (AST) and Alanine aminotransferase (ALT) (E) levels in DEN-injected mouse sera. For B–E, n = 15 mice for +/+, 12 mice for +/− and 11 mice for −/−. F. H&E and immunostaining for the indicated proteins in liver sections. Scale bar: 20 µm. For graphs, data represent mean ± SD. *: p<0.01.

Article Snippet: Mouse Igfbp7 ( {"type":"entrez-nucleotide","attrs":{"text":"NM_001159518","term_id":"226958452"}} NM_001159518 ) expression plasmid was obtained from Origene Technologies, Inc. (MR222256).

Techniques: Injection, Immunostaining

Journal: Cancer research

Article Title: IGFBP7 Deletion Promotes Hepatocellular Carcinoma

doi: 10.1158/0008-5472.CAN-16-2885

Figure Lengend Snippet: Loss of Igfbp7 generates an inflammatory and immunosuppressive environment. A. Immune profile of DEN-treated Igfbp7+/+ and Igfbp7−/− mice at 20 weeks. B. Levels of the indicated mRNAs in the livers of DEN-injected mice at 32 wks (top), in hepatocytes isolated from DEN-injected mice at 24 wks (middle), and in Kupffer cells isolated from DEN-injected mice at 5 wks (bottom). C. Levels of the indicated mRNAs in hepatocytes in vitro treated with DEN for 24 h (top). Levels of the indicated mRNAs in macrophages treated or not with IL-1β (10 ng/ml) for 6 or 12h. D. Immunofluorescence for p65 NF-κB in hepatocytes treated with LPS (500 ng/mL) for 30 min. E. NF-κB luciferase reporter activity was measured in Igfbp7+/+ and Igfbp7−/− hepatocytes. Firefly luciferase activity was normalized by Renilla luciferase activity. The activity of empty pGL3-basic vector was considered as 1. R.L.U.: Relative luciferase units. F. Western blot analysis for the indicated proteins in LPS-treated hepatocytes and bone marrow-derived macrophages. For graphs, data represent mean ± SD. *: p<0.01; **: p<0.05; #: p<0.04. For all analyses at least 3 mice or more were used per group. A.U.: arbitrary unit.

Article Snippet: Mouse Igfbp7 ( {"type":"entrez-nucleotide","attrs":{"text":"NM_001159518","term_id":"226958452"}} NM_001159518 ) expression plasmid was obtained from Origene Technologies, Inc. (MR222256).

Techniques: Injection, Isolation, In Vitro, Immunofluorescence, Luciferase, Activity Assay, Plasmid Preparation, Western Blot, Derivative Assay

Journal: Cancer research

Article Title: IGFBP7 Deletion Promotes Hepatocellular Carcinoma

doi: 10.1158/0008-5472.CAN-16-2885

Figure Lengend Snippet: Inhibition of immunosurveillance in Igfbp7−/− mice. A. Top biological pathways downregulated in naive Igfbp7−/− livers. B. Tap1 mRNA levels in total liver. C. Levels of CD86 and CD80 in bone marrow-derived dendritic cells (BMDCs). D. Donor pmel-17 T cells were co-cultured with BMDCs pulsed with gp100 at the indicated molar ratios (DC:TC) for 60 h with 2 h pre-treatment with LPS (500 ng/mL) for priming. 3H-thymidine (3HTdR) incorporation was measured (left). IL-12p40 (middle) and IL-2 (right) levels were measured in the media by ELISA. E. gp100-loaded Igfbp7−/− BMDCs were treated or not with rIGFBP7 protein for 3 h followed by OSI-906 (4 µmol/L) for 2 h and LPS (500 ng/mL) for 2 h. The cells were washed and co-cultured with pmel-17 T cells for 60 h. 3H-thymidine (3HTdR) incorporation was measured (left). IFN-γ (middle) and IL-2 (right) levels were measured in the media by ELISA. F. BMDCs were treated with LPS (500 ng/mL) for the indicated time points and Western blot analysis was performed for the indicated proteins. For graphs, data represent mean ± SD. *: p<0.01.

Article Snippet: Mouse Igfbp7 ( {"type":"entrez-nucleotide","attrs":{"text":"NM_001159518","term_id":"226958452"}} NM_001159518 ) expression plasmid was obtained from Origene Technologies, Inc. (MR222256).

Techniques: Inhibition, Derivative Assay, Cell Culture, Enzyme-linked Immunosorbent Assay, Western Blot

Journal: Cancer research

Article Title: IGFBP7 Deletion Promotes Hepatocellular Carcinoma

doi: 10.1158/0008-5472.CAN-16-2885

Figure Lengend Snippet: Overexpression of Igfbp7 decreases tumor growth and stimulates an anti-tumor immune response. A. IGFBP7 protein levels in the conditioned media (top) and Igfbp7 mRNA levels in the cells (bottom) of control and IGFBP7-overexpressing clone (BP7-OE) of Hepa1-6 cells. B. The indicated clones (1X106 cells) were injected s.c. in C57L mice (n=5 per group) and tumor volume was measured. C. Levels of the indicated proteins in tumor lysates at the end of the study. D. IFN-γ-producing CD8+ or CD4+ T cells and MDSCs in the tumors at the end of the study. E. Il12a (left), Ifng (middle) and Tap1 (right) mRNA levels in the tumors at the end of the study. F. BP7-OE cells were injected s.c. in C57L mice (n=5 per group) and treated with Control IgG and CD8+ or CD4+ neutralizing antibodies (200 µg i.p.). Tumor volume was measured. For all graphs, the data represent mean ± SD; *: p<0.01. G. Schematic representation of the molecular mechanism by which IGFBP7 suppresses tumors. IGFBP7, secreted from hepatocytes, inhibits IGF and NF-κB signaling in macrophages preventing inflammation. IGFBP7, secreted from the microenvironment cells, inhibits IGF and NF-κB signaling in hepatocytes thus impeding proliferation and inducing senescence. IGFBP7 promotes antigen presentation by dendritic cells resulting in recruitment of CD4+ and CD8+ cells. These effects collectively protect from HCC. In addition to the paracrine effects depicted in this diagram there might be autocrine effects regulating the same end-points.

Article Snippet: Mouse Igfbp7 ( {"type":"entrez-nucleotide","attrs":{"text":"NM_001159518","term_id":"226958452"}} NM_001159518 ) expression plasmid was obtained from Origene Technologies, Inc. (MR222256).

Techniques: Over Expression, Control, Clone Assay, Injection, Immunopeptidomics