Journal: Gene Therapy

Article Title: Efficacy of combining ING4 and TRAIL genes in cancer-targeting gene virotherapy strategy: first evidence in preclinical hepatocellular carcinoma

doi: 10.1038/gt.2017.86

Figure Lengend Snippet: Validation of TRAIL and ING4 gene expression in human HCC cells treated with Ad-ΔB/TRAIL and/or Ad-ΔB/ING4. ( a ) Schematic representation of genomic structure of generated oncolytic adenovirus (Ad-ΔB) armed with human ING4 gene (Ad-ΔB/ING4) or human TRAIL gene (Ad-ΔB/TRAIL). ING4 or TRAIL gene was incorporated in the E3 region of the viral backbone under the transcriptional control of the human cytomegalovirus (CMV) promoter. Next, viral replication and gene expression pattern of TRAIL and ING4 was validated on human HCC HuH7 cells treated with PBS, Ad-ΔB/TRAIL, Ad-ΔB/ING4 or Ad-ΔB/TRAIL + ING4 at a MOI of 5 for each virus. The harvested cells and their supplements were analyzed by qRT-PCR to measure the relative mRNA of TRAIL ( b ) and ING4 ( d ), ELISA; to measure TRAIL protein ( c ) and western blot ( e and f ); to measure the expression level of ING4, TRAIL and Ad production by measuring E1A protein. Data of ( b – d ) are represented as mean±s.e. *** P <0.001 versus PBS.

Article Snippet: Thereafter, the membranes were incubated overnight at 4 °C with rabbit polyclonal primary antibodies (Santa Cruz Biotechnology, Inc.) against ING4, Ad E1A, TRAIL and β-actin.

Techniques: Biomarker Discovery, Gene Expression, Generated, Control, Virus, Quantitative RT-PCR, Enzyme-linked Immunosorbent Assay, Western Blot, Expressing

Journal: Gene Therapy

Article Title: Efficacy of combining ING4 and TRAIL genes in cancer-targeting gene virotherapy strategy: first evidence in preclinical hepatocellular carcinoma

doi: 10.1038/gt.2017.86

Figure Lengend Snippet: In vitro effect of mono- and combined therapy with Ad-ΔB/TRAIL and Ad-ΔB/ING4 on human HCC and normal liver cells. ( a ) Quantitative results of methylthiazolyltetrazolium (MTT) assay showing the inhibitor effects on viability of human HCC (Hep3B) cells treated with 5 and 10 MOI per vector. ( b ) Quantitative results of MTT assay showing the inhibitor effects on viability of human HCC (HuH7) cells treated with 10 and 20 MOI per vector. Data are represented as mean±s.e. *** P <0.001 versus PBS. ( c ) Representatives of crystal violet staining assay demonstrating the cytotoxic effects on human HCC (Hep3B and HuH7) cells, and on normal human liver (WRL68) cells, at the indicated MOIs of each vector.

Article Snippet: Thereafter, the membranes were incubated overnight at 4 °C with rabbit polyclonal primary antibodies (Santa Cruz Biotechnology, Inc.) against ING4, Ad E1A, TRAIL and β-actin.

Techniques: In Vitro, MTT Assay, Plasmid Preparation, Staining

Journal: Gene Therapy

Article Title: Efficacy of combining ING4 and TRAIL genes in cancer-targeting gene virotherapy strategy: first evidence in preclinical hepatocellular carcinoma

doi: 10.1038/gt.2017.86

Figure Lengend Snippet: Antitumor effect of mono- and combined therapy with Ad-ΔB/TRAIL and Ad-ΔB/ING4 on orthotopic mouse of human HCC model. The model was established by intrahepatic implantation of 2 × 10 6 human HCC Hep3B/fluc cells into the liver lobes of athymic nude mice. As described in Methodology Part , confirmed positive HCC-bearing mice ( n =40) were randomly and equally assigned into four groups and systemically treated with PBS alone (Group 1), Ad-ΔB/TRAIL (Group 2), Ad-ΔB/ING4 (Group 3) and Ad-ΔB/TRAIL + ING4 (Group 4) at a dosage regimen of 1 × 10 10 VP in 200 μl PBS of each virus; repeated three times every other day. At day 3 post-treatment, mice of each group were further subdivided into two subgroups: the first subgroup (4 mice/group) was killed and their blood and liver tumor tissues were harvested and used for biochemical, histopathological and mechanistic explorations, while the second one (6 mice/group) was maintained and monitored by in vivo bioluminescence imaging (BLI) at days 0, 7, 14, 21, 28, and 35 post-treatment to determine the tumor response rate and the therapeutic effect of each tested treatment strategy. ( a ) Photographed BLI showing tumor growth and its response to each applied treatment. ( b ) BLI signals were calculated after background subtraction in total flux photons/s from a body region of interest. Data presented as mean±s.e. * P <0.05, ** P <0.01, *** P <0.001.

Article Snippet: Thereafter, the membranes were incubated overnight at 4 °C with rabbit polyclonal primary antibodies (Santa Cruz Biotechnology, Inc.) against ING4, Ad E1A, TRAIL and β-actin.

Techniques: Virus, In Vivo, Imaging

Journal: Gene Therapy

Article Title: Efficacy of combining ING4 and TRAIL genes in cancer-targeting gene virotherapy strategy: first evidence in preclinical hepatocellular carcinoma

doi: 10.1038/gt.2017.86

Figure Lengend Snippet: Validation of viral replication and TRAIL and ING4 gene expression in the harvested tumor tissues at day 3 post-treatment. The harvested liver tumor tissues of HCC-bearing mice treated with PBS, Ad-ΔB/TRAIL, Ad-ΔB/ING4 or Ad-ΔB/TRAIL+ ING4 were analyzed by qRT-PCR to measure the relative mRNA levels of TRAIL ( a ) and ING4 ( c ), while the expressed proteins of TRAIL ( b ) and ING4 ( d ) were measured using ELISA and western blot assay, respectively. The expression of Ad E1A protein (as an indicator of viral production) was also assessed by western blot assay ( d ). Data points of (a–c) are mean expression±s.e. *** P <0.001 versus PBS.

Article Snippet: Thereafter, the membranes were incubated overnight at 4 °C with rabbit polyclonal primary antibodies (Santa Cruz Biotechnology, Inc.) against ING4, Ad E1A, TRAIL and β-actin.

Techniques: Biomarker Discovery, Gene Expression, Quantitative RT-PCR, Enzyme-linked Immunosorbent Assay, Western Blot, Expressing

Journal: Gene Therapy

Article Title: Efficacy of combining ING4 and TRAIL genes in cancer-targeting gene virotherapy strategy: first evidence in preclinical hepatocellular carcinoma

doi: 10.1038/gt.2017.86

Figure Lengend Snippet: Histopathological, immunological, antiangiogenic and TUNEL findings in the harvested tumor tissues at day 3 post-treatment. The harvested liver tumor tissues of HCC-bearing mice treated with PBS, Ad-ΔB/TRAIL, Ad-ΔB/ING4 or Ad-ΔB/TRAIL + ING4 were subjected to histopathological (H & E staining), immunohistochemical (IHC) staining and in situ apoptosis detection by TUNEL assay; and for ELISA assay for intratumoral levels of IFN-γ and VEGF. ( a ) Representative photographs of H & E staining (panel i); IHC staining of CD31-positive vascular endothelial cells (panel ii); and TUNEL staining of apoptotic cells (panel iii) in the harvested tumor tissues of different groups. Original magnification: × 400. ( b – d ) are relative intratumoral levels of IFN-γ, VEGF and microvascular density, respectively. Results are mean ± s.e. * P <0.05, *** P <0.001 versus PBS. ( e ) Tumor tissues were stained with anti-DX5 (green) and anti-IFN-γ (red) antibodies to assess intratumoral infiltration of NK cells and their activation. Original magnification, × 200. ( f ) The mean intensity of DX5-positive cells was quantified from three independent fields within five different microscope images for each experimental group. Data are representative of three independent experiments. * P <0.05, ** P <0.01 or *** P <0.001 versus PBS-treated group. ( g ) The mean intensity of IFN-γ-positive cells was quantified from three independent fields within five different microscope images for each experimental group. Data are representative of three independent experiments. ** P <0.01 or *** P <0.001 versus PBS-treated group. ( h ) The percentage of DX5 + IFN-γ + NK cells was assessed by ImageJ software. The mean intensity of DX5 + IFN-γ + NK cells was quantified from three independent fields within five different microscope images for each experimental group. Data are representative of three independent experiments. ** P <0.01 or *** P <0.001 versus PBS-treated group.

Article Snippet: Thereafter, the membranes were incubated overnight at 4 °C with rabbit polyclonal primary antibodies (Santa Cruz Biotechnology, Inc.) against ING4, Ad E1A, TRAIL and β-actin.

Techniques: TUNEL Assay, Staining, Immunohistochemical staining, Immunohistochemistry, In Situ, Enzyme-linked Immunosorbent Assay, Activation Assay, Microscopy, Software

Journal: Gene Therapy

Article Title: Efficacy of combining ING4 and TRAIL genes in cancer-targeting gene virotherapy strategy: first evidence in preclinical hepatocellular carcinoma

doi: 10.1038/gt.2017.86

Figure Lengend Snippet: In vivo biochemical findings

Article Snippet: Thereafter, the membranes were incubated overnight at 4 °C with rabbit polyclonal primary antibodies (Santa Cruz Biotechnology, Inc.) against ING4, Ad E1A, TRAIL and β-actin.

Techniques: In Vivo

Journal: Gene Therapy

Article Title: Efficacy of combining ING4 and TRAIL genes in cancer-targeting gene virotherapy strategy: first evidence in preclinical hepatocellular carcinoma

doi: 10.1038/gt.2017.86

Figure Lengend Snippet: In vitro apoptotic findings and expression pattern of caspases-8 and-3 in HCC cells and tumor tissues. ( a ) In vitro flow cytometric analysis of apoptosis induction in HCC cells treated with Ad-ΔB/TRAIL, Ad-ΔB/ING4 or Ad-ΔB/TRAIL + ING4 by using Annexin V and propidium iodide (PI) fluorescence staining assay. Each scatter plot demonstrates the percentage of early and late apoptotic cells. *** P <0.001 versus PBS. Next, western blotting analysis of human HCC cells ( b ) and harvested tumor tissues ( c ) treated with PBS, Ad-ΔB/TRAIL, Ad-ΔB/ING4 or Ad-ΔB/TRAIL + ING4 showing the expression pattern of caspase-8 and cleaved caspase-3.

Article Snippet: Thereafter, the membranes were incubated overnight at 4 °C with rabbit polyclonal primary antibodies (Santa Cruz Biotechnology, Inc.) against ING4, Ad E1A, TRAIL and β-actin.

Techniques: In Vitro, Expressing, Fluorescence, Staining, Western Blot

Journal: Oncology Letters

Article Title: Enhanced anti-melanoma efficacy of interferon α-2b via overexpression of ING4 by enhanced Fas/FasL-mediated apoptosis

doi: 10.3892/ol.2018.8534

Figure Lengend Snippet: Synergistic effects of ING4 and IFN-α2b expression on A375 cells. (A) The expression of ING4 at 48 h following transfection with LV-control, LV-ING4-RNAi or LV-ING4 in A375 cells (left panel) or HT-144 cells (right panel). A375 cells were treated with LV-control, LV-ING4-RNAi or LV-ING4 for 48 h. The cells were subsequently treated with 5×104 IU/l IFN-α2b for (B) 24 or (C) 48 h. HT-144 Cells were treated with LV-control, LV-ING4-RNAi or LV-ING4 for 48 h, then treated with 5×104 IU/l IFN-α2b for (D) 24 or (E) 48 h. Cell viability was examined by MTT assay. *P<0.05, **P<0.01.

Article Snippet: The membranes were incubated with antibodies against ING4 (cat. no. sc-135742), PARP (cat. no. sc-136208), caspase 8 (cat. no. sc-6136), caspase 3 (cat. no. sc-271759), Fas (cat. no. sc-4856), FasL (cat. no. sc-71096) (dilution, 1:1,000; Santa Cruz Biotechnology, Inc.) and cleaved PARP (cat. no. 5625), anti-cleaved caspase-8 (cat. no. 8529) and cleaved caspase-3 (cat. no. 9661) (dilution, 1:1,000; Cell Signaling Technology, Inc., Danvers, MA, USA) for 1 h at room temperature.

Techniques: Expressing, Transfection, Control, MTT Assay

Journal: Oncology Letters

Article Title: Enhanced anti-melanoma efficacy of interferon α-2b via overexpression of ING4 by enhanced Fas/FasL-mediated apoptosis

doi: 10.3892/ol.2018.8534

Figure Lengend Snippet: Combination of ING4 and IFN-α2b overexpression increases the apoptosis of A375 melanoma cell lines more markedly compared with overexpression of either gene alone. Fluorescence-activated cell sorting analysis of A375 cells at 48 h following transfection with LV-control, LV-ING4-RNAi or LV-ING4. A375 cells were stained with Annexin V-fluorescein isothiocycanate and 7-AAD following transfection with LV-control, LV-ING4-RNAi or LV-ING4. The cells were either treated (A) without or (B) with 5×104 IU/l IFN-α2b. The percentages represent the proportion of cells that are Annexin V-positive/7-AAD-negative (early apoptotic) and Annexin V-positive/7-AAD-positive cells (apoptotic). IFN, interferon; ING4, inhibitor of growth family member 4; PBS, phosphate buffered saline; PE, phycoerythrin; 7-AAD, 7-aminoactinomycin D.

Article Snippet: The membranes were incubated with antibodies against ING4 (cat. no. sc-135742), PARP (cat. no. sc-136208), caspase 8 (cat. no. sc-6136), caspase 3 (cat. no. sc-271759), Fas (cat. no. sc-4856), FasL (cat. no. sc-71096) (dilution, 1:1,000; Santa Cruz Biotechnology, Inc.) and cleaved PARP (cat. no. 5625), anti-cleaved caspase-8 (cat. no. 8529) and cleaved caspase-3 (cat. no. 9661) (dilution, 1:1,000; Cell Signaling Technology, Inc., Danvers, MA, USA) for 1 h at room temperature.

Techniques: Over Expression, Fluorescence, FACS, Transfection, Control, Staining, Saline

Journal: Oncology Letters

Article Title: Enhanced anti-melanoma efficacy of interferon α-2b via overexpression of ING4 by enhanced Fas/FasL-mediated apoptosis

doi: 10.3892/ol.2018.8534

Figure Lengend Snippet: Western blot analysis of PARP, cleaved PARP, caspase 8, cleaved caspase 8, caspase 3, cleaved caspase 3, Fas and FasL in A375 cells that were treated with PBS, IFN-α2b, a combination of IFN-α2b and LV-ING4 or LV-ING4 alone. ING4, inhibitor of growth family member 4; PARP, poly(ADP-ribose) polymerase; FasL, Fas ligand; IFN, interferon.

Article Snippet: The membranes were incubated with antibodies against ING4 (cat. no. sc-135742), PARP (cat. no. sc-136208), caspase 8 (cat. no. sc-6136), caspase 3 (cat. no. sc-271759), Fas (cat. no. sc-4856), FasL (cat. no. sc-71096) (dilution, 1:1,000; Santa Cruz Biotechnology, Inc.) and cleaved PARP (cat. no. 5625), anti-cleaved caspase-8 (cat. no. 8529) and cleaved caspase-3 (cat. no. 9661) (dilution, 1:1,000; Cell Signaling Technology, Inc., Danvers, MA, USA) for 1 h at room temperature.

Techniques: Western Blot

Journal: Journal of Ovarian Research

Article Title: Combinatorial strategies based on CRAd-IL24 and CRAd-ING4 virotherapy with anti-angiogenesis treatment for ovarian cancer

doi: 10.1186/s13048-016-0248-5

Figure Lengend Snippet: Validation of CRAd-mediated expression of IL-24 and ING4 genes. a ) The concentrations of IL-24 protein following infection of the indicated OvCa cell lines and normal IOSE523 cells with CRAd-IL24 at the MOIs of 1 and 10 vp/cell were determined in cell culture supernatants 3 days postinfection using commercial ELISA kit with IL-24 concentration standards. Each bar represents the cumulative mean ± SD (* p ≤ 0.05). b) The relative levels of ING4 gene expression were determined following infection of SKOV3ip.1 cells with CRAd-ING4 at the MOIs of 100, 33, and 11 vp/cell. The ING4 protein band of 29 kDa was detected in cell lysates 3 days postinfection with 100, 33, and 11 vp/cell, but not in mock-infected cells (0 vp/cell) using Western blot with rabbit polyclonal Ab raised against ING4 internal region

Article Snippet: Electrophoretically resolved proteins were transferred to a polyvinylidene fluoride membrane and analyzed for the presence of ING4 polypeptides using polyclonal rabbit antibody raised against ING4 internal region (Assay Biotechnology Company, Inc) diluted 1:1000 for overnight incubation at 4 °C.

Techniques: Biomarker Discovery, Expressing, Infection, Cell Culture, Enzyme-linked Immunosorbent Assay, Concentration Assay, Gene Expression, Western Blot

Journal: Breast Cancer : Targets and Therapy

Article Title: Tumor suppressor ING4 inhibits estrogen receptor activity in breast cancer cells

doi: 10.2147/BCTT.S119691

Figure Lengend Snippet: ER+ breast cancer cells overexpressing ING4 are more sensitive to hormone deprivation and to tamoxifen treatment. Notes: T47D (A) or MCF7 (B) cells ectopically expressing the pMIG vector or ING4 were hormone-deprived in media containing CSS for 3 days (day 0, d0), followed by 7 days in media containing FS, CSS, CSS+10 nM E2, or CSS+E2 in the presence of OHT in 10, 100 nM, or 1 μM. Cell growth was assessed by SRB colorimetric assay. * p -value <0.001, ** p -value <0.002. Error bars represent minimal and maximal values. Abbreviations: ER+, estrogen receptor-positive; ING4, inhibitor of growth 4; CSS, charcoal-stripped serum; FS, full serum; E2, 17β-estradiol; OHT, 4-hydroxy tamoxifen; SRB, sulforhodamine B.

Article Snippet: Nuclear and cytoplasmic fractions were analyzed by Western blot using antibodies against ERα (Cell Signaling, Danvers, MA, USA), ING4 (EMD Millipore, Temecula, CA, USA), histone H3 (Cell Signaling), and tubulin (Cell Signaling), and phospho-extracellular signal-regulated kinase (ERK) (Cell Signaling) at 1:1,000 dilution.

Techniques: Expressing, Plasmid Preparation, Colorimetric Assay

Journal: Breast Cancer : Targets and Therapy

Article Title: Tumor suppressor ING4 inhibits estrogen receptor activity in breast cancer cells

doi: 10.2147/BCTT.S119691

Figure Lengend Snippet: ING4 inhibits ERα transcription activity without affecting ERα protein expression. Notes: (A) Amounts of the nuclear ERα protein were comparable between the pMIG vector and ING4 expressing T47D or MCF7 cells. Cells were treated for 24 h with FS, CSS, CSS+10 nM E2, CSS+10 nM E2+1 μM OHT, or CSS+10 nM E2+100 nM ICI182,780. Total lysate, cytosolic, and nuclear fractions were analyzed by Western blot with antibodies against ING4, ERα, or phospho-ERK. Antibodies against histone H3 (nuclear) and α-tubulin (cytosolic) were used for loading control. ( B ) ING4 inhibits the expression of an ER-target gene, PDZK1 . PDZK1 mRNA levels in the cells treated with the same assay conditions in (A) were quantified by qRTPCR normalized to GAPDH as sample control. Relative expression was normalized to pMIG (FS) samples. “-” represents no OHT or ICI added. Abbreviations: ING4, inhibitor of growth 4; CSS, charcoal stripped serum; FS, full serum; E2, 17β-estradiol; OHT, 4-hydroxy tamoxifen; ICI, ICI182,780; ER, estrogen receptor; qRTPCR, quantitative reverse transcription polymerase chain reaction; ERK, extracellular signal-regulated kinase.

Article Snippet: Nuclear and cytoplasmic fractions were analyzed by Western blot using antibodies against ERα (Cell Signaling, Danvers, MA, USA), ING4 (EMD Millipore, Temecula, CA, USA), histone H3 (Cell Signaling), and tubulin (Cell Signaling), and phospho-extracellular signal-regulated kinase (ERK) (Cell Signaling) at 1:1,000 dilution.

Techniques: Activity Assay, Expressing, Plasmid Preparation, Western Blot, Control, Reverse Transcription, Polymerase Chain Reaction

Journal: Breast Cancer : Targets and Therapy

Article Title: Tumor suppressor ING4 inhibits estrogen receptor activity in breast cancer cells

doi: 10.2147/BCTT.S119691

Figure Lengend Snippet: ING4 inhibits ligand-independent ER activity. Notes: (A) ICI treatment, not OHT, of the vector pMIG expressing T47D cells results in diminished PDZK1 expression comparable to ING4 expressing cells. Cells were treated with 1 μM OHT or 1 μM ICI in FS, CSS, or CSS+E2, for 24 h and harvested for total RNA isolation. PDZK1 mRNA was quantified by qRTPCR, normalized to GAPDH . ( B ) ICI, not OHT, suppresses hormone-independent growth of pMIG expressing T47D cells in a dose-dependent manner. Cells were grown in CSS media for 10 days with OHT in incremental concentrations of 100 nM, 1, or 10 μM, or with ICI in incremental concentrations of 100 nM, 1, or 10 μM. “-” represents no OHT or ICI added. Abbreviations: ING4, inhibitor of growth 4; CSS, charcoal stripped serum; FS, full serum; E2, 17β-estradiol; OHT, 4-hydroxy tamoxifen; ER, estrogen receptor; ICI, ICI182,780; qRTPCR, quantitative reverse transcription polymerase chain reaction.

Article Snippet: Nuclear and cytoplasmic fractions were analyzed by Western blot using antibodies against ERα (Cell Signaling, Danvers, MA, USA), ING4 (EMD Millipore, Temecula, CA, USA), histone H3 (Cell Signaling), and tubulin (Cell Signaling), and phospho-extracellular signal-regulated kinase (ERK) (Cell Signaling) at 1:1,000 dilution.

Techniques: Activity Assay, Plasmid Preparation, Expressing, Isolation, Reverse Transcription, Polymerase Chain Reaction

Journal: Breast Cancer : Targets and Therapy

Article Title: Tumor suppressor ING4 inhibits estrogen receptor activity in breast cancer cells

doi: 10.2147/BCTT.S119691

Figure Lengend Snippet: ING4 represses transcription of selective ER-target genes containing ERE. Notes: (A) Expression of a luciferase reporter construct containing an ERE promoter sequence in T47D cells. Cells were treated with CSS (−) for 2 days and additional 24 h with (E2) or without (−) 10 nM E2. ( B ) Relative mRNA expression of the ER-target genes, TFF1 , PDZK1 , MYC , and PGR in cells treated with CSS (−E2) or 10 nM E2 (+E2) for 4, 24, or 72 h. Abbreviations: ING4, inhibitor of growth 4; CSS, charcoal stripped serum; FS, full serum; E2, 17β-estradiol; ER, estrogen receptor; ERE, estrogen response element.

Article Snippet: Nuclear and cytoplasmic fractions were analyzed by Western blot using antibodies against ERα (Cell Signaling, Danvers, MA, USA), ING4 (EMD Millipore, Temecula, CA, USA), histone H3 (Cell Signaling), and tubulin (Cell Signaling), and phospho-extracellular signal-regulated kinase (ERK) (Cell Signaling) at 1:1,000 dilution.

Techniques: Expressing, Luciferase, Construct, Sequencing