ikk inhibitor bay 117085  (Cayman Chemical)

Journal: The Journal of Biological Chemistry

Article Title: Histone Deacetylase (HDAC) Inhibition Induces IκB Kinase (IKK)-dependent Interleukin-8/CXCL8 Expression in Ovarian Cancer Cells *

doi: 10.1074/jbc.M116.771014

Figure Lengend Snippet: Vorinostat-induced IL-8/CXCL8 expression is dependent on IKK activity and associated with IKKβ recruitment to the IL-8 promoter in ovarian cancer cells. A, Western blotting of CEs and NEs prepared from SKOV3 cells incubated with vorinostat for 48 h and analyzed by using p65, Lys-314/315 ac-p65, p50, IκBα, histone H3, Lys-9/14 ac-histone H3, and actin antibodies. Each lane corresponds to ∼5 × 104 cells. B, densitometric evaluation of IκBα levels in CEs and NEs (top panel) and of p65, Lys-314/315 ac-p65, p50, histone H3, and Lys-9/14 ac-histone H3 in NEs (bottom panel) of SKOV3 cells shown in A. The protein densities were normalized to actin. The values in untreated cells were arbitrarily set to 1, and the other values are presented relative to these values. The data represent the means of three experiments ± S.E. *, p < 0.05 compared with untreated cells. C and D, real-time RT-PCR analysis of IL-8 mRNA (C) and ELISA of the released IL-8 (D) in SKOV3 cells preincubated for 12 h with 5 μm Bay 117085, 5 mm aspirin, 10 μm SB 203580, or control DMSO and then treated for 48 h with 1.5 μm vorinostat. E, ChIP analysis of IKKα, IKKβ, and IKKϵ occupancy at the IL-8 promoter quantified by real-time PCR in SKOV3 cells incubated for 48 h with increasing concentrations of vorinostat. F, ChIP of IKKβ occupancy at the IL-8, TNFα, and IL-6 promoters in SKOV3 cells incubated for 48 h with vorinostat. The results in E and F are presented as -fold difference in occupancy over the human IGX1A (SA Biosciences) sequence control and represent the mean ± S.E. of three experiments. G, real-time RT-PCR analysis of IKKα, IKKβ, and IKKϵ mRNA levels in SKOV3 cells incubated for 48 h with increasing vorinostat. H, Western blotting analysis of IKKβ and control actin levels in WCEs of SKOV3 cells incubated for 48 h with increasing vorinostat (top panel). Each lane corresponds to ∼5 × 104 cells. Bottom panel, densitometric evaluation of IKKβ (shown in the top panel) normalized to actin. The values are presented relative to the value in untreated cells, which was set to 1. The data represent the means of three experiments ±S.E.

Article Snippet: The IKK inhibitor Bay 117085 was purchased from Cayman Chemicals (Ann Arbor, MI).

Techniques: Expressing, Activity Assay, Western Blot, Incubation, Quantitative RT-PCR, Enzyme-linked Immunosorbent Assay, Real-time Polymerase Chain Reaction, Sequencing

Journal: The Journal of Biological Chemistry

Article Title: Histone Deacetylase (HDAC) Inhibition Induces IκB Kinase (IKK)-dependent Interleukin-8/CXCL8 Expression in Ovarian Cancer Cells *

doi: 10.1074/jbc.M116.771014

Figure Lengend Snippet: Vorinostat induces p65, Lys-314/315-acetylated p65, and Lys-9/14-acetylated histone H3 recruitment to the IL-8/CXCL8 promoter in ovarian cancer cells. A, ChIP analysis of p65 occupancy at the IL-8, TNFα, and IL-6 promoters quantified by real-time PCR in SKOV3 cells incubated for 48 h with vorinostat. B, ChIP of p65 occupancy at the IL-8 promoter in SKOV3 cells preincubated 12 h with 5 μm Bay 117085 or control DMSO and treated for 48 h with increasing concentrations of vorinostat. C, ChIP of Lys-314/315 ac-p65 occupancy at the IL-8, TNFα, and IL-6 promoters in SKOV3 cells incubated for 48 h with vorinostat. D, ChIP of Lys-314/315 ac-p65 occupancy at the IL-8 promoter in SKOV3 cells preincubated for 12 h with 5 μm Bay 117085 or control DMSO and treated for 48 h with vorinostat. E, ChIP of Lys-9/14 ac-histone H3 occupancy at the IL-8, TNFα, and IL-6 promoters in SKOV3 cells incubated for 48 h with vorinostat. F, ChIP of Lys-9/14 ac-histone H3 occupancy at the IL-8 promoter in SKOV3 cells preincubated for 12 h with 5 μm Bay 117085 or control DMSO and treated for 48 h with vorinostat. G, time course of p65, Lys-314/315 ac-p65, Lys-9/14 ac-histone H3, and IKKβ occupancy at the IL-8/CXCL8 promoter in SKOV3 cells incubated with 1.5 μm vorinostat (Vor) or control DMSO and analyzed by ChIP. The data in A–G are presented as -fold difference in occupancy of the particular protein at the particular locus in comparison with the human IGX1A (SA Biosciences) locus and represent the mean ± S.E. of three experiments. *, p < 0.05; **, p < 0.01; ***, p < 0.001 compared with cells treated with DMSO.

Article Snippet: The IKK inhibitor Bay 117085 was purchased from Cayman Chemicals (Ann Arbor, MI).

Techniques: Real-time Polymerase Chain Reaction, Incubation

Journal: The Journal of Biological Chemistry

Article Title: Histone Deacetylase (HDAC) Inhibition Induces IκB Kinase (IKK)-dependent Interleukin-8/CXCL8 Expression in Ovarian Cancer Cells *

doi: 10.1074/jbc.M116.771014

Figure Lengend Snippet: Combination of vorinostat and Bay 117085 enhances vorinostat effectiveness in reducing tumor growth in nude mice implanted with ovarian cancer xenografts. A, average body weight of mice in four treatment groups (n = 7) (control, Bay 117085, vorinostat (Vor), and Bay 117085/vorinostat combination) over the course of 4 weeks. B, average tumor volumes in the four treatment groups (n = 7) over the course of 4 weeks. C, excised SKOV3 tumors implanted subcutaneously in mice (n = 7) after 4 weeks of treatment. D, average weight of the excised tumors (n = 7) at the end of the 4-week treatment period. The values represent the mean ± S.E. *, p < 0.05; **, p < 0.01.

Article Snippet: The IKK inhibitor Bay 117085 was purchased from Cayman Chemicals (Ann Arbor, MI).

Techniques:

Journal: The Journal of Biological Chemistry

Article Title: Histone Deacetylase (HDAC) Inhibition Induces IκB Kinase (IKK)-dependent Interleukin-8/CXCL8 Expression in Ovarian Cancer Cells *

doi: 10.1074/jbc.M116.771014

Figure Lengend Snippet: Combination of vorinostat and Bay 117085 decreases IL-8/CXCL8 levels in vivo. A, IL-8 and TGFβ1 mRNA levels analyzed by real-time RT-PCR in excised tumors from the four treatment groups (n = 7). Vor, vorinostat. B, IL-8 and TGFβ1 release measured by ELISA in mouse plasma samples in the four treatment groups (n = 7) at the end of the experiment. C, representative Western blotting of tumor WCEs analyzed using antibodies against IL-8/CXCL8, TGFβ1, murine neutrophil [7/4] antigen, and actin. D, densitometry evaluation of IL-8/CXCL8, TGFβ1, and the murine neutrophil [7/4] antigen in tumor samples; the band intensities were normalized to actin (n = 5). The values represent the mean ± S.E. *, p < 0.05; **, p < 0.01; ***, p < 0.001).

Article Snippet: The IKK inhibitor Bay 117085 was purchased from Cayman Chemicals (Ann Arbor, MI).

Techniques: In Vivo, Quantitative RT-PCR, Enzyme-linked Immunosorbent Assay, Western Blot