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.2017 Mar 24;292(12):5043-5054.
doi: 10.1074/jbc.M116.771014. Epub 2017 Feb 6.

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

Affiliations

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

Himavanth R Gatla et al. J Biol Chem..

Abstract

Overexpression of the pro-angiogenic chemokine IL-8 (CXCL8) is associated with a poor prognosis in several solid tumors, including epithelial ovarian cancer (EOC). Even though histone deacetylase (HDAC) inhibition has shown remarkable antitumor activity in hematological malignancies, it has been less effective in solid tumors, including EOC. Here we report results that may explain the decreased efficiency of HDAC inhibition in EOC, based on our data demonstrating that HDAC inhibition specifically induces expression of IL-8/CXCL8 in SKOV3, CAOV3, and OVCAR3 cells. Suppression or neutralization of vorinostat-induced IL-8/CXCL8 potentiates the vorinostat inhibitory effect on cell viability and proliferation. The IL-8/CXCL8 expression induced by vorinostat in EOC cells is dependent on IκB kinase (IKK) activity and associated with a gene-specific recruitment of IKKβ and IKK-dependent recruitment of p65 NFκB to the IL-8/CXCL8 promoter. In addition, HDAC inhibition induces acetylation of p65 and histone H3 and their IL-8/CXCL8 promoter occupancy.In vivo results demonstrate that combining vorinostat and the IKK inhibitor Bay 117085 significantly reduces tumor growth in nude mice compared with control untreated mice or either drug alone. Mice in the combination group had the lowest IL-8/CXCL8 tumor levels and the lowest tumor expression of the murine neutrophil [7/4] antigen, indicating reduced neutrophil infiltration. Together, our results demonstrate that HDAC inhibition specifically induces IL-8/CXCL8 expression in EOC cells and that the mechanism involves IKK, suggesting that using IKK inhibitors may increase the effectiveness of HDAC inhibitors when treating ovarian cancer and other solid tumors characterized by increased IL-8/CXCL8 expression.

Keywords: CXCL8; IκB kinase; chemokine; histone deacetylase (HDAC); histone deacetylase inhibitor (HDI); interleukin; interleukin-8; ovarian cancer.

© 2017 by The American Society for Biochemistry and Molecular Biology, Inc.

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Conflict of interest statement

The authors declare that they have no conflicts of interest with the contents of this article

Figures

FIGURE 1.
FIGURE 1.
HDAC inhibition induces IL-8/CXCL8 expression in ovarian cancer SKOV3 cells.A andB, real-time RT-PCR analysis of mRNA levels (A) and ELISA of cytokine release of IL-8, TNFα, and IL-6 (B) in SKOV3 cells incubated with vorinostat for 48 h.C andD, real-time RT-PCR analysis (C) of IL-8 mRNA levels and ELISA of IL-8 cytokine release (D) in SKOV3 cells incubated with 1.5 μm vorinostat or control DMSO for up to 72 h.E, RT-PCR analysis of IL-8 mRNA in SKOV3 cells incubated with romidepsin for 48 h.F, RT-PCR of IL-8 mRNA in SKOV3 cells incubated for 48 h with equal concentrations of vorinostat, romidepsin, or apicidin. The values represent the mean ± S.E. of four experiments. *,p < 0.05; **,p < 0.01; ***,p < 0.001 compared with control UT cells.
FIGURE 2.
FIGURE 2.
Regulation of NFκB-dependent genes by vorinostat in ovarian cancer cells.A andB, real-time RT-PCR analysis of mRNA levels of the NFκB-regulated genes IL-8/CXCL8, CXCL5, TGFβ1, cIAP-1, Bcl-2, p65, p50, IκBα, CXCR1, and CXCR2 in SKOV3 (A) and CAOV3 (B) cells incubated with 1.5 μm vorinostat or control DMSO for 48 h.C, RT-PCR of IL-8 mRNA in CAOV3 cells incubated with increasing vorinostat concentrations for 48 h.D, RT-PCR of the above NFκB-regulated genes in OVCAR3 cells incubated with 1.5 μm vorinostat or DMSO for 48 h.E, RT-PCR of IL-8 mRNA in OVCAR3 cells incubated with increasing vorinostat concentrations for 48 h. The values represent the mean ± S.E. of four experiments. *,p < 0.05; **,p < 0.01; ***,p < 0.001 compared with control UT cells.
FIGURE 3.
FIGURE 3.
Suppression of vorinostat-induced IL-8/CXCL8 expression enhances vorinostat proapoptotic and antiproliferative effects in OC cells.A andB, SKOV3 cells were transfected with control siRNA or IL-8-specific siRNA-1 or siRNA-2, treated for 48 h with 0, 2, and 5 μm vorinostat, and analyzed for IL-8 mRNA expression by real-time RT-PCR (A) and for IL-8 release by ELISA (B).C, cell viability was measured by trypan blue exclusion in cells transfected with control, IL-8 siRNA-1, or IL-8 siRNA-2 and incubated with vorinostat for 48 h.D andE, apoptosis was analyzed by cytoplasmic nucleosome enrichment assay (D), and the colony formation rate was evaluated as the percentage of plated cells that formed colonies (E) in cells transfected with control or IL-8 siRNA-1 and incubated with 0, 2, and 5 μm vorinostat for 48 h.F, cell proliferation was measured by CellTiter 96 One Solution cell proliferation assay in cells transfected with control or IL-8 siRNA-1 and incubated with 1.5 μm vorinostat or control DMSO. The values represent the mean ± S.E. of four experiments. *,p < 0.05; **,p < 0.01 compared with cells transfected with the corresponding control siRNA.
FIGURE 4.
FIGURE 4.
Neutralization of vorinostat-induced IL-8/CXCL8 potentiates the vorinostat inhibitory effect on OC cell viability and proliferation.A andB, SKOV3 and CAOV3 cells were incubated for 48 h with 0, 2, and 5 μm vorinostat in the presence of 2 μg/ml of control mouse IgG1 or 2 μg/ml of mouse anti-human IL-8 IgG1, and cell viability (A) and proliferation (B) were measured as described above.C, Western blotting analysis of CXCR1 and CXCR2 in WCEs of SKOV3 cells incubated with 0, 2, and 5 μm vorinostat for 48 h. Each lane corresponds to ∼5 × 104 cells.D, proliferation of SKOV3 cells incubated for 48 h with 0 and 5 μm vorinostat in the presence of 2 μg/ml of control mouse IgG1 or anti-IL-8 IgG1 with and without 0.5 μg/ml of exogenously added recombinant human IL-8/CXCL8 or CXCL5 proteins. The values represent the mean ± S.E. of three experiments. *,p < 0.05; **,p < 0.01 compared with cells incubated with control mouse IgG1.
FIGURE 5.
FIGURE 5.
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 inA. 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 andD, 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 inE andF 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 thetop 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.
FIGURE 6.
FIGURE 6.
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 inA–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.
FIGURE 7.
FIGURE 7.
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.
FIGURE 8.
FIGURE 8.
Combination of vorinostat and Bay 117085 decreases IL-8/CXCL8 levelsin 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).
FIGURE 9.
FIGURE 9.
Proposed model of IL-8 regulation by HDAC and IKK in ovarian cancer cells. In ovarian cancer cells, the IL-8 promoter is occupied by an HDAC, which limits IL-8 transcription. Inhibition of HDAC activity increases IKKβ recruitment to the IL-8 promoter, facilitating recruitment of p65 NFκB and its acetylation, resulting in increased IL-8 transcription, cell survival, and proliferation. Inhibition of IKK activity decreases p65 recruitment to the IL-8 promoter, suppressing HDI-induced IL-8 expression.
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