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We have recently shown that IFNγ, produced during cancer therapy, induces expression of the Bcl3 proto-oncogene in ovarian cancer (OC) cells, resulting in their increased proliferation, migration, and invasion, but the mechanisms are unknown. Here, we demonstrate that the IFNγ-induced Bcl3 expression is dependent on JAK1 and STAT1 signaling, and on p65 NFκB. Furthermore, the IFNγ-induced Bcl3 expression is associated with an increased occupancy of Ser-727 phosphorylated STAT1 and acetylated histone H3 at the Bcl3 promoter. Our data indicate that Bcl3 promotes expression of the pro-inflammatory chemokine interleukin-8 (IL-8) in OC cells. These findings identify Bcl3 as a novel target of IFNγ/JAK1/STAT1 signaling and suggest that targeting the JAK1/STAT1 pathway may suppress IFNγ-induced Bcl3 expression in OC., (© 2023 The Authors. FEBS Open Bio published by John Wiley & Sons Ltd on behalf of Federation of European Biochemical Societies.)

Interferon-γ (IFNγ) is a pleiotropic cytokine that has a crucial role in immune response and tumor immunity. Because of its anti-tumor effects, IFNγ has been used in cancer treatment. However, IFNγ also has tumor-promoting functions that are less well understood. Here, we show that IFNγ induces expression of the pro-inflammatory and pro-angiogenic chemokine interleukin-8 (IL-8, CXCL8) in ovarian cancer (OC) cells. The IFNγ-induced IL-8 expression is dependent on JAK1, STAT1, and p65 NFκB, and is associated with an increased occupancy of K314/315 acetylated p65 NFκB and Ser-727 phosphorylated STAT1 at the IL-8 promoter. Neutralization of IL-8 using anti-IL-8 antibody reduces IFNγ-induced migration of OC cells, and their invasion ability in 3D spheroids. Together, these findings identify IL-8 as a novel target induced by IFNγ/JAK1/STAT1/p65 NFκB signaling, and indicate that the IFNγ-induced IL-8 contributes to IFNγ pro-tumorigenic effects in ovarian cancer cells., (Copyright © 2021 Elsevier Ltd. All rights reserved.)

The pro-inflammatory and pro-angiogenic chemokine interleukin-8 (IL-8, CXCL8) induces proliferation and invasion of solid tumor cells. In many types of solid cancer, including triple-negative breast cancer (TNBC), the IL-8 expression is induced by proteasome inhibition. In this chapter, we describe a protocol for the analysis of TNBC cell invasion induced by IL-8 in response to proteasome inhibition by bortezomib (BZ). Using this approach, we show that BZ increases the invasion ability of TNBC cells, and that the BZ-increased TNBC cell invasion is suppressed by IκB kinase (IKK) inhibition, which also decreases the IL-8 expression. The experimental protocol includes the cell invasion assay, microscopic evaluation of the invading cells, and quantitative analysis of the obtained images. This protocol should be applicable also for measurement of chemokine-induced tumor cell invasion in other types of cancer cells.

Ovarian cancer (OC) is the most common cause of cancer deaths among gynecological malignancies. OC ascites contain multicellular spheroid aggregates, which exhibit increased pro-survival signaling, invasive behavior, and chemotherapeutic resistance. OC cells are characterized by an increased expression of the pro-inflammatory and pro-angiogenic chemokine interleukin-8 (IL-8, CXCL8), which increases their survival and migration, thus contributing to OC metastasis and angiogenesis. While previous studies have shown that IL-8 increases proliferation of OC cells grown in monolayer cultures, the effect of IL-8 on proliferation of OC cells grown in 3D spheroids has not been investigated. The spheroid 3D culture assays have been particularly useful in translational research since they allow cell-to-cell interactions that resemble tumor growth in vivo, while allowing easy cell manipulations and visualization. Here, we used the 3D spheroid culture assay to investigate the effect of IL-8 on OC cell proliferation. Using this assay, our results show that IL-8 significantly increases proliferation of OC cells grown in 3D spheroids.

Triple negative breast cancer (TNBC) cells express increased levels of the pro-inflammatory and pro-angiogenic chemokine interleukin-8 (IL-8, CXCL8), which promotes their proliferation and migration. Because TNBC patients are unresponsive to current targeted therapies, new therapeutic strategies are urgently needed. While proteasome inhibition by bortezomib (BZ) or carfilzomib (CZ) has been effective in treating hematological malignancies, it has been less effective in solid tumors, including TNBC, but the mechanisms are incompletely understood. Here we report that proteasome inhibition significantly increases expression of IL-8, and its receptors CXCR1 and CXCR2, in TNBC cells. Suppression or neutralization of the BZ-induced IL-8 potentiates the BZ cytotoxic and anti-proliferative effect in TNBC cells. The IL-8 expression induced by proteasome inhibition in TNBC cells is mediated by IκB kinase (IKK), increased nuclear accumulation of p65 NFκB, and by IKK-dependent p65 recruitment to IL-8 promoter. Importantly, inhibition of IKK activity significantly decreases proliferation, migration, and invasion of BZ-treated TNBC cells. These data provide the first evidence demonstrating that proteasome inhibition increases the IL-8 signaling in TNBC cells, and suggesting that IKK inhibitors may increase effectiveness of proteasome inhibitors in treating TNBC., Competing Interests: The authors have declared that no competing interests exist.

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

Bortezomib (BZ) is the first clinically approved proteasome inhibitor that has shown remarkable anticancer activity in patients with hematological malignancies. However, many patients relapse and develop resistance; yet, the molecular mechanisms of BZ resistance are not fully understood. We have recently shown that in solid tumors, BZ unexpectedly increases expression of the pro-inflammatory and pro-angiogenic chemokine interleukin-8 (IL-8), while it inhibits expression of other NFκB-regulated genes. Since monocytes and macrophages are major producers of IL-8, the goal of this study was to test the hypothesis that BZ increases the IL-8 expression in human monocytes and macrophages. Here, we show that BZ dramatically increases the IL-8 expression in lipopolysaccharide (LPS)-stimulated U937 macrophages as well as in unstimulated U937 monocytes and peripheral blood mononuclear cells, while it inhibits expression of IL-6, IL-1 and tumor necrosis factor-α. In addition, our results show that the underlying mechanisms involve p38 mitogen-activated protein kinase, which is required for the BZ-induced IL-8 expression. Together, these data suggest that the BZ-increased IL-8 expression in monocytes and macrophages may represent one of the mechanisms responsible for the BZ resistance and indicate that targeting the p38-mediated IL-8 expression could enhance the BZ effectiveness in cancer treatment., (Copyright © 2015 Elsevier Inc. All rights reserved.)

Proinflammatory and pro-angiogenic chemokine interleukin-8 (IL-8, CXCL8) contributes to ovarian cancer progression through its induction of tumor cell proliferation, survival, angiogenesis, and metastasis. Proteasome inhibition by bortezomib, which has been used as a frontline therapy in multiple myeloma, has shown only limited effectiveness in ovarian cancer and other solid tumors. However, the responsible mechanisms remain elusive. Here, we show that proteasome inhibition dramatically increases the IL-8 expression and release in ovarian cancer cells. The responsible mechanism involves an increased nuclear accumulation of IκB kinase β (IKKβ) and an increased recruitment of the nuclear IKKβ, p65-phosphorylated at Ser-536, and the transcription factor early growth response-1 (EGR-1) to the endogenous IL-8 promoter. Coimmunoprecipitation studies identified the nuclear EGR-1 associated with IKKβ and with p65, with preferential binding to S536P-p65. Both IKKβ activity and EGR-1 expression are required for the increased IL-8 expression induced by proteasome inhibition in ovarian cancer cells. Interestingly, in multiple myeloma cells the IL-8 release is not increased by bortezomib. Together, these data indicate that the increased IL-8 release may represent one of the underlying mechanisms responsible for the decreased effectiveness of proteasome inhibition in ovarian cancer treatment and identify IKKβ and EGR-1 as potential new targets in ovarian cancer combination therapies.

Interleukin-8 (IL-8), originally discovered as the neutrophil chemoattractant and inducer of leukocyte-mediated inflammation, contributes to cancer progression through its induction of tumor cell proliferation, survival, and migration. IL-8 expression is increased in many types of advanced cancers, including ovarian cancer, and correlates with poor prognosis. Bortezomib (BZ) is the first FDA-approved proteasome inhibitor that has shown remarkable antitumor activity in multiple myeloma and other hematological malignancies. In solid tumors, including ovarian carcinoma, BZ has been less effective as a single agent; however, the mechanisms remain unknown. We have recently shown that in ovarian cancer cells, BZ greatly increases IL-8 expression, while expression of other NFκB-regulated cytokines, IL-6 and TNF, is unchanged. In this chapter, we describe a protocol that uses real-time qRT-PCR to quantitatively analyze mRNA levels of IL-8 and IL-6 in BZ-treated ovarian cancer cells. The protocol can be easily modified and used for analysis of other cytokines in different cell types.

Increased expression and cellular release of inflammatory cytokines, interleukin-8 (IL-8; CXCL8), and high mobility group box-1 (HMGB1) are associated with increased cell proliferation, angiogenesis, and metastasis during cancer progression. In prostate and ovarian cancer cells, increased levels of IL-8 and HMGB1 correlate with poor prognosis. We have recently shown that proteasome inhibition by bortezomib (BZ) specifically increases IL-8 release from metastatic prostate and ovarian cancer cells. In this chapter, we describe a protocol to analyze the cytoplasmic and nuclear levels of IL-8 and HMGB1 in prostate and ovarian cancer cells by western blotting. IL-8 is localized in the cytoplasm in both cell types, and its protein levels are significantly increased by BZ. In contrast, HMGB1 is localized in the nucleus, and BZ increases its nuclear levels only in ovarian cancer cells. The protocol includes isolation of cytoplasmic and nuclear extracts, followed by SDS electrophoresis and western blotting, and can be easily modified to analyze the cytoplasmic and nuclear cytokine levels in other cell types.

Most cytokines are stored in the cytoplasm until their release into the extracellular environment; however, some cytokines have been reported to localize in the nucleus. Traditional whole cell extract preparation does not provide information about the intracellular localization of cytokines. Here, we describe how to prepare cytoplasmic and nuclear extracts that can be analyzed by immunoblotting. While in this chapter we use this method to analyze intracellular localization of interleukin-8 (IL-8) in human mononuclear leukocytes, this protocol is adaptable to any cell type or protein of interest.

Expression of the proinflammatory and proangiogenic chemokine IL-8, which is regulated at the transcriptional level by NF-κB, is constitutively increased in androgen-independent metastatic prostate cancer and correlates with poor prognosis. Inhibition of NF-κB-dependent transcription was used as an anticancer strategy for the development of the first clinically approved 26S proteasome inhibitor, bortezomib (BZ). Even though BZ has shown remarkable antitumor activity in hematological malignancies, it has been less effective in prostate cancer and other solid tumors; however, the mechanisms have not been fully understood. In this article, we report that proteasome inhibition by BZ unexpectedly increases IL-8 expression in androgen-independent prostate cancer PC3 and DU145 cells, whereas expression of other NF-κB-regulated genes is inhibited or unchanged. The BZ-increased IL-8 expression is associated with increased in vitro p65 NF-κB DNA binding activity and p65 recruitment to the endogenous IL-8 promoter. In addition, proteasome inhibition induces a nuclear accumulation of IκB kinase (IKK)α, and inhibition of IKKα enzymatic activity significantly attenuates the BZ-induced p65 recruitment to IL-8 promoter and IL-8 expression, demonstrating that the induced IL-8 expression is mediated, at least partly, by IKKα. Together, these data provide the first evidence, to our knowledge, for the gene-specific increase of IL-8 expression by proteasome inhibition in prostate cancer cells and suggest that targeting both IKKα and the proteasome may increase BZ effectiveness in treatment of androgen-independent prostate cancer.

The objective of this study was to determine the doses of dexamethasone (DEX), betamethasone (BET), and hydrocortisone (HC) that effectively inhibit the release of two potent proinflammatory chemokines, interleukin 8 (IL-8) and macrophage inflammatory protein alpha (MIP), from polymorphonuclear neutrophils (PMNs) of the newborn. Human PMNs were isolated from cord blood (n = 18). Chemokines were measured from PMN cell culture supernatants after 18 h of stimulation using tumor necrosis factor (1 ng/ml), with and without pretreatment by DEX (10(-10) to 10(-6) M) versus HC or BET (10(-10) to 10(-5) M). Maximal inhibitions of IL-8 release by BET, DEX, and HC were 97, 91, and 91%, respectively. For MIP, the maximal inhibitions by BET, DEX, and HC were 88, 69, and 70%, respectively. The 50% inhibitory concentrations by DEX, BET, and HC for IL-8 release were 3.4 +/- (SE) 1.6 x 10(-9), 1.8 +/- 7.4 x 10(-8), and 1.8 +/- 0.5 x 10(-7) M, respectively. The 50% inhibitory concentrations by DEX, BET, and HC for MIP release were 1.0 +/- (SE) 0.5 x 10(-8), 3.8 +/- 3.1 x 10(-8), and 4.8 +/- 1.6 x 10(-7) M, respectively. In vitro, these corticosteroids effectively inhibited the release of two structurally different chemokines that are found in the airway lavage fluids of infants developing bronchopulmonary dysplasia. When compared to plasma DEX levels previously reported during the treatment of bronchopulmonary dysplasia, our results suggest that the doses of DEX, and potentially BET, needed to treat chronic lung disease may be more than five to ten times lower than those of current DEX regimens., (Copyright 2002 S. Karger AG, Basel)

The increased expression of pro-inflammatory and pro-angiogenic chemokines contributes to ovarian cancer progression through the induction of tumor cell proliferation, survival, angiogenesis, and metastasis. The substantial potential of these chemokines to facilitate the progression and metastasis of ovarian cancer underscores the need for their stringent transcriptional regulation. In this Review, we highlight the key mechanisms that regulate the transcription of pro-inflammatory chemokines in ovarian cancer cells, and that have important roles in controlling ovarian cancer progression. We further discuss the potential mechanisms underlying the increased chemokine expression in drug resistance, along with our perspective for future studies. [ABSTRACT FROM AUTHOR]

Background: Neutrophils followed by monocytic cells are recruited into the lung during the early development of bronchopulmonary dysplasia (BPD). Objectives: We determined: (1) the capacity of polymorphonuclear leukocytes (PMNs) and peripheral blood monocytic cells (PBMCs) of the newborn to produce and release the anti-inflammatory cytokine, interleukin (IL)-10, after stimulation by lipopolysaccharide (LPS) or tumor necrosis factor (TNF), and (2) the levels of exogenous IL-10 and/or dexamethasone (DEX) needed to inhibit the release of the pro-inflammatory chemokine IL-8 from stimulated cells. Methods: PMNs and PBMCs were isolated from cord blood of healthy term infants. RT-PCR and ELISA were used to detect mRNA and cytokine levels from culture media, respectively. Results: We found that PMNs did not produce IL-10 mRNA or release IL-10 but did produce IL-8 mRNA by 1 h. PBMCs did produce IL-10 mRNA after 4 h (with IL-8 mRNA expression by 1 h). LPS-stimulated PBMCs released IL-10 to a maximum of 1,038 pg/ml/5 million cells (56 femtomolar). Equimolar doses of exogenous IL-10 or DEX produced up to 83% inhibition of IL-8 from PMNs. Exogenous IL-10 was more potent than DEX, on an equimolar basis, with regard to IL-8 release from PBMCs (90 vs. 33% respectively at a 10 nanomolar level). No inhibition of IL-8 release by IL-10 or DEX was observed at 100 femtomolar level. IL-10 and DEX did not have an additive inhibitory effect on IL-8 release. Conclusions: We conclude that for the newborn: (1) PBMCs produce IL-10 far below the level needed to inhibit a submaximal release of IL-8 from PMNs or PBMCs, and (2) exogenous IL-10 was equipotent or more potent than therapeutic levels of DEX on inhibition of IL-8 from these cells. Further studies are needed to determine if exogenous IL-10 may be useful in the treatment of BPD or other inflammatory disorders of the newborn. Copyright © 2007 S. Karger AG, Basel [ABSTRACT FROM AUTHOR]

Human neutrophils differ from other cells by containing high amount of IκBα in the nucleus, and this increased nuclear IκBα accumulation is associated with the inhibition of NFκB activity and increased apoptosis. However, the mechanisms regulating NFκB activation and IκBα degradation in human neutrophils are little understood. The objective of this study was to provide a further insight into the mechanisms regulating NFκB activity and IκBα degradation in human neutrophils. We show that okadaic acid (OA), an inhibitor of protein phosphatases PP1 and PP2A, induces sustained activation of NFκB and degradation of the nuclear IκBα, and increases interleukin-8 expression in the neutrophils. Furthermore, inhibitors of protein kinase C-δ (PKCδ) and IκB kinase (IKK) inhibit the OA-induced activation of NFκB. Collectively, our results indicate that in human neutrophils, the sustained activation of NFκB is regulated by a continuous phosphorylation and degradation of the nuclear IκBα. [Copyright &y& Elsevier]