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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.

Expression of programmed death ligand-1 (PD-L1, CD274) on cancer cells is regulated by interferon-γ (IFNγ) signaling as well as by epigenetic mechanisms. By binding to PD-1 on cytotoxic T cells, PD-L1 inhibits T cell-mediated antitumor responses, resulting in immune escape. This chapter describes analysis of the surface PD-L1 expression in ovarian cancer (OC) cells using flow cytometry (FC). Our data demonstrate that the surface PD-L1 expression in OC cells is induced by IFNγ as well as by the class I histone deacetylase (HDAC) inhibition by romidepsin, suggesting that class I HDAC inhibition might provide a useful strategy to modulate the PD-L1 levels on OC cells.

IFNγ is a pleiotropic cytokine that has both antitumor functions and pro-tumorigenic effects. Recent studies have shown that IFNγ induces expression of the immune checkpoint PD-L1 in ovarian cancer (OC) cells, resulting in their increased proliferation and tumor growth. Here, we tested the hypothesis that IFNγ induces migration of OC cells. Using the scratch wound healing assay, our results demonstrate that IFNγ promotes OC cell migration, thus adding to the complexities of IFNγ pro-tumorigenic mechanisms. This chapter describes analysis of the IFNγ-induced migration of OC cells by the wound healing assay followed by quantification of the obtained images using ImageJ software.

Expression of the immune checkpoint programmed death ligand 1 (PD-L1, CD274) is increased in many types of cancer, including ovarian cancer (OC), but the mechanisms that regulate the PD-L1 expression are not fully understood. In addition to binding to PD-1 on T cells, thus inhibiting T cell-mediated antitumor responses, PD-L1 has also tumor-intrinsic effects that include increased cancer cell survival and proliferation, and that might be in part mediated by the intracellular PD-L1. In this chapter, we describe a protocol for the analysis of the intracellular PD-L1 protein levels in OC cells by immunoblotting. Our results show that interferon-γ (IFNγ) induces the intracellular levels of PD-L1 and the proto-oncogene Bcl3 in OC cells. However, the PD-L1 expression is significantly decreased in OC cells stably transfected with Bcl3 shRNA, demonstrating that the IFNγ-induced PD-L1 expression in OC cells is mediated by Bcl3. These data identify the IFNγ-Bcl3-PD-L1 axis as a novel therapeutic target in OC, and suggest that targeting Bcl3 may provide a novel strategy to regulate the PD-L1 expression, and especially the tumor-intrinsic PD-L1 effects mediated by the intracellular PD-L1 in OC cells.

The immune checkpoint molecule, programmed death ligand 1 (PD-L1; B7-H1, CD274), induces T cell apoptosis and tolerance, thus inhibiting the antitumor immunity. PD-L1 expression is increased in many types of cancer, including ovarian cancer (OC), and correlates with poor prognosis. However, the mechanisms that regulate the PD-L1 expression in cancer cells are incompletely understood. The transcriptional regulation of PD-L1 expression is orchestrated by several transcription factors, including NFκB. The human PD-L1 promoter contains five NFκB-binding sites. Interferon-γ (IFNγ) stimulation of OC cells induces p65, and particularly K314/315 acetylated p65 recruitment to all five NFκB-binding sites in PD-L1 promoter, resulting in increased PD-L1 expression. In this chapter, we describe a protocol that uses chromatin immunoprecipitation (ChIP) to analyze the transcriptional regulation of PD-L1 by measuring recruitment of NFκB p65 and K314/315 acetylated p65 to PD-L1 promoter in human OC 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.

The immunosuppressive cytokines transforming growth factor β1 (TGFβ1) and interleukin-10 (IL-10) regulate a variety of biological processes including differentiation, proliferation, tissue repair, tumorigenesis, inflammation, and host defense. Aberrant expression of TGFβ1 and IL-10 has been associated with many types of autoimmune and inflammatory disorders, as well as with many types of cancer and leukemia. Patients with cutaneous T cell lymphoma (CTCL) have high levels of malignant CD4+ T cells expressing IL-10 and TGFβ1 that suppress the immune system and diminish the antitumor responses. The transcriptional regulation of TGFβ1 and IL-10 expression is orchestrated by several transcription factors, including NFκB. However, while the transcriptional regulation of pro-inflammatory and anti-apoptotic genes by NFκB has been studied extensively, much less is known about the NFκB regulation of immunosuppressive genes. In this chapter, we describe a protocol that uses chromatin immunoprecipitation (ChIP) to analyze the transcriptional regulation of TGFβ1 and IL-10 by measuring recruitment of NFκB p65, p50, c-Rel, Rel-B, and p52 subunits to TGFβ1 and IL-10 promoters in human CTCL Hut-78 cells.

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.

Interleukin-1β (IL-1) and tumor necrosis factor-α (TNF) are important pro-inflammatory cytokines involved in the mediation of the immune response, inflammation, tissue repair, and tumor progression. Regulation of IL-1 and TNF expression is mediated at the level of transcription by the transcription factor NFκB. Inhibition of NFκB activity by the proteasome inhibitor bortezomib (BZ) has been used as a frontline therapy in multiple myeloma and other hematological malignancies. In this chapter, we describe a protocol that uses chromatin immunoprecipitation (ChIP) to analyze the NFκB recruitment to endogenous IL-1 and TNF promoters in BZ-treated human macrophages. Corresponding to the BZ-suppressed mRNA levels of IL-1 and TNF, we show that BZ inhibits p65 NFκB recruitment to IL-1 and TNF promoters. This study specifically uses U937 macrophages, but the protocol could be easily modified to analyze the regulation of NFκB recruitment in other cell types.

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.

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.

Transcription factor NFκB is a key regulator of genes involved in immune and inflammatory responses, as well as genes regulating cell proliferation and survival. In addition to many inflammatory disorders, NFκB is constitutively activated in a variety of human cancers and leukemia. Thus, inhibition of NFκB DNA binding activity represents an important therapeutic approach for disorders characterized by high levels of constitutive NFκB activity. We have previously shown that NFκB DNA binding activity is suppressed by the nuclear translocation and accumulation of IκBα, which is induced by inhibition of the 26S proteasome. In this chapter, we describe a protocol that uses small inhibitory RNA (si RNA) interference followed by electrophoretic mobility shift assay (EMSA) to analyze the regulation of NFκB DNA binding by nuclear IκBα induced by the proteasome inhibitor MG132. Using this protocol, we show that in human leukemia Hut-78 cells that exhibit high levels of NFκB DNA binding activity, MG132 induces nuclear translocation and accumulation of IκBα, which then specifically inhibits NFκB DNA binding. This protocol uses human leukemia Hut-78 cells; however, it can be easily adapted for other cells exhibiting high levels of constitutive NFκB DNA binding.

Transcription factor NFκB comprises a family of proteins that serve as crucial regulators of genes involved in host immune and inflammatory responses, cell survival, proliferation, and differentiation. Since transcription of NFκB-dependent genes is increased in numerous inflammatory disorders as well as in many types of cancer and leukemia, inhibition of NFκB-dependent transcription thus represents an important therapeutic target. We have previously shown that in human leukocytes, transcription of NFκB-dependent genes is inhibited by the nuclear translocation and accumulation of IκBα, which can be induced by an inhibitor of CRM1-dependent nuclear export, leptomycin B (LMB). In this chapter, we describe a protocol that uses chromatin immunoprecipitation (ChIP) to analyze the regulation of NFκB recruitment to NFκB-dependent promoters by nuclear IκBα induced by LMB. We show that in lipopolysaccharide (LPS)-stimulated human U-937 macrophages, recruitment of NFκB p65 and p50 proteins to NFκB-dependent promoters of IκBα and cIAP2 genes is suppressed by the LMB-induced nuclear IκBα. Even though in this study we use U-937 macrophages, this protocol should be readily adaptable to analyze the regulation of NFκB recruitment by nuclear IκBα also in other cell types.

Controlled nucleocytoplasmic localization regulates activity of NF kappa B as well as other transcription factors. Analysis of the nucleocytoplasmic protein shuttling has been greatly facilitated by the use of leptomycin B (LMB), an inhibitor of CRM1-dependent nuclear export. The authors have previously shown that LMB inhibits NF kappa B activity in human neutrophils by increasing the nuclear accumulation of NF kappa B inhibitor, I kappa B alpha. In this chapter, the authors describe a protocol that uses LMB to study the nucleocytoplasmic shuttling of I kappa B alpha in human macrophage-like U937 cells, thus inhibiting NF kappa B activity. This protocol should be readily adaptable to analyze the nucleocytoplasmic shuttling of other proteins in human leukocytes.