Abstract
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.
Access this chapter
Tax calculation will be finalised at checkout
Purchases are for personal use only
References
Xu L, Fidler IJ (2000) Interleukin 8: an autocrine growth factor for human ovarian cancer. Oncol Res 12:97–106
Waugh DJ, Wilson C (2008) The interleukin-8 pathway in cancer. Clin Cancer Res 14:6735–6741
Balkwill F, Mantovani A (2001) Inflammation and cancer: back to Virchow? Lancet 357:539–545
Balkwill F (2004) Cancer and the chemokine network. Nat Rev Cancer 4:540–550
Mantovani A, Allavena P, Sica A et al (2008) Cancer-related inflammation. Nature 454:436–444
Araki S, Omori Y, Lyn D et al (2007) Interleukin-8 is a molecular determinant of androgen independence and progression in prostate cancer. Cancer Res 67:6854–6862
Singh RK, Lokeshwar BL (2011) The IL-8-regulated chemokine receptor CXCR7 stimulates EGFR signaling to promote prostate cancer growth. Cancer Res 71:3268–3277
Chen J, Xi B, Zhao Y et al (2012) High-mobility group protein B1 (HMGB1) is a novel biomarker for human ovarian cancer. Gynecol Oncol 126:109–117
Scaffidi P, Misteli T, Bianchi ME (2002) Release of chromatin protein HMGB1 by necrotic cells triggers inflammation. Nature 418:191–195
Lotze MT, Tracey KJ (2005) High-mobility group box 1 protein (HMGB1): nuclear weapon in the immune arsenal. Nat Rev Immunol 5:331–342
Bell CW, Jiang W, Reich CF III et al (2006) The extracellular release of HMGB1 during apoptotic cell death. Am J Physiol Cell Physiol 291:1318–1325
Sha Y, Zmijewski J, Xu Z et al (2008) HMGB1 develops enhanced proinflammatory activity by binding to cytokines. J Immunol 180:2531–2537
Tang D, Kang R, Cheh CW et al (2010) HMGB1 release and redox regulates autophagy and apoptosis in cancer cells. Oncogene 29:5299–5310
Tang D, Kang R, Livesey KM et al (2010) Endogenous HMGB1 regulates autophagy. J Cell Biol 190:881–892
Gnanasekar M, Kalyanasundaram R, Zheng G et al (2013) HMGB1: a promising therapeutic target for prostate cancer. Prostate Cancer 2013:1–8
Chen J, Liu X, Zhang J et al (2012) Targeting HMGB1 inhibits ovarian cancer growth and metastasis by lentivirus-mediated RNA interference. J Cell Physiol 227:3629–3638
Lu B, Nakamura T, Inouye K et al (2012) Novel role of PKR in inflammasome activation and HMGB1 release. Nature 488:670–674
Lu B, Wang H, Andersson U et al (2013) Regulation of HMGB1 release by inflammasomes. Protein Cell 4:163–167
Yang H, Antoine DJ, Andersson U et al (2013) The many faces of HMGB1: molecular structure-functional activity in inflammation, apoptosis, and chemotaxis. J Leukoc Biol 93:865–873
Guo ZS, Liu Z, Bartlett DL et al (2013) Life after death: targeting high mobility group box 1 in emergent cancer therapies. Am J Cancer Res 3:1–20
Li W, Zhu S, Li J et al (2011) EGCG stimulates autophagy and reduces cytoplasmic HMGB1 levels in endotoxin-stimulated macrophages. Biochem Pharmacol 81:1152–1163
Manna S, Singha B, Phyo SA et al (2013) Proteasome inhibition by bortezomib increases IL-8 expression in androgen-independent prostate cancer cells: the role of IKKα. J Immunol 191:2837–2846
Singha B, Gatla H, Manna S et al (2014) Proteasome inhibition increases recruitment of IKKβ, S536P-p65 and transcription factor EGR1 to interleukin-8 (IL-8) promoter, resulting in increased IL-8 production in ovarian cancer cells. J Biol Chem 289:2687–2700
Miskolci V, Ghosh CC, Rollins J et al (2006) TNFα release from peripheral blood leukocytes depends on a CRM1-mediated nuclear export. Biochem Biophys Res Commun 351:354–360
Acknowledgement
This work was supported by NIH grant CA173452 to I. Vancurova.
Author information
Authors and Affiliations
Corresponding author
Editor information
Editors and Affiliations
Rights and permissions
Copyright information
© 2014 Springer Science+Business Media, New York
About this protocol
Cite this protocol
Gatla, H.R., Singha, B., Persaud, V., Vancurova, I. (2014). Evaluating Cytoplasmic and Nuclear Levels of Inflammatory Cytokines in Cancer Cells by Western Blotting. In: Vancurova, I. (eds) Cytokine Bioassays. Methods in Molecular Biology, vol 1172. Humana Press, New York, NY. https://doi.org/10.1007/978-1-4939-0928-5_25
Download citation
DOI: https://doi.org/10.1007/978-1-4939-0928-5_25
Published:
Publisher Name: Humana Press, New York, NY
Print ISBN: 978-1-4939-0927-8
Online ISBN: 978-1-4939-0928-5
eBook Packages: Springer Protocols