title: Evaluating cytoplasmic and nuclear levels of inflammatory cytokines in cancer cells by western blotting.
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.
authors.affiliation: Department of Biology, St. John's University, 8000 Utopia Parkway, Queens, NY, 11439, USA.
authors.forename: Himavanth R
authors.initials: HR
authors.lastname: Gatla
authors.forename: Bipradeb
authors.initials: B
authors.lastname: Singha
authors.forename: Valerie
authors.initials: V
authors.lastname: Persaud
authors.forename: Ivana
authors.initials: I
authors.lastname: Vancurova
mesh_terms: D000970:Antineoplastic Agents / Q000494:pharmacology; D015153:Blotting, Western; D001897:Boronic Acids / Q000494:pharmacology; D000069286:Bortezomib; D002458:Cell Fractionation; D045744:Cell Line, Tumor; D002467:Cell Nucleus / Q000737:chemistry* / Q000187:drug effects / Q000378:metabolism; D045424:Complex Mixtures / Q000737:chemistry; D003593:Cytoplasm / Q000737:chemistry* / Q000187:drug effects / Q000378:metabolism; D004591:Electrophoresis, Polyacrylamide Gel; D005260:Female; D015870:Gene Expression; D024243:HMGB1 Protein / Q000235:genetics / Q000302:isolation & purification* / Q000378:metabolism; D006801:Humans; D016209:Interleukin-8 / Q000235:genetics / Q000302:isolation & purification* / Q000378:metabolism; D008297:Male; D011719:Pyrazines / Q000494:pharmacology
chemical_list: D000970:Antineoplastic Agents; D001897:Boronic Acids; C508606:CXCL8 protein, human; D045424:Complex Mixtures; D024243:HMGB1 Protein; C585491:HMGB1 protein, human; D016209:Interleukin-8; D011719:Pyrazines; D000069286:Bortezomib