Quantitative analysis of bortezomib-induced IL-8 gene expression in ovarian cancer cells

Methods Mol Biol. 2014:1172:295-304. doi: 10.1007/978-1-4939-0928-5_27.

Abstract

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.

Publication types

  • Research Support, N.I.H., Extramural

MeSH terms

  • Antineoplastic Agents / pharmacology*
  • Boronic Acids / pharmacology*
  • Bortezomib
  • Cell Line, Tumor
  • Female
  • Gene Expression / drug effects*
  • Humans
  • Interleukin-6 / genetics*
  • Interleukin-6 / metabolism
  • Interleukin-8 / genetics*
  • Interleukin-8 / metabolism
  • NF-kappa B / genetics
  • NF-kappa B / metabolism
  • Pyrazines / pharmacology*
  • RNA, Messenger / genetics*
  • RNA, Messenger / isolation & purification
  • RNA, Messenger / metabolism
  • Real-Time Polymerase Chain Reaction
  • Tumor Necrosis Factor-alpha / genetics
  • Tumor Necrosis Factor-alpha / metabolism

Substances

  • Antineoplastic Agents
  • Boronic Acids
  • IL6 protein, human
  • Interleukin-6
  • Interleukin-8
  • NF-kappa B
  • Pyrazines
  • RNA, Messenger
  • Tumor Necrosis Factor-alpha
  • Bortezomib