Expression of the pro-inflammatory and pro-angiogenic chemokine interleukin-8 (IL-8), which is regulated at the transcriptional level by NFκB, is constitutively increased in the androgen independent metastatic prostate cancer and correlates with poor prognosis. Inhibition of NFκB-dependent transcription was used as an anti-cancer strategy for the development of the first clinically approved 26S proteasome inhibitor, bortezomib (BZ). Even though BZ has shown remarkable anti-tumor activity in hematological malignancies, it has been less effective in prostate cancer and other solid tumors; however, the mechanisms have not been fully understood. Here we report that the proteasome inhibition by BZ unexpectedly increases the IL-8 expression in androgen independent prostate cancer PC3 and DU145 cells, while expression of other NFκB-regulated genes is inhibited or unchanged. The BZ-increased IL-8 expression is associated with increased in vitro p65 NFκB DNA binding activity and p65 recruitment to the endogenous IL-8 promoter. In addition, proteasome inhibition induces a nuclear accumulation of IKKα 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 for the gene specific increase of IL-8 expression by the proteasome inhibition in prostate cancer cells and suggest that targeting both IKKα and the proteasome may increase the BZ effectiveness in androgen independent prostate cancer treatment.

Ovarian cancer is associated with increased expression of the pro-angiogenic chemokine interleukin-8 (IL-8, CXCL8), which induces tumor cell proliferation, angiogenesis, and metastasis. Even though bortezomib (BZ) has shown remarkable anti-tumor activity in hematological malignancies, it has been less effective in ovarian cancer; however, the mechanisms are not understood. We have recently shown that BZ unexpectedly induces the expression of IL-8 in ovarian cancer cells in vitro, by IκB kinase (IKK)-dependent mechanism. Here, we tested the hypothesis that IKK inhibition reduces the IL-8 production and increases BZ effectiveness in reducing ovarian tumor growth in vivo. Our results demonstrate that the combination of BZ and the IKK inhibitor Bay 117085 significantly reduces the growth of ovarian tumor xenografts in nude mice when compared to either drug alone. Mice treated with the BZ/Bay 117085 combination exhibit smallest tumors, and lowest levels of IL-8. Furthermore, the reduced tumor growth in the combination group is associated with decreased tumor levels of S536P-p65 NFκB and its decreased recruitment to IL-8 promoter in tumor tissues. These data provide the first in vivo evidence that combining BZ with IKK inhibitor is effective, and suggest that using IKK inhibitors may increase BZ effectiveness in ovarian cancer treatment.

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.

Prostate cancer is one of the most common malignancies in men. One of the critical factors is the increased activity of NFκB, which induces synthesis of anti‐apoptotic and pro‐inflammatory genes, thus increasing cell survival, proliferation, and resistance to chemotherapy. The expression of NFκB‐regulated proinflammatory cytokines IL‐6 and IL‐8 is increased in prostate cancer, and correlates with poor prognosis. Bortezomib (BZ) is the first approved proteasome inhibitor that has a remarkable anti‐tumor activity in multiple myeloma. Although BZ has shown promising results also in the metastatic prostate cancer, it has not been used because of its limited effectiveness. However, the mechanisms are not understood. The goal of this study was to investigate the mechanism of bortezomib function in metastatic prostate cancer cells. Surprisingly, we found that while the expression of NFκB‐dependent anti‐apoptotic genes is inhibited by bortezomib, the expression of pro‐inflammatory cytokines IL‐6 and IL‐8 is significantly increased. Our data show that BZ has opposing effects on p65 and p50 recruitment to IL‐6 and IL‐8 promoters. In addition, our results indicate an involvement of IKK in the BZ‐increased IL‐6 and IL‐8 expression. These data suggest that the BZ‐increased IL‐6 and IL‐8 expression may represent one of the mechanisms responsible for the decreased BZ effectiveness in prostate cancer treatment.

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