R-Flurbiprofen, a Non-Steroidal Anti-Inflammatory Agent (NSAID) with Anti-Tumor Activity, Inhibits the Stromal-Induced Upregulation of Interleukin-8 in Chronic Lymphocytic Leukemia Cells.

NSAIDs have documented activity in the prevention or treatment of human epithelial cancers. Little information exists, however, to determine if similar anti-tumor effects can occur with hematologic malignancies. We have evaluated R-flurbiprofen (RFB), an enantiomer of an arylpropionic acid NSAID with known anti-tumor effects, as a potential treatment for CLL. PBMNCs from 7 CLL patients (WBC 30,000 –104,000/mcL; all CD5+/CD19+/CD38−) were exposed in short term culture to RFB. All samples showed caspase-dependent cell death following addition of RFB, with a median LD50 of approximately 90mcg/mL, a clinically achievable concentration. Addition of PGE2 or caffeic acid did not prevent RFB-induced cell death, demonstrating that the effect was not due to cyclooxygenase or lipooxygenase modulation. To identify molecular events resulting from RFB treatment, we performed microarray anaylsis (with Affymetrix U74v2A chips) on drug-treated CLL cells. PBMNCs were initially cultured in medium alone, or co-cultured with allogeneic primary marrow stromal cells (MSC). MSC co-culture increased or decreased (by >2-fold) approximately 13% of the 12,000 genes/probe sets interrogated by these microarrays (2 independent experiments), but had little effect on RFB cell kill. Approximately 35 genes (fusion of 2 independent experiments) were reproducibly up- or down-regulated by RFB in the non-adherent CLL cells cultured with MSC, but not in CLL cultured alone. Among the down-regulated genes were several chemokines and adhesion molecules. These included the gene for interleukin-8 (IL8). The expression of IL8 was markedly increased in CLL cells by co-cultivation with MSC. Furthermore, IL8 expression showed reproducible and dose-dependent down-regulation in CLL cells by RFB treatment. In contrast RFB did not affect expression of IL8 in CLL cells cultured without MSC. Intracellular IL8 was measured in CLL cells by flow cytometry; RFB markedly decreased expression of IL8 protein in addition to its effects on IL8 mRNA. IL8 has been identified as a growth/survival factor for CLL cells, and is a potent tumor-derived stimulator of angiogenesis. Furthermore, increased blood levels of IL8 have been identified as a poor prognostic sign in patients with CLL. RFB may modulate leukemia cell/MSC interactions by blocking the expression of chemokines and other pro-angiogenic mediators. RFB may play a supporting role as part of a multi-modality therapy regimen for CLL.