Abstract
4880
Introduction: Increased expression of the inducible, pro-inflammatory enzyme cyclooxygenase-2 (COX-2) is associated with cancer, but its role in hormone refractory prostate cancer (HRPC) is less clear and the direct involvement of tumor derived COX-2 is still lacking. We investigated the consequences of suppression of COX-2 by a silencing of tumor cell expression of COX-2, via Tetracycline-inducible antisense cDNA. Methods: Clones of PC-3ML cells, conditionally expressing COX-2 antisense, were created by transfecting PC-3ML cells with a plasmid containing Tetracycline-regulated COX-2 cDNA in antisense orientation (Tet-ON COX-2-Off). Commercial ELISA kits were used to assay COX-1 and COX-2 activity (prostaglandin-E2 (PGE2) production), apoptosis, IL-1β, IL-8, bFGF and VEGF-1 secretion. Cell cycle analysis was determined by flow cytometry. Invasive activity was determined in vitro bythe Matrigel invasion assay.Cytotoxicity of anticancer drugs was evaluated by by clonogenic and MTT assay. Tumor growth and response to antitumor drugs were evaluated in xenografts of PC-3ML or Tet-ON COX-2-Off tumor cells by measuring tumor volumes in athymic mice. Apoptotic cells in tumor sections were enumerated in explanted tumors by TUNELassay. Results: Suppression of tumor cell COX-2 expression by antisense cDNA(Tet-ON COX-2-Off clones) caused a significant decrease in COX-2 activity (87%) and ∼60% decrease in the COX-2 protein level as compared to PC-3ML cells. Cell-cycle arrest at G1 was observed in COX-2-OFF clones (+44%). Also, significant decreases in VEGF-1 (58%); bFGF-1 (30%) and IL-8 (80%) were observed in COX-2-OFF clones. The invasive activity of COX-2-Off cells decreased significantly (80%) as compared to PC-3ML cells. A 52% reduction in the tumor growth rate was observed in mice injected with COX-2-OFF cells as compared to the mice injected with PC-3ML cells. The incidence of apoptotic cells in COX-2-Off tumors was 8-fold higher than that of control. Treating mice bearing PC-3 COX-2OFF tumors, with Docetaxel (2.3 mg/kg, IP,3 x wk.) further slowed tumor growth by 93%. Conclusion: We provide a direct evidence for the involvement of COX-2 in HRPC tumor growth. Inhibition of tumor COX-2 activity significantly reduces tumor growth and increases the efficacy of established antitumor drugs, such as Docetaxel. Direct inhibition of tumor cell COX-2 activity promises to be of greater efficacy than that by systemic COX-2 inhbition,using COX-2 inhibitors. [Funded by NIH grant 2R01 CA 061038 and US Army Grant DAMD 179818526 (BLL)]
[Proc Amer Assoc Cancer Res, Volume 45, 2004]
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