Metabolism of arachidonic acid by either the cyclooxygenase (COX) or the lipoxygenase (LOX) pathways generates eicosanoids involved in tumor promotion, progression and metastasis of cancer. It has been pointed out that inhibition of one of the biosynthetic ways, either COX or LOX alone may not modulate the carcinogenesis metabolism of AA. Hence, both COX and LOX pathways need to be targeted simultaneously. Earlier studies indicate that licofelone (ML-3000) is a potent inhibitor of cyclooxygenase (COX) and lipoxygenase (5-LOX) enzymes in human and bovine thrombocytes and granulocytes in vitro. Most importantly, it was shown to exert analgesic and anti-inflammatory effects in several animal models of arthritis. Based on these concepts, in the present study, we tested for the first time the efficacy of licofelone, a novel inhibitor of COX /LOX enzymes, in a dose dependent manner, against metastatic prostate cancer. To determine the chemopreventive efficacy and cell growth inhibition by licofelone, we used 59R cells derived from metastatic prostate cancer of the transgenic adenocarcinoma of the mouse prostate (TRAMP).We also compared the effects of licofelone in cells derived from human benign prostate hyperplasia (BPH) which exhibits increased androgen responsiveness. Phase contrast microscopic observation of cells treated with licofelone revealed a dose dependent effect against cancer cell growth. An IC50 at10μM was observed within 48 h in 59R cells. Fewer adherent and more floating cells were recorded at doses of 30, 40 and 50 μM concentrations. A moderate inhibition of cell growth was observed with BPH cells at 10μM after 48h. Apoptotic cells were visualized by annexine V staining and confirmed with DAPI staining. Semi-quantification revealed the appearance of early apoptotic cells (65%) at 48 h with 10μM concentration. Western blot analysis of the total protein indicated a marked inhibition of COX-2, LOX-5 and vasculoendothelial growth factor (VEGF) by licofelone in 59R cells. As the base level expression of COX-2 protein in BPH cells was not much higher than the licofelone treated cells, at this point we conclude that there is no significant change. It is also important to mention here that we did not see significant changes on COX-1 protein expression in the licofelone treated cells. Further, licofelone reduced the level of PGE2 very effectively (P<0.005) and the level of PGE2 in 59R cell lysate in comparison to that in the cell culture medium was significantly lower. Real time RT-PCR analysis using specific primers for COX-2, PGE2, LOX-5, LOX-12and VEGFshowed reduced mRNA expression with licofelone compared to the untreated control. Overall, our preliminary findings indicate a promising role for licofelone against metastatic prostate cancer which lies in its ability inhibiting key enzymes involved in the AA metabolism and to induce anti-inflammatory effects.
[Fifth AACR International Conference on Frontiers in Cancer Prevention Research, Nov 12-15, 2006]