The effects of licofelone, a dual lipoxygenase and cyclooxygenase inhibitor, with and without rosiglitazone on human MDA-MB-231 breast cancer cells

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Clinical and preclinical studies suggest that nonsteridoal antiinflammatory drugs (NSAIDs) including cyclooxygenase (COX)-2 inhibitors reduce the risk of various cancers, however, their prolonged administration causes gastrointestinal toxicity and an increased cardiovascular risk. 5-Lipoxygenase (5-LOX), similar to COX-2, is over-expressed in breast cancer specimens. Thus, agents with 5-LOX/COX inhibition provide a practical approach for improving chemopreventive efficacy without unwanted side effects. COX and LOX inhibition have also been associated with an increase in PPAR γ expression. The present experiments were designed to test the effects of concomitant inhibition of 5-LOX and COX by licofelone in breast cancer cell lines and to assess whether dual inhibition of LOX and COX may potentiate the action of rosiglitazone. MDA-MB-231 cells were exposed to various sub-toxic concentrations of licofelone and rosiglitazone alone and in combination and analyzed for growth inhibition (MTT method), apoptosis (EB-AO and DAPI methods), cell-cycle analysis (flow-cytometry), and protein expression (immunoblot method). We found that treatment with licofelone and rosiglitazone inhibited cell growth in a dose-dependent manner. Licofelone induced apoptosis in breast cancer cells at >40 μM. At 80 μM the induction of apoptosis was >10 fold (P<0.0001). Rosiglitazone treatment induced apoptosis in breast cancer cells at higher concentrations (>100 μM). Importantly, the low dose combination of licofelone (20 μM) and rosiglitazone (40 μM) treatment induced apoptosis (~7 fold vs control) with significant induction at the higher dose combination (p<0.001 at 40 μM licofelone/80 μM rosiglitazone). Flow cytometric analysis showed that licofelone (>40 μM) and rosiglitazone (>80 μM) exhibited cell cycle arrest at the G0/G1 phase. The low dose combination did not promote cell cycle arrest while the higher dose combination therapy demonstrated significant inhibition (p <0.0009). Similarly, combination treatments resulted in decreased number of cells in the S and G2/M phases when compared to control. Western blot analysis showed a dose dependent increase in expression of p21^WAFI/CIPI with significantly increased expression noted in the combination treatments. Cyclin D1 and PCNA expressions were diminished with increasing drug concentrations, particularly in the combination treatments. Licofelone and rosiglitazone induced caspase-3 expression at higher concentrations, with even higher expression observed when both agents were applied simultaneously. Thus, our results suggest that licofelone, a novel dual 5-LOX/COX inhibitor, is a potential agent for prevention and treatment of breast cancer and the combination of low-doses of licofelone and rosiglitazone provide further promise in improving efficacy against breast cancer.