Targeting Wnt pathway in lymphoma and myeloma cells

The present study investigated the apoptotic effects of ethacrynic acid (EA) and the antifungal agent ciclopiroxolamine (cic), another drug that inhibits Wnt/beta-catenin signalling, on the myeloma cell line OPM-2 and three lymphoma cell lines OCI-LY8-LAM-53, SU-DHL-4 and Raji in vitro in comparison to normal peripheral blood mononuclear cells (PBMC) derived from healthy people. In contrast to PBMC, cic and EA led to a significant decrease of viability in lymphoma and myeloma cell lines. In conclusion, our results showed a significant selective induction of apoptosis by cic and EA in lymphoma and myeloma cells. The Wnt/beta-catenin pathway has been shown to play an important role in the regulation of cell proliferation, differentiation and apoptosis (Miller et al, 1999; You et al, 2002; Cadigan & Liu, 2006). It was recently demonstrated that the Wnt pathway is activated in lymphoma (Lu et al, 2004). Therefore, the Wnt/beta-catenin signalling molecules are attractive candidates for developing targeted therapies for lymphoma. Recently, we confirmed that the diuretic agent ethacrynic acid (EA) inhibited Wnt/beta-catenin signalling (J.X. Liu, T. Endo, I.G.H. Schmidt-Wolf, H. Zhou, J.E. Castro, T.J. Kipps, D.A. Carson and D. Lu, unpublished data). EA is already used clinically as a diuretic agent. Glutathione-Stransferase (GST), which is overexpressed in human tumours in form of GST-P, couples glutathione (GSH) with electrophilic compounds and detoxifies the cell (Aizawa et al, 2003). GSH acts as a reducing agent and antioxidant. The binding of EA to GSH can enhance the cytotoxicity of chemotherapeutic agents (Nagourney et al, 1990). Ciclopiroxolamine (cic) is a synthetic antifungal agent used topically for the treatment of yeast infections in humans and is degraded by glucoronidation (Hoffman et al, 1991). It acts as chelator of polyvalent metal cations (e.g. Feand Al) resulting in the inhibition of the metal-depending enzymes occurring in the metabolism of the cell. Furthermore, it blocks the cell cycle near the G1/S phase boundary (Hoffman et al, 1991). Recently, we used a 96-well plate-based TOPflash reporter system to screen the Gen-plus drug library (Microsource, Gaylordsville, CT, USA), which contained 960 compounds. This screen identified EA and cic as Wnt/beta-catenin inhibitors (J.X. Liu, T. Endo, I.G.H. Schmidt-Wolf, H. Zhou, J.E. Castro, T.J. Kipps, D.A. Carson and D. Lu, unpublished data). Given that the canonical Wnt signalling pathway is activated in lymphoma and myeloma cells (Lu et al, 2004), we investigated whether EA and cic could induce apoptosis and decrease viability of lymphoma and myeloma cell lines. EA and cic were titred in various lymphoma and myeloma cell lines and in PBMC derived from healthy individuals in order to check for toxicity. Titration of EA and cic revealed that 30 lmol/l EA and 10 lmol/l cic were the most effective concentrations to initialise cell death in lymphoma and myeloma cell lines without deteriorating the viability of normal PBMCs significantly (data not shown). The effect of dimethyl sulphoxide (DMSO) as a toxic solvent was only observed in the myeloma cell line OPM-2 (see below). As control, PBMC were investigated for fluorescence-activated cell sorting (FACS) analysis. A toxic effect toward cells induced a shift from viable, DiOC6-positive cells to apoptotic, DiOC6-negative cells. Viability of the cell lines and PBMC decreased slowly over time. After 72 h, the relative viability for the lymphoma cell lines SU-DHL-4, LAM-53 and Raji in the presence of EA (30 lmol/l) was 97Æ6 ± 1Æ1%, 91Æ0 ± 4Æ9% and 78Æ9 ± 2Æ1% respectively (Fig 1). These values were similar to values of the control of PBMC with 89Æ5 ± 3Æ1%. In contrast to PBMC and lymphoma cell lines, the myeloma cell line OPM-2 was highly sensitive towards DMSO and showed a decreased relative viability to 66Æ8 ± 2Æ3%. In the presence of cic (10 lmol/l), viability of the lymphoma cell lines was reduced to 55Æ9 ± 1Æ6% for SU-DHL-4, 32Æ4 ± 3Æ3% for LAM-53 and 21Æ7 ± 1Æ4% for Raji (all statistically significant with a P-value < 0Æ05) in contrast to PBMC, with 94Æ4 ± 1Æ8% viability. Similar to lymphoma cell lines, cic had a strong effect on cell vitality of the myeloma cell line OPM-2 reducing the relative viability by up to 29Æ4 ± 4Æ7% (P-value < 0Æ05). The decrease in viability was less pronounced using EA as compared to cic cell cultures (Fig 1). The combination of EA and cic showed similar values to those obtained with cic alone for SU-DHL-4 and LAM-53 cell lines. In the case of Raji cells, EA inhibited the toxic effect of cic from 21Æ7 ± 1Æ4% up to 59Æ3/)2Æ3%. In addition, in OPM myeloma cells the combination of cic and EA showed low values, with 27Æ9 ± 4Æ6% viability after 72 h of culture (P-value < 0Æ05; Fig 1). The effect of EA was studied in primary cultures derived from patients with chronic lymphocytic leukaemia. Similar data as for cell lines (Fig 1) were obtained for primary cells (data not shown). In conclusion, our results showed a significant induction of apoptosis by cic and EA in lymphoma and myeloma cells. Taken together with our previous results (Lu et al, 2004), our data suggest that EA and cic can inhibit Wnt/beta-catenin signalling in lymphoma and myeloma cell lines. In addition, EA was also shown to be effective in primary cultures derived from patients with chronic lymphocytic leukaemia. Our results are in accordance with a recent report (Sukhdeo et al, 2007) that the canonical Wnt signalling pathway is activated in multiple myeloma through constitutively active beta-catenin. Correspondence