Synergism between Rituximab, Xanthohumol and Simvastatin in B-NHL Lines In Vitro.

Rituximab acts in B-NHL via antibody-dependent cell-mediated cytotoxicity (ADCC), complement-dependent cytotoxicity (CDC) and apoptosis, and it also sensitizes tumor cells to the cytotoxic effects of chemotherapy via partial negative modulation of survival signal transduction pathways like constitutively activated, NF-κB and Raf-Mek1/2-Erk1/2-pathways, at saturating doses. Resistance still occurs and the introduction of non-chemotherapeutic agents which can reinforce the anti-apoptotic signal of Rtx should be explored.

Xanthohumol (X), a hop-derived flavonoid, with pro-apoptotic effects in breast cancer and B-CLL cells in vitro, has been described to interfere with NF-kappaB. Simvastatin (Sim) is known to induce growth arrest and apoptosis in various human cancer cell lines. In analogy with the functional complementation model described by Jazirehi et al., we studied synergy of X and Sim, on the effect of Rtx in NHL cell lines.

Methods: The effects of Rtx (Roche, Basel, Switzerland), X (purified hop extract) and Sim (Calbiochem, San Diego, CA) were evaluated by MTT-tests and Annexin V-propidium iodide staining on Raji and Ramos cells. Various concentrations of X, Sim and Rtx were tested in different combinations in triplo. All experiments were performed in RPMI supplemented with 5% pooled human serum as a complement source. Synergism was tested by the Observed/expected and by Calcusyn software. The influence of the different treatment combinations on NF-κB activity, was studied by Electroforetic Mobility Shift Assay (EMSA).

Results: Sim and X induce time and dose dependent apoptosis in Raji and Ramos cells (LD₅₀Sim 48h = 20 μM, LD₅₀X 48h = 25 μM). Simultaneous treatment with X and Sim synergistically augments the effect of the single drugs. After 48h of treatment of Ramos cells (39.06 μM X + 31.25 μM Sim) the observed viability is only 8% for an expected viability of 70%, and found a combination index (CI) by Calcusyn of 0.233, suggesting a strong synergism. In Raji cells, the lower concentration ranges (5–6.25 μM) of single drug treatment caused no observable cell death but in combination, X and Sim reached cell death percentages of up to 80%. Next, we added saturating concentrations of Rtx to the combination of X and Sim. For Ramos cells, the effect of Rtx in single treatment was too strong to observe any supplementary effects of X and Sim. Focussing on Raji cells, we observed synergism of X-Sim (15.625 μM X + 12.5 μM Sim 48h) and Rtx (5 μg/ml).

The influence of X-Sim on the effect of Rtx in Raji cells was reflected in the NF-κB pathway, on EMSA. On single treatment with Rtx, we already observed an inhibition of NF-κB, which was synergistically augmented after adding X and Sim. Synergism was further observed after combining Rtx, Sim and X in Raji cells and was confirmed by a very strong downregulation of NF-κB, suggesting that the combination of the compounds, studied in our experiments could be very useful in the management of NHL with constitutively activated NF-κB.

CONCLUSION Combination treatment with simvastatin and the NF-kappB inhibitor xanthohumol synergizes with rituximab in killing Raji and Ramos cells in vitro.