Synergistic Activity of Bendamustine in Combination with Doxorubicin and Bortezomib in Multiple Myeloma Cells

Abstract Bendamustine is a purine analogue/alkylator hybrid that has shown potent clinical activity against several human cancer cells including non-Hodgkin’s lymphoma (NHL) and multiple myeloma (MM). In combination with corticosteroids, bendamustine was superior to melphalan in newly diagnosed patients with MM (Ponisch et al. J Cancer Res Clin Oncol, 2006; 132:205–12). In addition, the combination of doxil and bortezomib had been shown to be an effective regimen in MM (Orlowski et al. J Clin Oncol2007; 25:3892–901). However, published data has shown that the combination of bendamustine with doxorubicin (or mitoxantrone) is antagonistic in lymphoma cells in vitro (Chow et al. Haematologica2001; 86:485–93), discouraging the investigation of bendamustine/doxorubicin combination therapy in MM. Here we investigate the in vitro effects of bendamustine (B) in combination with doxorubicin (D) and bortezomib (V) in MM cells. H929 and MM1R myeloma cell lines were cultured in vitro and cellular cytotoxicity was measured by MTS ([3-(4,5-dimethylthiazol-2-yl)-5-(3-carboxymethoxyphenyl)-2-(4-sulfophenyl)-2H-tetrazolium, inner salt) assay. The effect of the combination in terms of additive, antagonistic, or synergistic activity was assessed by Valeriote and Lin’s method (Valeriote and Lin, Cancer Chemother Rep1975; 59:895–900). In repeated experiments, bendamustine alone induced MM cell death in a time and dose-dependent manner, with a bendamustine concentration that was lethal to 50% of cells (LC50) after 48 hours of 34.9± 3.4 μg/ml. Further, when combined with doxorubicin, bendamustine showed no antagonistic effect when cells were exposed to drug for 48 hours. The measured cell survival for the combination of B+D was similar to the calculated expected cell survival based on individual drug activity, indicating an additive effect (Figure 1). In addition, bendamustine showed an additive effect when combined with bortezomib, and as previously shown doxorubicin and bortezomib were synergistic (Figure 1). To test the effect of the combination of all three drugs, cells were cultured with bendamustine (15μg/ml) and doxorubicin (0.03μg/ml) for 48 hours, and bortezomib (2.5nmol/L) was added in the last 24 hours, and cytotoxicity was assayed. The expected cell survival for the combination of B+(D+V) is lower than the product of the effects of B and D+V (Figure 1), indicating a synergistic effect of bendamustine when combined with doxorubicin and bortezomib. These results provide pre-clinical rationale for clinically testing the efficacy of combining bendamustine with the established combination of doxil and bortezomib, which may be a highly effective regimen in the treatment for MM. Figure 1: Representative experiment showing synergistic cytotoxic activity of bendamustine when combined with doxorubicin and bortezomib in H929 cells. B, 15μg/ml bendamustine; D, 0.03μg/ml doxorubicin; V, 2.5nmol/L bortezomib; B+D, 15μg/ml bendamustine and 0.03μg/ml doxorubicin; B+V, 15μg/ml bendamustine and 2.5nmol/L bortezomib; D+V, 0.03μg/ml doxorubicin and 2.5nmol/L bortezomib; B+D+V, the combination of 15μg/ml bendamustine with 0.03μg/ml doxorubicin and 2.5nmol/L bortezomib. Synergy of the drug combination was defined when the measured cell survival was lower than the product of the effect of each single drug. Figure 1:. Representative experiment showing synergistic cytotoxic activity of bendamustine when combined with doxorubicin and bortezomib in H929 cells. B, 15μg/ml bendamustine; D, 0.03μg/ml doxorubicin; V, 2.5nmol/L bortezomib; B+D, 15μg/ml bendamustine and 0.03μg/ml doxorubicin; B+V, 15μg/ml bendamustine and 2.5nmol/L bortezomib; D+V, 0.03μg/ml doxorubicin and 2.5nmol/L bortezomib; B+D+V, the combination of 15μg/ml bendamustine with 0.03μg/ml doxorubicin and 2.5nmol/L bortezomib. Synergy of the drug combination was defined when the measured cell survival was lower than the product of the effect of each single drug.