Combination of a Spicamycin Analogue KRN5500 and Chemotherapeutic Agents Synergistically Enhances Their Cytotoxicity in Human Myeloma Cells.

Multiple myeloma (MM) is a plasma cell malignancy characterized by the accumulation of neoplastic plasma cells in the bone marrow. Although new classes of agents such as thalidomide, lenalidomide, and bortezomib have shown marked anti-MM activity in clinical settings, MM remains an incurable disease due to increased resistance to these agents. Therefore, alternative approaches are necessary to overcome drug resistance in MM. KRN5500 is a new derivative of spicamycin produced by Streptomyces alanosinicus (Kirin Pharma, Tokyo, Japan). This drug potently decreases protein synthesis and inhibits cell growth in human tumor cell lines both in vitro and in vivo. Several phase I studies of KRN5500 were conducted in patients with solid tumors, which showed Cmax values of 1000–3000 nM at the maximum tolerated doses. However, no objective anti-tumor response to KRN5500 alone was observed in these patients. In this study, we examined the anti-tumor activity of KRN5500 against MM cells and evaluated its therapeutic potential in combination with other anti-MM agents. MM cell lines and freshly-isolated MM cells were incubated with various concentrations of KRN5500 for 24 hours. Cell proliferation assay showed marked inhibition of cell growth in MM cells such as RPMI 8226, KMS12-BM, and UTMC-2 (IC50 = 10–40 nM), and U266, MM.1S, and primary MM cells (IC50 = 500–1000 nM). Importantly, a chemotherapy-resistant subclone of RPMI 8226 had a similar sensitivity to KRN5500. Annexin V/propidium iodide staining confirmed that KRN5500 induced apoptosis of MM cells in a dose- and time-dependent manner. Moreover, cleavage of poly (ADP-ribose) polymerase (PARP) was detected after 24 hours with only modest activation of caspase-8, -9, and -3 by immunoblotting. Flow cytometric analysis of anti-apoptotic proteins revealed that apoptosis induced by KRN5500 was associated with down-regulation of Mcl-1 and Bcl-2 expression. To determine the effect of KRN5500 on the unfolded protein response (UPR), splicing of XBP-1 mRNA was analyzed by reverse transcription-polymerase chain reaction. In response to stimulation with KRN5500, splicing of XBP-1 mRNA occurred after 24 hours in RPMI 8226 cells, suggesting that KRN5500-induced apoptosis is mediated in part by the inhibition of UPR. Furthermore, synergistic effects on MM cells were observed when KRN5500 was combined with anti-MM agents including melphalan, dexamethasone, and bortezomib. These results suggest that KRN5500 induces apoptosis in MM cells mainly by the caspase-independent pathway and that its unique mechanism of action provides a valuable therapeutic option to overcome drug resistance in patients with MM.