Zoledronate Can Overcome Drug Resistance in a Dexamethasone Resistant Myeloma Cell Line.

<Introduction> Multiple myeloma (MM) is almost invariably fatal despite all available chemotherapeutic and supportive treatment. Initial treatment of MM with dexamethasone (Dex), a key drug for MM chemotherapy, effectively induces myeloma cell death. However, prolonged drug exposure results in the development of Dex-resistance and clinical failures. Therefore, one of the most important issues in myeloma therapy is to overcome resistance to Dex at the stage of clinical refractoriness. Several recent in-vitro studies have demonstrated an antitumor effect of nitrogen-containing amino-bisphosphonates (N-BPs) in some tumor cell lines, including myeloma cell lines. However, few data are available concerning the effects of N-BPs on Dex resistant myeloma cell lines. In this study, we have established a Dex-resistant human myeloma cell line and investigated the antitumor effect of the third generation bisphosphonate Zoledronate (ZOL) on the Dex-resistant subline.

<Methods> 1) Development of de novo resistance through continuous exposure to Dex. A Dex-resistant human myeloma cell line (Dex-R) was selected from the Dex-sensitive myeloma cell line RPMI8226 by continuously exposing cells to gradually increasing doses of Dex. Resistance was validated by flow cytometry. 2) Apoptosis assay. Dual-color FACS with propidium iodide (PI) and Annexin V was used to detect Dex-induced apoptosis in RPMI8226 and Dex-R. 3) Surface markers. Phenotypes of sensitive and resistant cells were compared by FACS analysis. 4) Cell Proliferation and DNA synthesis assay. RPMI8226 and Dex-R cells were cultured in 96 wells plates for 72 hours in the absence or presence of Dex (1 μM) or increasing concentrations of ZOL (2.5 to 50 μM). Cell growth was assessed by WST-8 assay kit. DNA synthesis was measured according to thymidine uptake. 5) Actin staining. RPMI8226 and Dex-R cells were cultured for 48 hours in the absence or presence of Dex (2 μM) or ZOL (40 μM). The distribution of f-actin stained by fluorescent phalloidin was investigated by confocal fluorescence microscopy.

<Results> The developed subline Dex-R showed reduced apoptotic and antiproliferative responses to Dex treatment. Flow cytometry on 24 hours Dex exposure revealed significantly low percentage of apoptotic (Annexin V positive and PI negative) population in Dex-R cells compared with RPMI8226 cells (6.7% vs. 29.9%). Our data demonstrate that Dex-R cells showed increased CD38, CD44, CD49d (VLA-4) expression, and decreased CD45, CD95 (Fas), CD138 (Syndecan-I) expression as compared to native RPMI8226 cells. ZOL induced apoptosis and inhibited DNA synthesis and cell proliferation in both RPMI8226 and Dex-R cell lines in a dose-dependant manner. Interestingly, some modifications of the cellular morphology were observed in ZOL-treated cells. Rhodamine-phalloidin staining of the f-actin cytoskeleton showed disruption of the cytoskeleton in these deformed cells. This fibroblast-like cellular morphology was common to Dex-R and RPMI8226 cells treated with ZOL, but was not detected under Dex treatment. This result might reflect different mechanism of antitumor effect between Dex and ZOL..

<Conclusion> Our results suggest that ZOL can induce myeloma cell death in vitro in a different mechanistic way than Dex. ZOL might be effective as an antitumor drug for Dex resistant myeloma.