SDX-101, the R-enantiomer of etodolac, induces cytotoxicity, overcomes drug resistance, and enhances the activity of dexamethasone in multiple myeloma

Abstract

In this study we report that R-etodolac (SDX-101), at clinically relevant concentrations, induces potent cytotoxicity in drug-sensitive multiple myeloma (MM) cell lines, as well as in dexamethasone (MM.1R)-, doxorubicin (Dox40/RPMI8226)-, and bortezomib (DHL4)-resistant cell lines. Immunoblot analysis demonstrates that R-etodolac induces apoptosis characterized by caspase-8, -9, and -3 and PARP (poly-ADP [adenosine diphosphate]-ribose polymerase) cleavage and down-regulation of cyclin D1 expression. Subcytotoxic doses of R-etodolac up-regulate myeloid cell leukemia-1 proapoptotic variant (Mcl-1S), while enhancing dexamethasone (Dex)-induced caspase activation and apoptosis. The combination of R-etodolac with Dex results in a highly synergistic cytotoxic effect. R-etodolac also induces apoptosis against primary cells isolated from patients with MM refractory to chemotherapy. Although interleukin 6 (IL-6) and insulin-like growth factor-1 (IGF-1) abrogate Dex-induced MM cell cytotoxicity, neither IL-6 nor IGF-1 protects against R-etodolac-induced cytotoxicity in MM cells. R-etodolac also inhibits viability of MM cells adherent to bone marrow stromal cells (BMSCs), thereby overcoming a mechanism of drug resistance commonly observed with other conventional chemotherapeutic agents. Our data, therefore, indicate that R-etodolac circumvents drug resistance in MM cells at clinically relevant concentrations, targets Mcl-1, and can be synergistically combined with Dex.

Figures

Figure 1.

R-etodolac induces growth inhibition in MM cell lines. (A) MM.1S (♦), U266 (▪), RPMI8226 (▴), INA-6 (•), and OPM1 (□) MM cells; (B) Dex-sensitive MM.1S (♦) and DEX-resistant MM.1R (▪) MM cells; (C) RPMI8226 (♦) and doxorubicin-resistant RPMI-Dox40 (▴) MM cells; (D) DHL4 (♦) and MM.1S (▴) cells; as well as (E) normal peripheral mononuclear cells from 3 healthy volunteers, no. 1 (♦), no. 2 (▪), and no. 3 (▴) were cultured for 48 hours in the presence of R-etodolac (0-2.5 mM). Cell growth was assessed by MTT assays, and data represent mean (± SD) of quadruplicate cultures.

Figure 2.

R-etodolac induces caspase cleavage and activation of apoptosis. MM.1S cells were cultured (A) for 24 hours with R-etodolac (0-1.25 mM) or (B) with R-etodolac (0.6 mM) for the indicated times. CL indicates cleaved band. (C) MM.1S cells were preincubated with Z-VAD-FMK (25 μM) for 30 minutes prior to treatment with R-etodolac (0.6 mM) for the indicated times. (D) RPMI8226 cells were also cultured with or without R-etodolac (0.6 mM) for indicated times. Total cell lysates were subjected to Western blotting using anti-caspase-8, -caspase-9, and -caspase-3, -PARP, and -α-tubulin Abs.

Figure 3.

R-etodolac induces down-regulation of cyclin D1 expression. (A) U266 cells were cultured with control media (□) or 1.0 mM R-etodolac (▪) at the indicated times. Assessed by trypan blue exclusion, the percentage of cell growth is shown in the bar graph; and the percentage of cell viability is depicted by line. Data represent mean (± SD) of quadruplicate cultures. (B) U266 cells were cultured with or without R-etodolac (1.0 mM) for the indicated times. Total cell lysates were subjected to Western blotting using anti-cyclin D1 and -α-tubulin Abs. (C) Cell-cycle profile was assessed by propidium iodide (PI) staining and flow cytometric analysis. The percentage on each panel indicates the percentage of cells in the sub-G1 region (horizontal bar).

Figure 4.

R-etodolac synergistically enhances Dex-induced cytotoxicity against MM cells. MM.1S cells were cultured for 24 hours with control media (□) and with 0.5 μM Dex () or 1.0 μM Dex (▪) in the presence or absence of R-etodolac (0.15-0.6 mM). Cell growth was assessed by [3H]-thymidine uptake (A) and MTT assays (B). CPM indicates counts per minute. (C) Dose dependency for inhibition of proliferation of MM1.S cells at 24 hours was analyzed by MTT assays, after culture with 18 to 600 μM R-etodolac alone (♦), 0.06 to 2 μM Dex alone (□), or both (▴). Data represent mean (± SD) of quadruplicate cultures.

Figure 5.

Low doses of R-etodolac induce up-regulation of Mcl-1s and Dex-induced apoptosis in MM cells. (A) MM.1S cells were cultured with R-etodolac at the indicated doses for 24 hours. (B) MM.1S cells were cultured with R-etodolac (0.6 mM) for the indicated times. (C) MM.1S cells were cultured for 24 hours with control media or 1.0 μM Dex, in the presence or absence of R-etodolac (0.15 or 0.3 mM). Total cell lysates were subjected to Western blotting using anti-caspase-8, -caspase-9, -PARP, -Bax, -Bcl-2, -Bcl-xL, -Mcl-1, and -α-tubulin Abs.

Figure 7.

R-etodolac overcomes the growth stimulatory effects of IL-6, IGF-1, and BMSCs. MM.1S (A) and RPMI8226 (B) MM cells were cultured for 24 hours with control media (□) and with 0.3 mM (), 0.6 mM (), or 1.25 mM (▪) R-etodolac, in the presence or absence of IL-6 (25 ng/mL) and IGF-1 (50 ng/mL). MM.1S cells, 2 different patient BMSCs (BMSC no. 1 [C] and BMSC no. 2 [D]), or both MM.1S cells and BMSCs were cultured for 24 hours with control media (□), and with 0.3 mM (), 0.6 mM (), or 1.25 mM (▪) R-etodolac. Cell growth was assessed by [3H]-thymidine uptake, and data represent mean (± SD) of quadruplicate cultures. *P < .01.

Figure 6.

R-etodolac induces apoptosis in cells of patients with MM. CD138+ cells were isolated from BM of patients with MM (MM no. 1 and MM no. 2) who had relapsed and were refractory to conventional therapies. (A) MM no. 1 (♦), MM no. 2 (▪), and MM.1S cell line (▴) were cultured for 48 hours in the presence of R-etodolac (0-2.5 mM). Cell growth was assessed by MTT assays, and data represent mean (± SD) of triplicate cultures. (B) MM no. 1 and MM no. 2 cells were cultured with R-etodolac (0.6 mM) for 24 hours. Total cell lysates were subjected to immunoblotting using anti-caspase-8 and anti-PARP Abs.