Azaspirane (N-N-diethyl-8,8-dipropyl-2-azaspiro [4.5] decane-2-propanamine) inhibits human multiple myeloma cell growth in the bone marrow milieu in vitro and in vivo

Abstract

Azaspirane (N-N-diethyl-8,8-dipropyl-2-azaspiro [4.5] decane-2-propanamine; trade name, Atiprimod) is an orally bioavailable cationic amphiphilic compound that significantly inhibits production of interleukin 6 (IL-6) and inflammation in rat arthritis and autoimmune animal models. We here characterize the effect of atiprimod on human multiple myeloma (MM) cells. Azaspirane significantly inhibited growth and induced caspase-mediated apoptosis in drug-sensitive and drug-resistant MM cell lines, as well as patient MM cells. IL-6, insulin-like growth factor 1 (IGF-1), or adherence of MM cells to bone marrow stromal cells (BMSCs) did not protect against atiprimod-induced apoptosis. Both conventional (dexamethasone, doxorubicin, melphalan) and novel (arsenic trioxide) agents augment apoptosis induced by atiprimod. Azaspirane inhibits signal transducer activator of transcription 3 (STAT3) and a PI3-K (phosphatidylinositol 3-kinase) target (Akt), but not extracellular signal-regulated kinase 1 and 2 (ERK1/2), inhibits phosphorylation triggered by IL-6, and also inhibits inhibitorkappaBalpha (IkappaBalpha) and nuclear factor kappaB (NFkappaB) p65 phosphorylation triggered by tumor necrosis factor alpha (TNF-alpha). Of importance, azaspirane inhibits both IL-6 and vascular endothelial growth factor (VEGF) secretion in BMSCs triggered by MM cell binding and also inhibits angiogenesis on human umbilical vein cells (HUVECs). Finally, azaspirane demonstrates in vivo antitumor activity against human MM cell growth in severe combined immunodeficient (SCID) mice. These results, therefore, show that azaspirane both induces MM cell apoptosis and inhibits cytokine secretion in the BM milieu, providing the framework for clinical trials to improve patient outcome in MM.

Figure 1.

Azaspirane induces growth inhibition in MM cell lines and patient MM cells. (A) MM.1S (♦), U266 (▪), and RPMI8226 (▴) MM cells; (B) Dex-sensitive MM.1S (♦) and Dex-resistant MM.1R (▪) MM cells; (C) drug-sensitive RPMI8226 (♦), melphalan-resistant RPMI-LR5 (▪), and doxorubicin-resistant RPMI-Dox40 (▴) cells; (D) OPM1 (▴), INA-6 (▪), and MM.1S (♦) cells; (E) freshly isolated tumor cells from patients with MM (n = 3; ▪, , □), as well as (F) peripheral blood mononuclear cells from healthy volunteers (n = 3; ▪, ▴, ♦) were cultured for 48 hours in the presence of azaspirane (0-5 μM). Cell growth was assessed by MTT assay, and data represent mean (± SD) of quadruplicate cultures.

Figure 2.

Azaspirane induces caspase-dependent apoptotic cell death. (A) MM.1S and U266 cells were cultured with azaspirane (5 μM) for 48 and 72 hours. Azaspirane-induced apoptosis was confirmed by the TUNEL method, using flow cytometry. Percentage demonstrated is TUNEL-negative fraction gated by horizontal bars. (B) MM.1S cells were cultured with azaspirane (5 μM) for 4 and 8 hours. Whole-cell lysates were subjected to caspase-3 colorimetric assay. The absorbance was measured at 405 nm, using a spectrophotometer, and data represent mean (± SD) of triplicate cultures. (C) MM.1S cells were cultured with azaspirane (5 μM) for 4, 8, and 12 hours. Whole-cell lysates were subjected to Western blotting, using anti–caspase-8, anti–caspase-3, PARP Abs, as well as anti–caspase-9, BAX, Bcl-2, and Mcl-1 Abs. (D) MM.1S cells were preincubated with Z-VAD-FMK (20 μM) for 1 hour prior to treatment with azaspirane (5 μM) for 4, 8, and 12 hours. Whole-cell lysates were subjected to Western blotting using anti-PARP Ab.

Figure 3.

Azaspirane enhances the growth inhibitory effect of conventional chemotherapeutic agents. MM.1S cells were cultured with control media (□) and with 0.6 μM () or 1.25 μM (▪) azaspirane in the absence or presence of Dex (0.5 μM), Dox (0.5 μM), and As₂O₃ (1 μM) for 24 hours. Cell growth was assessed by MTT assay, and data represent mean (± SD) of quadruplicate cultures. ^*P < .01.

Figure 4.

IL-6 and IGF-1 do not protect against azaspirane-induced apoptosis. MM.1S cells were cultured with azaspirane (0-8 μM) for 48 hours in the presence (▪) or absence (♦) of IL-6 (25 ng/mL) (A) or IGF-1 (50 ng/mL) (B). Cell growth was assessed by [³H]-thymidine uptake, and data represent mean (± SD) of quadruplicate cultures.

Figure 5.

Azaspirane inhibits phosphorylation of JAK2/STAT3 and Akt but not ERK1/2. MM.1S cells were pretreated with azaspirane (5 μM) for 2 hours and 4 hours. Cells were then stimulated with IL-6 (20 ng/mL) or TNF-α (10 ng/mL) for 10 minutes. Whole-cell lysates were subjected to Western blotting using anti–phospho-ERK/ERK, phospho-Akt/Akt, phospho-STAT3/STAT3, phospho-JAK2, phospho-IκBα/IκBα, phospho-NFκB p65/NFκB p65, and α-tubulin Abs (A); MM.1S cells were treated with azaspirane (5 μM) for 4 hours and 8 hours. Whole-cell lysates were subjected to Western blotting, using anti–Mcl-1 and α-tubulin Abs (B).

Figure 6.

Azaspirane inhibits MM cell growth, as well as IL-6 and VEGF secretion, triggered by tumor cell binding to BMSCs. MM.1S cells, BMSCs, or both MM.1S cells and BMSCs were cultured for 48 hours in the presence of DMSO control (□) or with 0.6 μM(), 1.25 μM(), and 2.5 μM(▪) azaspirane (A). DNA synthesis was assessed by [³H]-thymidine uptake, and data represent mean (± SD) of quadruplicate cultures. IL-6 (B) and VEGF (C) levels in culture supernatants were measured by ELISA. ^*P < .01.

Figure 7.

Azaspirane inhibits generation of capillary vessels on HUVECs. HUVECs were cultured in 96-well plates with ECMatrix with or without azaspirane (Aza; 1.25 and 2.5 μM) (magnification, × 40) (A). Cytotoxicity of azaspirane (0-5μM) against HUVECs was determined at 24 hours by MTT assay (B), and VEGF levels in culture supernatants were measured by ELISA (C), and data represent mean (± SD) of quadruplicate cultures. ^*P < .01.

Figure 8.

Azaspirane inhibits in vivo human MM cell growth in a SCID mouse model. CB-17 SCID mice were inoculated subcutaneously in the interscapular area with 5 × 10⁶ OPM1 MM cells. Three weeks later with detection of palpable tumors, mice were treated intravenously with azaspirane (50 mg/kg per day) or vehicle control (PBS) for 6 days. Tumor volume was calculated as described in “Materials and methods.” Figure demonstrates significant tumor regression at 3 to 6 days following treatment. Error bars represent ± SD of tumor volume.