Arsenic Trioxide Targets the Interleukin-6 Cascade in Multiple Myeloma by Promoting Lysosomal Degradation of the Interleukin-6 Receptor Complex.

Introduction. Treatment of multiple myeloma (MM), a B-cell neoplasm characterized by clonal expansion of plasma cells in the bone marrow, remains unsuccessful in a significant proportion of patients, so that innovative strategies are needed. Arsenic trioxide (As₂O₃) has shown notable efficacy against MM in vitro and in clinical studies. Multiple cellular pathways in MM are targeted by As₂O₃. As cellular growth of MM cells is interleukin-6 (IL-6) dependent, we investigated if As₂O₃ also targeted the IL-6 cascade. Materials and methods. The IL-6-dependent MM cell line U266 was used as an in vitro model. Cell growth was measured by MTT assay, and apoptosis by flow cytometry. Protein phosphorylation was studied by Western blotting with specific antibodies. Expression of IL-6 receptor (IL-6R) was investigated by Western blotting and flow cytometry. Gene expression was detected by quantitative polymerase chain reaction (Q-PCR). Results. As₂O₃ showed a time and dose related inhibition of U266 cellular proliferation by induction of apoptosis. At clinically achievable concentrations (2 – 4 μmol/L), As₂O₃-induced apoptosis was associated with inhibition of constitutive tyrosine phosphorylation of JAK2 and STAT3, in a time and dose-dependent manner. Furthermore, pre-treatment of U266 cells with As₂O₃ prevented rescue of phosphorylation of JAK2 and STAT3 by exogenous IL-6, implying that the IL-6 cascade was targeted. Using Western blot analysis, we showed that As₂O₃ induced a time and dose-dependent down-regulation of both components of the IL-6R complex: IL-6R alpha subunit (IL-6Rα) and gp130 signal transducer. These results were confirmed by flow cytometry, showing that As₂O₃ treatment led to a down-regulation of surface expression of the IL-6Rα. Interestingly, Q-PCR did not reveal any change in the mRNA levels of the two genes with As₂O₃ treatment, suggesting that As₂O₃ downregulated IL-6R complex via a post-transcriptional mechanism. It is known that under physiological conditions, the IL-6R complex is internalized upon ligand binding and is targeted to lysosomes for degradation. Treatment of the U266 cell line with the lysosome inhibitor ammonium chloride totally abrogated As₂O₃-induced degradation of IL-6Rα and gp130. These results suggested that As₂O₃ might promote lysosomal degradation of IL-6Rα and gp130 by inducing a ligand-independent internalization of the receptor complex. Conclusion. Our results demonstrated that As₂O₃ suppressed IL-6-induced JAK/STAT3 signaling pathway in MM cells and this might be, at least partly, mediated by promoting ligand-independent internalization and lysosomal degradation of the IL-6R complex. These results have significant implications on the use of As₂O₃ in the treatment of patients with MM and other malignancies that are IL-6 dependent.