Lycorine Downregulates HMGB1 to Inhibit Autophagy and Enhances Bortezomib Activity in Multiple Myeloma

Multiple myeloma (MM) is the second most prevalent hematologic malignancy, characterized by the infiltration of malignant plasma cells into bone marrow. In spite of current efficient therapeutic regimens, which have significantly increased patients overall survival, the major features inevitably present in MM are the intrinsic and acquired resistance with nearly universal relapse. In addition, the diverse heterogeneous characteristics of this largely incurable disease emphasize the importance of innovative therapies and identification of more effective drugs. Autophagy removes defective cellular organelles, protein aggregates, and intracellular microbes and is associated with cell survival and tumor maintenance. Inhibition of autophagy enhances sensitivity of a number of anticancer agents and induces cell death in MM. High-mobility group box-1 (HMGB1) protein plays an important subcellular localization-dependent role during autophagy. The importance of HMGB1 for induction of autophagy and tumor development has made this protein as a novel target for cancer therapy. Lycorine is a natural alkaloid with significant anti-cancer activity. While previous studies mainly showed lycorine as a potential apoptosis inducer, recent studies stated that apoptosis is not the primary underlying anti-proliferative mechanism of this compound. This led the interest to investigate the role of lycorine on other cell maintenance systems, such as autophagy. In addition single-agent efficacy of lycorine or in combination with other anti-MM agents has not been evaluated in vivo. Herein we investigated the anti-MM effect of lycorine and the role of this natural agent on regulation of autophagy in vitro and in vivo. We found that lycorine inhibits proliferation and induces apoptosis in MM cells with less sensitivity to the normal B-cell at the same concentrations. We also found that lycorine promisingly inhibits autophagy, the mechanism that MM cells use to survive and defeat treatment. We identified HMGB1, an important regulator of autophagy, as the most aberrantly expressed protein after lycorine treatment. Furthermore, we characterized HMGB1 as a critical mediator of lycorine activity against MM. Gene expression profiling (GEP) analysis showed that higher expression of HMGB1 is linked with the poor prognosis of MM. We further confirmed this correlation in human bone marrow CD138⁺ primary myeloma cells and MM cell lines. Mechanistically, by activating the proteasomal degradation of HMGB1, lycorine induces a rapid turnover of HMGB1. This led to decreased Bcl-2 phosphorylation by MEK-ERK pathway and increased association of Bcl-2 with Beclin-1 resulting in autophagy inhibition and growth attenuation. In addition, we observed higher HMGB1 expression in bortezomib resistant cells. The combination of bortezomib plus lycorine was highly efficient against MM cells and MM cells grown in bone marrow micro-environment. Lycorine showed the capability of inhibiting bortezomib induced autophagy as well as re-sensitizing resistant cells to bortezomib. In agreement with our in vitro observations, in vivo study using human MM xenograft model showed that lycorine is well tolerated, inhibits HMGB1 expression and thereby autophagy and induces enhanced bortezomib activity. These observations indicated lycorine as an effective autophagy inhibitor and revealed that lycorine alone or in combination with bortezomib is a potential therapeutic strategy. Our study supports the feasibility of lycorine in anti-MM treatment and provides a preclinical framework for combining lycorine with bortezomib in clinical setting.