石蒜碱抑制HMGB1介导的自噬增强多发性骨髓瘤化疗敏感性的作用机制研究

Autophagy is closely related to the survival of multiple myeloma (MM) cells and becoming a new hot spot of MM therapy.We first demonstrated that lycorine had a selective anti-multiple myeloma (MM) effect via autophagy inhibition and the enhancing effect on the sensitivity to chemotherapy drugs in MM cell lines. Lycorine treatments resulted in significant decrease of high mobility group box 1 (HMGB 1) protein level both in cytoplasm and nucleus,which was not shown through exocytosis pathways.HMGB1 is univerally enriched in tumors,and functions by translocating to cytoplasm from the nuleus, then binding to Beclin1 and resulting in the releasing of Beclin1 from complex Beclin1-Bcl2,which finally induces autophagy and enhances drug resistance in some other tumor cell lines.In this study, we hypothesize that lycorine inhibits cells autophagy via ubiquitination degradation of HMGB1. HMGB1 degradation blocks formation of HMGB1-Bcl-2 complex and induces Bcl-2 combining with Beclin1.To prove these hypotheses, we intend to clarify the molecular signalings of autophagy inhibition introduced by lycorine and HMGB1 functions involved, and explore the potential key role of HMGB1 in autophagy supression targeted by lycorine. This study will further elucidate new molecular mechanisms of lycorine and posssibility of HMGB1 as a new target of MM therapy, to provide new prospect in MM treatment and medcine design.

自噬增加与多发性骨髓瘤(MM)紧密相关，抑制自噬是治疗MM新策略。项目组首次发现石蒜碱(lycorine)能抑制自噬抵抗耐药发挥抗MM效应。且石蒜碱在蛋白水平下调胞核和胞质中HMGB1(高迁移率族蛋白1)，而HMGB1并非通过外泌途径减少。HMGBl在肿瘤中高表达，可从胞核转位至胞质与 Beclin 1作用，使得Beclin1从 Beclin1/Bcl-2复合物中解离出来而促进自噬，增强肿瘤细胞的耐药性。由此本项目提出科学假说：石蒜碱通过泛素化降解HMGB1，减少HMGB1与Beclin1结合，增加Beclinl/Bcl-2复合物抑制自噬抵抗耐药发挥抗MM的作用。探索HMGB1是否为石蒜碱关键效应分子, 研究石蒜碱下调HMGB1抑制MM自噬抵抗耐药的机制，有望阐明石蒜碱作用MM新机制，为发现MM治疗新靶标和新药物提供新策略。

多发性骨髓瘤（Multiple myeloma, MM）是骨髓内浆细胞恶性增生的血液肿瘤。尽管目前针对MM病人有许多种有效治疗策略，但由于MM细胞遗传性改变和骨髓微环境变化导致的耐药性是MM治疗面临的难题。石蒜碱（lycorine）是一种从石蒜科植物中分离得到的天然化合物。高迁移率族蛋白1 (HMGB1)是细胞自噬的重要参与者，与细胞凋亡抑制和耐药性有关。. 基因表达谱（GEP）分析表明HMGB1的高水平表达与MM不良预后有关，体外研究也证实了这一点。石蒜碱在蛋白水平下调胞核和胞质中HMGB1，而HMGB1并非通过外泌途径减少。进一步用蛋白合成抑制剂放线菌酮 (Cycloheximide, CHX) 来阻断HMGB1的合成，结合定量co-IP实验后证实石蒜碱能通过增强HMGB1的泛素-蛋白酶体系统降解途径来降低其蛋白水平。过表达HMGB1后明显降低了石蒜碱对MM细胞增殖和自噬的抑制作用。相应地，敲低HMGB1 后明显增强石蒜碱对MM细胞增殖的能力和自噬抑制作用。且石蒜碱能导致p-MEK、p-ERK1/2和p-Bcl-2显著降低，同时co-IP结果显示Bcl-2 与Beclin-1的结合增加。这些结果表明石蒜碱介导的HMGB1蛋白酶体降解抑制了MEK-ERK通路，继而抑制了Beclin-1从Bcl-2/Beclin-1复合体中解离，导致自噬抑制。. CCK-8实验结果表明石蒜碱能有效增强硼替佐米（BTZ）对MM细胞的增殖抑制能力。建立了人MM细胞异体移植瘤NOD/SCID小鼠模型，结果表明石蒜碱治疗后坏死与凋亡的细胞数显著增加，移植瘤细胞中LC3B、Beclin-1和HMGB1的表达显著下调，证实在体内石蒜碱仍然能有效性的降低HMGB1的蛋白水平，进而抑制自噬，同时也能增强BTZ的体内抗MM效应。. 综上，本研究深入阐明石蒜碱作用MM的分子机制和HMGB1作为MM治疗新靶点的可行性，为MM治疗和药物设计提供新前景。