苦参碱分步调控泛素蛋白酶体通路和融合蛋白自噬降解而诱导维甲酸耐药APL细胞再分化的作用研究

With the applicaion of differentiation inducing drug, ATRA, acute promyelocytic leukemia may achieve an CR rate of 85%, while the rapid development of resistance and high recurrence rate is still influence the long-term curative effect, and the 10-year cumulative relapse incidence is almost 33%. No drug with the effect of resistance reversal and synergistic differentiation is available in clinical. Our previous studies indicated that wiht the co-treatment of ATRA and Matrine, the ATRA-resistance APL cell (APL-LR1) recovers its response to ATRA. The leukemia cells differentiate again, and the PML/RARα fusion protein degraded. Matrine cooperated with ATRA can increase the ubiquitin level in resistant cell, which is similar to proteasome inhibitor and do favor to the expression of differentitation gene, while it may impair the the process of PML/RARα fusion protein degradation. We further explored that, matrine combined with ATRA significantly upregulated the expression of autophagy specific protein -- light chain 1 microtubule associated protein 3 (LC3), which indicated that the fusion protein degradation may mediated by autophagy, rather than the proteasome. This study is mainly on confirming whether the depth differentiation of NB4-LR1 induced by Matrine is mediated by its two steps regulation of ubiquitin proteasome and autophagy pathway, and we also mean to investigate the interaction between the two pathways.

ATRA诱导分化治疗APL可使其完全缓解率达85%以上，但快速发展的耐药与高复发率仍是影响长期疗效的两大障碍，10年的累积复发率近33%。目前临床上仍缺少确切有效的逆转耐药并协同分化的药物。我们的前期研究发现，联用中药有效成分苦参碱能使ATRA耐药APL细胞（NB4-LR1）重新获得对ATRA的反应，白血病细胞再次分化，并伴有PML/RARα融合蛋白特征性降解。苦参碱联用ATRA能上调耐药细胞内蛋白的泛素化水平，有类似蛋白酶体抑制剂的作用，这虽能促进细胞分化基因的表达，但并不利于融合蛋白的降解。我们进一步发现，苦参碱联合ATRA能显著上调自噬特异性蛋白——微管相关蛋白1的轻链3（LC3）表达，这提示融合蛋白降解可能是由自噬所介导，而非经蛋白酶体降解。本研究旨在进一步明确苦参碱是否通过分别调控泛素蛋白酶体及自噬通路诱导耐药细胞的深度分化，并探讨两通路间的交互作用。

ATRA诱导分化治疗APL可使其完全缓解率达85%以上，但快速发展的耐药与高复发率仍是影响长期疗效的两大障碍，10年的累积复发率近33%。目前临床上仍缺少确切有效的逆转耐药并协同分化的药物。我们的前期研究发现，联用中药有效成分苦参碱能使ATRA耐药APL细胞（NB4-LR1）重新获得对ATRA的反应，白血病细胞再次分化，并伴有PML/RARα融合蛋白特征性降解。本研究旨在进一步明确苦参碱在融合蛋白降解的具体机制，围绕泛素蛋白酶体通路及自噬通路进行了深入阐明。结果显示，苦参碱能直接上调维甲酸耐药细胞（NB4-LR1）的整体泛素化水平，而非通过抑制蛋白酶体活性起效；并能稳定维甲酸受体和蛋白酶体核心蛋白20S的表达，协同ATRA的作用；继而通过促进自噬流现象，PML经P62介导进入自噬溶酶体，最终融合蛋白得到降解。此外，研究同时发现雷帕霉素和STI571同样具有协同纠正维甲酸耐药的作用，其作用机制也与泛素及自噬通路有关；而蛋白酶体抑制剂（MG132）并不利于融合蛋白的降解，能抑制维甲酸敏感细胞的分化。本研究的开展为今后苦参碱在临床的应用提供了实验基础，同时也为其他药物的选择，包括雷帕霉素、STI571以及MG132提供了参考。