DADS下调calreticulin促进C/EBPα表达诱导人白血病细胞分化

Developing new differentiation-induced drugs for blocking leukemia is of clinical significance. Our previous research demonstrated that the main effective component of garlic, Diallyl disulphide (DADS), induces differentiation and increases the products of ROS in leukemia cells. Through proteomic analysis, we identified differentially expressed proteins during DADS-induced differentiation in HL-60 cells, and one of them is calreticulin. Calreticulin is an important regulator of tumor cell differentiation and its down-regulation promotes C/EBPα expression. We hypothesize that DADS causes down-regulation and translocation of calreticulin through the ROS pathway and subsequently promotes expression of C/EBPα, leading to induced differentiation in leukemia cells. We will address this hypothesis using the approaches including genetically engineering cell lines, tumor formation in SCID mice, RIP-Chip, FCM, qPCR, and Western blot. The result of our proposed study will lead to a better understanding of the molecular mechanisms of DADS-induced leukemia cell differentiation and provide essential knowledge for the development of differentiation inducers to treat leukemia.

白血病的诱导分化治疗具有重要意义。申请者前期研究结果显示，大蒜中的有效成分DADS能诱导人白血病细胞分化并能增加ROS的产生，筛选DADS诱导HL-60细胞分化的差异蛋白质，发现calreticulin表达下调。基于calreticulin对肿瘤细胞的分化具有重要的调控作用，而抑制calreticulin可促进C/EBPα的表达，我们提出了DADS可通过ROS信号通路下调calreticulin并引起其移位、促进C/EBPα 的表达而诱导人白血病细胞分化的科学假说。本课题通过构建重组细胞系、SCID小鼠成瘤，应用激光共聚焦、RIP-Chip、FCM、qPCR、western blot等实验技术，体内外研究calreticulin在DADS诱导人白血病细胞分化中的作用及分子机制，深入阐明DADS诱导白血病细胞分化的作用机制，为开发白血病细胞分化的诱导剂提供理论基础和实验依据。

白血病的诱导分化治疗具有重要意义。前期研究结果显示，大蒜中的有效成分二烯丙基二硫（Diallyl disulphide，DADS）能诱导人白血病细胞分化并能增加ROS的产生，筛选DADS诱导HL-60细胞分化的差异蛋白质，发现calreticulin表达下调。本研究在前期工作的基础上，通过临床病例分析、构建重组细胞系、SCID小鼠成瘤，应用免疫荧光、RIP-Chip、FCM、qPCR、western blot等实验技术，体内外研究calreticulin在DADS诱导人白血病细胞分化中的作用及分子机制。结果显示：治疗前组与对照组、治疗前组与治疗后组CRT水平及CEBPα水平的比较差异具有统计学意义，而治疗后组与对照组CRT水平及CEBPα水平的比较差异无统计学意义，且CRT与CEBPα表达水平具有相关性；DADS诱导人白血病HL-60细胞分化过程中，calreticulin表达明显下调，而C/EBPα的表达明显上调；calreticulin过表达能逆转DADS诱导白血病细胞的生长、增殖、侵袭以及分化等生物学行为，calreticulin过表达也能逆转DADS对白血病细胞C/EBPα表达的促进作用；calreticulin沉默能增强DADS诱导白血病细胞生长、增殖、侵袭、分化等生物学行为，干扰calreticulin表达能增强DADS对白血病细胞C/EBPα表达的上调作用； DADS对人白血病细胞SCID小鼠移植瘤成瘤、生长和分化有积极的影响，与对照组相比，DADS处理组瘤组织的calreticulin表达明显上调 ，C/EBPα表达明显下调；DADS处理HL-60细胞后，细胞的活性氧水平明显升高；用NAC预处理细胞抑制细胞内活性氧水平，DADS处理组相比，细胞calreticulin表达上调；免疫荧光结果显示，DADS处理后，calreticulin由细胞质转到细胞膜； DADS处理后，细胞内calreticulin水平降低，而C/EBPα mRNA水平增高。综上所述，本研究表明DADS通过ROS信号通路下调calreticulin，并引起calreticulin移位，从而解除calreticulin与C/EBPα mRNA的黏附，促进C/EBPα表达而诱导白血病细胞分化。该研究为开发白血病细胞分化的诱导剂提供理论基础和实验依据。