二烯丙基二硫通过Rac1/LIMK1通路下调与失活cofilin1诱导人白血病细胞分化

Leukemia is a common malignant tumor, which single or combined ATRA and chemotherapeutic agents are highly toxic with retinoic acid syndrome and are followed by relapse in a high proportion of patients. Therefore, development new antileukemias have important significance. Diallyl disulfide (DADS) is a allyl sulfur compound derived from garlic. Our previous works demonstrate that DADS can induce differentiation of acute myelocytic leukemia HL-60 cells, and analysis of differential expression proteins in differentiation of HL-60 cells induced DADS by proteome technique show down-regulation of cofilin1. Hitherto, cofilin1 effect differentiation in HL-60 cells induced by DADS not are investigate. The topic further study that DADS induce differentiation of HL-60 cells through down-regulation and inactivation of cofilin1 mediated Rac1/LIMK1 pathway using reconstituted cell line, HL-60/pcDNA3.1/cofilin1 and HL-60/shRNA cofilin1 cells, Real-time PCR, Western blot, immunofluorescence, confocal microscopy, MTT, NBT, morphology, flow cytometry, cell migration and invasion and formation of xenograft tumor in nude mice, etc, methods.

白血病是常见恶性肿瘤之一。ATRA治疗存在维甲酸综合征与耐药性，容易复发。因此，开发新一代的抗白血病药物具有重要意义。二烯丙基二硫（diallyl disulfide，DADS）是大蒜的一种有效成分。我们已经证实，DADS可诱导人白血病细胞分化，蛋白质组学分析DADS诱导HL-60细胞分化的差异蛋白质，发现cofilin1明显下调。迄今，尚未见cofilin1对DADS诱导人白血病细胞分化影响的研究。本课题在前期工作基础上，通过建立高表达HL-60/pcDNA3.1/cofilin1与低表达HL-60/shRNA cofilin1的HL-60细胞、Real-time PCR、Western blot、MTT、形态学、NBT、流式细胞术、细胞迁移、侵袭实验、裸鼠成瘤等技术，研究DADS由Rac1-LIMK1通路介导，下调或减少cofilin1磷酸化，从而诱导人白血病细胞分化。

背景与目的：本课题在前期研究二烯丙基二硫（DADS）可诱导HL-60细胞分化与蛋白质组学技术发现DADS诱导HL-60细胞分化的差异蛋白质中cofilin1明显下调的基础上，证实DADS可下调cofilin1诱导HL-60细胞分化，通过构建cofilin1高表达和低表达HL-60细胞、Real-time PCR、Western blot、细胞增殖、细胞迁移、细胞侵袭、细胞还原能力实验等技术，进一步研究DADS通过Rac1-Rock1-LIMK1通路，下调cofilin1，诱导HL-60细胞分化，阐明其作用的分子机制，证实cofilin1是DADS诱导HL-60细胞分化的关键靶分子。结果：DADS通过抑制Rac1-Rock1-LIMK1通路下调cofilin1表达与磷酸化。DADS和沉默cofilin1均可诱导HL-60细胞分化并明显抑制HL-60细胞增殖与侵袭，沉默cofilin1可增强DADS的诱导分化能力和抑制作用。而cofilin1高表达可增加cofilin1磷酸化水平，减低DADS对HL-60细胞的还原诱导能力，并削弱DADS抑制HL-60细胞增殖、迁移与侵袭的作用。结论：DADS通过拮抗Rac1-Rock1-LIMK1通路下调cofilin1与降低p-cofilin1抑制HL-60细胞增殖、迁移与侵袭及诱导分化，cofilin1可能是DADS作用的靶点。