Octa-arginine mediated delivery of wild-type Lnk protein inhibits TPO-induced M-MOK megakaryoblastic leukemic cell growth by promoting apoptosis.
Octa-arginine mediated delivery of wild-type Lnk protein inhibits TPO-induced M-MOK megakaryoblastic leukemic cell growth by promoting apoptosis.
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DOI：
10.1371/journal.pone.0023640
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发表时间：
2011
期刊：
影响因子：
3.7
通讯作者：
Kumaki S
中科院分区：
文献类型：
--
作者：
Looi CY;Imanishi M;Takaki S;Sato M;Chiba N;Sasahara Y;Futaki S;Tsuchiya S;Kumaki S
Lnk 通过负调节 TPO、SCF 或 EPO 的细胞因子信号传导发挥非冗余作用。 Lnk 的逆转录病毒表达已被证明可以抑制造血白血病细胞增殖，表明其在癌症治疗中的治疗价值。然而，逆转录病毒基因传递具有插入突变的风险。为了避免这种不良后果，我们将细胞渗透性肽八精氨酸与 Lnk 融合，并评估体外抑制白血病细胞增殖的功效。在本研究中，对野生型 (WT)、突变型 Lnk R8 或 BSA 处理的 M-MOK 细胞进行增殖测定、流式细胞术、蛋白质印迹分析。我们发现递送的WT而非突变型Lnk R8可阻断TPO诱导的M-MOK巨核细胞白血病细胞增殖。相反，WT Lnk R8对非造血HELA或COS-7细胞没有生长抑制作用。此外，我们证明WT Lnk R8对TPO诱导的M-MOK细胞生长的抑制是剂量依赖性的。穿透的 WT Lnk R8 诱导细胞周期停滞和细胞凋亡。免疫沉淀和蛋白质印迹数据表明，WT Lnk R8 与内源性 Jak2 相互作用，并在 TPO 刺激后下调 M-MOK 细胞中的 Jak-Stat 和 MAPK 磷酸化水平。用特定抑制剂（TG101348 和 PD98059）处理表明 Jak-Stat 和 MAPK 途径对于 TPO 诱导的 M-MOK 细胞增殖至关重要。使用 TF-1 和 HEL 白血病细胞系的进一步分析表明，WT Lnk R8 抑制 Jak2 依赖性细胞增殖。使用脐带血来源的 CD34+ 干细胞，我们发现递送的 WT Lnk R8 可在体外阻断 TPO 诱导的巨核细胞生成。 WT Lnk R8 融合蛋白的细胞内递送通过促进细胞凋亡有效抑制 TPO 诱导的 M-MOK 白血病细胞生长。 WT Lnk R8蛋白递送可能提供一种更安全、更实用的抑制白血病细胞生长的方法，值得进一步开发。
Lnk plays a non-redundant role by negatively regulating cytokine signaling of TPO, SCF or EPO. Retroviral expression of Lnk has been shown to suppress hematopoietic leukemic cell proliferation indicating its therapeutic value in cancer therapy. However, retroviral gene delivery carries risks of insertional mutagenesis. To circumvent this undesired consequence, we fused a cell permeable peptide octa-arginine to Lnk and evaluated the efficacy of inhibition of leukemic cell proliferation in vitro. In this study, proliferation assays, flow cytometry, Western Blot analyses were performed on wild-type (WT), mutant Lnk R8 or BSA treated M-MOK cells. We found that delivered WT, but not mutant Lnk R8 blocked TPO-induced M-MOK megakaryoblastic leukemic cell proliferation. In contrast, WT Lnk R8 showed no growth inhibitive effect on non-hematopoietic HELA or COS-7 cell. Moreover, we demonstrated that TPO-induced M-MOK cell growth inhibition by WT Lnk R8 was dose-dependent. Penetrated WT Lnk R8 induced cell cycle arrest and apoptosis. Immunoprecipitation and Western blots data indicated WT Lnk R8 interacted with endogeneous Jak2 and downregulated Jak-Stat and MAPK phosphorylation level in M-MOK cells after TPO stimulation. Treatment with specific inhibitors (TG101348 and PD98059) indicated Jak-Stat and MAPK pathways were crucial for TPO-induced proliferation of M-MOK cells. Further analyses using TF-1 and HEL leukemic cell-lines showed that WT Lnk R8 inhibited Jak2-dependent cell proliferation. Using cord blood-derived CD34+ stem cells, we found that delivered WT Lnk R8 blocked TPO-induced megakaryopoiesis in vitro. Intracellular delivery of WT Lnk R8 fusion protein efficiently inhibited TPO-induced M-MOK leukemic cell growth by promoting apoptosis. WT Lnk R8 protein delivery may provide a safer and more practical approach to inhibit leukemic cell growth worthy of further development.
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