Echinochrome A Promotes Ex Vivo Expansion of Peripheral Blood-Derived CD34+ Cells Potentially through Downregulation of ROS Production and Activation of the Src-Lyn-p110δ Pathway

摘要

Intracellular reactive oxygen species (ROS) play an important role in the proliferation and differentiation of hematopoietic stem and progenitor cells (HSPCs). HSPCs are difficult to be expanded ex vivo while maintaining their stemness when they are exposed to oxidative damage after being released from the bone marrow. There have been efforts to overcome this limitation by using various cytokine cocktails and antioxidants. In this study, we investigated the effects of echinochrome A (Ech A)-a well-established and non-toxic antioxidant-on the ex vivo expansion of HSPCs by analyzing a CD34⁺ cell population and their biological functions. We observed that Ech A-induced suppression of ROS generation and p38-MAPK/JNK phosphorylation causes increased expansion of CD34⁺ cells. Moreover, p38-MAPK/JNK inhibitors SB203580 and SP600125 promoted ex vivo expansion of CD34⁺ cells. We also demonstrated that the activation of Lyn kinase and p110δ is a novel mechanism for Ech A to enhance ex vivo expansion of CD34⁺ cells. Ech A upregulated phospho-Src, phospho-Lyn, and p110δ expression. Furthermore, the Ech A-induced ex vivo expansion of CD34⁺ cells was inhibited by pretreatment with the Src family inhibitor PP1 and p110δ inhibitor CAL-101; PP1 blocked p110δ upregulation and PI3K/Akt activation, whereas CAL-101 and PI3K/Akt pathway inhibitor did not block Src/Lyn activation. These results suggest that Ech A initially induces Src/Lyn activation, upregulates p110δ expression, and finally activates the PI3K/Akt pathway. CD34⁺ cells expanded in the presence of Ech A produced equal or more hematopoietic colony-forming cells than unexpanded CD34⁺ cells. In conclusion, Ech A promotes the ex vivo expansion of CD34⁺ cells through Src/Lyn-mediated p110δ expression, suppression of ROS generation, and p38-MAPK/JNK activation. Hence, Ech A is a potential candidate modality for the ex vivo, and possibly in vivo, expansion of CD34⁺ cells.