高三尖杉酯碱与三氧化二砷协同杀伤急性髓系白血病干细胞及其作用机制。

BACKGROUND: At present, it is generally believed that leukemia stem cells are the source of AML, so the killing of leukemia stem cells has become important. Previous studies have suggested that HHT combined with ATO can synergistically kill U937 cells, and HHT has also demonstrated the ability to kill leukemia stem cells. We evaluated whether HHT combined with ATO can systematically kill leukemia stem cells (LSCs) and explored the synergistic effect and molecular mechanism. METHODS: CCK-8 was used to detect cell viability. The changes of cell cycle (PI staining), apoptosis (Annexin V/PI) and surface markers (CD34, CD38, CD96, CD45) were detected by flow cytometry. The cells of CD34+ primary leukemia and CD38- KG-1, and TF-1 were separated by flow cytometry. High-throughput mRNA sequencing was used to analysis mRNA level changes after the application of the two drugs. Western blot was used to verify the changes of pathway protein expression. NRG mice were used as the receptor of xenograft model. Histological H&E staining assess the invaded ability of leukemia cells, and laser scanning confocal microscopy evaluated the molecule markers change. RESULTS: HHT and ATO synergistically killed KG-1 (CD34/CD96/CD38/) and Kasumi-1 (CD34/CD38) cells. Their combination had a stronger effect of inducing apoptosis and blocking the cell cycle than HHT or ATO administrator alone, meanwhile significantly reducing the numbers of LSCs. Further, CD34CD38 cells in KG-1, KG-1a, TF-1, and primary leukemia cells were more sensitive to HHT and ATO. High-throughput mRNA sequencing suggested that HHT alone could significantly upregulate molecules related to the Notch, P53, and NF-κB signaling pathways. When combined with ATO, HHT further upregulated P53, whereas HHT-induced NF-κB pathway activation was significantly suppressed. Western blot analysis verified the change of protein expression in the above pathways and further demonstrated that GSI, could eliminate these effects. In vivo, HHT combined with ATO significantly reduced the LSC burden, and weakened the expression of LSC markers. CONCLUSIONS: This is the first evidence that HHT combined with arsenic can synergistically kill LSCs in vitro and in vivo, along with identification of the underlying mechanism, highlighting a potentially effective treatment strategy.

背景：目前普遍认为白血病干细胞是 AML 的源头，因此杀伤白血病干细胞成为重要的治疗手段。前期研究表明，HHT 联合 ATO 可协同杀伤 U937 细胞，且 HHT 亦具有杀伤白血病干细胞的能力。我们评估了 HHT 联合 ATO 是否可以系统地杀伤白血病干细胞（LSCs），并探讨了协同作用及其分子机制。

方法：CCK-8 法检测细胞活力；流式细胞术检测细胞周期（PI 染色）、凋亡（Annexin V/PI）及表面标志物（CD34、CD38、CD96、CD45）的变化；采用流式细胞术分选 CD34+初发白血病及 CD38-KG-1、TF-1 细胞；高通量 mRNA 测序分析两药应用后 mRNA 水平变化；Western blot 验证通路蛋白表达变化；NRG 小鼠作为异种移植模型的受体，组织学 H&E 染色评估白血病细胞侵袭能力，激光共聚焦扫描显微镜评价分子标志物变化。

结果：HHT 和 ATO 协同杀伤 KG-1（CD34/CD96/CD38/）和 Kasumi-1（CD34/CD38）细胞，其联合应用诱导凋亡、阻滞细胞周期的作用强于单药 HHT 或 ATO，同时显著减少 LSCs 数量；进一步，KG-1、KG-1a、TF-1 及初发白血病细胞中的 CD34CD38 细胞对 HHT 和 ATO 更敏感；高通量 mRNA 测序提示 HHT 单药可显著上调 Notch、P53、NF-κB 等信号通路相关分子，联合 ATO 后 HHT 进一步上调 P53，而 HHT 诱导的 NF-κB 通路激活则被明显抑制；Western blot 分析验证了上述通路蛋白表达变化，并进一步证实 GSI 可消除这些作用；体内实验显示，HHT 联合 ATO 显著降低 LSC 负荷，减弱 LSC 标志物表达。

结论：这是首次证明 HHT 联合砷剂可在体内外协同杀伤 LSCs，并明确其作用机制，为白血病治疗提供了一种潜在有效的治疗策略。