[Daporinad Potently Induces Cell Death in Ph(+) Acute Lymphoblastic Leukemia SUP-B15 Cells Independent of the Intrinsic Apoptotic Pathway].

内容摘要

To investigate whether daporinad (APO866), a nicotinamide phosphoribosyltransferase (NAMPT) inhibitor, can potently kill Philadelphia chromosome-positive (Ph+) acute lymphoblastic leukemia (ALL) cells via an intrinsic apoptosis-independent pathway. Ph+ ALL SUP-B15 cells were treated with varying concentrations of APO866. After treatment, cell viability was measured via Deep Blue assay, while apoptosis and cell death were determined by Annexin V/7-AAD double staining. Cleavage of Caspase-3 and PARP1 was detected by Western blot, and rescue experiments with the pan-caspase inhibitor Z-VAD-FMK were performed to rule out apoptotic involvement. To determine whether APO866-induced cell killing depends on nicotinamide adenine dinucleotide (NAD+) depletion, the NAD+ precursor nicotinic acid (NA) was supplemented and NAD+consuming enzyme CD38 was knocked down. Furthermore, TP53 knockout and BAX/BAK double-knockout SUP-B15 cell models were used to verify that the cytotoxic effect of APO866 is independent of the intrinsic apoptotic pathway. Both Deep Blue and Annexin V/7-AAD staining assays showed that APO866 effectively kills SUP-B15 cells. This cytotoxic effect could not be abrogated by the pan-caspase inhibitor Z-VAD-FMK, and no cleavage of Caspase-3 and PARP1 was detected during the process of cell killing. Intracellular NAD+ levels were markedly decreased following APO866 treatment, and supplementation with NA or knockout of CD38 partially reversed such cytotoxicity. Neither TP53 knockout nor BAX/BAK double knockout impaired the killing efficiency of APO866 against SUP-B15 cells. APO866 could potently induce SUP-B15 cell death by depleting intracellular NAD+ levels, a process that lacks canonical features of the intrinsic apoptotic pathway. Moreover, its cytotoxicity persists even with deficiencies in key regulators of this pathway, indicating a mechanism of action that is independent of intrinsic apoptosis. 达珀利奈通过非内源性凋亡途径诱导Ph+急性淋巴细胞白血病SUP-B15细胞死亡. 探讨烟酰胺磷酸核糖转移酶（NAMPT）抑制剂达珀利奈（APO866）能否通过不依赖内源性凋亡的途径有效杀伤Ph+急性淋巴细胞白血病细胞。. 采用不同浓度的APO866处理Ph+急性淋巴白血病SUP-B15细胞。通过Deep Blue细胞活力检测试剂测定活细胞数量；Annexin V/7-AAD双染法检测细胞凋亡和死亡情况；采用Western blot检测Caspase-3与PARP1的切割情况，并使用凋亡抑制剂Z-VAD-FMK进行挽救实验，以排除凋亡途径；通过添加NAD+ 前体烟酸（NA）或敲除NAD分解酶CD38 验证APO866对SUP-B15细胞的杀伤是否依赖于NAD + 耗竭；利用 TP53 敲除及BAX/BAK双敲除的SUP-B15细胞模型，进一步验证APO866对SUP-B15细胞的杀伤作用是否独立于内源性凋亡通路。. Deep Blue 与Annexin V/7-AAD检测结果均表明APO866可有效杀伤SUP-B15细胞；该杀伤作用无法被凋亡抑制剂Z-VAD-FMK阻断，且细胞杀伤过程中未检测到Caspase-3和PARP1的切割现象。APO866处理后细胞内NAD+ 水平显著降低，补充烟酸或敲除 CD38 可部分逆转其杀伤效应； TP53敲除或BAX/BAK 双敲除均未降低APO866对SUP-B15细胞的杀伤效率。. APO866可通过降低NAD+水平有效诱导SUP-B15细胞死亡，该过程不具备经典凋亡特征；且该杀伤作用不受内源性凋亡关键基因缺陷的影响，提示其作用机制独立于该通路。.