Muduli Suchismita, Golan-Goldhirsh Avi, Gopas Jacob, Danilenko Michael
Department of Clinical Biochemistry & Pharmacology, Faculty of Health Sciences, Ben-Gurion University of the Negev, Beer Sheva 8410501, Israel.
The Jacob Blaustein Institutes for Desert Research (BIDR), French Associates Institute for Agriculture and Biotechnology of Drylands, Ben-Gurion University of the Negev, Sede Boqer Campus, Midreshet Ben Gurion 8499000, Israel.
Pharmaceuticals (Basel). 2022 Mar 28;15(4):410. doi: 10.3390/ph15040410.
Acute myeloid leukemia (AML) is an aggressive hematological malignancy characterized by uncontrolled proliferation of immature myeloid progenitors. Here, we report the in vitro antileukemic effects of the sesquiterpene thioalkaloid-enriched fraction of the leaf extract (NUP) and a purified thioalkaloid 6,6'-dihydroxythiobinupharidine (DTBN). Treatment with 0.3-10 µg/mL NUP caused a dose- and time-dependent reduction in proliferation and viability of human AML cells (KG-1a, HL60 and U937). This was associated with apoptosis induction manifested by annexin-V/propidium iodide binding as well as cleavage of caspases 8, 9, and 3 as well as poly (ADP-ribose) polymerase. Caspase-dependence of the apoptotic effect was confirmed using the pan-caspase inhibitor Q-VD-OPH. NUP induced significant biphasic changes in the cytosolic levels of reactive oxygen species (ROS) compared to untreated cells-a decrease at early time points (2-4 h) and an increase after a longer incubation (24 h). ROS accumulation was accompanied by lowering the cellular glutathione (GSH) levels. In addition, NUP treatment resulted in elevation of the cytosolic Ca (Ca) levels. The thiol antioxidant and glutathione precursor N-acetyl cysteine prevented NUP-induced ROS accumulation and markedly inhibited apoptosis. A similar antiapoptotic effect was obtained by Ca chelating using BAPTA. These data indicate that NUP-induced cell death is mediated, at least in part, by the induction of oxidative stress and Ca accumulation. However, a substantial apoptotic activity of pure DTBN (0.05-0.25 µg/mL), was found to be independent of cytosolic ROS or Ca, suggesting that alternative mechanisms are involved in DTBN-induced cytotoxicity. Notably, neither NUP nor DTBN treatment significantly induced cell death of normal human peripheral blood mononuclear cells. Our results provide the basis for further investigation of the antileukemic potential of NUP and its active constituents.
急性髓系白血病（AML）是一种侵袭性血液系统恶性肿瘤，其特征为未成熟髓系祖细胞的不受控制的增殖。在此，我们报告了叶提取物富含倍半萜硫生物碱的部分（NUP）和纯化的硫生物碱6,6'-二羟基硫代去甲斑蝥碱（DTBN）的体外抗白血病作用。用0.3 - 10 µg/mL的NUP处理导致人AML细胞（KG-1a、HL60和U937）的增殖和活力呈剂量和时间依赖性降低。这与通过膜联蛋白-V/碘化丙啶结合以及半胱天冬酶8、9和3以及聚（ADP-核糖）聚合酶的裂解所表现出的凋亡诱导有关。使用泛半胱天冬酶抑制剂Q-VD-OPH证实了凋亡效应的半胱天冬酶依赖性。与未处理的细胞相比，NUP诱导了细胞溶质活性氧（ROS）水平的显著双相变化——在早期时间点（2 - 4小时）降低，在较长孵育后（24小时）升高。ROS积累伴随着细胞内谷胱甘肽（GSH）水平的降低。此外，NUP处理导致细胞溶质钙（Ca）水平升高。硫醇抗氧化剂和谷胱甘肽前体N-乙酰半胱氨酸可防止NUP诱导的ROS积累并显著抑制凋亡。通过使用BAPTA进行钙螯合获得了类似的抗凋亡作用。这些数据表明，NUP诱导的细胞死亡至少部分是由氧化应激和钙积累的诱导介导的。然而，发现纯DTBN（0.05 - \alpha.25 µg/mL）的显著凋亡活性与细胞溶质ROS或钙无关，这表明DTBN诱导的细胞毒性涉及其他机制。值得注意的是，NUP和DTBN处理均未显著诱导正常人外周血单个核细胞的细胞死亡。我们的结果为进一步研究NUP及其活性成分的抗白血病潜力提供了基础。
