Department of Biology, Faculty of Natural Sciences, University of Tabriz, Tabriz, Iran.
Stem Cell Research Center, Tabriz University of Medical Sciences, Tabriz, Iran.
Anticancer Agents Med Chem. 2019;19(11):1340-1349. doi: 10.2174/1871520619666190313165130.
OBJECTIVE: Many experiments have revealed the anti-tumor activity of spiro-quinazolinone derivatives on different cell types. Exposing KG1-a cells to (4t-CHQ), as an active sub-component of spiroquinazolinone benzenesulfonamides, the experiment investigated the possible mechanisms that manifest the role of 4t-CHQ in leukemic KG1-a progenitor cells. Mechanistically, the inhibitory effects of 4t-CHQ on KG1-a cells emerge from its modulating function on the expression of Bax/Bcl2 and survinin proteins. METHODS: Cell viability was assessed using MTT assay. The IC value of cells was calculated to be 131.3μM, after 72h-incubation with 4t-CHQ, ranging from 10 to 150μM. Apoptotic changes were studied using Acridine Orange/Ethidium Bromide (AO/EB) staining. DNA fragmentation was analyzed by agarose gel electrophoresis method. To evaluate the percentage of apoptotic cells and cell growth dynamic apoptotic features, we performed Annexin V/PI double staining assay and cell cycle analysis by flow cytometry. RESULTS: According to the results, apoptosis induction was initiated by 4t-CHQ in the KG1-a cells (at IC value). Cell dynamic analysis revealed that the cell cycle at the G1 phase was arrested after treatment with 4t- CHQ. Western blotting analysis showed enhancement in the expression ratio of Bax/Bcl-2, while the expression of survinin protein decreased in a time-dependent manner in the KG1-a cells. According to the docking simulation data, the effectiveness of 4t-CHQ on KG1-a cells commenced by its reactions with the functional domain of BH3 and Bcl2 and BIR domains of survivin protein. CONCLUSION: These results demonstrate a remarkable role of 4t- CHQ in arresting leukemia KG1-a stem cells both by induction of apoptosis as well as by down-regulating survivin and Bcl2 proteins.
目的：许多实验已经揭示了螺旋喹唑啉酮衍生物在不同细胞类型上的抗肿瘤活性。将 KG1-a 细胞暴露于（4t-CHQ），作为螺旋喹唑啉酮苯磺酰胺的活性亚组分，实验研究了 4t-CHQ 在白血病 KG1-a 祖细胞中发挥作用的可能机制。从机制上讲，4t-CHQ 对 KG1-a 细胞的抑制作用源于其对 Bax/Bcl2 和 survinin 蛋白表达的调节作用。
方法：使用 MTT 测定法评估细胞活力。在 4t-CHQ 孵育 72 小时后，计算出细胞的 IC 值为 131.3μM，浓度范围为 10 至 150μM。使用吖啶橙/溴化乙锭（AO/EB）染色研究凋亡变化。通过琼脂糖凝胶电泳法分析 DNA 片段化。为了评估凋亡细胞的百分比和细胞生长动态凋亡特征，我们通过流式细胞术进行 Annexin V/PI 双重染色测定和细胞周期分析。
结果：根据结果，凋亡诱导是由 4t-CHQ 在 KG1-a 细胞（在 IC 值）中引发的。细胞动态分析表明，在 4t-CHQ 处理后，细胞周期在 G1 期被阻滞。Western blot 分析显示 Bax/Bcl-2 的表达比值增加，而 survinin 蛋白的表达则呈时间依赖性降低。根据对接模拟数据，4t-CHQ 对 KG1-a 细胞的有效性是通过其与 survivin 蛋白的 BH3 和 Bcl2 功能域以及 BIR 结构域的反应开始的。
结论：这些结果表明，4t-CHQ 在通过诱导凋亡以及下调 survivin 和 Bcl2 蛋白来阻止白血病 KG1-a 干细胞方面发挥了重要作用。
J Cell Physiol. 2020-11
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