Lestari Beni, Utomo Rohmad Yudi, Rahman Faaza Aulia, Putri Dyaningtyas Dewi Pamungkas, Zulfin Ummi Maryam, Suenaga Yusuke, Meiyanto Edy, Hippo Yoshitaka
Department Pharmacology and Therapy, Faculty of Medicine, Public Health and Nursing, Universitas Gadjah Mada, Yogyakarta, 55281, Indonesia.
Cancer Chemoprevention Research Center, Faculty of Pharmacy, Universitas Gadjah Mada, Sekip Utara, Sleman, Yogyakarta, 55281, Indonesia.
BMC Complement Med Ther. 2025 Jul 19;25(1):279. doi: 10.1186/s12906-025-05004-8.
Neuroblastomas evade apoptosis due to oncogene mutations and antiapoptotic proteins, necessitating novel therapeutics that work in concert with other forms of cell death. Pyroptosis has potential as a strategic cell death mechanism in neuroblastoma. This study aimed to identify compounds that modulate pyroptosis, specifically those that target gasdermin D (GSDMD) oligomerization.
The study employed computational analysis and in vitro screening. The COCONUT database provides the natural compound dataset. Drug-likeness analysis and pharmacophore fitting were applied to screen potential GSDMD oligomerization modulators. Hit compounds underwent molecular docking with MOE and molecular dynamic (MD) simulations with NAMD 2.14 to analyse structural changes. The computational screening results were corroborated by in vitro assays, including the WST-8 assay, Western blot, and immunofluorescence, which target pyroptosis-specific caspase-1.
Pharmacophore fitting, molecular docking, and molecular dynamics simulations identified the top five compounds, namely, quercetin, naringenin, hesperetin, curcumin, and galangin, as potent modulators of GSDMD oligomerization. Among these compounds, curcumin, quercetin, and galangin exerted potent cytotoxic effects on GSDMD-expressing neuroblastoma SK-N-AS cells, with IC values of 21, 37, and 49 µM, respectively. Curcumin and quercetin also promoted apoptosis via increased caspase-3 cleavage and reduced procaspase-7 and -8 levels, as shown by immunoblotting. Curcumin and galangin upregulated caspase-1 expression, as demonstrated by the detection of a fluorescent-labelled inhibitor of caspase-1 by immunostaining, suggesting that these two compounds could induce pyroptosis. Hesperetin and naringenin showed low cytotoxicity, had no effect on caspase activation, and did not exhibit signs of pyroptosis in SK-NA-S cells.
Our study successfully identified curcumin as a strong regulator of both apoptosis and pyroptosis, quercetin as a strong modulator of apoptosis, and galangin as a strong modulator of pyroptosis. Further research on these compounds is crucial for the development of novel therapeutic strategies for neuroblastoma treatment.
神经母细胞瘤因癌基因突变和抗凋亡蛋白而逃避凋亡，因此需要与其他形式的细胞死亡协同作用的新型疗法。焦亡作为神经母细胞瘤中的一种策略性细胞死亡机制具有潜力。本研究旨在鉴定调节焦亡的化合物，特别是那些靶向gasdermin D（GSDMD）寡聚化的化合物。
本研究采用计算分析和体外筛选。COCONUT数据库提供天然化合物数据集。应用类药性分析和药效团拟合来筛选潜在的GSDMD寡聚化调节剂。命中化合物与MOE进行分子对接，并与NAMD 2.14进行分子动力学（MD）模拟以分析结构变化。通过体外试验证实了计算筛选结果，包括针对焦亡特异性半胱天冬酶-1的WST-8试验、蛋白质印迹法和免疫荧光法。
药效团拟合、分子对接和分子动力学模拟确定了前五种化合物，即槲皮素、柚皮素、橙皮素、姜黄素和高良姜素，作为GSDMD寡聚化的有效调节剂。在这些化合物中，姜黄素、槲皮素和高良姜素对表达GSDMD的神经母细胞瘤SK-N-AS细胞具有强大的细胞毒性作用，IC值分别为21、37和49 μM。蛋白质印迹法显示，姜黄素和槲皮素还通过增加半胱天冬酶-3的切割并降低前半胱天冬酶-7和-8的水平来促进凋亡。免疫染色检测荧光标记的半胱天冬酶-1抑制剂表明，姜黄素和高良姜素上调了半胱天冬酶-1的表达，这表明这两种化合物可以诱导焦亡。柚皮素和橙皮素显示出低细胞毒性，对半胱天冬酶激活没有影响，并且在SK-NA-S细胞中没有表现出焦亡迹象。
我们的研究成功确定姜黄素是凋亡和焦亡的强效调节剂，槲皮素是凋亡的强效调节剂，高良姜素是焦亡的强效调节剂。对这些化合物的进一步研究对于开发神经母细胞瘤治疗的新型治疗策略至关重要。
