Zhang Y, Gao M, Zhu M, Li H, Ma T, Wu C
School of Laboratory Medicine, Bengbu Medical College, Bengbu 233030, China.
School of Pharmacy, Bengbu Medical College, Bengbu 233030, China.
Nan Fang Yi Ke Da Xue Xue Bao. 2022 Jun 20;42(6):878-885. doi: 10.12122/j.issn.1673-4254.2022.06.11.
To explore the effects of isobavachalcone (IBC) on cell death of human breast cancer MCF-7 cells and explore the possible mechanism.
MCF-7 cells were treated with different concentrations of IBC, and the changes in cell proliferation were assessed using MTT assay. Apoptosis of MCF-7 cells following treatment with 10, 20, and 40 μmol/L IBC was analyzed using flow cytometry with annexin V-FITC/PI double staining and fluorescence microscopy, and the expressions of apoptosis- and autophagy-related proteins (Bax, Bcl-2, Akt, p-Akt, p62, and LC3) were detected with Western blotting. Electron microscopy was used to observe the changes in submicrostructure of the cells following treatment with 40 μmol/L IBC. JC-1 assay kit, ATP assay kit, and reactive oxygen species (ROS) kit were used to determine the effect of IBC on mitochondrial function of the cells.
MTT assay showed that IBC significantly inhibited the proliferation of MCF-7 cells in a concentration- and time-dependent manner, with IC values of 38.46, 31.31, and 28.26 μmol/L at 24, 48, and 72 h, respectively. IBC also concentration-dependently induced apoptosis of MCF-7 cells. IBC-induced cell death was inhibited by z-VAD-fmk, a caspase inhibitor ( < 0.05), but not by the necroptosis inhibitor necrostatin-1 (Nec-1). Western blotting showed that IBC-induced MCF-7 cell apoptosis by increasing Bax expression and down-regulating the expressions of Bcl-2, Akt and p-Akt-473 (all < 0.05). With the increase of IBC concentration, the expression of autophagy-related protein p62 and the LC3-II/I ratio increased progressively. Electron microscopy revealed the presence of autophagic bodies in IBC-treated MCF-7 cells. IBC treatment also resulted in decreased mitochondrial membrane potential and intracellular ATP level and increased ROS accumulation in MCF-7 cells ( < 0.05).
IBC is capable of inducing both apoptosis and autophagy in MCF-7 cells, suggesting the potential value of IBC as a lead compound in the development of anti-breast cancer agents.
探讨异补骨脂查耳酮（IBC）对人乳腺癌MCF-7细胞死亡的影响并探究其可能机制。
用不同浓度的IBC处理MCF-7细胞，采用MTT法评估细胞增殖变化。使用膜联蛋白V-FITC/PI双染流式细胞术和荧光显微镜分析10、20和40 μmol/L IBC处理后MCF-7细胞的凋亡情况，并用蛋白质免疫印迹法检测凋亡和自噬相关蛋白（Bax、Bcl-2、Akt、p-Akt、p62和LC3）的表达。用电子显微镜观察40 μmol/L IBC处理后细胞亚微结构的变化。使用JC-1检测试剂盒、ATP检测试剂盒和活性氧（ROS）试剂盒测定IBC对细胞线粒体功能的影响。
MTT法显示，IBC以浓度和时间依赖性方式显著抑制MCF-7细胞增殖，在24、48和72 h时的IC值分别为38.46、31.31和28.26 μmol/L。IBC还以浓度依赖性方式诱导MCF-7细胞凋亡。IBC诱导的细胞死亡被半胱天冬酶抑制剂z-VAD-fmk抑制（<0.05），但未被坏死性凋亡抑制剂necrostatin-1（Nec-1）抑制。蛋白质免疫印迹法显示，IBC通过增加Bax表达并下调Bcl-2、Akt和p-Akt-473的表达诱导MCF-7细胞凋亡（均<0.05）。随着IBC浓度的增加，自噬相关蛋白p62的表达和LC3-II/I比值逐渐升高。电子显微镜显示IBC处理的MCF-7细胞中存在自噬体。IBC处理还导致MCF-7细胞线粒体膜电位降低、细胞内ATP水平下降和ROS积累增加（<0.05）。
IBC能够诱导MCF-7细胞凋亡和自噬，提示IBC作为抗乳腺癌药物开发的先导化合物具有潜在价值。
