Wu Qing, Lin Wei-Dong, Liao Guan-Qun, Zhang Li-Guo, Wen Shun-Qian, Lin Jia-Ying
Qing Wu, Wei-Dong Lin, Guan-Qun Liao, Li-Guo Zhang, Shun-Qian Wen, Jia-Ying Lin, Department of General Surgery, the Affiliated Foshan Hospital of Southern Medical University, Foshan 528000, Guangdong Province, China.
World J Gastroenterol. 2015 Jan 21;21(3):854-61. doi: 10.3748/wjg.v21.i3.854.
To investigate the antiproliferative activity of cinobufacini on human hepatocellular carcinoma HepG2 cells and the possible mechanism of its action.
HepG2 cells were treated with different concentrations of cinobufacini. Cell viability was measured by methylthiazolyl tetrazolium (MTT) assay. Cell cycle distribution was analyzed by flow cytometry (FCM). Cytoskeletal and nuclear alterations were observed by fluorescein isothiocyanate-phalloidin and DAPI staining under a laser scanning confocal microscope. Changes in morphology and ultrastructure of cells were detected by atomic force microscopy (AFM) at the nanoscale level.
MTT assay indicated that cinobufacini significantly inhibited the viability of HepG2 cells in a dose-dependent manner. With the concentration of cinobufacini increasing from 0 to 0.10 mg/mL, the cell viability decreased from 74.9% ± 2.7% to 49.41% ± 2.2% and 39.24% ± 2.1% (P < 0.05). FCM analysis demonstrated cell cycle arrest at S phase induced by cinobufacini. The immunofluorescence studies of cytoskeletal and nuclear morphology showed that after cinobufacini treatment, the regular reorganization of actin filaments in HepG2 cells become chaotic, while the nuclei were not damaged seriously. Additionally, high-resolution AFM imaging revealed that cell morphology and ultrastructure changed a lot after treatment with cinobufacini. It appeared as significant shrinkage and deep pores in the cell membrane, with larger particles and a rougher cell surface.
Cinobufacini inhibits the viability of HepG2 cells via cytoskeletal destruction and cell membrane toxicity.
探讨华蟾素对人肝癌HepG2细胞的增殖抑制活性及其可能的作用机制。
用不同浓度的华蟾素处理HepG2细胞。采用甲基噻唑基四氮唑（MTT）法检测细胞活力。通过流式细胞术（FCM）分析细胞周期分布。在激光扫描共聚焦显微镜下用异硫氰酸荧光素-鬼笔环肽和4',6-二脒基-2-苯基吲哚（DAPI）染色观察细胞骨架和细胞核的变化。用原子力显微镜（AFM）在纳米水平检测细胞形态和超微结构的变化。
MTT法表明，华蟾素以剂量依赖性方式显著抑制HepG2细胞的活力。随着华蟾素浓度从0增加到0.10 mg/mL，细胞活力从74.9%±2.7%降至49.41%±2.2%和39.24%±2.1%（P<0.05）。FCM分析表明，华蟾素诱导细胞周期阻滞于S期。细胞骨架和细胞核形态的免疫荧光研究表明，华蟾素处理后，HepG2细胞中肌动蛋白丝的规则重组变得混乱，而细胞核未受到严重损伤。此外，高分辨率AFM成像显示，华蟾素处理后细胞形态和超微结构发生了很大变化。表现为细胞膜明显收缩和出现深孔，细胞表面有较大颗粒且更粗糙。
华蟾素通过破坏细胞骨架和细胞膜毒性抑制HepG2细胞的活力。
