Chen Jiaorong, Huang Chunmei, Liu Fangfang, Xu Zihui, Li Li, Huang Zheng, Zhang Hongfeng
Department of Anatomy and Histology and Embryology, Basic Medical College, Hubei University of Traditional Chinese Medicine, Wuhan, China.
Pathology Department, The Central Hospital of Wuhan, Tongji Medical College, Huazhong University of Science and Technology, Wuhan, China.
Arch Med Sci. 2019 Jul;15(4):1056-1064. doi: 10.5114/aoms.2018.73711. Epub 2018 Apr 16.
The main purpose of the present research was to study the anticancer effects of methylwogonin in A375 human malignant melanoma cells by evaluating its effects on apoptosis, DNA fragmentation, cancer cell invasion and the mTOR/PI3K/AKT signalling pathway.
Effects on cell cytotoxicity were evaluated by MTT assay while a clonogenic assay determined the effects of methylwogonin on colony formation. Fluorescence microscopy evaluated apoptotic effects of methylwogonin in these cells using acridine orange/propidium iodide and Hoechst 33342 staining dyes. Gel electrophoresis evaluated the effects of methylwogonin on DNA fragmentation while the Matrigel invasion assay evaluated the effects of the drug on cancer cell invasion. Effects of methylwogonin on the mTOR/PI3K/AKT signalling pathway were evaluated by western blot assay.
Methylwogonin induces concentration-dependent as well as time-dependent growth inhibitory effects inducing significant cytotoxicity in these cancer cells. Methylwogonin led to dose-dependent inhibition of colony formation in A375 human malignant melanoma cells. The number of cell colonies decreased significantly as the methylwogonin dose increased from 0, 50, 150, to 300 μM. Methylwogonin treatment of cells at lower doses led to yellow fluorescence (early apoptosis), which changed to red/orange fluorescence, indicating late apoptosis at higher doses. Similar results were obtained using Hoechst 33342 staining, revealing that 50, 150 and 300 μM doses of methylwogonin led to significant morphological changes including chromatin condensation, fragmented nuclei and cellular shrinkage. DNA ladder formation was also observed, and this effect increased with increasing doses of methylwogonin. Methylwogonin also inhibited cancer cell invasion in a dose-dependent manner.
Different doses of methylwogonin led to concentration-dependent downregulation of phosphorylated PI3K, AKT and mTOR.
本研究的主要目的是通过评估甲基吴茱萸素对细胞凋亡、DNA片段化、癌细胞侵袭以及mTOR/PI3K/AKT信号通路的影响，来研究其对A375人恶性黑色素瘤细胞的抗癌作用。
通过MTT法评估对细胞毒性的影响，而克隆形成试验则确定甲基吴茱萸素对集落形成的影响。荧光显微镜使用吖啶橙/碘化丙啶和Hoechst 33342染色染料评估甲基吴茱萸素对这些细胞的凋亡作用。凝胶电泳评估甲基吴茱萸素对DNA片段化的影响，而基质胶侵袭试验评估该药物对癌细胞侵袭的影响。通过蛋白质免疫印迹法评估甲基吴茱萸素对mTOR/PI3K/AKT信号通路的影响。
甲基吴茱萸素诱导浓度依赖性以及时间依赖性的生长抑制作用，在这些癌细胞中诱导显著的细胞毒性。甲基吴茱萸素导致A375人恶性黑色素瘤细胞中集落形成的剂量依赖性抑制。随着甲基吴茱萸素剂量从0、50、150增加到300 μM，细胞集落数量显著减少。较低剂量的甲基吴茱萸素处理细胞导致黄色荧光（早期凋亡），在较高剂量时变为红色/橙色荧光，表明晚期凋亡。使用Hoechst 33342染色获得了类似结果，显示50、150和300 μM剂量的甲基吴茱萸素导致显著的形态学变化，包括染色质浓缩、核碎片化和细胞收缩。还观察到DNA梯带形成，并且这种效应随着甲基吴茱萸素剂量的增加而增强。甲基吴茱萸素还以剂量依赖性方式抑制癌细胞侵袭。
不同剂量的甲基吴茱萸素导致磷酸化PI3K、AKT和mTOR的浓度依赖性下调。
