Methylwogonin exerts anticancer effects in A375 human malignant melanoma cells through apoptosis induction, DNA damage, cell invasion inhibition and downregulation of the mTOR/PI3K/Akt signalling pathway
Authors: Zheng Huang
Jiaorong Chen
Chunmei Huang
Hongfeng Zhang
Zihui Xu
Li Li
Fangfang Liu
Journal: Archives of Medical Science (2019)
Discipline: CLINICAL MEDICINE
Keywords: apoptosis; cell invasion; fluorescence microscopy; human melanoma; methylwogonin.
Affiliations: 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., Endocrinology Department, The Central Hospital of Wuhan, Tongji Medical College, Huazhong University of Science and Technology, Wuhan, China.

Abstract:  Introduction: 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. Material and methods: 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. Results: 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. Conclusions: Different doses of methylwogonin led to concentration-dependent downregulation of phosphorylated PI3K, AKT and mTOR. 

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 Figure 3.  Inhibition of colony formation in A375 human malignant melanoma cells after treatment with 0 (A), 50 (B), 150 (C) and 300 (D) u00b5M doses of methylwogonin for 48 h . ![file] Coloured boxes mark some of the many repeated graphic elements. , 
 Figure 8.  Inhibition of cell invasion in A375 human malignant melanoma cells. The cells were treated with 0 (A), 50 (B), 150 (C) and 300 (D) u00b5M doses of methylwogonin for 48 h . ![file] The repeated sections at the upper-left corners of each panel invite speculation as to what was originally there that needed to be effaced. , 
 Figure 6.  Fluorescence microscopic (at 200u00d7 magnification) study of apoptosis in A375 human malignant melanoma cells using Hoechst 33342 staining dye. The cells were treated with 0 (A, untreated cells), 50 (B), 150 (C) and 300 (D) u00b5M concentrations of methylwogonin for 48 h and then stained with Hoechst 33342 before being analysed by fluorescence microscopy. Yellow arrows indicate cells with chromatin condensation, DNA fragmentation and cell shrinkage (characteristic of apoptosis) . ![file] I have not tried to annotate the repetitions within these panels exhaustively. , 
 Figure 9  Methylwogonin leads to downregulation of PI3K Akt signalling pathway in A375 human malignant melanoma cells. The cells were treated with 0, 50, 150 and 300 u03bcM doses of methylwogonin for 48 h and the protein expression was evaluated by western blot. u03b2-Actin served as a positive control . ![file] , 
 #2 Figure 8 again: ![] Compare with Fig 3 from [Xiao et al (2017)] . ![] , 
 *[right]* Figure 7 from [Chen et al (2019)] .  Methylwogonin induced DNA fragmentation in A375 human malignant melanoma cells. The cells were treated with 0 (control), 50, 150 and 300 u00b5M doses of the drug, incubated for 48 h and then harvested. The DNA was extracted and then analysed using 1.5% agarose gel electrophoresis . *[left]* Figure 5 from [Guo et al (2019)] .  5-Methoxypsoralen led to DNA fragmentation in U87MG human glioblastoma cells. The cells were treated with 0 (control), 5, 20 and 50 u00b5M doses of the drug, incubated for 48 h and then harvested. The DNA was extracted and then analyzed using 1.5% agarose gel electrophoresis . ![file] ,