Abstract:

Han Xiaodong*, Meng Songshu, Cheng Wei, Sun Zhe, Ni Jing, Zhang Yunfei, Lin Jingrong, Song Zhiqi. *Department of Dermatology, First Affiliated Hospital of Dalian Medical University, Dalian 116011, China Corresponding author: Song Zhiqi, Email: szqdalian@163.com 【Abstract】 Objective To evaluate the inhibitory effect of apigenin on malignant melanoma in vitro, and to investigate its mechanisms. Methods Human malignant melanoma cell lines A375 and C8161 were divided into test groups and control group separately. Cells in the test groups were treated with apigenin at different concentrations, while cells in the control group were treated with dimethyl sulfoxide, for different durations. Subsequently, methyl thiazolyl tetrazolium （MTT） assay, wound healing assay and Matrigel invasion assay were carried out to estimate cellular proliferative activity, migratory activity and invasive activity, respectively, and scanning electron microscopy （SEM） was used to observe morphology of melanocyte dendrites. Flow cytometry using annexin-V/propidium iodide （PI） staining was performed to detect cell apoptosis, propidium iodide （PI） staining to analyze cell cycle, and Western blot to measure the expressions of proteins related to apoptosis and involved in the extracellular signal-regulated kinase （ERK） signaling pathway. Results MTT assay showed significant differences in cellular proliferative activity between the test groups and control group （all P < 0.05）. The proliferation of A375 and C8161 cells was inhibited by apigenin in a dose-dependent manner when the concentration of apigenin was 10 － 40 mg/L, and in a time-dependent manner when the treatment duration varied from 0 to 48 hours. The half-maximal inhibitory concentration （IC50） of apigenin at 24 hours was 25 mg/L for both A375 and C8161 cells. Wound healing assay showed that the migration of A375 and C8161 cells was significantly decelerated after 24-hour treatment with apigenin of 10, 20 and 25 mg/L compared with the control cells （all P < 0.01）. Matrigel invasion assay demonstrated that the number of invasive cells was significantly smaller in A375 and C8161 cells treated with apigenin of 10, 20 and 25 mg/L for 72 hours than in the control cells （all P < 0.01）. SEM showed that the dendrits of both A375 and C8161 cells became thinner and longer after treatment with 25 mg/L apigenin for 24 hours, with the length of dendrits being （23.30 ± 2.62） μm and （16.50 ± 1.62） μm respectively, compared to （12.38 ± 2.27） μm and （9.36 ± 2.51） μm respectively in the control groups （both P < 0.01）. After treatment with apigenin of 10 and 25 mg/L for 24 hours, a significant increase was observed in apoptosis rate in both A375 cells （3.30% ± 0.82% vs. 0.40% ± 0.07%, P < 0.01; 10.00% ± 0.60% vs. 4.00% ± 0.70%, P < 0.01） and C8161 cells （13.10% ± 1.45% vs. 7.27% ± 1.31%; 25.77% ± 2.40% vs. 7.27% ± 1.31%; both P < 0.01） compared with the control cells. Both A375 and C8161 cells were arrested in G2/M phase after treatment with 25 mg/L apigenin for 24 hours, with the percentage of cells in G2/M phase being 48.70% ± 3.04% and 31.10% ± 1.90% respectively, compared to 21.30% ± 0.75% and 25.06% ± 2.12% respectively in the control groups （both P < 0.01）. Western blot showed an increase in the expressions of apoptosis-related proteins including cleaved caspase-3 and cleaved poly ADP-ribose polymerase （PARP） with the activation of ERK signaling pathway in both A375 and C8161 cells after 24-hour treatment with 25 mg/L apigenin compared with the control groups. Conclusions Apigenin can inhibit the proliferation, migration and invasion of, but induce apoptosis and cell cycle arrest in human malignant melanoma cells, likely by regulating the expression of ERK signaling pathway-related proteins.