Abstract:

Tu Yunhua, Kang Yingqian, Li Ming′e, Zhou Ying, Xue Yuecui, Ye Zhenyuan, Rong Dongyun, Zan Xuejuan, Pan Junling, Lu Hongguang, Cao Yu Department of Dermatology and Venereology, Affiliated Hospital of Guizhou Medical University, Guiyang 550004, China （Tu YH, Li ME, Xue YC, Ye ZY, Rong DY, Zan XJ, Pan JL, Lu HG, Cao Y）; Department of Microbiology, Guizhou Medical University, Guiyang 550004, China （Kang YQ）; Guizhou Provincial Chinese Medicine （Ethnic Medicine） Creation Engineering Center, College of Life Sciences, Guizhou University, Guiyang 550025, China （Zhou Y） Corresponding author: Cao Yu, Email: 382541077@qq.com 【Abstract】 Objective To investigate the effects of an ar?turmerone derivative （ATD） on the proliferation and apoptosis of A375 human melanoma cells. Methods Both A375 cells and human skin fibroblasts （HSFs） were cultured with different concentrations （5, 10, 20, 40 and 80 μmol/L） of ATD, vincristine and ar?turmerone, separately, for 48 hours in vitro. Subsequently, cell counting kit?8 （CCK?8） was used to evaluate cell proliferation, inverted microscopy to observe cell morphology after acridine orange/ethidium bromide （AO/EB） staining, and a colorimetric method to estimate caspase?3 activity. DNA fragmentation assay and flow cytometry were performed to assess cell apoptosis, and flow cytometry was conducted to analyze cell cycle. Results ATD, vincristine and Ar?turmerone all inhibited the proliferation of A375 cells in a dose?dependent manner （ATD: R2 = 0.99, F = 340.96, P < 0.05; vincristine: R2 = 0.99, F = 349.19, P < 0.05; ar?turmerone: R2 = 0.89, F = 25.41, P < 0.05）. The fifty percent inhibitory concentra?tions （IC50s） of ATD, vincristine and ar?turmerone against A375 cells were 15.96 ± 0.02 μmol/L, 77.00 ± 0.04 μmol/L and 356.95 ± 0.01 μmol/L respectively. When the drug concentrations were 5 and 10 μmol/L, the proliferation of HSFs was inhibited by 8% ± 0.06% and 25%±0.02% respectively by ATD, by 49% ± 0.09% and 34% ± 0.07% respectively by ar?turmerone, and by 33% ± 0.04% and 29% ± 0.08% respectively by vincristine, and the proliferation of A375 cells was inhibited by 26% ± 0.06% and 39% ± 0.02% respectively by ATD, by 6% ± 0.09% and 10% ± 0.07% respectively by ar?turmerone, and by 8% ± 0.04% and 17% ± 0.08% respectively by vincristine, with the inhibitory effects of the three drugs being significantly different from that of dimethyl sulfoxide （all P < 0.05）. ATD showed stronger inhibitory effects on the proliferation of A375 cells, but weaker cytotoxic effects on HSFs compared with ar?turmerone and vincristine （all P < 0.05）. Meanwhile, ATD, vincristine and ar?turmerone all induced the apoptosis of A375 cells （P < 0.05）, and caspase?3 activity increased with the increase in drug concentrations （ATD: R2 = 0.98, F = 162.30, P < 0.05; vincristine: R2 = 0.96, F = 94.39, P < 0.05; ar?turmerone：R2 = 0.95, F = 57.35, P < 0.05）. The effect of ATD on caspase?3 activity was strongest, followed by that of vincristine and ar?turmerone. As flow cytometry showed, all the three drugs induced cell apoptosis to different degrees, and ATD showed a relatively strong effect on cell apoptosis, especially late apoptosis, compared with the other two drugs. In the ATD group, the number of A375 cells in G1 phase gradually increased, while that in G2 phase and S phase significantly decreased with the increase in drug concentrations. Conclusions ATD exhibited proliferation?inhibiting and apoptosis?inducing effects on A375 cells, and the effects were stronger than those of vincristine and ar?turmerone. It is quite possible that ATD affects cell proliferation and differentiation by activating caspase?3 and arresting cell cycle in the G1 phase.