芳姜黄酮衍生物对A375人黑素瘤细胞增殖及凋亡作用的机制研究

Effects of an ar-turmerone derivative on the proliferation and apoptosis of A375 human melanoma cells and their mechanisms

目的：研究一种芳姜黄酮衍生物（ATD）对人皮肤黑色素瘤A375细胞增殖及凋亡的影响。方法不同浓度（5、10、20、40、80μmol/L）ATD、长春新碱及芳姜黄酮体外作用A375及人皮肤成纤维细胞（HSF）48 h。CCK？8法检测细胞增殖抑制率；吖啶橙/溴化乙锭（AO/EB）染色，倒置显微镜观察细胞凋亡形态；DNA片段化检测细胞凋亡；比色法检测半胱氨酸天冬氨酸蛋白酶3（caspase？3）活性；流式细胞仪检测细胞凋亡及周期。结果ATD、长春新碱及芳姜黄酮对A375细胞有抑制增殖作用，且呈剂量依赖性（ATD：R2=0.99，F=340.96；长春新碱：R2=0.99，F=349.19；芳姜黄酮：R2=0.89，F=25.41，均P〈0.05），三者IC50分别为（15.96±0.02）、（77.00±0.04）及（356.95±0.01）μmol/L。当药物浓度为5μmol/L及10μmol/L时，ATD对HSF增殖抑制率分别为（8±0.06）%和（25±0.02）%，长春新碱为（33±0.04）%和（29±0.08）%，芳姜黄酮为（49±0.09）%和（34±0.07）%；ATD对A375细胞抑制率分别为（26±0.06）%和（39±0.02）%，长春新碱为（8±0.04）%和（17±0.08）%，芳姜黄酮为（6±0.09）%和（10±0.07）%，与二甲基亚砜相比，差异均有统计学意义（P＜0.05），且ATD对A375细胞增殖抑制活性强于长春新碱及芳姜黄酮（P＜0.05），但对HSF细胞毒性却明显低于长春新碱及芳姜黄酮（P＜0.05）。ATD、长春新碱及芳姜黄酮均可诱导A375细胞凋亡，caspase？3活性随3种药物浓度增加而增强，且药效为ATD〉长春新碱〉芳姜黄酮。流式细胞仪检测证实，3种药物都能诱导细胞发生不同程度凋亡，同芳姜黄酮及长春新碱相比，ATD能显著诱导细胞凋亡，且以晚期凋亡为主。随药物浓度增加，ATD组G1期A375细胞逐渐增多，G2期及S期细胞数明显减少。结论 ATD对A375细胞有抑制增殖及促凋亡作用，该作用明显强于芳姜黄酮及长春新碱，其机制可能是激活caspase？3，使细胞周期阻滞在G1期，进而抑制肿瘤细胞分化与增殖。

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 80μmol/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 ± 0.02μmol/L, 77.00 ± 0.04μmol/L and 356.95 ± 0.01μmol/L respectively. When the drug concentrations were 5 and 10μmol/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.