姜黄挥发油联合顺铂对人皮肤鳞状细胞癌A431细胞增殖和凋亡的影响及机制

Effects of turmeric volatile oil combined with cisplatin on the proliferation and apoptosis of a human cutaneous squamous cell carcinoma cell line A431 and their mechanisms

目的 探讨姜黄挥发油（TVO）联合顺铂对人皮肤鳞状细胞癌（鳞癌）A431细胞增殖、凋亡的影响及机制。方法 取对数生长期A431细胞，用5、10、20、40、80 mg/L TVO处理，对照组用含有1％二甲基亚砜（DMSO）的DMEM高糖培养基处理，24 h后，CCK8法检测细胞增殖情况。另取部分A431细胞，分为4组，分别用含1％ DMSO的DMEM高糖培养基（对照组）、40 mg/L TVO（TVO组）、10 mg/L顺铂（顺铂组）、40 mg/L TVO和10 mg/L顺铂（TVO ＋ 顺铂组）处理24 h后，利用CCK8法检测细胞增殖活性；倒置显微镜观察细胞形态变化；吖啶橙（AO）/溴化乙锭（EB）双染色荧光显微镜观察细胞凋亡情况；比色法检测Caspase-3、Caspase-9酶活性；Western印迹检测Caspase-3、P糖蛋白表达。结果 5、10、20、40、80 mg/L TVO作用A431细胞24 h，对细胞增殖抑制率分别是（12.83 ± 6.4）%、（16.27 ± 11.4）%、（21.61 ± 9.1）%、（33.11 ± 2.0）%和（46.00 ± 3.3）%，与对照组（4.03 ± 1.4）%比较差异均有统计学意义（P 〈 0.05）；24 h时抑制50%细胞增殖的TVO浓度为（61.66 ± 1.03） mg/L；TVO组、顺铂组及TVO ＋ 顺铂组细胞增殖抑制率显著上升，与对照组比较差异均有统计学意义（P 〈 0.05），联合用药有协同作用，联合指数（CI）为1.366（P 〈 0.05）。TVO组、顺铂组细胞均不同程度变圆并出现凋亡，TVO ＋ 顺铂组细胞明显减少，出现大量细胞碎片。TVO组、顺铂组及TVO ＋ 顺铂组细胞Caspase-3酶活性分别是1.117 ± 0.095、1.381 ± 0.089、1.520 ± 0.115，Caspase-9酶活性分别是1.259 ± 0.059、1.829 ± 0.171、2.760 ± 0.297，Caspase-3蛋白表达量分别是1.156 ± 0.006、1.296 ± 0.021、1.482 ± 0.016，P糖蛋白表达量分别是1.311 ± 0.011、1.169 ± 0.012、0.528 ± 0.014，其中TVO ＋ 顺铂组细胞Caspase-3、Caspase-9酶活性及Caspase-3蛋白表达量显著高于TVO组和顺铂组，P糖蛋白表达量显著低于TVO组和顺铂组，差异均有统计学意义（P 〈 0.01）。结论 姜黄挥发油与顺铂联用可协同抑制人皮肤鳞癌A431细胞增殖并诱导其凋亡，其机制可能与活化Caspase系统并降低P糖蛋白表达相关。

Objective To evaluate the effects of turmeric volatile oil （TVO） combined with cisplatin on the proliferation and apoptosis of a human cutaneous squamous cell carcinoma cell line A431, and to explore their mechanisms. Methods Some cultured A431 cells at exponential growth phase were divided into several groups to be treated with 5, 10, 20, 40 and 80 mg/L TVO, as well as high-glucose Dulbecco′s modified Eagle′s medium （DMEM） containing 1% dimethyl sulfoxide （DMSO, control group）, respectively. After 24-hour treatment, cell counting kit 8 （CCK8） assay was performed to estimate the proliferative activity of A431 cells in the above groups. Some other A431 cells were divided into 4 groups： control group treated with high-glucose DMEM containing 1% DMSO, TVO group treated with 40 mg/L TVO, cisplatin group treated with 10 mg/L cisplatin, and TVO ＋ cisplatin group treated with 40 mg/L TVO and 10 mg/L cisplatin. After 24-hour treatment, CCK8 assay was performed to estimate the cellular proliferative activity, inverted microscopy to observe changes in cell morphology, fluorescence microscopy to detect cell apoptosis after acridine orange （AO）/ethidium bromide （EB） double-staining, colorimetry to evaluate the activity of Caspase-3 and Caspase-9, and Western blot analysis to determine the protein of Caspase-3 and p-glycoprotein. Results After 24-hour treatment with 5, 10, 20, 40 and 80 mg/L TVO, the cell proliferation rates were inhibited by （12.83 ± 6.4）%, （16.27 ± 11.4）%, （21.61 ± 9.1）%, （33.11 ± 2.0）% and （46.00 ± 3.3）% respectively, and the inhibition rates were all significantly higher in these groups than in the control group（4.03% ± 1.4%, all P 〈 0.05）. The 50% inhibitory concentration （IC50） of TVO at 24 hours was （61.66 ± 1.03） mg/L. Compared with the control group, the proliferation inhibition rates significantly increased in the TVO group, cisplatin group and TVO ＋ cisplatin group （all P 〈 0.05）, suggesting that the combination of TVO and cisplatin showed synergistic inhibitory effects with a combination index of 1.366. Moreover, A431 cells turned round to different extents and became apoptotic in the TVO group and cisplatin group, and the TVO ＋ cisplatin group showed obviously decreased number of cells and a large number of cell debris. The TVO ＋ cisplatin group also showed significantly increased activity of Caspase-3 （1.520 ± 0.115） and Caspase-9 （2.760 ± 0.297） as well as protein of Caspase-3 （1.482 ± 0.016） compared with the TVO group （Caspase-3 activity： 1.117 ± 0.095; Caspase-9 activity： 1.259 ± 0.059; Caspase-3 protein ： 1.156 ± 0.006, all P 〈 0.01） and cisplatin group （Caspase-3 activity： 1.381 ± 0.089; Caspase-9 activity： 1.829 ± 0.171; Caspase-3 protein ： 1.296 ± 0.021, all P 〈 0.01）, but significantly decreased p-glycoprotein （0.528 ± 0.014） compared with the TVO group （1.311 ± 0.011, P 〈 0.01） and cisplatin group （1.169 ± 0.012, P 〈 0.01）. Conclusion TVO combined with cisplatin can synergistically inhibit the proliferation of A431 cells and induce cell apoptosis, which may be associated with activation of the caspase system and decreased of p-glycoprotein.