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

Abstract

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 expression 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 expression 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 expression: 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 expression: 1.296 ± 0.021, all P < 0.01) , but significantly decreased p-glycoprotein expression (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 expression of p-glycoprotein. Key words: Neoplasms, squamous cell; Cell line, tumor; Curcuma longa; Cisplatin; Drug therapy, combination; Caspases; P-glycoprotein; A431 cells