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/LTVO,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 pglycoprotein.
ABSTRACT
Objectives To investigate the effect of proliferation and invasiveness by turmeri cvolatile oil on human neuroblastoma cell line SH-SY5Y. Methods Cells were incubated with different concentrations of TVO in vitro. Then cell survival rate was measured by MTT assay. The effect of 160 mg/L TVO on cell migration was assessed by cell scuffing test. Invasive ability of cell was detected by Transwell test. Apoptosis of cells was detected observed by flow cytometry assay. Results Survival rate of SH-SY5Y cells decreased and apoptisis rate was abated with elevated TVO concentration and prolonged cultivation time. Level of cell migration was lower than that in control group after being cultured with 160 mg/L TVO solution for 12 , 24 and 48h. With the in-crease of TVO concentration , the invasion ability of cells gradually decreased , and the invasive force and cis-platin had no obvious difference when the concentration of drug reached 160 mg/L. Conclusion The prolifera-tion of cells can be inhibited by inhibiting the proliferation and invasiveness ability with TVO.
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 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.