| Objective:To observe the effect of oxidative stress on TRAIL tolerance of ovarian cancer, and to explore whether regulation of oxidative stress can reverse the TRAIL tolerance and provide an important treatment strategy for cisplatin resistant ovarian cancer.Methods:Part one:1) cell viability detection:SKOV3 and SKOV3/DDP cells were treated by different concentrations of TRAIL and incubated in incubator for 48 h, using the full automatic enzyme standard instrument (wavelength 490nm) to detect the absorbance, then recorded and analyzed the data, and drew the curve of cell viability under different drug concentration.2) apoptosis detection:SKOV3 and SKOV3/DDP cells were cultured with 500 ng/ml TRAIL according to the specified time, and the apoptosis was detected by flow cytometry.Part two:1) the effects of cisplatin on cell viability and apoptosis of SKOV3: Pretreated the SKOV3 cells with different dose of cisplatin for 1 h, then treated with or without TRAIL (500 ng/ml) for another 24 h, Cell viability was measured by the MTT assay and the cells apoptosis was analyzed by flow cytometry, respectivly.2) levels of reactive oxygen species (ROS) detection:Ovarian cancer cells were treated for 1 h with different doses of cisplatin or H2O2, intracellular ROS level were measured by DCFH-DA staining and FACS.3) effects of H2O2 on SKOV3 cell viability and apoptosis:SKOV3 cells were pretreated with H2O2 for 1 h, then the cells treated with or without TRAIL (500 ng/ml) for 24 h, the cell viability was measured by MTT assay and the cells apoptosis was analyzed by flow cytometry.Part three:I) Oxidative stress regulation reverse the TRAIL tolerance in SKOV3 cells:Pretreatment with NAC (0 mM,10 mM) for 1 h, then the SKOV3 cells were treated with cisplatin (1 μM) or H2O2 (0.1 mM).0ne hour later, the cells were treated together with TRAIL(500 ng/ml) for another 24 h. DCFH-DA staining and FACS analysis of the intracellular ROS levels in SKOV3 cells, Measurement of the cell viability with the MTT assay, Flow cytometric analysis of cell apoptosis.2) Oxidative stress regulation reverse the TRAIL tolerance in SKOV3/DDP cells:SKOV3/DDP cells were pretreated with NAC (0 mM,10 mM,40 mM) for 1 h and then the cells were treated with or without TRAIL (500 ng/ml) for 24 h. DCFH-DA staining and FACS analysis of the intracellular ROS levels in SKOV3/DDP cells. Flow cytometric analysis of cell apoptosis.Results:Part one:1) the cell viability of the ovarian cancer cell lines was examined after treatment with TRAIL(0,10,50,100,250,500,1000 ng/ml) for 48 h. The cells viability of the SKOV3/DDP was significantly higher compared with the SKOV3 cells upon treatment with the same concentration of TRAIL (P<0.01, P<0.001).2)SKOV3 and SKOV3/DDP cells were incubated with 500 ng/ml of TRAIL for the indicated time (0,6,12,24 and 48 h), to detect cell apoptosis. The SKOV3/DDP cells exhibited a lower apoptosis level after TRAIL treatment than SKOV3 cells.(P< 0.01, P< 0.001).Part two:1) Pretreated SKOV3 cells with different concentrations of cisplatin (0 μM,1μM,10μM) for 1 h, before the cells were treated with TRAIL (500 ng/ml) or without for another 24 h. Measuring the cell viability revealed that pretreatment with the higher concentration of cisplatin(10μM) inhibited, but pretreatment with the lower concentration of cisplatin(1 μM) promoted the growth of SKOV3 cells upon TRAIL treatment(37.67±1.18%,125.11±6.29% vs 87.15±11.20%, P< 0.001). Comparing the cell apoptosis levels under the same culture conditions demonstrated that a high dose of cisplatin promoted TRAIL-induced apoptosis, while a low dose of cisplatin inhibits TRAIL-induced apoptosis(63.15±4.42%,5.55±1.78% vs 17.90±2.66%, P< 0.001, P< 0.01).2) SKOV3 and SKOV3/DDP cells were treated for 1 h with cisplatin (0 μM,1 μM,10μM) or wih H2O2 (0 mM,0.1 mM,1 mM), Intracellular ROS levels were measured. ROS levels in the SKOV3/DDP cells were generally higher than levels in the SKOV3 cells(193.6±8.2,23.7±3.2, P< 0.001). We observed a dose-dependent increase in the intracellular ROS levels upon cisplatin and H2O2 treatment in both the SKOV3 and SKOV3/DDP cells(P< 0.001, P< 0.01).3) SKOV3 cells were pretreated with H2O2 (0 mM,0.1 mM,1 mM) for 1 h, and then treated with or without TRAIL(500 ng/ml) for 24 h, Measuring the cell viability revealed that pretreatment with the higher concentration of H2O2 inhibited, but pretreatment with the lower concentration of H2O2 promoted the growth of SKOV3 cells upon TRAIL treatment(81.35±5.11%, 142.72±5.33% vs 87.15±11.20%, P< 0.05, P< 0.001). Comparing the cell apoptosis levels under the same culture conditions demonstrated that a high dose of H2O2 promoted TRAIL-induced apoptosis, while a low dose of H2O2 inhibits TRAIL-induced apoptosis(56.69±4.35%,6.25±1.47% vs 18.11±2.18%, P< 0.001, P< 0.01).Part three:1) After pretreatment with NAC (0 mM,10 mM) for 1 h, the SKOV3 cells were treated with cisplatin (1μM) or H2O2 (0.1 mM), one hour later, the cells were treated together with TRAIL(500 ng/ml) for another 24 h. Intracellular ROS levels were measured. NAC (10 mM) pretreatment inhibited the ROS levels of H2O2 (0.1 mM)+TRAIL and cisplatin (1μM)+TRAIL group(27.6±5.8 vs 87.5±6.5,26.3±5.3 vs 89.6±5.3, P< 0.001). Measuring the cell viability revealed that pretreatment with NAC (10 mM) inhibited the cell viability of H2O2 (0.1 mM)+TRAIL and cisplatin (1 μM)+TRAIL group(78.56±3.21% vs 142.72±5.33%,76.25±7.24% vs 125.18±6.63%, P < 0.001). Cell apoptosis was detected under the same culture conditions, NAC (10 mM) pretreatment promoted the apoptosis of H2O2 (0.1 mM)+TRAIL and cisplatin (1 μM)+TRAIL group(38.83±11% vs 6.54±1.38%, P< 0.01,33.13±10.60% vs 6.58±1.57%, P< 0.05).2) SKOV3/DDP cells were pretreated with oxidative stress scavenger NAC (0 mM,10 mM,40 mM) for 1 h and then the cells were treated with or without TRAIL(500 ng/ml) for 24 h, intracellular ROS levels were measured. NAC (10 mM) pretreatment inhibited the ROS levels of control and TRAIL group(170.0±5.6 vs 190.5±7.2,184.0±4.5 vs 203.0±3.2, P< 0.01). When concentration of NAC increased to 40 mM, which can further reduced the level of ROS in SKOV3/DDP cells, compared with the control group, ROS levels in the NAC (40 mM) group was inhibited by 74.8±4.5%, compared with the TRAIL group, ROS levels in the NAC (40 mM)+TRAIL group was inhibited by by 70.5±3.8%. Cell apoptosis was detected under the same culture conditions, the results of statistical analysis showed that NAC (10 mM)+TRAIL group apoptosis level was much higher than that in TRAIL group (5.75±2.67%, 3.77±1.59%, P> 0.05), while NAC (40 mM)+TRAIL group was significantly higher than that in TRAIL group(51.83±6.53%,3.77±1.59%, P< 0.001).Conclusions:Part one:SKOV3/DDP cells shown TRAIL tolerance.Part two:Mild oxidative stress induced by a low dose of cisplatin contributes to the tolerance of TRAIL in the ovarian cancer SKOV3 cell line.Part three:Regulation of oxidative stress can reverse TRAIL tolerance in SKOV3 and SKOV3/DDP cells. |
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