| Background:Tumor necrosis factor-related apoptosis-inducing ligand (TRAIL) is a new member of the tumor necrosis factor (TNF) family. Experiments show that TRAIL has selective apoptosis-inducing effect in tumor cells in vitro and in vivo, and no obvious toxicity to normal tissue was observed. The liver toxic effect of TRAIL of systematic administration is small. Thus it has a good application value prospect in the treatment of a variety of malignant tumors including gliomas. However, the findings of further study, showing that different kinds of tumor has diverse response to TRAIL and approximately half of the malignant tumor has potential or acquired resistance to TRAIL, limit the clinical application of its anti-tumor therapy. The current study focuses on revealing the factors influencing responses of different tumor cells and the mechanism of tumor cell resistance to TRAIL, so as to find new ways to enhance the anti-tumor effect of TRAIL. A number of studies have found that, TRAIL combined with other drugs or X-ray can improve its anti-tumor effect. It is an effective way to overcome tumor resistance to TRAIL. Many reagents are proved to be sensitizers to TRAIL on the induction of apoptosis of tumor cells, such as cytotoxic drugs, small molecular targeted drugs, and interferon, etc. These apoptosis sensitizers enhance the apoptosis-inducing effect of TRAIL by enhancement of endogenous and exogenous apoptotic signaling or inhibition of anti-apoptotic factors and stimulation of pro-apoptotic factors. At present, searching for effective apoptosis-inducing sensitizer, and revealing the related mechanism of sensitizing effect are important research directions of anti-tumor effect research of TRAIL. Nitric oxide (NO) is a regulator of apoptosis either in physiological and pathological condition, and it plays an important role in the bidirectional regulation of apoptosis of normal cells and tumor cells. Physiological level or lower level of NO can promote the cell resistance to some apoptosis inducing factors, and sustained high level of NO can induce cell apoptosis through activating caspase family, releasing cytochrome C (CytC), up-regulating p53gene and changing the expression of apoptosis-related proteins, such as Bcl-2. Sodium nitroprusside (SNP) is a traditional NO donor, which is widely used in the study of NO-related cell biology. Studies have shown that SNP can induce the apoptosis of tumor cells, and its mechanism mainly depends on the active molecules NO which induces cell apoptosis through activating multiple intracellular pro-apoptotic molecules. This study use human glioma cell line U251as the experimental model, and the SNP as sensitizer of TRAIL-induced apoptotic effect, to observe the respective and synergistic apoptosis-inducing effects of TRAIL and SNP on U251cells, to explore whether SNP has sensitizing effect on apoptosis induced by TRAIL and its possible mechanism through the detection of the expression of proteins of U251cells, and to find new ways of improving the anti-tumor effect of TRAIL and overcoming the resistance to TRAIL in vitro, so as to provide a research evidence for the treatment of glioma.Methods:1Cell culture:glioma cells were cultured in the medium of DMEM/F12which contained10%FBS under the condition of37℃,5%CO2. U251cells in logarithmic growth phase were chosen for the experiment.2Morphological changes of U251cells treated by TRAIL, SNP and their combination:U251cells were treated by TRAIL (800ng/ml), SNP (0.5mM) and their combination respectively. The morphology changes of U251cells were observed by inverted phase contrast microscope after12h and24h.3Detection of growth inhibition rate of U251cells by MTT method:3.1The inhibition rate of U251cells treated by TRAIL, SNP and their combination:U251cells were treated by TRAIL (800ng/ml), SNP (0.5mM) and their combination for24h, and the DMEM/F12was taken as blank control. MTT method was used to detect the OD value of each group. The cell growth inhibition rate%=(1-OD of the experimental group/OD of the control group)×100%.3.2Inhibition rate of U251cells treated by different concentrations of SNP:U251cells were treated by different concentrations of SNP (0.2,0.5,1,1.5,2mM) for24h, and DMEM/F12is taken as blank control. MTT method was used to detect the OD value of each group. The cell growth inhibition rate%=(1-OD of the experimental group/OD of the control group)×100%.4Detection of cellular NO content in U251cells by Griess method:Standard NaNO2solution was diluted into different concentrations (0,1,2,5,10,20,40,60,100μM) with DMEM/F12. Griess method was used to detect standard NO content and create a standard curve. U251cells were treated by different concentrations of SNP (0.2,0.5,1,1.5,2mM) for24h. The intracellular NO content was detected with Griess method.5Detection of apoptotic rate of U251cells by flow cytometry:U251cells were treated by TRAIL and SNP for24h according to different treatment groups:TRAIL (800ng/ml), SNP (0.5mM), the combination (800ng/ml of TRAIL+0.5mM of SNP), and DMEM/F12was used as blank control. Flow cytometry method was used to detect the apoptotic rate of U251cells in each group. The data was analyzed using Cell Quest software. Apoptotic cells were characterized by Annexin V-FITC+PI+and Annexin V-FITC+PI-. Apoptotic rate was calculated after10000cells were sorted.6The expression of DR5, caspase-3, survivin, Bcl-2proteins in U251cells by western blot method:U251cells were treated with TRAIL (800ng/ml), SNP (0.5mM) and their combination for24h, and the DMEM/F12was used as blank control. Western blot method was used to detect the expression of DR5, caspase-3, survivin, Bcl-2and β-Actin which was taken for internal reference. The data was analyzed by AlphaEase FC software to determine the gray levels of each protein in each group, and the relative content of proteins was calculated by the ratio of grey levels of target proteins and the one of the reference.7Statistical analysis: SPSS19.0software was used to analyze the data. The data was revealed by mean±standard deviation (X±s). Single factor variance analysis was used to analyze the data among multi-groups, and the significance between two specific groups were detected with q-test. Statistic difference was considered to be significant if P<0.05. Results:1Morphological changes of U251cells under the inverted phase contrast microscope:U251cells in control group were irregular fusiform and had much synapasis, good diopter, abundant cytoplasm and clear edge, and bonded to the culture plate closely. The U251cells treated by TRAIL for12h and24h became round and small, and the diopter was low, and adhesion was reduced. As the time went on, the changes of U251cells became more significant in number. The most of U251cells treated by the combination were in round and small shape. They were separated from the plate, with vague margins and decreased diopter, and fragments of apoptotic cells can be observed.2The growth inhibition of U251cells treated by TRAIL, SNP and their combination:Inhibition rate of U251cells were detected by MTT method. The results showed that the inhibition rate of U251cells treated by TRAIL (800ng/ml) was (59.272±3.197)%, which showed a significant inhibitory effect on the growth of U251cells. The inhibition rate of U251cells treated by SNP (0.5mM) was (35.467±3.091)%, which also showed inhibition effect of the growth of U251cells. The inhibition rate of the U251cell treated by the combination was (77.425±1.702)%. The inhibition rate rose significantly compared with TRAIL group and SNP group (F=351.522, P=0.000), which showed a significant difference between groups (P<0.01).3The intracellular NO content of U251cells treated by different concentrations of SNP (0.2,0.5,1.0,1.5,2.0mM) were (0.708±0.070)%,(2.673±0.246)%,(4.587±0.770)%,(6.965±0.141)%,(10.931±0.827)%respectively. The NO content rose with the increase of SNP concentration. The inhibition rate of U251cells treated by different concentrations (0.2,0.5,1.0,1.5,2.0mM) of SNP were (12.913±3.700)%,(35.993±2.808)%,(50.117±3.131)%,(61.141±2.355)%,(74.468±2.259)%respectively. The inhibition rate of U251cells rose with the increase of SNP concentration. Combined with the NO content, the inhibition rate showed that the increase of inhibition rates depends on the increase of NO concentration, and there was a significant correlation (P<0.01).4The apoptotic rate of U251cells treated by TRAIL, SNP and their combination:The rate of early apoptosis and late apoptosis showed that the apoptotic rate of control group was (8.20±0.81)%, the apoptotic rate of SNP group was (26.88±4.19)%, and the apoptotic rate of TRAIL group was (36.87±3.69)%. Both were significantly different from the control group (P<0.01). The apoptotic rate of combined group increased to (67.55±3.45)%, showing a significant difference between groups (P<0.01). Synergistic effect of TRAIL and SNP was observed (cofactor=1.06), showing that SNP can enhance the apoptotic effect of U251cells induced by TRAIL.5The expression of Bcl-2, caspase-3, surviving and DR5proteins:Western blot results showed that compared with the control group, the expression of Bcl-2protein in U251cells treated by TRAIL decreased significantly, and no significant difference was observed in caspase-3and DRS, while survivin expression was significantly increased. The expression in SNP group of Bcl-2and survivin significantly decreased, and the caspase-3expression did not change significantly, while the expression of DR5decreased significantly. The expression in the combination group of Bcl-2and surviving decreased significantly, while caspase-3and DR5expression increased significantly. The difference of protein expression in the combination group is significant (P<0.01).Conclusion:1TRAIL and SNP can significantly inhibit the proliferation of U251glioma cells in vitro and their combination can enhance the inhibitory effect in U2S1cells.2The inhibitory effect of SNP is dose-dependent, which means the inhibition rate of U251cells ascends with the increase of SNP concentration.3The inhibitory effect of SNP depends on the dose-dependent intracellular content of NO, which means the inhibition rate of U251cells ascends with the increase of NO concentration.4TRAIL and SNP can induce apoptotic effect of U251cells. The combination of TRAIL and SNP has a synergistic apoptosis inducing effect in U251cells. SNP can sensitize the TRAIL-induced apoptosis.5The synergistic effect of the combination of TRAIL and SNP relies on the up-regulation of caspase-8and down-regulation of Bcl-2. The sensitization of TRAIL-induced apoptosis by SNP relies on the up-regulation of DR5and down-regulation of Bcl-2and survivin. |
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