Study On Radiosensitizing Effect Of β-Elemene And Screening Of Related Target Genes

Lung cancer is one of the most common malignant tumor in our country.Radio-therapy is an important way to treat medium and advanced lung cancer.Radiosensiti-vity of the tumor influences the effect of radiotherapy.In order to improve the radiosensitivity of the tumor cell,people have been trying to explore high-effect and low-toxicity radiosensitizer.Research shows that althouth some agents had shown significient radiosensitizing effects,serious toxicity side effects has limited their further clinical applications.In the recent years,people pay more attention to the research of some traditional chinese medicines exhibiting radiosensitizing effects.Elemene is a kind of non-cytotoxic antineoplastic agents.It is the extract of common turmeric which pertains to zingiberaceous plants.It can enhance the radiosensitivity of the tumor cell with low side effects.Previous studies about radiosensitizing mechanism of P-elemene is mainly focus on the cell periodic variation and cell apoptosis.Whether it influences the expression of DSBs repair gene has not been reported.There is no reports about target gene screening about radiosensitizing of P-elemene.In our study,we exposed A549 cells in low concentration of P-elemene and radiated,observed the radiosensitizing effect of P-elemene to A549 cells,explored the influences fromβ-elemene on expression of apoptosis-related gene bcl-2,p53 and DSBs repair-related gene Ku70,DNA-PKcs.Oligonucleotide chip was used to detect genes related to radiosensitizing of P-elemene to A549 cells.We systematically analyzed functional gene related to radiosensitivity so as to make further research for molecular target and mechanism in radiosensitizing,provided the theoretical basis for clinical application of P-elemene.Objective: 1.The low concentration ofβ-elemene was used for clone forming assay to research the radiosensitizing effect on A549 in vitro.We studied the influences of P-elemene on the cell periodic variation and apoptosis by flow cytometry,and detected apoptosis related gene bcl-2 and p53 expression by RT-PCR and Western blot.2.We studied the influences ofβ-elemene on DSBs repair related gene DNA-PKcs and Ku70 expression in A549 cells,analyzed the correlations between DNA-PKcs,Ku70 expression and bcl-2,p53 expression to make a further study on the molecular mechanism of radiosensitizing.3.Oligonucleotide chip is used to detect gene expression changes overall of A549 cells withβ-elemene after radiation. Systematically analyzed functional gene and signaling pathway related to radiosensitivity,then make further research for molecular target and mechanism inβ-elemene radiosensitizing effects,provide the theoretical basis for clinical application ofβ-elemene.Methods:Part one:Detection of clone forming assay,cell cycle and apoptosis1.A549 cells in logarithmic growth phase were used for MTT assay to determine the 50% inhibiting concentration (IC50) value ofβ-elemene for 24 hours.2.10% IC50and 20% IC50 concentration of P-elemene were used for clone forming assay.A549 cells were exposed toβ-elemene for 24 hours,then were exposed to radiation at different dose.Clone formation assay was performed to determine survival fraction(SF).Then fitted the cell surving curve.Parameters Do,Dq and SF2 were obtained from the curve,sensitizer enhancement ratios of Do,Dq(SERDo,SERDq) were calculated to evaluate radiosensitivity.3.The cells were put into four groups:①control group(C)②radiation group (R):cells were exposed to 4 Gy radiation.③0.1xIC50 group:cells were exposed toβ-elemene of 10% IC50.④0.2×IC50 group:cells were exposed toβ-elemene of 20% IC50.⑤0.1×IC50+R group:cells were exposed toβ-elemene of 10% IC50 for 24 hours,then were exposed to 4 Gy radiation.⑥0.2×IC50+R group:cells were exposed toβ-elemene of 20% IC50 for 24 hours,then were exposed to 4 Gy radiation.Cells were treated as mentioned above.24 hours later,fluorescence microscope was used to observe cell apoptosis. Cell periodic variation and apoptosis rate were analyzed with flowcytometry.RT-PCR and Western blot were performed to detect the expression of bcl-2 and p53.Part two:Detection the relation between DSB repair gene and apoptosis related geneThe cells were put into four groups as the first part cells were treated as mentioned above.24 hours later,RT-PCR and Western blot were used to detect the expression of Ku70 and DNA-PKcs,then analyzed the correlation between the expression of Ku70,DNA-PKcs and the expression of bcl-2,p53 in 0.1×IC50+R group.Part three:Screening of target genes about radiosensitization by oligo gene chipl.Extrated the total RNA of the cellof 0.1×ICso+R group and R group,purified the mRNA,Chip-on-lab electrophoresis and agarose electrophoresis examined RNA purity and integrity,synthesised cDNA probe by RT-PCR,cDNA probe was hybridized with oligo gene chip with a set of 30968 human genes.Array image was screened and analyzed with array analyzing software.2.To confirm the reliability of the result from the genes expression profiles,we selected one of the up-regulated and one of the down-regulated gene to undergo analysis by RT-PCR.Results:Part one:1.Cell growth inhibition:.Different concentrations of P-elemene inhibitated the prolifiration of A549 cells, the inhibitory rate increased with the increasing concentration ofβ-elemene.The IC50 value ofβ-elemene for 24 hours in A549 cells is 120μg/ml.2.Radiosensitizing effect:With the same concentration ofβ-elemene,surving fraction of A549 cells decreased gradually with increasing dose of radiation. With the same dose radiation, surving fraction decreased gradually with increasing concentration of p-elemene.From the cell surving curve we obtained the value of Do,Dq and SF2.In control group,0.1×IC50 group and 0.2×IC50 group,the value of Do was 2.45±0.24 Gy,1.64±0.15 Gy and 1.55±0.13 Gy respectively.The value of Dq was 2.68±0.25 Gy,1.87±0.22 Gy and 1.53±0.11 Gy respectively.The value of SF2 was 84.6±20.9%,56.3±14.9% and 43.2±10.7% respectively.And the value of SERD0 and SERDq in 0.1×IC50 group was 1.54±0.20 and 1.43±0.15,in 0.2×IC50 group was 1.63±0.32 and 1.75±0.19 respectively.The value of D0,Dq and SF2 decreased with increasing concentration ofβ-elemene,the curve moved toward the left,the shoulder aera became narrow and straight slope increased gradually,,SER value increased gradually with increasing concentration ofβ-elemene. 3.Cell cycle:.In the aspect of cell periodic variation,compared with control group,the G2/M phase fraction in R group,0.1×IC50 group and 0.2×IC50 group had no obvious changes(P> 0.05).Compared with R group,G2/M phase fraction in 0.1×IC50+ R group and 0.2×IC50+R group increased obviously(P< 0.01),with the increasing concentr-ation ofβ-elemene,G2/M phase fraction increased gradually.4.Cell apoptosis:In the aspect of apoptosis, compared with control group,the apoptosis rate in R group increased,the difference had no statistical significance(P> 0.05),the apoptosis rate in 0.1×IC50 group and 0.2×IC50 group had no obvious change(P> 0.05),compared with R group,the apoptosis rate in 0.1×IC50+R group and 0.2×IC50+R group increased obviously(P< 0.01),and charac-teristic apoptosis peak was seen. Through fluorescence microscope cell apoptosis was seen much more in 0.1×IC50+R group and 0.2×IC50+R group than that in R group.5.Expression of bcl-2:Compared with control group,the expression of bcl-2 mRNA in R group 0.1×IC50 group and 0.2×IC50 group had no obvious changes(P> 0.05),compared with R group,the expression of bcl-2 mRNA reduced obviously(P< 0.05).The expression of bcl-2 protein showed the same trend.6.Expression of p53:Compared with control group,the expression of p53 mRNA in R group 0.1×IC50 group and 0.2×IC50 group had no obvious changes(P> 0.05).Cmpared with R group,the expression of p53 mRNA reduced obviously(P< 0.05).The expression of p53 protein showed the same trend.Part two:1.Expression of Ku70:Compared with control group,the expression of Ku70 mRNA in R group was slightly increased(P> 0.05),but the expression of Ku70 mRNA in 0.1×IC50 group and 0.2×IC50 group had no obvious changes(P> 0.05).Compared with R group,the expression of Ku70 mRNA reduced obviously in 0.1×IC50+R group and 0.2×IC5o+R group(P< 0.05), and the expression of Ku70 protein showed the same trend.2.Expression of DNA-PKcs:Compared with control group,the expression of DNA-PKcs mRNA in R group was slightly increased(P> 0.05),the expression of DNA-PKcs mRNA in 0.1×IC50 group and 0.2×IC50 group had no obvious changes(P > 0.05).Compared with R group, the expression of DNA-PKcs mRNA decreased obviously in 0.1×IC50+R group and 0.2×IC50+R group(P< 0.05),The expression of DNA-PKcs protein showed the same trend.3.Relation between DSBs repair gene and apoptosis gene:Correlation analysis of mRNA in 0.1×IC50+R group showed that there was a remarkable negative correlation between Ku70 and p53,DNA-PKcs and p53.(rKu70-p53=-0.758,P=0.024.rDNA-PKcs-p53=- 0.665,P=0.037),and a remarkable positive correlation betwee DNA-PKcs and bcl-2,Ku70 and bcl-2 (rKu70-bcl-2=0.847,P=0.013.rDNA-PKcs-bcl-2=0.861,P=0.010).Correlation analysis of protein showed that there was a remarkable negative correlation between Ku70 and p53,DNA-PKcs and p53.(rKu70-P53=-0.692,P=0.033. rKu70-p53=-0.569,P=0.040),and a remarkable positive correlation between Ku70 and bcl-2,DNA-PKcs and bcl-2 (rKu70-bcl-2=0.847,P=0.008.rDNA-PKcs-bcl-2=0.755,P=0.012).Part three:Screening of target genes about radiosensitization1.Chip-on-lab electrophoresis and agarose electrophoresis showed that RNA extracted from 0.1×IC50+R group and R group was pure and complete.2.The scatter diagram constructed by Cy5/Cy3 fluorescence signal showed repetitive R of the two chips was 0.95,the repeatability is well.3.Oligonucleotide chip screened out a total of 122 differential expression of genes,of which,99 were obviously up-regulated and 33 were obviously down-regulated.These genes involved in DNA damage and repair,cell cycle regulation,cell apoptosis,cell proliferation,signal transduction and transcription,cell adherence, immu-nity response and so on.4.The mRNA expression of Egr-1 in 0.1×IC50+R group which was up-regulated in gene chip was much higher than that in R group,mRNA expression of CyclinDl which was down-regulated in 0.1×IC50+R group was much lower than in R group, this result was in accordance with oligonucleotide chip result.Conclusion:Part one:Relation between radiosensitization effect and cell cycle,apoptosis1.Low concentration of P-elemene has a radiosensitizing effect on A549 cells, The SERD0 value of 10μg/mlβ-elemene and 20μg/mlβ-elemene is 1.54±0.20 and 1.63±0.32.The SERDq value of 10μg/mlβ-elemene and 20μg/ml P-elemene is 1.43±0.15 and 1.75±0.19.The value of SER increases and radiosensitizing effect enhances with the increasing concentration ofβ-elemene.2.β-elemene combined with radiation can influnce the distribution of A549 cell cycle distribution remarkblely.It can increase G2/M phase block and induce cell apoptosis.The ratio of G2/M phase fraction and apoptosis rate increase with the increasing ofβ-elemene concentration.Inducing G2/M phase block and apoptosis is one of the mechanism of radiosensitization 3.β-elemene combined with radiation can induce A549 cell apoptosis by down regulating the expression of bcl-2 and up regualating the expression of p53.The effect of inducing popotosis enhances with the increasing ofβ-elemene concentration.Part two:Relation between DSBs repair gene and apoptosis related gene1.β-elemene combined with radiation can increase the radiosensitivity of A549 by inhibiting the expression of Ku70 and DNA-PKcs.2.Inβ-elemene combined with radiation group,there was a significant negative correlation between the expression of Ku70,DNA-PKcs and p53.There was a signify-cant positive correlation between the expression of Ku70,DNA-PKcs and bcl-2.3.β-elemene has influences on the pathway of DSBs repair and apoptosis pathway simutaneously,p-elemene increase the radiosensitivity of A549 cells with the interaction of the two factors.Part three:Screening of target genes about radiosensitization effect1.Oligonucleotide chip screen out a total of 122 differential express genes which are associated with the increasing radiosensitivity ofβ-elemene to A549 cell.These genes involved in DNA damage and repair,cell cycle regulation,cell apoptosis,cell proliferation,signal transduction and transcription,cell adherence,immunity response and so on.2.The mechasim ofβ-elemene enhancing the radiosensitivity of A549 cells is the result of polygene together action.