A Study On Radiosensitivity Of β-elemene To Adenocarcinoma Cell Carcinoma Of Lung A549 Cell Line In Vitro

Radiotherapy is a very importmant way to treat the malignant tumor.About 70% patients with malignant tumor need to be treated by radiotherapy. But the result of radiotherapy is not satisfactory because of the biological characteristis and the local microenvironment of the tumor. In order to improve the raidotheraputic effect,people try to search for more effectinve radiosensitivity agents. Minsonidazole(MISO) ,a radiosensitivity agents,even though it has specific radiosensitizing effect on hypoxic cells,its serious toxic side effects has seriously limited its applications in clinical .In recent years, people find that some traditional chinese medicine have the radiosensitizing effect.β-elemene is used as a non-cytotoxicity anti-tumor drug, it is a kind of floristics new anti-cancerⅡtype drugs extracted from common turmeric.It has been showed thatβ-elemene is a broad-spectrum and relatively safe agent to be applied to treat the tumor. Researching of its function mechanism of radiosensitizing was mainly about proliferation,apoptosis and differentiation, the study on its new effect target is rare.We exposed A549 cells in low-cytotoxic concentrations,then observed the radiosititive effects on A549 cells in vitro and investigated the mechanism of radiosensitivity.It could be a new thought for clinical study and Provide study method for Chinese traditional radiationsensitizing agents. Objective:This study aims to evaluate the influence ofβ-elemene to human lung cancer A549 cells on radiosensitivity and to investigate that if there is correlation between radiosensitivity and the expression level of KU70 and KU80 gene.Method:Taking A549 cells as the research object,β-elemene was produced by Dalian Jingang Medicine Company. Cells were cultured in common RPMI-1640 culture medium.The test groups were exposed to the culture medium at different concentritions ofβ-elemene (0ug/ml 10ug/ml,20ug/ml,40ug/ml,80ug/ml,160ug/ml,320ug/ml).The control group had noβ- elemene in culture medium.Then they were exposed to radiation at different dose of X -ray(0Gy,2Gy,4Gy,6Gy,8Gy 10Gy).All indexes were detected after A549 cells culture finished. Inhibiting rate on cell proliferation exposed to different concentritions was evaluated with a way of MTT assay.,then calculate IC50 of 24h. Radiosensitivity of A549 cells was observed with the methed of MTT assay and clone forming assay. The expression level of KU80 and DNA-PKcs mRNA. were detected by Reverse Transcription-Polymerase Chain Reaction(RT-PCR).Results:1.The 24h IC50 value to A549 cells ofβ-elemene is 120ug/ml.2.Along with the increasing ofβ-elemene concentration, OD values gradually decreased, proliferation inhibition rate gradually increased.β-elemene is shown as possessing a proliferation inhibition effect in vitro in A549 cells. Statistical analysis has significant difference (p<0.05).3.A549 cells were exposed toβ-elemene in different concentrations (0ug/ml,20 ug/ml)for 24h and radiated,then were measured the OD value.The results were that at the same radiation dose,all of the OD data in 20ug/ml group is less than that in control group.After radiation (0Gy,2Gy,4Gy,6Gy,8Gy,10Gy), the OD data in 20ug/ml group was 0.964士0.025,0.825士0.012,0.758士0.042,0.623士0.011,0.544士0.031,0.376士0.015,and the OD data in 0ug/ml group was 0.828士0.009,0.650士0.022,0.562士0.016,0.474士0.011,0.345士0.015,0.205士0.014,Statistical analysis has significant difference (p<0.05).β-elemene is shown preliminarily as possessing a radiosensitizing effect in vitro in A549 cells.4.A549 cells were exposed toβ-elemene in the concentrations (10ug/ml,20ug/ml)cultured for 24h and radiated,then were measured the number of cell clone and survival rate. Compared to the control group,there was significant difference between 10ug/ml group and 20ug/ml group, (p<0.05), there was also significant difference between 10ug/ml group and 20ug/ml group (p<0.05) . After radiation (0Gy,2Gy,4Gy,6Gy,8Gy,10Gy),the number of cell clone reduced from 97士3.12,82士2.35,64士3.17,45士2.06,24士3.18,12士1.64 to 10ug/ml group 92士3.23,73士2.51,55士3.84,36士4.08,17士3.21,7士1.63;and 20ug/ml group88士3.09,65士4.25,42士3.87,28士1.67,12士1.59,4士1.10.respectively。Survival rate reduced accordingly.This further proved that the radio- sensitivity of A549 cells can be enhanced byβ-elemene, radiosensitization effect increased gradually with the increased concentration ofβ-elemene.5.A549 cells were exposed toβ-elemene in the concentrations 20ug/ml cultured for 24h and 48h,and radiated,then were measured the number of cell clone and survival rate. Compared to the control group,there was significant difference between 24h group and 48h group,(p<0.05),there was also significant difference between 24h group and 48h group (p<0.05).After radiation(0Gy,2Gy,4Gy,6Gy,8Gy,10Gy),the number of cell clone reduced from 97士3.12,82士2.35,64士3.17,45士2.06,24士3.18,12士1.64 to 24h group88士3.09,65士4.25,42士3.87,28士1.67,12士1.59,4士1.10 and 48h group 76士2.15,47士3.42,30士2.37,17士1.80,5士1.09,0士0.0.respectively.Survival rate reduced accordingly.This further proved that the radiosensitivity of A549 cells can be enhanced byβ-elemene,radiosensitization effect increased gradually with the prolongation of the action time.6.The expression of Ku80 and Ku70, the expression of Ku80 and Ku70 slightly up-regulated in the radiotherapy alone group, the expression of Ku80 and Ku70 slightly down-regulated in the simply drug group,. Statistical analysis has no significant difference (p>0.05).Compared to the radiotherapy alone group, the expression of Ku80 and Ku70 significantly down-regulated in theβ-elemene combined with radiotherapy group. Statistical analysis has significant difference (p<0.05)。There is significant difference between 10ug/ml and 20ug/mlβ-elemene combined with radiotherapy group.Statistical analysis has significant difference (p<0.05) Conclusion:1.β-elemene could significantly inhibit the proliferation of A549 cells,IC50 is 120ug/ml,proliferation inhibition rate increased along with increased concentrion ofβ-elemene.2 The radiosensitivity of A549 cells can be enhanced byβ-elemene, radiosensitization effect increased gradually with the prolongation of the action time and increment of concentrion.3. Neitherβ-elemene nor radiotherapy alone has significant influence on the expression of Ku80 and Ku70 of A549 cells,β-elemene combined with radiotherapy could significantly reduced the expression of Ku80 and Ku70 of A549 cells. Effect of radiosensitivity ofβ-elemene is probably associated with down-regulating expression of Ku80 and Ku70 genes.