A Study On Radiosensitivity Of β-elemene To Squamous Cell Carcinoma Of Oesophagus EC109 Cell Line In Vitro

Squamous cell carcinoma of oesophagus is the most common malignant tumor of digestive system that can be treated by radiotherapy in peroperation term. Improving the radiosensitivity, not only radiate by the dose planed, but also reduce the organise injure,is the target we always have been making great efforts to get.Reasearch Showed that the increasing of carcinoma cells apoptosis accompanies the enhancing of radiosensitivity. Some researchers have taken the apoptosis level of carcinoma cells as an index to judge radiosensitivity.In order to increase the radiosensitivity of cells,the reshearchers have been trying hard to find out the methods to increase cell apoptosis level.Radiobiology has confirmed that there is a big difference between different phases of cell cycle in cell radiosensitivity.The cells arrested in phase G2/M are the most radiosensitive. So it is an important method of enhancing carcinoma cell radiosensitivity and improving curative effect to induce cells in phase G₁/S to move forward and arrest in phase G2/M.Radiosensitizer is referred to some substances(chemosynthesised drugs, Chinese herbal medicine and biologic products) that could improve carcinoma cells' radiosensitivity and take less effect on normal cells. Since 1960s,many researches about radiosensitizer have been carried out and a series of in vivo and vitro protocols to test radiosensitization have been established. Though a great number of agents had shown significant radiosensitize effects, their clinical efficacies were not only disappointted, serious toxicity limite but also their further clinical application.So exploring a series of high-effect and low-toxicity radiosensitizer should have significantly practical value.β-elemene is a kind of floristics new anti-cancerâ…¡type drugs.There is a series of characteristic of low-toxicity in normal cells,little side-effect,little liver and kidney damage,no marrow inhibition.Above all, it obviously differs from other conventional chemotherapeutics. More and more literatures have showed thatβ-elemene is an effective and relatively safe agent for the therapy of many kinds of tumour. The main anti-cancer mechanism is thatβ-elemene reduces the expression of bcl-2 gene, induces cell apoptosis and changes cell cycle distribution.we exposed EC109 in low-cytotoxic concentrations,and then observed the radiosititive effects on EC109 in vitro, and investigated the mechanism of radiosensitivity. It could be a new thought for clinical study and provide study method for Chinese traditional radiation sensitizing agents.Objective:The studies aim to evaluate the influence ofβ-elemene on radiosensitivity of human squamous cell carcinoma of oesophagus. EC109 cell lines cultured in vitro and disscuss the relative mechanism. Firstly, ifβ-elemene could increase the radioscnsitivity of EC109 cell lines and whether there are any relations with various concentrations and different times ofβ-elemene effect, secondly, we tried to postulate the possible mechanisms of radiosensitizing effect ofβ-elemene.Materials and methods:EC109 cell lines was conserved by pathophysiology department of zhengzhou university medical college.β-elemene was produced by Dalian Jingang Medicine Company. EC109 cells were cultured in common RPMI-1640 culture medium.The test groups were exposed to the culture medium at different concentrions ofβ-elemene(20μg/mL,40μg/mL,60μg/mL,80μg/mL,120μg/mL).The control group had noβ-elemene in culture medium.Then they were exposed to, radiation at different dose(0Gy, 2Gy, 4Gy, 6Gy, 8Gy). All indexes were detected after EC109 cells culture finished. Inhibiting rate on cell proliferation exposed to different concentrions was evaluated with a way of MTT assay and clone forming assay. Radiosensitivity of EC 109 cells was judged according to results of MTT assay. Effect of Radiosensitivity was observed with the methed of clone forming assay. Change of cell cycle and apoptosis was analysed with flowcyometry(FCM). Immunocytochemical method combinated with indirectlyimmun- ofluorescene and FCM were used to detect the Bcl-2 and Bax expression. The statistical analysis was executed by SPSS. 10.0 software,analysis of variance(ANOVA),t-test and trend text. Statistically significant level was considered as "alpha less than 0.05".Results:1. After EC109 cells were exposed toβ-elemene for 24hs, the low concentration (20μg/mL) stimulated proliferation of EC109 cells instead. Above 40μg/mL, proliferation inhibition rate gradually increased along with the increasing ofβ-elemene concentration. Statistical analysis has significant difference(P＜0.05).2. EC109 cells were exposed toβ-elemene in different concentrations (0μg/mL, 40μg/mL) for a period of time and radiated, then were cultured in the common medium for 24hs and measured the OD vale.The results were that all of the OD data in 40μg/mL group is less than than the data of control group. After radiation(0Gy, 2Gy, 4Gy, 6Gy, 8Gy), the OD data reduced from 0.907±0.053, 0.838±0.027, 0.774±0.029, 0.680±0.013, 0.652±0.013 to 0.800±0.009, 0.680±0.009, 0.562±0.009, 0.454±0.009, 0.345±0.009. Statistical analysis has significant difference(P＜0.05).3. EC109 cells were exposed toβ-elemene in the concentrations(40μg/mL,60μg/mL) cultured for 24h and radiated by different dose, then were cultured in the common medium for 24h and measured the number of cell clone and survival rate. Compared to the control group, There was significant difference (P＜0.05) between 40μg/mL and 60μg/mL.After radiation (0Gy,2Gy,4Gy,6Gy,8Gy),the number of cell clone reduced from 88±2.65,74±2.65,55±2.65,48±3.61,38±3.46 to 78±2.00,60±3.61,49±1.00,31±1.00 to 71±2.65,53±1.73,43±2.65,25±5.00,13±3.61 respectively. Statistical analysis has significant difference(P＜0.05).4. After radiation by the same dose, there was significant difference(P＜0.05) in number of cell clone and survival rate between 48h group exposed toβ-elemene and 24h group.After radiation (0Gy, 2Gy, 4Gy, 6Gy, 8Gy), the number of cell clone reduced from 88±2.65,74±2.65,55±2.65,48±3.61,38±3.46 to 78±2.00,60±3.61,49±1.00,39±2.65,31±1.00 to 74±1.00,53±1.00,36±4.58,20±6.08,8±6.46 separately. There was radiosensitivity when EC109 cells was exposed to non-cytotoxic concentrations,the effected increasing along with exposed time increasing.5. Withβ-elemene concentration and time increasing, there was a increasing trenency EC109 cells Apoptosis from 0.1% to 17.6%. Statistical analysis had significant difference (P＜0.05). The apoptosis has positive correlation to concentration and exposed time.6. After treatment withβ-elemene,β-elemene could change distribution of EC109 cell cycle.G₂/M arresting happened.At the same time,increasing of G₂/M phase rate accompanied with increasing of concentration. Statistical analysis has significant difference(P＜0.05).7. After EC109 cells were exposed to 60μg/mLβ-elemene for 24h,Bcl-2 expression of EC109 cell decreased and cytoplasmic or nuclear staing weakened. Quantitative analysis showed that at the same time, after treatment with different concentration,decreasing of average fluorescene of Bcl-2 along with increasing of concentration (P＜0.05). At the same concentration, after treatment with different treatment time, the average fluorescene of Bcl-2 decreased along associated with increasing of treatment time (P＜0.05).8. After EC109 cells were exposed to 60μg/mLβ-elemene for 24h,Bax expression on EC109 cell increased, cytoplasmic staing enhanced. Quantitative analysis showed that at the same time, after treatment with different concentration, the average fluorescene reiforcement of Bax along with increasing of concentration (P＜0.05). At the same concentration, after treatment with different treatment time, the average fluorescene of Bcl-2 along with increasing of treated time (P＜0.05).Concision:1. The concentrations(40μg/mL～120μg/re)ofβ-elemene could inhibit proliferation of EC109 cells; and afeter treatment withβ-elemene, arrest of G₂/M phase has positive correlation with concentration, proliferation inhibition rate increased along with the increasing ofβ-elemene concentration.2. There was effect of radiosensitivity of EC109 cells exposed to low-cytotoxic concentrations, the effect increased along with increasing of concentrion and time.3. After treatment withβ-elemene, the expression of Bcl-2 has negative correlation with concentration and tread time.4. After treatment withβ-elemene, the expression of Bax has positive correlation with concentration and tread time.