Study On Cell Cycle And Apoptosis In Human Esophageal Carcinoma Cell Line Ec-9706 After Irradiation

Background and ObjectiveEsophageal carcinoma(EC) is a kind of malignant tumor of digestive tract frequently with poor prognosis.About 310,400 new cases of EC were diagnosed worldwide a year,more than half of which are lay in China.Standard management for EC still includes surgical resection and radiotherapy,the patient outcome has not been improved in recent decades.For late-stage EC,radiotherapy is the procedure of choice with a less than 10%5-year overall survival.With the advances in the image, computer and radiotheraputic techniques,new radiotheraputic methods such as late course accelerated hyper-fractionated radiotherapy(LCAHF) and three-dimensional conformal radiotherapy have been performed in EC,but patient outcome has not been improved significantly.The cell cycle,is an ordered set of events that take place in a eukaryotic cell leading to its replication.The cell cycle consists of four distinct phases:G₁ phase,S phase,G₂ phase(collectively known as interphase) and M phase.These events can be divided in two brief periods:interphase-during which the cell grows,accumulating nutrients needed for mitosis and duplicating its DNA-and the mitotic(M) phase, during which the cell splits itself into two distinct cells,often called "daughter cells". The cell-division cycle is a vital process by which a single-celled fertilized egg develops into a mature organism,Activation of each phase is dependent on the proper progression and completion of the previous one.The molecular events that control the cell cycle are ordered and directional;that is,each process occurs in a sequential fashion and it is impossible to "reverse" the cycle.Cell cycle checkpoints are used by the cell to monitor and regulate the progress of the cell cycle.Checkpoints prevent cell cycle progression at specific points,allowing verification of necessary phase processes and repair of DNA damage.Several checkpoints are designed to ensure that damaged or incomplete DNA is not passed on to daughter cells.Two main checkpoints exist:the G₁/S checkpoint and the G₂/M checkpoint.G₁/S transition is a rate-limiting step in the cell cycle and is also known as restriction point.The cells which are actively undergoing cell cycle are targeted in cancer therapy as the DNA is relatively exposed during cell division and hence susceptible to damage by drugs or radiation.Studies show that G₁ blockage,G₂ blockage,S delay and S/M uncoupling can be induced by ionizing radiation,which destroy the completion of cell genome,then cell cycle is altered and cell results in death or apoptosis.To explore the ionization-induced cell cycle alteration and apoptosis of human esophageal carcinoma cell,we conducted a study on cell cycle and apoptosis in human esophageal carcinoma cell line EC-9706 after irradiation.This study was designed to observe the relationship between vary doses of irradiation and cell cycle changes and apoptosis,then to seek the radiobiology of human EC.Materials and methods1 Cells:Human esophageal carcinoma cell line EC-97062 Methods:The human esophageal carcinoma cell line EC-9706 were in logarithmic growth phase,cells were digested with 0.25%trypsin and were made into single cell suspension and then inoculated into 90 Coming petri dish,which were divided into 4 treatment groups and non-treatment group(control group).The cells irradiated by 6-MV Medical Linear Accelerator with a dose rate of 2Gy/min,the radiation-doses were 2,5,10 and 15Gy,respectively.Cell cycles were analyzed by flow cytometry (FCM) at 6 hour,24 hour and 48 hour after irradiation,apoptosis was observed on the cells in a dose-dependent manner.We also compared the accuracy of FCM and Apoptosis Kit in measuring cell growth.Results1.Apoptosis of EC-9706 cells was induced and the process of cell cycles were altered by X-ray irradiation.The response diversified at varied irradiation doses,the proportion of G₂/M blockage was increased with the upgrade of irradiation dose, G₂/M blockage was peaked at 10Gy or 24 hour at all 4 treatment groups after irradiation,but the response was not significant with 2Gy or lower doses of radiation. The proportion of G₂/M blockage was buildup with the multiplication of irradiation doses and climaxing at 24 hour after irradiation,which was achieved 273%and 1084%at 48h and 6h,respectively,with 5Gy irradiation point.The proportions of G2/M blockage were 175%and 728%with 10 Gy irradiation,160%and 313%with 15Gy irradiation at 48h and 6h,respectively.2.G₂/M blockage and S delay of EC9706 cells were detected dominantly after irradiation,moreover,G₂/M blockage was decreased and S delay of EC9706 cells was increased with the step-up of irradiation doses.The difference of G₂/M blockage of EC9706 cells at 6h was significant between 15Gy group and other treatment groups,no difference was showen among 0Gy,2Gy,5Gy and 10Gy group.There was statistical difference of G₂/M blockage at 24h among 0Gy,2Gy and 5Gy group,no difference between 10Gy and 15Gy group.Statistical difference of G₂/M blockage at 48h was showen between 10Gy, 15Gy and 0Gy,2Gy,5Gy.According to time factor,there was statistical difference of G₂/M blockage between 6h and 24h,no difference between 6h and 48h,or 24h and 48h.3.There was dose-dependent with G₂/M blockage and S delay of EC9706 cells in the range of 0Gy to 15Gy,which was peaked at 24h.4.The apoptotic proportion of EC9706 cells was peaked at 6h,which was then declined with the lapse of time,and minimised at 48 hour.Morever,the apoptotic proportion was dose-dependent.Apoptosis Kit is more accuracy than FCM in measuring cell cycle. Conclusions1.Apoptosis and process of cell cycles of EC-9706 cells can be induced by X-ray irradiation.2.The process of cell cycles can be altered by X-ray irradiation,and G₂/M blockage and S delay of EC9706 cells are dominant.3.Dose-dependent of X-ray irradiation with G₂/M blockage and S delay of EC9706 cells is showen in the range of 0Gy to 15Gy,and peaked at 24h.4.Apoptosis Kit is more accuracy than FCM in measuring cell apoptosis,which can be a good choice of apparatus in vitro experimental.