The Effects Of ⁽180F-FDG On The Apoptosis Of Eca-109 Esophageal Cancer Cell Line

Esophageal cancer belongs to one of the common malignant tumor in China. Its incidence is increasing and the fatality is the fourth rate of cancer mortality. There are a lot of treatment method of esophageal cancer, including surgery, radiotherapy and chomotherapy, but the 5-year survivial of esophageal patients are not obviously improved[1] , so it is necessary to find new treatments. ¹⁸F-FDG is the most clincal tumor metabolic imaging agents, ¹⁸F decay positron-emitting, through when gamma radiation obliterated, 635keV energy. Because the primary malignant tumor and metastases can highly selective intake ¹⁸F-FDG, and tumor cells intake ¹⁸F-FDG rate with its degree of malignancy increased. So the higher level of malignant, therapeutic effect is better.Objectives: this topic though ¹⁸F-FDG radirate Eca-109 after 6h, 12h, 24h and 48h, observation the morphological changes, cellular proliferation and apoptosis, active oxygen level, the mitochondrial membrane potential changes.Discussing the possible mechanisms of different radioactive concentration of ¹⁸F-FDG induction apoptosis of Eca-109 cells. To provide experiment basis of ¹⁸F-FDG used for clinical tumor treatment.Methods: (1) Cell culture: Eca-109 cells were planted in RPMI1640 medium containing 10% FBS and 100U/ml streptomycin and penicillin (complete medium)at 37℃in 5%CO2. (2) Cell radiation: At 24h after cell subculture, Eca-109 cells at the logarithm growth stage were treated by ¹⁸F-FDG and then were used in the following experiments, whose final radioactive concentration was 0,9.25,¹⁸.5,37, 74 MBq/ml, respectively. (3) Cell morphology observation: Collect each ¹⁸F-FDG radiration 24h group and control group cells, observed under general inverted microscope for morphology changes and pictures were taken by digital camera. Cell Transmission electron microscope observation: collect 37 MBq/ml of ¹⁸F-FDG radiation 48h cells and control , trypsinized by 0.25% trypsin, centrifugated, washed with PBS, fixed in 2.5% glutaraldehyde precooled at 40C, photographed under transmission electron microscope for ultrastructure. (4) Determination of cell proliferation inhibition rate by MTT method: take exponential growth phases cells, trypsinized, counting, the cell density is adjusted as 3×10⁴/ml, grown in 96-well plates with 200ul per hole, cultured for 24h. ¹⁸F-FDG was added into experimental groups while the same volume RPMI1640 medium was added into blank groups. Continue to cultivate 12, 24, 48 h, per hole adding 20ul 5mg/ml MTT solution. After 4h then abandon it. Everyhole add DMSO 150ul. Detecting optical density value(OD) in each hole using the microplate reader (at wavelength 570nm), calculating the cell proliferation inhibiton rate. (5) 3^H-TdR incorporation method: 6×105/ml cells inoculated in 24-well plates, 2ml per hole. After 24h, ¹⁸F-FDG was added into experimental groups while the same volume RPMI1640 medium was added into blank groups. Continue to cultivate 24h, 20ul of 3^H-TdR(100uCi/ml) were added .24h after Eca-109 cells were collected and sampled by filtration.The liquid scintillation assay the CPM value of cells. (6)The cell apoptosis rate: Cultured 24h cells were trypsinized by 0.25% trypsin and collected and washed cell twice with PBS. Then cells were fixed by cold 70% ethanolat–20℃for 4h or overnight, and washed with PBS again. With 50ul Rnase (5mg/ml) at 37℃incubation 30min, PI solution dyeing. Cells were ready for test after 15min reaction in the dark. Flow cytometry analysis. (7)The Level of intracellular ROS: After ¹⁸F-FDG irradiation Eca-109 cells 6h, washed with PBS twice , incubated with 150μl of DCFH-DA (20μmol/L) 37℃in CO2 incubation 30 min, washed with PBS, fluorescence intensity was measured by flow cytometry, WinMDI software was used to analyze. Control cells were in the same procedure control.(8)Flow cytometry was used to test the intracellular mitoehondrial transmembrane Potential (△Ψm): After ¹⁸F-FDG irradiation Eca-109 cells 24h, washed with PBS twice, incubated with 10μl of Rhodamine123 (10mg/ml) work solution at 37℃in CO2 incubator for 30 min, washed with PBS, fluorescence intensity was measured by flow cytometry, 10000 cells were counted for each sample, WinMDI software was used to analyze.Results: (1) The apoptotic cells were observed under the optical microscope and transmission mirror in per ¹⁸F-FDG treated group. The cell shrinks, volume become smaller, cytoplasm condensed, small nuclei increased and pyknosis,etc. The density of apoptotic cells increased with the increase of ¹⁸F-FDG concentration. (2) MTT method and 3H-TdR incorporating show that Eca-109 cells inhibition rate increased with the increment of ¹⁸F-FDG radioactive concentration and the extension of the time . (3) Cell apoptosis rate increased gradually with the increase of ¹⁸F-FDG radioactive concentration. Apoptosis rate (%) of treated Eca-109 cells were 0.97±0.28,5.2±0.44,10.5±1.08,19.37±3.98,39.87±3.51 (compare to the apoptotic rate of control group 0.97±0.28, P <0.05) . (4) The content of ROS in cells in irradiated group was more than that control group (P<0.05). It shows that the content of ROS increased with the augmentation of ¹⁸F-FDG radioactive concentration. (5) Treated with different concentrations of ¹⁸F-FDG for 24 hours and processed by Rho123 , the mitoehondrial transmembrane Potential(△Ψm) in cells decreased with the augmentation of ¹⁸F-FDG radioactive concentration (P<0.05 compare to control group). It shows that the△Ψm level decreased with the increasing of ¹⁸F-FDG radioactive concentration. Conclusion: ¹⁸F-FDG irradiation can induce Eca-109 cells apoptosis, ¹⁸F can irradirate Eca-109 through beta+ rays and produce ROS, decrease the△Ψm level , causethe cells apoptosis and the apoptosis rate increased as the increment of its radioactive concentration.