Suppressive Effects Of Ceramide Analogs AD2646 On The Growth Of Lung Adenocarcinoma Cell Line A549 In Vivo And In Vitro

OBJECTIVES: To investigate the suppressive effects and its possible mechanisms of AD2646 on growth of lung adenocarcinoma cell line A549 in vitro.METHODS: A549 cells were treated with varying doses of AD2646 for up to 72 h respectively.The cell morphology was investigate by microscope and the cell viability was assessed using the MTT test.Flow cytometry were used for analysis of cell cycle.Effcetor caspase activity was measured with spectrofluorometer.The activity of GC and SM were assayed by TLC and the intracellular ceramide levels were determined by HPLC.RESULTS: A dose and time dependent proliferation inhibition was demonstrated in the treated A549 cell line with the morphology changes profoundly.Cell cycle analysis shows that cell ratio in G1 phases was decreased while that of the G2+M phase was increased.The IC50s increased when higher concentrations or the cells cultured with FCS.The activity of Caspase-3 can be measured increased early after giving AD2646,however,it was not a dose dependent increase with the dose augment of AD2646.There was a obviously decrease about the activety of both SM and GC,but the later shows much strongerly been reduced during the first hour after treating with AD2646.The intrinacellular ceramide also shows to be in a high concentration during first 2 hours and level up for at least 24 hours.CONCLUSION: Ceramide analogs AD2646 demonstrat can cause apoptosis process and changes of cell cycle of of A549 cells.There was a dose and time dependent reduction of the growth to A549.Besides Caspase-3,maybe the other signal molecules also involve in the process of AD2646 dependent cell death.The inhibition of GC/SM can be discovered soon after treat with AD2646 and GC showed be reduced much more. The increasing of intracellar ceramide with the inhibition of GC and SM indicate the ceramide analogs were entrance from AD2646.There wer no parallel changes among the increasing of Caspase-3 and ceramide concentration,and the low expression of GC/SM suggest that the other mechanism except for Caspase pathway participate the cell death of A549 incurred by AD2646.PART 2 SUPPRESSIVE EFFECTS OF CERAMIDE ANALOGS AD2646 ON THE GROWTH OF LUNG ADENOCARCINOMA CELL LINE A549 IN VIVOOBJECTIVES: To investigate the suppressive effects and its possible mechanisms of AD2646 on growth of lung adenocarcinoma cell line A549 in vivo tumor model.METHODS: Human lung adenocarcinoma A549 xenograft model was established in nude mice .The nude mice were divided into three groups.â‘ A D2646 group received 10mg/kg AD2646 every three days.â‘¡CE group group received the flux(CE)of AD2646 every three days.â‘¢NS group received NS every three days.Then observe the weight of the body, size of the tumor every time before injection. The cell morphology was investigate by microscope and the Flow cytometry were used for analysis of cell cycle.Effcetor caspase-3 activity was measured with by Western bloting,RT-PCR and spectrofluorometer.Results: 2 weeks after beginning of injection,the size of tumor increased in a same speed.Then the increasing of tumor size in the AD2646 group become slower than the other groups.Up to 5 weeks after injection,the tumor mass of AD2646 even got smaller than before.The cell morphology of heart ,lung,liver,kidney were almost the same as normal structures among the groups.The cell death rate in AD2646 group were at least 3 times increased compared with the control ones.RT-PCR and western bloting test shows the activity of Caspase in AD2646 group were also doubled to the other groups.Except for th decrease of the WBC in the blood,the other biochemical markers about the function of liver,kidney, were almost in a same level as in the CE group and NS group. CONCLUSION: In vivo lung adenocarcinoma A549model ,ceramide analogs AD2646 demonstrate to be a antiproliferative and proapoptotic agent to cause reduction of the tumor and cell death. The toxicity of AD2646 shows no obviously to the vital organs of mice,except inhibition of the bone marrow.AD26467 maybe have a good future in chemical therapy to lung adenocarcinoma.