Study Of The Antitumor Effects Of FTY720 And The Possible Mechanisms In Vitro

Objective:The malignant tumor has badly been threatening the health and life of human being.In recent half of a century,tumor treatment with Chinese herbal medicine having been accepted by more and more experts and patients,being the common way and one of the effective measures in the comprehensive treatment Clinical experiments prove that Chinese herbal medicine has the better effect of antitumor,not only decrease the toxicity and increase sensitivity or efficiency of radiotherapy and chemotherapy,but also prevent tumor from relapse,metabasis.In the manegant of malignant tumor,Chinese herbal medicine shows particular superiority for its less side effect,lower toxicity,abundant resources,lower price and being suitable to be used for a longer time.The researches of the effective component and mechanism of Chinese herbal medicine have been paid more and more attentions.Some drugs from herbs such as Vincristione(VCR)and Paclitaxel(PTX)have been used in the clinical treatment of tumor,which has gotten good clinical effect.However,effective components of Chinese herbal medicine used in clinic at the present time are still fewer,so exploiting and developing new effective antitumor drugs from Chinese herbal medicine becomes one of the important subjects of treatment of malignant tumor.FTY720,2-amino-2-(2-(4-octylphenyl)ethyl)-1,3-propanediol hydrochloride, was screened from chemically synthesized analogs of ISP-1,which was purified from culture filtrates of the fungus Isaria sinclairii,is a new immunosuppressant FTY720 can deal with some autoimmune disease.In addition there are some reports that FTY720 can induce cell apoptosis in some tumor to inhibit the cell proliferation So the effect and mechanism of the antitumor has a spacious perspective.To investigate the antineoplastic activity and the effect of inducing cell apoptosis of a new immunosuppressive FTY720 and its mechanism in vitro,we devise a serial experiment to provide the scientific evidence for developing the new antitumor drug.Method:1)Inhibition ratio of the different concentration of the FTY720 on Hep-2,Hela, A549,and SMMC-7721 cells was tested by MTT assay.2)With different concentration of the FTY720 on Hep-2 cell for six hours,the change of morphologic was observed by invert microscope.3)With different concentration of the FTY720 on Hep-2 cell for six hours,using hochest33342/PI fluorochrome,the change of morphologic was observed by fluorescent microscope.4)With different concentration of the FTY720 on Hep-2 cell for six hours,the apoptotic cells were detected by flow cytometry.5)With different concentration of the FTY720 on Hep-2 cell for six hours, the apoptosis rate was detected by flow cytometry.6)With different concentration of the FTY720 on Hep-2 cell for six hours,the activity of intracellular Caspase-9 was detected by spectrophotometric method.results1)The effect of growth inhibition of the FTY720 on Hep-2,Hela,A549, SMMC-7721 four types of tumor cells.â‘ FTY720 can remarkably inhibit the growth of Hep-2 cell:the difference of interclass have statistically significant,(F=454.726, P＜0.01,)Except 40μg/ml and 20μg/ml group,the IC₅₀was 4.05402μg/ml.â‘¡FTY720 can remarkably inhibit the growth of Hela cell:the difference of interclass have statistically significant,(F=2357.490,P＜0.01),Except 40μg/ml and 20μg/ml group,the IC₅₀was3.35259μg/ml.â‘¢FTY720 can remarkably inhibit the growth of A549 cell:the difference of interclass have statistically significant,(F=4941.806,P＜0. 01),Except 40μg/ml 20μg/ml and 10μg/ml group,the IC₅₀was 4.36825μg/ml.â‘£FTY720 can remarkably inhibit the growth of SMMC-7721 cell:the difference of the different groups have statistically significant(F=745.166,P＜0.01),IC₅₀was4.20633μg/ml.2)The typical Hep-2 cells apoptosis were observed by invert microscope after six hours,and the number of the apoptosis cell show a positive correlation with the dose of the FTY720.3)The typical Hep-2 cells apoptosis were observed by fluorescent microscope after six hours.The number of the apoptosis cell shows a positive correlation with the dose of the FTY720.4)The flow cytometry show the result:after FTY720 on Hep-2 cell six hours,we can see a remarkably apoptosis peak in G₂/M phase and the apoptosis ratio present a positive correlation with the dose of the FTY720.5)After acting Hep-2 cell for six hours,use the Annexin V/PI method show the result:the number of the apoptosis cell has a dose dependent with the FTY720.6)The spectrophotometric method show the result:after FTY720 on Hep-2 cell for six hours,the activity of intracellular Caspase-9 has a remarkably increasing,and present dose dependent.Conclusion1.The FTY720 can remarkably restrain the proliferation of Hep-2,Hela,A549, SMMC-7721 cells,and the effect of anti-proliferation is dose-dependent.The IC₅₀,of FTY720 on Hep-2,Hela,A549,SMMC-7721 cells have a litter different.2.The possible mechanism that FTY720 inhibit the proliferation of Hep-2 cells is May through inducing apoptosis.3.It is possible that the FTY720 induce the Hep-2 cells apoptosis on G₂M phase.4.The molecular mechanism of the FTY720 inducing apoptosis of Hep-2 is possible that activate the Caspase-9 of the intracellular,make the activity of the apoptosis associated protein caspase-9 increasing.