Study On The Synthetic Process And Anticancer Activity In Vitro Of Diacetyldianhydrogalactitol

Objective:（1）To study the synthetic process of Diacetyldianhydrogalactitol（D ADAG）,then to screen out the optimun process for synthesis of antitu mor drug DADAG.（2）To investigate the antitumor effect of DADAG chemotherapy in vitro on six hunman cancer cell lines,and screen out the sensitive cell lines for furtherr study.（3）To study the apoptosis induced by DADAG and its related mechanism in NCI-H460 cell.（4）To evaluate the effects of DADAG on embryos up-growth of zebrafish.Methods:（1）DADAG is synthesized through acethl chloride reaction from DAG.By changing the material ratio of the time,temperature,solwent,then to calculate the yield of DADAG under the different conditions.The structure of the compound was confirmed by IR,¹H-NMR,¹³C-NMR.（2）To investigate the inhibitory effects of DADAG on human lungcancer cell NCI-H460,A549,human liver cancer cell SMMC-7721,Hep G₂,human nasopharyngeal cancer cell HNE-1,human stomach cancer cell MGC-803 in vitro.（1）To establish thecultured system of the six cancer cells,then the cellular morphliogy after intervention of DADAG was observed under microscope.（2）Intervention using DADAG of differetconcentrations was conducted in NCI-H460,A549,SMMC-7721,Hep G₂,HNE-1 and MGC-803 cells for 48 hours.The concentrations of DADAG for NCI-H460,A549,SMMC-7721,Hep G₂ cells included0.00（blank group）,2.88,5.75,11.50,23.00,46.00μg/m L,and the concentrations for HNE-1、MGC-803 cells included 0.00（blank group）,11.50,23.00,46.00,69.00,92.00μg/m L.The proliferation of six types of tumor cells was detected using MTT bromide assay.The inhibitory rate of six tumor cells and half maximal inhibitory concentration(IC₅₀)of DADAG to six of tumor cells were calculated.Then screen out the sensitive cell lines for furtherr study.（3）Intervention using DADAG of different concentration（0.00,11.50,23.00,46.00,69.00μg/m L）was conducted in NCI-H460 cell,then colone formation assay was used to detect the ability of cell proliferation.（3）To study the effects of DADAG on the apopotosis in lung cancer cell NCI-H460.（1）Though AO/EB staining method to observed morphological changes of apopototic cell under fluorescent microscope.（2）To test the effects of DADAG on NCI-H460 cell apoptosis and cell cyclewas detected by flow cytometry.（4）Real Time-PCR（RT-PCR）was used to detect the effect of DADAG on Bcl-2,Bax,Caspase-3,Caspase-9 m RNA expression levels within the cell.（5）To evaluate the effects of DADAG on embryos upgrowth of zebrafish:zebrafish embryos of 6 hpf were treated with aqueous extraction of differentconcentrations DADAG,and then effects of DADAG on the zebrafish development were assessed under the microscope by assessing the24 hpf spontaneoμs movements,48 hpf heart rate,hatching rate embryo deformity rate and mortality rate.The drug solution was replaced every 24hours.（6）Statistical Methods:the data were expressed as mean±standard.SPSS17.0 statistical software was uesed for data analysis with method of one-way ANOVA,P﹤0.05,P﹤0.01 or P﹥0.05 was described with statistically signficant differece.Result:（1）The best optimun process for synthesis of antitumor drug diacetyldianhydrogalactitol was that the dosage ratio of acetyl chloride and DAG was 8:1,the reaction time and temperature were 4 hours and 30℃.The melting point of the targer compound was 103～104.5℃.The structure of the target compound was confirmed by IR and ¹H-NMR,¹³C-NMR,The total yield of target compound was 93.60%,the purity was 95%.（2）DADAG exhibited obvious proliferation inhibitory activity in NCI-H460,A549,SMMC-7721,Hep G₂,HNE-1,MGC-803 cells,after incubation for 48 hours,the IC₅₀ value was 24.79,47.75,145.39,28.60,49.36,141.29μg/m L.Compared with blank control group the group treated with DADAG inhibited cell prolifertion,（P﹤0.05）,all with significant difference,the number of colony formation decreases.（3）The effect of DADAG on apoptosis of NCI-H460 cells:AO/EB staining results showed that the cells of apoptosis were characterized by typical mor-phological changes such as swelling,shrinking and fragmentation.Flow cytometry detection results showed that the group treated with DADAG apoptosis rate was increases（P﹤0.05）,all with significant difference,the apoptotic rates of NCI-H460 cell were（13.13±0.87）%,（28.94±0.44）%,（40.06±0.91）%,the blank group was（5.71±0.88）%.Compared with blank control group the group treated with DADAG increased G₂ phase ratio,decrease G₁ phase ratio,all with significant difference（P﹤0.05,P﹤0.01）,and S phase have no signgicant changes（P﹥0.05）.（4）RT-PCR results indicated that affter treatment with DADAG on NCI-H460 cell for 48 hours,the expression of Bax,Caspase-3 was up-regulated,the expression of Bcl-2 was down-regulated.（5）Experimental results of zebrafish embryos:The zebrafish embryos were exposed in DADAG with different concentrations.Compared with the untreated embryos,the counts of spontaneous movements,heart rate and hatching rate had significant difference.The abnormality of zebrafish was observed for curved body axis,spinal curvature,pericardial edema,yolk sac edema and abnormal swimming.Deformity rate and mortality rate of zebrafish embryos were increased with the increasing concentration of DADAG solution and extended medication time.Conciusions:diacetyldianhydrogalactitol was that the dosage ratio of acetyl chloride and DAG was 8:1,the reaction time and temperature were 4 hours and 30℃,The total yield of target compound was 93.60%.（2）DADAG have dramatically growth inhibitory effect on NCI-H460,A549,SMMC-7721,Hep G₂,HNE-1,MGC-803 cells,and displays antitumor effect in vitro.DADAG can induce the apoptosis of NCI-H460 tumor cells,The apoptosis potential mechanism maybe associated with up-regulating the expression of Bax,Caspase-3,down-regulating the expression of Bcl-2.（3）DADAG has significant effect on the development of zebrafish embryos and presents a time and concentration dependent relationship.