Synthesis And Anti-tumor Activities Of The Rhein Derivatives In Vitro

Objective:To synthesize rhein derivatives and carry out hemolysis experiment in vitro,screen out the drug with the strongest anti-tumor activities and make a preliminary study on the anti-tumor activities in vitro.Methods:1.The lead compound rhein was as starting material and the concentrated sulfuric acid was as catalyst,reacted with 1,2-propanediol,1,3-propanediol and glycerol respectively to synthesize rhein derivatives at a certain temperature.The structures of rhein derivatives were confirmed by 1H-NMR and 13C-NMR.2.The hemolytic effect of rhein and its derivatives on 2%rabbit erythrocyte suspension was detected by hemolysis experiment in vitro.3.The inhibitory effects of rhein and rhein derivatives on human ovarian cancer cell line A2780,liver cancer cell line Bel-7402,lung cancer cell line H-460 and tongue cancer cell line TCA-8113 were detected by MTT assay.The drug with the strongest anti-tumor activities was screened out as drug candidate and the following experiments were carried out:(1)The effect of the drug candidate on the colony forming ability of cancer cell was detected by clone forming experiment.(2)The Fluorescence Hoechst 33258 method was used to observe the nuclear morphological changes of cell line after treating with the drug candidate.(3)The effect of drug candidate on cell apoptosis and cell cycle of cancer cell were detected by flow cytometry.(4)The expressions of cell apoptosis-related proteins BAX,BCL2 and Caspase 3 were detected after treating with the drug candidate by Western Blot.Results:1.Four rhein derivatives were synthesized.The drug BEC was a chemical compound drug,which composed of 2-hydroxypropyl-4,5-dihydroxy-9,10-dihydroanthracene-2-carboxylate(2-HDDC)and 1-hydroxypropyl-4,5-dihydroxy-9,10-dihydroanthracene-2-carboxylate(1-HDDC)in a ratio of about 3:2.The drug DBEC consisted of compound 3-hydroxypropyl-4,5-dihydroxy-9,10-dihydroanthracene-2-carboxylate(3-HDDC),and the drug DG consisted of compound 2,3-dihydroxypropyl-4,5-dihydroxy-9,10-dihydroanthracene-2-carboxylate(2,3-DDDC).2.Rhein and its derivatives had no hemolysis on 2%rabbit erythrocytes.3.Rhein and its derivatives had the inhibitory effects on cell proliferation of four cancer cell lines,the activities of rhein derivatives were stronger than rhein,and the inhibitory effects on ovarian cancer cell A2780 and lung cancer cell H-460 were more obvious,of which drug BEC had the strongest inhibitory effect on ovarian cancer cell A2780.The drug BEC was selected as the drug candidate.(1)After A2780 was treated with the drug BEC,the colony forming ability was significantly reduced(P<0.05,vs normal group).(2)After treated with the drug BEC on A2780,we could observe the increase of cells with strong blue fluorescence and the decrease of cells with weak blue fluorescence as the increase of concentration of the drug BEC by the fluorescence Hoechst 33258 method.(3)After intervened with the drug BEC for 48 h on A2780,the apoptosis rate of cells increased with the increase concentration of drud BEC,showed obvious dose dependence(P<0.01).After treated with the drug BEC for 24 h on A2780,drug BEC arrested cell A2780 in S and G2/M phase(P<0.01).(4)Western Blot results showed that the drug BEC could down-regulate the protein expression of BCL2 and up-regulate the protein of BAX and Caspase 3 on A2780.The difference was statistically significant compared with the normal group(P<0.05).Conclusions:1.Four rhein derivatives are successfully synthesized.Which compound 2-HDDC and 1-HDDC in a ratio of about 3:2 are chemical compound drug BEC;compound 3-HDDC constitutes drug DBEC;compound 2,3-DDDC constitutes drug DG.2.Rhein derivatives have no hemolytic effect on 2%rabbit erythrocytes.3.Rhein derivatives have good anti-tumor activity in vitro,the activities of them are stronger than the starting material rhein,and the drug BEC has the strongest inhibitory effect on ovarian cancer cell A2780.The drug BEC can inhibit colony formation,change nuclear morphology,arrest cell in S and G2/M phase.The drug BEC can induce apoptosis by inhibiting the protein expression of BCL2 and increasing the protein expression of BAX and Caspase 3.