The Inhibitory Mechanism And Material Basic Research Of Prostate Carcinoma Cells DU145 Of Marine Chinese Medicine Kandelia Candel

Objective:Using MTS and wound healing methods ensure the inhibitory effect of Kandelia Candel on human prostate carcinoma cells.Then,tracking the active component identify the most active fraction of the marine Chinese medicine,and further clarify the chemical constitute of the the fraction and its mechanism.Methods:1.Screening of anti-prostate cancer of various solvents extracts of Kandelia candle's twigs,leaves and fruits: We set up 5 drug concentration gradient groups of each extract of twigs,leaves,and fruitsof the K.candel,blank control group,paclitaxel positive control group,and 3 replicates in each group.After drug treating for 24 h,10 ?l MTS was added to each well,and the absorbance was determined at 490 nm.Calculated the cell viability to evulate the effect of the drug on the proliferation of human prostate cancer cells DU145 and PC3.2.Effects of EAKCT onthe proliferation and migration of DU145 cells:?1?Using MTS method to study the effect of 6 drug concentrations?50,100,150,200,300,400 ?g/ml?of EAKCT on the proliferation of DU145 at 24 and48 h.?2?DU145 cells in logarithmic growth phase were seeded into 96-well plates at 1.2×10⁴ cells/well,and cells were covered with 96-well plates for scratching on the 2nd day.The cells were washed with PBS for 2 times and the treated the drug.When the drug was treated,it was recorded as 0 h.Pictures were taken at 0,12 and 24 h after drug treated.The wound healing rate was calculated by the scratch area,and the effect of EAKCT on the migration ability of DU145 cells was evaluated.3.Screening of anti-prostate cancer activity of EAKCT fractions:EAKCT was purified by semi-preparative liquid chromatograph to prepare fractions.The effect of each fraction on the proliferation of DU145 was investigated by MTS method,and then the fractions with most active inhibitorywere further studied.4.Study on mechanism of EAKCT7 in Prostate Cancer:?1?Immuno cytochemistry and Western Blot were used to investigate the regulation of apoptosis protein expression of Bcl-2,Bax and cleaved-Caspase-3 by EAKCT7 in DU145 cells.?2?Using Click-i T Ed U Alexa Fluor^TM 488 HCS Assay Kit investigated the arresting of cell cycle and the effect o n the expression of cycle protein phosphorylation histone H3?p HH3?.5.Study of the chemical composition of EAKCT7: Using LC-MS analyzed the chemical composition of EAKCT7 and determine the pharmacodynamics material basis for the inhibition of DU145 by EAKCT.Results:1.?1?The four extracts of K.candel had certain inhibitory effects on human prostate cancer cell line DU145?P<0.05 or P<0.01?.In addition to the petroleum ether of K.candel,the other three crude extracts of K.candel had effects on human prostate cancer cell PC3.Proliferation had some inhibition?P<0.05 or P<0.01?.?2?The four crude extracts of K.candel had certain inhibitory effects on DU145 and PC3?P<0.05 or P<0.01?.?3?K.candel fruit ethanol extract can inhibit the proliferation of DU145?P <0.05 or P <0.01?,the other three crude extracts had no inhibitory effect on DU145;K.candel fruit ethanol extract,n-butanol extract can inhibit the proliferation of PC3?P<0.05 Or P<0.01?,while the petroleum ether fraction and ethyl acetate fraction did not inhibit proliferation of PC3.2.?1?After treating with EAKCT,with the increase of drug concentration and time,it began to observe that the number of DU145 cells decreased gradually,the cell outline was getting round,and finally collapsed into blocks.?2?Compared with the blank control group,after EAKCT treated12,24 h,the 75,100,and 150 ?g/ml concentrations of EAKCT can inhibit the wound healing rate significantly?P<0.05 or <0.01?.3.Nine fractions named EAKCT1-9 were isolated from EAKCT.Nine fractions had a certain inhibitory effect on the proliferation of DU145 cells,and EAKCT7 had the best effect with an IC50 of 56.71 ?g/ml.4.?1?The results of ICC experiments showed that EAKCT7 can make stronger Bax fluorescence intensityand less Bcl-2 expression.And Western Blot showed that after treated EAKCT7 the expression of cleaved-Caspase-3in DU145 cells tend to be increase.?2?EAKCT7 treated for 24 h,as the drug concentration increased,compared with the blank control group,the proportion of cells in M phase have no statistically difference?P>0.05?,but the proportion of S phase cells decreased and the average fluorescence intensity of Ed U markers decreased?P <0.05 or <0.01?.5.LC-MS technology was used to analyze several compounds that may be identified as flavonoids.EAKCT7 may contains terpine,saponins,pheols and flavonoid.The retention time of 1.75,2.01 and 2.68 min in mass spectrum may be procyanidine B3,epicatechin/catechinic acid and kaempferol-3-O-?-D-[3,6-dis-(hydroxy cinnamon hydroxy] glucoside,but further study is need to identify.Conclusion:1.Kandelia Candel's twigs,leaves,and fruits have certain inhibitory effects on the proliferation of human prostate cancer cells DU145 and PC3,of which EAKCT can be used for further research.2.EAKCT can inhibit the proliferation of DU145 cells in a time-concentration-dependent manner,and can inhibit the migration ability of DU145 cells.3.EAKCT isolated 9 fractions EAKCT1-9,9 fractions all have a certain inhibition on the proliferation of DU145,and EAKCT7 is most active.4.EAKCT7 tend to up-regulate the expression of Bax and cleaved Caspase-3,and down-regulate the expression of bcl-2.EAKCT7 can reduce the proportion of cells in S phase and arrest cell cycle in G1/S phase.5.The main active chemical constituents of EAKCT7 may be terpine,saponins,pheols and flavonoid,and the specific substance and structure needs further identification.