Effect And Mechanism Of Thalidomide On Gallbladder Cancer Cells

Chapterâ… The effect of thalidomide to Gallblader cancer GBC-SD cell line in vitroObjective:To investigate the sensibility and cell cycle of gallbladder cancer GBC-SD cell line by treatment of thalidomide and the allied effect of thalidomide and 5-Fu to GBC-SD cell line.Method:GBC-SD cell line were cultured in vitro,thalidodime was applied to interfere the growth of GBC-SD with different drug concentration. (1)MTT method was employed to investigate the effect of thalidomide and the allied effect of thalidomide and 5-Fu to GBC-SD cells.(2)Cell growth curve was assessed by trypan blue resisting assay.(3)We observe cell morphous and it's Ultrastructural changes through inversion light microscope and electron microscope.(4)We detect cell cycles and apoptosis ratio to thalidomide group by flow cytometry(FCM).Result:(1)Thalidomide can inhibit the growth of GBC-SD cell line obviously at different concentration with concentration-dependent.IC₅₀of thalidomide to GBC-SD is 19.44μmol/L.the CDI of thalidomide and 5-Fu are less than 1.(2)Cell growth curve show that the thalidomide at 10μmol/L has little effect to GBC-SD cell line,the thalidomide at 20μmol/L and 40μmol/L have obviously effect to GBC-SD cell line.The effect of thalidomide is the most obviously in the second day.(3)Observed through light microscope after 48 hours,we find out that viable counts of GBC-SD cells decrease and cyto-morph transform to round and shrink.When observed through electron microscope for treatment groups we see the increase of cells density,chromatic agglutination,chromatin margination,nuclear pyknosis,nuclear fragmentation and apoptotic body formation.(4)The result of FCM display for treatment groups that thalidomide mainly retard cell cycle phase in G₀/G₁ after 48 hours. The ratio of G₀/G₁ is different at the different concentration,with increase the concentration of thalidomide.The ratio of G₀/G₁ is increased and proliferation index(PI)is decreased.In same concentration,the apoptosis ratio at 24h is lower than those of 48h(P＜0.05).Conclusion:(1)thalidomide can inhibit the growth of GBC-SD cells,the inhibition is correlation with concentration and time.(2)thalidomide can induce the apoptosis of GBC-SD cells,the effect is related with concentration and time of thalidomide.(3)thalidomide have cooperation with 5-Fu to GBC-SD cells.Chapterâ…¡Investigation to the mechanisms of thalidomide to GBC-SD cell lineObjective:Investigation to the mechanisms of thalidomide to GBC-SD cell line. Method:RT-PCR were employed to investigate the expression of survivin,VEGF and MMP-9 in GBC-SD cell line.Result:With the effect of thalidomide,the ratio of survivin/β-actin of 0μmol/L,10μmol/L,20μmol/L and 40μmol/L are 1.86±0.50,1.39±0.43,0.36±0.24 and 0.15±0.07 respectively.the expression of survivin in GBC-SD cells are decreased with the increased concentration of thalidomide,the ratio of VEGF/β-actin of 0μmol/L,10μmol/L,20μmol/L and 40μmol/L are 2.47±0.76, 1.86±0.61,0.89±0.41 and 0.25±0.13 respectively.the expression of VEGF in GBC-SD cells are decreased with the increased concentration of thalidomide. the ratio of MMP-9/β-actin of 0μmol/L,10μmol/L,20μmol/L and 40μmol/L is 2.16±0.76,1.42±0.43,0.79±0.32 and 0.31±0.23 respectively.the expression of MMP-9 in GBC-SD cells are decreased with the increased concentration of thalidomidethe.Conclusion:thalidomide can inhibit the growth of GBC-SD cell,We concluded that thalidomide maybe regulate the effect by inducing cell apodtosis and inhibiting angiogenesis.Chapterâ…¢:Proteomics Research of Gallblader Cancer Cell line Treatment with ThalidomideObjective:To screen the relevant differential expression proteins after reatment with thalidomide,and to investigate the molecule mechanism of thalidomide to GBC-SD cell line.Method:We used proteomic Techniques and methods to separate the differential expression proteins in GBC-SD cells between treatment group by thalidomide and its control group,then analysis and identified it.Result:We select the differential expression proteins for between treatment group and control group which spots variance is over double to analyse with PDQuest software,and found out 36 protein spots at difference level of expression(p＜0.05),including 13 spots decreasing in treatment group, and 23 spots increasing in control groups.We choose 10 spot of them to analysis,which expression level are third double between two group,and 7 protein spot are identified.Among the identified 7 protein spots,the expression level of proteins which up-regulates in treatment group is 4 in total,including:triosephosphate isomerase 1,transgelin 2,neuregulin 3 and purine nucleoside phosphorylase;But 3 proteins in treatment group is down-regulation,which include heat shock protein 27,phosphoglycerate mutase 1 and endoplasmic reticulum protein29.Conclusion:(1)After gallbladder cancer cells were treated with thalidomide,we separate the 36 differential expression proteins and identify 7 protein spot.(2)the 7 protein are related with gallbladder cancer,they are maybe the point of action of thalidomide to gallbladder cancer.(3)many proteins are concerned with phosphoric acid or phosphorylation,such as HSP27,ERp29,PGM1,TPI,PNP,NRG3.phosphorylation of the proteins may be the mechanism of thalidomide for gallbladder cancer.