The Apoptotic Effects And Mechanism Of Eca-109 Cells Treated With Antisense Oligo-Deoxynucleotide Targeting Survivin And Trichosanthin

Abstract Objective:To study the effects of trichosanthin(TCS) on the cell proliferation of human esophageal carcinoma Eca -109 cells and its relation with survivin . On this basis, To explore the effects of the apoptosis of human esophageal carcinoma Eca -109 cells induced by antisense oligodeoxynucleotide targeting survivin and trichosanthin and its molecular mechanisms.Methods: After treatment with TCS,Inhibition of proliferation of Eca-109 cells was measured with MTT assay. The changes of ultra-structure of cells was observed by transmission electron microscope. The rate of early apoptosis was detected by AnnexinV-FITC/PI double stain.Cell cycle was analyzed by flow cytometry.The expression of survivin mRNA was detected by RT-PCR.The expression of survivin,caspase-3 and c-myc proteins were detected by Western blot.Telomerase activation was detected by TRAP-PCR kit. Furthemore , the Experimental Eca-109 cells were divided into six groups:untreated group, lipofectamine group,sense oligodeoxynucleotide targeting survivin group(SODN),antisense oligodeoxynucleotide targeting survivin group(ASODN),trichosanthin group,antisense oligodeoxynucleotide targeting survivin and trichosanthin group . Expression of survivin mRNA was detected by RT-PCR. expressions of Survivin,Bcl-2,Bax,Caspase-3 Proteins were detected by Western blot. The rate of early apoptosis was detected by AnnexinV-FITC/PI double stain.Results: TCS could inhibit the proliferation of Eca-109 cells on a time-and-dose-dependant manner.After treatment of TCS(33μg/ml) for 48 hours, as compared with untreated group, the rate of early apoptosis of TCS group was significantly increased(P<0.05) .S stage cell percentage of TCS group was significantly increased(P <0.05) . Expressions of survivin mRNA and survivin protein,c-myc protein were decreased as well as telomerase activation.Howover,expression of caspase-3 protein was increased.Differences were significant(P <0.05). After treatments of ever factor for 48 hours,Expressions of survivin mRNA and protein of the ASODN group,the TCS group and the combination group was significantly lower than the lipofectamine group, the SODN group and the untreated group(P<0.05).The lipofectamine group,the SODN group and the untreated group were compared each other. The differences were not significant (P> 0.05). Expressions of survivin mRNA and protein of the combination group was significantly lower than ASODN group and the TCS group.The differences were significant (P <0.05). The difference of the ASODN group and the TCS group was not significant (P >0.05). The early apoptosis rates and expression of caspase-3 protein of the ASODN group,the TCS group and the combination group were significantly higher than the lipofectin group, the SODN group and the untreated group.The differences were significant (P <0.05). The lipofectamine group,the SODN group and the untreated group were compared each other .The differences were not significant (P> 0.05). The early apoptosis rate and expression of caspase-3 protein of the combination group higher than the ASODN group and the TCS group.The differences were significant (P<0.05). The difference of the ASODN group and the TCS group was not significant (P >0.05). Expression of bcl-2 protein of the TCS group and the combination group lower than the other four groups.the differences were significant (P <0.05).However, expression of The bcl-2 protein was increased. The differences were significant (P <0.05). In the terms of expressions of the bcl-2 and bax protein ,the difference of the ASODN group and the TCS group was not significant (P >0.05). The lipofectamine group,the SODN group,the control group and the ASODN group were compared each other , the differences were not significant (P> 0.05).Conclusion: TCS can significantly inhibit the proliferation of Eca-109 cells and induce apoptosis .Suppressing the expression of survivin gene may be a crucial effect . Survivin antisense oligodeoxynucleotide targeting survivin and trichosanthin can collaboratively induce apoptosis of the Eca-109 cells. The molecular mechanism may be that two drugs can activate caspase cascade channels due to collaborative suppressing expression of survivin gene by two drugs and down-regulating expression of bcl-2 protein and up-regulating expression of bax protein by TCS.