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AIM:To investigate the anti-tumor mechanism of antisenseoligodeoxynucleotide cantide against hTERT.METHODS:Tumor cells were cultured overnight and grownto 50-60 % confluence.HepG2 and SMMC-7721 were treatedwith cantide mixed with lipofectin,or lipofectin alone.Afterinducted for 6 h at 37 ℃,10 % FCS in DMEM was replacedin each well.After the treatment repeated twice to threetimes in each concentration of cantide,hTERT mRNA andprotein expression were measured by RT-PCR and Westernblot analysis,respectively.Telomerase activity was determinedby TRAP-ELISA assay.CPP32-and ICE-like activity was alsoinvestigated using CasPACE assay system at 48 h aftercantide treatment,and apoptosis was evaluated using theDeadEnd assay at 24,48 and 72 h after cantide treatment.RESULTS:Compared to the control cells,the cells treatedwith cantide showed a dose-dependent decrease in hTERTmRNA levels at 24 h and in protein levels at 48 h respectively.The telomerase activity was decreased as the concentrationof cantide increased at 48 h.At the concentration of 800 nM,the telomerase activity in the treated HepG2 and SMMC-7721 cells was only 17.1% (P<0.01) and 20.3 % (P<0.01)of that in untreated cells.The levels of CPP32-like proteaseactivity in HepG2 and SMMC-7721 increased by 2.8-and3.0-fold (P<0.05) at 48 h,and the levels of ICE-like proteaseactivity also increased by 2.6-and 3.2-fold (P<0.05)respectively.The percentage of apoptosis in HepG2 andSMMC-7721 cells treated with 800 nM cantide at 72 h was63 % and 52 % (P<0.01),respectively.By contrast,8 %and 9 % of the cells were apoptosis after 72 h treatmentwith lipofectin alone.CONCLUSION:Cantide can decrease telomerase activityby inhibiting the expression of hTERT gene and has a rapidanti-tumor effect through inducing the Caspase-dependentapoptosis.The rapid inhibitory effect of cantide on tumorgrowth demonstrates its feasibility in cancer treatment.
AIM: To investigate the anti-tumor mechanism of antisense oligodeoxynucleotide cantide against hTERT.METHODS: Tumor cells were cultured overnight and grownto 50-60% confluence. HepG2 and SMMC-7721 were treated with cantide mixed with lipofectin, or lipofectin alone. Afterinducted for 6 h at 37 ° C, 10% FCS in DMEM was replaced in each well. After the treatment repeated twice to three times in each concentration of cantide, hTERT mRNA and protein expression were measured by RT-PCR and Western blot analysis, respectively. assay. CPP32-and ICE-like activity was also examined using CasPACE assay system at 48 h aftercantide treatment, and apoptosis was evaluated using theDeadEnd assay at 24, 48 and 72 h after cantide treatment .RESULTS: Compared to the control cells, the cells treatedwith cantide showed a dose-dependent decrease in hTERT mRNA levels at 24 h and in protein levels at 48 h respectively. The telomerase activity was decreased as the concentration of c Antide increased at 48 h.A concentration of 800 nM, the telomerase activity in the treated HepG2 and SMMC-7721 cells was only 17.1% (P <0.01) and 20.3% (P <0.01) of that in untreated cells. of CPP32-like protease activity in HepG2 and SMMC-7721 increased by 2.8-and 3.0-fold (P <0.05) at 48 h, and the levels of ICE- like protease activity also increased by 2.6- and 3.2- fold respectively. Percentage of apoptosis in HepG2 and SMMC-7721 cells treated with 800 nM cantide at 72 h was63% and 52% (P <0.01), respectively.By contrast, 8% and 9% of the cells were apoptosis after 72 h treatment with lipofectin alone. CONCLUSION: Cantide can decrease telomerase activity by inhibiting the expression of hTERT gene and has a rapid anti-tumor effect through inducing the Caspase-dependentapoptosis. the rapid inhibitory effect of cantide on tumorgrowth demonstrates its feasibility in cancer treatment.