Antitumor mechanism of antisense cantide targeting human telomerase reverse transcriptase

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 aclivity 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 treated with 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 cantide increased at 48 h. At the concentration of 800 nM,the telomerase activity in the treated HepG2 and SMMC7721 cells was only 17.1% (P＜0.01) and 20.3% (P＜0.01)of that in untreated cells. The levels 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 (P＜0.05)respectively. The percentage of apoptosis in HepG2 and SMMC-7721 cells treated with 800 nM cantide at 72 h was 63% 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-dependent apoptosis. The rapid inhibitory effect of cantide on tumor growth 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 grown to 50-60 % confluence.HepG2 and SMMC-7721 were treated with cantide mixed with lipofectin,or lipofectin alone.After inducted for 6 h at 37 ℃,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.Telomerase activity was determined by TRAP-ELISA assay.CPP32-and ICE-like activity was also investigated using CasPACE assay system at 48 h after cantide treatment,and apoptosis was evaluated using the DeadEnd assay at 24,48 and 72 h after cantide treatment. RESULTS:Compared to the control cells,the cells treated with 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 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 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 (P<0.05) respectively.The percentage of apoptosis in HepG2 and SMMC-7721 cells treated with 800 nM cantide at 72 h was 63 % 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-dependent apoptosis.The rapid inhibitory effect of cantide on tumor growth demonstrates its feasibility in cancer treatment.