Antitumor mechanism of antisense cantide targeting human telomerase reverse transcriptase.
OBJECTIVE
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 degrees, 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.


CONCLUSIONS
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.
Du
Qing-You
QY
Beijing Institute of Radiation Med-icine, Beijing 100850, China.
Wang
Xiao-Bo
XB
Chen
Xue-Jun
XJ
Zheng
Wei
W
Wang
Sheng-Qi
SQ
D000970:Antineoplastic Agents / Q000008:administration & dosage / Q000494:pharmacology*; D006528:Carcinoma, Hepatocellular / Q000201:enzymology* / Q000473:pathology; D004268:DNA-Binding Proteins; D004305:Dose-Response Relationship, Drug; D005240:Feasibility Studies; D006801:Humans; D008113:Liver Neoplasms / Q000201:enzymology* / Q000473:pathology; D020319:Oligodeoxyribonucleotides, Antisense / Q000494:pharmacology*; D020320:Oligoribonucleotides, Antisense / Q000008:administration & dosage / Q000494:pharmacology*; D054735:Phosphorothioate Oligonucleotides; D012333:RNA, Messenger / Q000037:antagonists & inhibitors; D019098:Telomerase / Q000235:genetics*; D014407:Tumor Cells, Cultured
D000970:Antineoplastic Agents; D004268:DNA-Binding Proteins; D020319:Oligodeoxyribonucleotides, Antisense; D020320:Oligoribonucleotides, Antisense; D054735:Phosphorothioate Oligonucleotides; D012333:RNA, Messenger; C478563:cantide; D019098:Telomerase