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AIM: To investigate synergism of inhibition of telomerase activity and proliferation of human colon cancer cells by combination of telomerase antisense oligonucleotides (ASODNs) simultaneously targeting human telomerase RNA (hTR) and human telomerase reverse transcriptase (hTERT)in vitro.METHODS: ASODN of hTR and ASODN of hTERT were transfected into human colon cancer SW480 cells by liposomal transfection reagents. Telomerase activity of SW480 cells was examined using telomeric repeat amplification protocol (TRAP)-enzyme-linked immunosorbent assay (PCR-ELISA). Proliferation activity of SW480 cells was tested by methyl thiazolyl tetrazolium assay. Apoptosis and cell cycle were analyzed by flow cytometry.RESULTS: The telomerase activity and cell survival rate in SW480 cells transfected with 0.2 μmol/L of ASODN of hTR or ASODN of hTERT for 24-72 h were significantly decreased in a time-dependent manner compared with those after treatment with sense oligonucleotides and untreated (telomerase activity: 24 h, 73%, 74% vs 99%,98%; 48 h, 61%, 55% vs 98%, 99%; 72 h, 41%, 37% vs 99%, 97%; P＜0.01; cell survival rate: 24 h, 88%, 86%vs94%, 98%; 48 h, 49%, 47% vs94%, 97%; 72 h, 44%,42% vs 92%, 96%; P＜0.01). Moreover, the telomerase activity and the cell survival rate in SW480 cells treated by the combination of telomerase anti-hTR and anti-hTERT were more significantly suppressed than single anti-hTR or anti-hTERT (telomerase activity: 24 h, 59% vs 73%,74%; 48 h, 43% vs61%, 55%; 72 h, 18% vs41%, 37%;P＜0.01; cell survival rate: 24 h, 64% vs88%, 86%; 48 h,37% vs49%, 47%; 72 h, 25% vs44%, 42%; P＜0.01).Meanwhile, the apoptosis rates in the combination group were markedly increased compared with those in the single group (24 h, 18.0% vs 7.2%, 7.4%; 48 h, 23.0%vs 13.0%, 14.0%; 72 h, 28.6% vs13.2%, 13.75; P＜0.01).Cells in combination group were arrested at G0/G1 phase.CONCLUSION: Telomerase anti-hRT and anti-hTERT suppress telomerase activity, and inhibit growth of human colon cancer cells probably via induction of apoptosis and retardation of cell cycle. Additionally, combined use of telomerase ASODNs targeting both hTR and hTERT yields synergistic action selective for human colon cancer.