A Study Of Inhabitive Effect Of HTERT DNAzyme On A549/DDP Cell Growth

Objective This study analyzed the relationship between telomerase activity and drug resistance in A549/DDP cell. With the decrease of telomerase activity in A549/DDP cell after hTERT DNAzyme treatment, cell growth was measured. The goal of this study was to show the possibility of telomerase as a new target for the treatment of drug resistance lung cancer cell.Method (1)Total RNA was harvested from cultured A549/DDP cell, and reverse transcription-polymerase chain reaction (RT-PCR) was performed to detect MRP gene expression. Bcl-2 protein was measured by western blot. (2) Total RNA was harvested from cultured A549/DDP cell, and reverse transcription-polymerase chain reaction (RT-PCR) was performed to detect telomerase RNA and telomerase reverse transcriptase (hTERT ) mRNA. Telomerase activity was measured by telomeric repeat amplification protocol (TRAP) modified from Kim's method. (3) Cultured A549/DDP cells were divided into five groups: control, cisplatin, liposome, liposome+hTERT DNAzyme, cisplatin+ liposome+hTERT DNAzyme , then treated for 72 hours. The concentrations of cisplatin, liposome and DNAzyme were 3ug/mK lug/ul, 0.25umol/L, respectively., Telomerase activity was measured by telomeric repeat amplification protocol (TRAP) and cells were counted by MTT to make cell-growth curve. Changes were compared among groups.Results (1) Both MRP mRNA and Bcl-2 protein were positive in A549/DDP cell. (2) Telomerase RNA and hTERT were expressed and telomerase activity was positive in A549/DDP cell.(3)A549/DDP cell telomerase activity was down-regulated by hTERT DNAzyme with a dose range from 4 to 500 nmol/L. (4)Compared with control, OD volume of A549/DDP cell was decreased after treatment of 0. 25umol/L hTERT DNAzyme for 72 hours. P<0. 01. (5)A549/DDP cell growth was significantly inhabited by 32.9% and 60.5% after treatment of hTERT DNAzyme at 0. 25 umol/L only and hTERT DNAzyme with 3 ug/ml cisplatin for 72 hours,respectively..Conclusion (1)MRP-. bcl~2> telomerase mRNA and hTERT have a positive expression in A549/DDP cell. It is suggested that the mechanism of A549/DDP MDR (multi-drug resistance) not only includes the MRP, but also the anti-apoptosis effect of bcl-2. Telomerase may contribute to this mechanism through anti-apoptosis with bcl-2. (2) Telomerase inhibitors may treat drug resistant lung cancer cell since the cell express telomerase activity. (3) hTERT inhibits A549/DDP cell growth and has a synergistic effect with cisplatin. It is suggested that telomerase might be a new target for the treatment of drug-resistance lung cancer cell.