hTERT-siRNA Could Potentiate the Cytotoxic Effect of Gemcitabine to Pancreatic Cancer Cells Bxpc-3

Abstract

Objectives: This study sought to observe transfection of pancreatic cancer cells BxPC-3 with recombinant plasmid pSilencer4.1-cytomegalovirus neo-hTERT-siRNA and examine the combined effect of gemcitabine and siRNA inhibition of telomerase on pancreatic cancer cells.

Materials and Methods: Transfected pancreatic cancer cells BxPC-3 with recombinant plasmid pSilencer4.1-cytomegalovirus neo-hTERT-siRNA were selected as target and divided into 9 groups: (1) T1 group (pSilencer4.1-CMV neo-hTERT1-siRNA), (2) T2 group (pSilencer4.1-CMV neo-hTERT2-siRNA), (3) L group (Lipofectamine) (4) M group (mismatch group pSilence4.1-CMV, as negative control), (5) C group (cell group without transfection), (6) blank and gemcitabine group, (7) mismatch siRNA and gemcitabine group, (8) hTERT1-siRNA and gemcitabine group, and (9) hTERT2-siRNA and gemcitabine group. Expression of hTERT mRNA was detected by reverse transcriptase polymerase chain reaction. Viability of cells was measured by colorimetric 3-(4,5-Dimethylthiazol-2-yl)-2, 5-Diphenyltetrazolium Bromide assay. Cell cycle and cell apoptosis were measured by flow cytometry. Expression of telomerase protein was measured by Western blot.

Results: Compared with the L group, M group, and C group, expression of hTERT-mRNA and the level of telomerase protein in T1 and T2 group was down-regulated significantly (P < .05), viability of BxPC-3 cells decreased significantly (P < .05), the ratio of cells in G(0)/G(1) stage increased, the ratio of cells in the S stage and the G(2)/M stage decreased, and the ratio of apoptotic cells increased significantly in the T1 and T2 groups. Gemcitabine treatment had a comparable effect. Combination hTERT siRNA and gemcitabine killed twice as many cancer cells, showing a cumulative effect of the treatments.

Conclusions: Transfection of pancreatic cancer cells BxPC-3 with recombinant plasmid pSilencer4.1-CMV neo-hTERT-siRNA represents good RNAi silencing and anti-pancreatic cancer effects in vitro and could potentiate the cytotoxic effect of gemcitabine to pancreatic cancer cells.