title: hTERT-siRNA could potentiate the cytotoxic effect of gemcitabine to pancreatic cancer cells Bxpc-3.
abstract: OBJECTIVE
  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.
  
  
  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.
authors: Division of Hepatopancreatobiliary, Second Affiliated Hospital, Kunming Medical College, Kunming 650101, China.
authors: Jing
authors: J
authors: Tan
authors: Xiaohu
authors: X
authors: Zhou
authors: Hong
authors: H
authors: Zhu
mesh_terms: D000964:Antimetabolites, Antineoplastic / Q000494:pharmacology*; D017209:Apoptosis / Q000187:drug effects; D015153:Blotting, Western; D045744:Cell Line, Tumor; D002470:Cell Survival / Q000187:drug effects; D003124:Colorimetry; D003587:Cytomegalovirus / Q000235:genetics; D003841:Deoxycytidine / Q000031:analogs & derivatives* / Q000494:pharmacology; D015536:Down-Regulation; D005434:Flow Cytometry; D059585:G1 Phase Cell Cycle Checkpoints / Q000187:drug effects; D015971:Gene Expression Regulation, Enzymologic; D015972:Gene Expression Regulation, Neoplastic; D005822:Genetic Vectors; D006801:Humans; D010190:Pancreatic Neoplasms / Q000201:enzymology / Q000235:genetics* / Q000473:pathology*; D034622:RNA Interference; D012333:RNA, Messenger / Q000378:metabolism; D034741:RNA, Small Interfering / Q000378:metabolism*; D020133:Reverse Transcriptase Polymerase Chain Reaction; D019098:Telomerase / Q000235:genetics* / Q000378:metabolism; D013997:Time Factors; D014162:Transfection; D000093542:Gemcitabine
chemical_list: D000964:Antimetabolites, Antineoplastic; D012333:RNA, Messenger; D034741:RNA, Small Interfering; D003841:Deoxycytidine; C509186:TERT protein, human; D019098:Telomerase; D000093542:Gemcitabine