Effect of gemcitabine on the expression of apoptosis-related genes in human pancreatic cancer cells.
OBJECTIVE
To investigate the expression of genes involved in the gemcitabine-induced cytotoxicity in human pancreatic cancer cells.


METHODS
A human pancreatic cancer cell line, PANC-1, was cultured. 1x10(4) PANC-1 cells were plated in 96-well microtiter plates. After being incubated for 24 h, gemcitabine was added to the medium at concentrations ranging 2.5-1000 mg/L. The AlamarBlue dye method was used for cell growth analysis. DNA fragmentation was quantitatively assayed using a DNA fragmentation enzyme-linked immunosorbent assay (ELISA) kit. PAP and TP53INP1 mRNA expression was determined using the reverse transcription-polymerase chain reaction with semi-quantitative analysis. The expression of GSK-3beta and phospho-GSK-3beta proteins was examined with Western blot analysis.


RESULTS
The IC50 for the drug after a 48-h exposure to gemcitabine was 16 mg/L. The growth of PANC-1 cells was inhibited by gemcitabine in a concentration-dependent manner (P<0.0001) and the cell growth was also inhibited throughout the time course (P<0.0001). The DNA fragmentation rate in the gemcitabine-treated group at 48 h was 44.7%, whereas it was 25.3% in the untreated group. The PAP mRNA expression was decreased after being treated with gemcitabine, whereas the TP53INP1 mRNA was increased by the gemcitabine treatment. Western blot analysis showed that phospho- GSK-3beta (ser9) was induced by the gemcitabine treatment.


CONCLUSIONS
Gemcitabine suppresses PANC-1 cell proliferation and induces apoptosis. Apoptosis is considered to be associated with the inhibition of PAP and GSK-3beta, and the activation of TP53INP1 and pospho-GSK-3beta (ser9).
Department of Internal Medicine and Medical Oncology, Cancer Research Institute, Kanazawa Medical University, 1-1 Daigaku, Uchinada, Ishikawa 920-0293, Japan.
Pei-Hong
PH
Jiang
Yoshiharu
Y
Motoo
Norio
N
Sawabu
Toshinari
T
Minamoto
D000951:Antigens, Neoplasm / Q000235:genetics; D000964:Antimetabolites, Antineoplastic / Q000494:pharmacology*; D017209:Apoptosis / Q000187:drug effects* / Q000235:genetics*; D051017:Apoptosis Regulatory Proteins / Q000235:genetics*; D014408:Biomarkers, Tumor / Q000037:antagonists & inhibitors / Q000235:genetics; D002352:Carrier Proteins / Q000235:genetics; D045744:Cell Line, Tumor; D049109:Cell Proliferation / Q000187:drug effects; D053938:DNA Fragmentation; D004273:DNA, Neoplasm / Q000032:analysis; D003841:Deoxycytidine / Q000031:analogs & derivatives* / Q000494:pharmacology; D015972:Gene Expression Regulation, Neoplastic / Q000187:drug effects; D038362:Glycogen Synthase Kinase 3 / Q000037:antagonists & inhibitors / Q000235:genetics; D000071679:Glycogen Synthase Kinase 3 beta; D006360:Heat-Shock Proteins / Q000235:genetics; D006801:Humans; D037181:Lectins, C-Type / Q000037:antagonists & inhibitors / Q000235:genetics; D010190:Pancreatic Neoplasms / Q000235:genetics* / Q000473:pathology; D000073718:Pancreatitis-Associated Proteins; D012333:RNA, Messenger / Q000032:analysis; D000093542:Gemcitabine
D000951:Antigens, Neoplasm; D000964:Antimetabolites, Antineoplastic; D051017:Apoptosis Regulatory Proteins; D014408:Biomarkers, Tumor; D002352:Carrier Proteins; D004273:DNA, Neoplasm; D006360:Heat-Shock Proteins; D037181:Lectins, C-Type; D000073718:Pancreatitis-Associated Proteins; C000612141:REG3A protein, human; D012333:RNA, Messenger; C429767:TP53INP1 protein, human; D003841:Deoxycytidine; C000605506:GSK3B protein, human; D000071679:Glycogen Synthase Kinase 3 beta; D038362:Glycogen Synthase Kinase 3; D000093542:Gemcitabine