Upregulation of BNIP3 by 5-aza-2'-deoxycytidine sensitizes pancreatic cancer cells to hypoxia-mediated cell death.
BACKGROUND
Pancreatic cancer cells often show resistance to hypoxia-mediated apoptosis, but the molecular mechanism underlying that resistance remains unknown. The purpose of the present study, therefore, was to examine the role of epigenetic gene alteration in the resistance to hypoxia-mediated apoptosis among pancreatic cancer cells.


METHODS
Reverse transcription-polymerase chain reaction (RT-PCR) was used to examine the expression of five genes associated with hypoxia-mediated apoptosis (PUMA, Caspase-8 [CASP8], APAF-1, BNIP3, and BNIP3L) in a panel of pancreatic cancer cell lines. Protein expression was examined by Western blot analysis, using lysates from cells incubated under normoxic or hypoxic conditions. The methylation status of the genes was determined using bisulfite-PCR and sequencing. The percentages of cells that were apoptotic were determined using flow cytometry.


RESULTS
Under normoxic conditions, the expression of the BNIP3 gene varied among the 12 pancreatic cancer cell lines tested, with 50% of them showing no BNIP3 expression at all, whereas expression of the other four genes was readily detected in all 12 cell lines. DNA methylation of BNIP3's CpG island in the region around the transcription start site of the gene was closely associated with its silencing. The expression of BNIP3 was restored by the methyltransferase inhibitor 5-aza-deoxycytidine (5-aza-dC), as was the hypoxia-mediated pancreatic cancer cell death.


CONCLUSIONS
BNIP3 expression is silenced in some pancreatic cancer cells by the methylation of its CpG island. Demethylation of BNIP3, using a methyltransferase inhibitor, restores the gene's expression and induces hypoxia-mediated cell death. BNIP3 may thus be a useful target for new therapies aimed at treating pancreatic cancer.
Abe
Tamaki
T
First Department of Internal Medicine, Sapporo Medical University, South 1, West 16, Chuo-ku, Sapporo, 060-8543, Japan.
Toyota
Minoru
M
Suzuki
Hiromu
H
Murai
Masafumi
M
Akino
Kimishige
K
Ueno
Masako
M
Nojima
Masanori
M
Yawata
Atsushi
A
Miyakawa
Hiroyuki
H
Suga
Toshihiro
T
Ito
Hideto
H
Endo
Takao
T
Tokino
Takashi
T
Hinoda
Yuji
Y
Imai
Kohzoh
K
D001483:Base Sequence; D015153:Blotting, Western; D016923:Cell Death / Q000187:drug effects*; D045744:Cell Line, Tumor / Q000187:drug effects / Q000378:metabolism; D003841:Deoxycytidine / Q000494:pharmacology*; D005434:Flow Cytometry; D005786:Gene Expression Regulation; D006801:Humans; D000860:Hypoxia; D008565:Membrane Proteins / Q000187:drug effects* / Q000235:genetics* / Q000378:metabolism; D008745:Methylation / Q000187:drug effects; D008969:Molecular Sequence Data; D010190:Pancreatic Neoplasms; D011518:Proto-Oncogene Proteins / Q000187:drug effects* / Q000235:genetics* / Q000378:metabolism; D020133:Reverse Transcriptase Polymerase Chain Reaction; D012680:Sensitivity and Specificity; D013447:Sulfites / Q000494:pharmacology*; D015854:Up-Regulation
C090121:BNIP3 protein, human; D008565:Membrane Proteins; D011518:Proto-Oncogene Proteins; D013447:Sulfites; D003841:Deoxycytidine; C042345:hydrogen sulfite