Effects of gemcitabine on APE/ref-1 endonuclease activity in pancreatic cancer cells, and the therapeutic potential of antisense oligonucleotides.
Apurinic/apyrimidinic endonuclease (APE) is a key enzyme involved in DNA base excision repair (BER) that is often expressed at elevated levels in human cancers. Pancreatic cancer cells treated with the nucleoside analogue gemcitabine (2', 2'-difluoro-2'deoxycytidine) showed increases in APE/redox effector factor (ref-1) protein levels (approximately two-fold for Panc-1 and six-fold for MiaPaCa-2), with corresponding increases in endonuclease activity. These results suggested that the activation of APE/ref-1 might be an adaptive response that contributes to gemcitabine resistance by facilitating BER. To test this hypothesis, we examined the effects of disrupting APE/ref-1 using antisense on gemcitabine toxicity. Antisense oligonucleotides decreased protein levels three-fold in MiaPaCa-2 and five-fold in Panc-1 in comparison to controls, associated with reduced endonuclease activity. Combination treatments with antisense oligonucleotides and gemcitabine partially suppressed the increase in APE/ref-1 activity seen in cells exposed to gemcitabine alone. While clonogenic assays showed only slight decreases in colony formation in cells treated with either antisense oligonucleotides or gemcitabine alone, the combination with APE/ref-1 antisense resulted in a 2-log enhancement of gemcitabine toxicity in Panc-1 cells. Overall these findings suggest that APE/ref-1 plays a significant role in gemcitabine resistance in some pancreatic cancer cells, and support the further investigation of novel treatments that target this protein.
Division of Experimental Therapeutics, Ontario Cancer Institute/Princess Margaret Hospital, 610 University Avenue, Toronto, Ontario, Canada M5G 2M9.
J P
JP
Lau
K L
KL
Weatherdon
V
V
Skalski
D W
DW
Hedley
D000964:Antimetabolites, Antineoplastic / Q000633:toxicity*; D001692:Biological Transport; D002455:Cell Division / Q000187:drug effects; D045744:Cell Line, Tumor; D002470:Cell Survival / Q000187:drug effects; D043603:DNA-(Apurinic or Apyrimidinic Site) Lyase / Q000187:drug effects / Q000302:isolation & purification / Q000378:metabolism*; D003841:Deoxycytidine / Q000031:analogs & derivatives* / Q000633:toxicity*; D019008:Drug Resistance, Neoplasm; D005434:Flow Cytometry; D006801:Humans; D007700:Kinetics; D016376:Oligonucleotides, Antisense / Q000493:pharmacokinetics / Q000494:pharmacology*; D010190:Pancreatic Neoplasms; D014162:Transfection; D000093542:Gemcitabine
D000964:Antimetabolites, Antineoplastic; D016376:Oligonucleotides, Antisense; D003841:Deoxycytidine; C085108:APEX1 protein, human; D043603:DNA-(Apurinic or Apyrimidinic Site) Lyase; D000093542:Gemcitabine