Enhanced oxaliplatin-induced apoptosis following antisense Bcl-xl down-regulation is p53 and Bax dependent: Genetic evidence for specificity of the antisense effect.
BACKGROUND
Oxaliplatin, licensed for colorectal cancer chemotherapy, damages DNA by generating intrastrand and interstrand cross-links and can induce apoptosis via a Bax-dependent pathway. Bcl-xl, an antiapoptotic Bcl-2 family member, regulates apoptosis and chemoresistance in several cancer models. Bcl-xl expression correlates with invasiveness in primary colorectal cancer. Bcl-xl may therefore represent a therapeutic target in this disease. We used the mismatch repair-deficient HCT116 colorectal cancer cell line (wild-type HCT116) and p53 null, Bax null, or p21/WAF1 null derivatives to identify genetic determinants of the response to oxaliplatin and tested the hypothesis that antisense-mediated Bcl-xl down-regulation would enhance the apoptotic response in a p53- or Bax-dependent manner.


RESULTS
At clinically relevant concentrations, oxaliplatin induced p53 and p53-dependent Bax, Bcl-xl, and p21/WAF1 protein accumulation. A minor degree of apoptosis resulted via a p53- and Bax-dependent pathway. The major response was a transient mixed G(1) and G(2) growth arrest. The G(1) arrest was p53 and p21/WAF1 dependent. A 2'-O-ribose methoxyethyl phosphorothioate antisense oligonucleotide reduced Bcl-xl protein expression by approximately 90% in HCT116 (Bcl-xl knockdown). Missense controls were inactive. Prior Bcl-xl knockdown enhanced the apoptotic and the global cytotoxic effect of oxaliplatin. The extent of enhancement of apoptosis depended on the integrity of the p53- and Bax-mediated apoptotic pathway, providing genetic evidence that the desired proapoptotic antisense effect is due to specific down-regulation of the Bcl-xl target.


CONCLUSIONS
The combination of oxaliplatin and Bcl-xl antisense merits testing in models of colorectal cancer in vivo.
Edinburgh Oncology Unit, Cancer Research UK, Edinburgh, United Kingdom. r.hayward@cancer.org.uk
Richard L
RL
Hayward
Janet S
JS
Macpherson
Jeff
J
Cummings
Brett P
BP
Monia
John F
JF
Smyth
Duncan I
DI
Jodrell
D017209:Apoptosis / Q000187:drug effects*; D002470:Cell Survival; D015179:Colorectal Neoplasms / Q000235:genetics / Q000473:pathology; D050759:Cyclin-Dependent Kinase Inhibitor p21; D016213:Cyclins / Q000378:metabolism; D016373:DNA, Antisense / Q000235:genetics / Q000378:metabolism; D015536:Down-Regulation; D005838:Genotype; D045325:HCT116 Cells; D006801:Humans; D016376:Oligonucleotides, Antisense / Q000235:genetics / Q000378:metabolism*; D009944:Organoplatinum Compounds / Q000494:pharmacology*; D000077150:Oxaliplatin; D019253:Proto-Oncogene Proteins c-bcl-2 / Q000235:genetics / Q000378:metabolism*; D013379:Substrate Specificity; D016159:Tumor Suppressor Protein p53 / Q000378:metabolism*; D051028:bcl-2-Associated X Protein; D051020:bcl-X Protein
C494826:BAX protein, human; C494811:BCL2L1 protein, human; C493956:CDKN1A protein, human; D050759:Cyclin-Dependent Kinase Inhibitor p21; D016213:Cyclins; D016373:DNA, Antisense; D016376:Oligonucleotides, Antisense; D009944:Organoplatinum Compounds; D019253:Proto-Oncogene Proteins c-bcl-2; D016159:Tumor Suppressor Protein p53; D051028:bcl-2-Associated X Protein; D051020:bcl-X Protein; D000077150:Oxaliplatin