Kinase-dead PKB gene therapy combined with hyperthermia for human breast cancer.
We have previously demonstrated that protein kinase B (PKB) is a mediator of heat-induced apoptosis for human breast cancer cells. To investigate the therapeutic potential of abrogating the function of this important survival protein, a novel replication-deficient adenovirus was constructed, wherein a mutant, kinase-inactive PKB gene (AAA) was inserted downstream of the CMV promoter. Two human breast cancer cell lines, MCF-7 and MDA-468, were treated, along with the MCF-10 serving as a "normal" mammary epithelial comparator. Apoptosis was increased with adv.AAA (25 PFU/cell) infection alone, but was significantly enhanced with the addition of heat exposure. Differential survival was observed with the MDA-468 cancer cells being more sensitive than the MCF-7 cells. The MCF-10 cells, in contrast, were most resistant to these treatments. Results from the clonogenic assay reflected the apoptosis data, with an apparent additive interaction between adv.AAA and hyperthermia treatments, again with greater differential sensitivity of the malignant, compared to the "normal" mammary epithelial cells. Heat or adv.beta-gal treatments led to phosphorylation of PKB and Forkhead, but this phosphorylation was reduced with adv.AAA therapy. In parallel, the combination of adv.AAA and heat treatment reduced PKB kinase activity more so than with either heat or adv.beta-gal alone. In conclusion, our results demonstrate that inhibition of the PKB-dependent survival pathway will promote apoptosis and thermosensitization in malignant breast cancer cells, with relative sparing of their normal counterpart, suggesting that a therapeutic gain could be achievable using this therapeutic strategy.
Ma
Nancy
N
Division of Experimental Therapeutics, Ontario Cancer Institute, University Health Network, Toronto, Ontario, Canada.
Szmitko
Paul
P
Brade
Anthony
A
Chu
Isabel
I
Lo
Alex
A
Woodgett
Jim
J
Klamut
Henry
H
Liu
Fei-Fei
FF
D000256:Adenoviridae / Q000235:genetics / Q000502:physiology; D017209:Apoptosis; D001943:Breast Neoplasms / Q000235:genetics* / Q000378:metabolism / Q000473:pathology / Q000628:therapy*; D045744:Cell Line, Tumor; D015316:Genetic Therapy / Q000379:methods*; D005822:Genetic Vectors / Q000235:genetics; D038362:Glycogen Synthase Kinase 3 / Q000378:metabolism; D006358:Hot Temperature; D006801:Humans; D006979:Hyperthermia, Induced; D009154:Mutation / Q000235:genetics; D010766:Phosphorylation; D017346:Protein Serine-Threonine Kinases / Q000009:adverse effects / Q000235:genetics* / Q000378:metabolism*; D011518:Proto-Oncogene Proteins / Q000009:adverse effects / Q000235:genetics* / Q000378:metabolism*; D051057:Proto-Oncogene Proteins c-akt
D011518:Proto-Oncogene Proteins; D017346:Protein Serine-Threonine Kinases; D051057:Proto-Oncogene Proteins c-akt; D038362:Glycogen Synthase Kinase 3