Kinase-dead PKB gene therapy combined with hyperthermia for human breast cancer

Abstract

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.b-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.b-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.

Tumor Cell Kill by c-MYC Depletion: Role of MYC-Regulated Genes that Control DNA Double-Strand Break Repair

“…DU145 and PC-3 prostate cancer cells and RKO colon carcinoma cells were obtained from American Type Culture Collection (2001,2001, and 2007, respectively) and cultured as previously described (17,18 , respectively, and were cultured as described previously (17)(18)(19)(20)(21)(22). MCF10A cells were a kind gift from Dr. Senthil Muthuswamy (2003; Ontario Cancer Institute/Princess Margaret Hospital, Toronto, Ontario, Canada) and were cultured as described previously either in full growth medium (23) or in growth factor withdrawal (0.05% horse serum, 10 μg/mL insulin) to downregulate endogenous MYC.…”

Correlation between the expression of Id-1 and hyperthermia-associated molecules in oral squamous cell carcinoma

“…Recent studies have also shown that inhibition of the PI3K/AKT pathway can significantly increase the sensitivity of hyperthermia 14 15…”

The Effect of Estradiol on in Vivo Tumorigenesis Is Modulated by the Human Epidermal Growth Factor Receptor 2/Phosphatidylinositol 3-Kinase/Akt1 Pathway

“…Introduction of an Akt-kinase dead mutant directly into tumor cells can also inhibit tumor formation in nude mice (52). However, these effects were not observed in normal cells (53) or the MCF10 normal counterpart (52). Thus, Akt inhibition may not be toxic to normal cells.…”

“…They showed that the Akt inhibitor geldamycin and its clinically relevant derivative, 17-allyl amino-17-demethoxygeldamicin, led to inhibition of MCF-7 and tamoxifen-resistant MCF-7 tumor xenografts in severe combined immunodeficient mice. Introduction of an Akt-kinase dead mutant directly into tumor cells can also inhibit tumor formation in nude mice (52). However, these effects were not observed in normal cells (53) or the MCF10 normal counterpart (52).…”