Objective: The inducible form of heat shock protein 70 is known to be overexpressed in tumors and seems to be necessary for the survival of tumor cells via an unknown mechanism. We therefore evaluated whether selective depletion of heat shock protein 70 induces cell death in lung cancer cells.
Methods: An adenovirus expressing antisense heat shock protein 70 and an adenovirus with beta-galactosidase were used for transduction of the lung cancer cell lines A549, NCI-H358, LXF-289, LOU-NH91, normal human bronchial epithelial cells, and normal lung fibroblasts IMR90. Cell death was determined by morphology, propidium iodide uptake, and trypan blue staining; DNA cleavage was assessed by terminal deoxynucleotidyl transferase-mediated dUTP nick end labeling. Expression of heat shock protein 70, heat shock cognate 70, and phosphorylated p53 was determined by Western blot analysis.
Results: Transduction of lung cancer cells with adenovirus expressing antisense heat shock protein 70 but not with adenovirus with beta-galactosidase resulted in extensive cell death after 96 hours (A549: 53.2 +/- 9.44% versus 12.9 +/- 6.6%; NCI-H358: 48.4 +/- 7.2% versus 25.2 +/- 1.4%; LXF-289: 58.8 +/- 6.5% versus 24.7 +/- 5.4%; LOU-NH91: 82.5 +/- 1.8% versus 38.5 +/- 2.6%). In contrast, adenovirus expressing antisense heat shock protein 70 showed much less cytotoxicity in normal human bronchial epithelial cells (16.0 +/- 0.5% versus 17.1 +/- 7.3%) and in normal lung fibroblasts IMR90 (17.2 +/- 3.6% versus 8.2 +/- 1.6%). After treatment with adenovirus expressing antisense heat shock protein 70, transactivation of p53 in A549 but not in NCI-H358, a cell line deleted for p53 has been observed. Furthermore, 22.0 +/- 3.0% of A549 cells treated for adenovirus expressing antisense heat shock protein 70 stained positive with terminal deoxynucleotidyl transferase-mediated dUTP nick end labeling versus 10.2 +/- 4.0% treated with control virus. This effect but not cell death itself was blocked by treatment with 10 micromol/L zVAD-fmk, a broad caspase inhibitor.
Conclusions: Selective down-regulation of heat shock protein 70 induces cell death in lung cancer but not in normal lung cells. The demonstrated effect is p53-independent and does not require DNA cleavage. The data suggest that gene transfer of antisense heat shock protein 70 might be useful in developing new strategies for the treatment of lung cancer.