Differential up-regulation of cytosolic and membrane-bound heat shock protein 70 in tumor cells by anti-inflammatory drugs

Purpose: Modulation of the heat shock protein (HSP) response affects sensitivity to therapeutic agents in cancer. Here, drugs with anti-inflammatory potential (cyclooxygenase 1/2 inhibitors) and peroxidase proliferator-activated receptor-γ agonists were analyzed for their capacity to affect Hsp70 expression in human cancer cells with a divergent Hsp70 membrane expression pattern. Experimental Design: In dose kinetics, the nonlethal concentration of acetyl-salicyl acid, celecoxib, rofecoxib, and the insulin-sensitizer pioglitazone was identified for the human adenocarcinoma cell line CX-. With the exception of CLX, which was diluted in DMSO, all reagents were dissolved in water. After treatment with the different compounds at nontoxic concentrations for 6 h, followed by a 1-h recovery period, the cytosolic Hsp70 levels were measured in CX-2 and CX- tumor cells by Western blot analysis. Fold increase was calculated in relation to the housekeeping protein tubulin. Membrane-bound Hsp70 was analyzed by flow cytometry using a FITC-labeled Hsp70-specific monoclonal antibody. Untreated cells and cells incubated with equivalent amounts of the diluting agents served as controls. The immunological function was tested in granzyme B apoptosis assays, standard ⁵¹Cr release assays, and antibody blocking studies. Results: Compared with aqua dest, the cytoplasmic amount of Hsp70 was equally enhanced in CX-2 and CX-cells by all compounds. An increase in membrane-bound Hsp70, detected selectively in CX- cells, corresponded to an enhanced sensitivity to granzyme B- and natural killer cell-mediated kill that was blockable by using a Hsp70-specific antibody. Conclusions: Although increase in cytosolic Hsp70 levels conferred resistance to further stress, membrane-bound Hsp70 rendered tumor cells more sensitive to the immunological attack mediated by granzyme B and natural killer cells. Our data provide a biological rational for combining anti-inflammatory drugs with immunotherapy in cancer therapy.