Differential up-regulation of cytosolic and membrane-bound heat shock protein 70 in tumor cells by anti-inflammatory drugs.
OBJECTIVE
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-gamma agonists were analyzed for their capacity to affect Hsp70 expression in human cancer cells with a divergent Hsp70 membrane expression pattern.


METHODS
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 (51)Cr 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.
Gehrmann
Mathias
M
Department of Hematology and Oncology, University Hospital Regensburg, Regensburg, Germany.
Brunner
Marion
M
Pfister
Karin
K
Reichle
Albrecht
A
Kremmer
Elisabeth
E
Multhoff
Gabriele
G
D000893:Anti-Inflammatory Agents / Q000494:pharmacology*; D000894:Anti-Inflammatory Agents, Non-Steroidal / Q000494:pharmacology; D000970:Antineoplastic Agents / Q000494:pharmacology; D017209:Apoptosis; D001241:Aspirin / Q000494:pharmacology; D000068579:Celecoxib; D045744:Cell Line, Tumor; D002462:Cell Membrane / Q000378:metabolism*; D002470:Cell Survival; D003593:Cytoplasm / Q000378:metabolism; D003600:Cytosol / Q000378:metabolism*; D004305:Dose-Response Relationship, Drug; D004591:Electrophoresis, Polyacrylamide Gel; D004791:Enzyme Inhibitors / Q000494:pharmacology; D005434:Flow Cytometry; D016650:Fluorescein-5-isothiocyanate / Q000494:pharmacology; D053804:Granzymes; D018840:HSP70 Heat-Shock Proteins / Q000378:metabolism*; D006801:Humans; D015151:Immunoblotting; D007167:Immunotherapy / Q000379:methods; D007328:Insulin / Q000378:metabolism; D007700:Kinetics; D007783:Lactones / Q000494:pharmacology; D008956:Models, Chemical; D009369:Neoplasms / Q000378:metabolism; D000077205:Pioglitazone; D011720:Pyrazoles; D012697:Serine Endopeptidases / Q000494:pharmacology; D013449:Sulfonamides / Q000494:pharmacology; D013450:Sulfones; D045162:Thiazolidinediones / Q000494:pharmacology; D015854:Up-Regulation
D000893:Anti-Inflammatory Agents; D000894:Anti-Inflammatory Agents, Non-Steroidal; D000970:Antineoplastic Agents; D004791:Enzyme Inhibitors; D018840:HSP70 Heat-Shock Proteins; D007328:Insulin; D007783:Lactones; D011720:Pyrazoles; D013449:Sulfonamides; D013450:Sulfones; D045162:Thiazolidinediones; C116926:rofecoxib; C509213:GZMB protein, human; D053804:Granzymes; D012697:Serine Endopeptidases; D016650:Fluorescein-5-isothiocyanate; D000068579:Celecoxib; D001241:Aspirin; D000077205:Pioglitazone