Differential Up-Regulation of Cytosolic and Membrane-Bound Heat Shock Protein 70 in Tumor Cells by Anti-Inflammatory Drugs

Abstract

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-␥ 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 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 cellmediated 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.

Lysosomal Rerouting of Hsp70 Trafficking as a Potential Immune Activating Tool for Targeting Melanoma

“…Moreover, facilitation of surface localization or release of Hsp70 has been shown to correlate with increased immunogenicity of several tumor cell types tested in mice or at in vitro conditions [19,[47][48][49][50] These observations together with our findings indicate that Hsp70 overexpression in B16 cells could generate an excellent immune activating milieu through an elevated level of surface and released Hsp70. Furthermore, we showed that B16 melanoma cells chronically expressing excess Hsp70 lost their ability to release Hsp70 through active transport mechanisms.…”

Lysosomal Rerouting of Hsp70 Trafficking as a Potential Immune Activating Tool for Targeting Melanoma

“…Moreover, facilitation of surface localization or release of Hsp70 has been shown to correlate with increased immunogenicity of several tumor cell types tested in mice or at in vitro conditions [19,[47][48][49][50] These observations together with our findings indicate that Hsp70 overexpression in B16 cells could generate an excellent immune activating milieu through an elevated level of surface and released Hsp70. Furthermore, we showed that B16 melanoma cells chronically expressing excess Hsp70 lost their ability to release Hsp70 through active transport mechanisms.…”

Impact of diabetes on alpha-crystallins and other heat shock proteins in the eye

“…Conditions that trigger their expression are as diverse as oxidative stress, heavy metals, amino acid manipulations or nutrient deprivation [7], UV and gamma irradiation [8], cytostatic and antiinflammatory drugs [8][9][10][11], bacterial and viral infections, and malignant transformation [12]. In line with this notion, the upregulation of heat shock proteins is also very present in the pathologies of many diseases including neurodegenerative and ocular disorders.…”