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Heat shock protein 70 promotes cell survival by inhibiting lysosomal membrane permeabilization.

ABSTRACT: Heat shock protein 70 (Hsp70) is a potent survival protein whose depletion triggers massive caspase-independent tumor cell death. Here, we show that Hsp70 exerts its prosurvival function by inhibiting lysosomal membrane permeabilization. The cell death induced by Hsp70 depletion was preceded by the release of lysosomal enzymes into the cytosol and inhibited by pharmacological inhibitors of lysosomal cysteine proteases. Accordingly, the Hsp70-mediated protection against various death stimuli in Hsp70-expressing human tumor cells as well as in immortalized Hsp70 transgenic murine fibroblasts occurred at the level of the lysosomal permeabilization. On the contrary, Hsp70 failed to inhibit the cytochrome c-induced, apoptosome-dependent caspase activation in vitro and Fas ligand-induced, caspase-dependent apoptosis in immortalized fibroblasts. Immunoelectron microscopy revealed that endosomal and lysosomal membranes of tumor cells contained Hsp70. Permeabilization of purified endo/lysosomes by digitonin failed to release Hsp70, suggesting that it is physically associated with the membranes. Finally, Hsp70 positive lysosomes displayed increased size and resistance against chemical and physical membrane destabilization. These data identify Hsp70 as the first survival protein that functions by inhibiting the death-associated permeabilization of lysosomes.

SUBMITTER: Nylandsted J

PROVIDER: S-EPMC2211935 | biostudies-literature | 2004 Aug

REPOSITORIES: biostudies-literature

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Heat shock protein 70 promotes cell survival by inhibiting lysosomal membrane permeabilization.

Nylandsted Jesper J Gyrd-Hansen Mads M Danielewicz Agnieszka A Fehrenbacher Nicole N Lademann Ulrik U Høyer-Hansen Maria M Weber Ekkehard E Multhoff Gabriele G Rohde Mikkel M Jäättelä Marja M

The Journal of experimental medicine 20040801 4

Heat shock protein 70 (Hsp70) is a potent survival protein whose depletion triggers massive caspase-independent tumor cell death. Here, we show that Hsp70 exerts its prosurvival function by inhibiting lysosomal membrane permeabilization. The cell death induced by Hsp70 depletion was preceded by the release of lysosomal enzymes into the cytosol and inhibited by pharmacological inhibitors of lysosomal cysteine proteases. Accordingly, the Hsp70-mediated protection against various death stimuli in H ...[more]

PMID: 15314073

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Project description:Brain metastasis is a major cause of mortality in melanoma patients. The blood-brain barrier (BBB) prevents most anti-tumor compounds from entering the brain, which significantly limits their use in the treatment of brain metastasis. One strategy in the development of new treatments is to assess the anti-tumor potential of drugs currently used in the clinic. Here, we tested the anti-tumor effect of the BBB-penetrating antipsychotic trifluoperazine (TFP) on metastatic melanoma. H1 and Melmet1 human metastatic melanoma cell lines were used in vitro and in vivo. TFP effects on viability and toxicity were evaluated in proliferation and colony formation assays. Preclinical, therapeutic efficacy was evaluated in NOD/SCID mice, after intracardial injection of tumor cells. Molecular studies using immunohistochemistry, western blots, immunofluorescence and transmission electron microscopy were used to gain mechanistic insight into the biological activity of TFP. Our results showed that TFP decreased cell viability and proliferation, colony formation and spheroid growth in vitro. The drug also decreased tumor burden in mouse brains and prolonged animal survival after injection of tumor cells (53.0 days vs 44.5 days), TFP treated vs untreated animals, respectively (P < 0.01). At the molecular level, TFP treatment led to increased levels of LC3B and p62 in vitro and in vivo, suggesting an inhibition of autophagic flux. A decrease in LysoTracker Red uptake after treatment indicated impaired acidification of lysosomes. TFP caused accumulation of electron dense vesicles, an indication of damaged lysosomes, and reduced the expression of cathepsin B, a main lysosomal protease. Acridine orange and galectin-3 immunofluorescence staining were evidence of TFP induction of lysosomal membrane permeabilization. Finally, TFP was cytotoxic to melanoma brain metastases based on the increased release of lactate dehydrogenase into media. Through knockdown experiments, the processes of TFP-induced lysosomal membrane permeabilization and cell death appeared to be STAT3 dependent. In conclusion, our work provides a strong rationale for further clinical investigation of TFP as an adjuvant therapy for melanoma patients with metastases to the brain.

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Heat shock protein 70 promotes cell survival by inhibiting lysosomal membrane permeabilization.

Publications

Heat shock protein 70 promotes cell survival by inhibiting lysosomal membrane permeabilization.

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