Design of a Flexible Cell-Based Assay for the Evaluation of Heat Shock Protein 70 Expression Modulators

Abstract

Heat shock protein 70 (Hsp70) is a chaperone protein that helps protect against cellular stress, a function that may be co-opted to fight human diseases. In particular, the upregulation of Hsp70 can suppress the neurotoxicity of misfolded proteins, suggesting possible therapeutic strategies in neurodegenerative diseases. Alternatively, in cancer cells where high levels of Hsp70 inhibit both intrinsic and extrinsic apoptotic pathways, a reduction in Hsp70 levels may induce apoptosis. To evaluate and identify, in a single assay format, small molecules that induce or inhibit endogenous Hsp70, we have designed and optimized a microtiter assay that relies on whole-cell immunodetection of Hsp70. The assay utilizes a minimal number of neuronal or cancer cells, yet is sufficiently sensitive and reproducible to permit quantitative determinations. We further validated the assay using a panel of Hsp70 modulators. In conclusion, we have developed an assay that is fast, robust, and cost efficient. As such, it can be implemented in most research laboratories. The assay should greatly improve the speed at which novel Hsp70 inducers and inhibitors of expression can be identified and evaluated.

Using the SNAP-Tag technology to easily measure and demonstrate apoptotic changes in cancer and blood cells with different dyes

“…Camptothecin is a well-characterized apoptosis inducing agent and often used as positive control [ 43 , 44 ]. Furthermore, heat shock treatment is a widely-used control method since eukaryotic cells form misfolded or aggregated proteins in the presence of thermal insults, thus inducing apoptosis [ 45 , 46 ].…”

Combined inhibition of heat shock proteins 90 and 70 leads to simultaneous degradation of the oncogenic signaling proteins involved in muscle invasive bladder cancer

“…Increased expression of HSP70 is a feature of N-terminal ATPase HSP90 inhibitors and is regarded as a biomarker of drug activity [ 24 ]. Some have suggested that this HSP70 overexpression may allow cells to overcome the effects of N-terminal ATPase HSP90 inhibitor drugs when used as monotherapy in clinical trials [ 25 ].…”