Inhibitors of heat-induced heat shock protein 70 (HSP70)a expression have the potential to enhance the therapeutic effectiveness of heat induced radiosensitization of tumors. Among known small molecule inhibitors, quercetin has the advantage of being easily modified for structure-activity studies. Herein, we report the ability of five mono-methyl and five carbomethoxymethyl derivatives of quercetin to inhibit heat-induced HSP70 expression and enhance HSP27 phosphorylation in human cells. While quercetin and several derivatives inhibit HSP70 induction and enhance HSP27 phosphorylation at Ser78, other analogs selectively inhibit HSP70 induction without enhancing HSP27 phosphorylation that would otherwise aid in cell survival. We also show that good inhibitors of HSP70 induction are also good inhibitors of both CK2 and CamKII, kinases that are known to activate HSP70 expression by phosphorylation of heat shock transcription factor 1. Derivatives that show poor inhibition of either or both kinases are not good inhibitors of HSP70 induction, suggesting that quercetin's effectiveness is due to its ability to inhibit both kinases.

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Cell penetrating peptides (CPPs) have been used to deliver the anti-apoptotic Bcl-xL-derived BH4 peptide to prevent injury-induced apoptosis both in vitro and in vivo. Here we demonstrate that the nuclear localization sequence (NLS) from the SV40 large T antigen has favorable properties for BH4 domain delivery to lymphocytes compared to sequences based on the HIV-1 TAT sequence. While both TAT-BH4 and NLS-BH4 protected primary human mononuclear cells from radiation-induced apoptotic cell death, TAT-BH4 caused persistent membrane damage and even cell death at the highest concentrations tested (5-10 μM) and correlated with in vivo toxicity as intravenous administration of TAT-BH4 caused rapid death. The NLS-BH4 peptide has significantly attenuated toxicity compared to TAT-BH4 and we established a dosing regimen of NLS-BH4 that conferred a significant survival advantage in a post-exposure treatment model of LD90 total body irradiation.

Aging is associated with an adverse decline in muscle function, often manifesting as decreased strength and increased muscle fatigability that negatively affects the overall health of the elderly. Heat shock proteins (HSPs), a family of stress inducible proteins known to protect cells from damage, are highly induced in muscle cells following exercise, but both basal and inducible levels decline with age. Utilizing young and old mice lacking HSP25 (Hsp25−/−) we tested the hypothesis that HSP25 is required to maintain normal muscle function and that age related decreases in HSP25 directly contribute to declining muscle function. Running wheel distances over 14 days for young Hsp25−/− mice were significantly lower than for the corresponding Hsp25+/+ genotype (81238 vs. 33956 AUC, respectively). While older groups both ran significantly less than young groups, in aged mice HSP25 loss did not lead to any additional decrease. Significantly lower myofibrillar (contractile) protein levels in young Hsp25−/− vs. Hsp25+/+ (15.7 ± 0.2 vs. 13.4 ± 0.3 mg/mg muscle) mice suggests HSP25 loss was associated with greater muscle breakdown during voluntary wheel running. In vivo, plantarflexor maximal isometric force was significantly decreased in aged vs. young mice, but the loss of HSP25 had no effect on either group. However, plantarflexor fatigability over 10 contractions was significantly higher in young Hsp25−/− vs. Hsp25+/+ mice (59 ± 3 vs. 49 ± 4% of initial force, respectively) but no similar effect of genotype was detected in the older groups. There was no difference in muscle caspase-3 activity between Hsp25−/− and Hsp25+/+ mice, whether young or old, but there was a significant genotype independent increase in activity with age. Overall, the results suggest that the absence of HSP25 primarily contributes to muscle fatigue resistance, rather than maximal force production, and that this effect is most evident in young compared to older mice.

In vitro studies have implicated the small heat shock protein HSPB1 in a range of physiological functions. However, its in vivo relevance is unclear as the phenotype of unstressed HSPB1−/− mice is unremarkable. To determine the impact of HSPB1 in injury, HSPB1−/− and wild type (WT) mice were subjected to cecal ligation and puncture, a model of polymicrobial sepsis. Ten-day mortality was significantly higher in HSPB1−/− mice following the onset of sepsis (65% vs. 35%). Ex vivo mechanical testing revealed that common carotid arteries from HSPB1−/− mice were more compliant than those in WT mice over pressures of 50–120 mm Hg. Septic HSPB1−/− mice also had increased peritoneal levels of IFN-γ and decreased systemic levels of IL-6 and KC. There were no differences in frequency of either splenic CD4+ or CD8+ T cells, nor were there differences in apoptosis in either cell type. However, splenic CD4+ T cells and CD8+ T cells from HSPB1−/− mice produced significantly less TNF and IL-2 following ex vivo stimulation. Systemic and local bacterial burden was similar in HSPB1−/− and WT mice. Thus while HSPB1−/− mice are uncompromised under basal conditions, HSPB1 has a critical function in vivo in sepsis, potentially mediated through alterations in arterial compliance and the immune response.