Inhibition of the activation of heat shock factor in vivo and in vitro by flavonoids.

Abstract

Transcriptional activation of human heat shock protein (HSP) genes by heat shock or other stresses is regulated by the activation of a heat shock factor (HSF). Activated HSF posttranslationally acquires DNA-binding ability. We previously reported that quercetin and some other flavonoids inhibited the induction of HSPs in HeLa and COLO 320DM cells, derived from a human colon cancer, at the level of mRNA accumulation. In this study, we examined the effects of quercetin on the induction of HSP70 promoterregulated chloramphenicol acetyltransferase (CAT) activity and on the binding of HSF to the heat shock element (HSE) by a gel mobility shift assay with extracts of COLO 320DM cells. Quercetin inhibited heat-induced CAT activity in COS-7 and COLO 320DM cells which were transfected with plasmids bearing the CAT gene under the control of the promoter region of the human HSP70 gene. Treatment with quercetin inhibited the binding of HSF to the HSE in whole-cell extracts activated in vivo by heat shock and in cytoplasmic extracts activated in vitro by elevated temperature or by urea. The binding of HSF activated in vitro by Nonidet P-40 was not suppressed by the addition of quercetin. The formation of the HSF-HSE complex was not inhibited when quercetin was added only during the binding reaction of HSF to the HSE after in vitro heat activation. Quercetin thus interacts with HSF and inhibits the induction of HSPs after heat shock through inhibition of HSF activation.Physiologic stress, including heat shock, enhances the synthesis of a limited number of intracellular proteins, the so-called heat shock proteins (HSPs) (19). The heat shock response has been observed in all cells so far tested, and some of the HSPs have been well conserved throughout evolution. In higher organisms, the induction of HSPs by heat shock or other stresses is regulated at the transcriptional and translational levels. The transcription of heat shock genes is regulated by the cis-acting heat shock element (HSE) in the promoter region and the trans-acting heat shock factor (HSF). The HSE consensus sequence was defined as the repeat of a 5-bp unit, NGAAN or NTTCN (2,28), where N is any nucleotide, and the molecular cloning of HSF from yeast and Drosophila cells has been reported (5,36,38). In Saccharomyces cerevisiae, HSF is already bound to the HSE under normal conditions, and transcriptional activation is induced after heat shock at least partly through the phosphorylation of HSF, whereas in Drosophila and mammalian cells, HSF acquires DNA-binding ability only after heat shock through posttranslational modification of HSF (18, 34).We have reported that quercetin and several other flavonoids inhibit the synthesis of HSPs, including HSP110, HSP90, HSP70, HSP47, HSP40, and HSP28, induced by heat shock, azetidine, or sodium arzenite treatment in two human cancer cell lines, HeLa and COLO 320DM cells (12). Quercetin inhibited the induction of HSP70 at the level of mRNA accumulation (12).Flavonoids are a group of dyes commonly contained in higher plants (...

Suppression of multidrug resistance via inhibition of heat shock factor by quercetin in MDR cells

“…Therefore, it is worthwhile to examine whether MDR phenotype could be restored by inhibition of a constitutively active HSF found in MDR cells. In order to answer this question, MDR cells were treated with quercetin, which is known to inhibit heat shock protein synthesis after heat shock in a human colon carcinoma cell line and Hela cells (Hosokawa et al, 1992) and also interfere with the formation of the complex between the HSE and HSF and downregulate the level of HSF1 (Nagai et al, 1995). In this study, quercetin appeared to inhibit dose-dependently the constitutive HSF DNA-binding activity and the sodium arsenite-induced HSF DNA-binding activity in the MDR cells.…”

“…Since FM3A/M and P388/M MDR cells showed a constitutive HSF DNA-binding activity under the non-stressed condition (Kim et al, 1997) and quercetin has been shown to interfere with the formation of the complex between the HSE and HSF, and to downregulate the level of HSF1 (Hosokawa et al, 1992;Nagai et al, 1995). It was examined whether the constitutive HSF activity in MDR cells could be downregulated by quercetin.…”

“…Therefore, modulation of HSF activity might be a useful target for overcoming MDR. Quercetin (3,3',4',5,, which is one of the most widely distributed flavonoids in nature, has been shown to interfere with the formation of the complex between the c i s-acting HSEs in the promoter region of the hsp70 gene and HSF, and to downregulate the level of HSF1 (Hosokawa et al ., 1992;Nagai et al ., 1995). Thus, quercetin is a good candidate molecule for downregulation of constitutive HSF DNA binding activity and thereby reversing the MDR phenotype in MDR cells.…”

Effects of heat stimulation via microwave applicator on cartilage matrix gene and HSP70 expression in the rabbit knee joint

“…15,16 The present study investigated the effects of quercetin on the acceleration of matrix Figure 4. The time course changes of HSP70 and the expression of extracellular matrix genes in articular cartilage after microwave (MW) irradiation.…”

Induction of 72‐kDa Inducible Heat Shock Protein (HSP72) in Cultured Rat Astrocytes After Energy Depletion

“…In brief, 108 astrocytes were exposed to hypoxia (24 h) and reoxygenated (6 h) in either the presence or the absence of quercetin (100 suM) (Hosokawa et al, 1992), followed by labeling with [3H] leucine (100~.rCi/ml) for 30 mm. Cells were homogenized in PBS containing NP-40 (0.5%), EDTA (1 mM), and cycloheximide (100~ug/ ml) using a Dounce homogenizer.…”