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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 (...

Keywords: flavonoids/heat shock proteins/human cancer cell lines ABSTRACT. Cells exposed to several forms of stress, such as heat shock, transiently synthesize a group of proteins called heat shock proteins (hsps). Although many stressors other than heat shock are known to induce hsps, inhibitors of hsp expression have never been reported. Here we show that quercetiri and several other flavonoids inhibit the synthesis of hsps induced by heat shock in two human cell lines, Hela cells and COLO320 DMcells. Quercetin inhibited the induction of hsp70 at the level of mRNAaccumulation. This is the first report to describe the inhibition of hsp expression by reagents.Whencells or organisms are exposed to heat shock,

Trans-1-amino-3-18 F-fluorocyclobutanecarboxylic acid (anti-18 F-FACBC) is an amino acid PET tracer that has shown promise for visualizing prostate cancer. Therefore, we aimed to clarify the anti-18 F-FACBC transport mechanism in prostate cancer cells. We also studied the fate of anti-18 F-FACBC after it is transported into cells. Methods: For convenience, because of their longer half-lives, 14 C compounds were used instead of 18 F-labeled tracers. Trans-1-amino-3-fluoro-1-14 C-cyclobutanecarboxylic acid ( 14 C-FACBC) uptake was examined in human prostate cancer DU145 cells with the following substrates of amino acid transporters: a-(methylamino) isobutyric acid (a system A-specific substrate) and 2-amino-2-norbornanecarboxylic acid (a system L-specific substrate). The messenger RNA expression of amino acid transporters in human prostate cancer specimens was analyzed by complementary DNA microarray and quantitative real-time reverse transcription polymerase chain reaction (qRT-PCR). Gene expression in DU145 cells was analyzed by qRT-PCR. We also examined the knockdown effect of the amino acid transporters system ASC transporter 2 (ASCT2) and sodium-coupled neutral amino acid transporter 2 (SNAT2) on 14 C-FACBC uptake. In addition, the possibility of 14 C-FACBC incorporation into proteins was examined. Results: 14 C-FACBC uptake by DU145 cells was markedly decreased to approximately 20% in the absence of Na 1 , compared with that in its presence, indicating that Na 1 -dependent transporters are mainly responsible for the uptake of this tracer. Moreover, 2-amino-2-norbornanecarboxylic acid inhibited the transport of 14 C-FACBC to the basal level in Na 1 -free buffer. In contrast, a-(methylamino) isobutyric acid did not inhibit 14 C-FACBC accumulation in DU145 cells. Human prostate tumor specimens and DU145 cells had similar messenger RNA expression patterns of amino acid transporter genes. Although SNAT2 and ASCT2 are 2 major amino acid transporters expressed in prostate tumor tissues and DU145 cells, ASCT2 knockdown using small interfering RNA was more effective in lowering 14 C-FACBC transport than SNAT2. Almost all intracellular 14 C-FACBC was recovered from the nonprotein fraction. Conclusion: ASCT2, which is a Na 1 -dependent amino acid transporter, and to a lesser extent Na 1 -independent transporters play a role in the uptake of 14 C-FACBC by DU145 cells. Among the Na 1 -independent transporters, system L transporters are also involved in the transport of 14 C-FACBC. Moreover, 14 C-FACBC is not incorporated into proteins in cells. These findings suggest a possible mechanism of anti-18 F-FACBC PET for prostate cancer.

Kojic acid (5-hydroxy-2-(hydroxymethyl)-4-pyrone), a fungal metabolic product, has increasingly been used as a skin-depigmenting agent in skin care products marketed in Japan since 1988. In order to determine its frequency of sensitization, during 1 year from October 1992 to September 1993, we performed patch testing with it in 220 female patients with suspected cosmetic-related contact dermatitis. Of the 220 patients, 8 used at least 1 skin care product containing kojic acid, 5 of whom reacted to kojic acid as well as to 1 or more their own products containing 1% kojic acid, but not to their other products not containing it, and 3 of whom were negative to kojic acid and all their own products. Patch testing with kojic acid in the remaining group of 212 patients, who had not previously used skin care products containing it, was negative without exception. The 5 kojic-acid-sensitive patients, aged 34 to 58 years, developed facial dermatitis 1-12 months after starting application of kojic-acid-containing products. Kojic acid is considered to have high sensitizing potential, as a comparatively high frequency of contact sensitivity was observed in patients using products containing it (5 out of 8).