Utjecaj strukturno srodnih flavonoida na ekspresiju hsp gena u ljudskim promieloidnim leukemijskim stanicama

Quercetin is a known specific inhibitor of hsp70 synthesis and thus might be a potent agent for enhancing the selective cytotoxicity of heat on tumour cells. A comparative analysis of the effects of quercetin and five structurally related flavonoids on hsp90α, hsp70A, hsp60 and hsp27 gene expression was carried out using human myeloid leukaemia cells (HL-60). The cells were preincubated with 50 μM quercetin, kaempferol, myricetin, taxifolin, isorhamnetin, methylquercetagetin or 0.1 % DMSO (controls) for 24 h at 37 °C before heat shock treatment (43 °C for 30 min). Total RNA was isolated from heat-stressed and unstressed cells and analysed by RT PCR. Hsp27 gene expression was inhibited by flavonoids more strongly than other hsp genes investigated in heat stressed as well as in unstressed cells. Among the hsp genes tested, only hsp60 was expressed above control level under the influence of taxifolin. Members of the hsp70 and hsp27 families are highly expressed in breast and lung cancer and leukaemias and they play a role in the acquired resistance to chemotherapy or radiation therapy combined with hyperthermia. Therefore, hsps present potential targets for cancer diagnosis and treatment. The present structure/activity study indicates that position, number and substitution of hydroxyl groups of the B ring and saturation of the C2-C3 bond are important factors affecting flavonoid activity on hsp gene expression. This study could help provide a basis for further design of specific inhibitors of hsp gene expression.Kvercetin je poznati specifični inhibitor sinteze hsp70 i kao takav mogući čimbenik povećanja selektivnog citotoksičnog učinka topline na tumorske stanice. Provedena je komparativna analiza učinka kvercetina i njemu strukturno srodnih flavonoida na ekspresiju hsp90α, hsp70A, hsp60 i hsp27 gena u stanicama humane mijeloidne leukemije (HL-60). Stanice su inkubirane s 50 μM kvercetina, kempferola, miricetina, taksifolina, izoramnetina, metilkvercetagetina ili 0,1% DMSO (kontrola) tijekom 24 h na 37 °C prije obrade toplinskim šokom (43 °C tijekom 30 minuta). Izolirana je ukupna RNA iz stanica koje su bile, kao i one koje nisu bile, podvrgnute toplinskom stresu te je provedena RT-PCR analiza. Ekspresija gena hsp27 bila je jače inhibirana flavonoidima nego ostali istraživani hsp geni, i to podjednako u stanicama podvrgnutim kao i u onima koje nisu bile podvrgnute toplinskom stresu. Među istraživanim hsp genima jedino je ekspresija hsp60 bila iznad kontrolne razine pod utjecajem taksifolina. Članovi porodica hsp70 i hsp27 snažno su ekspresirani kod raka dojke, pluća i leukemija te imaju bitnu ulogu u stečenoj rezistenciji stanica pri kemoterapiji ili terapiji zračenjem u kombinaciji s hipertermijom. Stoga bi hsp proteine trebalo istraživati pri dijagnosticiranju i liječenju raka. Prikazana studija strukture i aktivnosti upućuje na to da su položaj, broj i supstitucija hidroksilnih skupina na prstenu B kao i zasićeni vez C2–C3 kod flavonoida bitni čimbenici koji utječu na ekspresiju hsp gena. Ova bi studija mogla poslužiti kao osnova za daljnje strukturiranje specifičnih inhibitora ekspresije hsp gena

Tunable Protein Stabilization In Vivo Mediated by Shield-1 in Transgenic Medaka: Research Article

Techniques for conditional gene or protein expression are important tools in developmental biology and in the analysis of physiology and disease. On the protein level, the tunable and reversible expression of proteins can be achieved by the fusion of the protein of interest to a destabilizing domain (DD). In the absence of its specific ligand (Shield-1), the protein is degraded by the proteasome. The DD-Shield system has proven to be an excellent tool to regulate the expression of proteins of interests in mammalian systems but has not been applied in teleosts like the medaka. We present the application of the DD-Shield technique in transgenic medaka and show the ubiquitous conditional expression throughout life. Shield-1 administration to the water leads to concentration-dependent induction of a YFP reporter gene in various organs and in spermatogonia at the cellular level

Praćenje metabolizma flavonoida u humanim stanicama na temelju fluorescencije izazvane interakcijom kvercetina s proteinima

Despite the wealth of information concerning biological effects of flavonoids, a systematic approach to analyzing the molecular targets is still lacking and, for this reason, a rational evaluation of the risks or benefits of flavonoid-containing foods or of possible pharmaceutical applications is difficult. We have exploited the property of quercetin to elicit fluorescence when bound to specific target proteins and assayed several flavonoids with different modifications (methylation, hydroxylation, glycosylation). Quercetin target proteins can be visualized in living cells, but in vital human leukaemia cells (HL-60) the fluorescence decreases rapidly after labelling, while metabolically inactive apoptotic cells retain the fluorescence. These cytological differences were apparent under the fluorescent microscope and were quantified using flow cytometry. Metabolic conversion of quercetin in vital cells was confirmed and quantified by HPLC analysis. While apoptotic cells still contained considerable amounts of quercetin, vital cells rapidly metabolized the flavonoid (e.g., by methylation or glycosylation). Biochemical results are consistent with the cytological observations and support the conclusion that quercetin becomes rapidly converted to non-fluorogenic metabolites in vital cells. Loss of fluorescence in vital cells allows convenient monitoring and quantifying of the dynamics of quercetin metabolism in human cells.Unatoč mnoštvu informacija koje se odnose na biološke učinke flavonoida, sustavni pristup analizi njihovih ciljnih molekula još uvijek nedostaje. Iz toga razloga vrlo je teško racionalno vrednovati opasnosti ili koristi koje donosi hrana koja sadrži flavonoide kao i njihovu moguću farmakološku primjenu. Iskoristili smo svojstvo kvercetina da izazove fluorescenciju kada se veže za specifične ciljne proteine i analizirali nekoliko različito modificiranih flavonoida (metilacija, hidroksilacija, glikozilacija). Ciljni proteini za koje se kvercetin veže u živim stanicama mogu se vizualizirati na temelju fluorescencije. U živim stanicama humane leukemije (HL-60) fluorescencija naglo pada nakon označavanja flavonoidima, dok metabolički inaktivne apoptotične stanice zadržavaju fluorescenciju. Te su citološke razlike jasno zapažene pod fluorescencijskim mikroskopom, a kvantificirane su pomoću protočne citometrije. Metabolička pretvorba kvercetina u živim stanicama potvr|ena je i kvantificirana pomoću HPLC analiza. Dok apoptotične stanice zadržavaju značajnu količinu kvercetina, žive ga stanice brzo metaboliziraju (npr. metilacijom ili glikozilacijom). Ti su biokemijski rezultati u skladu s citološkim promatranjima i podupiru zaključak da se kvercetin u živim stanicama brzo pretvara u nefluorogene metabolite. Gubitak fluorescencije u živim stanicama omogućava praćenje i kvantifikaciju dinamike metabolizma kvercetina u humanim stanicama

Mechanism and specificity of pentachloropseudilin-mediated inhibition of myosin motor activity.

Here, we report that the natural compound pentachloropseudilin (PClP) acts as a reversible and allosteric inhibitor of myosin ATPase and motor activity. IC(50) values are in the range from 1 to 5 μm for mammalian class-1 myosins and greater than 90 μm for class-2 and class-5 myosins, and no inhibition was observed with class-6 and class-7 myosins. We show that in mammalian cells, PClP selectively inhibits myosin-1c function. To elucidate the structural basis for PClP-induced allosteric coupling and isoform-specific differences in the inhibitory potency of the compound, we used a multifaceted approach combining direct functional, crystallographic, and in silico modeling studies. Our results indicate that allosteric inhibition by PClP is mediated by the combined effects of global changes in protein dynamics and direct communication between the catalytic and allosteric sites via a cascade of small conformational changes along a conserved communication pathway

Effects of Structurally Related Flavonoids on hsp Gene Expression in Human Promyeloid Leukaemia Cells

Quercetin is a known specific inhibitor of hsp70 synthesis and thus might be a potent agent for enhancing the selective cytotoxicity of heat on tumour cells. A comparative analysis of the effects of quercetin and five structurally related flavonoids on hsp90α, hsp70A, hsp60 and hsp27 gene expression was carried out using human myeloid leukaemia cells (HL-60). The cells were preincubated with 50 μM quercetin, kaempferol, myricetin, taxifolin, isorhamnetin, methylquercetagetin or 0.1 % DMSO (controls) for 24 h at 37 °C before heat shock treatment (43 °C for 30 min). Total RNA was isolated from heat-stressed and unstressed cells and analysed by RT PCR. Hsp27 gene expression was inhibited by flavonoids more strongly than other hsp genes investigated in heat stressed as well as in unstressed cells. Among the hsp genes tested, only hsp60 was expressed above control level under the influence of taxifolin. Members of the hsp70 and hsp27 families are highly expressed in breast and lung cancer and leukaemias and they play a role in the acquired resistance to chemotherapy or radiation therapy combined with hyperthermia. Therefore, hsps present potential targets for cancer diagnosis and treatment. The present structure/activity study indicates that position, number and substitution of hydroxyl groups of the B ring and saturation of the C2-C3 bond are important factors affecting flavonoid activity on hsp gene expression. This study could help provide a basis for further design of specific inhibitors of hsp gene expression

Toxicity and Cell Cycle Effects of Synthetic 8-Prenylnaringenin and Derivatives in Human Cells

The estrogenic flavanone rac-8-prenylnaringenin (8-PN) and 3 derivatives (rac-7-(O-prenyl)naringenin-4′-acetate (7-O-PN), rac-5-(O-prenyl)naringenin-4′,7-diacetate (5-O-PN), and rac-6-(1,1-dimethylallyl)naringenin (6-DMAN) were prepared by chemical synthesis and analyzed with respect to their toxicity and possible cell cycle effects in human acute myeloid leukemia (HL-60) cells. With the exception of 5-O-PN, all the other naringenins showed only weak toxic effects at concentrations below 50 μmol/l. A cell cycle analysis over several cell generations up to 4 days was carried out using the fluorescent dye carboxyfluorescein diacetate N-succinimidyl ester (CFSE) followed by propidium iodide (PI) staining at the end of the experiment. The well-studied flavonol quercetin was included in the analysis as a reference substance. All flavonoids affected cell proliferation, but the extent and the resulting changes in the proliferation pattern were specific for each substance. In contrast to the radical scavenging activity of quercetin, the tested flavanones showed no anti-oxidative properties using several different test systems. Similarly, the mitochondrial membrane potential (ΔΨm) was hardly effected by these compounds, while both menadione and quercetin strongly reduced the potential after 1 h of treatment. The reported chemical modification of interesting lead substances (like the strongly estrogenic 8-PN) presents a promising approach to modulate the properties of a relevant substance in a pharmacologically desirable way. The low toxicity and weak cytostatic properties of the tested naringenin derivatives is encouraging for further studies on known naringenin target molecules.Dieser Beitrag ist mit Zustimmung des Rechteinhabers aufgrund einer (DFG-geförderten) Allianz- bzw. Nationallizenz frei zugänglich

Induced fluorescence of testis cells analyzed by flow cytometry.

<p>Primary testis cells of hemizygous Ola-Tg(DD-YFP)13 were analyzed for size (FSC), fluorescence (GFP) and DNA content (Gate R1; Hoechst staining, not shown). The quadrants Q1 and Q2 contain cells with a DNA content of 2C to 4C, i.e. somatic cells and germ cells prior to meiosis. The plots show the data of 129–154,000 single cells. (A) The fluorescence in a sample of a non-transgenic FLFII and was plotted against the cell size. (B) In the non-induced control virtually no additional fluorescence is observed in Q2 compared to FLFII. (C) Treatment with 1 μM Shield-1 causes a strong induction of fluorescence in the largest cells that comprise the stem cell fraction. Absolute fluorescence and number of cells in Q2 are increased. (D) For comparison, the GFP-positive stem cells in FLF-Tg(oct4-EGFP)18 are shown [<a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0131252#pone.0131252.ref017" target="_blank">17</a>]. In this line, the fluorescence decreases during diffentiation (arrow). In contrast, a cell fraction of similar size has an elevated fluorescence after induction (arrow in C). These cells represent the mitotically active type B spermatogonia that actively transcribe the <i>actb</i>-driven DD-YFP but not the <i>oct4</i>-driven EGFP.</p