Wedelactone (WL), a plant polyphenolic derivative of coumestan, represents a promising anti-cancer agent. The underlying mechanisms of its action are not fully understood and appear to involve interplay with copper ions. Herein, we examined coordination and redox interactions of WL with Cu2+ in phosphate buffer (pH 7), and in two breast cancer cell lines. EPR, UV-Vis and fluorescence spectroscopy showed that WL and Cu2+ build a coordination complex with 2 : 1 stoichiometry and distorted tetrahedral geometry. WL showed strong fluorescence that was quenched by Cu2+. The sequestration of the intracellular copper pool with neocuproine led to a significant drop in the cytotoxic effects of WL, whereas the co-application of Cu2+ and WL and the formation of an extracellular complex suppressed both the cytotoxic effects of WL and copper loading. Fluorescence microscopy showed that WL is mainly localized in the cytosol and significantly less in the nuclei. WL fluorescence was stronger in cells pretreated with neocuproine, implying that the complex of WL and Cu2+ is formed inside the cells. WL caused a two-fold increase in the lysosomal level of copper as well as copper-dependent lysosome membrane permeabilization. On the other hand, the protective effects of overexpression of thioredoxin 1 imply that WL exerts the main oxidative impact inside the nucleus. The interactions of WL with copper may be essential for therapeutic performance and selectivity against cancer cells, taking into account that a number of cancer types, including breast cancer, exhibit increased intratumoral copper levels or altered copper distribution.

• MeSH
• apoptóza MeSH
• komplexní sloučeniny metabolismus   MeSH
• kumariny farmakologie   MeSH
• lidé MeSH
• měď metabolismus   MeSH
• nádorové buňky kultivované MeSH
• nádory prsu farmakoterapie   metabolismus   patologie   MeSH
• protinádorové látky farmakologie   MeSH
• subcelulární frakce metabolismus   MeSH
• Check Tag
• lidé MeSH
• ženské pohlaví MeSH
• Publikační typ
• časopisecké články MeSH
• práce podpořená grantem MeSH

Wedelolactone is a multi-target natural plant coumestan exhibiting cytotoxicity towards cancer cells. Although several molecular targets of wedelolactone have been recognized, the molecular mechanism of its cytotoxicity has not yet been elucidated. In this study, we show that wedelolactone acts as an inhibitor of chymotrypsin-like, trypsin-like, and caspase-like activities of proteasome in breast cancer cells. The proteasome inhibitory effect of wedelolactone was documented by (i) reduced cleavage of fluorogenic proteasome substrates; (ii) accumulation of polyubiquitinated proteins and proteins with rapid turnover in tumor cells; and (iii) molecular docking of wedelolactone into the active sites of proteasome catalytic subunits. Inhibition of proteasome by wedelolactone was independent on its ability to induce reactive oxygen species production by redox cycling with copper ions, suggesting that wedelolactone acts as copper-independent proteasome inhibitor. We conclude that the cytotoxicity of wedelolactone to breast cancer cells is partially mediated by targeting proteasomal protein degradation pathway. Understanding the structural basis for inhibitory mode of wedelolactone might help to open up new avenues for design of novel compounds efficiently inhibiting cancer cells.

• MeSH
• inhibitory proteasomu chemie   farmakologie   toxicita   MeSH
• kumariny chemie   farmakologie   toxicita   MeSH
• lidé MeSH
• měď metabolismus   MeSH
• nádorové buněčné linie MeSH
• nádory prsu metabolismus   MeSH
• proteasomový endopeptidasový komplex chemie   metabolismus   MeSH
• proteolýza MeSH
• reaktivní formy kyslíku metabolismus   MeSH
• simulace molekulového dockingu MeSH
• ubikvitinace MeSH
• vazba proteinů MeSH
• viabilita buněk účinky léků   MeSH
• Check Tag
• lidé MeSH
• Publikační typ
• časopisecké články MeSH

Wedelolactone, a plant coumestan, was shown to act as anti-cancer agent for breast and prostate carcinomas in vitro and in vivo targeting multiple cellular proteins including androgen receptors, 5-lipoxygenase and topoisomerase IIα. It is cytotoxic to breast, prostate, pituitary and myeloma cancer cell lines in vitro at μM concentrations. In this study, however, a novel biological activity of nM dose of wedelolactone was demonstrated. Wedelolactone acts as agonist of estrogen receptors (ER) α and β as demonstrated by transactivation of estrogen response element (ERE) in cells transiently expressing either ERα or ERβ and by molecular docking of this coumestan into ligand binding pocket of both ERα and ERβ. In breast cancer cells, wedelolactone stimulates growth of estrogen receptor-positive cells, expression of estrogen-responsive genes and activates rapid non-genomic estrogen signalling. All these effects can be inhibited by pretreatment with pure ER antagonist ICI 182,780 and they are not observed in ER-negative breast cancer cells. We conclude that wedelolactone acts as phytoestrogen in breast cancer cells by stimulating ER genomic and non-genomic signalling pathways.

• MeSH
• aktivace transkripce genetika   MeSH
• alfa receptor estrogenů metabolismus   MeSH
• antagonisté estrogenového receptoru farmakologie   MeSH
• beta receptor estrogenů metabolismus   MeSH
• estradiol analogy a deriváty   farmakologie   MeSH
• estrogeny farmakologie   MeSH
• HEK293 buňky MeSH
• kumariny farmakologie   MeSH
• lidé MeSH
• MFC-7 buňky MeSH
• nádorové buněčné linie MeSH
• nádory prsu farmakoterapie   MeSH
• proliferace buněk účinky léků   MeSH
• protinádorové látky farmakologie   MeSH
• responzivní elementy genetika   MeSH
• signální transdukce účinky léků   MeSH
• simulace molekulového dockingu MeSH
• transkripční faktor AP-1 metabolismus   MeSH
• vazebná místa účinky léků   MeSH
• Check Tag
• lidé MeSH
• ženské pohlaví MeSH
• Publikační typ
• časopisecké články MeSH
• práce podpořená grantem MeSH

Wedelia calendulacea (L.) Less. je vytrvalá bylina z čeledi hvězdnicovitých (Asteraceae), původem z východní Asie. Areál rozšíření zahrnuje Čínu, Japonsko, Tchaj-wan, Indii, Indonésii, Vietnam, Filipíny a Srí Lanku. Listy a nať W. calendulacea se používají v tradiční čínské a ayurvédské medicíně zejména k léčbě jaterních onemocnění. Další uplatnění nacházejí při kašli, bolestech hlavy, respiračních infekcích, krvácení, zažívacích potížích, průjmu a některých kožních onemocněních. K léčebným účelům jsou rovněž využívány květy a plody. Rostlina obsahuje velké množství biologicky aktivních sloučenin. Hlavními obsahovými látkami jsou kumestany wedelolakton (7-methoxy-5,11,12-trihydroxy-kumestan) a demethylwedelo­lakton. Wedelolakton je přírodní polyfenolická látka, která byla kromě rostliny W. calendulacea izolována také z rostliny Eclipta prostrata. Na několika typech nádorových buněk byly prokázány antiproliferační a proapoptotické účinky wedelolaktonu in vitro a in vivo. V poslední době je W. calendulacea předmětem zájmu moderní medicíny a její léčivé účinky potvrzuje velké množství studií. Perspektivní je zejména hepatoprotektivní účinek extraktu z listů rostliny, který působí preventivně proti toxickému působení hepatotoxinů. Zjištěn byl také účinek protinádorový, neuroprotektivní, antimikrobiální, antioxidační, protizánětlivý a antiandrogenní. Intenzivní výzkum probíhá v oblasti využití obsahových látek rostliny v terapii nádorů. Výsledky jsou nadějné a je proto možné, že obsahové látky Wedelia calendulacea najdou své využití i v moderní medicíně.

Wedelia calendulacea (L.) Less. is a perennial herb of the family Asteraceae (Asteraceae), native to eastern Asia. Its distribution area includes China, Japan, Taiwan, India, Indonesia, Vietnam, the Philippines and Sri Lanka. The leaves and stems of W. calendulacea are used in traditional Chinese and Ayurvedic medicine, particularly for the treatment of liver diseases. The herb is also used to treat coughing, headaches, respirátory infections, bleeding, indigestion, diarrhoea and some skin diseases. The flowers and fruits are also used for therapeutic purposes. The plant contains high quantities of biologically active compounds. The main contained substances are the coumestans wedelolactone (7-methoxy-5,11,12-trihydroxy-coumestan) and demethylwedelo­lactone. Wedelolactone is a natural polyphenolic substance isolated besides W. calendulacea also from the plant Eclipta prostrata. In several types of cancer cells, the antiproliferative and proapoptotic effects of wedelolactone have been demonstrated both in vitro and in vivo. Recently, W. calendulacea has become the subject of interest of modern medicine, and its healing effects are confirmed by a large number of studies. Especially perspective is the hepatoprotective effect of the plant‘s leaf extract, as it protects against the harmful effects of hepatotoxins. In addition, it has been determined that it also has antitumor, neuroprotective, antimicrobial, antioxidant, anti-inflammatory and antiandrogen effects. Intensive research is being devoted to the use of the substances contained in the plant for the treatment of tumors. The results are promising, so the substances contained in Wedelia calendulacea might provide health benefits even in modern medicine.

• Klíčová slova
• Wedelia calendulacea, ayurvédská medicína, obsahové látky, biomedicína,
• MeSH
• Asteraceae růst a vývoj   účinky léků   MeSH
• farmakologické účinky MeSH
• farmakologie MeSH
• kumariny aplikace a dávkování   škodlivé účinky   terapeutické užití   MeSH
• léčivé rostliny MeSH
• lidé středního věku MeSH
• lidé MeSH
• myši MeSH
• tradiční lékařství MeSH
• Wedelia * růst a vývoj   MeSH
• zvířata MeSH
• Check Tag
• lidé středního věku MeSH
• lidé MeSH
• myši MeSH
• zvířata MeSH
• Publikační typ
• práce podpořená grantem MeSH

Wedelolactone is one of the active plant polyphenolic compounds. Anti-tumor effects of this drug have been demonstrated recently. We have described that wedelolactone acts as catalytic inhibitor of DNA topoisomerase IIα. The aim of this study was to further characterize the mechanism of its anti-tumor effects. We showed that wedelolactone inhibits binding of DNA topoisomerase IIα to plasmid DNA and antagonizes formation of etoposide-induced DNA cleavage complex. The inhibition of topoisomerase IIα by wedelolactone is reversible by excess of the enzyme but not DNA. The in vitro inhibitory effect of wedelolactone on the topoisomerase IIα activity is redox-dependent as it diminished in the presence of reducing agents. Cytotoxicity of wedelolactone was partially inhibited by N-acetylcysteine and glutathione ethyl ester in breast cancer MDA-MB-231 and MDA-MB-468 cells while the inhibitory effect of catalase was observed only in the former cell line. Finally, we found that wedelolactone can be oxidized in the presence of copper ions resulting in DNA strand break and abasic site formation in vitro. However, wedelolactone induced neither DNA damage in MDA-MB-231 cells nor mutations in bacterial cells detectable by Ames test suggesting that wedelolactone may not be an effective inducer of DNA damage. We conclude that the topoisomerase IIα inhibitory- and DNA damaging activities of wedelolactone in vitro depend on its redox state. Pro-oxidant activity could, however, explain only part of wedelolactone-induced cytotoxicity. Therefore, the major cellular target(s) of wedelolactone and the exact mechanism of wedelolactone-induced cytotoxicity still remain to be identified.

• MeSH
• acetylcystein MeSH
• antigeny nádorové metabolismus   MeSH
• DNA vazebné proteiny antagonisté a inhibitory   metabolismus   MeSH
• DNA-topoisomerasy typu II metabolismus   MeSH
• glutathion analogy a deriváty   MeSH
• imunoblotting MeSH
• katalasa MeSH
• kumariny chemie   metabolismus   farmakologie   MeSH
• lidé MeSH
• molekulární struktura MeSH
• NAD metabolismus   MeSH
• nádorové buněčné linie MeSH
• nádory prsu farmakoterapie   MeSH
• oxidace-redukce MeSH
• protinádorové látky chemie   metabolismus   farmakologie   MeSH
• techniky in vitro MeSH
• viabilita buněk účinky léků   MeSH
• Check Tag
• lidé MeSH
• ženské pohlaví MeSH
• Publikační typ
• časopisecké články MeSH
• práce podpořená grantem MeSH

The naturally occurring coumestan wedelolactone has been previously shown to reduce growth of various cancer cells. So far, the growth-suppressing effect of wedelolactone has been attributed to the inhibition of the NFκB transcription factor and/or androgen receptors. We found that wedelolactone suppressed growth and induced apoptosis of androgen receptor-negative MDA-MB-231 breast cancer cells at concentrations that did not inhibit the NFκB activity. The cells responded to wedelolactone by the S and G2/M phase cell cycle arrest and induction of the DNA damage signaling. Wedelolactone interacted with dsDNA and inhibited the activity of DNA topoisomerase IIα. We conclude that wedelolactone can act as growth suppressor independently of NFκB and androgen receptors.

• MeSH
• antigeny nádorové metabolismus   MeSH
• apoptóza účinky léků   MeSH
• buněčný cyklus účinky léků   MeSH
• buňky - růstové procesy účinky léků   MeSH
• DNA vazebné proteiny antagonisté a inhibitory   metabolismus   MeSH
• DNA-topoisomerasy typu II metabolismus   MeSH
• ELISA MeSH
• imunoblotting MeSH
• inhibitory topoisomeras farmakologie   MeSH
• kumariny farmakologie   MeSH
• lidé MeSH
• nádorové buněčné linie MeSH
• nádory prsu farmakoterapie   enzymologie   patologie   MeSH
• poškození DNA MeSH
• protinádorové látky farmakologie   MeSH
• signální transdukce MeSH
• viabilita buněk účinky léků   MeSH
• Check Tag
• lidé MeSH
• ženské pohlaví MeSH
• Publikační typ
• časopisecké články MeSH
• práce podpořená grantem MeSH