BACKGROUND: Natural bioproducts are invaluable resources in drug discovery. Isoquinoline alkaloids of Chelidonium majus constitute a structurally diverse family of natural products that are of great interest, one of them being their selectivity for human telomeric G-quadruplex structure and telomerase inhibition. METHODS: The study focuses on the mechanism of telomerase inhibition by stabilization of telomeric G-quadruplex structures by berberine, chelerythrine, chelidonine, sanguinarine and papaverine. Telomerase activity and mRNA levels of hTERT were estimated using quantitative telomere repeat amplification protocol (q-TRAP) and qPCR, in MCF-7 cells treated with different groups of alkaloids. The selectivity of the main isoquinoline alkaloids of Chelidonium majus towards telomeric G-quadruplex forming sequences were explored using a sensitive modified thermal FRET-melting measurement in the presence of the complementary oligonucleotide CT22. We assessed and monitored G-quadruplex topologies using circular dichroism (CD) methods, and compared spectra to previously well-characterized motifs, either alone or in the presence of the alkaloids. Molecular modeling was performed to rationalize ligand binding to the G-quadruplex structure. RESULTS: The results highlight strong inhibitory effects of chelerythrine, sanguinarine and berberine on telomerase activity, most likely through substrate sequestration. These isoquinoline alkaloids interacted strongly with telomeric sequence G-quadruplex. In comparison, chelidonine and papaverine had no significant interaction with the telomeric quadruplex, while they strongly inhibited telomerase at transcription level of hTERT. Altogether, all of the studied alkaloids showed various levels and mechanisms of telomerase inhibition. CONCLUSIONS: We report on a comparative study of anti-telomerase activity of the isoquinoline alkaloids of Chelidonium majus. Chelerythrine was most effective in inhibiting telomerase activity by substrate sequesteration through G-quadruplex stabilization. GENERAL SIGNIFICANCE: Understanding structural and molecular mechanisms of anti-cancer agents can help in developing new and more potent drugs with fewer side effects. Isoquinolines are the most biologically active agents from Chelidonium majus, which have shown to be telomeric G-quadruplex stabilizers and potent telomerase inhibitors.
- MeSH
- alkaloidy farmakologie MeSH
- benzofenantridiny farmakologie MeSH
- Chelidonium chemie MeSH
- cirkulární dichroismus MeSH
- G-kvadruplexy * MeSH
- isochinoliny farmakologie MeSH
- lidé MeSH
- MFC-7 buňky MeSH
- molekulární modely MeSH
- rezonanční přenos fluorescenční energie metody MeSH
- telomerasa antagonisté a inhibitory MeSH
- Check Tag
- lidé MeSH
- Publikační typ
- časopisecké články MeSH
- práce podpořená grantem MeSH
BACKGROUND: The search for new anticancer compounds is a crucial element of natural products research. PURPOSE: In this study the effects of naturally occurring homochelidonine in comparison to chelidonine on cell cycle progression and cell death in leukemic T-cells with different p53 status are described. METHODS: The mechanism of cytotoxic, antiproliferative, apoptosis-inducing effects and the effect on expressions of cell cycle regulatory proteins was investigated using XTT assay, Trypan blue exclusion assay, flow cytometry, Western blot analysis, xCELLigence, epi-fluorescence and 3D super resolution microscopy. A549 cells were used for xCELLigence, clonogenic assay and for monitoring microtubule stability. RESULTS: We found that homochelidonine and chelidonine displayed significant cytotoxicity in examined blood cancer cells with the exception of HEL 92.1.7 and U-937 exposed to homochelidonine. Unexpectedly, homochelidonine and chelidonine-induced cytotoxicity was more pronounced in Jurkat cells contrary to MOLT-4 cells. Homochelidonine showed an antiproliferative effect on A549 cells but it was less effective compared to chelidonine. Biphasic dose-depended G1 and G2/M cell cycle arrest along with the population of sub-G1 was found after treatment with homochelidonine in MOLT-4 cells. In variance thereto, an increase in G2/M cells was detected after treatment with homochelidonine in Jurkat cells. Treatment with chelidonine induced cell cycle arrest in the G2/M cell cycle in both MOLT-4 and Jurkat cells. MOLT-4 and Jurkat cells treated with homochelidonine and chelidonine showed features of apoptosis such as phosphatidylserine exposure, a loss of mitochondrial membrane potential and an increase in the caspases -3/7, -8 and -9. Western blots indicate that homochelidonine and chelidonine exposure activates Chk1 and Chk2. Studies conducted with fluorescence microscopy demonstrated that chelidonine and homochelidonine inhibit tubulin polymerization in A549 cells. CONCLUSION: Collectively, the data indicate that chelidonine and homochelidonine are potent inducers of cell death in cancer cell lines, highlighting their potential relevance in leukemic cells.
- MeSH
- apoptóza účinky léků MeSH
- benzofenantridiny farmakologie MeSH
- berberinové alkaloidy farmakologie MeSH
- Chelidonium chemie MeSH
- Jurkat buňky MeSH
- kaspasy metabolismus MeSH
- kontrolní body buněčného cyklu účinky léků MeSH
- lidé MeSH
- membránový potenciál mitochondrií účinky léků MeSH
- nádorové buněčné linie účinky léků MeSH
- Check Tag
- lidé MeSH
- Publikační typ
- časopisecké články MeSH
- práce podpořená grantem MeSH
The effect of aqueous and ether Chelidonium majus haulms extract on cervical HeLa tumor cells, mammary adenocarcinoma MCF 7 tumor cells and acute lymphoblastic leukemia CEM tumor cells in vitro have been studied. The purpose of this research was to compare the effect of aqueous and ether Chelidonium majus haulms extract on selected tumor cells. Colorimetric MTT assay have been used for the study of the antiproliferative effect of aqueous and ether haulms extract of Chelidonium majus on cell viability in vitro. The results of the experiments have shown the cytotoxic effect of the aqueous and the ether Chelidonium majus haulms extract on the individual tumor cells. The aqueous Chelidonium majus haulms extract was the most effective on CEM cells, it was less effective on MCF 7 cells and it was the least effective on HeLa cells. The ether haulms extract of Chelidonium majus was the most effective at all of studied concentrations on CEM cells and MCF 7 cells in comparison with HeLa cells, where it was significantly effective only at the highest concentration. Aqueous and ether haulms extract of Chelidonium majus tested in vitro indicated their cytotoxic activity. Both haulms extract of Chelidonium majus were more efficient on CEM cells. It is assumed that higher antiproliferative activity of ether haulms extract of Chelidonium majus is the result of higher antiproliferative activity of lipophilic substances. The lipophilic substances pass through membrane and bind to various proteins and change their biological activity.
- Klíčová slova
- vlaštovičník větší, Chelidonium majus, jmelí bílé, rosolovka mozkovitá, Tremella mezenterica,
- MeSH
- Chelidonium MeSH
- fytoterapie MeSH
- houby MeSH
- léčivé rostliny * MeSH
- lidé MeSH
- Santalaceae, Loranthaceae MeSH
- Viscum album MeSH
- Check Tag
- lidé MeSH
- MeSH
- benzofenantridiny farmakologie otrava toxicita MeSH
- Chelidonium chemie otrava růst a vývoj toxicita MeSH
- cholinesterasové inhibitory MeSH
- hypnotika a sedativa MeSH
- lidé MeSH
- manganistan draselný terapeutické užití MeSH
- obstipancia terapeutické užití MeSH
- rostlinné exsudáty škodlivé účinky toxicita MeSH
- Check Tag
- lidé MeSH
Intaktné rastliny čeľade Papaveraceae sú schopné produkovať celý rad benzylizochinolínových alkaloidov, ktoré majú využitie vo farmaceutickej praxi. In vitro kultúry odvodené z rastlín tejto čeľade si nezachovávajú schopnosť produkcie širokého spektra alkaloidov – aktívna je v nich iba biosyntetická dráha, vedúca k sanguinarínu. V rámci tejto práce boli odvodené in vitro kultúry z rastlín Papaver somniferum, Eschscholtzia californica, Chelidonium majus a Macleaya cordata a porovnaná ich schopnosť produkcie sanguinarínu. Najnižšie množstvá sanguinarínu vyprodukovali kultúry maku siateho (0,45–0,55 μg . g-1 čerstvej hmoty). Kultúry Eschscholtzia californica, Chelidonium majus a Macleaya cordata produkovali navzájom porovnateľné množstvá (18,0–22,7 μg; 20,5–26,3 μg; resp. 15,4 až 20,3 μg na 1 g č. hm.). Po elicitácii kultúr hydrolyzátom z Botrytis cinerea bola vo všetkých vzorkách zaznamenaná zvýšená produkcia sanguinarínu. K relatívne najvyššiemu zvýšeniu produkcie došlo v kultúrach maku siateho, avšak tieto kultúry ani po elicitácii nedosiahli úroveň produkcie ostatných troch kultúr bez ovplyvnenia. Kľúčové slová: Papaver somniferum • Eschscholtzia californica • Chelidonium majus • Macleaya cordata • suspenzné kultúry • elicitácia • sanguinarín
Intact plants of the Papaveraceae family are producers of a whole range of benzylisoquinoline alkaloids, which are used in pharmaceutical industry. In vitro cultures derived from plants of the Papaveraceae do not have the ability to produce such a broad spectrum of alkaloids, only the biosynthetic pathway leading to sanguinarine is active. This study deals with the preparation of in vitro cultures of Papaver somniferum, Eschscholtzia californica, Chelidonium majus and Macleaya cordata. Their sanguinarine production abilities were tested and compared. The lowest amounts of sanguinarine from all cultures tested were accumulated in suspension cultures of the opium poppy (0.45–0.55 μg in 1 g of fresh weight). Eschscholtzia californica, Chelidonium majus and Macleaya cordata cultures produced similar amounts of sanguinarine (18.0–22.7 μg; 20.5–26.3 μg; 15.4–20.3 μg in 1 g of fresh weight, resp.). The elicitation study used a biotic stressor, Botrytis cinerea hydrolysate. In all cultures treated, an increase in sanguinarine accumulation was observed. Of all cultures tested, the most intensive response was observed in the opium poppy cultures, although the amount of sanguinarine in the elicited poppy cultures was lower than in the non-elicited samples of the other cultures. Key words: Papaver somniferum • Eschscholtzia californica • Chelidonium majus • Macleaya cordata • suspension cultures • elicitation • sanguinarine Received 25 October 2012 / Accepted 17 November 2012
The roots and aerial parts of Chelidonium majus L. were extracted with EtOH and fractionated using CHCl3 and EtOH. Repeated column chromatography, preparative TLC and crystallization led to the isolation of five isoquinoline alkaloids, stylopine (3), chelidonine (4), homochelidonine (5), protopine (6), and allocryptopine (7), along with two isolation artifacts 6-ethoxydihydrosanguinarine (1) and 6-ethoxydihydrochelerythrine (2). All isolated compounds were tested for human blood acetylcholinesterase (HuAChE) and human plasma butyrylcholinesterase (HuBuChE) inhibitory activity. The isolation artifacts exhibited the highest activity against HuAChE and HuBuChE with IC50 values of 0.83 +/- 0.04 microM and 4.20 +/- 0.19 microM for 6-ethoxydihydrochelerythrine and 3.25 +/- 0.24 microM and 4.51 +/- 0.31 microM for 6-ethoxydihydrosanguinarine. The most active of the naturally-occurring alkaloids was chelidonine, which inhibited both HuAChE and HuBuChE in a dose-dependent manner with IC50 values of 26.8 +/- 1.2 microM and 31.9 +/- 1.4 microM, respectively.
- MeSH
- acetylcholinesterasa metabolismus MeSH
- butyrylcholinesterasa metabolismus MeSH
- Chelidonium chemie MeSH
- cholinesterasové inhibitory chemie farmakologie MeSH
- erytrocytární membrána účinky léků MeSH
- kořeny rostlin chemie MeSH
- lidé MeSH
- nadzemní části rostlin chemie MeSH
- Check Tag
- lidé MeSH
- Publikační typ
- časopisecké články MeSH
- práce podpořená grantem MeSH
Lastovičník väčší podobne ako iné rastliny z čeľade Papaveraceae produkuje benzylizochinolínové alkaloidy, predovšetkým benzofenantridíny. Polyfenoloxidáza (PPO) je pravdepodobne zapojená do tvorby dopamínu, ktorý je jedným z prekurzorov norkoklaurínu, prvého intermediátu s benzylizochinolínovou štruktúrou. V rámci práce bolo zistené, že PPO prítomná v latexe lastovičníka je lokalizovaná v organelách, ktoré slúžia k uskladneniu alkaloidov (tzv. 1000 g organely). Enzým bol purifikovaný afinitnou chromatografiou do elektroforetickej homogenity, má relatívnu molekulovú hmotnosť približne 65 kDa a vykazuje dve aktivity – monofenolázovú a difenolázovú. Použitím polymerázovej reťazovej reakcie sa podarilo amplifikovať časť génu PPO z oblasti aktívneho miesta.
Greater celandine, similarly as other plants of the family Papaveraceae, produces benzylisoquinoline alkaloids, primarily benzophenanthridines. Polyphenoloxidase (PPO) is most probably involved in the formation of dopamine, which is one of the precursors of norcoclaurine, the first intermediate with the benzylisoquinoline structure. This study has revealed that PPO present in the latex of greater celandine is localized in the organelles, which serve to store alkaloids (the so-called 1000 g organelles). The enzyme was purified by means of affinity chromatography into electrophoretic homogeneity. It possesses a relative molecular mass of approximately 65 kDa and exerts two activities, the monophenolase and diphenolase ones. With the use of a polymerase chain reaction, it was possible to amplify a part of the PPO gene from the region of the active site.
- MeSH
- alkaloidy chemie izolace a purifikace MeSH
- Chelidonium chemie MeSH
- chromatografie afinitní metody využití MeSH
- dopamin chemie MeSH
- elektroforéza v polyakrylamidovém gelu metody využití MeSH
- fenoly chemie MeSH
- financování organizované MeSH
- latex chemie MeSH
- lidé MeSH
- polymerázová řetězová reakce metody využití MeSH
- Check Tag
- lidé MeSH
