Mechanism of Action of Two Flavone Isomers Targeting Cancer Cells with Varying Cell Differentiation Status

Apoptosis can be triggered in two different ways, through the intrinsic or the extrinsic pathway. The intrinsic pathway is mediated by the mitochondria via the release of cytochrome C while the extrinsic pathway is prompted by death receptor signals and bypasses the mitochondria. These two pathways are closely related to cell proliferation and survival signaling cascades, which thereby constitute possible targets for cancer therapy. In previous studies we introduced two plant derived isomeric flavonoids, flavone A and flavone B which induce apoptosis in highly tumorigenic cancer cells of the breast, colon, pancreas, and the prostate. Flavone A displayed potent cytotoxic activity against more differentiated carcinomas of the colon (CaCo-2) and the pancreas (Panc28), whereas flavone B cytotoxic action is observed on poorly differentiated carcinomas of the colon (HCT 116) and pancreas (MIA PaCa). Apoptosis is induced by flavone A in better differentiated colon cancer CaCo-2 and pancreatic cancer Panc 28 cells via the intrinsic pathway by the inhibition of the activated forms of extracellular signal-regulated kinase (ERK) and pS6, and subsequent loss of phosphorylation of Bcl-2 associated death promoter (BAD) protein, while apoptosis is triggered by flavone B in poorly differentiated colon cancer HCT 116 and MIA PaCa pancreatic cancer cells through the extrinsic pathway with the concomitant upregulation of the phosphorylated forms of ERK and c-JUN at serine 73. These changes in protein levels ultimately lead to activation of apoptosis, without the involvement of AKT.     

Qualitative analysis of sequence specific binding of flavones to DNA using restriction endonuclease activity assays

Flavones, found in nature as secondary plant metabolites, have shown efficacy as anti‐cancer agents. We have examined the binding of two flavones, 5,7‐dihydroxy‐3,6,8‐trimethoxy‐2‐phenyl‐4H‐chromen‐4‐one (5,7‐dihydroxy‐3,6,8‐trimethoxy flavone; FlavA) and 3,5‐dihydroxy‐6,7,8‐trimethoxy‐2‐phenyl‐4H‐chromen‐4‐one (3,5‐dihydroxy‐6,7,8‐trimethoxy flavone; FlavB), to phiX174 RF DNA using restriction enzyme activity assays employing the restriction enzymes Alw44, AvaII, BssHII, DraI, MluI, NarI, NciI, NruI, PstI, and XhoI. These enzymes possess differing target and flanking sequences allowing for observation of sequence specificity analysis. Using restriction enzymes that cleave once with a mixture of supercoiled and relaxed DNA substrates provides for observation of topological effects on binding. FlavA and FlavB show differing sequence specificities in their respective binding to phiX. For example, with relaxed DNA, FlavA shows inhibition of cleavage with DraI (reaction site ^5′TTTAAA) but not BssHII (^5′GCGCGC) while FlavB shows the opposite results. Evidence for tolological specificity is also observed, Molecular modeling and conformational analysis of the flavones suggests that the phenyl ring of FlavB is coplanar with the flavonoid ring while the phenyl ring of FlavA is at an angle relative to the flavonoid ring. This may account for aspects of the observed sequence and topological specificities in the effects on restriction enzyme activity. © 2013 Wiley Periodicals, Inc. Biopolymers 99: 530–537, 2013.     

Methylenedioxy- and methoxyflavones from Melicope coodeana syn. Euodia simplex

Three new natural products, 3,8-dimethoxy-5,7-dihydroxy-3′,4′-methylenedioxyflavone, 3,6,8-trimethoxy-5,7-dihydroxy-3′,4′-methylenedioxyflavone and 3,6,8,3′,4′-pentamethoxy-5,7-dihydroxyflavone were isolated from Melicope coodeana syn. Euodia simplex (Rutaceae) along with 3,6,3′-trimethoxy-5,7,4′-trihydroxyflavone and 3,3′-dimethoxy-5,7,4′-trihydroxyflavone. The structural assignments are based on ¹H and ¹³C NMR data, including discussion of the chemical shifts of C-2 in 3,5-dihydroxy- and 3-methoxy-5-hydroxyflavones. The presence of highly methoxylated and methylenedioxyflavones is characteristic of the genus Melicope, and the present findings support the recent transfer of Euodia simplex to Melicope.     

Methylenedioxy- and methoxyflavones from Melicope coodeana syn. Euodia simplex

Three new natural products, 3,8-dimethoxy-5,7-dihydroxy-3′,4′-methylenedioxyflavone, 3,6,8-trimethoxy-5,7-dihydroxy-3′,4′-methylenedioxyflavone and 3,6,8,3′,4′-pentamethoxy-5,7-dihydroxyflavone were isolated from Melicope coodeana syn. Euodia simplex (Rutaceae) along with 3,6,3′-trimethoxy-5,7,4′-trihydroxyflavone and 3,3′-dimethoxy-5,7,4′-trihydroxyflavone. The structural assignments are based on ¹H and ¹³C NMR data, including discussion of the chemical shifts of C-2 in 3,5-dihydroxy- and 3-methoxy-5-hydroxyflavones. The presence of highly methoxylated and methylenedioxyflavones is characteristic of the genus Melicope, and the present findings support the recent transfer of Euodia simplex to Melicope.     

In vitro inhibition of cytosolic carbonic anhydrases I and II by some new dihydroxycoumarin compounds

A new series of 6, 7-dihydroxy-3-(methylphenyl) chromenones, including three new derivatives, i.e. 6,7-dihydroxy-3-(2-methylphenyl)-2H-chromen-2-one (OPC); 6,7-dihydroxy-3-(3-methylphenyl)-2H-chromen-2-one (MPC); 6,7-dihydroxy-3-(4-methylphenyl)-2H-chromen-2-one (PPC) and one previously described, namely 6,7-dihydroxy-3-phenyl-2H-chromen-2-one (DPC), were synthesized. These compounds were investigated as inhibitors of human carbonic anhydrase I (hCA-I) and human carbonic anhydrase II (hCA-II) which had been purified from human erythrocytes on an affinity gel comprised of L-tyrosine-sulfonamide-Sepharose 4B.     

In vitro inhibition of cytosolic carbonic anhydrases I and II by some new dihydroxycoumarin compounds

A new series of 6, 7-dihydroxy-3-(methylphenyl) chromenones, including three new derivatives, i.e. 6,7-dihydroxy-3-(2-methylphenyl)-2H-chromen-2-one (OPC); 6,7-dihydroxy-3-(3-methylphenyl)-2H-chromen-2-one (MPC); 6,7-dihydroxy-3-(4-methylphenyl)-2H-chromen-2-one (PPC) and one previously described, namely 6,7-dihydroxy-3-phenyl-2H-chromen-2-one (DPC), were synthesized. These compounds were investigated as inhibitors of human carbonic anhydrase I (hCA-I) and human carbonic anhydrase II (hCA-II) which had been purified from human erythrocytes on an affinity gel comprised of L-tyrosine-sulfonamide-Sepharose 4B.     

Iridoid glucosides and flavones from the aerial parts of Avicennia marina

Two new iridoid glucosides, namely, 2'-O-[(2E,4E)-5-phenylpenta-2,4-dienoyl]mussaenosidic acid (1; mussaenosidic acid = [1S-(1alpha,4aalpha,7alpha,7aalpha)]-1-(beta-D-glucopyranosyloxy)-1,4a,5,6,7,7a-hexahydro-7-hydroxy-7-methylcyclopenta[c]pyran-4-carboxylic acid) and 2'-O-(4-methoxycinnamoyl)mussaenosidic acid (2), were isolated from the aerial parts of the mangrove plant Avicennia marina. Beside that, one known iridoid glucoside, 2'-O-coumaroylmussaenosidic acid (3) and four known flavones (flavone = 2-phenyl-4H-1-benzopyran-4-one) including 4',5-dihydroxy-3',7-dimethoxyflavone (4), 4',5-dihydroxy-3',5',7-trimethoxyflavone (5), 4',5,7-trihydroxyflavone (6), and 3',4',5-trihydroxy-7-methoxyflavone (7) were also isolated and identified. The structures of these compounds were elucidated by NMR spectroscopy and by low- and high-resolution mass spectrometry. The chemotaxonomic significance of these findings was discussed. In addition, each isolated compound was evaluated for the ability of alpha,alpha-diphenyl-beta-picrylhydrazyl (DPPH) radical-scavenging activity.     

Microwell bioreactor system for cell-based high throughput proliferation and cytotoxicity assays

Cell-based high throughput proliferation and cytotoxicity assays are increasingly used in drug screening and bioprocess development. However, online monitoring of cell proliferation, pH, and dissolved oxygen (DO) has been a challenge in 3D cell-based assays. In this work, a 40-microwell bioreactor (40-MBR) system was developed from a 384-well plate for real-time, noninvasive monitoring of pH, DO, and cell proliferation in 3D microenvironments. Chinese hamster ovary (CHO) and MCF-07 breast cancer cells cultured in 40-MBR confirmed that the 40-MBR was capable of simultaneously monitoring DO and cell proliferation based on culture fluorescence and pH by measuring the absorbance of phenol red. Cytotoxicity studies of sodium butyrate on CHO cells demonstrated that 40-MBR with dynamic background fluorescence correction gave more reliable and highly reproducible growth kinetic data compared to conventional multiwells with static background correction. Furthermore, the dosage effects of two new anticancer drug candidates, 5,7-dihydroxy-2-(4-hydroxyphenyl)-8-[(E)-2-phenylethenyl]-3,4-dihydro-2H-1-benzopyran-4-one (DH-8P-DB) and 5,7-dihydroxy-2-(4-hydroxyphenyl)-6-[(E)-2-phenylethenyl]-3,4-dihydro-2H-1-benzopyran-4-one (DH-6P-DB), on HT-29 colon cancer cells were assessed using the 40-MBR, and the results indicated that DH-6P-DB would be a more potent drug in treating colon cancer than DH-8P-DB. These studies demonstrated that 40-MBR could serve as a high throughput platform for screening potential cancer drugs in early-stage drug discovery.     

Cis-2′, 3′-Dihydrodiol production on flavone B-Ring by Biphenyl Dioxygenase from Pseudomonas pseudoalcaligenes KF707 expressed in Escherichia coli

Escherichia coli JM109 strains expressing either toluene dioxygenase from Pseudomonas putida F1 or biphenyl dioxygenase from Pseudomonas pseudoalcaligenes KF707 were examined for their ability to catalyze flavones. Biphenyl dioxygenase produced metabolites from flavone and 5,7-dihydroxyflavone which were not found in the control experiments. The absorption maxima of UV-visible spectra for the metabolites from flavone and 5,7-dihydroxyflavone were found at 337 and 348 nm respectively by using a photodiode array detector in the HPLC. Liquid chromatography/mass spectroscopy (LC/MS) showed molecular weights 256 and 288 for the metabolites, respectively. The metabolite of flavone, which was isolated and purified from the bacterial culture, was further subjected to analysis by ¹H and ¹³C nuclear magnetic resonance (NMR) spectroscopy. Based on the LC/MS and NMR results, biphenyl dioxygenase inserted oxygen at C2′ and C3′ on the B-ring of flavone, resulting in the formation of flavone cis-2′, 3′-dihydrodiol (2-[3,4-dihydroxy-1.5-cyclohexadienyl]-4H-chromen-4-one). Since this product is not found in Chemical Abstracts, this compound is considered a novel one. In addition, biotransformation of flavones by biphenyl dioxygenase suggested a potential role of bacterial dioxygenase to synthesize novel compounds from plant secondary metabolites. This revised version was published online in June 2006 with corrections to the Cover Date.     

The chemical constituents of the fungus Stereum sp

Four new compounds, including a sesquiterpene and three aromatic compounds, and a known compound were isolated from a culture broth of the fungus Stereum sp. The novel sesquiterpene was determined to be stereumone A ((+)-2,3,4a,5,6,7,8a,9-octahydro-5-hydroxy-6,6,9-trimethyl-4,8a-epoxynaphtho[2,3-b]furan-8(8H)-one; 1), and the three new aromatic compounds were elucidated as 3,5-dihydroxy-4-(3-methylbut-2-enyl)benzene-1,2-dicarbaldehyde (2), 5,7-dihydroxy-6-(3-methylbut-2-enyl)isobenzofuran-1(3H)-one (3), butyl 2,4-dihydroxy-6-methylbenzoate (4), together with the known compound methyl 2,4-dihydroxy-6-methylbenzoate (5). The structures were established by spectroscopic methods including 2D-NMR techniques. Compounds 2 and 4 showed evident nematicidal activity against nematode Panagrellus redivivus.     

Homoisoflavanones from Disporopsis aspera

From cytotoxic extracts of the roots of Disporopsis aspera Engl. (Liliaceae) a homoisoflavanone, disporopsin (3-(2',4'-dihydroxy-benzyl)-5,7-dihydroxy-chroman-4-one) (1) and three rare methyl-homoisoflavanones, 3-(4'-hydroxy-benzyl)-5,7-dihydroxy-6-methyl-chroman-4-one (2), 3-(4'-hydroxy-benzyl)-5,7-dihydroxy-6,8-dimethyl-chroman-4-one (3) and 3-(4'-hydroxy-benzyl)-5,7-dihydroxy-6-methyl-8-methoxy-chroman-4- one (4) along with five other known compounds, N-trans-feruloyl tyramine (5), adenine (6), 5-(hydroxymethyl)-2-furfural (7), beta-sitosterol (8) and beta-sitosteryl glucopyranoside (9) were isolated. The structures of compounds 1-2 were elucidated by spectral data (1, 2-D NMR and EIMS). The four homoisoflavanones (1-4) were found to be cytotoxic against a series of human cancer cell lines (HCT15, T24S, MCF7, Bowes, A549 and K562) with IC(50) ranging from 15 to 200 microM. Possible biosynthesis routes for homoisoflavonoids (1-4) are discussed.     

Antibacterial and cytotoxic activity of prenylated bicyclic acylphloroglucinol derivatives from Hypericum amblycalyx

Two new bicyclic acylphloroglucinol derivatives, hypercalyxone A (1-[5,7-dihydroxy-2-methyl-3-(3-methyl-but-2-enyl)-2-(4-methyl-pent-3-enyl)-chroman-8-yl]-2-methyl-propan-1-one, 1) and B (1-[5,7-dihydroxy-2-methyl-3-(3-methyl-but-2-enyl)-2-(4-methyl-pent-3-enyl)-chroman-8-yl]-2-methyl-butan-1-one, 2), have been isolated from the petroleum ether extract of the aerial parts of Hypericum amblycalyx, together with two further compounds (1-[5,7-dihydroxy-2-methyl-2-(4-methyl-pent-3-enyl)-chroman-8-yl]-2-methyl-propan-1-one, 3 and 1-[5,7-dihydroxy-2-methyl-2-(4-methyl-pent-3-enyl)-chroman-8-yl]-2-methyl-butan-1-one, 4), which have been described only as semi-synthetic products. In addition, the known triterpene lup-20(29)-en-3-one was obtained. Structure elucidation was based on 1D and 2D NMR studies, as well as on data derived from mass spectrometry. The four acylphloroglucinol derivatives were evaluated for their cytotoxic and antibacterial activity. All compounds showed moderate cytotoxic activity against KB and Jurkat T cancer cells. Especially compounds 3 and 4 exhibited a strong antibacterial activity against different Gram-positive strains.     

Homoisoflavonoids from the medicinal plant Portulaca oleracea

Four homoisoflavonoids named portulacanones A−D, identified as 2′-hydroxy- 5,7-dimethoxy-3-benzyl-chroman-4-one, 2′-hydroxy-5,6,7-trimethoxy-3-benzyl-chroman-4-one, 5,2′-dihydroxy-6,7-dimethoxy-3-benzyl-chroman-4-one, and 5,2′-dihydroxy-7-methoxy-3-benzylidene-chroman-4-one, were isolated from aerial parts of the plant Portulaca oleracea along with nine other known metabolites. Their structures were established on the basis of extensive spectroscopic analyses. Portulacanones A−D is the first group of homoisoflavonoids so far reported from the family Portulacaceae. They represent a rare subclass of homoisoflavonoids in nature with a structural feature of a single hydroxyl group substituted at C-2′ rather than at C-4′ in ring B of the skeleton. Three homoisoflavonoids and the known compound 2,2′-dihydroxy-4′,6′-dimethoxychalcone selectively showed in vitro cytotoxic activities towards four human cancer cell lines. Especially 2,2′-dihydroxy-4′,6′-dimethoxychalcone showed cytotoxic activity against cell line SGC-7901 with an IC₅₀ value of 1.6 μg/ml, which was more potent than the reference compound mitomycin C (IC₅₀ 13.0 μg/ml).     

Homoisoflavonoids and xanthones from the tubers of wild and in vitro regenerated Ledebouria graminifolia and cytotoxic activities of some of the homoisoflavonoids

Eleven homoisoflavonoids and two xanthones were isolated and characterized from the bulbs of Ledebouria graminifolia. Five of the homoisoflavonoids are new compounds and were identified as: 5-hydroxy-7-methoxy-3-(4'-hydroxybenzyl)-4-chromanone, 5-hydroxy-6,7-dimethoxy-3-(4'-hydroxybenzyl)-4-chromanone, 5,7,8-trimethoxy-3-(4'-hydroxybenzyl)-4-chromanone, 5-hydroxy-3',4',7-trimethoxyspiro[2H-1-benzopyran-7'-bicyclo[4.2.0]octa-trien]-4-one, 5,7-dihydroxy-3',4'-dimethoxyspiro[2H-1-benzopyran-7'-bicyclo[4.2.0]octa-trien]-4-one. Structures were elucidated by extensive 1D, and 2D NMR spectroscopy and HRMS. A method for tissue culture was developed and the bulbs of mature plants were found to contain all the compounds isolated from the wild specimens of L. graminifolia.     

Homoisoflavonoids from Ophiopogon japonicus Ker-Gawler

From the ethyl acetate extract of the tuberous roots of Ophiopogon japonicus (Liliaceae) eight known and five new homoisoflavonoidal compounds were isolated. The new compounds are 5,7-dihydroxy-8-methoxy-6-methyl-3-(2'-hydroxy-4'-methoxybenzyl)chroman-4-one (1), 7-hydroxy-5,8-dimethoxy-6-methyl-3-(2'-hydroxy-4'-methoxybenzyl)chroman-4-one (2), 5,7-dihydroxy-6,8-dimethyl-3-(4'-hydroxy-3'-methoxybenzyl)chroman-4-one (3), 2,5,7-trihydroxy-6,8-dimethyl-3-(3',4'-methylenedioxybenzyl)chroman-4-one (4) and 2,5,7-trihydroxy-6,8-dimethyl-3-(4'-methoxybenzyl)chroman-4-one (5). Their structures have been elucidated by mass and NMR spectroscopy. Compounds 4 and 5 are the first isolated homoisoflavonoids with a hemiacetal function at position 2.     

Flavonoids from Goodyera schlechtendaliana

A flavonol glycoside, 3-[[6-O-(6-deoxy-alpha-L-mannopyranosyl)-beta-D- glucopyranosyl]oxi]-5,7-dihydroxy-8-[(4-hydroxy-3,5-dimethoxyphenyl)meth yl]- 2-(3,4-dihydroxypheny)-4H-1-benzopyran-4-one, trivially named goodyerin, was isolated from the whole plant of Goodyera schlechtendaliana, along with three known flavonoids, rutin, kaempferol-3-O-rutinoside and isorhamnetin-3-O-rutinoside. The structures were established by spectroscopic analysis.     

Assessment of estrogenic activity of flavonoids from Mediterranean plants using an in vitro short-term test

Six isoflavones, daidzein (4',7,-dihydroxyisoflavone), genistein (4',5,7-trihydroxyisoflavone), genistin (genistein 7-O-beta-D-glucopyranoside), isoprunetin (4',7-dihydroxy, 5-metoxyisoflavone), isoprunetin 7-O-beta-D-glucopyranoside, isoprunetin 4',7-di-O-beta-D-glucopyranoside and four flavones, luteolin (3',4',5,7-tetrahydroxyflavone), luteolin 7-O-beta-D-glucopyranoside, luteolin 4'-O-beta-D-glucopyranoside, licoflavone C (4',5,7-trihydroxy,8-isoprenylflavone) were purified from Mediterranean plants (Genista morisii and Genista ephedroides) and their estrogenic activity was assessed by a yeast reporter gene assay (Saccharomyces cerevisiae RMY326 ER-ERE). Licoflavone C showed a powerful estrogenic activity at 10(-7) M (0.0338 microg/ml) and it was 47.45% than 10(-8) M 17beta-estradiol (0.00272 microg/ml). The estrogenicity of this flavone was found to be comparable to the activity showed by genistein at 10(-6) M (0.27 microg/ml). This study points out that a glucose substituent in flavones and isoflavones modulates the hormone-like activity in a different way. Isoflavone aglycones showed a more estrogenic activity than the corresponding glucosides. Conversely, the glucosidation made estrogenic the flavone luteolin and the position of substitution differently influenced the estrogenic activity of compounds.     

Homoisoflavonoids from the inter-bulb surfaces of Scilla nervosa subsp. rigidifolia

Eight homoisoflavonoids, two of which are new: 3-(4′-methoxybenzyl)-5,6,7-trimethoxychroman-4-one (1); 3-(4′-methoxybenzyl)-5,7-dimethoxychroman-4-one (2); 3-(4′-methoxybenzyl)-7-hydroxy-5,6-dimethoxychroman-4-one (3); 3-(4′-methoxybenzyl)-6-hydroxy-5,7-dimethoxychroman-4-one (4); 3-(3′-hydroxy-4′-methoxybenzyl)-5,7-dihydroxy-6-methoxychroman-4-one (5); 3-(3′-hydroxy-4′-methoxybenzyl)-5,7-dihydroxychroman-4-one (6); 3-(4′-hydroxybenzylidene)-5,7-dihydroxy-6-methoxychroman-4-one (7) and 3-(4′-hydroxybenzylidene)-5,7-dihydroxychroman-4-one (8), were isolated from the yellow Inter-bulb deposits from Scilla nervosa. The structures of these compounds were elucidated and characterized by 1D- and 2D-NMR and mass spectrometry. The structures of the known compounds were compared to those ones in literature.     

Effective cytochrome P450 (CYP) inhibitor isolated from thyme (Thymus saturoides) purchased from a Japanese market

A highly polymethylated flavone that effectively inhibited cytochrome P450s (CYPs) 1A2 and 3A4 (IC(50) = 2.41 and 1.71 μM) in vitro was isolated from thyme leaves (Thymus saturoides) purchased from a Japanese market. Its structure was spectroscopically identified as 4',5-dihydroxy-3',6,7,8-tetramethoxy flavone (8-methoxycirsilineol, 1). This is the first report describing a strong inhibitor of CYP1A2 and 3A4 isolated from Thymus saturoides.     

细花线纹香茶菜的化学成分研究

用色谱技术对细花线纹香茶菜(Isodon lophanthoides var.graciliflora)全草水提物进行分离纯化,获得了11种单体化合物。通过光谱分析及与文献数据对照,分别鉴定为牡荆素(vitexin,1)、藿香黄酮醇(pachypodol,2)、猫眼草黄素(chrysoplenetin,3)、9-羟基-5,7-豆甾二烯-4-酮(9-hydroxy-5,7-megastigmadien-4-one,4)、3,9-二羟基-5,7-豆甾二烯-4-酮(3,9-dihydroxy-5,7-megastigmadien-4-one,5)、黑麦草内酯(loliolide,6)、4-乙酰-5-甲基二氢呋喃-2-酮(4-acetyl-5-methyldihydrofuran-2-one,7)、5-羟甲基糠醛(5-hydroxymethylfuraldehyde,8)、5-甲氧甲基-1H-吡咯-2-甲醛(5-methoxymethyl-1H-pyrrole-2-carbaldehyde,9)、3-吲哚甲醛(indole-3-aldehyde,10)和丁二酸单丁酯(succini cacid monobutyl ester,11)。这些化合物均为首次从线纹香茶菜中分离得到。