2,3��,4,4��,5��-Pentamethoxy-trans-stilbene, a resveratrol derivative, inhibits colitis-associated colorectal carcinogenesis in mice

Background and purpose:

Resveratrol, a naturally occurring polyphenolic antioxidant, has been shown to exhibit chemoprophylactic effects on cancer development. Previously, we reported that 2,3′,4,4′,5′-pentamethoxy-trans-stilbene (PMS), a methoxylated resveratrol derivative, exerted a highly potent anti-proliferative effect on human colon cancer cells as compared with its parent compound. In the present study, the chemopreventive effect of PMS was evaluated in a mouse model of colitis-associated colon carcinogenesis.

Experimental approach:

Seven-week-old Balb/c mice were injected i.p. with 10 mg·kg⁻¹ azoxymethane (AOM). After 1 week, 3% dextran sodium sulphate (DSS) was administered in the drinking water for 7 days followed by 14 days of tap water for recovery, and this cycle was repeated twice.

Key results:

Intragastric administration of PMS (25, 50 mg·kg⁻¹ body weight) for 16 weeks significantly reduced the multiplicity of colonic neoplasms by 15% and 35% (P < 0.01) respectively. Moreover, PMS at 50 mg·kg⁻¹ inhibited colon cancer cell proliferation and promoted apoptosis. Such changes were accompanied by reduction of Akt (protein kinase B) phosphorylation, inactivation of β-catenin and down-regulation of inducible nitric oxide synthase. In parallel, in vitro studies also demonstrated that PMS inhibited proliferation and induced apoptosis in the murine colon adenocarcinoma cell line Colon26 with concomitant inhibition of Akt phosphorylation and inactivation of β-catenin.

Conclusions and implications:

PMS effectively suppressed colon carcinogenesis in an AOM/DSS animal model and may merit further clinical investigation as a chemoprophylactic agent against colitis-associated colon cancer in humans.     

Hop proanthocyanidins induce apoptosis,protein carbonylation,and cytoskeleton disorganization in human colorectal adenocarcinoma cells via reactive oxygen species

Proanthocyanidins (PCs) have been shown to suppress the growth of diverse human cancer cells and are considered as promising additions to the arsenal of chemopreventive phytochemicals. An oligomeric mixture of PCs from hops (Humulus lupulus) significantly decreased cell viability of human colon cancer HT-29 cells in a dose-dependent manner. Hop PCs, at 50 or 100 μg/ml, exhibited apoptosis-inducing properties as shown by the increase in caspase-3 activity. Increased levels of intracellular reactive oxygen species (ROS) was accompanied by an augmented accumulation of protein carbonyls. Mass spectrometry-based proteomic analysis in combination with 2-alkenal-specific immunochemical detection identified β-actin and protein disulfide isomerase as major putative targets of acrolein adduction. Incubation of HT-29 cells with hop PCs resulted in morphological changes that indicated disruption of the actin cytoskeleton. PC-mediated hydrogen peroxide (H₂O₂) formation in the cell culture media was also quantified; but, the measured H₂O₂ levels would not explain the observed changes in the oxidative modifications of actin. These findings suggest new modes of action for proanthocyandins as anticarcinogenic agents in human colon cancer cells, namely, promotion of protein oxidative modifications and cytoskeleton derangement.     

Constituents of Corydalis heterocarpa and their anti-proliferative effects on human cancer cells

Two new coumarins, 1 and 2, along with four known coumarins (3–6) have been isolated from Corydalis heterocarpa. On the basis of spectroscopic and chemical methods, compounds 1 and 2 were elucidated as (2′S,7′S)-O-2-methylbutanoyl-columbianetin and (2′S)-columbianetin-3′-sulfate, respectively. The anti-proliferative activity against human cancer cells of compounds 1–6 isolated from C. heterocarpa was evaluated using a MTT assay and by mRNA expression of several factors related to apoptosis. Among them, compound 2 exerted the more potent anti-proliferative activity compared with the other compounds treated. The potent inhibitory effect of compound 2 was produced by induction of apoptosis through activating Bax, p53 and p21 expressions.     

Distinct interactions of 2′- and 3′-O-(N-methyl)anthraniloyl-isomers of ATP and GTP with the adenylyl cyclase toxin of Bacillus anthracis, edema factor

Anthrax disease is caused by the spore-forming bacterium, Bacillus anthracis. B. anthracis produces a calmodulin-activated adenylyl cyclase (AC) toxin, edema factor (EF). Through excessive cAMP accumulation EF disrupts host defence. In a recent study [Taha HM, Schmidt J, Göttle M, Suryanarayana S, Shen Y, Tang WJ, et al. Molecular analysis of the interaction of anthrax adenylyl cyclase toxin, edema factor, with 2′(3′)-O-(N-(methyl)anthraniloyl)-substituted purine and pyrimidine nucleotides. Mol Pharmacol 2009;75:693-703] we showed that various 2′(3′)-O-N-(methyl)anthraniloyl (MANT)-substituted nucleoside 5′-triphosphates are potent inhibitors (K_i values in the 0.1-5 μM range) of purified EF. Upon interaction with calmodulin we observed efficient fluorescence resonance energy transfer (FRET) between tryptophan and tyrosine residues of EF and the MANT-group of MANT-ATP. Molecular modelling suggested that both the 2′- and 3′-MANT-isomers can bind to EF. The aim of the present study was to examine the effects of defined 2′- and 3′-MANT-isomers of ATP and GTP on EF. 3′-MANT-2′-deoxy-ATP inhibited EF more potently than 2′-MANT-3′-deoxy-ATP, whereas the opposite was the case for the corresponding GTP analogs. Calmodulin-dependent direct MANT fluorescence and FRET was much larger with 2′-MANT-3′-deoxy-ATP and 2′-MANT-3′-deoxy-GTP compared to the corresponding 3′-MANT-2′-deoxy-isomers and the 2′(3′)-racemates. K_i values of MANT-nucleotides for inhibition of catalysis correlated with K_d values of MANT-nucleotides in FRET studies. Molecular modelling indicated different positioning of the MANT-group in 2′-MANT-3′-deoxy-ATP/GTP and 3′-MANT-2′-deoxy-ATP/GTP bound to EF. Collectively, EF interacts differentially with 2′- and 3′-MANT-isomers of ATP and GTP, indicative for conformational flexibility of the catalytic site and offering a novel approach for the development of potent and selective EF inhibitors. Moreover, our present study may serve as a general model of how to use MANT-nucleotide isomers for the analysis of the molecular mechanisms of nucleotide/protein interactions.     

Induction of apoptosis by phloroglucinol derivative from Ecklonia Cava in MCF-7 human breast cancer cells

Phloroglucinol derivatives, dioxinodehydroeckol (1) and 1-(3′,5′-dihydroxyphenoxy)-7-(2′′,4′′,6-trihydroxyphenoxy)-2,4,9-trihydroxydibenzo-1,4-dioxin (2), were isolated from Ecklonia Cava. Their ability to inhibit the proliferation of human breast cancer cells were evaluated by measuring cell death via induction of apoptosis. Compound 1 exerted a higher anti-proliferative activity in human breast cancer cells compared with compound 2. Furthermore, compound 1 induced a significant proliferative inhibition and apoptosis in a dose-dependent manner on MCF-7 human cancer cells. Treatment with compound 1 also induced the increase in caspase (-3 and -9) activity, DNA repair enzyme poly-(ADP-ribose) polymerase (PARP) cleavage, and pro-apoptotic gene and the decrease in anti-apoptotic gene. In addition, NF-κB family and -dependent activated genes were down-regulated by compound 1. These results indicated that the potential inhibitory effect of compound 1 against growth of MCF-7 human breast cancer cells might be associated with induction of apoptosis through NF-κB family and NF-κB dependent pathway. The present results suggest that compound 1 has a promising potential to be used as a valuable chemopreventive agent.     

Cis- and trans-1,2-diphenylaziridines: induction of xenobiotic-metabolizing enzymes in rat liver and mutagenicity in Salmonella typhimurium

trans-Stilbene imine (trans-1,2-diphenylaziridine) is the nitrogen analog of trans-stilbene oxide, a potent inducer of several microsomal and cytosolic xenobiotic-metabolizing enzymes. Although the acute toxicity of cis- and trans-stilbene imines prevents their application at the usual dose for trans-stilbene oxide (400 mg/kg/day), it is apparent that the imines nevertheless potently induce several xenobiotic-metabolizing enzymes in rat liver. The IP administration of trans-stilbene imine resulted in statistically significant increases in the activities of aminopyrine N-demethylase, microsomal epoxide hydrolase, glutathione transferase (toward 1-chloro-2,4-dinitrobenzene, 1,2-dichloro-4-nitrobenzene and ⁵-androstene-3,17-dione) and UDP-glucuronosyltransferase (toward testoster-one). cis-Stilbene imine was less potent in inducing these activities. Although trans-stilbene imine (total dose = 400 mg/kg) was more potent than trans-stilbene oxide (total dose = 1200 mg/kg) in inducing the activities of glutathione transferase (toward 1-chloro-2,4-dinitrobenzene) and UDP-glucuronosyltransferase (toward testosterone), both compounds belong to the class of substances which are more potent inducers of conjugating (phase II) enzymes.Because of their structural similarity with K-region arene imines which are potent mutagens, cis-stilbene imine and trans-stilbene imine were investigated for mutagenicity (reversion of his ^– strains of Salmonella typhimurium). cis-Stilbene imine and trans-stilbene imine were direct mutagens in the strain TA100. This result, and the finding that acenaphthene 1,2-imine efficiently reverts various strains of Salmonella typhimurium, demonstrates that not only K-region arene imines, but also other aziridines substituted at the two carbons with aromatic moieties, are mutagenic.Abbreviations CSI cis-stilbene imine (cis-2,3-diphenylaziridine) - CSO cis-stilbene oxide - GSH-Px Glutathion peroxidase (activity contributed by a selenium containing enzyme, E. C.1.11.1.9, and certain GST subunits) - GST glutathione transferase (E.C.2.5.1.18) - MC 3-methylcholanthrene - PB phenobarbital - TSI trans-stilbene imine (trans-2,3-diphenylaziridine) - TSO trans-stilbene oxide - UDP-GT uridine-5-diphosphate glucurono-syltransferase (E.C.2.4.1.17) Part of Ph. D. Theses of M. A., P. R., and H. S.     

Synthesis and pharmacological characterization of [I]MRS5127, a high affinity, selective agonist radioligand for the A3 adenosine receptor

A recently reported selective agonist of the human A₃ adenosine receptor (hA₃AR), MRS5127 (1′R,2′R,3′S,4′R,5′S)-4′-[2-chloro-6-(3-iodobenzylamino)-purine]-2′,3′-O-dihydroxy-bicyclo-[3.1.0]hexane, was radioiodinated and characterized pharmacologically. It contains a rigid bicyclic ring system in place of a 5′-truncated ribose moiety, and was selected for radiolabeling due to its nanomolar binding affinity at both human and rat A₃ARs. The radioiodination of the N⁶-3-iodobenzyl substituent by iododestannylation of a 3-(trimethylstannyl)benzyl precursor was achieved in 73% yield, measured after purification by HPLC. [¹²⁵I]MRS5127 bound to the human A₃AR expressed in membranes of stably transfected HEK 293 cells. Specific binding was saturable, competitive, and followed a one-site binding model, with a K_d value of 5.74 ± 0.97 nM. At a concentration equivalent to its K_d, non-specific binding comprised 27 ± 2% of total binding. In kinetic studies, [¹²⁵I]MRS5127 rapidly associated with the hA₃AR (t_1/2 = 0.514 ± 0.014 min), and the affinity calculated from association and dissociation rate constants was 3.50 ± 1.46 nM. The pharmacological profile of ligands in competition experiments with [¹²⁵I]MRS5127 was consistent with the known structure-activity-relationship profile of the hA₃AR. [¹²⁵I]MRS5127 bound with similar high affinity (K_d, nM) to recombinant A₃ARs from mouse (4.90 ± 0.77), rabbit (2.53 ± 0.11), and dog (3.35 ± 0.54). For all of the species tested, MRS5127 exhibited A₃AR agonist activity based on negative coupling to cAMP production. Thus, [¹²⁵I]MRS5127 represents a new species-independent agonist radioligand for the A₃AR. The major advantage of [¹²⁵I]MRS5127 compared with previously used A₃AR radioligands is its high affinity, low degree of non-specific binding, and improved A₃AR selectivity.     

Effects of Resveratrol and trans-3,5,4’-Trimethoxystilbene on Glutamate-Induced Cytotoxicity,Heme Oxygenase-1, and Sirtuin 1 in HT22 Neuronal Cells

Resveratrol (trans-3,5,4’-trihydroxystilbene) has received considerable attention recently for the potential neuroprotective effects in neurodegenerative disorders where heme oxygenase-1 (HO-1) and sirtuin 1 (SIRT1) represent promising therapeutic targets. Resveratrol has been known to increase HO-1 expression and SIRT1 activity. In this study, the effects of resveratrol and trans-3,5,4’-trimethoxystilbene (TMS), a resveratrol derivative, on cytotoxicity caused by glutamate-induced oxidative stress, HO-1 expression, and SIRT1 activation have been investigated by using murine hippocampal HT22 cells, which have been widely used as an in vitro model for investigating glutamate-induced neurotoxicity. Resveratrol protected HT22 neuronal cells from glutamateinduced cytotoxicity and increased HO-1 expression as well as SIRT1 activity in a concentration-dependent manner. Cytoprotec-tion afforded by resveratrol was partially reversed by the specific inhibition of HO-1 expression by HO-1 small interfering RNA and the nonspecific blockage of HO-1 activity by tin protoporphyrin IX, but not by SIRT1 inhibitors. Surprisingly, TMS, a resveratrol derivative with methoxyl groups in lieu of the hydroxyl groups, and trans-stilbene, a non-hydroxylated analog, failed to protect HT22 cells from glutamate-induced cytotoxicity and to increase HO-1 expression and SIRT1 activity. Taken together, our findings suggest that the cytoprotective effect of resveratrol was at least in part associated with HO-1 expression but not with SIRT1 activation and, importantly, that the presence of hydroxyl groups on the benzene rings of resveratrol appears to be necessary for cytoprotection against glutamate-induced oxidative stress, HO-1 expression, and SIRT1 activation in HT22 neuronal cells.     

A simple and sensitive HPLC-UV method for the quantification of piceatannol analog trans-3,5,3′,4′-tetramethoxystilbene in rat plasma and its application for a pre-clinical pharmacokinetic study

A simple and sensitive HPLC-UV method was developed and validated for the quantification of piceatannol analog trans-3,5,3′,4′-tetramethoxystilbene (M-PIC) in rat plasma. Following protein precipitation with three volumes of acetonitrile, the analytes were separated on a RP-HPLC column, which was protected by a guard column through gradient delivery of a mixture of acetonitrile–water at 40 °C. The UV absorbance at 325 nm was recorded to quantify M-PIC. The retention time of M-PIC and trans-3,5-dimethoxystilbene (internal standard) was 7.4 and 8.4 min, respectively. The calibration curves were linear (R² > 0.9989) with a lower limit of quantification of 15 ng/ml. The intra- and inter-day precisions, in terms of RSD, were all lower than 7.5%. The average analytical recovery ranged from 97.0 to 104.3% while the average absolute recovery ranged from 101.8 to 105.0%. This reliable HPLC method was subsequently applied to assess the pharmacokinetic profile of M-PIC in Sprague–Dawley rats using 2-hydroxypropyl-β-cyclodextrin as a dosing vehicle. The terminal elimination half-life (t_1/2λz) and clearance (Cl) of M-PIC were 313 ± 20 min and 33.1 ± 3.9 ml/min/kg, respectively; and its absolute oral bioavailability was as high as 50.7 ± 15.0%. M-PIC appeared to have a favorable pharmacokinetic profile and further pharmacological investigation on this phyto-stilbene was warranted.     

SL-01, an oral gemcitabine derivative, inhibited human cancer growth more potently than gemcitabine

SL-01, an oral gemcitabine derivative, was synthesized by introducing the moiety of 3-(dodecyloxycarbonyl)pyrazine-2-carbonyl at the N4-position on the cytidine ring of gemcitabine. Our goal in this study was to evaluate the efficacy of SL-01 on the growth of human cancers with gemcitabine as control. Experiments were performed on human non-small cell lung cancer NCI-H460 and colon cancer HCT-116 both in vitro and in vivo. In vitro assays, SL-01 significantly inhibited the growth of cancer cells as determined by the 3-[4, 5-dimethylthiazol-2-yl]-2, 5-diphenyltetrazolium bromide (MTT) assay. Further studies indicated that SL-01 induced the cancer cells to apoptosis showing chromatin condensation and externalization of phosphatidylserine. In in vivo studies, we evaluated the efficacy of SL-01 in nude mice bearing human cancer xenografts. SL-01 effectively delayed the growth of NCI-H460 and HCT-116 without significant loss of body weight. Molecular analysis indicated that the high efficacy of SL-01 was associated with its ability to induce apoptosis as evidenced by increase of terminal deoxynucleotidyl transferase-mediated dUTP nick end labeling (TUNEL) staining cells, activation of caspase-9, caspase-3 and cleaved poly ADP-ribose polymerase (PARP) in tumor tissues. SL-01 also increased Bax/Bcl-2 ratio in cancer cells. These biological activities of SL-01 were more potential than that of gemcitabine. Based on these in vitro and in vivo results, SL-01 is proposed as a potent oral anticancer agent that may supplant the use of gemcitabine in the clinic.     

A rapid HPLC method for the quantification of 3,5,4′-trimethoxy-trans-stilbene (TMS) in rat plasma and its application in pharmacokinetic study

A rapid HPLC-UV method had been developed and validated to quantify 3,5,4′-trimethoxy-trans-stilbene (TMS), a naturally occurring and pharmacologically active analog of resveratrol in rat plasma. The samples were mixed with three volumes of acetonitrile to precipitate protein. Chromatographic separation was achieved on a RP-HPLC column (Agilent ZORBAX Eclipse Plus C18: 250 mm × 4.6 mm i.d., 5 μm), which was protected by a guard column (Agilent ZORBAX Eclipse Plus C18: 12.5 mm × 4.6 mm i.d., 5 μm) through isocratic delivery of a mobile phase of acetonitrile: water (75:25, v/v) at a flow rate of 1.2 ml/min. The assay was executed at 30 °C and the UV absorbance at 320 nm was monitored. The retention time of TMS and trans-stilbene (internal standard) was 6.5 and 8.3 min, respectively. The calibration curve was linear within the range of 15–1000 ng/ml (R² > 0.998) and 15 ng/ml was the lower LOQ. The intra- and inter-day precisions were good and the RSD was all lower than 7.3%. The mean absolute recovery of TMS in plasma ranged from 99.2 to 104.1%. This HPLC method had been successfully applied to study the pharmacokinetics of TMS, which was fully dissolved with hydroxypropyl-β-cyclodextrin (HP-β-CyD). In comparison with resveratrol, TMS had greater plasma exposure, longer elimination half-life and lower clearance. As TMS had superior pharmacokinetic characteristics, its potential as a preventive or therapeutic agent in resveratrol-effective conditions or diseases should be considered.     

Physalin B not only inhibits the ubiquitin-proteasome pathway but also induces incomplete autophagic response in human colon cancer cells in vitro

Aim:

To investigate the effects of physalin B insolated from Physalis divericata on human colon cancer cells in vitro and its anticancer mechanisms.

Methods:

Human HCT116 colon cancer cell line was tested. Cell viability and apoptosis were detected, and relevant proteins were measured using Western blot analyses. Autophagosomes were observed in stable GFP-LC3 HCT116 cells. Localization of autophagosomes and lysosomes was evaluated in GFP-LC3/RFP-LAMP1-co-transfected cells. Microtubules and F-actin microfilaments were observed with confocal microscope. Mitochondrial ROS (mito-ROS) was detected with flow cytometry in the cells stained with MitoSox dye.

Results:

Physalin B inhibited the viability of HCT116 cells with an IC₅₀ value of 1.35 μmol/L. Treatment of the cells with physalin B (2.5–10 μmol/L) induced apoptosis and the cleavage of PARP and caspase-3. Meanwhile, physalin B treatment induced autophagosome formation, and accumulation of LC3-II and p62, but decreased Beclin 1 protein level. Marked changes of microtubules and F-actin microfilaments were observed in physalin B-treated cells, which led to the blockage of co-localization of autophagosomes and lysosomes. Physalin B treatment dose-dependently increased the phosphorylation of p38, ERK and JNK in the cells, whereas the p38 inhibitor SB202190, ERK inhibitor U0126 or JNK inhibitor SP600125 could partially reduce physalin B-induced PARP cleavage and p62 accumulation. Moreover, physalin B treatment dose-dependently increased mito-ROS production in the cells, whereas the ROS scavenger NAC could reverse physalin B-induced effects, including incomplete autophagic response, accumulation of ubiquitinated proteins, changes of microtubules and F-actin, activation of p38, ERK and JNK, as well as cell death and apoptosis.

Conclusion:

Physalin B induces mito-ROS, which not only inhibits the ubiquitin-proteasome pathway but also induces incomplete autophagic response in HCT116 cells in vitro.     

Quantification of 4′-geranyloxyferulic acid,a new natural colon cancer chemopreventive agent,by HPLC-DAD in grapefruit skin extract

Oxyprenylated natural products (isopentenyloxy-, geranyloxy- and the less spread farnesyloxy-compounds and their biosynthetic derivatives) represent a family of secondary metabolites that have been consider for years merely as biosynthetic intermediates of the most abundant C-prenylated derivatives. Many of the isolated oxyprenylated natural products were shown to exert in vitro and in vivo remarkable anti-cancer and anti-inflammatory effects. 4′-Geranyloxyferulic acid [3-(4′-geranyloxy-3′-methoxyphenyl)-2-trans-propenoic] has been discovered as a valuable chemopreventive agent of several types of cancer. After development of a high yield and “eco-friendly” synthetic scheme of this secondary metabolite, starting from cheap and non-toxic reagents and substrates, we developed a new HPLC-DAD method for its quantification in grapefruit skin extract. A preliminary study on C18 column showed the separation between GOFA and boropinic acid (having the same core but with an isopentenyloxy side chain), used as internal standard. The tested column were thermostated at 28 ± 1 °C and the separation was achieved in gradient condition at a flow rate of 1 mL/min with a starting mobile phase of H₂O:methanol (40:60, v/v, 1% formic acid). The limit of detection (LOD, S/N = 3) was 0.5 μg/mL and the limit of quantification (LOQ, S/N = 10) was 1 μg/mL. Matrix-matched standard curves showed linearity up to 75 μg/mL. In the analytical range the precision (RSD%) values were ≤2% and the accuracy (bias%) between ±12%. This method was used to evaluate for the first time the presence of this analyte in natural extract of grapefruit. In conclusion, this method showed LOQ values able to selective quantification of this analyte in grapefruit skin extract.     

Characterization of the microsomal cytochrome p-450 species induced in rat liver by trans-stilbene oxide

trans-Stilbene oxide differs from the classical inducers of drug-metabolizing enzymes, pheno-barbital and 3-methylcholanthrene, in that it induces the so-called phase II activities, epoxide hydrolase and glutathione S-transferase, to a much larger extent than it induces cytochrome P-450. Nonetheless, the level of cytochrome P-450 in liver microsomes from rats treated with trans-stilbene oxide is increased significantly to twice the control value.The existence of a number of different isozymes of cytochrome P-450 has now been clearly demonstrated and in the present study we have posed the question: What form(s) of cytochrome P-450 is induced by trans-stilbene oxide? A number of criteria including substrate specificity, pattern of benzo(a)pyrene metabolism, sensitivity to inhibitors, substrate binding spectra, ethylisocyanide binding spectra, sodium dodecyi sulfate-polyacrylamide gel electrophoresis, and crossed immunoelectrophoresis were used to answer this question. It seems clear that trans-stilbene oxide induces the same form(s) of cytochrome P-450 as phenobarbital.     

具有Src激酶和NO合酶双重抑制作用的4-芳杂胺-3-氰基喹啉类抗肿瘤化合物的设计、合成与生物活性研究

Src与iNOS为肿瘤发生、转移中位于不同通路的重要靶酶,本文采用分子拼合的药物设计原理,设计合成了全新的酪氨酸Src蛋白激酶与iNOS的双重抑制剂。所设计合成的化合物经过Src激酶和iNOS的抑制活性检测及体外抗肿瘤测试,实验结果表明大部分化合物对于两种靶酶均表现出一定的抑制活性,部分化合物对于多种肿瘤细胞的增殖有一定的抑制作用。其中化合物33对Src激酶和iNOS均有比较好的抑制活性,对于肝癌HepG2和结肠癌HT-29细胞的增殖也有明显的抑制作用。     

Effect of natural analogues of trans-resveratrol on cytochromes P4501A2 and 2E1 catalytic activities

The aim was to assess the inhibitory effect of a series of naturally occurring trans-resveratrol analogues on cytochromes P450, namely CYP1A2 and CYP2E1, in vitro in order to analyse any structure–activity relationships. 3,5-Dimethoxy-4′-hydroxy-trans-stilbene (pterostilbene), 3,4′,5-trimethoxy-trans-stilbene (TMS), 3,4′-dihydroxy-5-methoxy-trans-stilbene (3,4′-DH-5-MS) and 3,5-dihydroxy-4′-methoxy-trans-stilbene (3,5-DH-4′-MS) inhibited the activity of CYP1A2, with K_i?=?0.39, 0.79, 0.94 and 1.04?µM, respectively. Piceatannol (3,3′,4,5′-tetrahydroxy-trans-stilbene) was the least potent inhibitor of CYP1A2 with a K_i?=?9.67?µM. Piceatannol and TMS in the concentration range 1–100?µM did not inhibit CYP2E1 activity. The activity of this enzyme likewise was not significantly influenced by pterostilbene and 3,5-DH-4′-MS with IC₅₀?>?100?µM, whereas 3,4′-DH-5-MS appeared to be a moderately potent, competitive inhibitor of CYP2E1 (K_i?=?42.6?µM). Structure–activity relationship analysis leads to the conclusion that the substitution of hydroxy groups of resveratrol with methoxy groups increases the inhibition of CYP1A2, yet the number and position of methylation are not essential. However, the 4′-hydroxy group in trans-resveratrol and its analogues may play an important role in the interaction with a binding site of CYP2E1.     

(4-Methoxyphenyl)(3,4,5-trimethoxyphenyl)methanone inhibits tubulin polymerization,induces G2/M arrest,and triggers apoptosis in human leukemia HL-60 cells

(4-Methoxyphenyl)(3,4,5-trimethoxyphenyl)methanone (PHT) is a known cytotoxic compound belonging to the phenstatin family. However, the exact mechanism of action of PHT-induced cell death remains to be determined. The aim of this study was to investigate the mechanisms underlying PHT-induced cytotoxicity. We found that PHT displayed potent cytotoxicity in different tumor cell lines, showing IC₅₀ values in the nanomolar range. Cell cycle arrest in G₂/M phase along with the augmented metaphase cells was found. Cells treated with PHT also showed typical hallmarks of apoptosis such as cell shrinkage, chromatin condensation, phosphatidylserine exposure, increase of the caspase 3/7 and 8 activation, loss of mitochondrial membrane potential, and internucleosomal DNA fragmentation without affecting membrane integrity. Studies conducted with isolated tubulin and docking models confirmed that PHT binds to the colchicine site and interferes in the polymerization of microtubules. These results demonstrated that PHT inhibits tubulin polymerization, arrests cancer cells in G₂/M phase of the cell cycle, and induces their apoptosis, exhibiting promising anticancer therapeutic potential.     

Role of Annexin A5 on Mitochondria-Dependent Apoptosis Induced by Tetramethoxystilbene in Human Breast Cancer Cells

We have previously shown that 2,4,3′,5′-tetramethoxystilbene (TMS), a trans-stilbene analogue, induces apoptosis in human cancer cells. However, the detailed mechanisms of mitochondria-dependent apoptosis induced by TMS are not fully understood. In the present study, the possible roles of annexin A5 in TMS-mediated apoptosis were investigated in MCF7 human breast cancer cells. Quantitative real-time PCR analysis and Western blot analysis showed that the expression of annexin A5 was strongly increased in TMS-treated cells. TMS caused a strong translocation of annexin A5 from cytosol into mitochondria. Confocal laser scanning microscopic analysis clearly showed that TMS induced translocation of annexin A5 into mitochondria. TMS increased the expression and oligomerization of voltage-dependent anion channel (VDAC) 1, which may promote mitochondria-dependent apoptosis through disruption of mitochondrial membrane potential. When cells were treated with TMS, the levels of Bax, and Bak as well as annexin A5 were strongly enhanced. Moreover, we found that the cytosolic release of apoptogenic factors such as cytochrome c, or apoptosis-inducing factor (AIF) in mitochondria was markedly increased. Annexin A5 depletion by siRNA led to decreased proapoptotic factors such as Bax, Bak, and annexin A5. Taken together, our results indicate that annexin A5 may play an important role in TMS-mediated mitochondrial apoptosis through the regulation of proapoptotic proteins and VDAC1 expression.     

Effect of trans-resveratrol,a natural polyphenolic compound,on human polymorphonuclear leukocyte function

1. Polymorphonuclear leukocytes (PMN) may contribute to the pathogenesis of acute coronary heart disease (CHD).
2. Epidemiological and laboratory evidence suggests that red wine, by virtue of its polyphenolic constituents, may be more effective than other alcoholic beverages in reducing the risk of CHD mortality.
3. The aim of the present study was to investigate the effects of trans-resveratrol (3,4′,5-trihydroxy-trans-stilbene), a polyphenol present in most red wines, on functional and biochemical responses of PMN, upon in vitro activation.
4. trans-Resveratrol exerted a strong inhibitory effect on reactive oxygen species produced by PMN stimulated with 1 μM formyl methionyl leucyl phenylalamine (fMLP) (IC₅₀ 1.3±0.13 μM, mean±s.e.mean), as evaluated by luminol-amplified chemiluminescence.
5. trans-Resveratrol prevented the release of elastase and β-glucuronidase by PMN stimulated with the receptor agonists fMLP (1 μM, IC₅₀ 18.4±1.8 and 31±1.8 μM), and C5a (0.1 μM, IC₅₀ 41.6±3.5 and 42±8.3 μM), and also inhibited elastase and β-glucuronidase secretion (IC₅₀ 37.7±7 and 25.4±2.2 μM) and production of 5-lipoxygenase metabolites leukotriene B₄ (LTB₄), 6-trans-LTB₄ and 12-trans-epi-LTB₄ (IC₅₀ 48±7 μM) by PMN stimulated with the calcium ionophore A23187 (5 μM).
6. trans-Resveratrol significantly reduced the expression and activation of the β₂ integrin MAC-1 on PMN surface following stimulation, as revealed by FACS analysis of the binding of an anti-MAC-1 monoclonal antibody (MoAb) and of the CBRM1/5 MoAb, recognizing an activation-dependent epitope on MAC-1. Consistently, PMN homotypic aggregation and formation of mixed cell-conjugates between PMN and thrombin-stimulated fixed platelets in a dynamic system were also prevented by trans-resveratrol.
7. These results, indicating that trans-resveratrol interferes with the release of inflammatory mediators by activated PMN and down-regulates adhesion-dependent thrombogenic PMN functions, may provide some biological plausibility to the protective effect of red wine consumption against CHD.