Design and multi-step synthesis of chalcone-polyamine conjugates as potent antiproliferative agents.

The aim of this study is to synthesize chalcone-polyamine conjugates in order to enhance bioavailability and selectivity of chalcone core towards cancer cells, using polyamine-based vectors. 3-hydroxy-3',4,4',5'-tetramethoxychalcone (1) and 3',4,4',5'-tetramethoxychalcone (2) were selected as parent chalcones since they were found to be efficient anti-proliferative agents on various cancer cells. A series of ten chalcone-polyamine conjugates was obtained by reacting carboxychalcones with different polyamine tails. Chalcones 1 and 2 showed a strong cytotoxic activity against two prostatic cancer (PC-3 and DU-145) and two colorectal cancer (HT-29 and HCT-116) cell lines. Then, chalcone-spermine conjugates 7d and 8d were shown to be the most active of the series and could be considered as promising compounds for colon and prostatic cancer adjuvant therapy.

2'-Hydroxy-4-methylsulfonylchalcone enhances TRAIL-induced apoptosis in prostate cancer cells.

Prostate cancer is the most common malignant cancer in men and the second leading cause of cancer deaths. Previously, we have shown that 2'-hydroxy-4-methylsulfonylchalcone (RG003) induced apoptosis in prostate cancer cell lines PC-3 and DU145. Although tumor necrosis factor-related apoptosis-inducing ligand (TRAIL) is a promising anticancer agent, some cancer cells are resistant to TRAIL treatment. PC-3 and LNCaP prostatic cancer cell lines have been reported to be resistant to TRAIL-induced apoptosis. Here, we show for the first time that RG003 overcomes TRAIL resistance in prostate cancer cells. RG003 can enhance TRAIL-induced apoptosis through DR5 upregulation and downregulation of Bcl-2, PI3K/Akt, NF-κB, and cyclooxygenase-2 (COX-2) survival pathways. When used in combined treatment, RG003 and TRAIL amplified TRAIL-induced activation of apoptosis effectors and particularly activation of caspase-8 and the executioner caspase-3, leading to increased poly-ADP-ribose polymerase cleavage and DNA fragmentation in prostate cancer cells. Furthermore, we showed that RG003 reduced COX-2 expression in cells. Previously, we showed that COX-2 was involved in resistance to an apoptosis mechanism; then, its inhibition by RG003 could render cells more sensitive to TRAIL treatment. We showed that nuclear factor-κB activation was inhibited after RG003 treatment. This inhibition was correlated with reduction in COX-2 expression and induction of apoptosis. Overall, we conclude, for the first time, that RG003 can enhance TRAIL-induced apoptosis in human prostate cancer cells. The significance of our in-vitro study with RG003 and TRAIL combined is very encouraging, suggesting the relevance of testing this combined treatment in xenograft animal models.

Free radical scavenging by natural polyphenols: atom versus electron transfer.

Polyphenols (synthetically modified or directly provided by human diet) scavenge free radicals by H-atom transfer and may thus decrease noxious effects due to oxidative stress. Free radical scavenging by polyphenols has been widely theoretically studied from the thermodynamic point of view whereas the kinetic point of view has been much less addressed. The present study describes kinetic-based structure-activity relationship for quercetin. This compound is very characteristic of the wide flavonoid subclass of polyphenols. H-atom transfer is a mechanism based on either atom or electron transfer. This is analyzed here by quantum chemical calculations, which support the knowledge acquired from experimental studies. The competition between the different processes is discussed in terms of the nature of the prereaction complexes, the pH, the formation of activated-deprotonated forms, and the atom- and electron-transfer efficiency. The role of the catechol moiety and the 3-OH group of quercetin as scavengers of different types of free radicals (CH3OO(•), CH3O(•), (•)OH, and (•)CH2OH) is rationalized. Identifying the exact mechanism and accurately evaluating kinetics is of fundamental importance to understand antioxidant behavior in physiological environments.

Alternative to animal experimentation in pharmacology teaching: Development and validation of an equivalent digital learning tool.

Regarding animal experiments in pharmacology teaching, ethical considerations led us to examine an alternative approach to the use of living animals. This study aimed to assess whether digital tools could replace live animal experiments in terms of motivation and knowledge acquisition. The study was carried out with students enrolled in the 5th year of the industry/research stream at the Faculty of Pharmacy of the University of Limoges. The participants were randomly assigned to groups of traditional or digital teaching methods, with the common theme of the class being the effect of a diuretic agent (furosemide) in rats. The scenario and learning objectives were identical for the two groups. Before the class and after randomization, the acceptance of the digital educational material was assessed with a scale, which predicts the acceptability of users according to individual dimensions and social representations, followed by the assessment of the motivation by a situational motivation scale (SIMS) for both groups. After the class, the students' motivation was assessed by a questionnaire based on Deci and Ryan's self-determination theory. In the end, the participants were evaluated for homogeneity, based on general knowledge of renal pharmacology, and for knowledge acquisition concerning specific knowledge related to this teaching session. This study revealed a good acceptance of the digital tool and a good motivation toward the digital method among all the students. It found the two teaching methods (digital and traditional) to be equivalent in terms of motivation and knowledge acquisition. In our study, digital pedagogical tools as an alternative to live animals did not affect students' motivation and knowledge acquisition.

Dimerisation process of silybin-type flavonolignans: insights from theory.

Natural polyphenols are known to be oxidized by free radicals, which partially explains the antioxidant properties of a number of these compounds. This oxidation may also be used to synthesise new compounds of biological interest, for example, dimers. The present theoretical study describes the existing experimental evidence showing that silybin and dehydrosilybin [natural polyphenols isolated from milk thistle (Silybum marianum)] form dimers regioselectively. Based on DFT calculations, thermodynamic and kinetic values were computed in order to better understand the physicochemical behaviour of these dimerisation processes. Calculations showed that after H-atom transfer (from polyphenol to radical), dimerisation could proceed in two steps: 1) bond formation and, when possible, 2) tautomerisation reorganisation. The former step is the limiting step, while the latter is crucial for the process to be thermodynamically favourable (ΔG<0). If this rearrangement is impossible then dimerisation is not feasible, or at least becomes a minor process (e.g., dehydrosilybin dimerisation after H-atom abstraction from the 3-OH group). This explains the regioselectivity of polyphenol dimerisation.

Radiodermatitis prevention with sucralfate in breast cancer: fundamental and clinical studies.

BACKGROUND: Acute radiodermatitis induced by radiotherapy may affect the quality of life and in some cases requires withholding treatment. The present study concerns the protective effect of a 1% sucralfate lotion. We propose joint fundamental and clinical points of view. METHODS: The free radical scavenging capacity of sucralfate was measured with electron spin resonance and was supported by theoretical calculations. The clinical effects of sucralfate lotion were evaluated on 21 women treated for breast cancer. Breast skin response was evaluated at 0, 10, 20, 30, 40, and 50 Gy, according to (1) the radiation therapy oncology group (RTOG) acute toxicity scale and (2) spectrophotometry data obtained with X-Rite SP60. RESULTS AND CONCLUSIONS: Sucralfate appeared as a relatively poor free radical scavenger (compared to reference compounds such as vitamin E). The sucralfate-containing lotion used in the present study did not provide systematic radiodermatitis prevention. Spectrophotometric evaluation of the skin response to irradiation appeared to be a very effective and more sensitive technique than the RTOG scale. Its use should be recommended to study cutaneous radioprotective action.

Chemical composition and biological potential of Thymus Willdenowii Boiss. & Reut. essential oil.

The fresh aerial parts of Thymus willdenowii Boiss. & Reut. (syn. Thymus hirtus Willd.) were hydrodistilled in a Clevenger type apparatus and analyzed by GC and GC-MS. 44 Components were identified representing 97.3%, with 1,8-cineole (34.62%), camphor (18.55%), α-pinene (9.46%) and camphene (5.38%) as the main components. T. willdenowii essential oil was not cytotoxic (CC50 = 97.65 µg/mL) towards Vero non-tumoural cells, exhibiting good antibacterial and antiproliferative (30.8 ± 3.1% inhibition) potentials against four tested pathogenic bacteria and Human colorectal cell line HT-29, respectively. The essential oil did not show a DPPH radical scavenging activity, by Electron Spin Resonance spectroscopy (ESR), and it lacks antiviral effect towards coxsackievirus B3.

Mechanism of the antioxidant action of silybin and 2,3-dehydrosilybin flavonolignans: a joint experimental and theoretical study.

Flavonolignans from silymarin, the standardized plant extract obtained from thistle, exhibit various antioxidant activities, which correlate with the other biological and therapeutic properties of that extract. To highlight the mode of action of flavonolignans as free radical scavengers and antioxidants, 10 flavonolignans, selectively methylated at different positions, were tested in vitro for their capacity to scavenge radicals (DPPH and superoxide) and to inhibit the lipid peroxidation induced on microsome membranes. The results are rationalized on the basis of (i) the oxidation potentials experimentally obtained by cyclic voltammetry and (ii) the theoretical redox properties obtained by quantum-chemical calculations (using a polarizable continuum model (PCM)-density functional theory (DFT) approach) of the ionization potentials and the O-H bond dissociation enthalpies (BDEs) of each OH group of the 10 compounds. We clearly establish the importance of the 3-OH and 20-OH groups as H donors, in the presence of the 2,3 double bond and the catechol moiety in the E-ring, respectively. For silybin derivatives (i.e., in the absence of the 2,3 double bond), secondary mechanisms (i.e., electron transfer (ET) mechanism and adduct formation with radicals) could become more important (or predominant) as the active sites for H atom transfer (HAT) mechanism are much less effective (high BDEs).

Theoretical investigation of the formation of a new series of antioxidant depsides from the radiolysis of flavonoid compounds.

This paper deals with the formation of a series of antioxidant depsides obtained from flavonoid solutions irradiated with gamma rays. These reactions take place in radiolyzed alcohol solutions, a medium that is very rich in many different highly reactive species and that hosts specific reactions. We focus on the first step of those reactions, i.e., reactivity of the solute (flavonoid) with the alkoxy radicals CH(3)O(*) and CH(3)CH(2)O(*) formed in methanol and ethanol, respectively, and their carbon-centered isomers: the 1-hydroxy-methyl ((*)CH(2)OH) and the 1-hydroxy-ethyl (CH(3)(*)CHOH) radicals. Among the different flavonoid groups of molecules, only flavonols are transformed. To establish the structure-reactivity relationship that explains why the radiolytic transformation occurs only for those compounds, the process is rationalized theoretically, with Density Functional Theory calculations, taking into account the solvent effects by a Polarizable Continuum Model and a microhydrated environment (one or two water molecules surrounding the active center). The first redox reaction, occurring between the flavonol and the reactive species formed upon irradiation of the solvent, is studied in terms of (1) the O-H bond dissociation enthalpy of each OH group of the flavonoids and (2) electron abstraction from the molecule. We conclude that the reaction, initiated preferentially by the alkoxy radicals, first occurs at the 3-OH group of the flavonol. It is then followed by the formation of a peroxyl radical (after molecular oxygen or superoxide addition). The different cascades of reactions, which lead to the formation of depsides via C-ring opening, are discussed on the basis of the corresponding calculated energetic schemes.

Antioxidant Flavonoids from Asteriscus maritimus.

One new flavonol glycoside, patuletin-7-Ο-[6-Ο-caffeoyl-2-Ο-[(S)-3-hydroxyisobutanoyl]glucopyranoside] (astermaritimoside) (1) and four known flavonoids, patuletin-7-Ο-ß-[-6-Ο-[(S)-3-hydroxyisobutanoyl]glucopyranoside] (2), patuletin-7-Ο-[6-Ο-caffeoylglucopyranoside] (3), patuletin-7-Ο-ß-D- glucopyranoside (4) and quercetin-3-Ο-ß-rutinoside (5) have been isolated from Asteriscus maritinus (L.) Less. Chemical structures were elucidated by mass spectrometric, and ¹H NMR, ¹³C NMR, COSY, HMQC and HMBC spectroscopic techniques. The antioxidative effect was evaluated using an electronic spin resonance (ESR) method in order to visualize the inhibition of the DPPH radical. The n-butanol fraction was found to possess a remarkable antioxidant activity that was correlated with the total amount of phenolics. The isolated compounds exhibited good antioxidant activity.

Reactivity of chalcones with 1-hydroxymethyl radicals.

Reactivity of chalcones with reactive species issued from methanol radiolysis was investigated in the absence or presence of dioxygen. Chalcones are natural antioxidants that are present in fruit and vegetables. Their degradation in the radiolysed solutions was followed by HPLC, NMR, FAB-LSIMS mass spectroscopy and analytical TLC in deaerated solution. Among the 18 identified radiolytic compounds, 16 were new. The formation of the radiolytic products was not influenced by A- and B-ring substitutions. To explain the degradation process, we thus suggested that the primary step was an attack of the alpha,beta-double bond by the 1-hydroxymethyl radical, either at C(alpha) or at C(beta). This step was followed by addition, cyclization or bond dissociations. Different chemical pathways were discussed that implicate the reactive species issued from methanol radiolysis. This paper highlights the relative importance of the different radical species, especially the carbon-centered radical, 1-hydroxymethyl (HMR) and the corresponding oxygen-centered isomer. In addition, an interesting unusual role of dioxygen should be noted; indeed, in the presence of dioxygen, degradation of chalcones was inhibited.

Radiolytic transformation of 2,2',4'-trihydroxychalcone.

Radiolysis of 2,2',4'-trihydroxychalcone, a natural antioxidant present in fruit and vegetables, was performed in ethanol in the absence or in the presence of dioxygen. The degradation process of chalcone was followed in de-aerated solution by HPLC, NMR, FAB-LSIMS mass spectroscopy and analytical TLC. Under anaerobic conditions, six new products (three couples of diastereoisomers) were identified. Four of them kept the chalcone skeleton with OCH(2)CH(3), CH(OH)CH(3) and H substitutions on C(alpha) and C(beta). Thus the target was the alpha-beta double bond on which ethanol radicals were added. The two other compounds were formed in a second stage and exhibited a cyclization between the substituent on C(beta) and the carbonyl group. In the presence of dioxygen, these reactions were prevented and chalcone was protected. This study was the first step toward understanding of the behavior chalcone in irradiated fruits and vegetables.

Reactivity of flavonoids with 1-hydroxyethyl radical: a gamma-radiolysis study.

We have investigated the reactivity between 11 flavonoids and 1-hydroxyethyl radical (HER). HER was recently implicated in many liver injuries induced by ethanol intoxication. In this study, HER was generated by radiolysis; due to its reaction rate, HER is well known to be responsible for solute degradation in irradiated ethanol. Flavonoid ethanol solutions were irradiated with gamma-rays and the flavonoid degradation was followed by HLPC. We observed the degradation of flavonols while all other flavonoids (flavones, flavanones, dihydroflavonols, catechins) were not degraded after irradiation. The major radiolysis products were identified by NMR and LC-MS and we concluded that flavonols were essentially transformed into depsides. We proposed a reactivity mechanism between flavonols and HER. In a first step, H-transfer occurred from the 3-OH group to HER. Afterwards, C-ring opening occurred due to the presence of the 2,3-double bond in flavonols. Finally, we calculated the reaction constants in order to evaluate the antioxidant activity of flavonols against HER and to compare it with reference compounds.

Castanea sativa Mill. leaves as new sources of natural antioxidant: an electronic spin resonance study.

The antioxidant potential of Castanea sativa Mill. leaf (sweet chestnut) was explored as a new source of active extracts. The capacity of the different fractions issued from aqueous, methanol, and ethyl acetate extracts to inhibit the stable free radical 2,2-diphenyl-1-pycryl-hydrazyl, superoxide anion, and hydroxyl radical was measured by electronic spin resonance. Their scavenging potential was analyzed versus their amount of phenolic compounds. Among the active fractions, the most effective one was A6, an ethyl acetate fraction, which contained a high level of total phenolic compounds (29.1 g/100 g). Thus, a different extraction procedure was performed to concentrate the active compounds of A6 in the new C. sativa leaf extract (CSLE). Compared to reference antioxidants (quercetin and vitamin E) and standard extracts (Pycnogenol, from French Pinus maritima bark, and grape marc extract), it was observed that A6 and CSLE have high antioxidant potentials, equivalent to at least those of reference compounds.

Synergism of antioxidant action of vitamins E, C and quercetin is related to formation of molecular associations in biomembranes.

Vitamins E, C and polyphenols (flavonoids and non-flavonoids) are major natural antioxidants capable of preventing damage generated by oxidative stress. Here we show the capacity of these antioxidants to form non-covalent association within lipid bilayers close to the membrane/cytosol interface. Antioxidant regeneration is significantly enhanced in these complexes.

Radiolysis of kaempferol in water/methanol mixtures. Evaluation of antioxidant activity of kaempferol and products formed.

Oxidative reaction between hydroxymethyl radical ((*)CH(2)OH) and kaempferol, in methanol and methanol/water mixtures, was studied by gamma-radiolysis using a (60)Co source. Radiolysis was performed with concentrations and doses ranging from 5 x 10(-)(5) M to 5 x 10(-)(3) M and from 0.5 kGy to 14 kGy, respectively. Kaempferol degradation was followed by HPLC. Results showed that (*)CH(2)OH reacts with kaempferol at the 3-OH group and produces two depsides (K1 and K2) and other products including K3. K1, K2, and K3 were identified by NMR, LC-MS, and HRMS. The kaempferol degradation pathway leading to the K1, K2, and K3 formation is proposed. It was observed that the more water concentration in the irradiation medium increases, the more K2 concentration increases. Comprehension of food preservation is not clear because many phenomena occurring during irradiation are not established. Radiolysis of kaempferol in water/methanol mixtures helps to elucidate the phenomenon and it is possible that during the treatment of nutriments by gamma-irradiation, a series of products such as depside K2 could be formed. Antioxidant properties of kaempferol radiolysis products were evaluated according to their capacity to decrease the EPR DPPH (1,1-diphenyl-2-picrylhydrazil) signal and to inhibit superoxide radicals formed by the enzyme reaction "xanthine + xanthine oxidase".

Radiolysis of quercetin in methanol solution: observation of depside formation.

Radiolysis of the flavonol quercetin, a natural antioxidant, was performed in methanol. The degradation process was followed by HPLC analyses. The major product was identified as a depside (Q1) by NMR and LC-MS. The G(Q1) radiolytic factor was plotted versus the initial concentration of quercetin. This radiolytic process was attributed to the CH3O* radicals presented in the irradiated medium. The proposed mechanism invoked a stereospecific oxidation of the 3-hydroxyl group of quercetin which led to C-ring opening and to the formation of the depside Q1. In presence of water, Q1 was transformed into another depside, Q2, by an inverse esterification reaction. A chemical equilibrium was observed between Q1 and Q2. The comprehension of the radiolytic process of quercetin in methanol solution is of importance. Indeed, the same type of oxidative reactions could occur on flavonoids during preservation of food by ionizing radiation.

Acylglucuronide in alkaline conditions: migration vs. hydrolysis.

This work rationalizes the glucuronidation process (one of the reactions of the phase II metabolism) for drugs having a carboxylic acid moiety. At this stage, acylglucuronides (AG) metabolites are produced, that have largely been reported in the literature for various drugs (e.g., mycophenolic acid (MPA), diclofenac, ibuprofen, phenylacetic acids). The competition between migration and hydrolysis is rationalized by adequate quantum calculations, combing MP2 and density functional theory (DFT) methods. At the molecular scale, the former process is a real rotation of the drug around the glucuconic acid. This chemical-engine provides four different metabolites with various toxicities. Migration definitely appears feasible under alkaline conditions, making proton release from the OH groups. The latter reaction (hydrolysis) releases the free drug, so the competition is of crucial importance to tackle drug action and elimination. From the theoretical data, both migration and hydrolysis appear kinetically and thermodynamically favored, respectively.

Lipid bilayer membrane affinity rationalizes inhibition of lipid peroxidation by a natural lignan antioxidant.

Lipid peroxidation is a degenerative oxidative process that modifies the structure of membranes, influencing their biological functions. Lignans, natural polyphenolic antioxidants widely distributed in plants, can prevent this membrane damage by free-radical scavenging. Here, we rationalize the difference in lipid peroxidation inhibition activity of argenteane, a natural dilignan isolated from wild nutmeg, and 3,3'-dimethoxy-1,1'-biphenyl-2,2'-diol, which represents the central part of argenteane responsible for its antioxidant activity. Although both compounds have the same capacity to scavenge free radicals, argenteane is a more active inhibitor of lipid peroxidation. We show that both compounds penetrate into DOPC and PLPC lipid bilayers and adopt similar positions and orientations, which therefore does not explain the difference in their lipid peroxidation inhibition activity. However, free energy profiles indicate that argenteane has a significantly higher affinity to the lipid bilayer, and thus a higher effective concentration to scavenge radicals formed during lipid peroxidation. This finding explains the higher activity of argenteane to inhibit lipid peroxidation.

Novel methylsulfonyl chalcones as potential antiproliferative drugs for human prostate cancer: involvement of the intrinsic pathway of apoptosis.

Limited success has been achieved in extending the survival of patients with metastatic and hormone-refractory prostate cancer (HRPC). There is a strong need for novel agents in the treatment and prevention of HRPC. In the present study, the apoptotic mechanism of action of RG003 (2'-hydroxy-4-methylsulfonylchalcone) and RG005 (4'-chloro-2'-hydroxy-4-methylsulfonylchalcone) in association with intracellular signalling pathways was investigated in the hormone-independent prostate carcinoma cells PC-3 and DU145. We showed that these compounds induced apoptosis through the intrinsic pathway but not through the extrinsic one. We showed that synthetic chalcones induced an activation of caspase-9 but not caspase-8 in PC-3 cells. Even if both chalcones induced apoptosis in PC-3 cells, a dominant effect of RG003 treatment was observed resulting in a disruption of ∆ψm, caspase-9 and caspase-3 activation, PARP cleavage and DNA fragmentation. Furthermore, in regard to our results, it is clear that the simultaneous inhibition of Akt and NF-κB signalling can significantly contribute to the anticancer effects of RG003 and RG005 in PC-3 prostate cancer cells. NF-κB inhibition was correlated with the reduction of COX-2 expression and induction of apoptosis. Our results clearly indicate for the first time that RG003 and RG005 exert their potent anti­proliferative and pro-apoptotic effects through the modulation of Akt/NF-κB/COX-2 signal transduction pathways in PC-3 prostate cancer cells with a dominant effect for RG003.