Development and characterization of PLGA nanoparticles containing 1,3-dihydroxy-2-methylxanthone with improved antitumor activity on a human breast cancer cell line.

Interest on xanthones has been growing considerably due to their broad spectrum of biological activities. 1,3-Dihydroxy-2-methylxanthone (DHMXAN) showed a significant inhibitory effect on the growth of MCF-7 cancer cells. DHMXAN-loaded nanosphere and nanocapsule formulations were prepared by the solvent displacement technique with the aim of improving the delivery of this poorly water-soluble compound. Moreover, we investigated the usefulness of this nanotechnology-based approach on the improvement of compound's antitumor activity. Nanosphere and nanocapsule mean diameters ranged from 117 ± 8 to 138 ± 5 nm and from 266 ± 7 to 286 ± 22 nm, respectively, and both systems exhibited negative surface charge. Incorporation efficiency values of DHMXAN in nanospheres and nanocapsules were higher than 30% (30.9 ± 3.3 to 38.8 ± 3.6%) and 80% (85 ± 7 to 88 ± 6%), respectively. The release study of DHMXAN from nanocapsules suggests that drug release is mainly governed by its partition between the oil core and the external aqueous medium. The effect of different nanoparticulate formulations on the growth of human breast cancer cell line MCF-7 was evaluated using the sulforhodamine B (SRB) assay. Incorporation of DHMXAN in nanospheres and nanocapsules afforded a marked potentiation of the compound inhibitory effect.

The Antitumor Activity of a Lead Thioxanthone is Associated with Alterations in Cholesterol Localization.

The search for novel anticancer small molecules and strategies remains a challenge. Our previous studies have identified TXA1 (1-{[2-(diethylamino)ethyl]amino}-4-propoxy-9H- thioxanthen-9-one) as a hit compound, with in vitro antitumor potential by modulating autophagy and apoptosis in human tumor cell lines. In the present study, the mechanism of action and antitumor potential of the soluble salt of this molecule (TXA1.HCl) was further investigated using in vitro and mouse xenograft tumor models of NSCLC. Our results showed that TXA1.HCl affected steroid biosynthesis, increased RagD expression, and caused abnormal cellular cholesterol localization. In addition, TXA1.HCl treatment presented no toxicity to nude mice and significantly reduced the growth of human NSCLC cells xenografts in mice. Overall, this work provides new insights into the mechanism of action of TXA1, which may be relevant for the development of anticancer therapeutic strategies, which target cholesterol transport.

Modulation of Autophagy by a Thioxanthone Decreases the Viability of Melanoma Cells.

(1) Background: Our previous studies unveiled the hit thioxanthone TXA1 as an inhibitor of P-glycoprotein (drug efflux pump) and of human tumor cells growth, namely of melanoma cells. Since TXA1 is structurally similar to lucanthone (an autophagy inhibitor and apoptosis inducer) and to N10-substituted phenoxazines (isosteres of thioxanthones, and autophagy inducers), this study aimed at further assessing its cytotoxic mechanism and evaluating its potential as an autophagy modulator in A375-C5 melanoma cells; (2) Methods: Flow cytometry with propidium iodide (PI) for cell cycle profile analysis; Terminal deoxynucleotidyl transferase dUTP nick end labeling (TUNEL) assay, flow cytometry with Annexin V/PI labeling and Western blot for apoptosis analysis were conducted. A pharmacophore approach was used for mapping TXA1 onto pharmacophores for autophagy induction. Autophagy analyses included transmission electron microscopy for visualization of autophagic structures, fluorescence microscopy for observation of monodansylcadaverine (MDC) staining, pattern of LC3 expression in the cells and acridine orange staining, and Western blot for autophagic proteins expression; (3) Results: TXA1 induced autophagy of melanoma cells at the GI50 concentration (3.6 µM) and apoptosis at twice that concentration. Following treatment with TXA1, autophagic structures were observed, together with the accumulation of autophagosomes and the formation of autophagolysosomes. An increase in LC3-II levels was also observed, which was reverted by 3-methyladenine (3-MA) (an early stage autophagy-inhibitor) but further increased by E-64d/pepstatin (late-stage autophagy inhibitors). Finally, 3-MA also reverted the effect of TXA1 in cellular viability; (4) Conclusion: TXA1 decreases the viability of melanoma cells by modulation of autophagy and may, therefore, serve as a lead compound for the development of autophagy modulators with antitumor activity.

Screening a Small Library of Xanthones for Antitumor Activity and Identification of a Hit Compound which Induces Apoptosis.

Our previous work has described a library of thioxanthones designed to have dual activity as P-glycoprotein modulators and antitumor agents. Some of these compounds had shown a significant cell growth inhibitory activity towards leukemia cell lines, without affecting the growth of non-tumor human fibroblasts. However, their effect in cell lines derived from solid tumors has not been previously studied. The present work aimed at: (i) screening this small series of compounds from an in-house library, for their in vitro cell growth inhibitory activity in human tumor cell lines derived from solid tumors; and (ii) initiate a study of the effect of the most potent compound on apoptosis. The tumor cell growth inhibitory effect of 27 compounds was first analysed in different human tumor cell lines, allowing the identification of a hit compound, TXA1. Its hydrochloride salt TXA1·HCl was then synthesized, to improve solubility and bioavailability. Both TXA1 and TXA1·HCl inhibited the growth of MCF-7, NCI-H460, A375-C5, HeLa, 786-O, Caki-2 and AGS cell lines. The effect of TXA1·HCl in MCF-7 cells was found to be irreversible and was associated, at least in part, with an increase in cellular apoptosis.

New chiral derivatives of xanthones: synthesis and investigation of enantioselectivity as inhibitors of growth of human tumor cell lines.

A highly efficient and practical methodology for synthesis of new chiral derivatives of xanthones (CDXs) in enantiomerically pure form has been developed. According to this approach, thirty CDXs (3-32) were synthesized by coupling a carboxyxanthone (1) and a carboxymethoxyxanthone (2) with both enantiomers of commercially available chiral building blocks, namely six amino alcohols, one amine and one amino ester. The activation of the carboxylic acid group of the xanthonic scaffold was carried out with the coupling reagent O-(benzotriazol-1-yl)-N-N-N'-N'-tetramethyluronium tetrafluoroborate (TBTU), in the presence of a catalytic amount of TEA in anhydrous THF. The coupling reactions with the chiral blocks were performed at room temperature with short reactions times, excellent yields (ranging from 94% to 99%), and very high enantiomeric excess. The synthesized CDXs were evaluated for their effect on the in vitro growth of three human tumor cell lines, namely A375-C5 (melanoma), MCF-7 (breast adenocarcinoma), and NCI-H460 (non-small cell lung cancer). The most active compound was CDX 15 being active in all human tumor cell lines with values of GI50 of 32.15±2.03µM for A375-C5, 22.55±1.99µM for MCF-7, and 14.05±1.82µM for NCI-H460. Nevertheless, some CDXs showed cell-type selectivity. Furthermore, the growth inhibitory effects, in some cases, demonstrated to be depending on the stereochemistry of the CDXs. An interesting example was observed with the enantiomers 3 and 4, which demonstrated high enantioselectivity for MCF-7 and NCI-H460 cell lines. It can be inferred that the effects on the growth of the human tumor cell lines can be ascribed not only to the nature and positions of substituents on the xanthonic scaffold but also to the stereochemistry of the CDXs. Some considerations regarding structure-activity relationship within this class of compounds will be highlighted.

Multidimensional optimization of promising antitumor xanthone derivatives.

A promising antitumor xanthone derivative was optimized following a multidimensional approach that involved the synthesis of 17 analogues, the study of their lipophilicity and solubility, and the evaluation of their growth inhibitory activity on four human tumor cell lines. A new synthetic route for the hit xanthone derivative was also developed and applied for the synthesis of its analogues. Among the used cell lines, the HL-60 showed to be in general more sensitive to the compounds tested, with the most potent compound having a GI50 of 5.1 µM, lower than the hit compound. Lipophilicity was evaluated by the partition coefficient (K(p)) of a solute between buffer and two membrane models, namely liposomes and micelles. The compounds showed a logK(p) between 3 and 5 and the two membrane models showed a good correlation (r(2)=0.916) between each other. Studies concerning relationship between solubility and structure were developed for the hit compound and 5 of its analogues.

Butyrate activates the monocarboxylate transporter MCT4 expression in breast cancer cells and enhances the antitumor activity of 3-bromopyruvate.

Most malignant tumors exhibit the Warburg effect, which consists in increased glycolysis rates with production of lactate, even in the presence of oxygen. Monocarboxylate transporters (MCTs), maintain these glycolytic rates, by mediating the influx and/or efflux of lactate and are overexpressed in several cancer cell types. The lactate and pyruvate analogue 3-bromopyruvate (3-BP) is an inhibitor of the energy metabolism, which has been proposed as a specific antitumor agent. In the present study, we aimed at determining the effect of 3-BP in breast cancer cells and evaluated the putative role of MCTs on this effect. Our results showed that the three breast cancer cell lines used presented different sensitivities to 3-BP: ZR-75-1 ER (+)>MCF-7 ER (+)>SK-BR-3 ER (-). We also demonstrated that 3-BP reduced lactate production, induced cell morphological alterations and increased apoptosis. The effect of 3-BP appears to be cytotoxic rather than cytostatic, as a continued decrease in cell viability was observed after removal of 3-BP. We showed that pre-incubation with butyrate enhanced significantly 3-BP cytotoxicity, especially in the most resistant breast cancer cell line, SK-BR-3. We observed that butyrate treatment induced localization of MCT1 in the plasma membrane as well as overexpression of MCT4 and its chaperone CD147. Our results thus indicate that butyrate pre-treatment potentiates the effect of 3-BP, most probably by increasing the rates of 3-BP transport through MCT1/4. This study supports the potential use of butyrate as adjuvant of 3-BP in the treatment of breast cancer resistant cells, namely ER (-).

Studies on quinones. Part 42: Synthesis of furylquinone and hydroquinones with antiproliferative activity against human tumor cell lines.

The preparation of furyl-1,4-quinone and hydroquinones by reaction of 2-furaldehyde N,N-dimethylhydrazone with benzo- and naphthoquinones is reported. Access to furylnaphthoquinones from unactivated quinones requires acid-induced conditions, however oxidative coupling reactions of activated quinones proceed under neutral conditions. The in vitro cytotoxic activity of the prepared compounds against a panel of three human cancer cell lines has been studied. Most of the furyl-1,4-quinones exhibited good antiproliferative activity (GI(50)=6.5-33.5microm) against the MCF-7, NCI-H460, and SF-268 (CNS cancer) cell lines chosen for testing.

Multimilligram enantioresolution of low-solubility xanthonolignoids on polysaccharide chiral stationary phases using a solid-phase injection system.

Kielcorins are xanthonolignoids with protein kinase C inhibition and antitumor activities. Four racemates were enantioresolved at a multimilligram scale on tris-3,5-dimethylphenylcarbamate amylose phase using polar organic conditions as mobile phase. The low-solubility of these compounds conditioned the injection amount and consequently the productivity. A solid-phase injection system was developed to increase the production rate of the semipreparative process. The effects of the racemates and the related enantiomers on the in vitro growth of human breast cancer cell line MCF-7 were compared. Differences in their growth inhibitory activity were observed.

Effects of natural prenylated flavones in the phenotypical ER (+) MCF-7 and ER (-) MDA-MB-231 human breast cancer cells.

The effect of seven natural prenylated flavones in DNA synthesis of two human breast cancer cell lines, the estrogen-dependent ER (+) MCF-7 and the estrogen-independent ER (-) MDA-MB-231 cells, was evaluated. Flavones with an isopentenyl group at C-8 and a ring linking C-3 and C-2' presented a biphasic effect in DNA synthesis of ER (+) MCF-7 and displayed a stimulation at low concentrations (0.02-0.78 microM) whilst at higher concentrations (> 3.12 microM) inhibition was observed. No stimulation was observed in ER (-) MDA-MB-231. In contrast, all the flavones exhibited an antiproliferative effect in both ER (-) and ER (+). Curiously, the inhibition of DNA synthesis was accompanied by a high capacity of these cells to reduce MTT, which was concurrent with the appearance of an intense intracytoplasmic vacuolization. The accumulation of the formazan product in these vacuoles could justify the enhancements of MTT reduction. The characterization of these vacuoles with the autophagic marker monodansylcadaverine (MDC) is consistent with autophagic vacuoles, which led to the suggestion that these flavones could induce autophagy in both ER (+) and ER (-) breast cancer cell lines.

Psoralen analogues: synthesis, inhibitory activity of growth of human tumor cell lines and computational studies.

Eight psoralens have been evaluated for their ability to inhibit the in vitro growth of three human tumor cell lines representing different tumor types, MCF-7 (breast cancer), NCI-H460 (non-small cell lung cancer) and SF-268 (CNS cancer). The synthesis of four new psoralens (benzofurocoumarins) is presented as well as the results of the ab initio calculations to find the parameters that relate the structure with the antitumor activity. This work provides supplementary information that could allow the development of new psoralen analogues with this type of biological activity.

Artelastin is a cytotoxic prenylated flavone that disturbs microtubules and interferes with DNA replication in MCF-7 human breast cancer cells.

Artelastin, a novel prenylated flavone, previously isolated from the wood bark of Artocarpus elasticus, was evaluated for its capacity to inhibit the growth of fifty-two human tumor cell lines, representing nine different tumor types. A pronounced dose-dependent growth inhibitory effect was detected in all the cell lines, with GI50 values ranging from 0.8-20.8 microM. Studies to elucidate the basis of the growth inhibitory activity of artelastin were performed in the MCF-7 human breast cancer cell line (GI50 = 6.0 microM). We show that artelastin exerts a biphasic effect in the DNA synthesis of MCF-7 cells, a stimulatory effect at low concentrations (below GI50) for short times of exposition (6 h and 24 h), and an inhibitory effect at high concentrations (above GI50). Remarkably, treated cells that have DNA synthesis affected could be viable and metabolically active. Furthermore, artelastin acts as a cytotoxic rather than a cytostatic compound. Massive cytoplasmatic vacuoles were detected in cells after artelastin treatment. Together with these morphological alterations, cells show the presence of abnormal nuclear morphologies, and occasionally nuclear condensation, which were identified as apoptotic by TUNEL assay. Moreover, artelastin was shown to disturb the microtubule network while no effect was observed on the kinetochores. Flow cytometry analysis of cells treated with artelastin reveal an accumulation in S phase that interferes with the cell cycle progression. Additionally, according to BrdU patterns, studies with synchronized cells at G0 and at G1/S transition also suggest that artelastin delays DNA replication since progression of cells trough S-phase is perturbed.

Further halotyrosine derivatives from the marine sponge Suberea aff. praetensa.

Reexamination of the marine sponge Suberea aff. praetensa, (Row) from the Gulf of Thailand furnished in addition to bromotyrosine derivatives found previously 5-bromo- and 5-chlorocavernicolin, cavernicolins 1 and 2, two other brominated tyrosine metabolites, a known bisoxazolidone and a new unusual rearranged tyrosine metabolite subereatensin. Several of the metabolites exhibited significant inhibitory effects against five human cancer cell lines.

Evaluation of 2',4'-dihydroxy-3,4,5-trimethoxychalcone as antimitotic agent that induces mitotic catastrophe in MCF-7 breast cancer cells.

We previously reported the synthesis and the anti-proliferative action of 2',4'-dihydroxy-3,4,5-trimethoxychalcone. Here we reported its mechanism of action on MCF-7 cells. The compound induced aberrant spindles, and arrested cells at metaphase/anaphase boundary with accumulation of checkpoint proteins Mad2, Bub1 and BubR1. Live cell imaging revealed that the compound sustained a prolonged mitotic arrest, followed by massive cell death. The results indicate that 2',4'-dihydroxy-3,4,5-trimethoxychalcone exerts its anti-proliferative activity by affecting microtubules and causing mitotic catastrophe, and thus has the potential for antitumor activity.

Hair as an alternative matrix in bioanalysis.

Alternative matrices are steadily gaining recognition as biological samples for toxicological analyses. Hair presents many advantages over traditional matrices, such as urine and blood, since it provides retrospective information regarding drug exposure, can distinguish between chronic and acute or recent drug use by segmental analysis, is easy to obtain, and has considerable stability for long periods of time. For this reason, it has been employed in a wide variety of contexts, namely to evaluate workplace drug exposure, drug-facilitated sexual assault, pre-natal drug exposure, anti-doping control, pharmacological monitoring and alcohol abuse. In this article, issues concerning hair structure, collection, storage and analysis are reviewed. The mechanisms of drug incorporation into hair are briefly discussed. Analytical techniques for simultaneous drug quantification in hair are addressed. Finally, representative examples of drug quantification using hair are summarized, emphasizing its potentialities and limitations as an alternative biological matrix for toxicological analyses.

Pyranoxanthones: Synthesis, growth inhibitory activity on human tumor cell lines and determination of their lipophilicity in two membrane models.

The benzopyran and dihydrobenzopyran moieties can be considered as "privileged motifs" in drug discovery being good platforms for the search of new bioactive compounds. These moieties are commonly found fused to the xanthonic scaffold belonging to the biologically important family of the generally designated prenylated xanthones. Several pyranoxanthones have shown promising antitumor activity and since most of them are from natural origin, the biosynthetic pathway only allows a particular pattern of substitution which limits their structural diversity and renders any broad structure-activity study hard to be established. Accordingly, with the aim of rationalizing the importance of the fused ring orientation and oxygenation pattern in pyranoxanthones, this study describes the synthesis of 14 new pyranoxanthones and evaluation of their cell growth inhibitory activity in four human tumor cell lines as well as their lipophilicity in two membrane models. This systematic approach allowed establishing structure-activity and structure-lipophilicity relationships for the obtained compounds in combination with 6 previously described compounds. From this work an angular pyranoxanthone scaffold emerged as particularly promising, presenting a potent cell growth inhibitory activity and suitable drug-like lipophilicity.

4'-methoxy-2-styrylchromone a novel microtubule-stabilizing antimitotic agent.

4'-methoxy-2-styrylchromone, a new synthetic chromone was identified as a selective proliferation inhibitor of human tumor (MCF-7 and NCI-H460) cell lines than to non-tumor cells (MRC-5). The antiproliferative activity of this chromone was also extensive to peripheral human lymphocytes. 4'-Methoxy-2-styrylchromone was found to block tumor cells in the G2/M phase of the cell cycle. The G2/M arrest of NCI-H460 cells was dose- and time-dependent, reaching a maximum after 12-h treatment while MCF-7 cells reached the maximum value of G2/M accumulation only after a 24-h treatment. Chromone-treated cells evidenced a high frequency of cells in prometaphase, indicating progression beyond G2 and arrest early in mitosis. This mitotic arrest was associated with abnormal mitotic spindles characterized by the formation of a monopolar structure, suggesting that the chromone interferes with microtubules. The results of an in vitro tubulin polymerization assay showed that this chromone stabilizes microtubules in a manner similar to paclitaxel.

Cytotoxic activity of lupane-type triterpenes from Glochidion sphaerogynum and Glochidion eriocarpum two of which induce apoptosis.

Six known lupanes lupenone ( 1), 3- epi-lupeol ( 2), glochidone ( 4), glochidonol ( 5), glochidiol ( 6) and lup-20(29)-ene-1beta,3beta-diol ( 7) were isolated from the roots and stem wood of Glochidion eriocarpum and three, 5, 6 and lup-20(29)ene-3alpha,23-diol ( 3), were isolated from the roots and stem wood of Glochidion sphaerogynum. Compounds were identified by (1)H- and (13)C-NMR techniques. Triterpenes 2 - 7 were tested against the growth of three human tumor cell lines, MCF-7, NCI-H-460 and SF-268. Lupanes 3, 5, and 6 exhibited strong inhibitory effects against all three; thus GI (50) values for 3 were 12.7 +/- 3.7, 17.9 +/- 1.1 and 17.9 +/- 0.5, for 5 9.0 +/- 3.7, 4.9 +/- 0.2 and 9.8 +/- 0.5, and for 6 6.63 +/- 0.7, 7.5 +/- 0.5 and 9.7 +/- 0.3.3. Epilupeol was less active, with GI (50) values of 75.6 +/- 11.7, 86.1 +/- 12.4 and 80.9 +/- 2.6 while 7 was moderately active only against MCF-7 (GI (50) = 79.2 +/- 2.4). Additional studies indicated that triterpenes 5 and 6 exerted their antiproliferative activity through the involvement of apoptosis while triterpene 3 did not.

Xanthones as inhibitors of growth of human cancer cell lines and their effects on the proliferation of human lymphocytes in vitro.

Twenty-seven oxygenated xanthones have been assessed for their capacity to inhibit in vitro the growth of three human cancer cell lines, MCF-7 (breast cancer), TK-10 (renal cancer) and UACC-62 (melanoma). The effect of these xanthones on the proliferation of human T-lymphocytes was also evaluated. Differences on their potency towards the effect on the growth of the human cancer cell lines as well as on the proliferation of human T-lymphocytes can be ascribed to the nature and positions of the substituents on the xanthonic nucleus.

Euphopubescenol and euphopubescene, two new jatrophane polyesters, and lathyrane-type diterpenes from Euphorbia pubescens.

The structures of euphopubescenol and euphopubescene, two new macrocyclic jatrophane diterpene polyesters, isolated from the whole dried plant of Euphorbia pubescens, were established as 5alpha,8alpha,15beta-triacetoxy-3alpha-benzoyloxy -4alpha-hydroxy -9,14-dioxo-13beta H-jatropha-6(17),11 E-diene ( 1) and 3beta,7beta,8beta,9alpha,14alpha,15beta-hexaacetoxy-2beta H-jatropha-5 E,11 E-diene ( 2) by 1D- and 2D-NMR (COSY, HMQC, HMBC and NOESY), IR, EI-MS and EI-FTICR-MS. Two known lathyrane derivatives, jolkinol A ( 3) and jolkinol A ( 4), whose (13)C-NMR spectra were assigned, were also isolated. Compounds 1 - 3 have been evaluated for their ability to inhibit the in vitro growth of three human tumour cell lines representing different tumour types, MCF-7 (breast adenocarcinoma), NCI-H460 (non-small cell lung cancer) and SF-268 (CNS cancer). They inhibited both MCF-7 and NCI-H460 cell lines, with GI50 values ranging between 40.9 microM and 95.3 microM, but were found to be ineffective as growth inhibitors of the SF-268 cell line.