Chinese medicinal herb extract inhibits PQS-mediated quorum sensing system in Pseudomonas aeruginosa.

ETHNOPHARMACOLOGICAL RELEVANCE: Chinese medicinal herbs have long been recognized as important resources that can be used for the struggle against diseases and a significant component of health care system for thousands of years. AIM OF THE STUDY: In order to understand their roles in the treatment against bacterial infections, we examined the underlying mechanisms of one of the medicinal herb extracts (MHE) (Artemisiae argyi Folium, the root bark of Cortex dictamni and the root of Solanum melongena) on the human opportunistic pathogen Pseudomonas aeruginosa. MATERIALS AND METHODS: We combined phenotypic assays, transcriptional analysis and chemical investigations to identify the mechanisms underlying MHE inhibition. The standard sample was prepared and transcriptional reporters for quorum sensing systems were constructed. Electrophoretic mobility shift assays were used to clarify the mechanism. GC-MS and molecular docking were used to identify the chemicals in MHE and potential binding agents. RESULTS: We found that co-culturing of MHE with bacterial cells did not change the growth rate but substantially attenuate the production of virulence factors such as phenazine pyocyanin, siderophore pyoverdine and biofilm formation. Transcriptional responses of three major quorum sensing (QS) systems of P. aeruginosa to MHE showed that Pseudomonas quinolone signaling (PQS) system was completely repressed, rhlR/rhlI QS system was moderately inhibited, while lasR/lasI QS system was only slightly affected, suggesting that MHE might selectively target the PQS system to inhibit bacterial virulence. Furthermore, electrophoretic mobility shift assays (EMSA) showed that MHE inhibited the binding of MvfR the corresponding pqsA promoter region, suggesting that MHE serves as a competitive agent to quench the QS functionality in P. aeruginosa. CONCLUSION: We prove that MHE functions as an effective countermeasure against bacterial infections.

Induction of Secondary Metabolites from the Marine-Derived Fungus Aspergillus versicolor through Co-cultivation with Bacillus subtilis.

A new cyclic pentapeptide, cotteslosin C (1: ), a new aflaquinolone, 22-epi-aflaquinolone B (3: ), and two new anthraquinones (9: and 10: ), along with thirty known compounds (2, 4:  - 8, 11:  - 34: ) were isolated from a co-culture of the sponge-associated fungus Aspergillus versicolor with Bacillus subtilis. The new metabolites were only detected in the co-culture extract, but not when the fungus was grown under axenic conditions. Furthermore, the co-culture extract exhibited an enhanced accumulation of the known constituents versicolorin B (14: ), averufin (16: ), and sterigmatocyctin (19: ) by factors of 1.5, 2.0, and 4.7, respectively, compared to the axenic fungal culture. The structures of the isolated compounds were elucidated on the basis of 1D and 2D NMR spectra and mass spectrometry as well as by comparison with literature data. The absolute configuration of compounds 3, 9: , and 10: was determined by ECD (electronic circular dichroism) analysis aided by TDDFT-ECD (time-dependent density functional theory electronic circular dichroism) calculations. Compounds 15, 18:  - 21: , and 26: exhibited strong to moderate cytotoxic activity against the mouse lymphoma cell line L5178Y, with IC50 values ranging from 2.0 to 21.2 µM, while compounds 14, 16, 31, 32: , and 33: displayed moderate inhibitory activities against several gram-positive bacteria, with MIC values ranging from 12.5 to 50 µM.

2-Alkylquinolone alkaloid biosynthesis in the medicinal plant Evodia rutaecarpa involves collaboration of two novel type III polyketide synthases.

2-Alkylquinolone (2AQ) alkaloids are pharmaceutically and biologically important natural products produced by both bacteria and plants, with a wide range of biological effects, including antibacterial, cytotoxic, anticholinesterase, and quorum-sensing signaling activities. These diverse activities and 2AQ occurrence in vastly different phyla have raised much interest in the biosynthesis pathways leading to their production. Previous studies in plants have suggested that type III polyketide synthases (PKSs) might be involved in 2AQ biosynthesis, but this hypothesis is untested. To this end, we cloned two novel type III PKSs, alkyldiketide-CoA synthase (ADS) and alkylquinolone synthase (AQS), from the 2AQ-producing medicinal plant, Evodia rutaecarpa (Rutaceae). Functional analyses revealed that collaboration of ADS and AQS produces 2AQ via condensations of N-methylanthraniloyl-CoA, a fatty acyl-CoA, with malonyl-CoA. We show that ADS efficiently catalyzes the decarboxylative condensation of malonyl-CoA with a fatty acyl-CoA to produce an alkyldiketide-CoA, whereas AQS specifically catalyzes the decarboxylative condensation of an alkyldiketide acid with N-methylanthraniloyl-CoA to generate the 2AQ scaffold via C-C/C-N bond formations. Remarkably, the ADS and AQS crystal structures at 1.80 and 2.20 Å resolutions, respectively, indicated that the unique active-site architecture with Trp-332 and Cys-191 and the novel CoA-binding tunnel with Tyr-215 principally control the substrate and product specificities of ADS and AQS, respectively. These results provide additional insights into the catalytic versatility of the type III PKSs and their functional and evolutionary implications for 2AQ biosynthesis in plants and bacteria.

Adjuvant effect of cranberry proanthocyanidin active fraction on antivirulent property of ciprofloxacin against Pseudomonas aeruginosa.

Quorum sensing inhibitors (QSIs) act as antivirulent agents since quorum sensing (QS) plays a vital role in regulating pathogenesis of Pseudomonas aeruginosa. However, application of single QSI may not be effective as pathogen is vulnerable to successful mutations. In such conditions, combination of QSIs can be exploited as there can be synergistic or adjuvant action. In the present study, we evaluated the antivirulence efficacy of combination of Vaccinium macrocarpon proanthocyanidin active fraction (PAF) and ciprofloxacin (CIP) at their sub-MICs using standard methods followed by analysis of their mode of action on QS using TLC and molecular docking. There was significant improvement in action of CIP when it was combined with PAF in reducing the QS controlled virulence factors (p < 0.05), motilities and biofilm of P. aeruginosa. TLC profiles of QS signals [(Acyl homoserine lactone (AHL) and Pseudomonas quinolone signal (PQS)] indicated that CIP in combination with PAF, besides showing inhibitory action on production of AHLs, also modulated production and inactivation of PQS. Docking scores also supported the observation. We therefore hypothesize that PAF-CIP combination, having improved anti-virulence property; can be exploited as a potent drug pairing against P. aeruginosa.

Chemopreventive activity of systemically administered curcumin on oral cancer in the 4-nitroquinoline 1-oxide model.

Curcumin has therapeutic potential in preventing several types of cancer, including colon, liver, prostate, and breast. The goal of this study was to evaluate the chemopreventive activity of systemically administered curcumin on oral carcinogenesis induced by 4-nitroquinolone-1-oxide (4-NQO). A total of 50 male albino rats, Rattus norvegicus, (Holtzman), were divided into five groups (n = 10 per group). Four of these groups were exposed to 50 ppm 4-NQO in their drinking water ad libitum for 8 or 12 weeks, two groups were treated with curcumin by oral gavage at 30 or 100 mg/kg per day, and one group was treated with corn oil (vehicle) only. The negative control group was euthanized at baseline. Tongues of all animals were removed after euthanasia and used in the subsequent analysis because the tongue is the primary site of carcinogenesis in this model. Descriptive histological analysis and immunohistochemistry for PCNA, Bcl-2, SOCS1 e-3, and STAT3 were performed to assess the oncogenic process. The gene expression of Vimentin, E-cadherin, N-cadherin, or TWIST1 was assessed using RT-qPCR as a representative of epithelial-mesenchymal transition (EMT) events. The administration of curcumin at 100 mg/kg during the 12 weeks markedly decreased the expression of PCNA, Bcl-2, SOCS1 e -3, and STAT3. Curcumin also minimized the cellular atypia under microscopic analysis and diminished the expression of the genes associated with EMT. These findings demonstrate that the systemic administration of curcumin has chemopreventive activity during oral carcinogenesis induced by 4-NQO.

Discovery of thienoquinolone derivatives as selective and ATP non-competitive CDK5/p25 inhibitors by structure-based virtual screening.

Calpain mediated cleavage of CDK5 natural precursor p35 causes a stable complex formation of CDK5/p25, which leads to hyperphosphorylation of tau. Thus inhibition of this complex is a viable target for numerous acute and chronic neurodegenerative diseases involving tau protein, including Alzheimer's disease. Since CDK5 has the highest sequence homology with its mitotic counterpart CDK2, our primary goal was to design selective CDK5/p25 inhibitors targeting neurodegeneration. A novel structure-based virtual screening protocol comprised of e-pharmacophore models and virtual screening workflow was used to identify nine compounds from a commercial database containing 2.84 million compounds. An ATP non-competitive and selective thieno[3,2-c]quinolin-4(5H)-one inhibitor (10) with ligand efficiency (LE) of 0.3 was identified as the lead molecule. Further SAR optimization led to the discovery of several low micromolar inhibitors with good selectivity. The research represents a new class of potent ATP non-competitive CDK5/p25 inhibitors with good CDK2/E selectivity.

A drug-repositioning screening identifies pentetic acid as a potential therapeutic agent for suppressing the elastase-mediated virulence of Pseudomonas aeruginosa.

Pseudomonas aeruginosa, a Gram-negative bacterium of clinical significance, produces elastase as a predominant exoprotease. Here, we screened a library of chemical compounds currently used for human medication and identified diethylene triamine penta-acetic acid (DTPA, pentetic acid) as an agent that suppresses the production of elastase. Elastase activity found in the prototype P. aeruginosa strain PAO1 was significantly decreased when grown with a concentration as low as 20 µM DTPA. Supplementation with Zn(2+) or Mn(2+) ions restored the suppressive effect of DTPA, suggesting that the DTPA-mediated decrease in elastase activity is associated with ion-chelating activity. In DTPA-treated PAO1 cells, transcription of the elastase-encoding lasB gene and levels of the Pseudomonas quinolone signal (PQS), a molecule that mediates P. aeruginosa quorum sensing (QS), were significantly downregulated, reflecting the potential involvement of the PQS QS system in DTPA-mediated elastase suppression. Biofilm formation was also decreased by DTPA treatment. When A549 alveolar type II-like adenocarcinoma cells were infected with PAO1 cells in the presence of DTPA, A549 cell viability was substantially increased. Furthermore, the intranasal delivery of DTPA to PAO1-infected mice alleviated the pathogenic effects of PAO1 cells in the animals. Together, our results revealed a novel function for a known molecule that may help treat P. aeruginosa airway infection.

Structure revision of N-mercapto-4-formylcarbostyril produced by Pseudomonas fluorescens G308 to 2-(2-hydroxyphenyl)thiazole-4-carbaldehyde [aeruginaldehyde].

An antibiotic substance isolated from Pseudomonas fluorescens strain G308 was earlier assigned the structure of N-mercapto-4-formylcarbostyril, but computational predictions of the 1H and 13C NMR magnetic shielding tensors show this structure to be incompatible with the published spectroscopic data. The same is true for six quinoline derivatives related to N-mercapto-4-formylcarbostyril by permutation of the O and S atoms. In contrast, 2-(2-hydroxyphenyl)thiazole-4-carbaldehyde [aeruginaldehyde], isolated from Pseudomonas protegens Pf-5, together with the reduced derivative aeruginol, displays spectroscopic data identical with those of the alleged carbostyril derivative. In addition, the published 1H and 13C NMR data are in agreement with those calculated for aeruginaldehyde. We propose that aeruginaldehyde and aeruginol originate from the non-ribosomal peptide synthetase enzymes involved in the siderophores enantio-pyochelin (or pyochelin) biosynthetic pathways.

Design and synthesis of azaisoflavone analogs as phytoestrogen mimetics.

A series of azaisoflavone analogs were designed and synthesized and their transactivation activities and binding affinities for ERα and ERß were investigated. Among these compounds, 2b and 3a were the most potent with 6.5 and 1.1 µM of EC50, respectively. Molecular modeling study showed putative binding modes of the compound 3a in the active site of ERα and ERß, which were similar with that of genistein and provided insight of the effect of N-alkyl substitution of azaisoflavones on ERß activity. Also, a biphasic effect of azaisoflavone analogs on MCF-7 cell growth depending on their concentrations was investigated.

Vitiquinolone--a quinolone alkaloid from Hibiscus vitifolius Linn.

Phytochemical investigations of the powdered root of Hibiscus vitifolius Linn. (Malvaceae) was extracted successively with n-hexane and chloroform. Analysis of the n-hexane extract by GC-MS led to the identification of twenty-six components by comparison of their mass spectra with GC-MS library data. A novel quinolone alkaloid, vitiquinolone (5) together with eight known compounds viz. ß-Amyrin acetate (1), n-octacosanol (2), ß-Amyrin (3), stigmasterol (4), xanthyletin (6), alloxanthoxyletin (7), xanthoxyletin (8) and betulinic acid (9) were isolated from chloroform extract by column chromatography over silica gel. The structure of vitiquinolone was established on the basis of spectroscopic methods including UV, IR, 1D, 2D NMR and ESI-MS. The known compounds were identified on the basis of their physical and spectroscopic data as reported in the literature.

Selective androgen receptor modulators: in vitro and in vivo metabolism and analysis.

For future targeted screening in National Residue Control Programmes, the metabolism of seven SARMs, from the arylpropionamide and the quinolinone classes, was studied in vitro using S9 bovine liver enzymes. Metabolites were detected and identified with ultra-performance liquid chromatography (UPLC) coupled to time-of-flight mass spectrometry (ToF-MS) and triple quadrupole mass spectrometry (QqQ-MS). Several metabolites were identified and results were compared with literature data on metabolism using a human cell line. Monohydroxylation, nitro-reduction, dephenylation and demethylation were the main S9 in vitro metabolic routes established. Next, an in vivo study was performed by oral administration of the arylpropionamide ostarine to a male calf and urine samples were analysed with UPLC-QToF-MS. Apart from two metabolites resulting from hydroxylation and dephenylation that were also observed in the in vitro study, the bovine in vivo metabolites of ostarine resulted in glucuronidation, sulfation and carboxylation, combined with either a hydroxylation or a dephenylation step. As the intact mother compounds of all SARMs tested are the main compounds present after in vitro incubations, and ostarine is still clearly present in the urine after the in vivo metabolism study in veal calves, the intact mother molecules were selected as the indicator to reveal treatment. The analytical UPLC-QqQ-MS/MS procedure was validated for three commercially available arylpropionamides according to European Union criteria (Commission Decision 2002/657/EC), and resulted in decision limits ranging from 0.025 to 0.05 µg l⁻¹ and a detection capability of 0.025 µg l⁻¹ in all cases. Adequate precision and intra-laboratory reproducibility (relative standard deviation below 20%) were obtained for all SARMs and the linearity was 0.999 for all compounds. This newly developed method is sensitive and robust, and therefore useful for confirmation and quantification of SARMs in bovine urine samples for residue control programmes and research purposes.

Discovery of a novel series of quinolone α7 nicotinic acetylcholine receptor agonists.

High throughput screening led to the identification of a novel series of quinolone α7 nicotinic acetylcholine receptor (nAChR) agonists. Optimization of an HTS hit (1) led to 4-phenyl-1-(quinuclidin-3-ylmethyl)quinolin-2(1H)-one, which was found to be potent and selective. Poor brain penetrance in this series was attributed to transporter-mediated efflux, which was in turn due to high pKa. A novel 4-fluoroquinuclidine significantly lowered the pKa of the quinuclidine moiety, reducing efflux as measured by a Caco-2 assay.

Pharmacophore-based drug design and biological evaluation of novel ABCB1 inhibitors.

Overexpression of ABCB1 is one of major barriers for multidrug resistance in chemotherapy and limits drug oral bioavailability. Inhibition of ABCB1 would sensitize multidrug resistance in clinical cancer chemotherapy. With this aim, a 3D pharmacophore model was created based on known ABCB1 inhibitors with correlation coefficient of 0.94, comprising three hydrophobic features and one hydrogen bond acceptor. It was further validated and used to search our in-house 3D database for potential ABCB1 inhibitors. The inhibitory activities of the best hits were evaluated by several biological assays, such as rhodamine 123 accumulation assay, chemosensitization assay, multidrug resistance 1-Madin-Darby canine kidney cells/Madin-Darby canine kidney cells permeability assay. Finally, compounds YZ-3 and YZ-16 were identified as potential leads to be developed in the designing of novel potent ABCB1 inhibitors.

Quorum sensing signaling molecules produced by reference and emerging soft-rot bacteria (Dickeya and Pectobacterium spp.).

BACKGROUND: Several small diffusible molecules are involved in bacterial quorum sensing and virulence. The production of autoinducers-1 and -2, quinolone, indole and γ-amino butyrate signaling molecules was investigated in a set of soft-rot bacteria belonging to six Dickeya or Pectobacterium species including recent or emerging potato isolates. METHODOLOGY/PRINCIPAL FINDINGS: Using bacterial biosensors, immunoassay, and chromatographic analysis, we showed that soft-rot bacteria have the common ability to produce transiently during their exponential phase of growth the N-3-oxo-hexanoyl- or the N-3-oxo-octanoyl-l-homoserine lactones and a molecule of the autoinducer-2 family. Dickeya spp. produced in addition the indole-3-acetic acid in tryptophan-rich conditions. All these signaling molecules have been identified for the first time in the novel Dickeya solani species. In contrast, quinolone and γ-amino butyrate signals were not identified and the corresponding synthases are not present in the available genomes of soft-rot bacteria. To determine if the variations of signal production according to growth phase could result from expression modifications of the corresponding synthase gene, the respective mRNA levels were estimated by reverse transcriptase-PCR. While the N-acyl-homoserine lactone production is systematically correlated to the synthase expression, that of the autoinducer-2 follows the expression of an enzyme upstream in the activated methyl cycle and providing its precursor, rather than the expression of its own synthase. CONCLUSIONS/SIGNIFICANCE: Despite sharing the S-adenosylmethionine precursor, no strong link was detected between the production kinetics or metabolic pathways of autoinducers-1 and -2. In contrast, the signaling pathway of autoinducer-2 seems to be switched off by the indole-3-acetic acid pathway under tryptophan control. It therefore appears that the two genera of soft-rot bacteria have similarities but also differences in the mechanisms of communication via the diffusible molecules. Our results designate autoinducer-1 lactones as the main targets for a global biocontrol of soft-rot bacteria communications, including those of emerging isolates.

Dolutegravir (S/GSK1349572) exhibits significantly slower dissociation than raltegravir and elvitegravir from wild-type and integrase inhibitor-resistant HIV-1 integrase-DNA complexes.

The integrase inhibitor (INI) dolutegravir (DTG; S/GSK1349572) has significant activity against HIV-1 isolates with raltegravir (RAL)- and elvitegravir (ELV)-associated resistance mutations. As an initial step in characterizing the different resistance profiles of DTG, RAL, and ELV, we determined the dissociation rates of these INIs with integrase (IN)-DNA complexes containing a broad panel of IN proteins, including IN substitutions corresponding to signature RAL and ELV resistance mutations. DTG dissociates slowly from a wild-type IN-DNA complex at 37°C with an off-rate of 2.7 × 10(-6) s(-1) and a dissociative half-life (t(1/2)) of 71 h, significantly longer than the half-lives for RAL (8.8 h) and ELV (2.7 h). Prolonged binding (t(1/2), at least 5 h) was observed for DTG with IN-DNA complexes containing E92, Y143, Q148, and N155 substitutions. The addition of a second substitution to either Q148 or N155 typically resulted in an increase in the off-rate compared to that with the single substitution. For all of the IN substitutions tested, the off-rate of DTG from IN-DNA complexes was significantly slower (from 5 to 40 times slower) than the off-rate of RAL or ELV. These data are consistent with the potential for DTG to have a higher genetic barrier to resistance, provide evidence that the INI off-rate may be an important component of the mechanism of INI resistance, and suggest that the slow dissociation of DTG may contribute to its distinctive resistance profile.

Does Pseudomonas fluorescens produce N-mercapto-4-formylcarbostyril?

The claim that N-mercapto-4-formylcarbostyril was isolated from Pseudomonas fluorescens strain G308 is questioned since N-thiols appear to be unstable compounds at ambient temperature.

Synthesis and biotransformation of 2-alkyl-4(1H)-quinolones by recombinant Pseudomonas putida KT2440.

2-Alkyl-4(1H)-quinolones (AQs) and related derivatives, which exhibit a variety of biological properties, are secondary metabolites produced by, e.g., Pseudomonas and Burkholderia spp. Due to their main role as signaling molecules in the quorum sensing system of Pseudomonas aeruginosa, 2-heptyl-4(1H)-quinolone (HHQ) and its 3-hydroxy derivative, termed the "Pseudomonas quinolone signal" (PQS), have received considerable attention. Since chemical synthesis of different AQs is complex, we assessed the applicability of recombinant P. putida KT2440 strains for the biosynthetic production of AQs. In mineral salts medium supplemented with octanoate and anthranilate, batch cultures of P. putida KT2440 [pBBR-pqsABCD] produced about 45 µM HHQ, 30% and 70% of which were localized in the culture supernatant and methanolic cell extract, respectively. 2,4-Dihydroxyquinoline and minor amounts of C3- to C13-saturated and C7:1 to C13:1 monounsaturated AQs were formed as by-products. Mass spectrometry and nuclear magnetic resonance analyses spectroscopy indicated that unsaturated AQs having the same molecular mass are cis and trans isomers rather than position isomers, with the double bond located between the α and ß carbon of the alkyl chain. Supplementing the cultures with hexanoate instead of octanoate shifted the AQ profile towards increased formation of C5-AQ. Individual AQs can be prepared from concentrated methanolic extracts by preparative high-performance liquid chromatography (HPLC). Regioselective hydroxylation of HHQ to PQS can be achieved in > 90% yield by biotransformation with P. putida KT2440 [pBBR-pqsH]. PQS can be isolated from methanolic cell extracts by HPLC, or be precipitated as Fe(III)-PQS complex. Preparation of a library of AQs will facilitate studies on the biological functions of these compounds.

Partial agonists in schizophrenia--why some work and others do not: insights from preclinical animal models.

While dopamine D2 receptor partial agonists (PAs) have been long considered for treating schizophrenia, only one, aripiprazole, is clinically available for therapeutic use. This raises critically important questions as to what is unique about aripiprazole and to what extent animal models can predict therapeutic success. A number of PAs whose clinical fate is known: aripiprazole, preclamol, terguride, OPC-4392 and bifeprunox were compared to haloperidol (a reference antipsychotic) in several convergent preclinical animal models; i.e. amphetamine-induced locomotion (AIL) and conditioned avoidance response (CAR), predictive of antipsychotic effects; unilateral nigrostriatal lesioned rats, a model of hypo-dopaminergia; striatal Fos induction, a molecular marker for antipsychotic activity; and side-effects common to this class of drugs: catalepsy (motor side-effects) and prolactaemia. The results were compared across drugs with reference to their measured striatal D2 receptor occupancy. All the PAs occupied striatal D2 receptors in a dose dependent manner, inhibited AIL and CAR, and lacked motor side-effects or prolactinaemia despite D2 receptor occupancy exceeding 80%. At comparative doses, aripiprazole distinguished itself from the other PAs by causing the least rotation in the hypo-dopaminergic model (indicating the least intrinsic activity) and showed the highest Fos expression in the nucleus accumbens (indicating functional D2 antagonism). Although a number of PAs are active in antipsychotic animal models, not all of them succeed. Given that only aripiprazole is clinically available, it can be inferred that low functional intrinsic activity coupled with sufficient functional antagonism as reflected in the animal models may be a marker of success.

Xenobiotic biotransformation of 4-methoxy-N-methyl-2-quinolone, isolated from Zanthoxylum monophyllum.

Phytochemical evaluation of Zanthoxylum monophyllum has led to the isolation of the alkaloid 4-methoxy-N-methyl-2-quinolone (1) with a significant activity against methicillin-resistant Staphylococcus aureus (MRSA), with an IC50 value of 1.5 microg/mL. Xenobiotic biotransformation of 1 has been conducted with the general goal of increasing the bioactivity of the compound and contributing new leads for further pharmacological research. Twenty-nine microorganisms were used for screening and two (Aspergillus flavus and Cunninghamella echinulata var. echinulata) were able to transform compound 1 to 4-methoxy-2-quinolone (2). Structural identification of the compounds was based on NMR, IR, and MS data.

Cytotoxic effects of haplamine and its major metabolites on human cancer cell lines.

Haplamine, extracted from Haplophyllum perforatum, is widely used in Central Asia for treating various diseases, including testicular cancer. The purpose of the present study was to investigate in vitro the cytotoxic properties of haplamine and its major metabolites (trans/cis-3,4-dihydroxyhaplamine) on human pancreatic cancer, colorectal cancer and hepatic cancer cell lines. The efficacy of haplamine was compared with those of the respective reference drugs for treating digestive cancers (e. g., 5-FU, gemcitabine). Finally, the implication of apoptosis in haplamine-induced cell death was investigated. The IC50 values of of haplamine were 52.5 +/- 2.6, 24.3 +/- 0.7; 41.5 +/- 2.5, 72 +/- 2, 32 +/- 2.2 and 59.7 +/- 2.1 microM in human pancreatic cancer (Capan1 and Capan2), colorectal cancer (LS174T, HT29, and SW620) and hepatic cancer (HepG2) cells, respectively. The IC50 values of trans/cis-3,4-dihydroxyhaplamine were both > 200 microM, thus suggesting that the previously reported cytotoxic efficacy of haplamine was supported by the parent drug only. Besides, our data showed that haplamine leads to cell death through the induction of early/late apoptosis in the target cells. Interestingly, we found that haplamine showed significant antiproliferative efficacy on resistant SW620 colorectal cells, whereas the reference drug 5-FU was ineffective (32 vs. 73 microM, p < 0.01 t- test), thus suggesting that haplamine could be of interest for treating digestive cancers resistant to standard fluoropyrimidines. Similarly, haplamine proved to be significantly more potent in pancreatic cells than gemcitabine, the reference cytotoxic drug for treating pancreatic carcinomas. Overall, these results confirm the anticancer properties of haplamine suggested by its traditional use, and indicate that it could be further considered in various other solid tumours frequently encountered in adults, including those resistant to standard chemotherapy.