Tryptamine, a Microbial Metabolite in Fermented Rice Bran Suppressed Lipopolysaccharide-Induced Inflammation in a Murine Macrophage Model.

Fermentation is thought to alter the composition and bioavailability of bioactive compounds in rice bran. However, how this process affects the anti-inflammatory effects of rice bran and the bioactive compounds that might participate in this function is yet to be elucidated. This study aimed to isolate bioactive compounds in fermented rice bran that play a key role in its anti-inflammatory function. The fermented rice bran was fractionated using a succession of solvent and solid-phase extractions. The fermented rice bran fractions were then applied to lipopolysaccharide (LPS)-activated murine macrophages to evaluate their anti-inflammatory activity. The hot water fractions (FRBA), 50% ethanol fractions (FRBB), and n-hexane fractions (FRBC) were all shown to be able to suppress the pro-inflammatory cytokine expression from LPS-stimulated RAW 264.7 cells. Subsequent fractions from the hot water fraction (FRBF and FRBE) were also able to reduce the inflammatory response of these cells to LPS. Further investigation revealed that tryptamine, a bacterial metabolite of tryptophan, was abundantly present in these extracts. These results indicate that tryptamine may play an important role in the anti-inflammatory effects of fermented rice bran. Furthermore, the anti-inflammatory effects of FRBE and tryptamine may depend on the activity of the aryl hydrocarbon receptor.

Tentative identification of in vitro metabolites of O-acetylpsilocin (psilacetin, 4-AcO-DMT) by UHPLC-Q-Orbitrap MS.

4-Acetoxy-N,N-dimethyltryptamine (4-AcO-DMT, psilacetin, O-acetylpsilocin) is a synthetic tryptamine with psychedelic properties. Psilacetin may also act as precursor drug of psilocin, similar to psilocybin, but little is known about its metabolism. In this study, the phase I and phase II in vitro metabolism of 4-AcO-DMT was investigated with pooled human liver microsomes, and the reaction mixture was analyzed using liquid chromatography-quadrupole/electrostatic field orbitrap mass spectrometry. Fifteen metabolites were formed after incubation of pooled human liver microsomes with 4-AcO-DMT (12 phase I metabolites and 3 phase II metabolites). The proposed metabolite structures were based on accurate mass analysis and MS/MS fragmentation patterns. The biotransformations included hydrolysis, hydroxylation, N-demethylation, oxidation, and conjugation with glucuronic acid. The hydrolysis metabolite was the most abundant compound. For the development of new methods for the identification of 4-AcO-DMT consumption, the beta-hydroxylation metabolite of 4-AcO-DMT (M2-1) is recommended as a biomarker. The data reported in this work might be applicable to metabolic transformation of 4-AcO-DMT in vivo and also forensically helpful.

ß-Methyltryptamine Provoking the Crucial Role of Strictosidine Synthase Tyr151-OH for Its Stereoselective Pictet-Spengler Reactions to Tryptoline-type Alkaloids.

Strictosidine synthase (STR), the gate enzyme for monoterpenoid indole alkaloid biosynthesis, catalyzes the Pictet-Spengler reaction (PSR) of various tryptamine derivatives with secologanin assisted by "indole sandwich" stabilization. Continuous exploration with ß-methyltryptamine (IPA) stereoselectively delivered the C6-methylstrictosidines and C6-methylvincosides by enzymatic and nonenzymatic PSR, respectively. Unexpectedly, the first "nonindole sandwich" binding mode was witnessed by the X-ray structures of STR1-ligand complexes. Site-directed mutagenesis revealed the critical cryptic role of the hydroxyl group of Tyr151 in IPA biotransformation. Further computational calculations demonstrated the adjustable IPA position in STR1 upon the binding of secologanin, and Tyr151-OH facilitates the productive PSR binding mode via an advantageous hydrogen-bond network. Further chemo-enzymatic manipulation of C6-methylvincosides successfully resulted in the discovered antimalarial framework (IC50 = 0.92 µM).

Characterization of Arylalkylamine N-Acyltransferase from Tribolium castaneum: An Investigation into a Potential Next-Generation Insecticide Target.

The growing issue of insecticide resistance has meant the identification of novel insecticide targets has never been more important. Arylalkylamine N-acyltransferases (AANATs) have been suggested as a potential new target. These promiscuous enzymes are involved in the N-acylation of biogenic amines to form N-acylamides. In insects, this process is a key step in melanism, hardening of the cuticle, removal of biogenic amines, and in the biosynthesis of fatty acid amides. The unique nature of each AANAT isoform characterized indicates each organism accommodates an assembly of discrete AANATs relatively exclusive to that organism. This implies a high potential for selectivity in insecticide design, while also maintaining polypharmacology. Presented here is a thorough kinetic and structural analysis of AANAT found in one of the most common secondary pests of all plant commodities in the world, Tribolium castaneum. The enzyme, named TcAANAT0, catalyzes the formation of short-chain N-acylarylalkylamines, with short-chain acyl-CoAs (C2-C10), benzoyl-CoA, and succinyl-CoA functioning in the role of acyl donor. Recombinant TcAANAT0 was expressed and purified from E. coli and was used to investigate the kinetic and chemical mechanism of catalysis. The kinetic mechanism is an ordered sequential mechanism with the acyl-CoA binding first. pH-rate profiles and site-directed mutagenesis studies identified amino acids critical to catalysis, providing insights about the chemical mechanism of TcAANAT0. A crystal structure was obtained for TcAANAT0 bound to acetyl-CoA, revealing valuable information about its active site. This combination of kinetic analysis and crystallography alongside mutagenesis and sequence analysis shines light on some approaches possible for targeting TcAANAT0 and other AANATs for novel insecticide design.

Effect of orange juice and tryptamine on the behavior and c-fos expression of Wistar rats.

Recent reports have shown that commercial orange juice is rich in biogenic amines. Consumption of foods containing large amounts of biogenic amines increase hypertensive crisis and high levels of histamine and tyramine, which have been implicated as causative agents in a number of food poisoning episodes. In addition, accumulation of tryptamine in plasma may be associated with mood disorders. The aim of this study was to determine whether chronic administration of orange juice extract and tryptamine affects the behavior and c-fos expression in the rat. For this purpose, Wistar male rats were injected with saline solution, tryptamine or orange juice extract. Sucrose preference test and elevated plus maze were evaluated to determine hedonic and anxiety behavior, respectively. Rats treated with orange juice extract showed increased anxiety behavior and sucrose consumption, similar to those treated with tryptamine. In addition, dorsal raphe nucleus, accumbens nucleus, and hippocampus showed an increase of c-fos positive cells in rats treated with orange juice extract. In conclusion, the chronic and lengthy consumption of orange juice or their derivatives in the diet could be a factor responsible to induce mood disorders and may promote excess caloric consumption.

Evaluation of Organic Anion Transporter 1A2-knock-in Mice as a Model of Human Blood-brain Barrier.

The present study aimed to establish a humanized mouse model with which to explore OATP1A2-mediated transcellular transport of drug substrates across the blood-brain barrier (BBB) and to evaluate the usefulness of the humanized mice in preclinical studies. Sulpiride, amisulpride, sultopride, and triptans were used as probes to discriminate OATP1A2 and Oatp1a4. We generated a mouse line humanized for OATP1A2 by introducing the coding region downstream of the Oatp1a4 promoter using the CRISPR/Cas9 technique. In the mice generated, OATP1A2 mRNA in the brain was increased corresponding to disappearance of Oatp1a4. OATP1A2 was localized on both the luminal and abluminal sides of the BBB. Unfortunately, study in vivo employing sulpiride, sumatriptan, and zolmitriptan as probes did not indicate any difference in their brain-to-plasma ratio between the control and humanized mice. Quantitative targeted absolute proteomic analysis of the BBB fraction from the humanized mice revealed that almost all analyzed transporters and membrane proteins were expressed at similar levels to those in control mice. The quantitative levels of OATP1A2 differed depending on the peptide quantified, which suggests that incomplete translation or posttranslational modification may occur. The blood-to-brain transport of zolmitriptan, determined by brain perfusion in situ, was 1.6-fold higher in the humanized mice than in the controls, whereas that of sulpiride was not significantly changed. To our knowledge, we established a mouse line humanized for a BBB uptake transporter for the first time. Unfortunately, because of limited impact, there is still room for improvement of the model system.

Cryo-EM structure of the serotonin 5-HT1B receptor coupled to heterotrimeric Go.

G-protein-coupled receptors (GPCRs) form the largest family of receptors encoded by the human genome (around 800 genes). They transduce signals by coupling to a small number of heterotrimeric G proteins (16 genes encoding different α-subunits). Each human cell contains several GPCRs and G proteins. The structural determinants of coupling of Gs to four different GPCRs have been elucidated1-4, but the molecular details of how the other G-protein classes couple to GPCRs are unknown. Here we present the cryo-electron microscopy structure of the serotonin 5-HT1B receptor (5-HT1BR) bound to the agonist donitriptan and coupled to an engineered Go heterotrimer. In this complex, 5-HT1BR is in an active state; the intracellular domain of the receptor is in a similar conformation to that observed for the ß2-adrenoceptor (ß2AR) 3 or the adenosine A2A receptor (A2AR) 1 in complex with Gs. In contrast to the complexes with Gs, the gap between the receptor and the Gß-subunit in the Go-5-HT1BR complex precludes molecular contacts, and the interface between the Gα-subunit of Go and the receptor is considerably smaller. These differences are likely to be caused by the differences in the interactions with the C terminus of the Go α-subunit. The molecular variations between the interfaces of Go and Gs in complex with GPCRs may contribute substantially to both the specificity of coupling and the kinetics of signalling.

Gut Microbiota-Derived Tryptophan Metabolites Modulate Inflammatory Response in Hepatocytes and Macrophages.

The gut microbiota plays a significant role in the progression of fatty liver disease; however, the mediators and their mechanisms remain to be elucidated. Comparing metabolite profile differences between germ-free and conventionally raised mice against differences between mice fed a low- and high-fat diet (HFD), we identified tryptamine and indole-3-acetate (I3A) as metabolites that depend on the microbiota and are depleted under a HFD. Both metabolites reduced fatty-acid- and LPS-stimulated production of pro-inflammatory cytokines in macrophages and inhibited the migration of cells toward a chemokine, with I3A exhibiting greater potency. In hepatocytes, I3A attenuated inflammatory responses under lipid loading and reduced the expression of fatty acid synthase and sterol regulatory element-binding protein-1c. These effects were abrogated in the presence of an aryl-hydrocarbon receptor (AhR) antagonist, indicating that the effects are AhR dependent. Our results suggest that gut microbiota could influence inflammatory responses in the liver through metabolites engaging host receptors.

Substrate selection of adenylation domains for nonribosomal peptide synthetase (NRPS) in bacillamide C biosynthesis by marine Bacillus atrophaeus C89.

Nonribosomal peptide synthetases (NRPSs) are multi-modular enzymes involved in the biosynthesis of natural products. Bacillamide C was synthesized by Bacillus atrophaeus C89. A nonribosomal peptide synthetase (NRPS) cluster found in the genome of B. atrophaeus C89 was hypothesized to be responsible for the biosynthesis of bacillamide C using alanine and cysteine as substrates. Here, the structure analysis of adenylation domains based on homologous proteins with known crystal structures indicated locations of the substrate-binding pockets. Molecular docking suggested alanine and cysteine as the potential substrates for the two adenylation domains in the NRPS cluster. Furthermore, biochemical characterization of the purified recombinant adenylation domains proved that alanine and cysteine were the optimum substrates for the two adenylation domains. The results provided the in vitro evidence for the hypothesis that the two adenylation domains in the NRPS of B. atrophaeus C89 preferentially select alanine and cysteine, respectively, as a substrate to synthesize bacillamide C. Furthermore, this study on substrates selectivity of adenylation domains provided basis for rational design of bacillamide analogs.

Insights Into the Molecular Mechanism of Triptan Transport by P-glycoprotein.

The P-glycoprotein (Pgp) transporter reduces the penetration of a chemically diverse range of neurotherapeutics at the blood-brain barrier, but the molecular features of drugs and drug-Pgp interactions that drive transport remain to be clarified. In particular, the triptan neurotherapeutics, eletriptan (ETT) and sumatriptan (STT), were identified to have a >10-fold difference in transport rates despite being from the same drug class. Consistent with these transport differences, ETT activated Pgp-mediated ATP hydrolysis ∼2-fold, whereas STT slightly inhibited Pgp-mediated ATP hydrolysis by ∼10%. The interactions between them were also noncompetitive, suggesting that they occupy different binding sites on the transporter. Despite these differences, protein fluorescence spectroscopy revealed that the drugs have similar affinity to the transporter. NMR with Pgp and the drugs showed that they have distinct interactions with the transporter. Tertiary conformational changes probed by acrylamide quenching of Pgp tryptophan fluorescence with the drugs and a nonhydrolyzable ATP analog implied that the STT-bound Pgp must undergo larger conformational changes to hydrolyze ATP than ETT-bound Pgp. These results and previous transport studies were used to build a conformationally driven model for triptan transport with Pgp where STT presents a higher conformational barrier for ATP hydrolysis and transport than ETT.

Recombinant flavin-dependent halogenases are functional in tobacco chloroplasts without co-expression of flavin reductase genes.

Halogenation of natural compounds in planta is rare. Herein, a successful engineering of tryptophan 6-halogenation into the plant context by heterologous expression of the Streptomyces toxytricini Stth gene and localization of its enzymatic product in various tobacco cell compartments is described. When co-expressed with the flavin reductase rebF from Lechevalieria aerocolonigenes, Stth efficiently produced chlorinated tryptophan in the cytosol. Further, supplementation of KBr yielded the brominated metabolite. More strikingly, targeting of the protein to the chloroplasts enabled effective halogenation of tryptophan even in absence of the partner reductase, providing crucial evidence for sufficient, organelle-specific supply of the FADH2 cofactor to drive halogen integration. Incorporation of an alternative enzyme, the 7-halogenase RebH from L. aerocolonigenes, into the metabolic set-up resulted in the formation of 6,7-dichlorotryptophan. Finally, expression of tryptophan decarboxylase (tdc) in concert with stth led to the generation of 6-chlorotryptamine, a new-to-nature precursor of monoterpenoid indole alkaloids. In sum, the report highlights the tremendous application potential of plants as a unique chassis for the engineering of rare and valuable halogenated natural products, with chloroplasts as the cache of reduction equivalents driving metabolic reactions.

A new scaffold of topoisomerase I inhibitors: Design, synthesis and biological evaluation.

The synthesis of a new hexacyclic system was realized starting from tryptamines and exploiting as a key step a sequential Pd-catalyzed N-arylation/acylation reaction. Having topoisomerases as biological target and the campthotecins class as benchmark, the new scaffold was decorated with substituents having different polarity and tested as Topoisomerase I inhibitors.

Solute Carrier Family of the Organic Anion-Transporting Polypeptides 1A2- Madin-Darby Canine Kidney II: A Promising In Vitro System to Understand the Role of Organic Anion-Transporting Polypeptide 1A2 in Blood-Brain Barrier Drug Penetration.

Organic anion-transporting polypeptide (OATP) 1A2 has the potential to be a target for central nervous system drug delivery due to its luminal localization at the human blood-brain barrier and broad substrate specificity. We found OATP1A2 mRNA expression in the human brain to be comparable to breast cancer resistance protein and OATP2B1 and much higher than P-glycoprotein (P-gp), and confirmed greater expression in the brain relative to other tissues. The goal of this study was to establish a model system to explore OATP1A2-mediated transcellular transport of substrate drugs and the interplay with P-gp. In vitro (human embryonic kidney 293 cells stably expressing Oatp1a4, the closest murine isoform) and in vivo (naïve and Oatp1a4 knock-out mice) studies with OATP1A2 substrate triptan drugs demonstrated that these drugs were not Oatp1a4 substrates. This species difference demonstrates that the rodent is not a good model to investigate the active brain uptake of potential OATP1A2 substrates. Thus, we constructed a novel OATP1A2 expressing Madin-Darby canine kidney (MDCK) II wild type and an MDCKII-multidrug resistance protein 1 (MDR1) system using BacMam virus transduction. The spatial expression pattern of OATP1A2 after transduction in MDCKII-MDR1 cells was superimposed to P-gp, confirming apical membrane localization. OATP1A2-mediated uptake of zolmitriptan, rosuvastatin, and fexofenadine across monolayers increased with increasing OATP1A2 protein expression. OATP1A2 counteracted P-gp efflux for cosubstrates zolmitriptan and fexofenadine. A three-compartment model incorporating OATP1A2-mediated influx was used to quantitatively describe the time- and concentration-dependent apical-to-basolateral transcellular transport of rosuvastatin across OATP1A2 expressing the MDCKII monolayer. This novel, simple and versatile experimental system is useful for understanding the contribution of OATP1A2-mediated transcellular transport across barriers, such as the blood-brain barrier.

Myeloperoxidase catalyzes the conjugation of serotonin to thiols via free radicals and tryptamine-4,5-dione.

Serotonin (5-hydroxytryptamine; 5HT) is a favorable substrate for myeloperoxidase and is likely to be oxidized by this heme enzyme during inflammation. In this study, we have investigated how serotonin becomes conjugated to amino acid residues and proteins when it is oxidized by myeloperoxidase. 5HT formed three adducts with N-acetylcysteine (NAC) when it was incubated with myeloperoxidase, xanthine oxidase, and acetaldehyde. One of the adducts was identified as 5HT-NAC, and the others were conjugates of NAC and tryptamine-4,5-dione (TD). There was no evidence for coupling of oxidized serotonin to amine residues. Glyceraldehyde-3-phosphate dehydrogenase (GAPDH) was exposed to 5HT with the enzymatic system or synthetic TD. Both caused a loss of thiols on GAPDH and covalent attachment of quinones derived from TD to the protein. Biotin-labeled 5HT was used instead of 5HT to confirm the conjugation of 5HT to GAPDH. It was incorporated into the GAPDH when oxidized by myeloperoxidase. Analysis of tryptic peptides of human GAPDH by liquid chromatography with mass spectrometry revealed that an adduct of TD was formed with the peptide containing Cys(152) and Cys(156). Our results indicate that myeloperoxidase can oxidize serotonin to species that form adducts with low molecular weight thiols and cysteine residues in proteins. Low molecular weight conjugates will redox cycle and fuel oxidative stress. Conjugation of serotonin to proteins will affect their function and may provide useful biomarkers of serotonin oxidation during inflammatory events.

Hydrophilic anti-migraine triptans are substrates for OATP1A2, a transporter expressed at human blood-brain barrier.

OATP1A2 is expressed in the luminal membrane of human blood-brain barrier (BBB). The human tissue with the highest OATP1A2 mRNA expression is the brain. We have established a robust BacMam2-OATP1A2 transduced HEK293 system. Among the 36 central nervous system (CNS) marketed drugs tested, hydrophilic triptans, 5-HT(1B/1D) receptor agonists for the treatment of migraine attacks, were identified as OATP1A2 substrates. Kinetics (K(m) and V(max)) were determined for six marketed triptans. Structure-activity relationship (SAR) obtained from 18 triptan structural analogs revealed that the positively charged basic amine atom was essential for efficient OATP1A2-mediated triptan uptake and uptake rate was in the order of tertiary > secondary > primary. Preliminary quantitative SAR analysis of the triptan analogs demonstrated positive correlation between OATP1A2-mediated uptake rate and van der Waals volume (vdw_vol). OATP1A2 was specifically expressed on the apical side of MDCKII monolayer after BacMam2-OATP1A2 transduction and can facilitate transport of triptans across the MDCKII monolayer from apical to basolateral side. Involvement of OATP1A2 for brain penetration of triptans in human requires further investigation.

Two new tryptamine derivatives, leptoclinidamide and (-)-leptoclinidamine B, from an Indonesian ascidian Leptoclinides dubius.

Two new tryptamine-derived alkaloids, named as leptoclinidamide (1) and (-)-leptoclinidamine B (2), were isolated from an Indonesian ascidian Leptoclinides dubius together with C²-α-D-mannosylpyranosyl-L-tryptophan (3). The structure of 1 was assigned on the basis of spectroscopic data for 1 and its N-acetyl derivative (4). Compound 1 was an amide of tryptamine with two ß-alanine units. Although the planar structure of 2 is identical to that of the known compound (+)-leptoclinidamine B (5), compound 2 was determined to be the enantiomer of 5 based on amino acid analysis using HPLC methods. Compounds 1 to 4 were evaluated for cytotoxicity against two human cancer cell lines, HCT-15 (colon) and Jurkat (T-cell lymphoma) cells, but none of the compounds showed activity.

Serotonin, but not N-methyltryptamines, activates the serotonin 2A receptor via a ß-arrestin2/Src/Akt signaling complex in vivo.

Hallucinogens mediate many of their psychoactive effects by activating serotonin 2A receptors (5-HT(2A)R). Although serotonin is the cognate endogenous neurotransmitter and is not considered hallucinogenic, metabolites of serotonin also have high affinity at 5-HT(2A)R and can induce hallucinations in humans. Here we report that serotonin differs from the psychoactive N-methyltryptamines by its ability to engage a ß-arrestin2-mediated signaling cascade in the frontal cortex. Serotonin and 5-hydroxy-L-tryptophan (5-HTP) induce a head-twitch response in wild-type (WT) mice that is a behavioral proxy for 5-HT(2A)R activation. The response in ß-arrestin2 knock-out (ßarr2-KO) mice is greatly attenuated until the doses are elevated, at which point, ßarr2-KO mice display a head-twitch response that can exceed that of WT mice. Direct administration of N-methyltryptamines also produces a greater response in ßarr2-KO mice. Moreover, the inhibition of N-methyltransferase blocks 5-HTP-induced head twitches in ßarr2-KO mice, indicating that N-methyltryptamines, rather than serotonin, primarily mediate this response. Biochemical studies demonstrate that serotonin stimulates Akt phosphorylation in the frontal cortex and in primary cortical neurons through the activation of a ß-arrestin2/phosphoinositide 3-kinase/Src/Akt cascade, whereas N-methyltryptamines do not. Furthermore, disruption of any of the components of this cascade prevents 5-HTP-induced, but not N-methyltryptamine-induced, head twitches. We propose that there is a bifurcation of 5-HT(2A)R signaling that is neurotransmitter and ß-arrestin2 dependent. This demonstration of agonist-directed 5-HT(2A)R signaling in vivo may significantly impact drug discovery efforts for the treatment of disorders wherein hallucinations are part of the etiology, such as schizophrenia, or manifest as side effects of treatment, such as depression.

Thermodynamics of peptide and non-peptide interactions with the human 5HT1a receptor.

The human serotonin 1a receptor (H5HT1aR) is a highly studied member of the 7 transmembrane G protein-coupled receptors. This model receptor, negatively coupled to adenylyl cyclase via Gi, is linked to physiological processes such as cognition and mood regulation and to associated disorders like anxiety and depression. Gibb's free energies, enthalpies, and entropies were calculated for the agonist [(3)H]8-OH-DPAT in the presence of synthetic peptides derived from sequences of intracellular loops 2 and 3 of the H5HT1aR. For comparative purposes, the thermodynamic parameters were also determined in the presence of a limited number of ligand-binding site substances (the partial agonist dipropyltryptamine [DPT], and the full agonist [(3)H]8-OH-DPAT alone). All of these thermodynamic measurements were based on binding data accumulated over a range of temperatures (0-35 degrees C). Representative examples of binding constant experiments and van't Hoff plots are shown to establish the thermodynamic variables. Although differences exist between the peptides themselves and the non-peptide agonists, in all situations the binding events are highly entropy driven. Differences between this information and published data for rat 5HT1aR are discussed, as are relationships to other receptor systems. Overall, the conclusions should be useful in further defining a comprehensive model of 5HT1aR, and for future development of binding-site and non-binding-site directed agents for the receptor.

Molecular and cellular pharmacological properties of 5-methoxycarbonylamino-N-acetyltryptamine (MCA-NAT): a nonspecific MT3 ligand.

5-Methoxycarbonylamino-N-acetyltryptamine (MCA-NAT) has been initially described as a ligand at non MT(1), non MT(2) melatonin binding site (MT3) selective versus MT(1) and MT(2), two membrane melatonin receptors. MCA-NAT activity has been reported by others in different models, in vivo, particularly in the intra-ocular pressure (IOP) models in rabbits and monkeys. Its activity was systematically linked to either MT3 or to a new, yet unknown, melatonin receptor. In this article, the melatonin receptor pharmacology of MCA-NAT is described. MCA-NAT has micromolar range affinities at the melatonin receptors MT(1) and MT(2), while in functional studies, MCA-NAT proved to be a powerful MT(1)/MT(2) partial agonist in the sub-micromolar range. These data strongly suggest that MCA-NAT actions might be mediated by these receptors in vivo. Finally, as described by others, we show that MCA-NAT is unable to elicit any type of receptor-like functional responses from Chinese hamster ovary cells over-expressing quinone reductase 2, the MT3.

Luzindole but not 4-phenyl-2- propionamidotetralin (4P-PDOT) diminishes the inhibitory effect of melatonin on murine Colon 38 cancer growth in vitro.

OBJECTIVE: Our earlier studies have shown that MLT exerts the inhibitory effect on murine cancer via membrane and nuclear receptors. We have found that the antagonist of MT1 receptors does not diminish the antiproliferative effect of MLT on Colon 38 cells, and the contribution of MT2 receptors has been suggested to be responsible. Therefore, in the present study we have examined the influence of the 4-phenyl-2-propionamidotetralin (4P-PDOT), which is a selective antagonist of MT2 membrane receptor, and luzindole - an antagonist of both membrane receptors, on an oncostatic action of MLT. MATERIALS AND METHODS: The murine cancer cell line Colon 38 was used in the experiments. In 48 hrs cell culture the effects of MLT, 4P-PDOT and luzindole administered alone and MLT applied jointly with either 4P-PDOT or luzindole were examined. The growth of cancer cells was assessed using the modified colorimetric Mosmann method. RESULTS: Melatonin at both examined concentrations (10-7, 10-9 M) significantly decreased the viability of cancer cells. The selective antagonist of MT2 membrane receptor, namely 4P-PDOT and luzindole applied separately did not have an effect on the growth of Colon 38 cells. The addition of 4P-PDOT to MLT did not change the inhibitory effect of MLT, whereas luzindole given together with MLT diminished, but failed to block totally, the oncostatic properties of MLT. CONCLUSIONS: The obtained data and our previous studies conducted on Colon 38 cancer indicate that membrane melatonin receptors are not indispensable to the oncostatic action of melatonin and thus other pathways such as nuclear signaling and receptor-independent mechanism may be also involved.