Inhibition of DYRK1A attenuates vascular remodeling in pulmonary arterial hypertension via suppressing STAT3/Pim-1/NFAT pathway.

Pulmonary arterial hypertension (PAH) is characterized by progressive vascular remodeling caused by the excessive proliferation and survival of pulmonary artery smooth muscle cells (PASMCs). Dual-specificity tyrosine regulated kinase 1A (DYRK1A) is a pleiotropic kinase involved in the regulation of multiple biological functions, including cell proliferation and survival. However, the role and underlying mechanisms of DYRK1A in PAH pathogenesis remain unclear. We found that DYRK1A was upregulated in PASMCs in response to hypoxia, both in vivo and in vitro. Inhibition of DYRK1A by harmine significantly attenuated hypoxia-induced pulmonary hypertension and pulmonary artery remodeling. Mechanistically, we found that DYRK1A promoted pulmonary arterial remodeling by enhancing the proliferation and survival of PASMCs through activating the STAT3/Pim-1/NFAT pathway, because STAT3 gain-of-function via adeno-associated virus serotype 2 (AAV2) carrying the constitutively active form of STAT3 (STAT3C) nearly abolished the protective effect of harmine on PAH. Collectively, our results reveal a significant role for DYRK1A in pulmonary arterial remodeling and suggest it as a drug target with translational potential for the treatment of PAH.

Discovery of YH677 as a cancer stemness inhibitor that suppresses triple-negative breast cancer growth and metastasis by regulating the TGFß signaling pathway.

Triple-negative breast cancer (TNBC) has a poor prognosis due to the lack of specific and highly effective therapeutic agents. Cancer stem cells (CSCs) are one of the main factors contributing to TNBC relapse and metastasis. Therefore, targeting CSCs selectively with small molecules is a novel strategy for drug development. In this study, the natural product harmine (HM) was identified as a hit compound from 2632 natural product monomers based on phenotypic screening of a 2D assay and patient-derived organoid (PDO) model that was established from a patient who had multiple drug resistance and various visceral and contralateral breast metastases. Next, harmine was further modified and optimized to obtain a lead compound (YH677) with a tetrahydro-ß-carboline scaffold. YH677 showed potent antiproliferative and antimigratory activities against several TNBC cell lines in vitro. In addition, YH677 inhibited epithelial mesenchymal transition (EMT) and stem cell marker expression in a dose-dependent manner. More importantly, YH677 suppressed breast cancer growth and metastasis in orthotopic, metastatic xenograft and patient-derived xenograft (PDX) models in vivo. Mechanistic studies showed that YH677 inhibits the expansion of CSCs by regulating the TGFß/Smad signaling pathway. These preclinical data provide a basis for the development of YH677 as a lead compound for TNBC treatment.

Structure-activity insights of harmine targeting DNA, ROS inducing cytotoxicity with PARP mediated apoptosis against cervical cancer, anti-biofilm formation and in vivo therapeutic study.

Harmine exhibits pH dependent structural equilibrium and possesses numerous biological and pharmacological activities. Mode and mechanism of DNA binding and its cytotoxicity were studied by multiple spectroscopic, calorimetric, molecular docking and in vitro apoptotic as well as in vivo biochemical and histological studies. It exists as cationic (structure I) and decationic form (structure II) in the pH range 3.0-7.8 and 8.5-12.4, respectively, with a pKa of 8.0. Structure I at pH 6.8 binds strongly to DNA with a cooperative mode of binding of Kiω 1.03 × 106 M-1and stoichiometry of 5.0 nucleotide phosphates. Structure I stabilized DNA by 10 °C, showed85%quenching of fluorescence intensity, perturbation in circular dichroism, partial intercalation and enthalpy driven exothermic binding. While, structure II at pH 8.5 has very weak interaction with CT DNA. Cytotoxic potencies of structure I was tested on four different cancer cell lines along with normal embryonic cell. It showed maximum cytotoxicity with GI50of 20 µM, against HeLa causing several apoptotic induction abilities. Harmine exhibited G2M arrest with ROS induced effective role in PARP mediated apoptosis as well as anti-inflammatory action on HeLa cells. Harmine further presented MIC and antibiofilm activity against Staphylococcus aureus in presence of <160 and 30 µg/ml, respectively. Mice with post harmine treatment (30 mg/kg b.w., I.P.) showed maximum recovery from damaged to near normal architecture of cervical epithelial cells. This study may be of prospective use in a framework to design novel beta carboline compounds for improved therapeutic applications in future against cervical cancer. HighlightsHarmine exists in structure I and structure II forms in the pH 6.8 and 8.5with a pKa of 8.0.Structure I at pH 6.8 binds strongly to DNA compared to structure II.Structure I showed maximum cytotoxicity with GI50 of 20 µM against HeLa.ROS mediated cytotoxicitywithG2M arrest with PARP mediated apoptosis was studied.Harmine (30µg/ml) exhibited antibiofilm activity against Staphylococcus aureus.Post harmine dose (30 mg/kg b.w., I.P.) in mice showed recovery of cervical epithelial cells.Communicated by Ramaswamy H. Sarma.

Harmine inhibits the proliferation and migration of glioblastoma cells via the FAK/AKT pathway.

AIMS: Glioblastoma is one of the most invasive tumors of the central nervous system, and has a high degree of malignancy and poor prognosis. Harmine, an active ingredient extracted from perennial herbs, has been reported to have obvious antitumor effects on various tumors. However, the effects of harmine on glioblastoma growth remain unknown. We here explored the effects of harmine on glioblastoma and its underlying molecular mechanisms related to tumorigenesis. MATERIALS AND METHODS: CCK-8 and immunofluorescent assay were performed to measure anti-proliferative effect of harmine on U251-MG and U373-MG cells. Wound healing assay was performed to measure the effects of harmine on cell migration. qRT-PCR and western blot were performed to detect the protein/gene expression. BALB/c nude mice bearing U251-MG xenografts was used to measure the effects of harmine on the growth of glioblastoma in vivo. KEY FINDINGS: Harmine treatment significantly suppressed the proliferation of U251-MG and U373-MG cells in a dose and time-dependent way. Mechanistically, harmine reduced the basal and EGF-enhanced the phosphorylation level of FAK and AKT. Moreover, harmine inhibited the cell viability of U251-MG and U373-MG cells by downregulating the phosphorylation of the FAK/AKT pathway. Besides, harmine significantly suppressed the migration of U251-MG cells by suppressing the expression of MMP2, MMP9 and VEGF. Subsequently, orthotopic xenograft models revealed that harmine treatment dramatically inhibited the growth of glioblastoma in vivo. SIGNIFICANCE: In conclusion, these results suggest that harmine suppresses the proliferation and migration of U251-MG and U373-MG cells by inhibiting the FAK/AKT signaling pathway. Our findings elucidate harmine could be a promising drug for glioblastoma therapy.

Pharmacologic and genetic approaches define human pancreatic ß cell mitogenic targets of DYRK1A inhibitors.

Small molecule inhibitors of dual specificity, tyrosine phosphorylation-regulated kinase 1A (DYRK1A), including harmine and others, are able to drive human ß cell regeneration. While DYRK1A is certainly a target of this class, whether it is the only or the most important target is uncertain. Here, we employ a combined pharmacologic and genetic approach to refine the potential mitogenic targets of the DYRK1A inhibitor family in human islets. A combination of human ß cell RNA sequencing, DYRK1A inhibitor kinome screens, pharmacologic inhibitors, and targeted silencing of candidate genes confirms that DYRK1A is a central target. Surprisingly, however, DYRK1B also proves to be an important target: silencing DYRK1A results in an increase in DYRK1B. Simultaneous silencing of both DYRK1A and DYRK1B yields greater ß cell proliferation than silencing either individually. Importantly, other potential kinases, such as the CLK and the GSK3 families, are excluded as important harmine targets. Finally, we describe adenoviruses that are able to silence up to 7 targets simultaneously. Collectively, we report that inhibition of both DYRK1A and DYRK1B is required for induction of maximal rates of human ß cell proliferation, and we provide clarity for future efforts in structure-based drug design for human ß cell regenerative drugs.

The Human Fecal Microbiota Metabolizes Foodborne Heterocyclic Aromatic Amines by Reuterin Conjugation and Further Transformations.

SCOPE: Heterocyclic aromatic amines (HAAs) are process-induced food contaminants with high mutagenic and/or carcinogenic potential. Although the human gut microbiota is known to affect the metabolism of dietary constituents, its impact on HAA metabolism and toxicity has been little studied. Here, the glycerol-dependent metabolism of seven foodborne HAAs (AαC, Trp-P-1, harman, norharman, PhIP, MeIQx, and MeIQ) by the human fecal microbiota is investigated. METHODS AND RESULTS: As analyzed by HPLC-DAD/FLD, the extent of conversion is strongly dependent on glycerol supplementation and HAA structure. AαC (60-100%) and the 2-aminoimidazoazarenes (up to 58%) are especially prone to microbial conversion. Based on high-resolution MS and/or NMR spectroscopy data, 70 fecal metabolites are identified in total, mainly formed by chemical reactions with one or two molecules of microbially derived reuterin. Moreover, it has been demonstrated that the human fecal microbiota can further transform reuterin adducts by reduction and/or hydroxylation reactions. Upon isolation, some reuterin-induced HAA metabolites appear to be partially unstable, complicating structural identification. CONCLUSION: The formation of microbial metabolites needs to be incorporated into risk assessment considerations for HAAs in human health. In this study, several HAA metabolites, mainly reuterin-dependent, are identified in vitro, providing the basis for future human studies investigating microbial HAA metabolism.

Crystal Structure of Human Dual-Specificity Tyrosine-Regulated Kinase 3 Reveals New Structural Features and Insights into its Auto-phosphorylation.

Dual-specificity tyrosine-regulated kinases (DYRKs) auto-phosphorylate a critical tyrosine residue in their activation loop and phosphorylate their substrate on serine and threonine residues. The auto-phosphorylation occurs intramolecularly and is a one-off event. DYRK3 is selectively expressed at a high level in hematopoietic cells and attenuates erythroblast development, leading to anemia. In the present study, we determined the crystal structure of the mature form of human DYRK3 in complex with harmine, an ATP competitive inhibitor. The crystal structure revealed a phosphorylation site, residue S350, whose phosphorylation increases the stability of DYRK3 and enhances its kinase activity. In addition, our structural and biochemical assays suggest that the N-terminal auto-phosphorylation accessory domain stabilizes the DYRK3 protein, followed by auto-phosphorylation of the tyrosine of the activation loop, which is important for kinase activity. Finally, our docking analysis provides information for the design of novel and potent therapeutics to treat anemia.

A novel harmine derivative, N-(4-(hydroxycarbamoyl)benzyl)-1-(4- methoxyphenyl)-9H-pyrido[3,4-b]indole-3-carboxamide (HBC), as histone deacetylase inhibitor: in vitro antiproliferation, apoptosis induction, cell cycle arrest, and antimetastatic effects.

This study aims to design and synthesize a novel harmine derivative N-(4-(hydroxycarbamoyl) benzyl)-1-(4-methoxyphenyl)-9H-pyrido [3,4-b]indole-3-carboxamide (HBC) as histone deacetylase (HDAC) inhibitor, and evaluate its antitumor activities and anti-metastasis mechanism. HBC not only exerted significant ant-proliferation activity against five human cancer cell lines, especially for HepG2 cell with an IC50 value of 2.21 µM, which is nearly three-fold lower than SAHA (IC50 = 6.26 µM), but also showed selective HDAC1/6 inhibitory effects in vitro. However, HBC had little effect on normal hepatic cells LO2. Furthermore, HBC simultaneously increased the acetylation of histone H3, H4, and α-tubulin, induced hypochromism by electrostatical interaction with CT-DNA, triggered more significant cancer cell apoptosis and cell cycle arrest at G2/M than SAHA by inhibition of both CDK1 and cyclin B in a concentration dependent manner. In addition, scratch and invasion assay showed that HBC also dose-dependently suppressed migration and invasion capacities of highly metastatic HCC HepG2 cells through down-regulated the expression of tumor metastasis related proteins MMP-2 and MMP-9, significantly better than SAHA. Finally, HBC showed low acute toxicity to mice and significant growth inhibition of the hepatoma tumor in vivo. These results demonstrate that novel harmine-based HDAC inhibitor HBC not only exhibited selective HDAC1/6 inhibitory activity and significant in vitro and in vivo antitumor activity, but also possessed DNA binding effect, apoptosis induction, cell cycle arrest effects, and potent anti-metastasis mechanisms, which may hold great promise as therapeutic agent targeting HDAC1/6 for the intervention of human cancers.

A harmine-derived beta-carboline displays anti-cancer effects in vitro by targeting protein synthesis.

Growing evidence indicates that protein synthesis is deregulated in cancer onset and progression and targeting this process might be a selective way to combat cancers. While harmine is known to inhibit DYRK1A and intercalate into the DNA, tri-substitution was shown previously to modify its activity profile in favor of protein synthesis inhibition. In this study, we thus evaluated the optimized derivative CM16 in vitro anti-cancer effects unfolding its protein synthesis inhibition activity. Indeed, the growth inhibitory profile of CM16 in the NCI 60-cancer-cell-line-panel correlated with those of other compounds described as protein synthesis inhibitors. Accordingly, CM16 decreased in a time- and concentration-dependent manner the translation of neosynthesized proteins in vitro while it did not affect mRNA transcription. CM16 rapidly penetrated into the cell in the perinuclear region of the endoplasmic reticulum where it appears to target translation initiation as highlighted by ribosomal disorganization. More precisely, we found that the mRNA expression levels of the initiation factors EIF1AX, EIF3E and EIF3H differ when comparing resistant or sensitive cell models to CM16. Additionally, CM16 induced eIF2α phosphorylation. Those effects could explain, at least partly, the CM16 cytostatic anti-cancer effects observed in vitro while neither cell cycle arrest nor DNA intercalation could be demonstrated. Therefore, targeting protein synthesis initiation with CM16 could represent a new promising alternative to current cancer therapies due to the specific alterations of the translation machinery in cancer cells as recently evidenced with respect to EIF1AX and eIF3 complex, the potential targets identified in this present study.

Spectroscopic and structural studies on the interaction of an anticancer ß-carboline alkaloid, harmine with GC and AT specific DNA oligonucleotides.

Harmine, a tricyclic ß-carboline alkaloid possesses anticancer properties. Thus, its binding studies with DNA are considerably important because mechanism of action of anticancer drug involves DNA binding. On the other hand, the DNA binding study is also useful in drug designing and synthesis of new compounds with enhanced biological properties. Hence, the binding of harmine with sequence specific DNA oligonucleotides has been studied using various biophysical techniques i.e. absorption, fluorescence and molecular docking techniques. UV absorption study, Fluorescence quenching and Iodide quenching experiments revealed intercalation type of binding of harmine with short sequence specific DNA oligonucleotides. Fluorescence and absorption studies also concluded binding constants of harmine with GC rich DNA sequence in the order of 105 M-1 while with AT rich sequences it was in the order of 103 M-1 which clearly indicated that harmine showed greater intercalation with GC rich sequences as compared to AT rich sequences. From thermodynamic studies, it was concluded that harmine-DNA complex formation was spontaneous, exothermic and energetically favorable process. Molecular docking studies confirmed that harmine intercalates between the base pairs of DNA structure but energetically prefers intercalation between GC base pairs. Molecular docking studies and the calculated thermodynamic parameters, i.e. Gibbs free energy (ΔG), Enthalpy change (ΔH) and Entropy change (ΔS) indicated that H-bonds, van der Waals interactions and hydrophobic interactions play a major role in the binding of harmine to DNA oligomers.

Harmine blocks herpes simplex virus infection through downregulating cellular NF-κB and MAPK pathways induced by oxidative stress.

Herpes simplex virus types 1 and 2 (HSV-1 and -2) are highly prevalent in many populations and therapeutic options are limited. Both viruses can establish latency by maintaining viral genomes in neurons of sensory ganglia. Primary or recurrent HSV infections may lead to deleterious outcomes: HSV-1 infection may result in corneal blindness and encephalitis and HSV-2 infection leads to herpes genitalis. While no effective vaccine is available, acyclovir is widely used for therapy, which targets and inhibits viral DNA polymerase. Although acyclovir is of low toxicity, resistant strains arise due to persistent use, mainly in immune compromised patients. In our effort to identify new HSV inhibitory molecules, harmine was found to potently inhibit HSV infection. Harmine, a beta-carbon alkaloid with an indole core structure and a pyridine ring, is widely distributed in plants. Earlier studies showed that harmine exhibited pharmacological activities such as antifungal, antimicrobial, antitumor, antiplasmodial and antioxidants. In the current study, we showed that harmine was a potent inhibitor of HSV-2 infection in vitro assays with EC50 value at around 1.47µM and CC50 value at around 337.10µM. The HSV RNA transcription, protein synthesis, and virus titers were reduced by the presence of harmine in a dose dependent manner. Further study on the mechanism of the anti-HSV activity showed that harmine blocked HSV-induced ROS production and the upregulated cytokine/chemokine expression, but our evidence showed that the inhibition of viral replication was unlikely mediated by the blocking of ROS production. We demonstrated that harmine significantly reduced HSV-2-induced NF-κB activation, as well as IκB-α degradation and p65 nuclear translocation. We found that harmine also inhibited HSV-2-mediated p38 kinase and c-Jun N-terminal kinases (JNK) phosphorylation.

Monoamine oxidase A binding in the prefrontal and anterior cingulate cortices during acute withdrawal from heavy cigarette smoking.

CONTEXT: Greater prefrontal cortex and anterior cingulate cortex monoamine oxidase A (MAO-A) binding is associated with depressed mood. Substances in cigarette smoke, such as harman, inhibit MAO-A, and cigarette withdrawal is associated with depressed mood. Dysphoria during cigarette withdrawal predicts relapse. It is unknown whether MAO-A binding increases during early cigarette withdrawal. OBJECTIVES: To measure prefrontal and anterior cingulate cortex MAO-A binding during acute cigarette withdrawal and to assess the relationship with smoking severity, plasma levels of harman, and severity of depression. DESIGN: Study via positron emission tomography of healthy control and cigarette-smoking individuals. PATIENTS: Twenty-four healthy nonsmoking and 24 otherwise healthy cigarette-smoking individuals underwent positron emission tomography with harmine labeled with carbon 11. Healthy nonsmoking individuals underwent scanning once. Cigarette-smoking individuals underwent scanning after acute withdrawal and after active cigarette smoking. Cigarette smoking was heavy (≥25 cigarettes per day) or moderate (15-24 cigarettes per day). SETTING: Tertiary care psychiatric hospital. MAIN OUTCOME MEASURE: An index of MAO-A density, MAO-A V(T), was measured in the prefrontal and anterior cingulate cortices. RESULTS: In heavy-smoking individuals, prefrontal and anterior cingulate cortex MAO-A V(T) was greater during withdrawal (23.7% and 33.3%, respectively; repeated-measures multivariate analysis of variance, F(1,22) = 25.58, P < .001). During withdrawal from heavy smoking, prefrontal and anterior cingulate cortex MAO-A V(T) was greater than in healthy controls (25.0% and 25.6%, respectively; multivariate analysis of variance, F(2,33) = 6.72, P = .004). The difference in MAO-A V(T) in the prefrontal cortex and anterior cingulate cortex between withdrawal and active, heavy smoking covaried with change in plasma harman levels in the prefrontal cortex and anterior cingulate cortex (multivariate analysis of covariance, F(1,10) = 9.97, P = .01). The change in MAO-A V(T) between withdrawal and active, heavy smoking also covaried with severity of depression (multivariate analysis of covariance, F(1,10) = 11.91, P = .006). CONCLUSIONS: The increase in prefrontal and anterior cingulate cortex MAO-A binding and associated reduction in plasma harman level represent a novel, additional explanation for depressed mood during withdrawal from heavy cigarette smoking. This finding resolves a longstanding paradox regarding the association of cigarette smoking with depression and suicide and argues for additional clinical trials on the effects of MAO-A inhibitors on quitting heavy cigarette smoking.

Modulation of prototropic activity and rotational relaxation dynamics of a cationic biological photosensitizer within the motionally constrained bio-environment of a protein.

The present work describes the interaction of a promising cancer cell photosensitizer, harmane (HM), with a model transport protein, Bovine Serum Albumin (BSA). The studied molecule of interest (HM) belongs to the family of naturally occurring fluorescent drug-binding alkaloids, the ß-carbolines. A combined use of steady-state and time-resolved fluorescence techniques is applied to follow and characterize the binding interaction. The polarity-dependent prototropic activity of HM is found to be responsible for the commendable sensitivity of the probe to the protein environments and is distinctly reflected on the emission profile. Steady-state fluorescence anisotropy study reveals the impartation of a considerable degree of motional restriction on the drug molecule as a result of binding to the protein. Contrary to the single-exponential nature of fluorescence anisotropy decay of HM in aqueous buffer, they are found to be biexponential in the protein environment. The rotational relaxation dynamics of HM within the protein has been interpreted on the lexicon of the Two-Step and Wobbling-in-Cone model. The probable binding location for the cationic drug is found to be the hydrophilic binding zone of BSA, i.e., domain I (characterized by a net negative charge). The AutoDock-based blind docking simulation has been explored for evaluating an unbiased result of the probable interaction site of HM in the protein. To unfold the effect of binding of the drug on the secondary structural content of the protein, circular dichroism (CD) spectroscopy has been exploited to see that binding of the drug accompanies some decrease in α-helical content of BSA, and the effect gradually saturates toward a higher drug/protein molar ratio.

Harmine is an ATP-competitive inhibitor for dual-specificity tyrosine phosphorylation-regulated kinase 1A (Dyrk1A).

Harmine is a ß-carboline alkaloid. The compound is a potent inhibitor of dual-specificity tyrosine phosphorylation-regulated kinase 1A (Dyrk1A), a kinase implicated in Down syndrome. In this study, we show that harmine functions as an ATP-competitive inhibitor against Dyrk1A. Our conclusion is supported by kinetic analysis of harmine inhibition as well as by the characterization of a Dyrk1A mutation conferring significant resistance to harmine. The mutation, V306A, is located next to the highly conserved D307 residue in kinases known to coordinate the phosphate groups of ATP through a Mg²+ ion. The V306A mutation offers harmine resistance by differentially altering Dyrk1A affinity for harmine and ATP. The V306A mutation causes no apparent alteration to Dyrk1A activity except for the reduction in ATP affinity. This deficiency could be fully compensated by supplying ATP with a concentration in the physiological range. Our results reveal that harmine inhibits Dyrk1A activity by interacting with residues in the ATP-binding pocket and displacing ATP. Our results also suggest that harmine will be a good lead compound for further designing of selective ATP-competitive Dyrk1A inhibitors through exploration of the ATP-binding pocket of Dyrk1A.

Mitochondrial dysfunction and biotransformation of ß-carboline alkaloids, harmine and harmaline, on isolated rat hepatocytes.

The cytotoxic effects and biotransformation of harmine and harmaline, which are known ß-carboline alkaloids and potent hallucinogens, were studied in freshly isolated rat hepatocytes. The exposure of hepatocytes to harmine caused not only concentration (0-0.50mM)- and time (0-3h)-dependent cell death accompanied by the formation of cell blebs and the loss of cellular ATP, reduced glutathione, and protein thiols but also the accumulation of glutathione disulfide. Of the other analogues examined, the cytotoxic effects of harmaline and harmol (a metabolite of harmine) at a concentration of 0.5mM were less than those of harmine. The loss of mitochondrial membrane potential and generation of oxygen radical species in hepatocytes treated with harmine were greater than those with harmaline and harmol. In the oxygen consumption of mitochondria isolated from rat liver, the ratios of state-3/state-4 respiration of these ß-carbolines were decreased in a concentration-dependent manner. In addition, harmine resulted in the induction of the mitochondrial permeability transition (MPT), and the effects of harmol and harmaline were less than those of harmine. At a weakly toxic level of harmine (0.25mM), it was metabolized to harmol and its monoglucuronide and monosulfate conjugates, and the amounts of sulfate rather than glucuronide predominantly increased with time. In the presence of 2,5-dichloro-4-nitrophenol (50µM; an inhibitor of sulfotransferase), harmine-induced cytotoxicity was enhanced, accompanied by decrease in the amount of harmol-sulfate conjugate, due to an increase in the amount of unconjugated harmol and the inhibition of harmine loss. Taken collectively, these results indicate that (a) mitochondria are target organelles for harmine, which elicits cytotoxicity through mitochondrial failure related to the induction of the MPT, mitochondrial depolarization, and inhibition of ATP synthesis; and (b) the toxic effects of harmine are greater than those of either its metabolite harmol or its analogue harmaline, suggesting that the onset of harmine-induced cytotoxicity may depend on the initial and/or residual concentrations of harmine rather than on those of its metabolites.

Harman induces CYP1A1 enzyme through an aryl hydrocarbon receptor mechanism.

Harman is a common compound in several foods, plants and beverages. Numerous studies have demonstrated its mutagenic, co-mutagenic and carcinogenic effects; however, the exact mechanism has not been fully identified. Aryl hydrocarbon receptor (AhR) is a transcription factor regulating the expression of the carcinogen-activating enzyme; cytochrome P450 1A1 (CYP1A1). In the present study, we examined the ability of harman to induce AhR-mediated signal transduction in human and rat hepatoma cells; HepG2 and H4IIE cells. Our results showed that harman significantly induced CYP1A1 mRNA in a time- and concentration-dependent manner. Similarly, harman significantly induced CYP1A1 at protein and activity levels in a concentration-dependent manner. Moreover, the AhR antagonist, resveratrol, inhibited the increase in CYP1A1 activity by harman. The RNA polymerase inhibitor, actinomycin D, completely abolished the CYP1A1 mRNA induction by harman, indicating a transcriptional activation. The role of AhR in CYP1A1 induction by harman was confirmed by using siRNA specific for human AhR. The ability of harman to induce CYP1A1 was strongly correlated with its ability to stimulate AhR-dependent luciferase activity and electrophoretic mobility shift assay. At post-transcriptional and post-translational levels, harman did not affect the stability of CYP1A1 at the mRNA and the protein levels, excluding other mechanisms participating in the obtained effects. We concluded that harman can directly induce CYP1A1 gene expression in an AhR-dependent manner and may represent a novel mechanism by which harman promotes mutagenicity, co-mutagenicity and carcinogenicity.

The small molecule phenamil is a modulator of adipocyte differentiation and PPARgamma expression.

We previously described the use of a cell-based screening approach to identify small molecules that regulate adipocyte differentiation. Here we identify the amiloride derivative phenamil as an adipogenic compound. Phenamil acutely induces expression of the key transcription factor of adipogenesis, peroxisome proliferator-activated receptor gamma (PPARgamma) and, consequently, promotes the differentiation of multiple preadipocyte cell lines, including 3T3-L1 and F442A. Interestingly, the adipogenic action of phenamil is distinct from and additive with both PPARgamma ligands and the previously identified adipogenic small molecule harmine. To identify signaling pathways mediating phenamil's effects, we performed transcriptional profiling of 3T3-F442A preadipocytes. ETS variant 4 (ETV4) was identified as a gene rapidly induced by phenamil but not by other adipogenic small molecules or PPARgamma agonists. Transient expression of ETV4 in preadipocytes enhances the expression of PPARgamma. Stable overexpression of ETV4 promotes expression of PPARgamma and its downstream target genes and enhances morphological differentiation. Finally, knockdown of PPARgamma expression by shRNA blocks the effects of phenamil on adipocyte differentiation and gene expression, but it does not block phenamil induction of ETV4, which suggests that ETV4 acts upstream of PPARgamma in differentiation processes. These results identify a phenamil as new small molecule tool for the probing of adipocyte differentiation that acts, at least in part, through induction of ETV4 expression.

Apparent differences in mechanisms of harmol sulfate biliary excretion in mice and rats.

Previous experiments demonstrated that the biliary excretion of harmol sulfate (HS) was mediated by breast cancer resistance protein (Bcrp) and not by multidrug resistance-associated protein (Mrp)2 or P-glycoprotein in mice. However, recent reports suggested that species differences in hepatic canalicular transport mechanisms for a given substrate exist between mice and rats. In the present study, biliary excretion of HS was examined in perfused livers from mice and rats in the absence or presence of the P-glycoprotein and Bcrp inhibitor N-(4-[2-(1,2,3,4-tetrahydro-6,7-dimethoxy-2-isoquinolinyl)ethyl]-phenyl)-9,10-dihydro-5-methoxy-9-oxo-4-acridine carboxamide (GF120918). As expected, in mouse liver perfusions, the biliary excretion of HS was decreased approximately 3.5-fold by GF120918, consistent with previous reports of Bcrp-mediated HS biliary excretion. However, despite sufficient hepatic unbound concentrations of GF120918 to achieve extensive inhibition of Bcrp, the biliary excretion of HS was not decreased significantly in wild-type (50 +/- 12 versus 41 +/- 6%) or TR(-) (18 +/- 2 versus 16 +/- 3%) Wistar rats. In summary, biliary excretion of HS was mediated by a GF120918-sensitive mechanism in mice, previously elucidated as Bcrp. In contrast, the pathway responsible for HS biliary excretion in rats was not impaired by GF120918. Thus, transport mechanism(s) responsible for harmol sulfate biliary excretion appear to differ between mice and rats.

Oxidative metabolism of the bioactive and naturally occurring beta-carboline alkaloids, norharman and harman, by human cytochrome P450 enzymes.

Norharman and harman are naturally occurring beta-carboline alkaloids exhibiting a wide range of biological, psychopharmacological, and toxicological actions. They occur in foods and tobacco smoke and also appear endogenously in humans. In this research, metabolic and kinetic studies with cytochrome P450 enzymes and human liver microsomes showed that beta-carbolines were efficiently oxidized to several ring-hydroxylated and N-oxidation products that were subsequently identified and quantified. 6-Hydroxy- beta-carboline (6-hydroxynorharman and 6-hydroxyharman) was a major metabolite efficiently produced (high kcat and low Km) by P450 1A2 and 1A1 and to a minor extent by P450 2D6, 2C19 and 2E1. 3-Hydroxy-beta-carboline (3-hydroxynorharman and 3-hydroxyharman), another major metabolite, was specifically produced by P450 1A2 and 1A1, whereas beta-carboline-N(2)-oxide (harman-2-oxide and norharman-2-oxide) was produced by P450 2E1. The same pattern of metabolism was confirmed for human liver microsomes. Oxidative metabolism for harman was slightly higher than norharman, but norharman showed lower Km values. The oxidation of beta-carbolines is a detoxication route performed mainly by P450 1A2 and 1A1, with the participation of P450 2D6, 2C19, and 2E1, as additional contributors. Then, individual variations in the levels and activity of these P450s may influence biotransformation of beta-carboline alkaloids and their ultimate biological effects. beta-Carbolines were previously reported as comutagens and/or inhibitors of mutagens activated by P450 1A enzymes such as heterocyclic amines and polycyclic hydrocarbons. Results in this work show that beta-carbolines are good ligands and substrates for P450 1A2/1A1, contributing to the explanation of some of their toxicological effects.

Differential effects of dopamine melanin on norharman-induced toxicity in PC12 cells.

The food contaminant norharman structurally resembles MPTP a compound that selectively damages pigmented brain areas. Both compounds are sequestered and retained in melanin-containing neurons. The aim of the study was to examine whether intracellular melanin can modulate the toxicity of norharman in melanin-loaded PC12 cells. Dopamine melanin protected against norharman-induced upregulation of grp78, activation of caspase 3 and necrosis at low concentrations (5 and 50 microM). In contrast, at a high conentration (500 microM) there was a significantly increased expression of grp78, hsp90 and caspase 3 and a disassociation of melanin aggregates leading to dispersal of granules to swollen neurite terminals. In human populations, a long-term low-level exposure to toxicants with a high affinity to melanin will probably result in accumulation in melanin-containing neurons in vivo. Our data suggest that accumulation of a neurotoxicant in melanin-loaded cells may lead to increased cell stress, apoptotic signaling and disassociation of melanin aggregates.