Activation of β2-adrenergic receptor (β2AR) and deorphanized GPR55 has been shown to modulate can... more Activation of β2-adrenergic receptor (β2AR) and deorphanized GPR55 has been shown to modulate cancer growth in diverse tumor types in vitro and in xenograft models in vivo. (R,R′)-4′-methoxy-1-naphthylfenoterol [(R,R′)-MNF] is a bivalent compound that agonizes β2AR but inhibits GPR55-mediated pro-oncogenic responses. Here, we investigated the molecular mechanisms underlying the anti-tumorigenic effects of concurrent β2AR activation and GPR55 blockade in C6 glioma cells using (R,R′)-MNF as a marker ligand. Our data show that (R,R′)-MNF elicited G1-phase cell cycle arrest and apoptosis, reduced serum-inducible cell motility, promoted the phosphorylation of PKA target proteins, and inhibited constitutive activation of ERK and AKT in the low nanomolar range, whereas high nanomolar levels of (R,R′)-MNF were required to block GPR55-mediated cell motility. siRNA knockdown and pharmacological inhibition of β2AR activity were accompanied by significant upregulation of AKT and ERK phosphorylation, and selective alteration in (R,R′)-MNF responsiveness. The effects of agonist stimulation of GPR55 on various readouts, including cell motility assays, were suppressed by (R,R′)-MNF. Lastly, a significant increase in phosphorylation-mediated inactivation of β-catenin occurred with (R,R′)-MNF, and we provided new evidence of (R,R′)-MNF-mediated inhibition of oncogenic β-catenin signaling in a C6 xenograft tumor model. Thus, simultaneous activation of β2AR and blockade of GPR55 may represent a novel therapeutic approach to combat the progression of glioblastoma cancer.
Various species of the Carlina genus have been used in traditional medicine in many countries to ... more Various species of the Carlina genus have been used in traditional medicine in many countries to treat numerous skin disorders, including cancer. The objective of this work was to assess the anticancer properties of root and leaf extracts from Carlina acaulis subsp. caulescens and C. acanthifolia subsp. utzka. Anti-tumor properties of the extracts were explored using a tetrazolium-based cell viability assay and flow cytometric apoptosis analysis, followed by immunodetection of phosphoactive ERK1/2 in UACC-903, C32, and UACC-647 human melanoma cell lines. Normal human fibroblasts were used as a control. Leaf extracts inhibited the viability of all tested melanoma cell lines in a dose-dependent fashion while the fibroblasts were less sensitive to such extract. The root extracts inhibited the proliferation of UACC-903 and UACC-647 cells only at the highest doses (300 µg/mL). However, the C32 and fibroblast cells exhibited an increase in the cellular proliferation rate and no caspase activity was observed in response to the root extracts (100 µg/mL). An increase in caspase activity was observed in melanoma cells treated with the leaf extracts of both Carlina species. Leaf extracts from C. acaulis subsp. caulescens (100 µg/mL) inhibited proliferatory ERK1/2 in UACC-903 and C32 cells, as demonstrated by the decrease in ERK1/2 phosphorylation. No reduction in phospho-ERK1/2 was observed in the tested cell lines treated with the root extracts, apart from UACC-647 after incubation with the C. acanthifolia subsp. utzka root extract (100 µg/mL). There was no change in ERK1/2 phosphorylation in the fibroblasts. The extracts from the leaves and roots were analyzed by HPLC and the analysis showed the presence of triterpenes and phenolic acids as the main extract components. The research demonstrated that the extracts from the leaves of the plants were cytotoxic against the human melanoma line and induced apoptosis of the cells. The triterpene fraction present in the tested extracts may be responsible for this activity.
Tree saps are nourishing biological media commonly used for beverage and syrup production. Althou... more Tree saps are nourishing biological media commonly used for beverage and syrup production. Although the nutritional aspect of tree saps is widely acknowledged, the exact relationship between the sap composition, origin, and effect on the metabolic rate of human cells is still elusive. Thus, we collected saps from seven different tree species and conducted composition-activity analysis. Saps from trees of Betulaceae, but not from Salicaceae, Sapindaceae, nor Juglandaceae families, were increasing the metabolic rate of HepG2 cells, as measured using tetrazolium-based assay. Content of glucose, fructose, sucrose, chlorides, nitrates, sulphates, fumarates, malates, and succinates in sap samples varied across different tree species. Grade correspondence analysis clustered trees based on the saps' chemical footprint indicating its usability in chemotaxonomy. Multiple regression modeling showed that glucose and fumarate present in saps from silver birch (Betula pendula Roth.), black alder (Alnus glutinosa Gaertn.), and European hornbeam (Carpinus betulus L.) are positively affecting the metabolic activity of HepG2 cells.
Purpose
Kynurenine aminotransferases (KATs) catalyze the synthesis of kynurenic acid (KYNA), a co... more Purpose Kynurenine aminotransferases (KATs) catalyze the synthesis of kynurenic acid (KYNA), a compound of significant biological activity. The aim of this study is to investigate the presence and distribution of KAT immunoreactivity in the healthy human cornea.
Methods Data on gene expression in human eye structures were extracted from public microarray experiments using Genevestigator software. Immunohistochemistry was conducted using polyclonal antibodies against KAT I, II, and III on sections of eight enucleated eyes from patients with choroidal melanoma.
Results Bioinformatics analysis showed that all four KAT isoforms were actively transcribed in the cornea and the conjunctiva. Immunohistochemical analysis revealed the presence of KAT I, II, and III in all examined corneal sections. The corneal endothelium showed the strongest reactivity for all three KAT isoforms. There was a slight positive staining of the corneal stroma for KAT I and II. KAT III immunoreactivity was found only in the stroma of the limbal region. In the corneal epithelium, the expression of all three KAT isoforms showed a specific pattern of the stain with fine squatter granules throughout the cytoplasm. This reactivity was more pronounced in the basal cell layers. The intermediate cell layers showed only faint immunoreactivity, and occasionally, there was no staining. KAT I, II, and III were also present in the adjacent limbal conjunctiva.
Conclusions The results indicate that kynurenine can be metabolized to KYNA in the corneal epithelium, stroma, and endothelium.
The amount of secondary metabolites in plants can be enhanced or reduced by various external fact... more The amount of secondary metabolites in plants can be enhanced or reduced by various external factors. In this study, the effect of strontium ions on the production of phytoestrogens in soybeans was investigated. The plants were treated with Hoagland's solution, modified with Sr2+ with concentrations ranging from 0.5 to 3.0 mM, and were grown for 14 days in hydroponic cultivation. After harvest, soybean plants were separated into roots and shoots, dried, and pulverized. The plant material was extracted with methanol and hydrolyzed. Phytoestrogens were quantified by HPLC. The significant increase in the concentration of the compounds of interest was observed for all tested concentrations of strontium ions when compared to control. Sr2+ at a concentration of 2 mM was the strongest elicitor, and the amount of phytoestrogens in plant increased ca. 2.70, 1.92, 3.77 and 2.88-fold, for daidzein, coumestrol, genistein and formononetin, respectively. Moreover, no cytotoxic effects were observed in HepG2 liver cell models after treatment with extracts from 2 mM Sr2+-stressed soybean plants when compared to extracts from non-stressed plants. Our results indicate that the addition of strontium ions to the culture media may be used to functionalize soybean plants with enhanced phytoestrogen content.
Angiotensin II receptor blockers (ARBs) are one of the most frequently recommended antihypertensi... more Angiotensin II receptor blockers (ARBs) are one of the most frequently recommended antihypertensive drugs. Apart from their activity towards the circulatory system, ARBs also penetrate the blood-brain barrier and display neuroprotective effects. Kynurenic acid (KYNA) is an endogenous metabolite of tryptophan produced by kynurenine aminotransferase II (KAT II) in the brain. Antagonism towards all ionotropic glutamate (GLU) receptors is the main mechanism of KYNA action. An elevated brain level of KYNA is linked with memory impairment and psychotic symptoms. The aim of this study was to examine the influence of three ARBs: irbesartan, losartan, and telmisartan on KYNA production and KAT II activity in rat brain. The effect of ARBs on KYNA production was analyzed in rat brain cortical slices and on isolated KAT II enzyme. Irbesartan, losartan, and telmisartan decreased KYNA production and KAT II activity in a dose-dependent manner in rat brain cortex in vitro. Molecular docking suggested that the examined ARBs could bind to an active site of KAT II. In conclusion, ARBs decrease KYNA production in rat brain by direct inhibition of KAT II enzymatic activity. This novel mechanism of ARBs action may be advantageous in the treatment of cognitive impairment or the management of schizophrenia.
In a recent issue of Journal of Cancer Research and Clinical Oncology, Coelho et al. (2017) revie... more In a recent issue of Journal of Cancer Research and Clinical Oncology, Coelho et al. (2017) reviewed preclinical and epidemiological reports regarding changes in cellular proliferation in response to β-adrenergic receptor (β-AR) ligands. The authors indicated that cancer cells commonly express both β1 and β2 adrenoceptors and that the agonist-mediated activation of these receptors leads to increased proliferation of cultured cells of different origin. Consequently, β-blockers (β-AR antagonists) were proposed as adjuvants for cancer management. This was supported by numerous pharmacoepidemiological studies indicating that cancer patients experience beneficial effects like prolonged survival or reduced risk of metastasis due to chronic β-blocker intake for cancer-unrelated heart conditions (Barron et al. 2011; Lemeshow et al. 2011). As noted by the authors, the amount of data generated over time strongly supports the notion that blockage of β-ARs may constitute a novel therapeutic option for cancer. However, a non-negligible body of evidence indicates that opposite measures—i.e. activation of β-AR—may also be effective in suppressing the growth of cancer cells, at least in some cases (Strell et al. 2012). Thus, the purpose of this letter is to highlight the studies demonstrating antitumorigenic effects of β-AR activation, as opposed to β-blocker usage.
Previous studies have shown positive effects of long-term resveratrol (RSV) supplementation in pr... more Previous studies have shown positive effects of long-term resveratrol (RSV) supplementation in preventing pancreatic beta cell dysfunction, arterial stiffening and metabolic decline induced by high-fat/high-sugar (HFS) diet in nonhuman primates. Here, the analysis was extended to examine whether RSV may reduce dietary stress toxicity in the cerebral cortex of the same cohort of treated animals. Middle-aged male rhesus monkeys were fed for 2 years with HFS alone or combined with RSV, after which whole-genome microarray analysis of cerebral cortex tissue was carried out along with ELISA, immunofluorescence, and biochemical analyses to examine markers of vascular health and inflammation in the cerebral cortices. A number of genes and pathways that were differentially modulated in these dietary interventions indicated an exacerbation of neuroinflammation (e.g., oxidative stress markers, apoptosis, NF-κB activation) in HFS-fed animals and protection by RSV treatment. The decreased expres...
To determine whether 3-furan-2-yl-N-p-tolyl-acrylamide (PAM-2), a positive allosteric modulator o... more To determine whether 3-furan-2-yl-N-p-tolyl-acrylamide (PAM-2), a positive allosteric modulator of α7 nicotinic acetylcholine receptors (nAChRs), improves memory processes, passive avoidance tests were conducted in male mice after acute and chronic treatments. To determine the neuronal mechanisms underlying the promnesic activity elicited by PAM-2, the effect of this ligand on α7 nAChR up-regulation and ERK1/2 phosphorylation was assessed in the hippocampus and prefrontal cortex. The results indicate that: (1) PAM-2 improves memory acquisition/consolidation after acute treatment (Day 2) and memory consolidation after chronic treatment (Day 22). Although no effect was observed on α7 nAChR up-regulation, the chronic, but not acute, PAM-2 treatment increases ERK1/2 kinase phosphorylation, (2) the promnesic activity of PAM-2 was inhibited by methyllycaconitine, a selective α7-antagonist, confirming the role of α7 nAChRs, (3) a synergistic (acute) effect was observed between inactive doses of PAM-2 (0.1mg/kg) and DMXBA (0.3mg/kg), a selective α7-agonist, and (4) PAM-2 reversed the memory impairment elicited by scopolamine, a muscarinic antagonist. The results demonstrate that PAM-2 presents promnesic activity mediated by α7 nAChRs, and is able to trigger ERK1/2 phosphorylation only after chronic treatment.
The amount of secondary metabolites in plants can be enhanced or reduced by various external fact... more The amount of secondary metabolites in plants can be enhanced or reduced by various external factors. In this study, the effect of strontium ions on the production of phytoestrogens in soybeans was investigated. The plants were treated with Hoagland's solution, modified with Sr(2+) with concentrations ranging from 0.5 to 3.0 mM, and were grown for 14 days in hydroponic cultivation. After harvest, soybean plants were separated into roots and shoots, dried, and pulverized. The plant material was extracted with methanol and hydrolyzed. Phytoestrogens were quantified by HPLC. The significant increase in the concentration of the compounds of interest was observed for all tested concentrations of strontium ions when compared to control. Sr(2+) at a concentration of 2 mM was the strongest elicitor, and the amount of phytoestrogens in plant increased ca. 2.70, 1.92, 3.77 and 2.88-fold, for daidzein, coumestrol, genistein and formononetin, respectively. Moreover, no cytotoxic effects were observed in HepG2 liver cell models after treatment with extracts from 2 mM Sr(2+)-stressed soybean plants when compared to extracts from non-stressed plants. Our results indicate that the addition of strontium ions to the culture media may be used to functionalize soybean plants with enhanced phytoestrogen content.
Journal of enzyme inhibition and medicinal chemistry, Jan 30, 2015
Drug resistance has become a major concern in the field of infection management, therefore search... more Drug resistance has become a major concern in the field of infection management, therefore searching for new antibacterial agents is getting more challenging. Our study presents an optimized and eco-friendly synthesis scheme for a panel of nitroalkenes bearing various functional groups in the aromatic moiety and bromine or cyano substituents in 1 position of nitrovinyl moiety. The presence of nitrolefine group outside the ring minimalizes genotoxic properties while conjugation of aryl group with nitrovinyl moiety increases stability of the compounds. Then our research focused on evaluation of biological properties of such obtained (E)-2-aryl-1-cyano-1-nitroethenes. As they exhibit strong bacteriostatic and bactericidal activities against reference bacteria and yeast species with no detectable cytotoxicity towards cultured human HepG2 and HaCaT cells, they could be promising candidates for the replacement of traditional nitrofurane-containing antibacterial drugs. Nevertheless, valida...
(R,R')-4'-methoxy-1-naphthylfenoterol [(R,R')-MNF] is a highly-selective β2 adrenergic receptor (... more (R,R')-4'-methoxy-1-naphthylfenoterol [(R,R')-MNF] is a highly-selective β2 adrenergic receptor (β2-AR) agonist. Incubation of a panel of human-derived melanoma cell lines with (R,R')-MNF resulted in a dose- and time-dependent inhibition of motility as assessed by in vitro wound healing and xCELLigence migration and invasion assays. Activity of (R,R')-MNF positively correlated with the β2-AR expression levels across tested cell lines. The anti-motility activity of (R,R')-MNF was inhibited by the β2-AR antagonist ICI-118,551 and the protein kinase A inhibitor H-89. The adenylyl cyclase activator forskolin and the phosphodiesterase 4 inhibitor Ro 20-1724 mimicked the ability of (R,R')-MNF to inhibit migration of melanoma cell lines in culture, highlighting the importance of cAMP for this phenomenon. (R,R')-MNF caused significant inhibition of cell growth in β2-AR-expressing cells as monitored by radiolabeled thymidine incorporation and xCELLigence system. The MEK/ERK cascade functions in cellular proliferation, and constitutive phosphorylation of MEK and ERK at their active sites was significantly reduced upon β2-AR activation with (R,R')-MNF. Protein synthesis was inhibited concomitantly both with increased eEF2 phosphorylation and lower expression of tumor cell regulators, EGF receptors, cyclin A and MMP-9. Taken together, these results identified β2-AR as a novel potential target for melanoma management, and (R,R')-MNF as an efficient trigger of anti-tumorigenic cAMP/PKA-dependent signaling in β2-AR-expressing lesions.
(R,R′)-4′-Methoxy-1-naphthylfenoterol (MNF) promotes growth inhibition and apoptosis of human Hep... more (R,R′)-4′-Methoxy-1-naphthylfenoterol (MNF) promotes growth inhibition and apoptosis of human HepG2 hepatocarcinoma cells via cannabinoid receptor (CBR) activation. The synthetic CB1R inverse agonist, AM251, has been shown to block the anti-mitogenic effect of MNF in these cells; however, AM251 is also an agonist of the recently deorphanized, lipid-sensing receptor, GPR55, whose upregulation contributes to carcinogenesis. Here, we investigated the role of MNF in GPR55 signaling in human HepG2 and PANC-1 cancer cell lines in culture by focusing first on internalization of the fluorescent ligand Tocrifluor 1117 (T1117). Initial results indicated that cell pretreatment with GPR55 agonists, including the atypical cannabinoid O-1602 and l-α-lysophosphatidylinositol, dose-dependently reduced the rate of cellular T1117 uptake, a process that was sensitive to MNF inhibition. GPR55 internalization and signaling mediated by O-1602 was blocked by MNF in GPR55-expressing HEK293 cells. Pretreatment of HepG2 and PANC-1 cells with MNF significantly abrogated the induction of ERK1/2 phosphorylation in response to AM251 and O-1602. Moreover, MNF exerted a coordinated negative regulation of AM251 and O-1602 inducible processes, including changes in cellular morphology and cell migration using scratch wound healing assay. This study shows for the first time that MNF impairs GPR55-mediated signaling and, therefore, may have therapeutic potential in the management of cancer.
The genus β human papillomavirus (HPV) type 8 is associated with nonmelanoma skin cancer in patie... more The genus β human papillomavirus (HPV) type 8 is associated with nonmelanoma skin cancer in patients with epidermodysplasia verruciformis, and evidence for its protumorigenic potential in the general population increases. To date, strategies to suppress genus β HPV infections are limited. Interferon regulatory factors IRF-3 and IRF-7 play key roles in the activation of the innate immune response to viral infections. In this study, we show for the first time that both IRF-3 and IRF-7 regulate transcription of a papillomavirus, but with opposing effects. IRF-7, expressed in the suprabasal layers of human epidermis, increased HPV8 late promoter activity via direct binding to viral DNA. UV-B light-induced activation of the HPV8 promoter involved IRF-7 as a downstream effector. In contrast, IRF-3, expressed in all layers of human epidermis, induced strong HPV8 suppression in primary keratinocytes. IRF-3-mediated suppression prevailed over IRF-7-induced HPV8 transcription. Unlike the E6 oncoprotein of the mucosal high-risk HPV16, the HPV8 E6 protein did not bind to IRF-3 and only weakly antagonized its activity. Strong antiviral activity was also observed, when keratinocytes were treated with potent IRF-3 activators, poly(I:C) or RNA bearing 5' phosphates. In conclusion, we show that IRF-3 activation induces a state of cell-autonomous immunity against HPV in primary human keratinocytes. Our study suggests that local application of IRF-3-activating compounds might constitute an attractive novel therapeutic strategy against HPV8-associated diseases, particularly in epidermodysplasia verruciformis patients.
(R,R')-4-methoxy-1-naphthylfenoterol (MNF) inhibits cancer cell proliferation in vitro through ce... more (R,R')-4-methoxy-1-naphthylfenoterol (MNF) inhibits cancer cell proliferation in vitro through cell-type specific modulation of β2-adrenergic receptor and/or cannabinoid receptor function. Here, we report an investigation into antitumor activity of MNF in rat C6 glioma cells. The potent antiproliferative action of MNF in these cells (IC50 of ∼1 nmol/L) was refractory to pharmacological inhibition of β2-adrenergic receptor while a synthetic inverse agonist of cannabinoid receptor 1 significantly blocked MNF activity. The antitumor activity of MNF was then assessed in a C6 glioblastoma xenograft model in mice. Three days after subcutaneous implantation of C6 cells into the lower flank of nude mice, these animals were subjected to i.p. injections of saline or MNF (2 mg/kg) for 19 days and tumor volumes were measured over the course of the experiment. Gene expression analysis, quantitative RT-PCR and immunoblot assays were performed on the tumors after treatment. Significant reduction in mean tumor volumes was observed in mice receiving MNF when compared with the saline-treated group. We identified clusters in expression of genes involved in cellular proliferation, as well as molecular markers for glioblastoma that were significantly downregulated in tumors of MNF-treated mice as compared to saline-injected controls. The efficacy of MNF against C6 glioma cell proliferation in vivo and in vitro was accompanied by marked reduction in the expression of cell cycle regulator proteins. This study is the first demonstration of MNF-dependent chemoprevention of a glioblastoma xenograft model and may offer a potential mechanism for its anticancer action in vivo.
The β2-adrenergic receptor (β2AR) is the prototypic member of G protein-coupled receptors (GPCRs)... more The β2-adrenergic receptor (β2AR) is the prototypic member of G protein-coupled receptors (GPCRs) involved in the production of physiological responses to adrenaline and noradrenaline. Research done in the past few years vastly demonstrated that β2AR can form homo- and hetero-oligomers. Despite the fact that currently this phenomenon is widely accepted, the spread and relevance of β2AR oligomerization are still a matter of debate. This review considers the progress achieved in the field of β2AR oligomerization with focus on the implications of the receptor-receptor interactions to β2AR trafficking, pharmacology and downstream signal transduction pathways.
Activation of β2-adrenergic receptor (β2AR) and deorphanized GPR55 has been shown to modulate can... more Activation of β2-adrenergic receptor (β2AR) and deorphanized GPR55 has been shown to modulate cancer growth in diverse tumor types in vitro and in xenograft models in vivo. (R,R′)-4′-methoxy-1-naphthylfenoterol [(R,R′)-MNF] is a bivalent compound that agonizes β2AR but inhibits GPR55-mediated pro-oncogenic responses. Here, we investigated the molecular mechanisms underlying the anti-tumorigenic effects of concurrent β2AR activation and GPR55 blockade in C6 glioma cells using (R,R′)-MNF as a marker ligand. Our data show that (R,R′)-MNF elicited G1-phase cell cycle arrest and apoptosis, reduced serum-inducible cell motility, promoted the phosphorylation of PKA target proteins, and inhibited constitutive activation of ERK and AKT in the low nanomolar range, whereas high nanomolar levels of (R,R′)-MNF were required to block GPR55-mediated cell motility. siRNA knockdown and pharmacological inhibition of β2AR activity were accompanied by significant upregulation of AKT and ERK phosphorylation, and selective alteration in (R,R′)-MNF responsiveness. The effects of agonist stimulation of GPR55 on various readouts, including cell motility assays, were suppressed by (R,R′)-MNF. Lastly, a significant increase in phosphorylation-mediated inactivation of β-catenin occurred with (R,R′)-MNF, and we provided new evidence of (R,R′)-MNF-mediated inhibition of oncogenic β-catenin signaling in a C6 xenograft tumor model. Thus, simultaneous activation of β2AR and blockade of GPR55 may represent a novel therapeutic approach to combat the progression of glioblastoma cancer.
Various species of the Carlina genus have been used in traditional medicine in many countries to ... more Various species of the Carlina genus have been used in traditional medicine in many countries to treat numerous skin disorders, including cancer. The objective of this work was to assess the anticancer properties of root and leaf extracts from Carlina acaulis subsp. caulescens and C. acanthifolia subsp. utzka. Anti-tumor properties of the extracts were explored using a tetrazolium-based cell viability assay and flow cytometric apoptosis analysis, followed by immunodetection of phosphoactive ERK1/2 in UACC-903, C32, and UACC-647 human melanoma cell lines. Normal human fibroblasts were used as a control. Leaf extracts inhibited the viability of all tested melanoma cell lines in a dose-dependent fashion while the fibroblasts were less sensitive to such extract. The root extracts inhibited the proliferation of UACC-903 and UACC-647 cells only at the highest doses (300 µg/mL). However, the C32 and fibroblast cells exhibited an increase in the cellular proliferation rate and no caspase activity was observed in response to the root extracts (100 µg/mL). An increase in caspase activity was observed in melanoma cells treated with the leaf extracts of both Carlina species. Leaf extracts from C. acaulis subsp. caulescens (100 µg/mL) inhibited proliferatory ERK1/2 in UACC-903 and C32 cells, as demonstrated by the decrease in ERK1/2 phosphorylation. No reduction in phospho-ERK1/2 was observed in the tested cell lines treated with the root extracts, apart from UACC-647 after incubation with the C. acanthifolia subsp. utzka root extract (100 µg/mL). There was no change in ERK1/2 phosphorylation in the fibroblasts. The extracts from the leaves and roots were analyzed by HPLC and the analysis showed the presence of triterpenes and phenolic acids as the main extract components. The research demonstrated that the extracts from the leaves of the plants were cytotoxic against the human melanoma line and induced apoptosis of the cells. The triterpene fraction present in the tested extracts may be responsible for this activity.
Tree saps are nourishing biological media commonly used for beverage and syrup production. Althou... more Tree saps are nourishing biological media commonly used for beverage and syrup production. Although the nutritional aspect of tree saps is widely acknowledged, the exact relationship between the sap composition, origin, and effect on the metabolic rate of human cells is still elusive. Thus, we collected saps from seven different tree species and conducted composition-activity analysis. Saps from trees of Betulaceae, but not from Salicaceae, Sapindaceae, nor Juglandaceae families, were increasing the metabolic rate of HepG2 cells, as measured using tetrazolium-based assay. Content of glucose, fructose, sucrose, chlorides, nitrates, sulphates, fumarates, malates, and succinates in sap samples varied across different tree species. Grade correspondence analysis clustered trees based on the saps' chemical footprint indicating its usability in chemotaxonomy. Multiple regression modeling showed that glucose and fumarate present in saps from silver birch (Betula pendula Roth.), black alder (Alnus glutinosa Gaertn.), and European hornbeam (Carpinus betulus L.) are positively affecting the metabolic activity of HepG2 cells.
Purpose
Kynurenine aminotransferases (KATs) catalyze the synthesis of kynurenic acid (KYNA), a co... more Purpose Kynurenine aminotransferases (KATs) catalyze the synthesis of kynurenic acid (KYNA), a compound of significant biological activity. The aim of this study is to investigate the presence and distribution of KAT immunoreactivity in the healthy human cornea.
Methods Data on gene expression in human eye structures were extracted from public microarray experiments using Genevestigator software. Immunohistochemistry was conducted using polyclonal antibodies against KAT I, II, and III on sections of eight enucleated eyes from patients with choroidal melanoma.
Results Bioinformatics analysis showed that all four KAT isoforms were actively transcribed in the cornea and the conjunctiva. Immunohistochemical analysis revealed the presence of KAT I, II, and III in all examined corneal sections. The corneal endothelium showed the strongest reactivity for all three KAT isoforms. There was a slight positive staining of the corneal stroma for KAT I and II. KAT III immunoreactivity was found only in the stroma of the limbal region. In the corneal epithelium, the expression of all three KAT isoforms showed a specific pattern of the stain with fine squatter granules throughout the cytoplasm. This reactivity was more pronounced in the basal cell layers. The intermediate cell layers showed only faint immunoreactivity, and occasionally, there was no staining. KAT I, II, and III were also present in the adjacent limbal conjunctiva.
Conclusions The results indicate that kynurenine can be metabolized to KYNA in the corneal epithelium, stroma, and endothelium.
The amount of secondary metabolites in plants can be enhanced or reduced by various external fact... more The amount of secondary metabolites in plants can be enhanced or reduced by various external factors. In this study, the effect of strontium ions on the production of phytoestrogens in soybeans was investigated. The plants were treated with Hoagland's solution, modified with Sr2+ with concentrations ranging from 0.5 to 3.0 mM, and were grown for 14 days in hydroponic cultivation. After harvest, soybean plants were separated into roots and shoots, dried, and pulverized. The plant material was extracted with methanol and hydrolyzed. Phytoestrogens were quantified by HPLC. The significant increase in the concentration of the compounds of interest was observed for all tested concentrations of strontium ions when compared to control. Sr2+ at a concentration of 2 mM was the strongest elicitor, and the amount of phytoestrogens in plant increased ca. 2.70, 1.92, 3.77 and 2.88-fold, for daidzein, coumestrol, genistein and formononetin, respectively. Moreover, no cytotoxic effects were observed in HepG2 liver cell models after treatment with extracts from 2 mM Sr2+-stressed soybean plants when compared to extracts from non-stressed plants. Our results indicate that the addition of strontium ions to the culture media may be used to functionalize soybean plants with enhanced phytoestrogen content.
Angiotensin II receptor blockers (ARBs) are one of the most frequently recommended antihypertensi... more Angiotensin II receptor blockers (ARBs) are one of the most frequently recommended antihypertensive drugs. Apart from their activity towards the circulatory system, ARBs also penetrate the blood-brain barrier and display neuroprotective effects. Kynurenic acid (KYNA) is an endogenous metabolite of tryptophan produced by kynurenine aminotransferase II (KAT II) in the brain. Antagonism towards all ionotropic glutamate (GLU) receptors is the main mechanism of KYNA action. An elevated brain level of KYNA is linked with memory impairment and psychotic symptoms. The aim of this study was to examine the influence of three ARBs: irbesartan, losartan, and telmisartan on KYNA production and KAT II activity in rat brain. The effect of ARBs on KYNA production was analyzed in rat brain cortical slices and on isolated KAT II enzyme. Irbesartan, losartan, and telmisartan decreased KYNA production and KAT II activity in a dose-dependent manner in rat brain cortex in vitro. Molecular docking suggested that the examined ARBs could bind to an active site of KAT II. In conclusion, ARBs decrease KYNA production in rat brain by direct inhibition of KAT II enzymatic activity. This novel mechanism of ARBs action may be advantageous in the treatment of cognitive impairment or the management of schizophrenia.
In a recent issue of Journal of Cancer Research and Clinical Oncology, Coelho et al. (2017) revie... more In a recent issue of Journal of Cancer Research and Clinical Oncology, Coelho et al. (2017) reviewed preclinical and epidemiological reports regarding changes in cellular proliferation in response to β-adrenergic receptor (β-AR) ligands. The authors indicated that cancer cells commonly express both β1 and β2 adrenoceptors and that the agonist-mediated activation of these receptors leads to increased proliferation of cultured cells of different origin. Consequently, β-blockers (β-AR antagonists) were proposed as adjuvants for cancer management. This was supported by numerous pharmacoepidemiological studies indicating that cancer patients experience beneficial effects like prolonged survival or reduced risk of metastasis due to chronic β-blocker intake for cancer-unrelated heart conditions (Barron et al. 2011; Lemeshow et al. 2011). As noted by the authors, the amount of data generated over time strongly supports the notion that blockage of β-ARs may constitute a novel therapeutic option for cancer. However, a non-negligible body of evidence indicates that opposite measures—i.e. activation of β-AR—may also be effective in suppressing the growth of cancer cells, at least in some cases (Strell et al. 2012). Thus, the purpose of this letter is to highlight the studies demonstrating antitumorigenic effects of β-AR activation, as opposed to β-blocker usage.
Previous studies have shown positive effects of long-term resveratrol (RSV) supplementation in pr... more Previous studies have shown positive effects of long-term resveratrol (RSV) supplementation in preventing pancreatic beta cell dysfunction, arterial stiffening and metabolic decline induced by high-fat/high-sugar (HFS) diet in nonhuman primates. Here, the analysis was extended to examine whether RSV may reduce dietary stress toxicity in the cerebral cortex of the same cohort of treated animals. Middle-aged male rhesus monkeys were fed for 2 years with HFS alone or combined with RSV, after which whole-genome microarray analysis of cerebral cortex tissue was carried out along with ELISA, immunofluorescence, and biochemical analyses to examine markers of vascular health and inflammation in the cerebral cortices. A number of genes and pathways that were differentially modulated in these dietary interventions indicated an exacerbation of neuroinflammation (e.g., oxidative stress markers, apoptosis, NF-κB activation) in HFS-fed animals and protection by RSV treatment. The decreased expres...
To determine whether 3-furan-2-yl-N-p-tolyl-acrylamide (PAM-2), a positive allosteric modulator o... more To determine whether 3-furan-2-yl-N-p-tolyl-acrylamide (PAM-2), a positive allosteric modulator of α7 nicotinic acetylcholine receptors (nAChRs), improves memory processes, passive avoidance tests were conducted in male mice after acute and chronic treatments. To determine the neuronal mechanisms underlying the promnesic activity elicited by PAM-2, the effect of this ligand on α7 nAChR up-regulation and ERK1/2 phosphorylation was assessed in the hippocampus and prefrontal cortex. The results indicate that: (1) PAM-2 improves memory acquisition/consolidation after acute treatment (Day 2) and memory consolidation after chronic treatment (Day 22). Although no effect was observed on α7 nAChR up-regulation, the chronic, but not acute, PAM-2 treatment increases ERK1/2 kinase phosphorylation, (2) the promnesic activity of PAM-2 was inhibited by methyllycaconitine, a selective α7-antagonist, confirming the role of α7 nAChRs, (3) a synergistic (acute) effect was observed between inactive doses of PAM-2 (0.1mg/kg) and DMXBA (0.3mg/kg), a selective α7-agonist, and (4) PAM-2 reversed the memory impairment elicited by scopolamine, a muscarinic antagonist. The results demonstrate that PAM-2 presents promnesic activity mediated by α7 nAChRs, and is able to trigger ERK1/2 phosphorylation only after chronic treatment.
The amount of secondary metabolites in plants can be enhanced or reduced by various external fact... more The amount of secondary metabolites in plants can be enhanced or reduced by various external factors. In this study, the effect of strontium ions on the production of phytoestrogens in soybeans was investigated. The plants were treated with Hoagland's solution, modified with Sr(2+) with concentrations ranging from 0.5 to 3.0 mM, and were grown for 14 days in hydroponic cultivation. After harvest, soybean plants were separated into roots and shoots, dried, and pulverized. The plant material was extracted with methanol and hydrolyzed. Phytoestrogens were quantified by HPLC. The significant increase in the concentration of the compounds of interest was observed for all tested concentrations of strontium ions when compared to control. Sr(2+) at a concentration of 2 mM was the strongest elicitor, and the amount of phytoestrogens in plant increased ca. 2.70, 1.92, 3.77 and 2.88-fold, for daidzein, coumestrol, genistein and formononetin, respectively. Moreover, no cytotoxic effects were observed in HepG2 liver cell models after treatment with extracts from 2 mM Sr(2+)-stressed soybean plants when compared to extracts from non-stressed plants. Our results indicate that the addition of strontium ions to the culture media may be used to functionalize soybean plants with enhanced phytoestrogen content.
Journal of enzyme inhibition and medicinal chemistry, Jan 30, 2015
Drug resistance has become a major concern in the field of infection management, therefore search... more Drug resistance has become a major concern in the field of infection management, therefore searching for new antibacterial agents is getting more challenging. Our study presents an optimized and eco-friendly synthesis scheme for a panel of nitroalkenes bearing various functional groups in the aromatic moiety and bromine or cyano substituents in 1 position of nitrovinyl moiety. The presence of nitrolefine group outside the ring minimalizes genotoxic properties while conjugation of aryl group with nitrovinyl moiety increases stability of the compounds. Then our research focused on evaluation of biological properties of such obtained (E)-2-aryl-1-cyano-1-nitroethenes. As they exhibit strong bacteriostatic and bactericidal activities against reference bacteria and yeast species with no detectable cytotoxicity towards cultured human HepG2 and HaCaT cells, they could be promising candidates for the replacement of traditional nitrofurane-containing antibacterial drugs. Nevertheless, valida...
(R,R')-4'-methoxy-1-naphthylfenoterol [(R,R')-MNF] is a highly-selective β2 adrenergic receptor (... more (R,R')-4'-methoxy-1-naphthylfenoterol [(R,R')-MNF] is a highly-selective β2 adrenergic receptor (β2-AR) agonist. Incubation of a panel of human-derived melanoma cell lines with (R,R')-MNF resulted in a dose- and time-dependent inhibition of motility as assessed by in vitro wound healing and xCELLigence migration and invasion assays. Activity of (R,R')-MNF positively correlated with the β2-AR expression levels across tested cell lines. The anti-motility activity of (R,R')-MNF was inhibited by the β2-AR antagonist ICI-118,551 and the protein kinase A inhibitor H-89. The adenylyl cyclase activator forskolin and the phosphodiesterase 4 inhibitor Ro 20-1724 mimicked the ability of (R,R')-MNF to inhibit migration of melanoma cell lines in culture, highlighting the importance of cAMP for this phenomenon. (R,R')-MNF caused significant inhibition of cell growth in β2-AR-expressing cells as monitored by radiolabeled thymidine incorporation and xCELLigence system. The MEK/ERK cascade functions in cellular proliferation, and constitutive phosphorylation of MEK and ERK at their active sites was significantly reduced upon β2-AR activation with (R,R')-MNF. Protein synthesis was inhibited concomitantly both with increased eEF2 phosphorylation and lower expression of tumor cell regulators, EGF receptors, cyclin A and MMP-9. Taken together, these results identified β2-AR as a novel potential target for melanoma management, and (R,R')-MNF as an efficient trigger of anti-tumorigenic cAMP/PKA-dependent signaling in β2-AR-expressing lesions.
(R,R′)-4′-Methoxy-1-naphthylfenoterol (MNF) promotes growth inhibition and apoptosis of human Hep... more (R,R′)-4′-Methoxy-1-naphthylfenoterol (MNF) promotes growth inhibition and apoptosis of human HepG2 hepatocarcinoma cells via cannabinoid receptor (CBR) activation. The synthetic CB1R inverse agonist, AM251, has been shown to block the anti-mitogenic effect of MNF in these cells; however, AM251 is also an agonist of the recently deorphanized, lipid-sensing receptor, GPR55, whose upregulation contributes to carcinogenesis. Here, we investigated the role of MNF in GPR55 signaling in human HepG2 and PANC-1 cancer cell lines in culture by focusing first on internalization of the fluorescent ligand Tocrifluor 1117 (T1117). Initial results indicated that cell pretreatment with GPR55 agonists, including the atypical cannabinoid O-1602 and l-α-lysophosphatidylinositol, dose-dependently reduced the rate of cellular T1117 uptake, a process that was sensitive to MNF inhibition. GPR55 internalization and signaling mediated by O-1602 was blocked by MNF in GPR55-expressing HEK293 cells. Pretreatment of HepG2 and PANC-1 cells with MNF significantly abrogated the induction of ERK1/2 phosphorylation in response to AM251 and O-1602. Moreover, MNF exerted a coordinated negative regulation of AM251 and O-1602 inducible processes, including changes in cellular morphology and cell migration using scratch wound healing assay. This study shows for the first time that MNF impairs GPR55-mediated signaling and, therefore, may have therapeutic potential in the management of cancer.
The genus β human papillomavirus (HPV) type 8 is associated with nonmelanoma skin cancer in patie... more The genus β human papillomavirus (HPV) type 8 is associated with nonmelanoma skin cancer in patients with epidermodysplasia verruciformis, and evidence for its protumorigenic potential in the general population increases. To date, strategies to suppress genus β HPV infections are limited. Interferon regulatory factors IRF-3 and IRF-7 play key roles in the activation of the innate immune response to viral infections. In this study, we show for the first time that both IRF-3 and IRF-7 regulate transcription of a papillomavirus, but with opposing effects. IRF-7, expressed in the suprabasal layers of human epidermis, increased HPV8 late promoter activity via direct binding to viral DNA. UV-B light-induced activation of the HPV8 promoter involved IRF-7 as a downstream effector. In contrast, IRF-3, expressed in all layers of human epidermis, induced strong HPV8 suppression in primary keratinocytes. IRF-3-mediated suppression prevailed over IRF-7-induced HPV8 transcription. Unlike the E6 oncoprotein of the mucosal high-risk HPV16, the HPV8 E6 protein did not bind to IRF-3 and only weakly antagonized its activity. Strong antiviral activity was also observed, when keratinocytes were treated with potent IRF-3 activators, poly(I:C) or RNA bearing 5' phosphates. In conclusion, we show that IRF-3 activation induces a state of cell-autonomous immunity against HPV in primary human keratinocytes. Our study suggests that local application of IRF-3-activating compounds might constitute an attractive novel therapeutic strategy against HPV8-associated diseases, particularly in epidermodysplasia verruciformis patients.
(R,R')-4-methoxy-1-naphthylfenoterol (MNF) inhibits cancer cell proliferation in vitro through ce... more (R,R')-4-methoxy-1-naphthylfenoterol (MNF) inhibits cancer cell proliferation in vitro through cell-type specific modulation of β2-adrenergic receptor and/or cannabinoid receptor function. Here, we report an investigation into antitumor activity of MNF in rat C6 glioma cells. The potent antiproliferative action of MNF in these cells (IC50 of ∼1 nmol/L) was refractory to pharmacological inhibition of β2-adrenergic receptor while a synthetic inverse agonist of cannabinoid receptor 1 significantly blocked MNF activity. The antitumor activity of MNF was then assessed in a C6 glioblastoma xenograft model in mice. Three days after subcutaneous implantation of C6 cells into the lower flank of nude mice, these animals were subjected to i.p. injections of saline or MNF (2 mg/kg) for 19 days and tumor volumes were measured over the course of the experiment. Gene expression analysis, quantitative RT-PCR and immunoblot assays were performed on the tumors after treatment. Significant reduction in mean tumor volumes was observed in mice receiving MNF when compared with the saline-treated group. We identified clusters in expression of genes involved in cellular proliferation, as well as molecular markers for glioblastoma that were significantly downregulated in tumors of MNF-treated mice as compared to saline-injected controls. The efficacy of MNF against C6 glioma cell proliferation in vivo and in vitro was accompanied by marked reduction in the expression of cell cycle regulator proteins. This study is the first demonstration of MNF-dependent chemoprevention of a glioblastoma xenograft model and may offer a potential mechanism for its anticancer action in vivo.
The β2-adrenergic receptor (β2AR) is the prototypic member of G protein-coupled receptors (GPCRs)... more The β2-adrenergic receptor (β2AR) is the prototypic member of G protein-coupled receptors (GPCRs) involved in the production of physiological responses to adrenaline and noradrenaline. Research done in the past few years vastly demonstrated that β2AR can form homo- and hetero-oligomers. Despite the fact that currently this phenomenon is widely accepted, the spread and relevance of β2AR oligomerization are still a matter of debate. This review considers the progress achieved in the field of β2AR oligomerization with focus on the implications of the receptor-receptor interactions to β2AR trafficking, pharmacology and downstream signal transduction pathways.
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Papers by Artur Wnorowski
Kynurenine aminotransferases (KATs) catalyze the synthesis of kynurenic acid (KYNA), a compound of significant biological activity. The aim of this study is to investigate the presence and distribution of KAT immunoreactivity in the healthy human cornea.
Methods
Data on gene expression in human eye structures were extracted from public microarray experiments using Genevestigator software. Immunohistochemistry was conducted using polyclonal antibodies against KAT I, II, and III on sections of eight enucleated eyes from patients with choroidal melanoma.
Results
Bioinformatics analysis showed that all four KAT isoforms were actively transcribed in the cornea and the conjunctiva. Immunohistochemical analysis revealed the presence of KAT I, II, and III in all examined corneal sections. The corneal endothelium showed the strongest reactivity for all three KAT isoforms. There was a slight positive staining of the corneal stroma for KAT I and II. KAT III immunoreactivity was found only in the stroma of the limbal region. In the corneal epithelium, the expression of all three KAT isoforms showed a specific pattern of the stain with fine squatter granules throughout the cytoplasm. This reactivity was more pronounced in the basal cell layers. The intermediate cell layers showed only faint immunoreactivity, and occasionally, there was no staining. KAT I, II, and III were also present in the adjacent limbal conjunctiva.
Conclusions
The results indicate that kynurenine can be metabolized to KYNA in the corneal epithelium, stroma, and endothelium.
Kynurenine aminotransferases (KATs) catalyze the synthesis of kynurenic acid (KYNA), a compound of significant biological activity. The aim of this study is to investigate the presence and distribution of KAT immunoreactivity in the healthy human cornea.
Methods
Data on gene expression in human eye structures were extracted from public microarray experiments using Genevestigator software. Immunohistochemistry was conducted using polyclonal antibodies against KAT I, II, and III on sections of eight enucleated eyes from patients with choroidal melanoma.
Results
Bioinformatics analysis showed that all four KAT isoforms were actively transcribed in the cornea and the conjunctiva. Immunohistochemical analysis revealed the presence of KAT I, II, and III in all examined corneal sections. The corneal endothelium showed the strongest reactivity for all three KAT isoforms. There was a slight positive staining of the corneal stroma for KAT I and II. KAT III immunoreactivity was found only in the stroma of the limbal region. In the corneal epithelium, the expression of all three KAT isoforms showed a specific pattern of the stain with fine squatter granules throughout the cytoplasm. This reactivity was more pronounced in the basal cell layers. The intermediate cell layers showed only faint immunoreactivity, and occasionally, there was no staining. KAT I, II, and III were also present in the adjacent limbal conjunctiva.
Conclusions
The results indicate that kynurenine can be metabolized to KYNA in the corneal epithelium, stroma, and endothelium.