Humoral immune response of Galleria mellonella after mono- and co-injection with Hypericum perforatum extract and Candida albicans.

Galleria mellonella is used as a model organism to study the innate immune response of insects. In this study, the humoral immune response was assessed by examining phenoloxidase activity, fungal burden, and the expression of phenoloxidase and antimicrobial peptide genes at different time point following separate and combined injections of Hypericum perforatum extract and a nonlethal dose of Candida albicans. The administration of a plant extract at low doses increased phenoloxidase activity, while higher doses had no effect. Similarly, co-injection of a low dose of the extract with the pathogen allowed half of the yeast cells to survive after 24 h. Co-injection of plant extract with the pathogen decreased the phenoloxidase activity at the end of 4 h compared to C. albicans mono-injection. The phenoloxidase gene expressions was reduced in all experimental conditions with respect to the control. When plant extracts and the pathogen were administered together, gallerimycin and hemolin gene expressions were considerably higher compared to mono-injections of plant extracts and the pathogen. The results of this study reveal that gene activation and regulatory mechanisms may change for each immune gene, and that recognition and signaling pathways may differ depending on the involved immunoregulator.

Exploring the effect of butyric acid, a metabolite from periodontopathic bacteria, on primary human melanocytes: An in vitro study.

Effects of butyric acid, a bacterial metabolite implicated in periodontitis progression, have never been examined on oral melanocytes. Herein, primary human epidermal melanocytes were used as a model for oral melanocytes. Results show the adverse effects of butyric acid (sodium butyrate; NaB) on them, which comprise marked cytotoxicity at higher concentrations (>1 mM) and robust differentiation at lower nontoxic concentrations. NaB did not alter MITF protein levels; however, it stimulated tyrosinase protein synthesis and inhibited tyrosinase activity, with no changes in cellular melanin. NaB did not affect oxidative stress, although it induced significant levels of the pro-inflammatory cytokine IL-6.

Melanogrit potentiates melanogenesis by escalating cellular tyrosinase activity and MITF levels via pERK inhibition.

Vitiligo is characterized by the development of white patches on the skin either due to the loss of functional melanocytes or perturbations in the melanogenesis pathway. In the present study, we investigated the therapeutic potential of herbo-mineral formulation, Melanogrit in neutralizing the white patches in the skin. The study utilized UPLC/MS-QToF technique to determine the diversified phytochemical profile in Melanogrit. The murine B16F10 cells when treated with Melanogrit underwent morphological changes, including increased angularity, enlarged cell size, and greater dendritic protrusions. To establish an equivalent model to study melanogenesis, we carefully optimized the dosage of α-melanocyte stimulating hormone (αMSH) in B16F10 cells as an alternative to using melanocyte-keratinocyte cocultures. The study determined a sub-optimal dose of αMSH (0.2 nM) in B16F10 cells that does not manifest any measurable effects on melanogenesis. In contrast, Melanogrit when used in conjunction with 0.2 nM αMSH, induced a dose-dependent increase in extracellular and intracellular melanin levels. Melanogrit transcriptionally up-regulated the decisive genes of the melanogenesis pathway, MITF, TYR, and TRP1, which was evident from the increased cellular tyrosine activity. Our findings also demonstrated that Melanogrit ameliorated the MITF protein levels by inhibiting pERK; notably without involving GSK3ß in the process. Taken together, our findings strongly suggest that Melanogrit has the potential to stimulate melanogenesis, making it a promising candidate for clinical applications in the treatment of white skin patches that develop in vitiligo patients.

Antioxidant, Volatile Compounds; Antimicrobial, Anti-Inflammatory, and Dermatoprotective Properties of Cedrus atlantica (Endl.) Manetti Ex Carriere Essential Oil: In Vitro and In Silico Investigations.

Cedrus atlantica (Endl.) Manetti ex Carriere is an endemic tree possessing valuable health benefits which has been widely used since time immemorial in international traditional pharmacopoeia. The aim of this exploratory investigation is to determine the volatile compounds of C. atlantica essential oils (CAEOs) and to examine their in vitro antimicrobial, antioxidant, anti-inflammatory, and dermatoprotective properties. In silico simulations, including molecular docking and pharmacokinetics absorption, distribution, metabolism, excretion, and toxicity (ADMET), and drug-likeness prediction were used to reveal the processes underlying in vitro biological properties. Gas chromatography-mass spectrophotometry (GC-MS) was used for the chemical screening of CAEO. The antioxidant activity of CAEO was investigated using four in vitro complementary techniques, including ABTS and DPPH radicals scavenging activity, ferric reductive power, and inhibition of lipid peroxidation (ß-carotene test). Lipoxygenase (5-LOX) inhibition and tyrosinase inhibitory assays were used for testing the anti-inflammatory and dermatoprotective properties. GC-MS analysis indicated that the main components of CAEO are ß-himachalene (28.99%), α-himachalene (14.43%), and longifolene (12.2%). An in vitro antimicrobial activity of CAEO was examined against eleven strains of Gram-positive bacteria (three strains), Gram-negative bacteria (four strains), and fungi (four strains). The results demonstrated high antibacterial and antifungal activity against ten of them (>15 mm zone of inhibition) using the disc-diffusion assay. The microdilution test showed that the lowest values of MIC and MBC were recorded with the Gram-positive bacteria in particular, which ranged from 0.0625 to 0.25 % v/v for MIC and from 0.5 to 0.125 % v/v for MBC. The MIC and MFC of the fungal strains ranged from 0.5 to 4.0% (MIC) and 0.5 to 8.0% v/v (MFC). According to the MBC/MIC and MFC/MIC ratios, CAEO has bactericidal and fungicidal activity. The results of the in vitro antioxidant assays revealed that CAEO possesses remarkable antioxidant activity. The inhibitory effects on 5-LOX and tyrosinase enzymes was also significant (p < 0.05). ADMET investigation suggests that the main compounds of CAEO possess favorable pharmacokinetic properties. These findings provide scientific validation of the traditional uses of this plant and suggest its potential application as natural drugs.

Mechanism of skin whitening through San-Bai decoction-induced tyrosinase inhibition and discovery of natural products targeting tyrosinase.

Melanin deposition is the main cause of skin darkening, which can lead to severe physical and psychological distress, necessitating the development of approaches for preserving skin health and fairness. Tyrosinase (TYR) is the rate-limiting enzyme in melanin synthesis, and its activity directly determines the degree of melanin accumulation in the skin, which in turn affects skin color. Currently, TYR inhibitors derived from natural products are widely used for skin whitening. San-Bai decoction (SBD) is effective for skin whitening and softening, but its mechanism of action, efficacy and high efficiency TYR inhibitors for skin whitening remain poorly understood. Here, we employed systems biology and network pharmacology to analyze the active compounds and targets of SBD, using the follow databases: TCMIP, TCMID, and BATMAN-TCM. Construct a molecular network centered on the regulation of TYR by SBD in skin whitening, using STRING database and cytoscape. Enrichment analysis using KOBAS database and ClusterProfiler. Virtual screening of candidate TYR inhibitors using Molecular Operating Environment software and Amber 18 software. SBD may act through tyrosine metabolism, melanogenesis, and other signaling pathways to regulate TYR activity and inhibit melanogenesis. We identified TYR and ESR1 as possible key targets for the whitening effect of SBD and screened out pentagalloylglucose, 1,3,6-tri-O-galloyl-beta-D-glucose, 1,2,4,6-tetragalloylglucose, and liquiritigenin 4',7-diglucoside as inhibitors of TYR, in addition to glycyrrhizic acid, pachymic acid methyl ester, nicotiflorin, gamma-sitosterol, and isoliensinine as inhibitors of ESR1. We also performed virtual drug screening of a library of natural small-molecule compounds (19,505 in total) and screened out lycopsamine, 2-phenylethyl b-D-glucopyranoside, and 6-beta-hydroxyhyoscyamine as inhibitors of TYR. We identified natural compounds with the potential for skin whitening through inhibition of TYR, thus advancing research on SBD and its applications.

Pterostilbene inhibits melanogenesis, melanocyte dendricity and melanosome transport through cAMP/PKA/CREB pathway.

Pterostilbene is a trans stilbene compound, which is an effective component of herbaceous plants such as Dalbergia woods and Vaccinium. Although pterostilbene has many uses in anti-inflammatory, anti-oxidant and anti-tumor, its whitening effect is drawing more and more attention, the mechanism of melanogenesis and melanosome transport still needs further study. In this research, we tried to further investigate how melanocyte melanogenesis is affected by pterostilbene and whether pterostilbene play a part in melanin transport. Our results showed that pterostilbene has a potent inhibitory effect on melanogenesis in B16F10 cells (3 µM, p < 0.001), in-vitro human skin (10 µM, p < 0.05) and zebrafish embryos (3 µM, p < 0.01). Besides, pterostilbene not only inhibited melanogenesis, but also inhibited melanocyte dendritic development and melanosome transport. Pterostilbene mainly plays a role by inhibiting cAMP/PKA/CREB signal pathway. After the cAMP/PKA/CREB signaling pathway was inhibited, tyrosinase activity and the expression of MITF, TYR, Rab27A, Rab17 and gp100 were decreased, which in turn suppressed melanogenesis, melanocyte dendritic development and melanosome transport. Our findings showed that pterostilbene can potently inhibit melanogenesis and melanosome transport, suggesting the applicability of pterostilbene in skin lightning. Therefore, a novel pharmacologic way to treat hyperpigmentation has been proposed.

Silibinin promotes melanogenesis through the PKA and p38 MAPK signaling pathways in melanoma cells.

Silibinin is a flavonolignan isolated from milk thistle (Silybum marianum). Silibinin has been reported to possess multiple biological activities; however, its effect on melanogenesis remains unclear. This study investigated the effect of silibinin on melanogenesis in melanoma cells and the associated molecular mechanism. Our findings demonstrated that silibinin markedly increased melanin content in murine B16-F1 and human HMV-II melanoma cells. Silibinin activated intracellular tyrosinase activity and expression of tyrosinase, tyrosinase-related protein (TRP)-1, TRP-2, and microphthalmia-associated transcription factor (MITF). Furthermore, silibinin enhanced the phosphorylation of cyclic AMP-responsive element-binding protein (CREB), protein kinase A (PKA), and p38 mitogen-activated protein kinase (MAPK) but not of Akt and extracellular signal-regulated kinase (ERK). The specific PKA (H-89) and p38 (SB203580) inhibitors significantly attenuated silibinin-mediated melanin synthesis. These results suggest that silibinin is an effective stimulator of melanogenesis through upregulation of the protein expression of melanogenic enzymes activated by the PKA and p38 pathways, leading to CREB phosphorylation and MITF expression. Therefore, silibinin may have potential for use in the treatment of hypopigmentation disorders.

Effects of keratinocyte-derived and fibroblast-derived exosomes on human epidermal melanocytes.

BACKGROUND: Exosomes have been demonstrated to carry proteins, membrane lipids, mRNAs and microRNAs which can be transferred to surrounding cells and regulate the functions of those recipient cells. OBJECTIVES: The objective of the study was to investigate the effects of exosomes released by keratinocytes and fibroblasts on the proliferation, tyrosinase activity and melanogenesis of melanocytes. METHODS: Melanocytes, keratinocytes and fibroblasts obtained from human foreskin were cultured and exosomes secreted by keratinocytes and fibroblasts were harvested from the culture supernatants by ultracentrifugation. Each exosome fraction was divided into two parts; one part was subjected to high-throughput sequencing using an Illumina HiSeq sequencer to characterize the microRNA expression profiles, while the other part was labeled with the fluorescent dye PKH67 and was then co-cultivated with epidermal melanocytes. RESULTS: High-throughput sequencing analysis showed 168 differentially expressed microRNA within exosomes derived from keratinocytes and from fibroblasts, 97 of those being up-regulated with the other 71 down-regulated. Gene ontology analysis showed that the target genes responsible for these differentially expressed microRNAs were mainly enriched in the protein-binding region of molecular functions. Kyoto Encyclopedia of Genes and Genomes (KEGG) pathway analysis showed that target genes regulated by differentially expressed microRNA were mainly involved in mitogen-activated protein kinase (MAPK) signaling pathway, Ras signaling pathway, cAMP signaling pathway and Wnt signaling pathway. Keratinocyte-derived exosomes were taken up by melanocytes co-cultured with them and promoted the proliferation, tyrosinase activity and melanin synthesis of those melanocytes. However, fibroblast-derived exosomes had no similar effects on melanocytes. CONCLUSION: Keratinocyte-derived exosomes but not fibroblast-derived exosomes were taken up by melanocytes in co-culture and significantly stimulated their proliferation, tyrosinase activity and melanin synthesis. Those different effects may be mainly due to the differential expression of microRNAs in exosomes derived from the different types of cells. LIMITATIONS: Electron microscopy of the obtained exosomes and in-depth study of apparently differentially expressed microRNAs were not performed.

Effect of Biocides on the Activity of Extracellular Enzymes Produced by Degrader Fungi from Technical Objects Used in Tropical Climates (Vietnam).

Activity of extracellular enzymes was assessed in 20 strains of microscopic fungi involved in biodegradation of technical objects exploited under tropical climate conditions (Vietnam). It was found that 19 strains possessed catalase activity, 18 strains had phenol oxidase activity, and eight strains had protease activity. The effect of industrial biocides on the activity of these enzymes was also assessed. The biocides Bior-1, Bioneutral A 10, and Bioneutral A 101 were shown to inhibit the enzymatic activity to various extent. All biocides inhibited extracellular catalase activity in most fungal strains studied. The inhibition of protease and phenol oxidase activity of same test strains was less pronounced. The response to biocides varied at the strain level; its characteristics could differ significantly even between strains of the same species. In several cases, it was observed that exposure to biocides resulted in an increase in enzyme activity.

K-Nearest Neighbor and Random Forest-Based Prediction of Putative Tyrosinase Inhibitory Peptides of Abalone Haliotis diversicolor.

Skin pigment disorders are common cosmetic and medical problems. Many known compounds inhibit the key melanin-producing enzyme, tyrosinase, but their use is limited due to side effects. Natural-derived peptides also display tyrosinase inhibition. Abalone is a good source of peptides, and the abalone proteins have been used widely in pharmaceutical and cosmetic products, but not for melanin inhibition. This study aimed to predict putative tyrosinase inhibitory peptides (TIPs) from abalone, Haliotis diversicolor, using k-nearest neighbor (kNN) and random forest (RF) algorithms. The kNN and RF predictors were trained and tested against 133 peptides with known anti-tyrosinase properties with 97% and 99% accuracy. The kNN predictor suggested 1075 putative TIPs and six TIPs from the RF predictor. Two helical peptides were predicted by both methods and showed possible interaction with the predicted structure of mushroom tyrosinase, similar to those of the known TIPs. These two peptides had arginine and aromatic amino acids, which were common to the known TIPs, suggesting non-competitive inhibition on the tyrosinase. Therefore, the first version of the TIP predictors could suggest a reasonable number of the TIP candidates for further experiments. More experimental data will be important for improving the performance of these predictors, and they can be extended to discover more TIPs from other organisms. The confirmation of TIPs in abalone will be a new commercial opportunity for abalone farmers and industry.

Tissue adhesive, rapid forming, and sprayable ECM hydrogel via recombinant tyrosinase crosslinking.

We report on a tissue adhesive hydrogel based on novel recombinant tyrosinase mediated crosslinking. The adhesive hydrogels were fabricated by the site-directed coupling of tyramine-conjugated hyaluronic acid (HA_t, 1% w/v) and gelatin (3% w/v) (HG_gel) with novel tyrosinase derived from Streptomyces avermitilis (SA_Ty). The enzyme-based crosslinking by SA_Ty was fast, with less than 50 s for complete gelation, and the SA_Ty based crosslinking enhanced the physical properties and adhesive strength of the hydrogel significantly with the native tissue samples. Furthermore, by optimizing the injection conditions, we tailored the enzyme-based crosslinking hydrogels to be injectable and sprayable with a medical syringe and commercial airbrush nozzle, respectively. An in vivo analysis of the adhesive hydrogel showed a negligible immune reaction. In this study, demonstrate that the novel enzyme-based crosslinking hydrogel has a robust potential in tissue engineering and regenerative medicine.

Multiple pharmacological approaches on Fibigia eriocarpa extracts by in vitro and computational assays.

The ethyl acetate, methanolic, and water extracts of Fibigia eriocarpa were assessed for a panoply of bioactivities. Total phenolic and flavonoid content were quantified as well as individual phenolic compounds by HPLC-DAD. The in vitro antioxidant and enzyme (acetylcholinesterase (AChE), butyrylcholinesterase (BChE), tyrosinase, α-amylase, and α-glucosidase) inhibitory potential of the extracts were evaluated. In silico molecular docking was used to investigate possible interaction between dominant compounds and selected enzymes. Vanillin (303 µg/g extract), apigenin (270 µg/g extract), and kaempferol (180 µg/g extract) were the main compounds in the ethyl acetate extract, while the methanolic extract was characterized by the presence of vanillin, rutin, and apigenin (616, 616 and 252 µg/g extract, respectively). (+)-catechin (1422 µg/g extract) was the main compound in the water extracts. The ethyl acetate extract was found to be a superior source of antioxidant compounds and enzyme inhibitors against above-mentioned enzymes. Docking studies revealed that p-hydroxybenzoic and (+)-catechin have the best scores for tyrosinase, while kaempferol and apigenin showed the best binding pose for α-glucosidase, AChE, and BChE. Results amassed herein are the first report on the phytochemical and biological attributes of F. eriocarpa, which tend to validate the pharmacological uses of this plant as an alternative medicine.

Phenoloxidase, an effective bioactivity target for botanical insecticide screening from green walnut husks.

Phenoloxidase, a critical enzyme in insects, may serve as a promising target in botanical insecticide development. In an effort to identify active ingredients with insecticidal properties in green walnut husks, juglone and plumbagin were isolated from the chloroform extract using phenoloxidase as bioactive target with the IC50 of 0.247 g/L and 0.256 g/L, respectively. After an artificial diet feeding of the juglone or plumbagin, more than 50% corrected mortality in stomach toxicity form was observed in Pieris rapae Linne larvae and Helicoverpa armigera Hübner larvae at the concentration ≥0.01 g/L, the LC50 of juglone and plumbagin for two kinds of insects were determined as 0.012, 0.011 and 0.022, 0.030 g/L, respectively. This research indicated the significance of PO as bioactive target in pesticides identification and also shed light on the development of phenoloxidase inhibitor as promising botanical insecticides in the future.

Phenolic compounds from the leaves of Breynia officinalis and their tyrosinase and melanogenesis inhibitory activities.

From the EtOAc-soluble fraction of a MeOH extract of the leaves of Breynia officinalis, five new compounds (1-5) along with 11 known compounds (6-16) were isolated. The structures of the new compounds were elucidated by spectroscopic methods and compounds 1-3 were found to be acylated hydroquinone apiofuranosylglucopyranosides, while compound 4 was an acylated hydroquinone glucopyranoside. Compound 5 was shown to be butyl p-coumarate and this seems to be its first isolation from a natural source. The tyrosinase inhibitory activity of all of the isolated compounds was assayed, and the activity was significant in p-coumarate derivatives. The most active compound, compound 3, also inhibited melanogenesis in an in vivo whole animal model, zebrafish.

The light subunit of mushroom Agaricus bisporus tyrosinase: Its biological characteristics and implications.

The light subunit of mushroom Agaricus bisporus tyrosinase (LSMT) is a protein of unknown function that was discovered serendipitously during the elucidation of the crystal structure of the enzyme. The protein is non-immunogenic and can penetrate the intestinal epithelial cell barrier, and thus, similar to its structural homologue HA-33 from Clostridium botulinum, may be potentially absorbable by the intestine. LSMT also shares high structural homology with the ricin-B-like lectin from the mushroom Clitocybe nebularis (CNL), which has been shown to display biological activity against leukemic cancer cells and dendritic cells. Therefore, we evaluated the biological activity of LSMT. An in vitro assay suggested that LSMT presentation to most of the cancer cell lines studied has a negligible effect on their proliferation. However, inhibition of cell growth and a slight stimulation of cell proliferation were observed with breast cancer and macrophage cells, respectively. LSMT appeared to be relatively resistant against proteolysis by trypsin and papain, but not bromelain. Challenges with gastric and intestinal juice suggested that the protein is resistant to gastrointestinal tract conditions. This is the first report on the biological characteristics and implication of LSMT.

Design and synthesis of reduced graphene oxide based supramolecular scaffold: A benign microbial resistant network for enzyme immobilization and cell growth.

The present study describes the synthesis, characterization and biological application of reduced graphene oxide - chitosan (GC) based benign supramolecular scaffold (SMS). Various spectroscopic and microscopic analyses established the supramolecular interaction in between rGO and chitosan. The active performance of the developed material towards microbial resistivity, in vitro cell growth and as a scaffold for enzyme immobilizing matrix illustrates its unique implementation. Immobilization of polyphenol oxidase (PPO) onto GC lowers the Michaelis- Menten constant (Km) value and facilitates to achieve maximum velocity at low substrate concentration. Importantly GC shows no noteworthy cytotoxicity towards Wistar rat macrophage cells. Moreover, incorporation of gold nanoparticle further strengthens the microbial resistance properties of GC as well as improves its biocompatibility by reducing cytotoxicity. Therefore these unique features may inspire it to appear in large scale for industrial utilization.

Dopamine inhibits lipopolysaccharide-induced nitric oxide production through the formation of dopamine quinone in murine microglia BV-2 cells.

Dopamine (DA) has been suggested to modulate functions of glial cells including microglial cells. To reveal the regulatory role of DA in microglial function, in the present study, we investigated the effect of DA on lipopolysaccharide (LPS)-induced nitric oxide (NO) production in murine microglial cell line BV-2. Pretreatment with DA for 24 h concentration-dependently attenuated LPS-induced NO production in BV-2 cells. The inhibitory effect of DA on LPS-induced NO production was not inhibited by SCH-23390 and sulpiride, D1-like and D2-like DA receptor antagonists, respectively. In addition, pretreatment with (-)-(6aR,12bR)-4,6,6a,7,8,12b-Hexahydro-7-methylindolo[4,3-a]phenanthridin (CY 208-243) and bromocriptine, D1-like and D2-like DA receptor agonists, respectively, did not affect the LPS-induced NO production. N-Acetylcysteine, which inhibits DA oxidation, completely inhibited the effect of DA. Tyrosinase, which catalyzes the oxidation of DA to DA quionone (DAQ), accelerated the inhibitory effect of DA on LPS-induced NO production. These results suggest that DA attenuates LPS-induced NO production through the formation of DAQ in BV-2 cells.

T-Cell Responses to Tyrosinase-Derived Self-Peptides in Patients with Leukoderma Induced by Rhododendrol: Implications for Immunotherapy Targeting Melanoma.

BACKGROUND: Rhododendrol, a phenolic compound contained in lightening/whitening cosmetics, can bind and inhibit tyrosinase and was reported to induce leukoderma in Japan. Only 2% of the cosmetics users are affected, and tacrolimus is effective in treatment of the condition. OBJECTIVE: To test the hypothesis that the disease is an autoimmune disorder. METHODS: Short-term T-cell lines were established using peripheral blood mononuclear cells from 8 patients with human melanoma-associated and tyrosinase-derived synthetic peptides. The effects of rhododendrol on melanoma immunization were also examined. RESULTS: Seven out of 8 patients were positive for HLA-DR4. Both class I- and class II-restricted and tyrosinase peptide-specific T-cell responses were observed. Immunization of mice with rhododendrol-treated and irradiated B16 melanoma cells successfully delayed the growth of melanoma cells in vivo. CONCLUSION: Rhododendrol-induced leukoderma is an autoimmune disorder, with rhododendrol as an environmental factor and HLA-DR4 as a genetic factor. Rhododendrol might be effective in treating melanomas.

Bioactivation of luteolin by tyrosinase selectively inhibits glutathione S-transferase.

Glutathione S-transferase (GST) plays a significant role in the metabolism and detoxification of drugs used in treatment of melanoma, resulting in a decrease in drug efficacy. Tyrosinase is an abundant enzyme found in melanoma. In this study, we used a tyrosinase targeted approach to selectively inhibit GST. In the presence of tyrosinase, luteolin (10 µM) showed 87% GST inhibition; whereas in the absence of tyrosinase, luteolin led to negligible GST inhibition. With respect to GSH, both luteolin-SG conjugate and luteolin-quinone inhibited ≥90% of GST activity via competitive reversible and irreversible mixed mechanisms with Ki of 0.74 µM and 0.02 µM, respectively. With respect to CDNB, the luteolin-SG conjugate inhibited GST activity via competitive reversible mechanism and competitively with Ki of 0.58 µM, whereas luteolin-quinone showed irreversible mixed inhibition of GST activity with Ki of 0.039 µM. Luteolin (100 µM) inhibited GST in mixed manner with Ki of 53 µM with respect to GSH and non-competitively with respect to CDNB with Ki of 38 µM. Luteolin, at a concentration range of 5-80 µM, exhibited 78-99% GST inhibition in human SK-MEL-28 cell homogenate. Among the 3 species of intact luteolin, luteolin-SG conjugate, and luteoline-quinone, only the latter two have potential as drugs with Ki < 1 µM, which is potentially achievable in-vivo as therapeutic agents. The order of GST inhibition was luteolin-quinone >> luteolin-SG conjugate >>> luteolin. In summary, our results suggest that luteolin was bioactivated by tyrosinase to form a luteolin-quinone and luteolin-glutathione conjugate, which inhibited GST. For the first time, in addition to intracellular GSH depletion, we demonstrate that luteolin acts as a selective inhibitor of GST in the presence of tyrosinase. Such strategy could potentially be used to selectively inhibit GST, a drug detoxifying enzyme, in melanoma cells.

Stimulation of erythrocyte cell membrane scrambling by mushroom tyrosinase.

BACKGROUND: Mushroom tyrosinase, a copper containing enzyme, modifies growth and survival of tumor cells. Mushroom tyrosinase may foster apoptosis, an effect in part due to interference with mitochondrial function. Erythrocytes lack mitochondria but are able to undergo apoptosis-like suicidal cell death or eryptosis, which is characterized by cell shrinkage and cell membrane scrambling leading to phosphatidylserine-exposure at the erythrocyte surface. Signaling involved in the triggering of eryptosis include increase of cytosolic Ca2+-activity ([Ca2+]i) and activation of sphingomyelinase with subsequent formation of ceramide. The present study explored, whether tyrosinase stimulates eryptosis. METHODS: Cell volume has been estimated from forward scatter, phosphatidylserine-exposure from annexin V binding, [Ca2+]i from Fluo3-fluorescence, and ceramide abundance from binding of fluorescent antibodies in flow cytometry. RESULTS: A 24 h exposure to mushroom tyrosinase (7 U/mL) was followed by a significant increase of [Ca2+]i, a significant increase of ceramide abundance, and a significant increase of annexin-V-binding. The annexin-V-binding following tyrosinase treatment was significantly blunted but not abrogated in the nominal absence of extracellular Ca2+. Tyrosinase did not significantly modify forward scatter. CONCLUSIONS: Tyrosinase triggers cell membrane scrambling, an effect, at least partially, due to entry of extracellular Ca2+ and ceramide formation.