The combined effects of preservative chemicals in consumer products: An analysis using embryonic and adult zebrafish.

Benzisothiazolinone (BIT) and propyl paraben (PP) are preservatives in cleaning products; however, their toxicities are not well understood. In this study, zebrafish embryos were exposed to BIT, PP, and mixtures of both for 96 h to investigate the effects on growth hormone (GH), insulin-like growth factor-1 (IGF-1), and the transcription of 19 genes related to the GH/IGFs axis. Concentrations of BIT and PP were measured in the whole body of larvae. Zebrafish pairs were also exposed to BIT, PP, and mixtures for 21 d to evaluate the effects on sex hormones, histology in gonad, and transcription of 22 genes related to the hypothalamus-pituitary-gonad axis and vitellogenin. The mixtures had potentiation effects on development, reproduction, hormones, and gene transcripts than individual exposure. Larvae exposed to 229 µg L-1 BIT, 64.5 µg L-1 PP, and mixtures showed reduced growth. Decreased GH and IGF-1 levels were supported by gene regulation associated with the GH/IGFs axis. In larvae, reactive oxygen species, superoxide dismutase, catalase, and glutathione peroxidase levels were increased under all exposures. The gonadosomatic index in males and number of eggs decreased after mixture exposure. In females exposed to mixtures, the percentage of atretic follicle in ovary was significantly increased. The significant decrease in testosterone in males and significant decrease in 17ß-estradiol in females exposed to mixtures suggest anti-estrogenic and anti-androgenic potential. Thus, preservative mixtures in consumer products may be more toxic than the individual substances, which is important for managing the risks of mixing preservatives.

Identifying the external N and Hg inputs to the estuary ecosystem based on the triple isotopic information (δ15NNO3, Δ17ONO3 and δ18ONO3).

The supply and sources of N and Hg in the Geum estuary of the western coast of Korea were evaluated. Triple isotope proxies (δ15NNO3, Δ17ONO3 and δ18ONO3) of NO3- combined with conservative mixing between river and ocean waters were used to improve isotope finger-printing methods. The N pool in the Geum estuary was primarily influenced by the Yellow Sea water, followed by riverine discharge (821 × 106 mol yr-1) and atmospheric deposition (51 × 106 mol yr-1). The influence of the river was found to be greater for Hg than that of the atmosphere. The triple isotope proxies revealed that the riverine and atmospheric inputs of N have been affected by septic wastes and fossil fuel burning, respectively. From the inner estuary towards offshore region, the influence of the river diminishes, thus increasing the relative impact of the atmosphere. Moreover, the isotope proxies showed a significant influence of N assimilation in February and nitrification in May.

Urolithin and Reduced Urolithin Derivatives as Potent Inhibitors of Tyrosinase and Melanogenesis: Importance of the 4-Substituted Resorcinol Moiety.

We previously reported (E)-ß-phenyl-α,ß-unsaturated carbonyl scaffold ((E)-PUSC) played an important role in showing high tyrosinase inhibitory activity and that derivatives with a 4-substituted resorcinol moiety as the ß-phenyl group of the scaffold resulted in the greatest tyrosinase inhibitory activity. To examine whether the 4-substituted resorcinol moiety could impart tyrosinase inhibitory activity in the absence of the α,ß-unsaturated carbonyl moiety of the (E)-PUSC scaffold, 10 urolithin derivatives were synthesized. To obtain more candidate samples, the lactone ring in synthesized urolithins was reduced to produce nine reduced urolithins. Compounds 1c (IC50 = 18.09 ± 0.25 µM), 1h (IC50 = 4.14 ± 0.10 µM), and 2a (IC50 = 15.69 ± 0.40 µM) had greater mushroom tyrosinase-inhibitory activities than kojic acid (KA) (IC50 = 48.62 ± 3.38 µM). The SAR results suggest that the 4-substituted resorcinol motif makes an important contribution to tyrosinase inhibition. To investigate whether these compounds bind to human tyrosinase, a human tyrosinase homology model was developed. Docking simulations with mushroom and human tyrosinases showed that 1c, 1h, and 2a bind to the active site of both tyrosinases with higher binding affinities than KA. Pharmacophore analyses showed that two hydroxyl groups of the 4-substituted resorcinol entity act as hydrogen bond donors in both mushroom and human tyrosinases. Kinetic analyses indicated that these compounds were all competitive inhibitors. Compound 2a inhibited cellular tyrosinase activity and melanogenesis in α-MSH plus IBMX-stimulated B16F10 melanoma cells more strongly than KA. These results suggest that 2a is a promising candidate for the treatment of skin pigment disorders, and show the 4-substituted resorcinol entity importantly contributes to tyrosinase inhibition.

In silico and in vitro insights into tyrosinase inhibitors with a 2-thioxooxazoline-4-one template.

The ß-phenyl-α,ß-unsaturated carbonyl (PUSC) scaffold confers tyrosinase inhibitory activity, and in the present study, 16 (Z)-5-(substituted benzylidene)-3-phenyl-2-thioxooxazolidin-4-one analogues containing this scaffold were synthesized. Mushroom tyrosinase inhibitory activities were examined. Compound 1c (IC50 = 4.70 ± 0.40 µM) and compound 1j (IC50 = 11.18 ± 0.54 µM) inhibited tyrosinase by 4.9 and 2.1-fold, respectively, and did so more potently than kojic acid (IC50 = 23.18 ± 0.11 µM). Kinetic analysis of tyrosinase inhibition revealed that 1c and 1j inhibited tyrosinase competitively. Results of docking simulation with mushroom tyrosinase using four docking programs suggested that 1c and 1j bind more strongly than kojic acid to the active site of tyrosinase and supported kinetic findings that both compounds are competitive inhibitors. The docking results of human tyrosinase homology model indicated that 1c and 1j can also strongly inhibit human tyrosinase. EZ-cytox assays revealed 1c and 1j were not cytotoxic to B16F10 melanoma cells. The effects of 1c and 1j on cellular tyrosinase activity and melanin production were also investigated in α-MSH- and IBMX-co-stimulated these cells. Both compounds significantly and dose-dependently reduced tyrosinase activity, and at 10 µM were more potent than kojic acid at 20 µM. Compounds 1c and 1j also inhibited melanogenesis, which suggested that the inhibitory effects of these compounds on melanin production were mainly attributable to their inhibitions of tyrosinase. These results indicate that compounds 1c and 1j with the PUSC scaffold have potential use as whitening agents for the treatment of hyperpigmentation-associated diseases.

(E)-1-(Furan-2-yl)-(substituted phenyl)prop-2-en-1-one Derivatives as Tyrosinase Inhibitors and Melanogenesis Inhibition: An In Vitro and In Silico Study.

A series of (E)-1-(furan-2-yl)prop-2-en-1-one derivatives (compounds 1-8) were synthesized and evaluated for their mushroom tyrosinase inhibitory activity. Among these series, compound 8 (2,4-dihydroxy group bearing benzylidene) showed potent tyrosinase inhibitory activity, with respective IC50 values of 0.0433 µM and 0.28 µM for the monophenolase and diphenolase as substrates in comparison to kojic acid as standard compound 19.97 µM and 33.47 µM. Moreover, the enzyme kinetics of compound 8 were determined to be of the mixed inhibition type and inhibition constant (Ki) values of 0.012 µM and 0.165 µM using the Lineweaver-Burk plot. Molecular docking results indicated that compound 8 can bind to the catalytic and allosteric sites 1 and 2 of tyrosinase to inhibit enzyme activity. The computational molecular dynamics analysis further revealed that compound 8 interacted with two residues in the tyrosinase active site pocket, such as ASN260 and MET280. In addition, compound 8 attenuated melanin synthesis and cellular tyrosinase activity, simulated by α-melanocyte-stimulating hormone and 1-methyl-3-isobutylxanthine. Compound 8 also decreased tyrosinase expressions in B16F10 cells. Based on in vitro and computational studies, we propose that compound 8 might be a worthy candidate for the development of an antipigmentation agent.

A New Histone Deacetylase Inhibitor, MHY4381, Induces Apoptosis via Generation of Reactive Oxygen Species in Human Prostate Cancer Cells.

Histone deacetylase (HDAC) inhibitors represent a novel class of anticancer agents, which can be used to inhibit cell proliferation and induce apoptosis in several types of cancer cells. In this study, we investigated the anticancer activity of MHY4381, a newly synthesized HDAC inhibitor, against human prostate cancer cell lines and compared its efficacy with that of suberoylanilide hydroxamic acid (SAHA), a well-known HDAC inhibitor. We assessed cell viability, apoptosis, cell cycle regulation, and other biological effects in the prostate cancer cells. We also evaluated a possible mechanism of MHY4381 on the apoptotic cell death pathway. The IC50 value of MHY4381 was lower in DU145 cells (IC50=0.31 µM) than in LNCaP (IC50=0.85 µM) and PC-3 cells (IC50=5.23 µM). In addition, the IC50 values of MHY4381 measured in this assay were significantly lower than those of SAHA against prostate cancer cell lines. MHY4381 increased the levels of acetylated histones H3 and H4 and reduced the expression of HDAC proteins in the prostate cancer cell lines. MHY4381 increased G2/M phase arrest in DU145 cells, and G1 arrest in LNCaP cells. It also activated reactive oxygen species (ROS) generation, which induced apoptosis in the DU145 and LNCaP cells by increasing the ratio of Bax/Bcl-2 and releasing cytochrome c into the cytoplasm. Our results indicated that MHY4381 preferentially results in antitumor effects in DU145 and LNCaP cells via mitochondria-mediated apoptosis and ROS-facilitated cell death pathway, and therefore can be used as a promising prostate cancer therapeutic.

Pro-apoptotic effect of the novel benzylidene derivative MHY695 in human colon cancer cells.

The induction of apoptosis is a useful strategy in anti-cancer research. Various Moon Hyung Yang (MHY) compounds have been developed as novel anti-cancer drug candidates; in the present study, the pro-apoptotic effects of (Z)-5-(3-ethoxy-4- hydroxybenzylidene)-2-thioxothiazolidin-4-one (MHY695) on HCT116 human colon cancer cells were assessed. MTT assays were performed to investigate the dose-dependent cytotoxic effects of MHY695 on HCT116 cells. Immunofluorescence staining and flow cytometry analyses were performed to identify apoptotic cell death, and western blot analysis was used to investigate the apoptotic-signaling pathways. A mouse xenograft model was also used to determine the effects of MHY695 in vivo. MHY695 decreased the viability of HCT116 cells and induced apoptotic cytotoxicity. The apoptotic mechanisms induced by MHY695 involved the dephosphorylation of Bcl-2-associated agonist of cell death protein following protein kinase B inactivation, induced myeloid leukaemia cell differentiation protein and BH3-interacting domain death agonist truncation, caspase-3 and -9 activation and poly (ADP-ribose) polymerase cleavage. In addition, MHY695 significantly suppressed tumor growth in the mouse xenograft model, compared with the vehicle control. Notably, MHY695 exhibited potent anti-cancer effects in four different types of human colon cancer cell line, including Caco-2, DLD-1, HT-29 and HCT116. Additionally, MHY695 showed reduced cytotoxicity in NCM460, normal colonic epithelial cells. Furthermore, MHY-induced cytotoxicity in colon cancer cells was independent of the tumor suppressor protein p53. Collectively, these observations suggested that MHY695 may be a novel drug for the treatment of colon cancer.

Inhibitory effects of N-(acryloyl)benzamide derivatives on tyrosinase and melanogenesis.

Targeting of tyrosinase has proven to be the best means of identifying safe, efficacious, and potent tyrosinase inhibitors for whitening skin. We designed and synthesized ten NAB (N-(acryloyl)benzamide) derivatives (1a-1j) using the Horner-Wadsworth-Emmons olefination of diethyl (2-benzamido-2-oxoethyl)phosphonate and appropriate benzaldehydes. A mushroom tyrosinase inhibitory assay showed compounds 1a (36.71 ±â€¯2.14% inhibition) and 1j (25.99 ±â€¯2.77% inhibition) inhibited tyrosinase more than the other eight NAB derivatives and kojic acid (21.56 ±â€¯2.93% inhibition), and docking studies indicated 1a (-6.9 kcal/mole) and 1j (-7.5 kcal/mole) had stronger binding affinities for tyrosinase than kojic acid (-5.7 kcal/mole). At a concentration of 25 µM, 1a and 1j were nontoxic in B16F10 melanoma cells and exhibited stronger tyrosinase inhibition (59.70% and 76.77%, respectively) than kojic acid (50.30% inhibition) or arbutin (41.78% inhibition at 400 µM). Similarly, in B16F10 melanoma cells, compounds 1a and 1j at 25 µM decreased total melanin content by 47.97% and 61.77%, respectively (kojic acid; 38.98%). Similarities between inhibitions of tyrosinase activity and melanin contents suggested the anti-melanogenic effects of 1a and 1j were due to tyrosinase inhibition. The excellent DPPH scavenging activity of 1j suggests it might enhance in vivo effect on melanin contents. The study suggests compound 1j offers a potential starting point for the development of safe, potent tyrosinase inhibitors.

Antioxidant, anti-tyrosinase and anti-melanogenic effects of (E)-2,3-diphenylacrylic acid derivatives.

During our continued search for strong skin whitening agents over the past ten years, we have investigated the efficacies of many tyrosinase inhibitors containing a common (E)-ß-phenyl-α,ß-unsaturated carbonyl scaffold, which we found to be essential for the effective inhibition of mushroom and mammalian tyrosinases. In this study, we explored the tyrosinase inhibitory effects of 2,3-diphenylacrylic acid (2,3-DPA) derivatives, which also possess the (E)-ß-phenyl-α,ß-unsaturated carbonyl motif. We synthesized fourteen (E)-2,3-DPA derivatives 1a-1n and one (Z)-2,3-DPA-derivative 1l' using a Perkin reaction with phenylacetic acid and appropriate substituted benzaldehydes. In our mushroom tyrosinase assay, 1c showed higher tyrosinase inhibitory activity (76.43 ±â€¯3.53%, IC50 = 20.04 ±â€¯1.91 µM) with than the other 2,3-DPA derivatives or kojic acid (21.56 ±â€¯2.93%, IC50 = 30.64 ±â€¯1.27 µM). Our mushroom tyrosinase inhibitory results were supported by our docking study, which showed compound 1c (-7.2 kcal/mole) exhibited stronger binding affinity for mushroom tyrosinase than kojic acid (-5.7 kcal/mole). In B16F10 melanoma cells (a murine cell-line), 1c showed no cytotoxic effect up to a concentration of 25 µM and exhibited greater tyrosinase inhibitory activity (68.83%) than kojic acid (49.39%). In these cells, arbutin (a well-known tyrosinase inhibitor used as the positive control) only inhibited tyrosinase by 42.67% even at a concentration of 400 µM. Furthermore, at 25 µM, 1c reduced melanin contents in B16F10 melanoma cells by 24.3% more than kojic acid (62.77% vs. 38.52%). These results indicate 1c is a promising candidate treatment for pigmentation-related diseases and potential skin whitening agents.

Tyrosinase inhibition and anti-melanin generation effect of cinnamamide analogues.

Abnormal melanogenesis results in excessive production of melanin, leading to pigmentation disorders. As a key and rate-limiting enzyme for melanogenesis, tyrosinase has been considered an important target for developing therapeutic agents of pigment disorders. Despite having an (E)-ß-phenyl-α,ß-unsaturated carbonyl scaffold, which plays an important role in the potent inhibition of tyrosinase activity, cinnamic acids have not attracted attention as potential tyrosinase inhibitors, due to their low tyrosinase inhibitory activity and relatively high hydrophilicity. Given that cinnamic acids' structure intrinsically features this (E)-scaffold and following our experience that minute changes in the chemical structure can powerfully affect tyrosinase activity, twenty less hydrophilic cinnamamide derivatives were designed as potential tyrosinase inhibitors and synthesised using a Horner-Wadsworth-Emmons reaction. Four of these cinnmamides (4, 9, 14, and 19) exhibited much stronger mushroom tyrosinase inhibition (over 90% inhibition) at 25 µM compared to kojic acid (20.57% inhibition); crucially, all four have a 2,4-dihydroxy group on the ß-phenyl ring of the scaffold. A docking simulation using tyrosinase indicated that the four cinnamamides exceeded the binding affinity of kojic acid, and bound more strongly to the active site of tyrosinase. Based on the strength of their tyrosinase inhibition, these four cinnamamides were further evaluated in B16F10 melanoma cells. All four cinnamamides, without cytotoxicity, exhibited higher tyrosinase inhibitory activity (67.33 - 79.67% inhibition) at 25 µM than kojic acid (38.11% inhibition), with the following increasing inhibitory order: morpholino (9) = cyclopentylamino (14) < cyclohexylamino (19) < N-methylpiperazino (4) cinnamamides. Analysis of tyrosinase activity and melanin content in B16F10 cells showed that the four cinnamamides dose-dependently inhibited both cellular tyrosinase activity and melanin content and that their inhibitory activity at 25 µM was much better than that of kojic acid. The results of melanin content analysis well matched those of the cellular tyrosinase activity analysis, indicating that tyrosinase inhibition by the four cinnamamides is a major factor in the reduction of melanin production. These results imply that these four cinnamamides with a 2,4-dihydroxyphenyl group can act as excellent anti-melanogenic agents in the treatment of pigmentation disorders.

Synthesis of cinnamic amide derivatives and their anti-melanogenic effect in α-MSH-stimulated B16F10 melanoma cells.

Of the three enzymes that regulate the biosynthesis of melanin, tyrosinase and its related proteins TYRP-1 and TYRP-2, tyrosinase is the most important because of its ability to limit the rate of melanin production in melanocytes. For treating skin pigmentation disorders caused by an excess of melanin, the inhibition of tyrosinase enzyme is by far the most established strategy. Cinnamic acid is a safe natural product with an (E)-ß-phenyl-α,ß-unsaturated carbonyl motif that we have previously shown to play an important role in high tyrosinase inhibition. Since cinnamic acid is relatively hydrophilic, which hinders its absorption on the skin, fifteen less hydrophilic cinnamic amide derivatives (1-15) were designed as safe and more potent tyrosinase inhibitors and were synthesized through a Horner-Wadsworth-Emmons reaction. The use of conc-HCl and acetic acid for debenzylation of the O-benzyl-protected cinnamic amides 40-54 produced the following three results. 1) Cinnamic amides 43, 48, and 53 with a 2,4-dibenzyloxyphenyl group, irrespective of the amine type of the amides, produced complex compounds with high polarity. 2) Cinnamic amides 40-42, 44, 50-52, and 54 with a benzylamino, or diethylamino group produced the desired debenzylated cinnamic amides 1-3, 5, 10-13, and 15. 3) Cinnamic amides 45-47, and 49 with an anilino moiety provided 3,4-dihydroquinolinones 16-19 through intramolecular Michael addition of the anilide group. Notably, the use of BBr3 as an alternative debenzylating agent for debenzylation of cinnamic amides 45-49 with the anilino moiety provided our desired cinnamic amides 6-10 without inducing the intramolecular Michael addition. Debenzylation of cinnamic amides 43, 48, and 53 with a 2,4-dibenzyloxyphenyl group was also successfully accomplished using BBr3 to give 4, 9, and 14. Among the nine compounds that inhibited mushroom tyrosinase more potently at 25 µM than kojic acid, four cinnamic amides 4, 5, 9, and 14 showed 3-fold greater tyrosinase inhibitory activity than kojic acid. The docking simulation using tyrosinase indicated that these four cinnamic amides (-6.2 to -7.9 kcal/mol) bind to the active site of tyrosinase with stronger binding affinity than kojic acid (-5.7 kcal/mol). All four cinnamic amides inhibited melanogenesis and tyrosinase activity more potently than kojic acid in α-MSH-stimulated B16F10 melanoma cells in a dose-dependent manner without cytotoxicity. The strong correlation between tyrosinase activity and melanin content suggests that the anti-melanogenic effect of cinnamic amides is due to tyrosinase inhibitory activity. Considering that the cinnamic amides 4, 9, and 14, which exhibited strong inhibition on mushroom tyrosinase and potent anti-melanogenic effect in B16F10 cells, commonly have a 2,4-dihydroxyphenyl substituent, the 2,4-dihydroxyphenyl substituent appears to be essential for high anti-melanogenesis. These results support the potential of these four cinnamic amides as novel and potent tyrosinase inhibitors for use as therapeutic agents with safe skin-lightening efficiency.

Evaluation of the Novel Synthetic Tyrosinase Inhibitor (Z)-3-(3-bromo-4-hydroxybenzylidene)thiochroman-4-one (MHY1498) In Vitro and In Silico.

Tyrosinase is a key enzyme in melanin synthesis, catalyzing the initial rate-limiting steps of melanin synthesis. Abnormal and excessive melanin synthesis is the primary cause of serious skin disorders including melasma, senile lentigo, freckles, and age spots. In attempts to find potent and safe tyrosinase inhibitors, we designed and synthesized a novel compound, (Z)-3-(3-bromo-4-hydroxybenzylidene)thiochroman-4-one (MHY1498), and evaluated its tyrosinase inhibitory activity in vitro and in silico. The chemical structures of (Z)-3-benzylidenethiochroman-4-one analogues, including the novel compound MHY1498, were rationally designed and synthesized as hybrid structures of reported potent tyrosinase inhibitors, which were confirmed both in vitro and in vivo: (Z)-5-(substituted benzylidene)thiazolidine-2,4-diones (Compound A) and 2-(substituted phenyl)benzo[d]thiazoles (Compound B). During screening, MHY1498 showed a strong dose-dependent inhibitory effect on mushroom tyrosinase. The IC50 value of MHY1498 (4.1 ± 0.6 µM) was significantly lower than that of the positive control, kojic acid (22.0 ± 4.7 µM). In silico molecular multi-docking simulation and inhibition mechanism studies indicated that MHY1498 interacts competitively with the tyrosinase enzyme, with greater affinity for the active site of tyrosinase than the positive control. Furthermore, in B16F10 melanoma cells treated with α-melanocyte-stimulating hormone, MHY1498 suppressed both melanin production and tyrosinase activity. In conclusion, our data demonstrate that MHY1498, a synthesized novel compound, effectively inhibits tyrosinase activity and has potential for treating hyperpigmentation and related disorders.

Design, synthesis and anti-melanogenic effect of cinnamamide derivatives.

Pigmentation disorders are attributed to excessive melanin which can be produced by tyrosinase. Therefore, tyrosinase is supposed to be a vital target for the treatment of disorders associated with overpigmentation. Based on our previous findings that an (E)-ß-phenyl-α,ß-unsaturated carbonyl scaffold can play a key role in the inhibition of tyrosinase activity, and the fact that cinnamic acid is a safe natural substance with a scaffolded structure, it was speculated that appropriate cinnamic acid derivatives may exhibit potent tyrosinase inhibitory activity. Thus, ten cinnamamides were designed, and synthesized by using a Horner-Emmons olefination as the key step. Cinnamamides 4 (93.72% inhibition), 9 (78.97% inhibition), and 10 (59.09% inhibition) with either a 2,4-dihydroxyphenyl, or 4-hydroxy-3-methoxyphenyl substituent showed much higher mushroom tyrosinase inhibition at 25 µM than kojic acid (18.81% inhibition), used as a positive control. Especially, the two cinnamamides 4 and 9 having a 2,4-dihydroxyphenyl group showed the strongest inhibition. Docking simulation with tyrosinase revealed that these three cinnamamides, 4, 9, and 10, bind to the active site of tyrosinase more strongly than kojic acid. Cell-based experiments carried out using B16F10 murine skin melanoma cells demonstrated that all three cinnamamides effectively inhibited cellular tyrosinase activity and melanin production in the cells without cytotoxicity. There was a close correlation between cellular tyrosinase activity and melanin content, indicating that the inhibitory effect of the three cinnamamides on melanin production is mainly attributed to their capability for cellular tyrosinase inhibition. These results imply that cinnamamides having the (E)-ß-phenyl-α,ß-unsaturated carbonyl scaffolds are promising candidates for skin-lighting agents.

The tyrosinase inhibitory effects of isoxazolone derivatives with a (Z)-ß-phenyl-α, ß-unsaturated carbonyl scaffold.

Thirteen (Z)-4-(substituted benzylidene)-3-phenylisoxazol-5(4H)-ones were designed to confirm the geometric effect of the double bond of the ß-phenyl-α, ß-unsaturated carbonyl scaffold on tyrosinase inhibitory activity. Compounds 1a-1m, which all possessed the (Z)-ß-phenyl-α, ß-unsaturated carbonyl scaffold, were synthesized using a tandem reaction consisting of an isoxazolone ring formation and a Knoevenagel condensation, and three starting materials, ethyl benzoylacetate, hydroxylamine and benzaldehydes. Some of the compounds showed inhibitory activity against mushroom tyrosinase as potent as compounds containing the "(E)"-ß-phenyl-α, ß-unsaturated carbonyl scaffold. Compounds 1c and 1m showed greater inhibitory activity than kojic acid: IC50 = 32.08 ±â€¯2.25 µM for 1c; IC50 = 14.62 ±â€¯1.38 µM for 1m; and IC50 = 37.86 ±â€¯2.21 µM for kojic acid. A kinetic study indicated that 1m inhibited tyrosinase in a competitive manner and that it probably binds to the enzyme's active site. In silico docking simulation supported binding of 1m (-7.6 kcal/mol) to the active site of tyrosinase with stronger affinity than kojic acid (-5.7 kcal/mol). Similar results were obtained using cell-based assays, and in B16F10 cells, compound 1m dose-dependently inhibited tyrosinase activity and melanogenesis. These results indicate the anti-melanogenic effect of compound 1m is due to the inhibition of tyrosinase and (Z)-isomer of the ß-phenyl-α, ß-unsaturated carbonyl scaffold can, like its congener the (E)-isomer, act as an excellent scaffold for tyrosinase inhibition.

MHY440, a Novel Topoisomerase Ι Inhibitor, Induces Cell Cycle Arrest and Apoptosis via a ROS-Dependent DNA Damage Signaling Pathway in AGS Human Gastric Cancer Cells.

We investigated the antitumor activity and action mechanism of MHY440 in AGS human gastric cancer cells. MHY440 inhibited topoisomerase (Topo) Ι activity and was associated with a DNA damage response signaling pathway. It exhibited a stronger anti-proliferative effect on AGS cells relative to Hs27 human foreskin fibroblast cells, and this effect was both time- and concentration-dependent. MHY440 also increased cell arrest in the G2/M phase by decreasing cyclin B1, Cdc2, and Cdc25c, and upregulating p53 and p73. MHY440 induced AGS cell apoptosis through the upregulation of Fas-L, Fas, and Bax as well as the proteolysis of BH3 interacting-domain death agonist and poly(ADP-ribose) polymerase. It also contributed to the loss of mitochondrial membrane potential. The apoptotic cell death induced by MHY440 was inhibited by pretreatment with Z-VAD-FMK, a pan-caspase inhibitor, indicating that apoptosis was caspase-dependent. Moreover, the apoptotic effect of MHY440 was reactive oxygen species (ROS)-dependent, as evidenced by the inhibition of MHY440-induced PARP cleavage and ROS generation via N-acetylcysteine-induced ROS scavenging. Taken together, MHY440 showed anticancer effects by inhibiting Topo I, regulating the cell cycle, inducing apoptosis through caspase activation, and generating ROS, suggesting that MHY440 has considerable potential as a therapeutic agent for human gastric cancer.