Gallate-induced nanoparticle uptake by tumor cells: Structure-activity relationships.

How nanoparticles interact with biological systems determines whether they can be used in theranostic applications. It has been demonstrated that tea catechins, may enhance interactions of magnetic nanoparticles (MNPs) with tumor cells and the subsequent cellular internalization of MNPs. As part of the chemical structure of the major tea catechins, gallates are found in a variety of plants and thus food components. We asked whether the structure of gallate might act as a pharmacophore in the enhancement of the effects of MNP-cell interactions. Uptake of dextran-coated MNPs by glioma cells and cell-associated MNPs (MNPcell) were respectively analyzed by confocal microscopy and a colorimetric iron assay. Co-incubation of MNPs and gallates, such as gallic acid and methyl gallate, induced a concentration-dependent increase in MNPcell, which was associated with co-localization of internalized MNPs and lysosomes. An analysis of the structure-activity relationship (SAR) revealed that the galloyl moiety exerted the most prominent enhancement effects on MNPcell which was further potentiated by the application of magnetic force; catechol coupled with a conjugated carboxylic acid side chain displayed comparable effects to gallate. Blockade or reduction in the number of hydroxyl groups rendered these compounds less effective, but without inducing cytotoxicity. The SAR results suggest that neighboring hydroxyl groups on the aromatic ring form an essential scaffold for the uptake effects; a similar SAR on antioxidant activities was also observed using a free radical-scavenging method. The results provide pivotal information for theranostic applications of gallates by facilitating nanoparticle-cell interactions and nanoparticle internalization by tumor cells.

Melanogenesis Inhibitors from the Rhizoma of Ligusticum Sinense in B16-F10 Melanoma Cells In Vitro and Zebrafish In Vivo.

The rhizoma of Ligusticum sinense, a Chinese medicinal plant, has long been used as a cosmetic for the whitening and hydrating of the skin in ancient China. In order to investigate the antimelanogenic components of the rhizoma of L. sinense, we performed an antimelanogenesis assay-guided purification using semi-preparative HPLC accompanied with spectroscopic analysis to determine the active components. Based on the bioassay-guided method, 24 compounds were isolated and identified from the ethyl acetate layer of methanolic extracts of L. sinense, and among these, 5-[3-(4-hydroxy-3-methoxyphenyl)allyl]ferulic acid (1) and cis-4-pentylcyclohex-3-ene-1,2-diol (2) were new compounds. All the pure isolates were subjected to antimelanogenesis assay using murine melanoma B16-F10 cells. Compound 1 and (3S,3aR)-neocnidilide (8) exhibited antimelanogenesis activities with IC50 values of 78.9 and 31.1 µM, respectively, without obvious cytotoxicity. Further investigation showed that compound 8 demonstrated significant anti-pigmentation activity on zebrafish embryos (10‒20 µM) compared to arbutin (20 µM), and without any cytotoxicity against normal human epidermal keratinocytes. These findings suggest that (3S,3aR)-neocnidilide (8) is a potent antimelanogenic and non-cytotoxic natural compound and may be developed potentially as a skin-whitening agent for cosmetic uses.

Isolation and synthesis of a bioactive benzenoid derivative from the fruiting bodies of Antrodia camphorata.

A new enynyl-benzenoid, antrocamphin O (1,4,7-dimethoxy-5-methyl-6-(3'-methylbut-3-en-1-ynyl)benzo[d][1,3]dioxide), and the known benzenoids antrocamphin A and 7-dimethoxy-5-methyl-1,3-benzodioxole, were isolated from the fruiting bodies of Antrodia camphorata (Taiwanofungus camphoratus). The structure of antrocamphin O was unambiguously assigned by the analysis of spectral data (including 1D and 2D NMR, high-resolution MS, IR, and UV) and total synthesis. Compound 1 was prepared through the Sonogashira reaction of 5-iodo-4,7-dimethoxy-6-methylbenzene and 2-methylbut-1-en-3-yne as the key step. The benzenoids were tested for cytotoxicity against the HT29, HTC15, DLD-1, and COLO 205 colon cancer cell lines, and activities are reported herein.

Determination of nimetazepam and 7-aminonimetazepam in human urine by using liquid chromatography-tandem mass spectrometry.

We report determination of metabolites of popular drugs of abuse, including nimetazepam and nitrazepam, in urine by using liquid chromatography/mass spectrometry. Nimetazepam and its metabolites, 7-aminonimetazepam and nitrazepam, were extracted by solid-phase extraction using a DAU cartridge. An ammonium acetate buffer solution (pH 4) and a Luna polar-RP column were selected as the mobile and stationary phase, respectively, for liquid chromatography. Mass spectrometry was used for analysis and was optimized for operation in the positive mode for all analytes. The urine specimens were screened for the presence of nimetazepam and its metabolites nitrazepam and 7-aminonimetazepam at a concentration of 0.1ng/mL. Presence of 7-aminonimetazepam in the urine was an indicator of the subject being a probable abuser of nimetazepam.

Vitamin K3-2,3-epoxide induction of apoptosis with activation of ROS-dependent ERK and JNK protein phosphorylation in human glioma cells.

2-Methyl-1,4-naphthoquinone (menadione or vitamin K3; EPO) and K3-2,3-epoxide (EPO1), but not vitamin K3-3-OH (EPO2), exhibited cytotoxicity that caused DNA fragmentation and chromatin condensation in U87 and C6 cells. EPO1 showed more-potent cytotoxicity than EPO, and the IC(50) values of EPO and EPO1 in U87 cells were 37.5 and 15.7µM, respectively. Activation of caspase 3 enzyme activity with cleavage of caspase 3 protein was detected in EPO1-treated U87 and C6 cells, and the addition of the caspase 3 peptidyl inhibitor, DEVD-FMK, reduced the cytotoxic effect of EPO1. An increase in the intracellular ROS level by EPO1 was observed in the DCHF-DA analysis, and EPO1-induced apoptosis and caspase 3 protein cleavage were prevented by adding the antioxidant, N-acetyl-cysteine (NAC), with decreased ROS production elicited by EPO1. Activation of ERK and JNK, but not p38, via phosphorylation induction was identified in EPO1- but not EPO- or EPO2-treated U87 and C6 cells, and this was blocked by adding NAC. However, the ERK inhibitor, PD98059, and the JNK inhibitor, SP600125, showed no effect on EPO1-induced cytotoxicity in either cell type. Our findings demonstrate that 2,3-epoxide substitution significantly potentiates the apoptotic effect of vitamin K3 via stimulating ROS production, which may be useful in the chemotherapy of glioblastoma cells.

5,5-Diphenyl-2-thiohydantoin-N10 (DPTH-N10) suppresses proliferation of cultured colon cancer cell line COLO-205 by inhibiting DNA synthesis and activating apoptosis.

The aim of this study was to investigate the potential applications of 5,5-diphenyl-2-thiohydantoin-N10 (DPTH-N10) in the treatment of human colon cancer. Subcultured human colon cancer cell line, COLO-205, was used for examining the antiproliferation effect of DPTH-N10 on colon cancer. Thymidine incorporation and cell count were conducted to examine the antiproliferation effect of DPTH-N10. Western blot analysis was performed to examine the protein levels of cell cycle-related proteins. DNA fragmentation assay was performed to examine the occurrence of apoptosis. DPTH-N10 at a range of concentrations (0-30 microM) inhibits the proliferation but did not cause the cell death of COLO-205, indicating that it may have an inhibitory effect on the cell proliferation in COLO-205. The apoptosis was observed in COLO-205 when the DPTH-N10 concentrations were higher than 30 muM. Western blot analysis showed that the protein level of the cell cycle inhibitory protein, p21, in COLO-205 increased after DPTH-N10 treatment. Immunoprecipitation showed that the formation of the cyclin-dependent kinase (CDK)2-p21 complex was increased in the DPTH-N10-treated COLO-205. Kinase assay further demonstrated that the CDK2 activity was decreased in the DPTH-N10-treated COLO-205. DPTH-N10 caused growth inhibition in COLO-205 by inhibiting DNA synthesis and activating apoptosis. The findings from our previous in vitro studies in DPTH-N10-induced anti-angiogenic effect and from the present in vitro studies in DPTH-N10-induced antiproliferation effect on colon cancer cell line strongly suggest the potential applications of DPTH-N10 in the treatment of human colon cancer.

Anti-angiogenic action of 5,5-diphenyl-2-thiohydantoin-N10 (DPTH-N10).

Previously, we demonstrated that 5,5-diphenyl-2-thiohydantoin (DPTH) exerts an anti-proliferation effect on subcultured human umbilical vein endothelial cells (HUVEC). In the present study, we show that 2(naphthalen-2-ylmethylsulfanyl)-5,5-diphenyl-1,5-dihydro-imidazol-4-one (DPTH-N10), a derivative compound of DPTH, exerts a 5 times stronger inhibition of [3H]thymidine incorporation into HUVEC as compared with DPTH and at very low concentrations (0-20 microM) inhibited DNA synthesis and decreased cell number in cultured HUVEC in a concentration- and time-dependent manner, but not in human fibroblasts. [3H]thymidine incorporation analysis demonstrated that treatment of HUVEC with DPTH-N10 arrested the cell at the G0/G1 phase of the cell cycle. Western blot analysis revealed that the protein level of p21 in HUVEC increased after DPTH-N10 treatment. In contrast, the protein levels of p27, p53, cyclins A, D1, D3 and E, cyclin-dependent kinase (CDK)2, and CDK4 in HUVEC were not changed significantly after DPTH-N10 treatment. Immunoprecipitation showed that the formation of the CDK2-p21 complex, but not the CDK2-p27, CDK4-p21, and CDK4-p27 complex, was increased in the DPTH-N10-treated HUVEC. Kinase assay further demonstrated that CDK2, but not CDK4, kinase activity was decreased in the DPTH-N10-treated HUVEC. Pretreatment of HUVEC with a p21, but not p27, antisense oligonucleotide reversed the DPTH-N10-induced inhibition of [3H]thymidine incorporation into HUVEC. Taken together, these data suggest that DPTH-N10 inhibits HUVEC proliferation by increasing the level of p21 protein, which in turn inhibits CDK2 kinase activity, and finally interrupts the cell cycle. Capillary-like tube formation, aortic ring culture, and chick embryo chorioallantoic membrane (CAM) assays further demonstrated the anti-angiogenic effect of DPTH-N10.

Structure and anti-proliferation function of 5,5-diphenyl-2-thiohydantoin (DPTH) derivatives in vascular endothelial cells.

Previously, we identified DPTH, an analogue of antiepileptic drug phenytoin (5,5-diphenylhydantoin, DPT), capable of retarding the cell cycle in the human vascular endothelial cells. Our data suggest that DPTH inhibits human umbilical venous endothelial cells (HUVEC) proliferation by increasing the level of p21 protein, which in turn inhibits the activities of cyclin-dependent kinase (CDK)2 and CDK4, and finally interrupts the cell cycle. To search chemicals with more potency in anti-angiogenic activity, we designed and synthesized several chemical compounds based on the structure-activity relationship consideration. We evaluated the anti-angiogenic activity of these compounds by examining their effects on DNA synthesis, cell number, p21 induction and capillary-like tube formation. Our results showed that introduction of side chain containing an aromatic ring structure with right spatial arrangement at sulfur atom of DPTH enhanced the anti-angiogenic activity in HUVEC.

Anti-proliferation effect of 3-amino-2-imino-3,4-dihydro-2H-1,3-benzothiazin-4-one (BJ-601) on human vascular endothelial cells: G0/G1 p21-associated cell cycle arrest.

The aim of this study was to examine the anti-proliferation effect of 3-amino-2-imino-3,4-dihydro-2H-1,3-benzothiazin-4-one (BJ-601) on human vascular endothelial cells and its possible molecular mechanism underlying. Our data showed that BJ-601 at a range of concentrations (0-40 microM) dose- and time-dependently decreased cell number in cultured human dermal microvascular endothelial cells (HDMVECs), but not human fibroblasts. The BJ-601-induced growth inhibition in HDMVECs was reversible. [3H]thymidine incorporation demonstrated that BJ-601 arrested the HDMVECs at the G0/G1 phase of the cell cycle. Western blot analysis revealed that BJ-601 (0-40 microM) dose-dependently increased the levels of the protein p21, but not of p27, p53, cyclins A, D1, D3 and E, cyclin-dependent kinase 2 (CDK2), and CDK4 in HDMVECs. Immunoprecipitation showed that the formation of the CDK2-p21 complex, but not CDK2-p27, CDK4-p21 and CDK4-p27 complexes, was increased in the BJ-601-treated HDMVECs. Kinase assay further demonstrated that CDK2, but not CDK4, kinase activity was decreased in a dose-dependent manner in the BJ-601-treated HDMVECs. Pretreatment of HDMVECs with a p21 antisense oligonucleotide, which blocked the expression of p21 protein, reversed the BJ-601-induced inhibition of [3H]thymidine incorporation into HDMVECs. Moreover, cotreatment of the endothelial cells with protein kinase C (PKC) inhibitor, staurosporine, prevented the BJ-601-induced decrease of [3H]thymidine incorporation into HDMVECs. Administration of BJ-601 dose-dependently inhibited capillary-like tube formation of HDMVECs in Matrigel. In conclusion, these data suggest that BJ-601 inhibits HDMVECs proliferation by increasing the level of p21 protein, which in turn inhibits CDK2 kinase activity, and finally causes retardation of the cell cycle at the G0/G1 phase.

Anti-proliferation effect of 5,5-diphenyl-2-thiohydantoin (DPTH) in human vascular endothelial cells.

The aim of this study was to examine the anti-proliferation effect of 5,5-diphenyl-2-thiohydantoin (DPTH), an analogue of antiepileptic drug phenytoin (5,5-diphenylhydantoin), on human umbilical vein endothelial cells (HUVEC) and its possible molecular mechanism underlying. Here we demonstrated that DPTH at a range of concentrations (12.5-50 microM) dose- and time-dependently inhibited DNA synthesis and decreased cell number in cultured HUVEC, but not human fibroblasts. DPTH was not cytotoxic at these concentrations. [3H]Thymidine incorporation and flow cytometry analyses demonstrated that treatment of HUVEC with DPTH arrested the cell at the G0/G1 phase of the cell cycle. Western blot analysis revealed that the protein level of p21 increased after DPTH treated. In contrast, the protein levels of p27, p53, cyclins A, D1, D3 and E, cyclin-dependent kinase (CDK)2, and CDK4 in HUVEC were not changed significantly after DPTH treatment. Immunoprecipitation showed that the formations of the CDK2-p21 and CDK4-p21 complex, but not the CDK2-p27 and CDK4-p27 complex, were increased in the DPTH-treated HUVEC. Kinase assay further demonstrated that both CDK2 and CDK4 kinase activities were decreased in the DPTH-treated HUVEC. Pretreatment of HUVEC with a p21 antisense oligonucleotide reversed the DPTH-induced inhibition of [3H]thymidine incorporation into HUVEC. In conclusion, these data suggest that DPTH inhibits HUVEC proliferation by increasing the level of p21 protein, which in turn inhibits CDK2 and CDK4 kinase activities, and finally interrupts the cell cycle. The findings from the present study suggest that DPTH might have the potential to inhibit the occurrence of angiogenesis.