Biosynthesis and evaluation of a novel highly water-soluble quercetin glycoside derivative.

Quercetin (1) was converted into quercetin 7-O-succinyl glucoside (2) by used Bacillus amyloliquefaciens FJ18 as a solvent-resistant whole-cell biocatalyst. The structure of the new compound was confirmed by LC-MS analysis and NMR spectroscopy. The water-solubility of this novel quercetin 7-O-succinyl glucoside (2) was approximately 1000 times higher than that of native quercetin (2). Quercetin (1) and quercetin 7-O-succinyl glucoside (2) exhibited significant DPPH scavenging capacity with IC50 values of 23.55 and 36.05 µM, respectively. Both compounds showed moderate cytotoxic effects against the two human cancer cell lines (MCF-7 and HepG2) with IC50 values ranging from 39.45-63.38 µM.

Synthesis of novel functional ionic liquids and their application in biomass.

A series of dicationic ionic liquids (ILs) including [PF6][(PYR)C4(MIM)][Cl], [PF6][(PYR)C4(PYR)][Cl], [PF6][(PYR)C5(MIM)][Cl], and [PF6][(PYR)C5(PYR)][Cl], and monocationic ILs including [(PYR)C4Cl][PF6], [(PYR)C5Cl][PF6], [(MIM)C2COOH][PF6] and [(PYR)C2COOH][PF6] were synthesized. Their thermal stability and melting points were determined. Their solubility with organic solvents and the miscibility with water were investigated. These functional ILs are hydrophilic at high temperatures and they are hydrophobic at low temperatures, which enable the effective isolation of the resulting reducing sugar. High yields of reducing sugar were obtained for corn stalk after 8 h (20.73%) and potato starch after 6 h (72.50%) by the treatment with the mixture of [PF6][(PYR)C4(PYR)][Cl] and [(PYR)C2COOH][PF6]. The reuse of dicationic and monocationic ILs was successfully performed and no significant reduction in yields of reducing sugar was observed. These functional ILs have important implications in the design of homogeneous and heterogeneous systems with water and organic solvents, which could be used to satisfy some specific applications.

Screening of potential IL-tolerant cellulases and their efficient saccharification of IL-pretreated lignocelluloses.

Lignocellulosic biomass as one of the most abundant and renewable resources has great potential for biofuel production. The complete conversion of biomass to biofuel is achieved through the effective pretreatment process and the following enzyme saccharification. Ionic liquids (ILs) are considered as a green solvent for lignocellulose pretreatment. However, ILs exhibit an inhibitory effect on cellulase activity, leading to a subsequent decrease in the efficiency of saccharification. The screening of new potential IL-tolerant cellulases is important. In the current study, a fungal strain with a relatively high cellulase production was isolated and identified as Penicillium oxalicum HC6. The culture conditions were optimized using corn stover and peptone as the carbon source and nitrogen source at pH 4.0 and 30 °C with an inoculation size of 2% (v/v) for 8 days. It was found that P. oxalicum HC6 exhibited potential salt tolerance with the increase of the enzyme production at a salt concentration of 5.0% (w/v). In addition, high enzyme activities were obtained at pH 4.0-6.0 and 50-65 °C. The crude enzyme from P. oxalicum HC6 with good thermal stability was also stable in the presence of salt and ILs. Good yields of reducing sugar were obtained by the crude enzyme from P. oxalicum HC6 after the saccharification of corn stover that was pretreated by ILs. P. oxalicum HC6 with potentially salt-tolerant and IL-tolerant enzymes has great potential application in the enzymatic saccharification of lignocellulose.

Endogenous angiogenesis inhibitor blocks tumor growth via direct and indirect effects on tumor microenvironment.

Tissue inhibitor of matrix metalloproteinase-2 (TIMP-2) belongs to a small family of endogenous proteins that inhibits a group of enzymes, the matrix metalloproteinases (MMPs). TIMP-2 inhibits endothelial cell proliferation and migration in vitro and angiogenesis in vivo, through MMP-dependent and -independent mechanisms. However, little is known regarding the contribution of these mechanisms to the antitumor effects of TIMP-2. Using a retroviral delivery system, we stably overexpressed TIMP-2 and its mutant Ala+TIMP-2 (devoid of MMP inhibitory activity) in human adenocarcinoma A549 cells. Using real time PCR, and enzyme-linked immunosorbent assay (ELISA), we confirmed enhanced TIMP-2 expression and its MMP inhibitory activity by reverse zymography. In vitro, growth assays suggested that TIMP-2 and Ala+TIMP-2 did not alter basal cell proliferation rates, however, tumor cell migration and invasion were inhibited. In vivo, both TIMP-2 and Ala+TIMP-2 A549 xenografts exhibited reduced growth rate, CD31 immunostaining indicated decreased intratumoral microvascular density, and TUNEL demonstrated enhanced tumor cell apoptosis. Immunoblotting and immunohistochemical analyses of A549 xenograft tissues with either phospho-FAK (Tyr397) or phospho-AKT (Ser473) showed decreased activation in both TIMP-2 and Ala+TIMP-2 tumor cells. We conclude that TIMP-2-mediated inhibition of tumor growth occurs, at least in part, independently of MMP inhibition, and is a consequence of both direct effects of TIMP-2 on tumor cells and modulation of the tumor microenvironment.

The role of Id-1 in chemosensitivity and epirubicin-induced apoptosis in bladder cancer cells.

Recurrence and progression are the major problems in the treatment of bladder cancer. Increased expression of Id-1, a basic helix-loop-helix transcription factor, has recently been shown in several types of advanced cancer. Some studies have provided evidence to suggest that Id-1 can be considered a potential therapeutic target. The objective of this study was to investigate the role of Id-1 in the chemosensitivity of bladder cancer cells, and the effect of Id-1 on chemotherapeutic drug-induced apoptosis in bladder cancer cells. We compared the different sensitivity to epirubicin in RT112 and MGH-U1 cell lines with different Id-1 expression. Then, we transfected different vectors into RT112 and MGH-U1 respectively, and generated the stable Id-1 up-regulation and down-regulation transfectants. The results of cell viability assay showed up-regulation of Id-1 in RT112 leading to increased sensitivity in response to epirubicin, and down-regulation of Id-1 increased cellular sensitivity to epirubicin. Furthermore, the analysis of apoptosis related protein revealed that up-regulation of Id-1 suppressed epirubicin-induced apoptosis and down-regulation of Id-1 leading to increased epirubicin-induced apoptosis. Wound closure assay showed up-regulation of Id-1 leading to improved migration abilities of bladder cancer cells under chemotherapy. Our results suggest that up-regulation of Id-1 in bladder cancer cells lead to increased cell viability in response to epirubicin by its improved anti-apoptotic role, and down-regulation of Id-1 increases cellular sensitivity to epirubicin by increased anticancer drug-induced apoptosis.

CDC25A functions as a novel Ar corepressor in prostate cancer cells.

Androgen receptor (AR) is a ligand-dependent transcription factor and its activity is regulated by numerous AR coregulators. Aberrant expression of AR coregulators in prostate cancer cells has an important role in the development and progression of prostate cancer. We report here that CDC25A, a cell cycle-promoting phosphatase over-expressed in a number of cancers, functions as an AR coregulator suppressing the AR transcriptional activity. In this study, we found that CDC25A is upregulated in human prostate cancer and its expression level is positively associated with the Gleason score and disease metastasis. More importantly, we showed that CDC25A can physically interact with AR through its putative catalytic domain. In addition, ectopic expression of CDC25A in prostate cancer cell lines suppresses PSA and Probasin promoter activities significantly, indicating that CDC25A may function as an AR corepressor. This was further confirmed by knockdown of endogenous CDC25A expression using small interfering RNA (siRNA), which resulted in upregulation of PSA promoter activity. Moreover, a truncated mutant that does not interact with AR fails to suppress the PSA promoter activity, indicating that CDC25A downregulates androgen-responsive promoter by physically interacting with AR. Taken together, our results demonstrated a novel function of CDC25A in the regulation of androgen signaling in human prostate cancer cells.

Coumarins from campylotropis hirtella (FRANCH.) SCHINDL. and their inhibitory activity on prostate specific antigen secreted from LNCaP cells.

Campylotropis hirtella (FRANCH.) SCHINDL. was used as a folk medicine for the treatment of benign prostate hyperplasia (BPH) in China. In this study, two new coumarins, 7,2',4'-trihydroxy-5-methoxy-3-arylcoumarin (1), 6-[(1S,2S)-2-angeloyloxy-1,3-dihydroxy-3-methylbutyl]-7-methoxycoumarin, named angelol M (2), together with eleven known related compounds (3-13) were isolated from this plant under the bioassay guided fractionation. All the compounds showed activity with different levels in inhibiting prostate specific antigen (PSA) secreted from androgen dependent prostate cancer cell line, LNCaP cells, using ELISA method.

MAD2 interacts with DNA repair proteins and negatively regulates DNA damage repair.

MAD2 (mitotic arrest deficient 2) is a key regulator of mitosis. Recently, it had been suggested that MAD2-induced mitotic arrest mediates DNA damage response and that upregulation of MAD2 confers sensitivity to DNA-damaging anticancer drug-induced apoptosis. In this study, we report a potential novel role of MAD2 in mediating DNA nucleotide excision repair through physical interactions with two DNA repair proteins, XPD (xeroderma pigmentosum complementation group D) and ERCC1. First, overexpression of MAD2 resulted in decreased nuclear accumulation of XPD, a crucial step in the initiation of DNA repair. Second, immunoprecipitation experiments showed that MAD2 was able to bind to XPD, which led to competitive suppression of binding activity between XPD and XPA, resulting in the prevention of physical interactions between DNA repair proteins. Third, unlike its role in mitosis, the N-terminus domain seemed to be more important in the binding activity between MAD2 and XPD. Fourth, phosphorylation of H2AX, a process that is important for recruitment of DNA repair factors to DNA double-strand breaks, was suppressed in MAD2-overexpressing cells in response to DNA damage. These results suggest a negative role of MAD2 in DNA damage response, which may be accounted for its previously reported role in promoting sensitivity to DNA-damaging agents in cancer cells. However, the interaction between MAD2 and ERCC1 did not show any effect on the binding activity between ERCC1 and XPA in the presence or absence of DNA damage. Our results suggest a novel function of MAD2 by interfering with DNA repair proteins.

Lignans isolated from Campylotropis hirtella (Franch.) Schindl. decreased prostate specific antigen and androgen receptor expression in LNCaP cells.

Accumulating epidemiological data suggest that Asian men have lower incidences of prostate cancer and benign prostate hyperplasia (BPH) compared with American and European populations and may have benefited from their higher intake of phytoestrogens in their diet. However, how these phytochemicals affect prostatic diseases is still unclear. In this study, we isolated six lignans from a plant, Campylotropis hirtella (Franch.) Schindl., which has been used as a folk medicine for treatment of BPH in China, through bioassay guided fractionation. They were dehydrodiconiferyl alcohol (C1), 4-[(-6-hydroxy-2,3-dihydro-1-benzofuran-3-yl)methyl]-5-methoxybenzene-1,3-diol (C2), erythro-guaiacylglycerol-beta-O-4'-coniferyl ether (C3), threo-guaiacylglycerol-beta-O-4'-coniferyl ether (C4), secoisolariciresinol (C5), and prupaside (C6), where C2 was identified as a new lignan analog. Their IC50 values for inhibition of prostate specific antigen (PSA) secretion were 19, 45, 110, 128, 137, and 186 microM, respectively, from C1 to C6 in LNCaP cells. Further study showed that C1-5 down-regulated cellular PSA expression and C1-4 also decreased androgen receptor (AR) expression in LNCaP cells. Furthermore, we investigated the proapoptotic effect of C1 on LNCaP cells. The active forms of caspase 3 associated with the specific proteolysis of poly (ADP-ribose) polymerase (PARP) were detected, and the antiapoptotic protein Bcl-2 was down-regulated after the treatment with C1. These results collectively indicated that these lignans may have chemopreventive or therapeutic actions for prostate cancer through suppressing AR signaling pathway and inducing apoptosis.

Sesquilignans and dilignans from Campylotropis hirtella (Franch.) Schindl.

A new sesquilignan, erythro-Guaiacylglycerol-beta-O-4'-(5')-methoxylariciresinol, together with six known compounds including three sesquilignans and three dilignans, have been isolated from Campylotropis hirtella (Franch.) Schindl. that was used as traditional Chinese medicine for treatment of benign prostate hyperplasia. All these known compounds were found for the first time from the family leguminosae. The structure of the new compound and its relative configurations were elucidated on the basis of detailed spectroscopic analysis (NMR and MS). These compounds showed no obvious inhibition effect on prostate specific antigen secretion in LNCaP cells by ELISA method.