Bing Hung Chen

Puerariae radix isoflavones and their metabolites inhibit growth and induce apoptosis in breast cancer cells

Puerariae radix (PR) is a popular natural herb and a traditional food in Asia, which has antithrombotic and anti-allergic properties and stimulates estrogenic activity. In the present study, we investigated the effects of the PR isoflavones puerarin, daidzein, and genistein on the growth of breast cancer cells. Our data revealed that after treatment with PR isoflavones, a dose-dependent inhibition of cell growth occurred in HS578T, MDA-MB-231, and MCF-7 cell lines. Results from cell cycle distribution and apoptosis assays revealed that PR isoflavones induced cell apoptosis through a caspase-3-dependent pathway and mediated cell cycle arrest in the G2/M phase. Furthermore, we observed that the serum metabolites of PR (daidzein sulfates/glucuronides) inhibited proliferation of the breast cancer cells at a 50% cell growth inhibition (GI(50)) concentration of 2.35 microM. These results indicate that the daidzein constituent of PR can be metabolized to daidzein sulfates or daidzein glucuronides that exhibit anticancer activities. The protein expression levels of the active forms of caspase-9 and Bax in breast cancer cells were significantly increased by treatment with PR metabolites. These metabolites also increased the protein expression levels of p53 and p21. We therefore suggest that PR may act as a chemopreventive and/or chemotherapeutic agent against breast cancer by reducing cell viability and inducing apoptosis.

Anonaine Induces DNA Damage and Inhibits Growth and Migration of Human Lung Carcinoma H1299 Cells

The anticancer effects of (-)-anonaine were investigated in this current study. (-)-Anonaine at concentration ranges of 50-200 μM exhibited significant inhibition to cell growth and migration activities on human lung cancer H1299 cells at 24 h, albeit cell cycle analyses showed that (-)-anonaine at the above concentration ranges did not cause any significant changes in cell-cycle distributions. Significant nuclear damages of H1299 cells were observed with 10-200 μM (-)-anonaine treatment in a comet assay, whereas higher concentrations (6 and 30 mM) of (-)-anonaine concentrations were required to cause DNA damages in an in vitro plasmid cleavage assay. In summary, our results demonstrated that (-)-anonaine exhibited dose-dependent antiproliferatory, antimigratory, and DNA-damaging effects on H1299 cells. We inferred that (-)-anonaine can cause cell-cycle arrest and DNA damage to hamper the physiological behavior of cancer cells at 72 h, and therefore, it can be useful as one of the potential herbal supplements for chemoprevention of human lung cancer.

Antiallergic potential on RBL-2H3 cells of some phenolic constituents of Zingiber officinale (ginger)

In the present study on five pure phenolic compounds (1-5) isolated from the rhizomes of Zingiber officinale (ginger) and investigated for their antiallergic potency, rat basophilic leukemia (RBL-2H3) cells were incubated with these compounds and the release of beta-hexosaminidase was measured kinetically. The data obtained suggest that ginger rhizomes harbor potent compounds capable of inhibiting allergic reactions and may be useful for the treatment and prevention of allergic diseases.

Association of the lumican gene functional 3'-UTR polymorphism with high myopia.

PURPOSE The lumican gene (LUM) encodes a major extracellular component of the fibrous mammalian sclera. Alteration in the expression levels of extracellular matrix components may influence scleral shape, which in turn could affect visual acuity. Single-nucleotide polymorphisms (SNPs) in the LUM gene were determined in an investigation of whether LUM gene polymorphisms correlate with high myopia. METHODS Sequences spanning all three exons, intron-exon boundaries, and promoter regions were determined in 50 normal individuals. Five SNPs were identified, one of which was found to be a newly identified polymorphism. Genomic DNA was prepared from peripheral blood obtained from 201 patients with high myopia and 86 control subjects. Genotypes of the SNPs -1554 T/C (rs3759223), -628 A/-(rs17018757), -59 CC/-(rs3832846), c.601 T/C (rs17853500), and the novel SNP c.1567 C>T were determined by polymerase chain reaction. RESULTS Of the five SNPs, one showed a significant difference between patients and control subjects (c.1567 C>T, P = 0.0016). Haplotype analysis revealed a significantly higher presence of polymorphisms in patients with myopia (P < 0.0001). Moreover, the c.1567 T polymorphism was determined to have lower reporter gene activity than that of c.1567 C. CONCLUSIONS These observations suggest that LUM gene polymorphisms contribute to the development of high myopia.

Type I IFN induced IL1‐Ra expression in hepatocytes is mediated by activating STAT6 through the formation of STAT2: STAT6 heterodimer

The biological activities of type I interferons (IFNs) are mediated by their binding to a heterodimer receptor complex (IFNAR1 and IFNAR2), resulting in the activation of the JAK (JAK1 and TYK2)‐STAT (1, 2, 3, 5 isotypes) signalling pathway. Although several studies have indicated that IFN‐α and IFN‐β can activate complexes containing STAT6, the biological role of this activation is still unknown. We found that exposure of hepatoma cells (HuH7 and Hep3B) to IFN‐α or IFN‐β led to the activation of STAT6. Activated STAT6 in turn induced the formation of STAT2: STAT6 complexes, which led to the secretion of IL‐1Ra. The activation of STAT6 by type I IFN in hepatocytes was mediated by JAK1 and Tyk2. In addition, IFN‐α or IFN‐β significantly enhanced the stimulatory effect of IL‐1β on production of IL‐1Ra. The present study suggests a novel function of IFN‐α and IFN‐β signalling in human hepatocytes. Our results provide evidence for the mechanism how IFN‐α and IFN‐β modulate inflammatory responses through activation of STAT6 and production of secreted IL‐1Ra.

Versatile Phosphoramidation Reactions for Nucleic Acid Conjugations with Peptides, Proteins, Chromophores, and Biotin Derivatives

Chemical conjugations of nucleic acids with macromolecules or small molecules are common approaches to study nucleic acids in chemistry and biology and to exploit nucleic acids for medical applications. The conjugation of nucleic acids such as oligonucleotides with peptides is especially useful to circumvent cell delivery and specificity problems of oligonucleotides as therapeutic agents. However, current approaches are limited and inefficient in their ability to afford peptide-oligonucleotide conjugates (POCs). Here, we report an effective and reproducible approach to prepare POCs and other nucleic acid conjugates based on a newly developed nucleic acid phosphoramidation method. The development of a new nucleic acid phosphoramidation reaction was achieved by our successful synthesis of a novel amine-containing biotin derivative used to systematically optimize the reactions. The improved phosphoramidation reactions dramatically increased yields of nucleic acid-biotin conjugates up to 80% after 3 h reaction. Any nucleic acids with a terminal phosphate group are suitable reactants in phosphoramidation reactions to conjugate with amine-containing molecules such as biotin and fluorescein derivatives, proteins, and, most importantly, peptides to enable the synthesis of POCs for therapeutic applications. Polymerase chain reactions (PCRs) to study incorporation of biotin or fluorescein-tagged DNA primers into the reaction products demonstrated that appropriate controls of nucleic acid phosphoramidation reactions incur minimum adverse effects on inherited base-pairing characteristics of nucleotides in nucleic acids. The phosphoramidation approach preserves the integrity of hybridization specificity in nucleic acids when preparing POCs. By retaining integrity of the nucleic acids, their effectiveness as therapeutic reagents for gene silencing, gene therapy, and RNA interference is ensured. The potential for POC use was demonstrated by two-step phosphoramidation reactions to successfully synthesize nucleic acid-tetraglycine conjugates. In addition, phosphoramidation reactions provided a facile approach to prepare nucleic acid-BSA conjugates with good yields. In summary, the new approach to phosphoramidation reactions offers a universal method to prepare POCs and other nucleic acid conjugates with high yields in aqueous solutions. The methods can be easily adapted to typical chemistry or biology laboratory setups which will expedite the applications of POCs for basic research and medicine.

Biological Properties of Acidic Cosmetic Water from Seawater

This current work was to investigate the biological effects of acidic cosmetic water (ACW) on various biological assays. ACW was isolated from seawater and demonstrated several bio-functions at various concentration ranges. ACW showed a satisfactory effect against Staphylococcus aureus, which reduced 90% of bacterial growth after a 5-second exposure. We used cultured human peripheral blood mononuclear cells (PBMCs) to test the properties of ACW in inflammatory cytokine release, and it did not induce inflammatory cytokine release from un-stimulated, normal PBMCs. However, ACW was able to inhibit bacterial lipopolysaccharide (LPS)-induced inflammatory cytokine TNF-α released from PBMCs, showing an anti-inflammation potential. Furthermore, ACW did not stimulate the rat basophilic leukemia cell (RBL-2H3) related allergy response on de-granulation. Our data presented ACW with a strong anti-oxidative ability in a superoxide anion radical scavenging assay. In mass spectrometry information, magnesium and zinc ions demonstrated bio-functional detections for anti-inflammation as well as other metal ions such as potassium and calcium were observed. ACW also had minor tyrosinase and melanin decreasing activities in human epidermal melanocytes (HEMn-MP) without apparent cytotoxicity. In addition, the cell proliferation assay illustrated anti-growth and anti-migration effects of ACW on human skin melanoma cells (A375.S2) indicating that it exerted the anti-cancer potential against skin cancer. The results obtained from biological assays showed that ACW possessed multiple bioactivities, including anti-microorganism, anti-inflammation, allergy-free, antioxidant, anti-melanin and anticancer properties. To our knowledge, this was the first report presenting these bioactivities on ACW.

A Novel Interaction Between Interferon-Inducible Protein p56 and Ribosomal Protein L15 in Gastric Cancer Cells

Type I interferons (IFNs) are potent inducers of antiviral and antiproliferative activities in vertebrates. IFNs cause activation of genes encoding antiviral proteins, such as p56 from the IFN-stimulated gene family. There are six tetratricopeptide repeat (TPR) motifs located at the N-terminal sequence of p56. Since TPR motifs are known to participate in protein-protein interactions, p56 may associate with various large protein complexes to modify their functions. Using a T7 phage display library, we identified ribosomal protein L15 (RPL15) as a novel interacting partner of p56. The p56-RPL15 interaction was confirmed by pull-down assays. Overexpression of p56 exhibited strong inhibition on the growth of RPL15-overexpressing cancer cells. Small interfering RNA targeting RPL15 not only reduced the growth rate of gastric cancer cells but also sensitized these cells to type I IFN-induced proliferative inhibition. Using site-directed mutagenesis, we also mapped the TPRs 1-4 of p56 as crucial domains to interact with RPL15. Taken together, our results demonstrated a novel interaction between p56 and RPL15. Differential regulation of p56 and RPL15 expression contributes to the antiproliferative capacity on gastric cancer cells, and further elucidation of their interaction may facilitate the development of new anticancer regimens.

Additive effects of C2-ceramide on paclitaxel-induced premature senescence of human lung cancer cells

AIMS the aims of the study are to investigate the additive effect of exogenous short-carbon chain phospholipids, C(2)-ceramide, on an anti-cancer drug paclitaxel (PTX)-induced senescence of human non-small cell lung cancer (NSCLC) cells deficient in functional p53 and p16, and to examine whether mitogen-activated protein kinase (MAPK) plays a role in ceramide-sensitized senescence of NSCLC cells. MAIN METHODS to determine whether exogenous C(2)-ceramide renders lung cancer cells more sensitive to PTX treatment, techniques employing a flow cytometry-based cell cycle analysis and acidic β-galactosidase staining for senescent cells were used. Furthermore, to elucidate the role of MAPK proteins in modulating senescence, assays for protein levels of selective MAPKs and Bcl-2 family members, and detection of transcriptional levels senescence-associated genes were used in the study. KEY FINDINGS a sub-lethal dose of C(2)-ceramide sensitized the NSCLC H1299 cells to PTX treatment. The additive effects of C(2)-ceramide and PTX resulted in proliferative inhibition, G(2)-phase arrest of cell cycle, activation of p38 and eventually premature senescence. Importantly, neither p53, p21(waf1/cip1) nor p16(ink4) was shown to be involved in C(2)-ceramide-sensitized proliferative inhibition and senescence of H1299 cells by PTX in our study. SIGNIFICANCE our study demonstrates that the short-carbon chain C(2)-ceramide can effectively sensitize PTX-induced senescence of H1299 cells via both p21(waf1/cip1)- and p16(ink4)-independent pathways.

Feruloyl-l-arabinose attenuates migration, invasion and production of reactive oxygen species in H1299 lung cancer cells

Ferulic acid (FA), a phenolic compound, is an abundant dietary antioxidant and exerts the mitogenic effect on cells. Recently, we isolated an active FA derivative, namely feruloyl-L-arabinose (FAA), from coba husk. The aim of this study was to investigate the effects of FAA on the proliferation, migration and invasion of H1299 human lung cancer cells. Our results showed a strong antioxidant potential of FAA. Additionally, FAA inhibited the migration and invasion ability, while causing a significant accumulation of G2/M-population, of H1299 tumor cells in a dose-dependent manner, whereas no significant change on cell proliferation was observed. Results from the wound healing assay revealed that cell migration ability was markedly inhibited by FAA treatments. Similarly, results of gelatin zymography study showed that FAA treatments significantly decreased the activities of matrix metalloproteinase (MMP)-2 and MMP-9, suggesting that FAA-mediated inhibition on migration and invasion of lung cancer cells may be achieved by the down-regulation of the MMPs activities. Taken together, our present work provides a new insight into the novel inhibitory function of FAA on cell migration in H1299 cells, suggesting its promising role in the chemoprevention of lung cancer.