Hypericin-photodynamic therapy leads to interleukin-6 secretion by HepG2 cells and their apoptosis via recruitment of BH3 interacting-domain death agonist and caspases

Photodynamic therapy (PDT) has emerged as a capable therapeutic modality for the treatment of cancer. PDT is a targeted cancer therapy that reportedly leads to tumor cell apoptosis and/or necrosis by facilitating the secretion of certain pro-inflammatory cytokines and expression of multiple apoptotic mediators in the tumor microenvironment. In addition, PDT also triggers oxidative stress that directs tumor cell killing and activation of inflammatory responses. However, the cellular and molecular mechanisms underlying the role of PDT in facilitating tumor cell apoptosis remain ambiguous. Here, we investigated the ability of PDT in association with hypericin (HY) to induce tumor cell apoptosis by facilitating the induction of reactive oxygen species (ROS) and secretion of Th1/Th2/Th17 cytokines in human hepatocellular liver carcinoma cell line (HepG2) cells. To discover if any apoptotic mediators were implicated in the enhancement of cell death of HY-PDT-treated tumor cells, selected gene profiling in response to HY-PDT treatment was implemented. Experimental results showed that interleukin (IL)-6 was significantly increased in all HY-PDT-treated cells, especially in 1 μg/ml HY-PDT, resulting in cell death. In addition, quantitative real-time PCR analysis revealed that the expression of apoptotic genes, such as BH3-interacting-domain death agonist (BID), cytochrome complex (CYT-C) and caspases (CASP3, 6, 7, 8 and 9) was remarkably higher in HY-PDT-treated HepG2 cells than the untreated HepG2 cells, entailing that tumor destruction of immune-mediated cell death occurs only in PDT-treated tumor cells. Hence, we showed that HY-PDT treatment induces apoptosis in HepG2 cells by facilitating cytotoxic ROS, and potentially recruits IL-6 and apoptosis mediators, providing additional hints for the existence of alternative mechanisms of anti-tumor immunity in hepatocellular carcinoma, which contribute to long-term suppression of tumor growth following PDT.     

Solution Structures of the Huntington's Disease DNA Triplets, (CAG)n

Summary

Highly polymorphic DNA triplet repeats, (CAG)_n, are located inside the first exon of the Huntington's disease gene. Inordinate expansion of this repeat is correlated with the onset and progression of the disease. NMR spectroscopy, gel electrophoresis, digestion by single-strand specific PI enzyme, and in vitro replication assay have been used to investigate the structural basis of (CAG)_n expansion. Nondenaturing gel electrophoresis and ID ¹H NMR studies of (CAG)₅ and (CAG)₆ reveal the presence of hairpins and mismatched duplexes as the major and minor populations respectively. However, at high DNA concentrations (i.e., 1.0–2.0 mM that is typically required for 2D NMR experiments) both (CAG)₅ and (CAG)₆ exist predominantly in mismatched duplex forms. Mismatched duplex structures of (CAG)₅and (CAG)₆ are useful, because they adequately model the stem of the biologically relevant hairpins formed by (CAG).,. We, therefore, performed detailed NMR spectroscopic studies on the duplexes of (CAG)₅ and (CAG)₆. We also studied a model duplex, (CGCAGCG)₂ that contains the underlined building block of the duplex. This duplex shows the following structural characteristics: (i) all the nucleotides are in (C2′-endo, anti) conformations, (ii) mismatched A?A base pairs are flanked by two Watson-Crick G?C base pairs and (iii) A?A base pairs are stably stacked (and intra-helical) and are formed by a single N6-H—N1 hydrogen bond. The nature of A?A pairing is confirmed by temperature-dependent HMQC and HMQC-NOESY experiments on the [(CA*G)₅]₂ duplex where the adenines are ¹⁵N-labeled at N6. Temperature-and pH-dependent imino proton spectra, nondenaturing electrophoresis, and PI digestion data demonstrate that under a wide range of solution conditions longer (CAG)_n repeats (n>10) exist exclusively in hairpin conformation with two single-stranded loops. Finally, an in vitro replication assay with (CAG)₈₂₁ inserts in the Ml3 single-stranded DNA templates shows a replication bypass for the (CAG)₂₁ insert but not for the (CAG)₈ insert in the template. This demonstrates that for a sufficiently long insert (n=21 in this case), a hairpin is formed by the (CAG)., even in presence of its complementary strand. This observation implies that the formation of hairpin by the (CAG)_n may cause slippage during replication and thus may explain the observed length polymorphism.     

The stereochemistry of N-methyl and aryl substituents determine the biological activities of 3-aryl-8-methyl-8-azabicyclo[3.2.1]oct-2,3-enes

Aryl substituted tropanes and their 2,3-ene analogs are highly selective inhibitors of monoamine uptake. The solution structures of a series of aryl tropanes were determined using NMR spectroscopy and molecular modeling to identify conformational preferences that may determine the overall activity. The majority of these analogs undergo nitrogen inversion, and the rate of interconversion between the axial and equatorial N-methyl conformers is fast on the NMR timescale at room temperature but slow between 217 and 243 K allowing us to determine the thermodynamic parameters of interconversion using dynamic and magnetization transfer NMR. The biological activities correlate strongly with the nature and the orientation of the aryl group. The relative orientation of the N-methyl further modulates the activity by directly influencing the ligand interaction in the protein binding pocket and/or by forcing a favorable orientation for the aryl substituent to fit in the binding pocket.     

Multiple sulfatase deficiency is caused by mutations in the gene encoding the human C(alpha)-formylglycine generating enzyme

C(alpha)-formylglycine (FGly) is the catalytic residue in the active site of eukaryotic sulfatases. It is posttranslationally generated from a cysteine in the endoplasmic reticulum. The genetic defect of FGly formation causes multiple sulfatase deficiency (MSD), a lysosomal storage disorder. We purified the FGly generating enzyme (FGE) and identified its gene and nine mutations in seven MSD patients. In patient fibroblasts, the activity of sulfatases is partially restored by transduction of FGE encoding cDNA, but not by cDNA carrying an MSD mutation. The gene encoding FGE is highly conserved among pro- and eukaryotes and has a paralog of unknown function in vertebrates. FGE is localized in the endoplasmic reticulum and is predicted to have a tripartite domain structure.     

Desmoplastic Infantile Ganglioglioma: cytologic findings and differential diagnosis on aspiration material

BACKGROUND: Desmoplastic infantile ganglioglioma (DIG) is a rare WHO Grade I tumor of infancy that is characterized by large volume, superficial location, invariable supratentoriality, fronto-parietal lobe predilection and morphologically, by an admixture of astroglial and neuroepithelial elements in a desmoplastic milieu. With over 50 cases described, the histologic and radiographic spectrum of DIG has been well-characterized. The superficial location of DIGs may render them greatly amenable to preoperative assessment utilizing aspiration cytology; however, the cytologic features of this rare tumor have only been reported once previously. CASE PRESENTATION: We present herein cytomorphologic findings from the intraoperative aspiration of a typical case of DIG diagnosed in a 1-year-old male. As evaluated on a single liquid-based preparation, the specimen showed low cellularity and was comprised predominantly of a population of dispersed (occasionally clustered) large neuronal cells with eccentrically located hyperchromatic nuclei (which were occasionally binucleated) and abundant unipolar cytoplasm. Rare smaller astroglial cells were intermixed. Despite the tumor's characteristic desmoplastic histologic appearance, no stromal fragments were identified on the aspiration material. CONCLUSIONS: A differential diagnosis is presented and analyzed in detail and it is concluded that when these large neuronal cells are encountered in an aspirate of a brain mass in a child, a combination of clinical, radiologic and immunohistochemical parameters can eliminate most of the differential possibilities.     

Inhibition of FEN-1 processing by DNA secondary structure at trinucleotide repeats

The mechanism by which trinucleotide expansion occurs in human genes is not understood. However, it has been hypothesized that DNA secondary structure may actively participate by preventing FEN-1 cleavage of displaced Okazaki fragments. We show here that secondary structure can, indeed, play a role in expansion by a FEN-1-dependent mechanism. Secondary structure inhibits flap processing at CAG, CGG, or CTG repeats in a length-dependent manner by concealing the 5' end of the flap that is necessary for both binding and cleavage by FEN-1. Thus, secondary structure can defeat the protective function of FEN-1, leading to site-specific expansions. However, when FEN-1 is absent from the cell, alternative pathways to simple inhibition of flap processing contribute to expansion.     

Identification of cyclin D3 as a new interaction partner of lamin A/C

Lamin A/C is a major component of the nuclear lamina. An intact nuclear lamina has been proposed to be necessary for muscle differentiation. Cyclin D3 is known to be upregulated in differentiated muscle cells and to form insoluble complexes with cell-cycle regulatory factors in these cells. We have examined the possibility of direct binding interactions between lamin A/C and cyclin D3 by in vitro binding assays and co-immunoprecipitation studies with muscle cells. Our results indicate that cyclin D3 binds specifically to amino acid residues 383-474 of lamin A/C and associates with lamin A/C in muscle cells. The identification of cyclin D3 as a novel binding partner of lamin A/C has important implications for a role for lamin A/C in muscle differentiation.