Visualization of the structures of the hepatitis C virus replication complex

Hepatitis C viral RNA synthesis has been demonstrated to occur on a lipid raft membrane structure. Lipid raft membrane fraction purified by membrane flotation analysis was observed using transmission electron microscopy and atomic force microscopy. Particles around 0.7 um in size were found in lipid raft membrane fraction purified from hepatitis C virus (HCV) replicon but not their parental HuH7 cells. HCV NS5A protein was associated with these specialized particles. After several cycles of freezing-thawing, these particles would fuse into larger sizes up to 10 um. Knockdown of seven proteins associated with lipid raft (VAPA, COPG, RAB18, COMT, CDC42, DPP4, and KDELR2) of HCV replicon cells reduced the observed number of these particles and suppressed the HCV replication. Results in this study indicated that HCV replication complexes with associated lipid raft membrane form distinct particle structures of around 0.7 um as observed from transmission electron microscopy and atomic force microscopy.     

Potentiating the Efficacy of Molecular Targeted Therapy for Hepatocellular Carcinoma by Inhibiting the Insulin-Like Growth Factor Pathway

Insulin-like growth factor (IGF) signaling pathway is an important regulatory mechanism of tumorigenesis and drug resistance in many cancers. The present study explored the potential synergistic effects between IGF receptor (IGFR) inhibition and other molecular targeted agents (MTA) in HCC cells. HCC cell lines (Hep3B, PLC5, and SK-Hep1) and HUVECs were tested. The MTA tested included sorafenib, sunitinib, and the IGFR kinase inhibitor NVP-AEW541. The potential synergistic antitumor effects were tested by median dose effect analysis and apoptosis assay in vitro and by xenograft models in vivo. The activity and functional significance of pertinent signaling pathways and expression of apoptosis-related proteins were measured by RNA interference and Western blotting. We found that IGF can activate IGFR and downstream AKT signaling activities in all the HCC cells tested, but the growth-stimulating effect of IGF was most prominent in Hep3B cells. NVP-AEW541 can abrogate IGF-induced activation of IGFR and AKT signaling in HCC cells. IGF can increase the resistance of HCC cells to sunitinib. The apoptosis-inducing effects of sunitinib, but not sorafenib, were enhanced when IGFR signaling activity was inhibited by NVP-AEW541 or IGFR knockdown. Chk2 kinase activation was found contributory to the synergistic anti-tumor effects between sunitinib and IGFR inhibition. Our data indicate that the apoptosis-potentiating effects of IGFR inhibition for HCC may be drug-specific. Combination therapy of IGFR inhibitors with other MTA may improve the therapeutic efficacy in HCC.     

The HDAC inhibitor,MPT0E028, enhances erlotinib-induced cell death in EGFR-TKI-resistant NSCLC cells

Epidermal growth factor receptor (EGFR), which promotes cell survival and division, is found at abnormally high levels on the surface of many cancer cell types, including many cases of non-small cell lung cancer. Erlotinib (Tarceva), an oral small-molecule tyrosine kinase inhibitor, is a so-called targeted drug that inhibits the tyrosine kinase domain of EGFR, and thus targets cancer cells with some specificity while doing less damage to normal cells. However, erlotinib resistance can occur, reducing the efficacy of this treatment. To develop more effective therapeutic interventions by overcoming this resistance problem, we combined the histone deacetylase inhibitor, MPT0E028, with erlotinib in an effort to increase their antitumor effects in erlotinib-resistant lung adenocarcinoma cells. This combined treatment yielded significant growth inhibition, induced the expression of apoptotic proteins (PARP, γH2AX, and caspase-3), increased the levels of acetylated histone H3, and showed synergistic effects in vitro and in vivo. These effects were independent of the mutation status of the genes encoding EGFR or K-Ras. MPT0E028 synergistically blocked key regulators of the EGFR/HER2 signaling pathways, attenuating multiple compensatory pathways (e.g., AKT, extracellular signal-regulated kinase, and c-MET). Our results indicate that this combination therapy might be a promising strategy for facilitating the effects of erlotinib monotherapy by activating various networks. Taken together, our data provide compelling evidence that MPT0E028 has the potential to improve the treatment of heterogeneous and drug-resistant tumors that cannot be controlled with single-target agents.     

Anti-liver fibrotic lignans from the fruits of Schisandra arisanensis and Schisandra sphenanthera

Three new polyoxygenated C₁₈-dibenzocyclooctadiene lignans, arisanschinins M and N (1 and 2) and schisphenin A (3), together with eight related metabolites (4–11), were isolated from the fruits of Schisandra arisanensis and Schisandra sphenanthera, respectively. The structures of 1–3 were elucidated on the basis of extensive spectroscopic and 2D NMR (HSQC, HMBC, and NOESY) analyses. The configuration of the biphenyl moiety in the octadiene ring was determined by circular dichroism (CD). Compound 1 possessed an unprecedented 3-(1-hydroxypropan-2-yl)-3-methyl-1,4-dioxo-2-one lactonide ring system attaching at C-6/C-14. Pharmacological studies revealed that compounds 3, 4, 6, 7, and 10 exhibited significant anti-hepatic fibrosis activity, while 9 and 11 showed cytotoxicity against HSC-T6 cells. The biogenetic pathway for compound 1 was also proposed.     

Direct measurement of DNA bending by type IIA topoisomerases: implications for non-equilibrium topology simplification

Type IIA topoisomerases modify DNA topology by passing one segment of duplex DNA (transfer or T–segment) through a transient double-strand break in a second segment of DNA (gate or G–segment) in an ATP-dependent reaction. Type IIA topoisomerases decatenate, unknot and relax supercoiled DNA to levels below equilibrium, resulting in global topology simplification. The mechanism underlying this non-equilibrium topology simplification remains speculative. The bend angle model postulates that non-equilibrium topology simplification scales with the bend angle imposed on the G–segment DNA by the binding of a type IIA topoisomerase. To test this bend angle model, we used atomic force microscopy and single-molecule Förster resonance energy transfer to measure the extent of bending imposed on DNA by three type IIA topoisomerases that span the range of topology simplification activity. We found that Escherichia coli topoisomerase IV, yeast topoisomerase II and human topoisomerase IIα each bend DNA to a similar degree. These data suggest that DNA bending is not the sole determinant of non-equilibrium topology simplification. Rather, they suggest a fundamental and conserved role for DNA bending in the enzymatic cycle of type IIA topoisomerases.     

Different NK cell developmental events require different levels of IL-15 trans-presentation

NK cell development requires IL-15, which is "trans-presented" to IL-15Rβγ on NK cells by IL-15Rα on other cells. In this study, we report that different levels of IL-15 trans-presentation are required for different NK cell developmental events to reach full maturation status. Because the IL-15Rα intracellular domain has the capacity to recruit signaling molecules, we generated knockin and transgenic (Tg) mice that lack the intracellular domain to assess the role of the IL-15 trans-presentation level independent of the function of this domain. The level of IL-15Rα on various cells of these mice follows the order WT > Tg6 > knockin > Tg1 ≥ knockout. Bone marrow (BM)-derived dendritic cells prepared from these mice induced Stat5 phosphorylation in NK cells. The level of phospho-Stat5 correlated with the level of IL-15Rα on BMDCs, thus offering the opportunity to study quantitative effects of IL-15 trans-presentation on NK cell development in vivo. We found that NK cell homeostasis, mature NK cell differentiation, and acquisition of Ly49 receptor and effector functions require different levels of IL-15 trans-presentation input to achieve full status. All NK cell developmental events examined were quantitatively regulated by the IL-15Rα level of BM-derived and radiation-resistant accessory cells, but not by IL-15Rα of NK cells. We also found that IL-15Rα of radiation-resistant cells was more potent than IL-15Rα of BM-derived accessory cells in support of stage 2 to stage 3 splenic mNK differentiation. In summary, each examined developmental event required a particular level of IL-15 trans-presentation by accessory cells.