Therapeutic potential of α,β‐thujone through metabolic reprogramming and caspase‐dependent apoptosis in ovarian cancer cells

The therapeutic potential of α,β‐thujone, a functional compound found in many medicinal plants of the Cupressaceae, Asteraceae, and Lamiaceae families, has been demonstrated, including in inflammation and cancers. However, its pharmacological functions and mechanisms of action in ovarian cancer remain unclear. We investigated the anticancer properties of α,β‐thujone in ES2 and OV90 human ovarian cancer cells and its effect on sensitization to cisplatin. α,β‐thujone inhibited cancer cell proliferation and induced cell death through caspase‐dependent intrinsic apoptotic pathways. Moreover, α,β‐thujone‐mediated endoplasmic reticulum stress was associated with the loss of mitochondrial functions and altered metabolic landscape of ovarian cancer cells. α,β‐Thujone attenuated blood vessel formation in transgenic zebrafish, implying it has significant antiangiogenic potential. In addition, α,β‐thujone sensitized ovarian cancer cells to cisplatin, causing synergistic pharmacological effects. Collectively, our results suggest that α,β‐thujone has therapeutic potential in human ovarian cancer and functions via regulating multiple intracellular stress‐associated metabolic reprogramming and caspase‐dependent apoptotic pathways.     

The structure of the human RNase H2 complex defines key interaction interfaces relevant to enzyme function and human disease

Ribonuclease H2 (RNase H2) is the major nuclear enzyme involved in the degradation of RNA/DNA hybrids and removal of ribonucleotides misincorporated in genomic DNA. Mutations in each of the three RNase H2 subunits have been implicated in a human auto-inflammatory disorder, Aicardi-Goutières Syndrome (AGS). To understand how mutations impact on RNase H2 function we determined the crystal structure of the human heterotrimer. In doing so, we correct several key regions of the previously reported murine RNase H2 atomic model and provide biochemical validation for our structural model. Our results provide new insights into how the subunits are arranged to form an enzymatically active complex. In particular, we establish that the RNASEH2A C terminus is a eukaryotic adaptation for binding the two accessory subunits, with residues within it required for enzymatic activity. This C-terminal extension interacts with the RNASEH2C C terminus and both are necessary to form a stable, enzymatically active heterotrimer. Disease mutations cluster at this interface between all three subunits, destabilizing the complex and/or impairing enzyme activity. Altogether, we locate 25 out of 29 residues mutated in AGS patients, establishing a firm basis for future investigations into disease pathogenesis and function of the RNase H2 enzyme.     

Testing the role of genetic factors across multiple independent invasions of the shrub Scotch broom (Cytisus scoparius)

Knowledge of the introduction history of invasive plants informs on theories of invasiveness and assists in the invasives management. For the highly successful invasive shrub Scotch broom, Cytisus scoparius, we analysed a combination of nuclear and chloroplast microsatellites for eight native source regions and eight independent invasion events in four countries across three continents. We found that two exotic Australian populations came from different sources, one of which was derived from multiple native populations, as was an invasive sample from California. An invasive population from New Zealand appeared to be predominantly sourced from a single population, either from the native or exotic ranges. Four invasive populations from Chile were genetically differentiated from the native range samples analysed here and so their source of introduction could not be confirmed, but high levels of differentiation between the Chilean populations suggested a combination of different sources. This extensive global data set of replicated introductions also enabled tests of key theories of invasiveness in relation to genetic diversity. We conclude that invasive populations have similar levels of high genetic diversity to native ranges; levels of admixture may vary across invasive populations so admixture does not appear to have been an essential requirement for invasion; invasive and native populations exhibit similar level of genetic structure indicating similar gene flow dynamics for both types of populations. High levels of diversity and multiple source populations for invasive populations observed here discount founder effects or drift as likely explanations for previously observed seed size differences between ranges. The high levels of genetic diversity, differential and source admixture identified for most exotic populations are likely to limit the ability to source biocontrol agents from the native region of origin of invasive populations.     

Regulation of c-Myc Expression by Ahnak Promotes Induced Pluripotent Stem Cell Generation

We have previously reported that Ahnak-mediated TGFβ signaling leads to down-regulation of c-Myc expression. Here, we show that inhibition of Ahnak can promote generation of induced pluripotent stem cells (iPSC) via up-regulation of endogenous c-Myc. Consistent with the c-Myc inhibitory role of Ahnak, mouse embryonic fibroblasts from Ahnak-deficient mouse (Ahnak^−/− MEF) show an increased level of c-Myc expression compared with wild type MEF. Generation of iPSC with just three of the four Yamanaka factors, Oct4, Sox2, and Klf4 (hereafter 3F), was significantly enhanced in Ahnak^−/− MEF. Similar results were obtained when Ahnak-specific shRNA was applied to wild type MEF. Of note, expressionof Ahnak was significantly induced during the formation of embryoid bodies from embryonic stem cells, suggesting that Ahnak-mediated c-Myc inhibition is involved in embryoid body formation and the initial differentiation of pluripotent stem cells. The iPSC from 3F-infected Ahnak^−/− MEF cells (Ahnak^−/−-iPSC-3F) showed expression of all stem cell markers examined and the capability to form three primary germ layers. Moreover, injection of Ahnak^−/−-iPSC-3F into athymic nude mice led to development of teratoma containing tissues from all three primary germ layers, indicating that iPSC from Ahnak^−/− MEF are bona fide pluripotent stem cells. Taken together, these data provide evidence for a new role for Ahnak in cell fate determination during development and suggest that manipulation of Ahnak and the associated signaling pathway may provide a means to regulate iPSC generation.     

Oxidative stress increases levels of endogenous amyloid-beta peptides secreted from primary chick brain neurons

Oxidative damage is associated with Alzheimer's disease and mild cognitive impairment, but its relationship to the development of neuropathological lesions involving accumulation of amyloid-beta (Abeta) peptides and hyperphosphorylated tau protein remains poorly understood. We show that inducing oxidative stress in primary chick brain neurons by exposure to sublethal doses of H(2)O(2 )increases levels of total secreted endogenous Abeta by 2.4-fold after 20 h. This occurs in the absence of changes to intracellular amyloid precursor protein or tau protein levels, while heat-shock protein 90 is elevated 2.5-fold. These results are consistent with the hypothesis that aging-associated oxidative stress contributes to increasing Abeta generation and up-regulation of molecular chaperones in Alzheimer's disease.     

Cyclic AMP blocks cell growth through Raf-1-dependent and Raf-1-independent mechanisms

It is widely accepted that cyclic AMP (cAMP) can block cell growth by phosphorylating Raf-1 on serine 43 and inhibiting signaling to extracellular signal-regulated protein kinase. We show that the suppression of Raf-1 by cAMP is considerably more complex than previously reported. When cellular cAMP is elevated, Raf-1 is phosphorylated on three residues (S43, S233, and S259), which work independently to block Raf-1. Both Ras-dependent and Ras-independent processes are disrupted. However, when cAMP-insensitive versions of Raf-1 are expressed in NIH 3T3 cells, their growth is still strongly suppressed when cAMP is elevated. Thus, although Raf-1 appears to be an important cAMP target, other pathways are also targeted by cAMP, providing alternative mechanisms that lead to suppression of cell growth.