ARHI (DIRAS3)-mediated autophagy-associated cell death enhances chemosensitivity to cisplatin in ovarian cancer cell lines and xenografts

Autophagy can sustain or kill tumor cells depending upon the context. The mechanism of autophagy-associated cell death has not been well elucidated and autophagy has enhanced or inhibited sensitivity of cancer cells to cytotoxic chemotherapy in different models. ARHI (DIRAS3), an imprinted tumor suppressor gene, is downregulated in 60% of ovarian cancers. In cell culture, re-expression of ARHI induces autophagy and ovarian cancer cell death within 72 h. In xenografts, re-expression of ARHI arrests cell growth and induces autophagy, but does not kill engrafted cancer cells. When ARHI levels are reduced after 6 weeks, dormancy is broken and xenografts grow promptly. In this study, ARHI-induced ovarian cancer cell death in culture has been found to depend upon autophagy and has been linked to G1 cell-cycle arrest, enhanced reactive oxygen species (ROS) activity, RIP1/RIP3 activation and necrosis. Re-expression of ARHI enhanced the cytotoxic effect of cisplatin in cell culture, increasing caspase-3 activation and PARP cleavage by inhibiting ERK and HER2 activity and downregulating XIAP and Bcl-2. In xenografts, treatment with cisplatin significantly slowed the outgrowth of dormant autophagic cells after reduction of ARHI, but the addition of chloroquine did not further inhibit xenograft outgrowth. Taken together, we have found that autophagy-associated cancer cell death and autophagy-enhanced sensitivity to cisplatin depend upon different mechanisms and that dormant, autophagic cancer cells are still vulnerable to cisplatin-based chemotherapy.Autophagy has a well-defined role in cellular physiology, removing senescent organelles and catabolizing long-lived proteins.^{1, 2} Under nutrient-poor conditions, the fatty acids and amino acids produced by hydrolysis of lipids and proteins in autophagolysosomes can provide energy to sustain starving cells. Prolonged autophagy is, however, associated with caspase-independent type II programmed cell death. Although the mechanism of autophagy-associated cell death has not been adequately characterized, programmed necrosis or necroptosis has been implicated in some studies.^{3, 4}Given the ability to sustain or kill cells, the role of autophagy in cancer is complex and dependent on the context of individual studies. During oncogenesis in genetically engineered mice, reduced hemizygous expression of genes required for autophagy (BECN1, Atg4, ATG5, Atg7) can accelerate spontaneous or chemically induced tumor formation,^{5, 6} suggesting that autophagy can serve as a tumor suppressor. Other observations with established cancers suggest that autophagy can sustain metabolically challenged neoplasms, particularly in settings with inadequate vascular access.^{7, 8} Autophagy has also been shown to protect cancer cells from the lethal effects of some cytotoxic drugs.^{9, 10}Our group has found that cancer cell proliferation,^{11, 12, 13} motility,¹⁴ autophagy and tumor dormancy^{15, 16} can be regulated by an imprinted tumor suppressor gene, ARHI (DIRAS3), that is downregulated in 60% of ovarian cancers by multiple mechanisms,^{17, 18} associated with shortened progression-free survival.¹⁹ Ovarian cancer cell sublines have been developed with tet-inducible expression of ARHI. In cell culture, re-expression of ARHI induces autophagy and clonogenic ovarian cancer cell death within 72 h.¹⁶ In xenografts, re-expression of ARHI arrests cell growth, inhibits angiogenesis and induces autophagy, but does not kill engrafted cancer cells. When ARHI levels are reduced after 6 weeks of induction, dormancy is broken, vascularization occurs and xenografts grow promptly. Treatment of dormant xenografts with chloroquine (CQ), a functional inhibitor of autophagy, delays tumor outgrowth, suggesting that autophagy facilitates survival of poorly vascularized, nutrient-deprived ovarian cancer cells. The relevance of this model to human disease is supported by the recent observation that small deposits of dormant ovarian cancer found on the peritoneal surface at ‘second look'' operations following initial surgery and chemotherapy exhibit autophagy and increased expression of ARHI in >80% of cases.²⁰Ovarian cancer develops in >22 000 women each year in the United States.²¹ Over the past four decades, the 5-year survival has increased from 37% to ∼50% with optimal cytoreductive surgery and combination chemotherapy using taxane- and platinum-based regimens,^{21, 22} but long-term survival and cure stand at ∼30% for all stages, due, in large part, to the persistence and recurrence of dormant, drug-resistant ovarian cancer cells. For the past two decades, standard chemotherapy for ovarian cancer has included a combination of a platinum compound and a taxane. Carboplatin and cisplatin are alkylating agents that bind covalently to DNA producing intra- and inter-strand crosslinks that, if not repaired, induce apoptosis and cell death.^{23, 24} Our previous studies suggest that ∼20% of primary ovarian cancers exhibit punctate immunohistochemical staining for LC3, a biomarker for autophagy that decorates autophagosome membranes, whereas >80% of cancers that have survived platinum-based chemotherapy exhibit punctate LC3.²⁰ Consequently, autophagy might provide one mechanism of resistance to platinum-based therapy.In this report, we have explored mechanism(s) by which ARHI induces autophagy-associated cell death and enhances cisplatin cytotoxicity. Cisplatin has been found to trigger apoptosis by inducing caspase-3 activation and PARP cleavage in ovarian cancer cells.^{25, 26} We hypothesized that autophagy-associated cell death and autophagy-enhanced sensitivity to cisplatin depend upon different mechanisms and that dormant, autophagic cancer cells might still be vulnerable to platinum-based chemotherapy.     

Global transcriptional response of pig brain and lung to natural infection by Pseudorabies virus

Background  

Pseudorabies virus (PRV) is an alphaherpesviruses whose native host is pig. PRV infection mainly causes signs of central nervous system disorder in young pigs, and respiratory system diseases in the adult.     

Defining the healthy "core microbiome" of oral microbial communities

Background  

Most studies examining the commensal human oral microbiome are focused on disease or are limited in methodology. In order to diagnose and treat diseases at an early and reversible stage an in-depth definition of health is indispensible. The aim of this study therefore was to define the healthy oral microbiome using recent advances in sequencing technology (454 pyrosequencing).     

Nectar replenishment and pollen receipt interact in their effects on seed production of Penstemon roseus

Resource supply and pollen delivery are often thought to equally limit seed production in animal-pollinated plants. At equilibrium, plants should show no response to experimental pollen supplementation because resources limit seed set above the current level of pollen attraction, while experimental reduction in pollen deposition below the equilibrium level would reduce seed set. The predicted equilibrium may be disrupted, however, if plants expend additional energy to replenish removed nectar. We investigated the combined effects of nectar removal and pollen delivery on female reproductive success of Penstemon roseus (Plantaginaceae), a hummingbird-pollinated plant that replenishes removed nectar. We first documented that the frequency of experimental nectar removal was correlated with total nectar secretion; and increased frequency of nectar removal resulted in increased female reproductive costs to the plant. Trade-offs between investing resources in nectar and investing resources in seeds were then investigated in two contrasting natural populations by removing nectar from flowers at increasing frequencies while simultaneously hand-pollinating flowers with increasing amounts of pollen. Seed set was lowest at low levels of pollen deposition, highest at medium-sized pollen loads, and intermediate when pollen loads were highest. At both sites, the frequency of nectar removal and pollen deposition had an interactive effect on seed production, in that intermediate levels of nectar removal result in the absolute highest seed set, but only at intermediate pollen loads. At high pollen loads, seed set was higher following little to no nectar removal, and at low pollen loads, all rates of nectar removal affected fecundity equally. Seed mass responded to nectar removal and pollination differently than did seed set. High levels of nectar removal and pollen delivery both lowered seed mass, with little interaction between main effects. Our findings are among the first to demonstrate that nectar replenishment costs and pollination intensity jointly affect seed production. This conflict between nectar replenishment costs and pollen-limiting factors results in trade-offs between pollinator attraction and seed provisioning. Thus, resource allocation towards nectar production should more often be considered in future studies of pollen limitation.     

Phylogeography of Podocarpus matudae (Podocarpaceae): pre‐Quaternary relicts in northern Mesoamerican cloud forests

Aim Cloud forests of northern Mesoamerica represent the northern and southern limit of the contact zone between species otherwise characteristic of North or South America. Several phylogeographic studies featuring temperate conifer species have improved our understanding of species responses to environmental changes. In contrast, conifer species that presumably colonized northern Mesoamerica from South America are far less studied. A phylogeographic study of Podocarpus matudae (Podocarpaceae) was conducted to identify any major evolutionary divergences or disjunctions across its range and to determine if its current distribution is associated with pre‐Quaternary climatic and/or long‐distance dispersal events. Location Northern Mesoamerica (Mexico and Guatemala). Methods Sixteen populations (157 individuals) of P. matudae were screened for variation at two plastid DNA markers. The intra‐specific phylogenetic relationships among haplotypes were reconstructed using Bayesian inference. Population genetic analyses were undertaken to gain insight into the evolutionary history of these populations. To test whether genetic divergence among populations occurred at different time‐scales plastid DNA sequence data and fossil‐ and coalescent‐based calibrations were integrated. Results The combination of plastid markers yielded 11 haplotypes. Differentiation among populations based on DNA variation (G_ST) (0.707, SE 0.0807) indicated a clear population structure in P. matudae. Differentiation for ordered alleles (N_ST) (0.811, SE 0.0732) was higher than that for G_ST, indicating phylogeographical structure in P. matudae. Most of the total variation (81.3%, P < 0.0001) was explained by differences among populations. The estimated divergence time between the unique haplotypes from a Guatemalan population and the two most common haplotypes from the Sierra Madre Oriental in Mexico was between 10 and 20 Ma, and further haplotype divergence in the poorly resolved clade of the Sierra Madre Oriental occurred between 3 and 0.5 Ma. Main conclusions Divergence estimations support the hypothesis that extant Podocarpus matudae populations are pre‐Quaternary relicts. This finding is consistent with fossil and pollen data that support a Miocene age for temperate floristic elements in Mesoamerican cloud forests, whereas further haplotype divergence within the Sierra Madre Oriental, Chiapas and Guatemala occurred more recently, coinciding with Pleistocene cloud forest refugia.     

Thioether benzenesulfonamide inhibitors of carbonic anhydrases II and IV: Structure-based drug design,synthesis, and biological evaluation

A novel series of potent thioether benzenesulfonamide inhibitors of carbonic anhydrases II and IV was discovered using structure-based drug design. Synthesis, structure–activity relationship, and optimization of physicochemical properties are described. Low nanomolar potency was achieved, and selected compounds with improved thermodynamic solubility showed promising in vitro inhibition of carbonic anhydrase activity in rabbit iris ciliary body homogenate.     

A novel surface protein of Trichomonas vaginalis is regulated independently by low iron and contact with vaginal epithelial cells

Background  

Trichomonosis caused by Trichomonas vaginalis is the number one, non-viral sexually transmitted disease (STD) that affects more than 250 million people worldwide. Immunoglobulin A (IgA) has been implicated in resistance to mucosal infections by pathogens. No reports are available of IgA-reactive proteins and the role, if any, of this class of antibody in the control of this STD. The availability of an IgA monoclonal antibody (mAb) immunoreactive to trichomonads by whole cell (WC)-ELISA prompted us to characterize the IgA-reactive protein of T. vaginalis.     

Phylogenetic analysis of the tenascin gene family: evidence of origin early in the chordate lineage

Background  

Tenascins are a family of glycoproteins found primarily in the extracellular matrix of embryos where they help to regulate cell proliferation, adhesion and migration. In order to learn more about their origins and relationships to each other, as well as to clarify the nomenclature used to describe them, the tenascin genes of the urochordate Ciona intestinalis, the pufferfish Tetraodon nigroviridis and Takifugu rubripes and the frog Xenopus tropicalis were identified and their gene organization and predicted protein products compared with the previously characterized tenascins of amniotes.