Lysosomal drug sequestration as a mechanism of drug resistance in vascular sarcoma cells marked by high CSF-1R expression

Human angiosarcoma and canine hemangiosarcoma are thought to arise from vascular tissue or vascular forming cells based upon their histological appearance. However, recent evidence indicates a hematopoietic or angioblastic cell of origin for these tumors. In support of this idea, we previously identified an endothelial-myeloid progenitor cell population with high expression of endothelial cell markers and the myeloid cell marker, colony stimulating factor 1 receptor (CSF-1R). Here, we further characterized these cells to better understand how their cellular characteristics may impact current therapeutic applications. We performed cell enrichment studies from canine hemangiosarcoma and human angiosarcoma cell lines to generate cell populations with high or low CSF-1R expression. We then utilized flow cytometry, side population and cell viability assays, and fluorescence based approaches to elucidate drug resistance mechanisms and to determine the expression of hematopoietic and endothelial progenitor cell markers. We demonstrated that cells with high CSF-1R expression enriched from hemangiosarcoma and angiosarcoma cell lines are more drug resistant than cells with little or no CSF-1R expression. We determined that the increased drug resistance may be due to increased ABC transporter expression in hemangiosarcoma and increased drug sequestration within cellular lysosomes in both hemangiosarcoma and angiosarcoma. We identified drug sequestration within cellular lysosomes as a shared drug resistance mechanism in human and canine vascular sarcomas marked by high CSF-1R expression. Taken together, our results demonstrate that studies in highly prevalent canine hemangiosarcoma may be especially relevant to understanding and addressing drug resistance mechanisms in both the canine and human forms of this disease

Propranolol Sensitizes Vascular Sarcoma Cells to Doxorubicin by Altering Lysosomal Drug Sequestration and Drug Efflux

Angiosarcoma is a rare cancer of blood vessel–forming cells with a high patient mortality and few treatment options. Although chemotherapy often produces initial clinical responses, outcomes remain poor, largely due to the development of drug resistance. We previously identified a subset of doxorubicin-resistant cells in human angiosarcoma and canine hemangiosarcoma cell lines that exhibit high lysosomal accumulation of doxorubicin. Hydrophobic, weak base chemotherapeutics, like doxorubicin, are known to sequester within lysosomes, promoting resistance by limiting drug accessibility to cellular targets. Drug synergy between the beta adrenergic receptor (β-AR) antagonist, propranolol, and multiple chemotherapeutics has been documented in vitro, and clinical data have corroborated the increased therapeutic potential of propranolol with chemotherapy in angiosarcoma patients. Because propranolol is also a weak base and accumulates in lysosomes, we sought to determine whether propranolol enhanced doxorubicin cytotoxicity via antagonism of β-ARs or by preventing the lysosomal accumulation of doxorubicin. β-AR-like immunoreactivities were confirmed in primary tumor tissues and cell lines; receptor function was verified by monitoring downstream signaling pathways of β-ARs in response to receptor agonists and antagonists. Mechanistically, propranolol increased cytoplasmic doxorubicin concentrations in sarcoma cells by decreasing the lysosomal accumulation and cellular efflux of this chemotherapeutic agent. Equivalent concentrations of the receptor-active S-(−) and -inactive R-(+) enantiomers of propranolol produced similar effects, supporting a β-AR-independent mechanism. Long-term exposure of hemangiosarcoma cells to propranolol expanded both lysosomal size and number, yet cells remained sensitive to doxorubicin in the presence of propranolol. In contrast, removal of propranolol increased cellular resistance to doxorubicin, underscoring lysosomal doxorubicin sequestration as a key mechanism of resistance. Our results support the repurposing of the R-(+) enantiomer of propranolol with weak base chemotherapeutics to increase cytotoxicity and reduce the development of drug-resistant cell populations without the cardiovascular and other side effects associated with antagonism of β-ARs

Identification of a Novel Two-Component System in Streptococcus gordonii V288 Involved in Biofilm Formation

Streptococcus gordonii is a pioneer colonizer of the teeth, contributing to the initiation of the oral biofilm called dental plaque. To identify genes that may be important in biofilm formation, a plasmid integration library of S. gordonii V288 was used. After screening for in vitro biofilm formation on polystyrene, a putative biofilm-defective mutant was isolated. In this mutant, pAK36 was inserted into a locus encoding a novel two-component system (bfr [biofilm formation related]) with two cotranscribed genes that form an operon. bfrA encodes a putative response regulator, while bfrB encodes a receptor histidine kinase. The bfr mutant and wild-type strain V288 showed similar growth rates in Todd-Hewitt broth (THB). A bfr-cat fusion strain was constructed. During growth in THB, the reporter activity (chloramphenicol acetyltransferase) was first detected in mid-log phase and reached a maximum in stationary phase, suggesting that transcription of bfr was growth stage dependent. After being harvested from THB, the bfr mutant adhered less effectively than did wild-type strain V288 to saliva-coated hydroxyapatite (sHA). To simulate pioneer colonization of teeth, S. gordonii V288 was incubated with sHA for 4 h in THB with 10% saliva to develop biofilms. RNA was isolated, and expression of bfrAB was estimated. In comparison to that of cells grown in suspension (free-growing cells), bfr mRNA expression by sessile cells on sHA was 1.8-fold greater and that by surrounding planktonic cells was 3.5-fold greater. Therefore, bfrAB is a novel two-component system regulated in association with S. gordonii biofilm formation in vitro

Streptococcus gordonii Hsa Environmentally Constrains Competitive Binding by Streptococcus sanguinis to Saliva-Coated Hydroxyapatite

Competition between pioneer colonizing bacteria may determine polymicrobial succession during dental plaque development, but the ecological constraints are poorly understood. For example, more Streptococcus sanguinis than Streptococcus gordonii organisms are consistently isolated from the same intraoral sites, yet S. gordonii fails to be excluded and survives as a species over time. To explain this observation, we hypothesized that S. gordonii could compete with S. sanguinis to adhere to saliva-coated hydroxyapatite (sHA), an in vitro model of the tooth surface. Both species bound similarly to sHA, yet 10- to 50-fold excess S. gordonii DL1 reduced binding of S. sanguinis SK36 by 85 to >95%. S. sanguinis, by contrast, did not significantly compete with S. gordonii to adhere. S. gordonii competed with S. sanguinis more effectively than other species of oral streptococci and depended upon the salivary film on HA. Next, putative S. gordonii adhesins were analyzed for contributions to interspecies competitive binding. Like wild-type S. gordonii, isogenic mutants with mutations in antigen I/II polypeptides (sspAB), amylase-binding proteins (abpAB), and Csh adhesins (cshAB) competed effectively against S. sanguinis. By contrast, an hsa-deficient mutant of S. gordonii showed significantly reduced binding and competitive capabilities, while these properties were restored in an hsa-complemented strain. Thus, Hsa confers a selective advantage to S. gordonii over S. sanguinis in competitive binding to sHA. Hsa expression may, therefore, serve as an environmental constraint against S. sanguinis, enabling S. gordonii to persist within the oral cavity, despite the greater natural prevalence of S. sanguinis in plaque and saliva

Involvement of Streptococcus gordonii Beta-Glucoside Metabolism Systems in Adhesion, Biofilm Formation, and In Vivo Gene Expression

Streptococcus gordonii genes involved in beta-glucoside metabolism are induced in vivo on infected heart valves during experimental endocarditis and in vitro during biofilm formation on saliva-coated hydroxyapatite (sHA). To determine the roles of beta-glucoside metabolism systems in biofilm formation, the loci of these induced genes were analyzed. To confirm the function of genes in each locus, strains were constructed with gene inactivation, deletion, and/or reporter gene fusions. Four novel systems responsible for beta-glucoside metabolism were identified, including three phosphoenolpyruvate-dependent phosphotransferase systems (PTS) and a binding protein-dependent sugar uptake system for metabolizing multiple sugars, including beta-glucosides. Utilization of arbutin and esculin, aryl-beta-glucosides, was defective in some mutants. Esculin and oligochitosaccharides induced genes in one of the three beta-glucoside metabolism PTS and in four other genetic loci. Mutation of genes in any of the four systems affected in vitro adhesion to sHA, biofilm formation on plastic surfaces, and/or growth rate in liquid medium. Therefore, genes associated with beta-glucoside metabolism may regulate S. gordonii in vitro adhesion, biofilm formation, growth, and in vivo colonization

Inactivation of Streptococcus gordonii SspAB Alters Expression of Multiple Adhesin Genes

SspA and SspB (antigen I/II family proteins) can bind Streptococcus gordonii to other oral bacteria and also to salivary agglutinin glycoprotein, a constituent of the salivary film or pellicle that coats the tooth. To learn if SspA and SspB are essential for adhesion and initial biofilm formation on teeth, S. gordonii DL1 was incubated with saliva-coated hydroxyapatite (sHA) for 2 h in Todd-Hewitt broth with 20% saliva to develop initial biofilms. Sessile cells attached to sHA, surrounding planktonic cells, and free-growing cells were recovered separately. Free-growing cells expressed more sspA-specific mRNA and sspB-specific mRNA than sessile cells. Free-growing cells expressed the same levels of sspA and sspB as planktonic cells. Surprisingly, an SspA(−) SspB(−) mutant strain showed 2.2-fold greater biofilm formation on sHA than wild-type S. gordonii DL1. To explain this observation, we tested the hypothesis that inactivation of sspA and sspB genes altered the expression of other adhesin genes during initial biofilm formation in vitro. When compared to wild-type cells, expression of scaA and abpB was significantly up-regulated in the SspA(−) SspB(−) strain in sessile, planktonic, and free-growing cells. Consistent with this finding, ScaA antigen was also overexpressed in planktonic and free-growing SspA(−) SspB(−) cells compared to the wild type. SspA/B adhesins, therefore, were strongly suggested to be involved in the regulation of multiple adhesin genes

S100A8/A9 (calprotectin) negatively regulates G2/M cell cycle progression and growth of squamous cell carcinoma.

Malignant transformation results in abnormal cell cycle regulation and uncontrolled growth in head and neck squamous cell carcinoma (HNSCC) and other cancers. S100A8/A9 (calprotectin) is a calcium-binding heterodimeric protein complex implicated in cell cycle regulation, but the specific mechanism and role in cell cycle control and carcinoma growth are not well understood. In HNSCC, S100A8/A9 is downregulated at both mRNA and protein levels. We now report that downregulation of S100A8/A9 correlates strongly with a loss of cell cycle control and increased growth of carcinoma cells. To show its role in carcinogenesis in an in vitro model, S100A8/A9 was stably expressed in an S100A8/A9-negative human carcinoma cell line (KB cells, HeLa-like). S100A8/A9 expression increases PP2A phosphatase activity and p-Chk1 (Ser345) phosphorylation, which appears to signal inhibitory phosphorylation of mitotic p-Cdc25C (Ser216) and p-Cdc2 (Thr14/Tyr15) to inactivate the G2/M Cdc2/cyclin B1 complex. Cyclin B1 expression then downregulates and the cell cycle arrests at the G2/M checkpoint, reducing cell division. As expected, S100A8/A9-expressing cells show both decreased anchorage-dependent and -independent growth and mitotic progression. Using shRNA, silencing of S100A8/A9 expression in the TR146 human HNSCC cell line increases growth and survival and reduces Cdc2 inhibitory phosphorylation at Thr14/Tyr15. The level of S100A8/A9 endogenous expression correlates strongly with the reduced p-Cdc2 (Thr14/Tyr14) level in HNSCC cell lines, SCC-58, OSCC-3 and UMSCC-17B. S100A8/A9-mediated control of the G2/M cell cycle checkpoint is, therefore, a likely suppressive mechanism in human squamous cell carcinomas and may suggest new therapeutic approaches

RNA-sequencing studies identify genes differentially regulated during inflammation-driven lung tumorigenesis and targeted by chemopreventive agents

YK:n piirissä on 2013 vuodesta alkaen keskusteltu autonomisista aseista tai tavanomaisemmin tappajaroboteista. Vuodesta 2014 alkaen keskustelu on käyty YK:n eräitä tavanomaisia aseita koskevan yleissopimuksen epävirallisissa istunnoissa, mutta vuodesta 2017 eteenpäin sen virallisissa GGE-muotoisissa kokouksissa. Tutkielmassa analysoidaan Yhdysvaltojen käyttämää retoriikkaa puheenvuoroissaan näissä kokouksissa vuosina 2017-2019. Aineisto on rajattu niihin kokouksiin, joissa aiheena on ollut autonomiset asejärjestelmät (LAWS). Puheista on käytetty Yhdysvaltojen Geneven edustuston sivuilla julkaistuja litteroituja versioita. Puheenvuoroja on analysoitu retorisen analyysin konventioon kuuluvan Chaïm Perelmanin uuden retoriikan avulla. Tämän avulla on puheista pyritty löytämään humanitaariseen oikeuteen ja ihmisoikeuksiin perustuvaa asejärjestelmiä puolustelevaa tai oikeuttavaa argumentaatiota. Aseita on oikeutettu historiallisesti valtioiden puolustuskykyyn ja iskukapasiteettiin vedoten. Hyveellisen sodankäynnin teoria puoltaa aseita oikeuttavan retoriikan analysointia ihmisoikeuksiin perustuvan oikeutuksen näkökulmasta. Autonomisista aseista käyty tutkimuskeskustelu ja YK:ssa kokousosapuolien näkökulmat ovat ristiriitaisia ja edes perustavanlaatuisten termien määrittelystä käydään väittelyä. Tässä kontekstissa Yhdysvaltojen argumentaatio pyrkii luomaan yleisesti hyväksyttävän, oman doktriininsa mukaisen keskustelun. Tutkimus osoittaa argumentoinnin luovan kuvaa autonomisista asejärjestelmistä hyveellisinä sekä humanitaarista oikeutta toteuttavina ja jopa sitä vahvistavana tekijänä. Yhtäältä tämä osoittaa mahdollista muutosta tavassa puhua asekehityksestä. Toisaalta aseiden ’hyveellisyys’ ja ’oikeudellisuus’ ovat humanitaaristen interventioiden ja sodankäynnin oikeutuksen kannalta tässä valossa kriittisen tarkastelun kohteina, sillä puheet eivät välttämättä vastaa tekoja

S100A8/A9 expression induced cell cycle and mitotic arrest at G2/M.

<p>(A) Cell cycle analysis of KB, KB-EGFP and KB-S100A8/A9 cells post-synchrony. Cells cultured under standard conditions were serum-starved overnight, synchronized at G1/S with aphidicolin treatment and stimulated to re-enter cell cycle. Synchronized cells were stained with propidium iodide DNA staining solution and analyzed by flow cytometry for changes in DNA content following release from G1/S blockage. (B) Mitotic analysis of synchronized cells stained with phospho-Histone H3 (Ser10) and analyzed by flow cytometry. (C) Percentage of cells in G₂/M. KB, KB-EGFP and KB-S100A8/A9 cells were analyzed over time post-synchrony and reported as mean ±SEM; n = 2 independent experiments (each analysis performed in duplicate); *p<0.05. (D) Percentage of mitotic cells post-synchrony, representing the mean of two independent repeat experiments. KB-S100A8/A9 cells showed fewer mitotic cells as shown by lower phospho-Histone H3 (Ser10) staining.</p

Expression of S100A8 and S100A9 in human head and neck tissues and carcinoma cell lines.

<p>The mRNA expression levels of S100A8 and S100A9 in (A) normal adjacent (NAT) and HNSCC tissues and (B) TR146 HNSCC and KB cells were measured by qRT-PCR and normalized to GAPDH; dotted line shown as a threshold for detection. Total RNA extracted from matching NAT and HNSCC tissues from each of three patients was pooled separately for gene expression analysis. Cell lines cultured under standard conditions were harvested and analyzed at approximately 70% confluency. Data presented as mean ±SD (n = 2). Representative immunoblots of (C) S100A8 and S100A9 in KB-S100A8/A9 transfected cells and (D) TR146-shRNA-S100A8/A9 knockdown cells compared to wild type and negative transfection controls. β-actin was used as loading control for immunoblotting analysis separated in 10% SDS-PAGE gels.</p