Single-step doxorubicin-selected cancer cells overexpress the ABCG2 drug transporter through epigenetic changes

Understanding the mechanisms of multidrug resistance (MDR) could improve clinical drug efficacy. Multidrug resistance is associated with ATP binding cassette (ABC) transporters, but the factors that regulate their expression at clinically relevant drug concentrations are poorly understood. We report that a single-step selection with low doses of anti-cancer agents, similar to concentrations reported in vivo, induces MDR that is mediated exclusively by ABCG2. We selected breast, ovarian and colon cancer cells (MCF-7, IGROV-1 and S-1) after exposure to 14 or 21 nM doxorubicin for only 10 days. We found that these cells overexpress ABCG2 at the mRNA and protein levels. RNA interference analysis confirmed that ABCG2 confers drug resistance. Furthermore, ABCG2 upregulation was facilitated by histone hyperacetylation due to weaker histone deacetylase 1-promoter association, indicating that these epigenetic changes elicit changes in ABCG2 gene expression. These studies indicate that the MDR phenotype arises following low-dose, single-step exposure to doxorubicin, and further suggest that ABCG2 may mediate early stages of MDR development. This is the first report to our knowledge of single-step, low-dose selection leading to overexpression of ABCG2 by epigenetic changes in multiple cancer cell lines

Chronic Citalopram Administration Causes a Sustained Suppression of Serotonin Synthesis in the Mouse Forebrain

BACKGROUND:Serotonin (5-HT) is a neurotransmitter with important roles in the regulation of neurobehavioral processes, particularly those regulating affect in humans. Drugs that potentiate serotonergic neurotransmission by selectively inhibiting the reuptake of serotonin (SSRIs) are widely used for the treatment of psychiatric disorders. Although the regulation of serotonin synthesis may be an factor in SSRI efficacy, the effect of chronic SSRI administration on 5-HT synthesis is not well understood. Here, we describe effects of chronic administration of the SSRI citalopram (CIT) on 5-HT synthesis and content in the mouse forebrain. METHODOLOGY/PRINCIPAL FINDINGS:Citalopram was administered continuously to adult male C57BL/6J mice via osmotic minipump for 2 days, 14 days or 28 days. Plasma citalopram levels were found to be within the clinical range. 5-HT synthesis was assessed using the decarboxylase inhibition method. Citalopram administration caused a suppression of 5-HT synthesis at all time points. CIT treatment also caused a reduction in forebrain 5-HIAA content. Following chronic CIT treatment, forebrain 5-HT stores were more sensitive to the depleting effects of acute decarboxylase inhibition. CONCLUSIONS/SIGNIFICANCE:Taken together, these results demonstrate that chronic citalopram administration causes a sustained suppression of serotonin synthesis in the mouse forebrain. Furthermore, our results indicate that chronic 5-HT reuptake inhibition renders 5-HT brain stores more sensitive to alterations in serotonin synthesis. These results suggest that the regulation of 5-HT synthesis warrants consideration in efforts to develop novel antidepressant strategies

Generational distribution of a Candida glabrata population: Resilient old cells prevail, while younger cells dominate in the vulnerable host.

Similar to other yeasts, the human pathogen Candida glabrata ages when it undergoes asymmetric, finite cell divisions, which determines its replicative lifespan. We sought to investigate if and how aging changes resilience of C. glabrata populations in the host environment. Our data demonstrate that old C. glabrata are more resistant to hydrogen peroxide and neutrophil killing, whereas young cells adhere better to epithelial cell layers. Consequently, virulence of old compared to younger C. glabrata cells is enhanced in the Galleria mellonella infection model. Electron microscopy images of old C. glabrata cells indicate a marked increase in cell wall thickness. Comparison of transcriptomes of old and young C. glabrata cells reveals differential regulation of ergosterol and Hog pathway associated genes as well as adhesion proteins, and suggests that aging is accompanied by remodeling of the fungal cell wall. Biochemical analysis supports this conclusion as older cells exhibit a qualitatively different lipid composition, leading to the observed increased emergence of fluconazole resistance when grown in the presence of fluconazole selection pressure. Older C. glabrata cells accumulate during murine and human infection, which is statistically unlikely without very strong selection. Therefore, we tested the hypothesis that neutrophils constitute the predominant selection pressure in vivo. When we altered experimentally the selection pressure by antibody-mediated removal of neutrophils, we observed a significantly younger pathogen population in mice. Mathematical modeling confirmed that differential selection of older cells is sufficient to cause the observed demographic shift in the fungal population. Hence our data support the concept that pathogenesis is affected by the generational age distribution of the infecting C. glabrata population in a host. We conclude that replicative aging constitutes an emerging trait, which is selected by the host and may even play an unanticipated role in the transition from a commensal to a pathogen state.post-print10768 K

Candida glabrata : a review of its features and resistance

Candida species belong to the normal microbiota of the oral cavity and gastrointestinal and vaginal tracts, and are responsible for several clinical manifestations, from mucocutaneous overgrowth to bloodstream infections. Once believed to be non-pathogenic, Candida glabrata was rapidly blamable for many human diseases. Year after year, these pathological circumstances are more recurrent and problematic to treat, especially when patients reveal any level of immunosuppression. These difficulties arise from the capacity of C. glabrata to form biofilms and also from its high resistance to traditional antifungal therapies. Thus, this review intends to present an excerpt of the biology, epidemiology, and pathology of C. glabrata, and detail an approach to its resistance mechanisms based on studies carried out up to the present.The authors are grateful to strategic project PTDC/SAU-MIC/119069/2010 for the financial support to the research center and for Celia F. Rodrigues' grant

Enhanced Drug Delivery into Cell Cytosol via Glycoprotein H-Derived Peptide Conjugated Nanoemulsions

The key role of nanocarriers in improving the pharmacological properties of commonly used drugs is recognized worldwide. It is also known that in the development of new effective nanocarriers the use of targeting moieties integrated on their surface is essential. Herein, we propose a nanocarrier based on an oil in water nanoemulsion coated with a membranotropic peptide derived from the glycoprotein H of Herpes simplex virus I, known as gH625, in order to reduce endolysosomal accumulation and to enhance cytosolic localization. In addition, we show an enhanced anti-inflammatory activity of curcumin, a bioactive compound isolated from the Curcuma longa plant, when loaded into our engineered nanocarriers. This effect is a consequence of a higher uptake combined with a high curcumin preservation exerted by the active nanocapsules compared to control ones. When loaded into our nanocapsules, indeed, curcumin molecules are directly internalized into the cytosol rather than into lysosomes. Further, in order to extend the in vitro experimental setting with a more complex model and to explore the possibility to use our nanocarriers for further biological applications, we tested their performance in a 3D sprouting angiogenesis model. Finally, we show promising preliminary in vivo results by assessing the anti-inflammatory properties of the proposed nanocarrier