Drug and Alcohol Rehabilitation: Non-Invasive Imaging of Phosphoinositide-3-Kinase-Catalytic-Subunit-Alpha (PIK3CA) Promoter Modulation in Small Animal Models.

Non-Invasive Imaging of Phosphoinositide-3-Kinase-Catalytic-Subunit-Alpha (PIK3CA) Promoter Modulation in Small Animal Models.

Filed under: Drug and Alcohol Rehabilitation

PLoS One. 2013; 8(2): e55971
Gaikwad SM, Gunjal L, Junutula AR, Astanehe A, Gambhir SS, Ray P

Activation of the PI3K/Akt pathway, a critical step for survival in cancer cells is often associated with decreased sensitivity to several chemotherapeutic drugs. PIK3CA gene amplification is observed in 16-24% of epithelial ovarian cancer (EOC) patients in conjunction with p53 mutations. A 900 bp long PIK3CA promoter is shown to be negatively regulated by p53 in ovarian surface epithelial cells but the consequence of chemotherapeutic drug treatments on this promoter in ovarian cancer cells is largely unknown. We aim to study the modulation of this promoter by cisplatin using an improved fusion reporter in ovarian cancer cells and tumor xenografts by non-invasive imaging approach. A PIK3CA sensor was developed using a bi-fusion reporter from a newly constructed library of bi- and tri-fusion vectors comprising of two mutant far red fluorescent proteins (mcherry/mch and tdTomato/tdt), a mutant firefly luciferase (fluc2), and a PET reporter protein (ttk). In vivo imaging of mice implanted with 293T cells transiently expressing these bi- and tri-fusion reporters along with respective controls revealed comparable activity of each reporter in the fusion background and fluc2-tdt as the most sensitive one. Repression of the PIK3CA sensor by drugs was inversely proportional to cellular p53 level in a germline (PA1) and in an EOC (A2780) cell line but not in a p53 deficient EOC (SKOV3) cell line. Bioluminescence imaging of tumor xenografts stably expressing the PIK3CA sensor in PA1 and A2780 cells exhibited attenuating activity without any change in SKOV3 tumors expressing the PIK3CA sensor after cisplatin treatment. Sequential mutation at p53 binding sites showed gradual increase in promoter activity and decreased effects of the drugs. These newly developed PIK3CA-fluc2-tdt and the mutant reporter sensors thus would be extremely useful for screening new drugs and for functional assessment of PIK3CA expression from intact cells to living subjects.
HubMed – drug

 

Chronic endocannabinoid system stimulation induces muscle macrophage and lipid accumulation in type 2 diabetic mice independently of metabolic endotoxaemia.

Filed under: Drug and Alcohol Rehabilitation

PLoS One. 2013; 8(2): e55963
Geurts L, Muccioli GG, Delzenne NM, Cani PD

Obesity and type 2 diabetes are characterised by low-grade inflammation, metabolic endotoxaemia (i.e., increased plasma lipopolysaccharides [LPS] levels) and altered endocannabinoid (eCB)-system tone. The aim of this study was to decipher the specific role of eCB-system stimulation or metabolic endotoxaemia in the onset of glucose intolerance, metabolic inflammation and altered lipid metabolism.Mice were treated with either a cannabinoid (CB) receptor agonist (HU210) or low-dose LPS using subcutaneous mini-pumps for 6 weeks. After 3 weeks of the treatment under control (CT) diet, one-half of each group of mice were challenged with a high fat (HF) diet for the following 3-week period.Under basal conditions (control diet), chronic CB receptor agonist treatment (i.e., 6 weeks) induced glucose intolerance, stimulated metabolic endotoxaemia, and increased macrophage infiltration (CD11c and F4/80 expression) in the muscles; this phenomenon was associated with an altered lipid metabolism (increased PGC-1? expression and decreased CPT-1b expression) in this tissue. Chronic LPS treatment tended to increase the body weight and fat mass, with minor effects on the other metabolic parameters. Challenging mice with an HF diet following pre-treatment with the CB agonist exacerbated the HF diet-induced glucose intolerance, the muscle macrophage infiltration and the muscle’s lipid content without affecting the body weight or the fat mass.Chronic CB receptor stimulation under basal conditions induces glucose intolerance, stimulates metabolic inflammation and alters lipid metabolism in the muscles. These effects worsen following the concomitant ingestion of an HF diet. Here, we highlight the central roles played by the eCB system and LPS in the pathophysiology of several hallmarks of obesity and type 2 diabetes.
HubMed – drug

 

A Compensatory Mutation Provides Resistance to Disparate HIV Fusion Inhibitor Peptides and Enhances Membrane Fusion.

Filed under: Drug and Alcohol Rehabilitation

PLoS One. 2013; 8(2): e55478
Wood MP, Cole AL, Ruchala P, Waring AJ, Rohan LC, Marx P, Tarwater PM, Gupta P, Cole AM

Fusion inhibitors are a class of antiretroviral drugs used to prevent entry of HIV into host cells. Many of the fusion inhibitors being developed, including the drug enfuvirtide, are peptides designed to competitively inhibit the viral fusion protein gp41. With the emergence of drug resistance, there is an increased need for effective and unique alternatives within this class of antivirals. One such alternative is a class of cyclic, cationic, antimicrobial peptides known as ?-defensins, which are produced by many non-human primates and exhibit broad-spectrum antiviral and antibacterial activity. Currently, the ?-defensin analog RC-101 is being developed as a microbicide due to its specific antiviral activity, lack of toxicity to cells and tissues, and safety in animals. Understanding potential RC-101 resistance, and how resistance to other fusion inhibitors affects RC-101 susceptibility, is critical for future development. In previous studies, we identified a mutant, R5-tropic virus that had evolved partial resistance to RC-101 during in vitro selection. Here, we report that a secondary mutation in gp41 was found to restore replicative fitness, membrane fusion, and the rate of viral entry, which were compromised by an initial mutation providing partial RC-101 resistance. Interestingly, we show that RC-101 is effective against two enfuvirtide-resistant mutants, demonstrating the clinical importance of RC-101 as a unique fusion inhibitor. These findings both expand our understanding of HIV drug-resistance to diverse peptide fusion inhibitors and emphasize the significance of compensatory gp41 mutations.
HubMed – drug

 

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