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The rapid emergence of antibiotic-resistant pathogenic bacteria has accelerated the search for new antibiotics. Many clinically used antibacterials were discovered through culturing a single microbial species under nutrient-rich conditions, but in the environment, bacteria constantly encounter poor nutrient conditions and interact with neighboring microbial species. In an effort to recapitulate this environment, we generated a nine-strain actinomycete community and used 16S rDNA sequencing to deconvolute the stochastic production of antimicrobial activity that was not observed from any of the axenic cultures. We subsequently simplified the community to just two strains and identified sp. AA4 as the producing strain and M145 as an inducing strain. Bioassay-guided isolation identified amycomicin (AMY), a highly modified fatty acid containing an epoxide isonitrile warhead as a potent and specific inhibitor of Amycomicin targets an essential enzyme (FabH) in fatty acid biosynthesis and reduces infection in a mouse skin-infection model. The discovery of AMY demonstrates the utility of screening complex communities against specific targets to discover small-molecule antibiotics.
The response to radiation of polymer gel dosimeters has previously been measured by magnetic resonance imaging (MRI) in terms of changes in the water transverse relaxation rate (R ) or magnetization transfer (MT) parameters. Here we report a new MRI approach, based on detecting nuclear Overhauser enhancement (NOE) mediated saturation transfer effects, which can also be used to detect radiation and measure dose distributions in MAGIC-f (Methacrylic and Ascorbic Acid and Gelatin Initiated by Copper Solution with formaldehyde) polymer gels. Results show that the NOE effects produced by low powered radiofrequency (RF) irradiation at specific frequencies offset from water may be quantified by appropriate measurements and over a useful range depend linearly on the radiation dose. The NOE effect likely arises from the polymerization of methacrylic acid monomers which become less mobile, facilitating dipolar through-space cross-relaxation and/or relayed magnetization exchange between polymer and water protons. Our study suggests a potential new MRI method for polymer gel dosimetry.
Modifications of cardiolipin (CL) levels or compositions are associated with changes in mitochondrial function in a wide range of pathologies. We have made the discovery that acetaminophen remodels CL fatty acids composition from tetralinoleoyl to linoleoyltrioleoyl-CL, a remodeling that is associated with decreased mitochondrial respiration. Our data show that CL remodeling causes a shift in electron entry from complex II to the β-oxidation electron transfer flavoprotein quinone oxidoreductase (ETF/QOR) pathway. These data demonstrate that electron entry in the respiratory chain is regulated by CL fatty acid composition and provide proof-of-concept that pharmacological intervention can be used to modify CL composition.
Copyright © 2016 Elsevier B.V. and Mitochondria Research Society. All rights reserved.
BACKGROUND - Ovarian cancer is the most lethal gynecologic malignancy, with limited treatment options for chemoresistant disease. An important link between inflammation and peritoneal spread of ovarian cancer is NF-κB signaling. Thymoquinone (TQ) exerts multiple anti-tumorigenic cellular effects, including NF-κB inhibition. We aimed to investigate the therapeutic potential of TQ in an established murine syngeneic model of ovarian cancer.
METHODS - ID8-NGL mouse ovarian cancer cells stably expressing an NF-κB reporter transgene were injected intra-peritoneally into C57BL/6 mice, and mice were treated with TQ or vehicle for 10 or 30 days. TQ was combined with the macrophage depleting drug, liposomal clodronate, in selected experiments. Effects on peritoneal tumor burden were measured by volume of ascites, number of peritoneal implants and mesenteric tumor mass. NF-κB reporter activity and markers of proliferation and apoptosis were measured in tumors and in confirmatory in vitro experiments. Protein or mRNA expression of M1 (anti-tumor) and M2 (pro-tumor) macrophage markers, and soluble cytokine profiles, were examined from harvested ascites fluid, peritoneal lavages and/or tumor sections. 2-tailed Mann-Whitney tests were used for measuring differences between groups in in vivo experiments.
RESULTS - Consistent with its effects in vitro, TQ reduced proliferation and increased apoptosis in ID8-NGL tumors after 10 and 30 day treatment. Prolonged TQ treatment did not significantly alter tumor number or mass compared to vehicle, but rather exerted an overall deleterious effect by stimulating ascites formation. Increased ascites was accompanied by elevated NF-κB activity in tumors and macrophages, increased pro-tumor M2 macrophages and expression of pro-tumorigenic soluble factors such as VEGF in ascites fluid, and increased tumor infiltration of M2 macrophages. In contrast, a 10 day exposure to TQ produced no ascites, and reduced tumor NF-κB activity, M2 macrophages and soluble VEGF levels. Peritoneal macrophage depletion by clodronate significantly reduced tumor burden. However, TQ-stimulated ascites was further enhanced by co-treatment with clodronate, with macrophages present overwhelmingly of the M2 phenotype.
CONCLUSIONS - Our findings show that pro-tumorigenic microenvironmental effects limited the efficacy of TQ in a syngeneic mouse model of ovarian cancer, and provide caution regarding its potential use in clinical trials in ovarian cancer patients.
BACKGROUND - Ovarian cancer is the most lethal gynecologic malignancy characterized by the frequent development of resistance to platinum chemotherapy. Finding new drug combinations to overcome platinum resistance is a key clinical challenge. Thymoquinone (TQ) is a component of black seed oil that exerts multiple anti-tumorigenic effects on cells, including inhibition of NF-κB and promotion of DNA damage. We aimed to determine whether TQ enhances cisplatin cytotoxicity in cultured ovarian cancer cells and in an established murine syngeneic model of ovarian cancer.
METHODS - Ovarian cancer cell viability in vitro was measured by sulforhodamine B (SRB) assays, and drug interactions tested for synergism by isobologram analysis. ID8-NGL mouse ovarian cancer cells stably expressing an NF-κB reporter transgene were injected intra-peritoneally into C57BL/6 mice. After 30 day TQ and/or cisplatin treatment, we measured the following indices: tumor burden (ascites volume, number of peritoneal implants and mesenteric tumor mass); NF-κB reporter activity (luciferase assay); protein expression of the double-strand DNA break marker, pH2AX(ser139), the proliferation markers, Ki67/mib-1 and PCNA, and the apoptosis markers, cleaved caspase-3, cleaved PARP and Bax; and mRNA expression of NF-κB targets, TNF-α and IL-1β. Two-tailed Mann-Whitney tests were used for measuring differences between groups in mouse experiments.
RESULTS - In SRB assays, TQ and cisplatin synergized in ID8-NGL cells. In mice, cisplatin significantly reduced cell proliferation and increased apoptosis in tumors, resulting in decreased overall tumor burden. Combining TQ with cisplatin further decreased these indices, indicating co-operative effects between the drugs. TQ treatment promoted cisplatin-induced pH2AX expression in cultured cells and in tumors. While NF-κB inhibition by TQ induced anti-tumor effects in vitro, we made the unexpected observation that TQ alone increased both tumor NF-κB activity and formation of ascites in vivo.
CONCLUSIONS - TQ enhanced cisplatin-mediated cytoxicity in ovarian cancer cells in vitro and in a mouse syngeneic model, effects associated with increased DNA damage. However, our results strongly caution that TQ treatment alone may have an overall deleterious effect in the immunocompetent host through stimulation of ascites. Since TQ is a potential candidate for future clinical trials in ovarian cancer patients, this finding has considerable potential relevance to the clinic.
IMPORTANCE - The PIK3CA mutation is one of the most common mutations in head and neck squamous cell carcinoma (HNSCC). Through this research we attempt to elicit the role of oncogene dependence and effects of targeted therapy on this PIK3CA mutation.
OBJECTIVES - (1) To determine the role of oncogene dependence on PIK3CA-one of the more common and targetable oncogenes in HNSCC, and (2) to evaluate the consequence of this oncogene on the effectiveness of newly developed targeted therapies.
DESIGN, SETTING, AND PARTICIPANTS - This was a cell culture-based, in vitro study performed at an academic research laboratory assessing the viability of PIK3CA-mutated head and neck cell lines when treated with targeted therapy.
EXPOSURES - PIK3CA-mutated head and neck cell lines were treated with 17-AAG, GDC-0941, trametinib, and BEZ-235.
MAIN OUTCOMES AND MEASURES - Assessment of cell viability of HNSCC cell lines characterized for PIK3CA mutations or SCC25 cells engineered to express the PIK3CA hotspot mutations E545K or H1047R.
RESULTS - Surprisingly, in engineered cell lines, the hotspot E545K and H1047R mutations conferred increased, rather than reduced, IC50 assay measurements when treated with the respective HSP90, PI3K, and MEK inhibitors, 17-AAG, GDC-0941, and trametinib, compared with the SCC25 control cell lines. When treated with BEZ-235, H1047R-expressing cell lines showed increased sensitivity to inhibition compared with control, whereas those expressing E545K showed slightly increased sensitivity of unclear significance.
CONCLUSIONS AND RELEVANCE - (1) The PIK3CA mutations within our engineered cell model did not lead to enhanced oncogene-dependent cell death when treated with direct inhibition of the PI3K enzyme yet did show increased sensitivity compared with control with dual PI3K/mTOR inhibition. (2) Oncogene addiction to PIK3CA hotspot mutations, if it occurs, is likely to evolve in vivo in the context of additional molecular changes that remain to be identified. Additional study is required to develop new model systems and approaches to determine the role of targeted therapy in the treatment of PI3K-overactive HNSCC tumors.
Scarring is a significant medical burden; financially to the health care system and physically and psychologically for patients. Importantly, there have been numerous case reports describing the occurrence of cancer in burn scars. Currently available therapies are not satisfactory due to their undesirable side-effects, complex delivery routes, requirements for long-term use and/or expense. Radix Arnebiae (Zi Cao), a perennial herb, has been clinically applied to treat burns and manage scars for thousands of years in Asia. Shikonin, an active component extracted from Radix Arnebiae, has been demonstrated to induce apoptosis in cancer cells. Apoptosis is an essential process during scar tissue remodelling. It was therefore hypothesized that Shikonin may induce apoptosis in scar-associated cells. This investigation presents the first detailed in vitro study examining the functional responses of scar-associated cells to Shikonin, and investigates the mechanisms underlying these responses. The data obtained suggests that Shikonin inhibits cell viability and proliferation and reduces detectable collagen in scar-derived fibroblasts. Further investigation revealed that Shikonin induces apoptosis in scar fibroblasts by differentially regulating the expression of caspase 3, Bcl-2, phospho-Erk1/2 and phospho-p38. In addition, Shikonin down-regulates the expression of collagen I, collagen III and alpha-smooth muscle actin genes hence attenuating collagen synthesis in scar-derived fibroblasts. In summary, it is demonstrated that Shikonin induces apoptosis and decreases collagen production in scar-associated fibroblasts and may therefore hold potential as a novel scar remediation therapy.
Copyright © 2015 Elsevier Ireland Ltd. All rights reserved.
Culture extracts from the cave-derived actinomycete Nonomuraea specus were investigated, resulting in the discovery of a new S-bridged pyronaphthoquinone dimer and its monomeric progenitors designated hypogeamicins A-D (1-4). The structures were elucidated using NMR spectroscopy, and the relative stereochemistries of the pyrans were inferred using NOE and comparison to previously reported compounds. Absolute stereochemistry was determined using quantum chemical calculations of specific rotation and vibrational and electronic circular dichroism spectra, after an extensive conformational search and including solute-solvent polarization effects, and comparing with the corresponding experimental data for the monomeric congeners. Interestingly, the dimeric hypogeamicin A (1) was found to be cytotoxic to the colon cancer derived cell line TCT-1 at low micromolar ranges, but not bacteria, whereas the monomeric precursors possessed antibiotic activity but no significant TCT-1 cytotoxicity.
Hsp90 is widely overexpressed in cancer cells and believed to be essential for the maintenance of malignant phenotypes. Targeting Hsp90 by small molecules has shown promise in solid and hematologic malignancies, which likely involves degradation of client oncoproteins in a cell-type-specific manner. In this study, we found that structurally unrelated Hsp90 inhibitors induce DNA damage and apoptosis via p53-dependent induction of PUMA, which indirectly triggers Bax activation and mitochondrial dysfunction in colon cancer cells. Deficiency in PUMA, BAX, or p53, at lesser extent, abrogated 17-allylamino-17-demethoxygeldanamycin (17-AAG)-induced apoptosis and mitochondrial dysfunction, and enhanced clonogenic cell survival. Furthermore, suppression of p53-dependent p21 induction or enhanced p53 activation synergized with 17-AAG to induce PUMA-dependent apoptosis. Finally, PUMA was found to mediate apoptotic and therapeutic responses to the 17-AAG analog 17-DMAG in xenografts. These results show an important role of the p53/PUMA/Bax axis in Hsp90 inhibitor-induced killing of p53 wild-type cells, and have important implications for their clinical applications.
Proteomic analyses of protein-electrophile adducts generally employ affinity capture of the adduct moiety, which enables global analyses, but is poorly suited to targeted studies of specific proteins. We describe a targeted molecular probe approach to study modifications of the molecular chaperone heat-shock protein 90 (Hsp90), which regulates diverse client proteins. Noncovalent affinity capture with a biotinyl analog of the HSP90 inhibitor geldanamycin enables detection of the native protein isoforms Hsp90α and Hsp90β and their phosphorylated forms. We applied this probe to map and quantify adducts formed on Hsp90 by 4-hydroxynonenal (HNE) in RKO cells. This approach was also applied to measure the kinetics of site-specific adduction of selected Hsp90 residues. A protein-selective affinity capture approach is broadly applicable for targeted analysis of electrophile adducts and their biological effects.