The antioxidant tempol transforms gut microbiome to resist obesity in female C3H mice fed a high fat diet.

The nitroxide, Tempol, prevents obesity related changes in mice fed a high fat diet (HFD). The purpose of this study was to gain insight into the mechanisms that result in such changes by Tempol in female C3H mice. Microarray methodology, Western blotting, bile acid analyses, and gut microbiome sequencing were used to identify multiple genes, proteins, bile acids, and bacteria that are regulated by Tempol in female C3H mice on HFD. The effects of antibiotics in combination with Tempol on the gut microflora were also studied. Adipose tissue, from Tempol treated mice, was analyzed using targeted gene microarrays revealing up-regulation of fatty acid metabolism genes (Acadm and Acadl > 4-fold, and Acsm3 and Acsm5 > 10-fold). Gene microarray studies of liver tissue from mice switched from HFD to Tempol HFD showed down-regulation of fatty acid synthesis genes and up-regulation of fatty acid oxidation genes. Analyses of proteins involved in obesity revealed that the expression of aldehyde dehydrogenase 1A1 (ALDH1A1) and fasting induced adipose factor/angiopoietin-like protein 4 (FIAF/ANGPTL4) was altered by Tempol HFD. Bile acid studies revealed increases in cholic acid (CA) and deoxycholic acid (DCA) in both the liver and serum of Tempol treated mice. Tempol HFD effect on the gut microbiome composition showed an increase in the population of Akkermansia muciniphila, a bacterial species known to be associated with a lean, anti-inflammatory phenotype. Antibiotic treatment significantly reduced the total level of bacterial numbers, however, Tempol was still effective in reducing the HFD weight gain. Even after antibiotic treatment Tempol still positively influenced several bacterial species such as as Akkermansia muciniphila and Bilophila wadsworthia. The positive effects of Tempol moderating weight gain in female mice fed a HFD involves changes to the gut microbiome, bile acids composition, and finally to changes in genes and proteins involved in fatty acid metabolism and storage.

Pharmacological Inhibition of HSP90 Radiosensitizes Head and Neck Squamous Cell Carcinoma Xenograft by Inhibition of DNA Damage Repair, Nucleotide Metabolism, and Radiation-Induced Tumor Vasculogenesis.

PURPOSE: Recent preclinical studies suggest combining the HSP90 inhibitor AT13387 (Onalespib) with radiation (IR) against colon cancer and head and neck squamous cell carcinoma (HNSCC). These studies emphasized that AT13387 downregulates HSP90 client proteins involved in oncogenic signaling and DNA repair mechanisms as major drivers of enhanced radiosensitivity. Given the large array of client proteins HSP90 directs, we hypothesized that other key proteins or signaling pathways may be inhibited by AT13387 and contribute to enhanced radiosensitivity. Metabolomic analysis of HSP90 inhibition by AT13387 was conducted to identify metabolic biomarkers of radiosensitization and whether modulations of key proteins were involved in IR-induced tumor vasculogenesis, a process involved in tumor recurrence. METHODS AND MATERIALS: HNSCC and non-small cell lung cancer cell lines were used to evaluate the AT13387 radiosensitization effect in vitro and in vivo. Flow cytometry, immunofluorescence, and immunoblot analysis were used to evaluate cell cycle changes and HSP90 client protein's role in DNA damage repair. Metabolic analysis was performed using liquid chromatography-Mass spectrometry. Immunohistochemical examination of resected tumors post-AT13387 and IR treatment were conducted to identify biomarkers of IR-induced tumor vasculogenesis. RESULTS: In agreement with recent studies, AT13387 treatment combined with IR resulted in a G2/M cell cycle arrest and inhibited DNA repair. Metabolomic profiling indicated a decrease in key metabolites in glycolysis and tricarboxylic acid cycle by AT13387, a reduction in Adenosine 5'-triphosphate levels, and rate-limiting metabolites in nucleotide metabolism, namely phosphoribosyl diphosphate and aspartate. HNSCC xenografts treated with the combination exhibited increased tumor regrowth delay, decreased tumor infiltration of CD45 and CD11b+ bone marrow-derived cells, and inhibition of HIF-1 and SDF-1 expression, thereby inhibiting IR-induced vasculogenesis. CONCLUSIONS: AT13387 treatment resulted in pharmacologic inhibition of cancer cell metabolism that was linked to DNA damage repair. AT13387 combined with IR inhibited IR-induced vasculogenesis, a process involved in tumor recurrence postradiotherapy. Combining AT13387 with IR warrants consideration of clinical trial assessment.

Abemaciclib, a Selective CDK4/6 Inhibitor, Enhances the Radiosensitivity of Non-Small Cell Lung Cancer In Vitro and In Vivo.

Purpose: To characterize the ionizing radiation (IR) enhancing effects and underlying mechanisms of the CDK4/6 inhibitor abemaciclib in non-small cell lung cancer (NSCLC) cells in vitro and in vivoExperimental Design: IR enhancement by abemaciclib in a variety of NSCLC cell lines was assessed by in vitro clonogenic assay, flow cytometry, and target inhibition verified by immunoblotting. IR-induced DNA damage repair was evaluated by γH2AX analysis. Global metabolic alterations by abemaciclib and IR combination were evaluated by LC/MS mass spectrometry and YSI bioanalyzer. Effects of abemaciclib and IR combination in vivo were studied by xenograft tumor regrowth delay, xenograft lysate immunoblotting, and tissue section immunohistochemistry.Results: Abemaciclib enhanced the radiosensitivity of NSCLC cells independent of RAS or EGFR status. Enhancement of radiosensitivity was lost in cell lines deficient for functional p53 and RB protein. After IR, abemaciclib treatment inhibited DNA damage repair as measured by γH2AX. Mechanistically, abemaciclib inhibited RB phosphorylation, leading to cell-cycle arrest. It also inhibited mTOR signaling and reduced intracellular amino acid pools, causing nutrient stress. In vivo, abemaciclib, when administered in an adjuvant setting for the second week after fractionated IR, further inhibited vasculogenesis and tumor regrowth, with sustained inhibition of RB/E2F activity, mTOR pathway, and HIF-1 expression. In summary, our study signifies inhibiting the CDK4/6 pathway by abemaciclib in combination with IR as a promising therapeutic strategy to treat NSCLC.Conclusions: Abemaciclib in combination with IR enhances NSCLC radiosensitivity in preclinical models, potentially providing a novel biomarker-driven combination therapeutic strategy for patients with NSCLC. Clin Cancer Res; 24(16); 3994-4005. ©2018 AACR.

Guggulsterone-mediated enhancement of radiosensitivity in human tumor cell lines.

PURPOSE: To observe the effect of guggulsterone (GS) on the radiation response in human cancer cell lines. MATERIALS AND METHODS: The radiation response of cancer cells treated with GS was observed by cell survival studies, cell growth assay, NF-κB activity assay, western blotting of some key growth promoting receptors, the DNA repair protein γH2AX, and flow cytometry for DNA analyses. RESULTS: GS inhibited radiation induced NF-κB activation and enhanced radiosensitivity in the pancreatic cell line, PC-Sw. It reduced both cell cycle movement and cell growth. GS reduced ERα protein in MCF7 cells and IGF1-Rß protein in colon cancer cells and pancreatic cancer cells and inhibited DNA double strand break (DSB) repair following radiation. CONCLUSION: GS induced radiation sensitization may be due to several different mechanisms including the inhibition of NF-κB activation and reductions in IGF1-Rß. In addition, GS induced γH2AX formation, primarily in the S-phase, indicates that DNA DSB's in the S-phase may be another reason for GS induced radiosensitivity. ERα down-regulation in response to GS suggests that it can be of potential use in the treatment of estrogen positive tumors that are resistant to tamoxifen.

Halofuginone enhances the radiation sensitivity of human tumor cell lines.

Transforming growth factor beta (TGF-beta) is implicated in radiation-induced fibrosis of normal tissues in patients receiving radiotherapy. Inhibiting the TGF-beta signaling pathway by various means has been shown to reduce radiation-induced fibrosis in pre-clinical studies. The present study evaluated the effects of interfering with the TGF-beta signaling pathway on the radiosensitivity of selected human tumor cell lines using the plant-derived alkaloid, halofuginone. Halofuginone treatment inhibited cell growth, halted cell cycle progression, decreased radiation-induced DNA damage repair, and decreased TGF-beta receptor II protein levels, leading to increased cellular radiosensitization. These data further support the goal of manipulating the TGF-beta pathway to achieve a positive increase in the therapeutic gain in clinical radiotherapy.

Factors influencing nitroxide reduction and cytotoxicity in vitro.

Nitroxides have been shown to be effective antioxidants, radiation protectors, and redox-active probes for functional electron paramagnetic resonance (EPR) imaging. More recently, the nitroxide 4-hydroxy-2,2,6,6-tetramethylpiperidinyl-N-oxyl (Tempol) has been shown to exert differential cytotoxicity to tumor compared with normal cell counterparts. Nitroxides are readily reduced in tissues to their respective hydroxylamines, which exhibit less cytotoxicity in vitro and do not provide radiation protection or an EPR-detectable signal for imaging. In order to better understand factors that influence nitroxide reduction, the rate of reduction of Tempol in mouse and human cell lines and in primary cultures of tumor cells was measured using EPR spectroscopy. Additionally, the cytotoxicity of high concentrations of Tempol and the hydroxylamine of Tempol (Tempol-H) was evaluated in wild-type and glucose-6-phosphate dehydrogenase (G6PD)-deficient Chinese hamster ovary cells. The results show that in general Tempol was reduced at a faster rate when cells were under hypoxic compared with aerobic conditions. Neither depletion of intracellular glutathione nor treatment of cells with sodium cyanide influenced Tempol reduction rates. G6PD-deficient cells were found to reduce Tempol at a significantly slower rate than wild-type cells. Likewise, Tempol-induced cytotoxicity was markedly less for G6PD-deficient cells compared with wild-type cells. Tempol-H exhibited no cytotoxicity to either cell type. Tempol-mediated cytotoxicity was enhanced by glutathione depletion and inhibition of 6-phosphogluconate dehydrogenase in wild-type cells, but was unaltered in G6PD-deficient cells. Collectively, the results indicate that while the bioreduction of Tempol can be influenced by a number of factors, the hexose monophosphate shunt appears to be involved in both nitroxide reduction as well as cytotoxicity induced by high levels of exposure to Tempol.