Multiomic-Based Molecular Landscape of FaDu Xenograft Tumors in Mice after a Combinatorial Treatment with Radiation and an HSP90 Inhibitor Identifies Adaptation-Induced Targets of Resistance and Therapeutic Intervention.

Treatments involving radiation and chemotherapy alone or in combination have improved patient survival and quality of life. However, cancers frequently evade these therapies due to adaptation and tumor evolution. Given the complexity of predicting response based solely on the initial genetic profile of a patient, a predetermined treatment course may miss critical adaptation that can cause resistance or induce new targets for drug and immunotherapy. To address the timescale for these evasive mechanisms, using a mouse xenograft tumor model, we investigated the rapidity of gene expression (mRNA), molecular pathway, and phosphoproteome changes after radiation, an HSP90 inhibitor, or combination. Animals received radiation, drug, or combination treatment for 1 or 2 weeks and were then euthanized along with a time-matched untreated group for comparison. Changes in gene expression occur as early as 1 week after treatment initiation. Apoptosis and cell death pathways were activated in irradiated tumor samples. For the HSP90 inhibitor and combination treatment at weeks 1 and 2 compared with Control Day 1, gene-expression changes induced inhibition of pathways including invasion of cells, vasculogenesis, and viral infection among others. The combination group included both drug-alone and radiation-alone changes. Our data demonstrate the rapidity of gene expression and functional pathway changes in the evolving tumor as it responds to treatment. Discovering these phenotypic adaptations may help elucidate the challenges in using sustained treatment regimens and could also define evolving targets for therapeutic efficacy.

The effect of modulation of gut microbiome profile on radiation-induced carcinogenesis and survival.

Non-lethal doses of ionizing radiation (IR) delivered to humans because of terrorist events, nuclear accidents or radiotherapy can result in carcinogenesis. Means of protecting against carcinogenesis are lacking. We questioned the role of the gut microbiome in IR-induced carcinogenesis. The gut microbiome was modulated by administering broad spectrum antibiotics (Ab) in the drinking water. Mice were given Ab 3 weeks before and 3 weeks after 3 Gy total body irradiation (TBI) or for 6 weeks one month after TBI. Three weeks of Ab treatment resulted in a 98% reduction in total 16S rRNA counts for 4 out of 6 of the phylum groups detected. However, 3 more weeks of Ab treatment (6 weeks total) saw an expansion in the phylum groups Proteobacteria and Actinobacteria. The Ab treatment altered the bacteria diversity in the gut, and shortened the lifespan when Ab were administered before and after TBI. Mortality studies indicated that the adverse Ab lifespan effects were due to a decrease in the time in which solid tumors started to appear and not to any changes in hematopoietic or benign tumors. In contrast, when Ab were administered one month after TBI, lifespan was unchanged compared to the control TBI group. Use of broad-spectrum antibiotics to simulate the germ-free condition did not afford an advantage on carcinogenesis or lifespan.

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.

Radiation-Induced Senescence Reprograms Secretory and Metabolic Pathways in Colon Cancer HCT-116 Cells.

Understanding the global metabolic changes during the senescence of tumor cells can have implications for developing effective anti-cancer treatment strategies. Ionizing radiation (IR) was used to induce senescence in a human colon cancer cell line HCT-116 to examine secretome and metabolome profiles. Control proliferating and senescent cancer cells (SCC) exhibited distinct morphological differences and expression of senescent markers. Enhanced secretion of pro-inflammatory chemokines and IL-1, anti-inflammatory IL-27, and TGF-ß1 was observed in SCC. Significantly reduced levels of VEGF-A indicated anti-angiogenic activities of SCC. Elevated levels of tissue inhibitors of matrix metalloproteinases from SCC support the maintenance of the extracellular matrix. Adenylate and guanylate energy charge levels and redox components NAD and NADP and glutathione were maintained at near optimal levels indicating the viability of SCC. Significant accumulation of pyruvate, lactate, and suppression of the TCA cycle in SCC indicated aerobic glycolysis as the predominant energy source for SCC. Levels of several key amino acids decreased significantly, suggesting augmented utilization for protein synthesis and for use as intermediates for energy metabolism in SCC. These observations may provide a better understanding of cellular senescence basic mechanisms in tumor tissues and provide opportunities to improve cancer treatment.

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.

Molecular Imaging of the Tumor Microenvironment Reveals the Relationship between Tumor Oxygenation, Glucose Uptake, and Glycolysis in Pancreatic Ductal Adenocarcinoma.

Molecular imaging approaches for metabolic and physiologic imaging of tumors have become important for treatment planning and response monitoring. However, the relationship between the physiologic and metabolic aspects of tumors is not fully understood. Here, we developed new hyperpolarized MRI and electron paramagnetic resonance imaging procedures that allow more direct assessment of tumor glycolysis and oxygenation status quantitatively. We investigated the spatial relationship between hypoxia, glucose uptake, and glycolysis in three human pancreatic ductal adenocarcinoma tumor xenografts with differing physiologic and metabolic characteristics. At the bulk tumor level, there was a strong positive correlation between 18F-FDG-PET and lactate production, while pO2 was inversely related to lactate production and 18F-2-fluoro-2-deoxy-D-glucose (18F-FDG) uptake. However, metabolism was not uniform throughout the tumors, and the whole tumor results masked different localizations that became apparent while imaging. 18F-FDG uptake negatively correlated with pO2 in the center of the tumor and positively correlated with pO2 on the periphery. In contrast to pO2 and 18F-FDG uptake, lactate dehydrogenase activity was distributed relatively evenly throughout the tumor. The heterogeneity revealed by each measure suggests a multimodal molecular imaging approach can improve tumor characterization, potentially leading to better prognostics in cancer treatment. SIGNIFICANCE: Novel multimodal molecular imaging techniques reveal the potential of three interrelated imaging biomarkers to profile the tumor microenvironment and interrelationships of hypoxia, glucose uptake, and glycolysis.

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.

Selective enhancement of hypoxic cell killing by tempol-regulated suicide gene expression.

The presence of hypoxic regions within solid tumors is caused by an imbalance between cell proliferation and angiogenesis. Such regions may facilitate the onset of recurrence after radiation therapy and chemotherapy, as hypoxic cells show resistance to these treatments. We found that tempol, a nitroxide, strongly induces the accumulation of hypoxia-inducible factor (HIF)-1α, particularly under conditions of hypoxia. We, therefore, evaluated whether tempol enhances the gene expression via HIF-1α, potentially leading to various applications for cancer gene therapy targeting hypoxic cells. Consequently, following treatment with tempol under hypoxia, the luciferase (Luc) activity in the cells transfected with the plasmid containing the luc gene with the oxygen-dependent degradation domain and a promoter composed of hypoxia-responsive elements increased up to approximately 10-fold compared to that observed in cells treated identically with the exception of tempol. The plasmid constructed by replacing the luc gene with the fcy::fur fusion gene as a suicide gene, strongly induced the accumulation of the Fcy::Fur fusion protein, only when incubated in the presence of the hypoxic mimic CoCl2 and tempol. The transfected cells were successfully killed with the addition of 5-fluorocytosine to the cell culture according to the fcy::fur fusion gene expression. As similar but lesser enhancement of the Luc activity was also observed in solid tumor tissues in nude mice, this strategy may be applied for hypoxic cancer eradication.

Radiation Enhancement of Head and Neck Squamous Cell Carcinoma by the Dual PI3K/mTOR Inhibitor PF-05212384.

PURPOSE: Radiation remains a mainstay for the treatment of nonmetastatic head and neck squamous cell carcinoma (HNSCC), a malignancy characterized by a high rate of PI3K/mTOR signaling axis activation. We investigated the ATP-competitive dual PI3K/mTOR inhibitor, PF-05212384, as a radiosensitizer in preclinical HNSCC models. EXPERIMENTAL DESIGN: Extent of radiation enhancement of two HNSCC cell lines (UMSCC1-wtP53 and UMSCC46-mtP53) and normal human fibroblast (1522) was assessed by in vitro clonogenic assay with appropriate target inhibition verified by immunoblotting. Radiation-induced DNA damage repair was evaluated by γH2AX Western blots with the mechanism of DNA double-strand break repair abrogation investigated by cell cycle analysis, immunoblotting, and RT-PCR. PF-05212384 efficacy in vivo was assessed by UMSCC1 xenograft tumor regrowth delay, xenograft lysate immunoblotting, and tissue section immunohistochemistry. RESULTS: PF-05212384 effectively inhibited PI3K and mTOR, resulting in significant radiosensitization of exponentially growing and plateau-phase cells with 24-hour treatment following irradiation, and variable radiation enhancement with 24-hour treatment before irradiation. Tumor cells radiosensitized to a greater extent than normal human fibroblasts. Postirradiation PF-05212384 treatment delays γH2AX foci resolution. PF-05212384 24-hour exposure resulted in an evident G1-S phase block in p53-competent cells. Fractionated radiation plus i.v. PF-05212384 synergistically delayed nude mice bearing UMSCC1 xenograft regrowth, with potential drug efficacy biomarkers identified, including pS6, pAkt, p4EBP1, and Ki67. CONCLUSIONS: Taken together, our results of significant radiosensitization both in vitro and in vivo validate the PI3K/mTOR axis as a radiation modification target and PF-05212384 as a potential clinical radiation modifier of nonmetastatic HNSCC.

Pyruvate induces transient tumor hypoxia by enhancing mitochondrial oxygen consumption and potentiates the anti-tumor effect of a hypoxia-activated prodrug TH-302.

BACKGROUND: TH-302 is a hypoxia-activated prodrug (HAP) of bromo isophosphoramide mustard that is selectively activated within hypoxic regions in solid tumors. Our recent study showed that intravenously administered bolus pyruvate can transiently induce hypoxia in tumors. We investigated the mechanism underlying the induction of transient hypoxia and the combination use of pyruvate to potentiate the anti-tumor effect of TH-302. METHODOLOGY/RESULTS: The hypoxia-dependent cytotoxicity of TH-302 was evaluated by a viability assay in murine SCCVII and human HT29 cells. Modulation in cellular oxygen consumption and in vivo tumor oxygenation by the pyruvate treatment was monitored by extracellular flux analysis and electron paramagnetic resonance (EPR) oxygen imaging, respectively. The enhancement of the anti-tumor effect of TH-302 by pyruvate treatment was evaluated by monitoring the growth suppression of the tumor xenografts inoculated subcutaneously in mice. TH-302 preferentially inhibited the growth of both SCCVII and HT29 cells under hypoxic conditions (0.1% O2), with minimal effect under aerobic conditions (21% O2). Basal oxygen consumption rates increased after the pyruvate treatment in SCCVII cells in a concentration-dependent manner, suggesting that pyruvate enhances the mitochondrial respiration to consume excess cellular oxygen. In vivo EPR oxygen imaging showed that the intravenous administration of pyruvate globally induced the transient hypoxia 30 min after the injection in SCCVII and HT29 tumors at the size of 500-1500 mm(3). Pretreatment of SCCVII tumor bearing mice with pyruvate 30 min prior to TH-302 administration, initiated with small tumors (∼ 550 mm(3)), significantly delayed tumor growth. CONCLUSIONS/SIGNIFICANCE: Our in vitro and in vivo studies showed that pyruvate induces transient hypoxia by enhancing mitochondrial oxygen consumption in tumor cells. TH-302 therapy can be potentiated by pyruvate pretreatment if started at the appropriate tumor size and oxygen concentration.

EPR oxygen imaging and hyperpolarized 13C MRI of pyruvate metabolism as noninvasive biomarkers of tumor treatment response to a glycolysis inhibitor 3-bromopyruvate.

The hypoxic nature of tumors results in treatment resistance and poor prognosis. To spare limited oxygen for more crucial pathways, hypoxic cancerous cells suppress mitochondrial oxidative phosphorylation and promote glycolysis for energy production. Thereby, inhibition of glycolysis has the potential to overcome treatment resistance of hypoxic tumors. Here, EPR imaging was used to evaluate oxygen dependent efficacy on hypoxia-sensitive drug. The small molecule 3-bromopyruvate blocks glycolysis pathway by inhibiting hypoxia inducible enzymes and enhanced cytotoxicity of 3-bromopyruvate under hypoxic conditions has been reported in vitro. However, the efficacy of 3-bromopyruvate was substantially attenuated in hypoxic tumor regions (pO2<10 mmHg) in vivo using squamous cell carcinoma (SCCVII)-bearing mouse model. Metabolic MRI studies using hyperpolarized 13C-labeled pyruvate showed that monocarboxylate transporter-1 is the major transporter for pyruvate and the analog 3-bromopyruvate in SCCVII tumor. The discrepant results between in vitro and in vivo data were attributed to biphasic oxygen dependent expression of monocarboxylate transporter-1 in vivo. Expression of monocarboxylate transporter-1 was enhanced in moderately hypoxic (8-15 mmHg) tumor regions but down regulated in severely hypoxic (<5 mmHg) tumor regions. These results emphasize the importance of noninvasive imaging biomarkers to confirm the action of hypoxia-activated drugs.

Antiangiogenic agent sunitinib transiently increases tumor oxygenation and suppresses cycling hypoxia.

Structural and functional abnormalities in tumor blood vessels impact the delivery of oxygen and nutrients to solid tumors, resulting in chronic and cycling hypoxia. Although chronically hypoxic regions exhibit treatment resistance, more recently it has been shown that cycling hypoxic regions acquire prosurvival pathways. Angiogenesis inhibitors have been shown to transiently normalize the tumor vasculatures and enhance tumor response to treatments. However, the effect of antiangiogenic therapy on cycling tumor hypoxia remains unknown. Using electron paramagnetic resonance imaging and MRI in tumor-bearing mice, we have examined the vascular renormalization process by longitudinally mapping tumor partial pressure of oxygen (pO(2)) and microvessel density during treatments with a multi-tyrosine kinase inhibitor sunitinib. Transient improvement in tumor oxygenation was visualized by electron paramagnetic resonance imaging 2 to 4 days following antiangiogenic treatments, accompanied by a 45% decrease in microvessel density. Radiation treatment during this time period of improved oxygenation by antiangiogenic therapy resulted in a synergistic delay in tumor growth. In addition, dynamic oxygen imaging obtained every 3 minutes was conducted to distinguish tumor regions with chronic and cycling hypoxia. Sunitinib treatment suppressed the extent of temporal fluctuations in tumor pO(2) during the vascular normalization window, resulting in the decrease of cycling tumor hypoxia. Overall, the findings suggest that longitudinal and noninvasive monitoring of tumor pO(2) makes it possible to identify a window of vascular renormalization to maximize the effects of combination therapy with antiangiogenic drugs.

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.

Low-field magnetic resonance imaging to visualize chronic and cycling hypoxia in tumor-bearing mice.

Tumors exhibit fluctuations in blood flow that influence oxygen concentrations and therapeutic resistance. To assist therapeutic planning and improve prognosis, noninvasive dynamic imaging of spatial and temporal variations in oxygen partial pressure (pO(2)) would be useful. Here, we illustrate the use of pulsed electron paramagnetic resonance imaging (EPRI) as a novel imaging method to directly monitor fluctuations in oxygen concentrations in mouse models. A common resonator platform for both EPRI and magnetic resonance imaging (MRI) provided pO(2) maps with anatomic guidance and microvessel density. Oxygen images acquired every 3 minutes for a total of 30 minutes in two different tumor types revealed that fluctuation patterns in pO(2) are dependent on tumor size and tumor type. The magnitude of fluctuations in pO(2) in SCCVII tumors ranged between 2- to 18-fold, whereas the fluctuations in HT29 xenografts were of lower magnitude. Alternating breathing cycles with air or carbogen (95% O(2) plus 5% CO(2)) distinguished higher and lower sensitivity regions, which responded to carbogen, corresponding to cycling hypoxia and chronic hypoxia, respectively. Immunohistochemical analysis suggests that the fluctuation in pO(2) correlated with pericyte density rather than vascular density in the tumor. This EPRI technique, combined with MRI, may offer a powerful clinical tool to noninvasively detect variable oxygenation in tumors.

Inhibition of adipogenesis by Tempol in 3T3-L1 cells.

Obesity is highly associated with an increased risk of serious health conditions including hypertension, cardiovascular disease, diabetes, and cancer. Changes in redox status with increased oxidative stress have been linked with obesity. Previous studies have shown that administration of the antioxidant Tempol in the food of mice prevents obesity, causing significant weight loss without toxicity. To gain a better understanding of the molecular mechanism(s) underlying this effect, the influence of Tempol on the differentiation of mouse 3T3-L1 preadipocytes was studied. Tempol inhibited differentiation of 3T3-L1 cells, resulting in a reduction in cellular lipid storage, down-regulation of protein levels of key adipogenesis transcription factors (PPARgamma and PPARalpha), down-regulation of prolyl hydroxylase, and up-regulation of HIF-1alpha. Mice on a Tempol diet demonstrated reduced systemic levels of IGF-1, in qualitative agreement with in vitro observations in 3T3-L1 cells, which also showed lower IGF-1 levels as a result of Tempol treatment. These results show that treatment of 3T3-L1 cells with Tempol inhibits the expression of key adipogenesis factors, adipose differentiation, and lipid storage and may underlie, at least in part, some of the in vivo effects of Tempol on body weight.

Improvement and induction property of radiation-responsive promoter through DNA shuffling of 5'-flanking regions of the human p21 gene.

A promoter that augments gene expression in response to stimulation of ionizing radiation would be a desired tool for radiogenetic therapy, a combination of radiotherapy and gene therapy. Although various promoters occurring naturally or artificially have been used for researches, one showing higher reactivity to ionizing radiation is desirable. In the present study, we attempted to improve a radiation-responsive promoter of the p21 through a technique called DNA shuffling. A library of DNA fragments was constructed by re-ligation of randomly digested promoter fragments and improved promoters were chosen out of the library. We repeated this process twice to obtain a promoter showing 2.6-fold better reactivity to ionizing radiation compared with its parent, p21 promoter after 10 Gy gamma-ray irradiation. Nucleotide sequence analyses revealed that the obtained promoter was densely packed with some of the cis-acting elements including binding sites for p53, NF-kappaB, NRF-2, AP-1 and NF-Y more than p21 promoter. In addition, it was shown that its induction by ionizing radiation was dependent upon p53 status of a cell line, suggesting that the promoter retained properties of the p21 promoter. This technique is simple and efficient to improve a promoter responsive to other stimulus of interest besides IR.

In vitro and in vivo radiation sensitization of human tumor cells by a novel checkpoint kinase inhibitor, AZD7762.

PURPOSE: Inhibition of checkpoint kinase 1 has been shown to enhance the cytotoxicity of DNA-damaging targeted chemotherapy through cell cycle checkpoint abrogation and impaired DNA damage repair. A novel checkpoint kinase 1/2 inhibitor, AZD7762, was evaluated for potential enhancement of radiosensitivity for human tumor cells in vitro and in vivo xenografts. EXPERIMENTAL DESIGN: Survival of both p53 wild-type and mutant human cell lines was evaluated by clonogenic assay. Dose modification factors (DMF) were determined from survival curves (ratio of radiation doses for control versus drug treated at 10% survival). Flow cytometry, Western blot, and radiation-induced tumor regrowth delay assays were conducted. RESULTS: AZD7762 treatment enhanced the radiosensitivity of p53-mutated tumor cell lines (DMFs ranging from 1.6-1.7) to a greater extent than for p53 wild-type tumor lines (DMFs ranging from 1.1-1.2). AZD7762 treatment alone exhibited little cytotoxicity to any of the cell lines and did not enhance the radiosensitivity of normal human fibroblasts (1522). AZD7762 treatment abrogated radiation-induced G(2) delay, inhibited radiation damage repair (assessed by gamma-H2AX), and suppressed radiation-induced cyclin B expression. HT29 xenografts exposed to five daily radiation fractions and to two daily AZD7762 doses exhibited significant radiation enhancement compared with radiation alone. CONCLUSIONS: AZD7762 effectively enhanced the radiosensitivity of mutated p53 tumor cell lines and HT29 xenografts and was without untoward toxicity when administered alone or in combination with radiation. The results of this study support combining AZD7762 with radiation in clinical trials.

Loratadine dysregulates cell cycle progression and enhances the effect of radiation in human tumor cell lines.

BACKGROUND: The histamine receptor-1 (H1)-antagonist, loratadine has been shown to inhibit growth of human colon cancer xenografts in part due to cell cycle arrest in G2/M. Since this is a radiation sensitive phase of the cell cycle, we sought to determine if loratadine modifies radiosensitivity in several human tumor cell lines with emphasis on human colon carcinoma (HT29). METHODS: Cells were treated with several doses of loratadine at several time points before and after exposure to radiation. Radiation dose modifying factors (DMF) were determined using full radiation dose response survival curves. Cell cycle phase was determined by flow cytometry and the expression of the cell cycle-associated proteins Chk1, pChk1(ser345), and Cyclin B was analyzed by western blot. RESULTS: Loratadine pre-treatment of exponentially growing cells (75 microM, 24 hours) increased radiation-induced cytotoxicity yielding a radiation DMF of 1.95. However, treatment of plateau phase cells also yielded a DMF of 1.3 suggesting that mechanisms other than cell cycle arrest also contribute to loratadine-mediated radiation modification. Like irradiation, loratadine initially induced G2/M arrest and activation of the cell-cycle associated protein Chk1 to pChk1(ser345), however a subsequent decrease in expression of total Chk1 and Cyclin B correlated with abrogation of the G2/M checkpoint. Analysis of DNA repair enzyme expression and DNA fragmentation revealed a distinct pattern of DNA damage in loratadine-treated cells in addition to enhanced radiation-induced damage. Taken together, these data suggest that the observed effects of loratadine are multifactorial in that loratadine 1) directly damages DNA, 2) activates Chk1 thereby promoting G2/M arrest making cells more susceptible to radiation-induced DNA damage and, 3) downregulates total Chk1 and Cyclin B abrogating the radiation-induced G2/M checkpoint and allowing cells to re-enter the cell cycle despite the persistence of damaged DNA. CONCLUSIONS: Given this unique possible mechanism of action, loratadine has potential as a chemotherapeutic agent and as a modifier of radiation responsiveness in the treatment of cancer and, as such, may warrant further clinical evaluation.

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.

Simultaneous imaging of tumor oxygenation and microvascular permeability using Overhauser enhanced MRI.

Architectural and functional abnormalities of blood vessels are a common feature in tumors. A consequence of increased vascular permeability and concomitant aberrant blood flow is poor delivery of oxygen and drugs, which is associated with treatment resistance. In the present study, we describe a strategy to simultaneously visualize tissue oxygen concentration and microvascular permeability by using a hyperpolarized (1)H-MRI, known as Overhauser enhanced MRI (OMRI), and an oxygen-sensitive contrast agent OX63. Substantial MRI signal enhancement was induced by dynamic nuclear polarization (DNP). The DNP achieved up to a 7,000% increase in MRI signal at an OX63 concentration of 1.5 mM compared with that under thermal equilibrium state. The extent of hyperpolarization is influenced mainly by the local concentration of OX63 and inversely by the tissue oxygen level. By collecting dynamic OMRI images at different hyperpolarization levels, local oxygen concentration and microvascular permeability of OX63 can be simultaneously determined. Application of this modality to murine tumors revealed that tumor regions with high vascular permeability were spatio-temporally coincident with hypoxia. Quantitative analysis of image data from individual animals showed an inverse correlation between tumor vascular leakage and median oxygen concentration. Immunohistochemical analyses of tumor tissues obtained from the same animals after OMRI experiments demonstrated that lack of integrity in tumor blood vessels was associated with increased tumor microvascular permeability. This dual imaging technique may be useful for the longitudinal assessment of changes in tumor vascular function and oxygenation in response to chemotherapy, radiotherapy, or antiangiogenic treatment.