Cheryl H. Baker

Cerium oxide nanoparticles protect gastrointestinal epithelium from radiation-induced damage by reduction of reactive oxygen species and upregulation of superoxide dismutase 2

UNLABELLED The ability of rare earth cerium oxide (CeO(2)) nanoparticles to confer radioprotection against gastrointestinal epithelium was examined. The pretreatment of normal human colon cells (CRL 1541) with varying concentrations of CeO(2) nanoparticles 24 hours before single-dose radiation exposure conferred protection from radiation-induced cell death by reducing the amount of reactive oxygen species produced and increasing the expression of superoxide dismutase 2 (SOD2), in a dose-dependent manner. In subsequent experiments athymic nude mice were pretreated with intraperitoneal injections of CeO(2) nanoparticles before a single dose of radiation to the abdominal area. Immunohistochemical analysis show a decrease in TUNEL- and caspase 3-positive cells in the colonic crypt, 4 hours after radiation. In sharp contrast, a significant increase in SOD2 expression was observed. In the end, these studies suggest that CeO(2) nanoparticles protect the gastrointestinal epithelium against radiation-induced damage by (1) acting as free-radical scavengers and (2) increasing the production of SOD2 before radiation insult. FROM THE CLINICAL EDITOR In this study, the ability of rare earth cerium oxide (CeO(2)) nanoparticles to confer radioprotection was examined. The results suggest that CeO(2) nanoparticles protect the gastrointestinal epithelium against radiation-induced damage both by acting as free-radical scavengers and by increasing the production of SOD2 before radiation insult.

Development of Biologic Markers of Response and Assessment of Antiangiogenic Activity in a Clinical Trial of Human Recombinant Endostatin

PURPOSE Angiogenesis is a target for the treatment of cancer and other diseases, and its complex biology suggests that establishing the appropriate dose and schedule for antiangiogenic treatment will require extensive study. We present the initial results of a dose-finding clinical trial of recombinant human endostatin (rh-Endo) that examined potential surrogates for response to antiangiogenic therapy. PATIENTS AND METHODS Twenty-five patients were treated with escalating doses of rh-Endo. Positron emission tomography (PET) was used to assess tumor blood flow (with [15O]H2O) and metabolism (with [18F]fluorodeoxyglucose) before the start of therapy and then every 4 weeks. To directly assess the effects of rh-Endo on endothelial cells within the tumors, biopsy specimens of tumor tissue were obtained before therapy and again at 8 weeks and evaluated for endothelial cell and tumor cell apoptosis. RESULTS Tumor blood flow and metabolism as measured by PET scans generally decreased with increasing doses of rh-Endo; however, the effects were complex and in some analyses nonlinear. Tumor biopsy analysis revealed a significant increase in tumor cell apoptosis (P =.027) and endothelial cell apoptosis (P =.027) after 8 weeks of therapy. However, there was no statistically significant relationship between rh-Endo dose and induction of tumor cell or endothelial cell apoptosis. CONCLUSION These initial data suggest that rh-Endo has measurable effects on tumor blood flow and metabolism and induces endothelial and tumor cell apoptosis even in the absence of demonstrable anticancer effects. Further study and validation of these biomarkers in the context of antiangiogenic therapy will be required.

Protection from radiation-induced pneumonitis using cerium oxide nanoparticles

In an effort to combat the harmful effects of radiation exposure, we propose that rare-earth cerium oxide (CeO(2)) nanoparticles (free-radical scavengers) protect normal tissue from radiation-induced damage. Preliminary studies suggest that these nanoparticles may be a therapeutic regenerative nanomedicine that will scavenge reactive oxygen species, which are responsible for radiation-induced cell damage. The effectiveness of CeO(2) nanoparticles in radiation protection in murine models during high-dose radiation exposure is investigated, with the ultimate goal of offering a new approach to radiation protection, using nanotechnology. We show that CeO(2) nanoparticles are well tolerated by live animals, and they prevent the onset of radiation-induced pneumonitis when delivered to live animals exposed to high doses of radiation. In the end, these studies provide a tremendous potential for radioprotection and can lead to significant benefits for the preservation of human health and the quality of life for humans receiving radiation therapy.

Differential effects of vascular endothelial growth factor receptor-2 inhibitor ZD6474 on circulating endothelial progenitors and mature circulating endothelial cells: Implications for use as a surrogate marker of antiangiogenic activity

Purpose: Circulating endothelial cells (CEC) comprise at least two distinct populations: bone marrow–derived circulating endothelial progenitors (CEP) and mature CECs derived from existing vasculature. We hypothesized that antiangiogenic agents may have differential effects on CEPs and mature CECs and that these changes may serve as a marker of biological activity. Experimental Design: The effect of angiogenesis inhibitors on CECs was evaluated by flow cytometry after vascular endothelial growth factor (VEGF)–induced mobilization and in mice bearing Lewis lung carcinoma (LLC). Tumor angiogenesis was evaluated in parallel by immunohistochemistry. Results: In nontumor-bearing mice, VEGF administration increased both mature CECs and CEPs. This increase was inhibited by the VEGF receptor 2 inhibitor ZD6474 as well as the VEGF inhibitor–soluble Flt-1. ZD6474 had no significant effect on CECs in the absence of exogenous VEGF stimulation. In contrast, LLC-bearing mice had an increase in mature CECs but not CEPs after 3 days of treatment with ZD6474. The increase in mature CECs was dose-dependent, accompanied by a decrease in tumor microvessel density, and preceded reduction in tumor volume. Treatment of LLC-bearing mice with the vascular targeting agent ZD6126 also increased mature CECs. Conclusions: VEGF inhibitors can have differential effects on mature CECs and CEPs, and agents inhibiting tumor angiogenesis may cause a concomitant increase in mature CECs. This increase occurs in tumor-bearing but not in nontumor-bearing mice, suggesting that tumor endothelium is a potential source of mature CECs. Therefore, assessing both mature CECs and CEPs may provide insights into the mechanism of antiangiogenic agents and serve as an early surrogate marker of biological activity.

Blockade of Epidermal Growth Factor Receptor Signaling on Tumor Cells and Tumor-Associated Endothelial Cells for Therapy of Human Carcinomas

The purpose of this study was to determine whether the expression of epidermal growth factor receptor (EGF-R) and activated EGF-R by tumor-associated endothelial cells is influenced by interaction with specific growth factors in the microenvironment. Different human carcinoma cell lines expressing EGF-R with low or high levels of EGF/transforming growth factor (TGF)-alpha were implanted into orthotopic organs of nude mice. In the EGF/TGF-alpha-positive bladder cancer (253J-BV), pancreatic cancer (L3.6pl), and renal cancer (RBM1-IT) but not in the EGF/TGF-alpha-negative renal cancer SN12-PM6, tumor-associated endothelial cells expressed EGF-R and activated EGF-R. Mice were implanted with human 253J-BV bladder tumors (EGF+) or human SN12-PM6 renal tumors (EGF-). Treatment with oral PKI 166 (a specific inhibitor of EGF-R phosphorylation) alone, intraperitoneal paclitaxel alone (253J-BV), gemcitabine alone (SN12-PM6), or combination of PKI 166 and chemotherapy produced a 60%, 32%, or 81% reduction in the volume of 253J-BV bladder tumors, respectively, and 26%, 23%, or 51% reduction in the volume of SN12-PM6 kidney tumors, respectively. Immunohistochemical analyses demonstrated down-regulation of activated EGF-R in EGF/TGF-alpha-positive and EGF/TGF-alpha-negative lesions from mice treated with PKI 166, although apoptosis of tumor-associated endothelial cells was found only in EGF/TGF-alpha-positive tumors. Collectively, these data suggest that expression of activated EGF-R by tumor-associated endothelial cells provides an important target for therapy.

Sensitization of pancreatic cancer cells to radiation by cerium oxide nanoparticle-induced ROS production.

UNLABELLED Side effect of radiation therapy (RT) remains the most challenging issue for pancreatic cancer treatment. In this report we determined whether and how cerium oxide nanoparticles (CONPs) sensitize pancreatic cancer cells to RT. CONP pretreatment enhanced radiation-induced reactive oxygen species (ROS) production preferentially in acidic cell-free solutions as well as acidic human pancreatic cancer cells. In acidic environments, CONPs favor the scavenging of superoxide radical over the hydroxyl peroxide resulting in accumulation of the latter whereas in neutral pH CONPs scavenge both. CONP treatment prior to RT markedly potentiated the cancer cell apoptosis both in culture and in tumors and the inhibition of the pancreatic tumor growth without harming the normal tissues or host mice. Taken together, these results identify CONPs as a potentially novel RT-sensitizer as well as protectant for improving pancreatic cancer treatment. FROM THE CLINICAL EDITOR Pancreatic tumors remain some of the most notoriously treatment-unresponsive malignancies. Cerium oxide nanoparticles may be capable of sensitizing such cells to radiotherapy, as demonstrated in this study.

Noninvasive bioluminescence imaging of luciferase expressing intracranial U87 xenografts: correlation with magnetic resonance imaging determined tumor volume and longitudinal use in assessing tumor growth and antiangiogenic treatment effect.

OBJECTIVE: Outcome studies in rodent tumor models rely on both histological and noninvasive study end points. Intracranial models require special tools to observe tumor growth over time noninvasively, such as magnetic resonance imaging (MRI), computed tomographic scanning, or cranial window techniques. These techniques share disadvantages in terms of cost, technical expertise required, and overall animal throughput for analysis. In this report, we sought to validate the use of the relatively newer technique of bioluminescence imaging (BLI) of intracranial glioblastoma xenograft growth by comparing it with gadolinium-enhanced MRI. METHODS: U87MG glioma cell lines genetically engineered to express the firefly luciferase gene were stereotactically injected into nude mice in the left frontal lobe. Weekly BLI and MRI were performed after the inoculation of tumor cells. For BLI, tumor growth was assessed as the peak BLI after systemic injection of luciferin substrate. MRI-based growth curves were created by three-dimensional volumetric reconstruction of axial gadolinium-enhanced MRI data covering the whole brain. In a separate experiment, mice were treated with adenoviruses encoding antiangiogenic soluble vascular endothelial growth factor receptors, and treatment effect was monitored by BLI. RESULTS: Untreated tumor growth was readily detected and observed over time by serial BLI measurements. Furthermore, tumor-derived light emission was highly correlated with volume of tumor as assessed by MRI. Furthermore, the tested antiangiogenic treatment effect was readily detected using this technique, suggesting the power of the technique for sensitive monitoring of novel therapeutics. CONCLUSION: BLI offers a simple and rapid technique for assessing intracranial glioblastoma growth in rodent models noninvasively, which correlates well with MRI. The speed of the BLI technique can increase experimental throughput, allows for targeted histological analysis in animals showing the greatest treatment effects, and provides new insights into the kinetics of intracranial tumor growth in the setting of different treatments.

Inhibition of growth and metastasis of human pancreatic cancer growing in nude mice by PTK 787/ZK222584, an inhibitor of the vascular endothelial growth factor receptor tyrosine kinases.

Since vascular endothelial growth factor (VEGF) plays a major role in tumor angiogenesis, we determined whether blockage of VEGF receptor signaling using a novel tyrosine kinase inhibitor (PTK 787) decreases the growth and metastasis of human pancreatic carcinoma growing orthotopically in nude mice. Human pancreatic L3.6pl cells were injected into the pancreas of nude mice. Seven days later, groups of mice were given daily oral administrations of PTK 787 alone, twice weekly i.p. injections of gemcitabine, or combination therapy. The mice were necropsied when control mice became moribund (day 35). Therapy with PTK 787 alone, gemcitabine alone, or the combination of both agents produced respectively 60%, 70%, and 81% inhibition in the volume of pancreatic cancers. The combination therapy significantly decreased the incidence of lymph node and liver metastasis, leading to a significant increase in survival. Microvessel density (MVD) was significantly decreased in tumors treated with either PTK 787 alone or PTK 787 plus gemcitabine. MVD directly correlated with tumor cell proliferation and inversely correlated with apoptosis of tumor cells and associated endothelial cells. Collectively, our results demonstrate that blockade of VEGF-R signaling may provide an additional approach to the therapy of pancreatic cancer.

Simultaneous Inhibition of EGFR, VEGFR, and Platelet-Derived Growth Factor Receptor Signaling Combined with Gemcitabine Produces Therapy of Human Pancreatic Carcinoma and Prolongs Survival in an Orthotopic Nude Mouse Model

Although gemcitabine has been approved as the first-line chemotherapeutic reagent for pancreatic cancer, its response rate is low and average survival duration is still only marginal. Because epidermal growth factor receptor (EGFR), vascular endothelial growth factor receptor (VEGFR), and platelet-derived growth factor receptor (PDGFR) modulate tumor progression, we hypothesized that inhibition of phosphorylation of all three on tumor cells, tumor-associated endothelial cells, and stroma cells would improve the treatment efficacy of gemcitabine in an orthotopic pancreatic tumor model in nude mice and prolong survival. We implanted L3.6pl, a human pancreatic cancer cell, in the pancreas of nude mice. We found that tumor-associated endothelial cells in this model highly expressed phosphorylated EGFR, VEGFR, and PDGFR. Oral administration of AEE788, a dual tyrosine kinase inhibitor against EGFR and VEGFR, decreased phosphorylation of EGFR and VEGFR. PDGFR phosphorylation was inhibited by STI571. Although i.p. injection of gemcitabine did not inhibit tumor growth, its combination with AEE788 and STI571 produced >80% inhibition of tumor growth and prolonged survival in parallel with increases in number of tumor cells and tumor-associated endothelial cell apoptosis, decreased microvascular density, decreased proliferation rate, and prolonged survival. STI571 treatment also decreased pericyte coverage on tumor-associated endothelial cells. Thus, inhibiting phosphorylation of EGFR, VEGFR, and PDGFR in combination with gemcitabine enhanced the efficacy of gemcitabine, resulting in inhibition of experimental human pancreatic cancer growth and significant prolongation of survival.

Levetiracetam enhances p53-mediated MGMT inhibition and sensitizes glioblastoma cells to temozolomide

Antiepileptic drugs (AEDs) are frequently used to treat seizures in glioma patients. AEDs may have an unrecognized impact in modulating O(6)-methylguanine-DNA methyltransferase (MGMT), a DNA repair protein that has an important role in tumor cell resistance to alkylating agents. We report that levetiracetam (LEV) is the most potent MGMT inhibitor among several AEDs with diverse MGMT regulatory actions. In vitro, when used at concentrations within the human therapeutic range for seizure prophylaxis, LEV decreases MGMT protein and mRNA expression levels. Chromatin immunoprecipitation analysis reveals that LEV enhances p53 binding on the MGMT promoter by recruiting the mSin3A/histone deacetylase 1 (HDAC1) corepressor complex. However, LEV does not exert any MGMT inhibitory activity when the expression of either p53, mSin3A, or HDAC1 is abrogated. LEV inhibits malignant glioma cell proliferation and increases glioma cell sensitivity to the monofunctional alkylating agent temozolomide. In 4 newly diagnosed patients who had 2 craniotomies 7-14 days apart, prior to the initiation of any tumor-specific treatment, samples obtained before and after LEV treatment showed the inhibition of MGMT expression. Our results suggest that the choice of AED in patients with malignant gliomas may have an unrecognized impact in clinical practice and research trial design.