Pharmacological Ascorbate as a Means of Sensitizing Cancer Cells to Radio-Chemotherapy While Protecting Normal Tissue.

Chemoradiation has remained the standard of care treatment for many of the most aggressive cancers. However, despite effective toxicity to cancer cells, current chemoradiation regimens are limited in efficacy due to significant normal cell toxicity. Thus, efforts have been made to identify agents demonstrating selective toxicity, whereby treatments simultaneously sensitize cancer cells to protect normal cells from chemoradiation. Pharmacological ascorbate (intravenous infusions of vitamin C resulting in plasma ascorbate concentrations ≥20 mM; P-AscH-) has demonstrated selective toxicity in a variety of preclinical tumor models and is currently being assessed as an adjuvant to standard-of-care therapies in several early phase clinical trials. This review summarizes the most current preclinical and clinical data available demonstrating the multidimensional role of P-AscH- in cancer therapy including: selective toxicity to cancer cells via a hydrogen peroxide (H2O2)-mediated mechanism; action as a sensitizing agent of cancer cells to chemoradiation; a protectant of normal tissues exposed to chemoradiation; and its safety and tolerability in clinical trials.

Persistent Oxidative Stress in Vestibular Schwannomas After Stereotactic Radiation Therapy.

OBJECTIVE: Stereotactic radiation therapy is increasingly used to treat vestibular schwannomas (VSs) primarily and to treat tumor remnants following microsurgery. Little data are available regarding the effects of radiation on VS cells. Tyrosine nitrosylation is a marker of oxidative stress following radiation in malignant tumors. It is not known how long irradiated tissue remains under oxidative stress, and if such modifications occur in benign neoplasms such as VSs treated with significantly lower doses of radiation. We immunostained sections from previously radiated VSs with an antibody that recognizes nitrosylated tyrosine residues to assess for ongoing oxidative stress. STUDY DESIGN: Immunohistochemical analysis. METHODS: Four VSs, which recurred after excision, were treated with stereotactic radiation therapy. Ultimately each tumor required salvage reresection for regrowth. Histologic sections of each tumor before and after radiation were immunolabeled with a monoclonal antibody specific to nitrotyrosine and compared. Two VSs that underwent reresection of a growing tumor remnant without previous radiation therapy served as additional controls. RESULTS: Irradiated tumors enlarged in volume by 3.16 to 8.62 mL following radiation. Preradiation sections demonstrated little to no nitrotyrosine immunostaining. Three of four of irradiated VSs demonstrated increased nitrotyrosine immunostaining in the postradiation sections compared with preradiation tumor sections. Nonirradiated VSs did not label with the antinitrotyrosine antibody. CONCLUSIONS: VSs exhibit oxidative stress up to 7 years after radiotherapy, yet these VSs continued to enlarge. Thus, VSs that grow following radiation appear to possess mechanisms for cell survival and proliferation despite radiation-induced oxidative stress.

Pharmacologic ascorbate (P-AscH-) suppresses hypoxia-inducible Factor-1α (HIF-1α) in pancreatic adenocarcinoma.

HIF-1α is a transcriptional regulator that functions in the adaptation of cells to hypoxic conditions; it strongly impacts the prognosis of patients with cancer. High-dose, intravenous, pharmacological ascorbate (P-AscH-), induces cytotoxicity and oxidative stress selectively in cancer cells by acting as a pro-drug for the delivery of hydrogen peroxide (H2O2); early clinical data suggest improved survival and inhibition of metastasis in patients being actively treated with P-AscH-. Previous studies have demonstrated that activation of HIF-1α is necessary for P-AscH- sensitivity. We hypothesized that pancreatic cancer (PDAC) progression and metastasis could be be targeted by P-AscH- via H2O2-mediated inhibition of HIF-1α stabilization. Our study demonstrates an oxygen- and prolyl hydroxylase-independent regulation of HIF-1α by P-AscH-. Additionally, P-AscH- decreased VEGF secretion in a dose-dependent manner that was reversible with catalase, consistent with an H2O2-mediated mechanism. Pharmacological and genetic manipulations of HIF-1α did not alter P-AscH--induced cytotoxicity. In vivo, P-AscH- inhibited tumor growth and VEGF expression. We conclude that P-AscH- suppresses the levels of HIF-1α protein in hypoxic conditions through a post-translational mechanism. These findings suggest potential new therapies specifically designed to inhibit the mechanisms that drive metastases as a part of PDAC treatment.

Redox active metals and H2O2 mediate the increased efficacy of pharmacological ascorbate in combination with gemcitabine or radiation in pre-clinical sarcoma models.

Soft tissue sarcomas are a histologically heterogeneous group of rare mesenchymal cancers for which treatment options leading to increased overall survival have not improved in over two decades. The current study shows that pharmacological ascorbate (systemic high dose vitamin C achieving ≥ 20mM plasma levels) is a potentially efficacious and easily integrable addition to current standard of care treatment strategies in preclinical models of fibrosarcoma and liposarcoma both in vitro and in vivo. Furthermore, enhanced ascorbate-mediated toxicity and DNA damage in these sarcoma models were found to be dependent upon H2O2 and intracellular labile iron. Together, these data support the hypothesis that pharmacological ascorbate may represent an easily implementable and non-toxic addition to conventional sarcoma therapies based on taking advantage of fundamental differences in cancer cell oxidative metabolism.

O2⋠- and H2O2-Mediated Disruption of Fe Metabolism Causes the Differential Susceptibility of NSCLC and GBM Cancer Cells to Pharmacological Ascorbate.

Pharmacological ascorbate has been proposed as a potential anti-cancer agent when combined with radiation and chemotherapy. The anti-cancer effects of ascorbate are hypothesized to involve the autoxidation of ascorbate leading to increased steady-state levels of H2O2; however, the mechanism(s) for cancer cell-selective toxicity remain unknown. The current study shows that alterations in cancer cell mitochondrial oxidative metabolism resulting in increased levels of O2⋅- and H2O2 are capable of disrupting intracellular iron metabolism, thereby selectively sensitizing non-small-cell lung cancer (NSCLC) and glioblastoma (GBM) cells to ascorbate through pro-oxidant chemistry involving redox-active labile iron and H2O2. In addition, preclinical studies and clinical trials demonstrate the feasibility, selective toxicity, tolerability, and potential efficacy of pharmacological ascorbate in GBM and NSCLC therapy.

Fluorine-18-Labeled Thymidine Positron Emission Tomography (FLT-PET) as an Index of Cell Proliferation after Pharmacological Ascorbate-Based Therapy.

Pharmacological ascorbate (AscH(-)) induces cytotoxicity and oxidative stress selectively in pancreatic cancer cells compared with normal cells. Positron emission tomography (PET) with the thymidine analog 3'-deoxy-3'-((18)F) fluorothymidine (FLT) enables noninvasive imaging and quantification of the proliferation fraction of tumors. We hypothesized that the rate of tumor proliferation determined by FLT-PET imaging, would be inversely proportional to tumor susceptibility to pharmacological AscH(-)-based treatments. Indeed, there was decreased FLT uptake in human pancreatic cancer cells treated with AscH(-) in vitro, and this effect was abrogated by co-treatment with catalase. In separate experiments, cells were treated with AscH(-), ionizing radiation or a combination of both. These studies demonstrated that combined AscH(-) and radiation treatment resulted in a significant decrease in FLT uptake that directly correlated with decreased clonogenic survival. MicroPET (18)F-FLT scans of mice with pre-established tumors demonstrated that AscH(-) treatment induced radiosensitization compared to radiation treatment alone. These data support testing of pharmacological ascorbate as a radiosensitizer in pancreatic cancer as well as the use of FLT-PET to monitor response to therapy.

Pharmacological Ascorbate Radiosensitizes Pancreatic Cancer.

The toxicity of pharmacologic ascorbate is mediated by the generation of H2O2 via the oxidation of ascorbate. Because pancreatic cancer cells are sensitive to H2O2 generated by ascorbate, they would also be expected to become sensitized to agents that increase oxidative damage such as ionizing radiation. The current study demonstrates that pharmacologic ascorbate enhances the cytotoxic effects of ionizing radiation as seen by decreased cell viability and clonogenic survival in all pancreatic cancer cell lines examined, but not in nontumorigenic pancreatic ductal epithelial cells. Ascorbate radiosensitization was associated with an increase in oxidative stress-induced DNA damage, which was reversed by catalase. In mice with established heterotopic and orthotopic pancreatic tumor xenografts, pharmacologic ascorbate combined with ionizing radiation decreased tumor growth and increased survival, without damaging the gastrointestinal tract or increasing systemic changes in parameters indicative of oxidative stress. Our results demonstrate the potential clinical utility of pharmacologic ascorbate as a radiosensitizer in the treatment of pancreatic cancer.

Ketogenic diets as an adjuvant cancer therapy: History and potential mechanism.

Cancer cells, relative to normal cells, demonstrate significant alterations in metabolism that are proposed to result in increased steady-state levels of mitochondrial-derived reactive oxygen species (ROS) such as O2(•-)and H2O2. It has also been proposed that cancer cells increase glucose and hydroperoxide metabolism to compensate for increased levels of ROS. Given this theoretical construct, it is reasonable to propose that forcing cancer cells to use mitochondrial oxidative metabolism by feeding ketogenic diets that are high in fats and low in glucose and other carbohydrates, would selectively cause metabolic oxidative stress in cancer versus normal cells. Increased metabolic oxidative stress in cancer cells would in turn be predicted to selectively sensitize cancer cells to conventional radiation and chemotherapies. This review summarizes the evidence supporting the hypothesis that ketogenic diets may be safely used as an adjuvant therapy to conventional radiation and chemotherapies and discusses the proposed mechanisms by which ketogenic diets may enhance cancer cell therapeutic responses.

Extracellular superoxide dismutase suppresses hypoxia-inducible factor-1α in pancreatic cancer.

Hypoxia-inducible factor-1 (HIF-1) is a heterodimeric transcription factor that governs cellular responses to reduced oxygen availability by mediating crucial homeostatic processes and is a major survival determinant for tumor cells growing in a low-oxygen environment. Clinically, HIF-1α seems to be important in pancreatic cancer, as HIF-1α correlates with metastatic status of the tumor. Extracellular superoxide dismutase (EcSOD) inhibits pancreatic cancer cell growth by scavenging nonmitochondrial superoxide. We hypothesized that EcSOD overexpression leads to changes in the O2(-)/H2O2 balance modulating the redox status affecting signal transduction pathways. Both transient and stable overexpression of EcSOD suppressed the hypoxic accumulation of HIF-1α in human pancreatic cancer cells. This suppression of HIF-1α had a strong inverse correlation with levels of EcSOD protein. Coexpression of the hydrogen peroxide-removing protein glutathione peroxidase did not prevent the EcSOD-induced suppression of HIF-1α, suggesting that the degradation of HIF-1α observed with high EcSOD overexpression is possibly due to a low steady-state level of superoxide. Hypoxic induction of vascular endothelial growth factor (VEGF) was also suppressed with increased EcSOD. Intratumoral injections of an adenoviral vector containing the EcSOD gene into preestablished pancreatic tumors suppressed both VEGF levels and tumor growth. These results demonstrate that the transcription factor HIF-1α and its important gene target VEGF can be modulated by the antioxidant enzyme EcSOD.

NOTCH3 is a prognostic factor that promotes glioma cell proliferation, migration and invasion via activation of CCND1 and EGFR.

Using a GWA analysis of a comprehensive glioma specimen population, we identified whole gain of chromosome 19 as one of the major chromosomal aberrations that correlates to patients' outcomes. Our analysis of significant loci revealed for the first time NOTCH3 as one of the most significant amplification. NOTCH3 amplification is associated with worse outcome compared to tumors with non-amplified locus. NOTCH receptors (NOTCH1-4) are key positive regulators of cell-cell interactions, angiogenesis, cell adhesion and stem cell niche development which have been shown to play critical roles in several human cancers. Our objective is to determine the molecular roles of NOTCH3 in glioma pathogenesis and aggressiveness. Here we show for the first time that NOTCH3 plays a major role in glioma cell proliferation, cell migration, invasion and apoptosis. Therefore, our study uncovers the prognostic value and the oncogenic function of NOTCH3 in gliomagenesis and supports NOTCH3 as a promising target of therapy in high grade glioma. Our studies allowed the identification of a subset of population that may benefit from GSI- or anti-NOTCH3- based therapies. This may lead to the design of novel strategies to improve therapeutic outcome of patients with glioma by establishing medical and scientific basis for personalized chemotherapies.

Manganoporphyrins increase ascorbate-induced cytotoxicity by enhancing H2O2 generation.

Renewed interest in using pharmacological ascorbate (AscH-) to treat cancer has prompted interest in leveraging its cytotoxic mechanism of action. A central feature of AscH- action in cancer cells is its ability to act as an electron donor to O2 for generating H2O2. We hypothesized that catalytic manganoporphyrins (MnP) would increase AscH- oxidation rates, thereby increasing H2O2 fluxes and cytotoxicity. Three different MnPs were tested (MnTBAP, MnT2EPyP, and MnT4MPyP), exhibiting a range of physicochemical and thermodynamic properties. Of the MnPs tested, MnT4MPyP exerted the greatest effect on increasing the rate of AscH- oxidation as determined by the concentration of ascorbate radical [Asc•-] and the rate of oxygen consumption. At concentrations that had minimal effects alone, combining MnPs and AscH- synergized to decrease clonogenic survival in human pancreatic cancer cells. This cytotoxic effect was reversed by catalase, but not superoxide dismutase, consistent with a mechanism mediated by H2O2. MnPs increased steady-state concentrations of Asc•- upon ex vivo addition to whole blood obtained either from mice infused with AscH- or patients treated with pharmacologic AscH-. Finally, tumor growth in vivo was inhibited more effectively by combining MnT4MPyP with AscH-. We concluded that MnPs increase the rate of oxidation of AscH- to leverage H2O2 flux and ascorbate-induced cytotoxicity.

Tumor derived mutations of protein tyrosine phosphatase receptor type k affect its function and alter sensitivity to chemotherapeutics in glioma.

Poor prognosis and resistance to therapy in malignant gliomas is mainly due to the highly dispersive nature of glioma cells. This dispersive characteristic results from genetic alterations in key regulators of cell migration and diffusion. A better understanding of these regulatory signals holds promise to improve overall survival and response to therapy. Using mapping arrays to screen for genomic alterations in gliomas, we recently identified alterations of the protein tyrosine phosphatase receptor type kappa gene (PTPRK) that correlate to patient outcomes. These PTPRK alterations are very relevant to glioma biology as PTPRK can directly sense cell-cell contact and is a dephosphorylation regulator of tyrosine phosphorylation signaling, which is a major driving force behind tumor development and progression. Subsequent sequencing of the full length PTPRK transcripts revealed novel PTPRK gene deletion and missense mutations in numerous glioma biopsies. PTPRK mutations were cloned and expressed in PTPRK-null malignant glioma cells. The effect of these mutations on PTPRK anti-oncogenic function and their association with response to anti-glioma therapeutics, such as temozolomide and tyrosine kinase inhibitors, was subsequently analyzed using in vitro cell-based assays. These genetic variations altered PTPRK activity and its post-translational processing. Reconstitution of wild-type PTPRK in malignant glioma cell lines suppressed cell growth and migration by inhibiting EGFR and ß-catenin signaling and improved the effect of conventional therapies for glioma. However, PTPRK mutations abrogated tumor suppressive effects of wild-type PTPRK and altered sensitivity of glioma cells to chemotherapy.

Erlotinib-mediated inhibition of EGFR signaling induces metabolic oxidative stress through NOX4.

Redox regulation of epidermal growth factor receptor (EGFR) signaling helps protect cells against oxidative stress. In this study, we investigated whether the cytotoxicity of an EGFR tyrosine kinase inhibitor, erlotinib (ERL), was mediated by induction of oxidative stress in human head and neck cancer (HNSCC) cells. ERL elicited cytotoxicity in vitro and in vivo while increasing a panel of oxidative stress parameters which were all reversible by the antioxidant N-acetyl cysteine. Knockdown of EGFR by using siRNA similarly increased these oxidative stress parameters. Overexpression of mitochondrial targeted catalase but not superoxide dismutase reversed ERL-induced cytotoxicity. Consistent with a general role for NADPH oxidase (NOX) enzymes in ERL-induced oxidative stress, ERL-induced cytotoxicity was reversed by diphenylene iodonium, a NOX complex inhibitor. ERL reduced the expression of NOX1, NOX2, and NOX5 but induced the expression of NOX4. Knockdown of NOX4 by using siRNA protected HNSCC cells from ERL-induced cytotoxicity and oxidative stress. Our findings support the concept that ERL-induced cytotoxicity is based on a specific mechanism of oxidative stress mediated by hydrogen peroxide production through NOX4 signaling.

Trends in brain cancer incidence and survival in the United States: Surveillance, Epidemiology, and End Results Program, 1973 to 2001.

OBJECT: An increasing incidence of brain cancer has been reported for the last three decades. In this study of brain cancer incidence and patient survival in the US, the authors attempt to update information on trends by examining data provided by the Surveillance, Epidemiology, and End Results (SEER) Program. METHODS: Population-based data from the SEER Program were used to calculate the incidence of and survival rates for people with brain cancer. The approximate Poisson method was used to calculate relative risks for brain cancer and to determine a 95% confidence interval. Annual age-standardized incidence rates were calculated, and time-trend analysis was conducted using joinpoint regression analysis. The relative risks of brain cancer were 1.48 for men compared with women, 3.18 for elderly persons compared with young adults, 1.86 for Caucasian patients compared with African-American patients, and 1.35 for those in metropolitan counties compared with those in nonmetropolitan counties. The incidence of brain cancer increased until 1987, when the annual percentage of change reversed direction, decreasing from 1.68 to 20.44%. The elderly experienced an increase until 1985, but their rates were stable thereafter. Rising trends were noticed for glioblastoma multiforme (GBM), oligodendroglioma, anaplastic astrocytoma, medulloblastoma, and mixed glioma, and falling trends were observed for astrocytoma not otherwise specified and malignant glioma. The survival rate for patients with GBM has not shown improvement in the last two decades. CONCLUSIONS: Increased risk of brain cancer is associated with being male, Caucasian, elderly, and residing in a metropolitan county. The incidence rate of brain cancer in the US is gradually declining, but the rising trend of GBM combined with its poor survival rate is disconcerting and needs further exploration.

Genetic characterization of commonly used glioma cell lines in the rat animal model system.

OBJECT: Animal models have been used extensively to discern the molecular biology of diseases and to gain insight into treatments. Nevertheless, discrepancies in the effects of treatments and procedures have been encountered during the transition from these animal models to application of the information to clinical trials in humans. To assess the genetic similarities between human gliomas and four cell lines used routinely in animal models, the authors used microarray technology to characterize the similarities and differences in gene expression. METHODS: To define the changes in gene expression, normal rat astrocytes were compared with four rat glioma cell lines (C6, 9L, F98, and RG2). The data were analyzed using two different methods: fold-change analysis and statistical analysis with t statistics. The gene products that were highlighted after intersecting the lists generated by the two methods of analysis were scrutinized against changes in gene expression reported in the literature. Tumorigenesis involves three major steps: the accumulation of genetic alterations, uncontrolled growth, and selected survival of transformed cells. The discussion of the results focuses attention on genes whose primary function is in pathways involved in glioma proliferation, infiltration, and neovascularization. A comparative microarray analysis of differentially expressed genes for four of the commonly used rat tumor cell lines is presented here. CONCLUSIONS: Due to the variances between the cell lines and results from analyses in humans, caution must be observed in interpreting as well as in the translation of information learned from animal models to its application in human trials.