H.A. Curry

Intracellular oxidation/reduction status in the regulation of transcription factors NF-κB and AP-1

The eukaryotic cell contains a multitude of pathways coupling environmental stimuli to the specific regulation of gene expression. Two early response transcriptional complexes, NF-kappaB and AP-1, appear to respond to environmental stressors by inducing the expression of response specific downstream genes. Both are well-characterized transcriptional regulatory factors that are induced by a wide variety of seemingly unrelated exogenous and endogenous agents and serve important roles in cell growth and differentiation, immunity, inflammation, and other preprogrammed cellular genetic processes. The activities of NF-kappaB and AP-1 are also affected following exposure to chemicals, drugs, or other agents that appear to alter the cellular oxidation/reduction (redox) status. From these observations, it has been suggested that changes in cellular oxidation/reduction status, communicated via a series of cellular redox-sensitive signaling circuitry employing metal- and thiol-containing proteins, serve as common mechanisms linking environmental stressors to adaptive cellular responses. As such, these transcription factors are ideal paradigms to study the mechanism and possible physiological significance of early response genes in the cellular response to changes in cellular redox status. In this article we summarize the evidence suggesting that cellular redox regulates these transcription factors.

Heat Shock Inhibits Radiation-induced Activation of NF-κB via Inhibition of I-κB Kinase

Radiation stimulates signaling cascades that result in the activation of several transcription factors that are believed to play a central role in protective response(s) to ionizing radiation (IR). It is also well established that heat shock alters the regulation of signaling cascades and transcription factors and is a potent radiosensitizing agent. To explore the hypothesis that heat disrupts or alters the regulation of signaling factors activated by IR, the effect of heat shock on IR-induced activation of NF-κB was determined. Irradiated HeLa cells demonstrated transient increases in NF-κB DNA binding activity and NF-κB protein nuclear localization. In addition, irradiated cells demonstrated increased I-κB phosphorylation and decreased I-κBα cytoplasmic protein levels, corresponding temporally with the increase of NF-κB DNA binding. Heat shock prior to IR inhibited the increase in NF-κB DNA binding activity, nuclear localization of NF-κB, and the phosphorylation and subsequent degradation of I-κB. I-κB kinase (IKK) immunoprecipitation assays demonstrated an increase in IKK catalytic activity in response to IR that was inhibited by pretreatment with heat. Kinetic experiments determined that heat-induced inhibition of NF-κB activation in response to IR decayed within 5 h after heating. Furthermore, pretreatment with cycloheximide, to blockde novo protein synthesis, did not alter heat shock inhibition of IR induction of NF-κB. These experiments demonstrate that heat shock transiently inhibits IR induction of NF-κB DNA binding activity by preventing IKK activation and suggests a mechanism independent of protein synthesis.

Radiosensitizing and anti-proliferative effects of resveratrol in two human cervical tumor cell lines

Resveratrol is a polyphenol isolated from the skins of grapes that has been shown to significantly alter the cellular physiology of tumor cells, as well as block the process of initiation and progression. At least one mechanism for the intracellular actions of resveratrol involves the suppression of prostaglandin (PG) biosynthesis. The involvement of PGs and other eicosanoids in the development of human cancer is well established. PGs are synthesized from arachidonic acid via the cyclooxygenase pathway and have multiple physiological and pathological functions. In addition, evidence has arisen suggesting that PGs may be implicated in the cytotoxic and/or cytoprotective response of tumor cells to ionizing radiation (IR). As such, we hypothesized that tumor cells may exhibit changes in the cellular response to IR following exposure to resveratrol, a naturally occurring compound that inhibits cyclooxygenase-1 (COX-1) activity. Thus, clonogenic cell survival assays were performed using irradiated HeLa and SiHa cells pretreated with resveratrol prior to IR exposure, and resulted in enhanced tumor cell killing by IR in a dose-dependent manner. Further analysis of COX-1 inhibition indicated that resveratrol pretreatment: (1), inhibited cell division as assayed by growth curves; and (2), induced an early S phase cell cycle checkpoint arrest, as demonstrated by fluorescence-activated cell sorting, as well as bromodeoxyuridine pulse-chase analysis. These results suggest that resveratrol alters both cell cycle progression and the cytotoxic response to IR in two cervical tumor cell lines.

Heat shock and the activation of AP-1 and inhibition of NF-κB DNA-binding activity: possible role of intracellular redox status

The early response genes comprising the AP-1 and NF-κB transcription factors are induced by environmental stress and thought to modulate responses to injury processes through the induction of target genes. Exposure to heat and ionizing radiation (IR) has been shown to affect signalling machinery involved in AP-1 and NF-κB activation. Furthermore, regulation of the signalling pathways leading to the activation of these transcription factors has been linked to changes in intracellular oxidation/reduction (redox) reactions. The hypothesis is proposed that exposure to thermal stress and/or IR might alter metabolic processes impacting upon cellular redox state and thereby modify the activity of redox-sensitive transcription factors such as AP-1 and NF-κB. Gel electromobility shift assays (EMSA) demonstrated that heat shock-induced AP-1 DNA-binding activity but inhibited IR-induced activation of NF-κB. A time course showed that activation of the AP-1 complex occurs between 4 and 5 h following thermal stress, and inhibition of IR-induced NF-κB activation also occurs during this time interval. Using a redox-sensitive fluorescent probe [5-(and -6)-carboxy-2′,7′-dichlorodihydrofluorescein diacetate], a shift to 40% less intracellular dye oxidation was observed in HeLa cells 0–4 h post-heat shock (45°C, 15 min) relative to cells held at 37°C. This was followed by a shift to greater dye oxidation between 4 and 12 h after treatment (about 1.8-fold) that returned to control levels by 24 h post-heating. These results show changes in DNA-binding activity closely paralleled apparent heat-induced changes in the intracellular redox state. Taken together, these results provide correlative evidence for disruption of redox-sensitive IR-induced signalling pathways by heat shock and support the hypothesis that this mechanism might play a role in heat-induced alterations in radiation response.

Inhibition of cyclooxygenase‐2 with NS‐398 and the prevention of radiation‐induced transformation, micronuclei formation and clonogenic cell death in C3H 10T1/2 cells

Purpose: Abnormally high levels of the cyclooxygenase (COX)‐2 isozyme as well as the prostaglandin metabolites produced by the COX pathway have been observed in a variety of malignancies, including cancers of the skin, pancreas, colon, breast, cervix, prostate, and head and neck. Furthermore, exogenous genotoxic agents, including ionizing radiation (IR), have been shown to induce cellular transformation and to elevate COX‐2 activity, whereas exposure to agents that specifically inhibit COX‐2 activity have been shown to inhibit transformation. These data suggest a possible role of COX‐2 both in IR‐mediated cellular transformation processes and cell death. Materials and methods: C3H 10T1/2 and/or HeLa cells were treated with N‐[2‐(cyclohexyloxy)‐4‐nitrophenyl]‐methanesulfonamide (NS‐398) and/or exposed to IR. Following treatment, cells were assayed for neoplastic transformation, clonogenicity, growth rates, cell cycle distribution, micronuclei formation and DNA damage by established methodologies. Statistical tests were performed on data as described. Results: In the present study, experiments in normal murine fibroblast C3H 10T1/2 cells demonstrated that the chemical inhibition of COX‐2 activity with moderate doses of NS‐398 abrogated IR‐induced transformation events by fourfold and protected irradiated C3H 10T1/2 cells from clonogenic cell death. Considering that these doses of NS‐398 had no significant effect on cellular proliferation or cell cycle distribution in C3H 10T1/2 cells, the results suggest that inhibition of COX‐2 either increases DNA repair or prevents the accumulation of DNA damage. In supplemental experiments, treatment with NS‐398 caused a 1.5‐fold reduction in IR‐induced micronuclei formation and a significant decrease in DNA damage. Conclusions: These results suggest a role for COX‐2 inhibitors in the normal tissue response to IR when administered at therapeutically achievable doses and therefore may have clinical implications for radiation oncology patients in the prevention of IR‐induced malignancy.

Overutilization of IMRT/IGRT in treatment of rectal cancer: Cost implications of deviation from evidence-based practices.

34 Background: Current evidence-based guidelines for management of rectal cancer (RC) caution against routine use of IMRT and do not address the role of IGRT. To explore patterns of care and cost implications for treatment of RC in commercially insured patients, we assessed treatment requests submitted for preauthorization through eviti Connect. METHODS A proprietary web-based application enables oncology providers to obtain automated precertification for patients insured by payers across the US that use the platform. All requests for pelvic radiation for treatment of RC submitted from 6/1/11-5/31/14 were reviewed. Treatment delivery costs for 3D CRT + weekly port films and for IMRT + IGRT were calculated based on average reimbursement rates from 3 payers for a typical course of 50.4 Gy/28 fractions. RESULTS A total of 195 cases for treatment of RC were submitted. At submission, 50.3% (98/195) of cases met evidence based standards and received automated preauthorization; 49.7% required treatment justification. Ninety-eight percent of deviations involved use of IMRT and/or IGRT. Upon review, 34.9% (68/195) had a medical rationale for the variance. Justification for IMRT/IGRT use included treatment volumes comparable to anal cancer, inadequate bowel displacement by routine techniques, and obesity. Fifteen percent (29/195) contained unwarranted deviations. In 23/29 cases peer to peer discussion resulted in the provider altering the plan to be compliant. Providers did not agree to changes in the other 6 cases. Cost for a course of 3D CRT + weekly port films was

Compliance with evidence-based guidelines for radiation of vertebral metastases.

6,591 vs.

Adoption of Hypofractionated Radiation Therapy for Breast Cancer After Publication of Randomized Trials

32,292 for IMRT + daily IGRT. For these 195 cases, the estimated cost of overutilization of IMRT/IGRT was

Postirradiation Morphea: A Case Report With a Review of the Literature and Summary of the Clinicopathologic Differential Diagnosis.

745,000 (

Utilization of Hypofractionated Regimens for the Treatment of Bone Metastases Before and After Initiation of ASTRO’s “Choosing Wisely” Campaign

25,700 X 29). CONCLUSIONS Despite lack of endorsement by consensus group guidelines, IMRT and IGRT were prescribed for treatment of RC in nearly 50% of cases. Case review and peer to peer discussion clarified the rationale for treatment deviations from guidelines and allowed providers to bring plans into compliance with evidence based practices, reducing inappropriate use of IMRT/IGRT from 15% to 3%. Reduction in unwarranted use of high cost technologies can improve quality and yield significant cost savings.