Jennifer Fazzari

Bystander eff ects in tumor-free and tumor-bearing rat brains following irradiation by synchrotron X-rays

Abstract Purpose: Synchrotron microbeam radiation therapy (MRT) is a radiosurgery concept in the preclinical stage, developed mainly for brain tumor treatment. Experimental studies suggest that with MRT a better therapeutic index can be obtained than with homogenous field radiotherapy, but the underlying cellular mechanisms need further understanding. The aim of this study was to investigate the dynamics of radiation-induced bystander effects (RIBE) in rats after exposing one brain hemisphere to either MRT or homogenous synchrotron radiation (HSR). Materials and methods: Healthy and tumor-bearing Wistar rats were exposed to doses of 17.5, 35, 70 or 350 Gy, applied either as MRT or HSR to the right cerebral hemisphere. Rats were euthanized at 4, 8 and 12 hours after irradiation to assess the release of bystander signals. Brains and urinary bladders were dissected, and explants for bystander clonogenic reporter assays were set up. Results: Clonogenic survival showed that RIBE occurred in both the non-irradiated brain hemisphere and in bladder of normal and tumor-bearing rats, while the irradiated hemisphere showed the direct effects of radiation. Conclusion: The RIBE observed in our reporter cells shows that both MRT and HSR yield a demonstrable abscopal effect after high doses of irradiation; presumably as part of a systemic response.

TRANSMISSION OF SIGNALS FROM RATS RECEIVING HIGH DOSES OF MICROBEAM RADIATION TO CAGE MATES: AN INTER-MAMMAL BYSTANDER EFFECT

Inter-animal signaling from irradiated to non-irradiated organisms has been demonstrated for whole body irradiated mice and also for fish. The aim of the current study was to look at radiotherapy style limited exposure to part of the body using doses relevant in preclinical therapy. High dose homogenous field irradiation and the use of irradiation in the microbeam radiation therapy mode at the European Synchrotron Radiation Facility (ESRF) at Grenoble was tested by giving high doses to the right brain hemisphere of the rat. The right and left cerebral hemispheres and the urinary bladder were later removed to determine whether abscopal effects could be produced in the animals and also whether effects occurred in cage mates housed with them. The results show strong bystander signal production in the contra-lateral brain hemisphere and weaker effects in the distant bladder of the irradiated rats. Signal strength was similar or greater in each tissue in the cage mates housed for 48hrs with the irradiated rats. Our results support the hypothesis that proximity to an irradiated animal induces signalling changes in an unirradiated partner. If similar signaling occurs between humans, the results could have implications for caregivers and hospital staff treating radiotherapy patients.

Evidence for a physical component to the radiation-induced bystander effect?

Abstract Purpose: The nature of the transferrable factor which goes from irradiated objects to bystander objects remains undefined. Most agree that a chemical entity is the likely ‘factor’ although some authors have produced in vitro evidence for the involvement of a physical component or a very potent volatile capable of traveling through air distances. In this paper we test the hypothesis that the communicated signal may be physical at least in part. Methods: The in vivo fish model was used to allow signal production and response to occur in organisms in vivo without any shared blood or central nervous system (CNS) connections. A reporter assay and calcium flux measurements were used to detect signal production when irradiated fish were separated from unirradiated fish by (a) a plastic container, and (b) a foil-wrapped plastic container. Results: The unirradiated fish showed bystander effects in both cases. The use of foil excludes the possibility of a light signal and although a highly active volatile could travel from one tank to another, the arrangement of sham and irradiated tanks makes it highly unlikely that this could explain our result. Conclusion: We conclude that there must be a physical component in the mechanism such as a weak acoustic or electromagnetic signal.

Effect of 5-hydroxytryptamine (serotonin) receptor inhibitors on the radiation-induced bystander effect

Abstract Purpose: To test the importance of serotonin as a signaling molecule involved in the production and response of radiation-induced bystander effects. Materials and methods: HPV-G human keratinocyte cultures were spiked with various concentrations of Granisetron or Ketanserin and subject to either 0 Gy or 0.5 Gy X-irradiation to observe the inhibitors effects on bystander signal production. Medium from these cultures was harvested and introduced to non- irradiated cultures of the same cell line to determine the clonogenic bystander response. Separate HPV-G cultures were set up for subsequent calcium measurements in response to irradiated cell conditioned medium (ICCM) in the presence or absence of Granisetron in an attempt to block bystander signal response. Results: Granisetron and Ketanserin produced a dose-dependent propagation of the bystander effect in recipient cultures. Granisetron completely abolished the characteristic calcium pulse observed when non-irradiated cultures are exposed to irradiated cell medium in the presence of this drug. Conclusions: Serotonin-dependent mechanisms appear to be involved in bystander signal production and response to radiation in this system.

Inhibitors of glutamate release from breast cancer cells; new targets for cancer-induced bone-pain

Glutamate is an important signaling molecule in a wide variety of tissues. Aberrant glutamatergic signaling disrupts normal tissue homeostasis and induces several disruptive pathological conditions including pain. Breast cancer cells secrete high levels of glutamate and often metastasize to bone. Exogenous glutamate can disrupt normal bone turnover and may be responsible for cancer-induced bone pain (CIBP). CIBP is a significant co-morbidity that affects quality of life for many advanced-stage breast cancer patients. Current treatment options are commonly accompanied by serious side-effects that negatively impact patient care. Identifying small molecule inhibitors of glutamate release from aggressive breast cancer cells advances a novel, mechanistic approach to targeting CIBP that could advance treatment for several pathological conditions. Using high-throughput screening, we investigated the ability of approximately 30,000 compounds from the Canadian Compound Collection to reduce glutamate release from MDA-MB-231 breast cancer cells. This line is known to secrete high levels of glutamate and has been demonstrated to induce CIBP by this mechanism. Positive chemical hits were based on the potency of each molecule relative to a known pharmacological inhibitor of glutamate release, sulfasalazine. Efficacy was confirmed and drug-like molecules were identified as potent inhibitors of glutamate secretion from MDA-MB-231, MCF-7 and Mat-Ly-Lu cells.

Behavioural Effects of Using Sulfasalazine to Inhibit Glutamate Released by Cancer Cells: A Novel target for Cancer-Induced Depression

Despite the lack of robust evidence of effectiveness, current treatment options for cancer-induced depression (CID) are limited to those developed for non-cancer related depression. Here, anhedonia-like and coping behaviours were assessed in female BALB/c mice inoculated with 4T1 mammary carcinoma cells. The behavioural effects of orally administered sulfasalazine (SSZ), a system xc− inhibitor, were compared with fluoxetine (FLX). FLX and SSZ prevented the development of anhedonia-like behaviour on the sucrose preference test (SPT) and passive coping behaviour on the forced swim test (FST). The SSZ metabolites 5-aminosalicylic acid (5-ASA) and sulfapyridine (SP) exerted an effect on the SPT but not on the FST. Although 5-ASA is a known anti-inflammatory agent, neither treatment with SSZ nor 5-ASA/SP prevented tumour-induced increases in serum levels of interleukin-1β (IL-1β) and IL-6, which are indicated in depressive disorders. Thus, the observed antidepressant-like effect of SSZ may primarily be attributable to the intact form of the drug, which inhibits system xc−. This study represents the first attempt at targeting cancer cells as a therapeutic strategy for CID, rather than targeting downstream effects of tumour burden on the central nervous system. In doing so, we have also begun to characterize the molecular pathways of CID.

Biological Entanglement–Like Effect After Communication of Fish Prior to X-Ray Exposure:

The phenomenon by which irradiated organisms including cells in vitro communicate with unirradiated neighbors is well established in biology as the radiation-induced bystander effect (RIBE). Generally, the purpose of this communication is thought to be protective and adaptive, reflecting a highly conserved evolutionary mechanism enabling rapid adjustment to stressors in the environment. Stressors known to induce the effect were recently shown to include chemicals and even pathological agents. The mechanism is unknown but our group has evidence that physical signals such as biophotons acting on cellular photoreceptors may be implicated. This raises the question of whether quantum biological processes may occur as have been demonstrated in plant photosynthesis. To test this hypothesis, we decided to see whether any form of entanglement was operational in the system. Fish from 2 completely separate locations were allowed to meet for 2 hours either before or after which fish from 1 location only (group A fish) were irradiated. The results confirm RIBE signal production in both skin and gill of fish, meeting both before and after irradiation of group A fish. The proteomic analysis revealed that direct irradiation resulted in pro-tumorigenic proteomic responses in rainbow trout. However, communication from these irradiated fish, both before and after they had been exposed to a 0.5 Gy X-ray dose, resulted in largely beneficial proteomic responses in completely nonirradiated trout. The results suggest that some form of anticipation of a stressor may occur leading to a preconditioning effect or temporally displaced awareness after the fish become entangled.

TUMOUR-DERIVED GLUTAMATE: LINKING ABERRANT CANCER CELL METABOLISM TO PERIPHERAL SENSORY PAIN PATHWAYS.

Background Chronic pain is a major symptom that develops in cancer patients, most commonly emerging during advanced stages of the disease. The nature of cancer-induced pain is complex, and the efficacy of current therapeutic interventions is restricted by the dose-limiting side-effects that accompany common centrally targeted analgesics. Methods This review focuses on how up-regulated glutamate production and export by the tumour converge at peripheral afferent nerve terminals to transmit nociceptive signals through the transient receptor cation channel, TRPV1, thereby initiating central sensitization in response to peripheral disease-mediated stimuli. Results Cancer cells undergo numerous metabolic changes that include increased glutamine catabolism and over-expression of enzymes involved in glutaminolysis, including glutaminase. This mitochondrial enzyme mediates glutaminolysis, producing large pools of intracellular glutamate. Up-regulation of the plasma membrane cystine/glutamate antiporter, system xc-, promotes aberrant glutamate release from cancer cells. Increased levels of extracellular glutamate have been associated with the progression of cancer-induced pain and we discuss how this can be mediated by activation of TRPV1. Conclusion With a growing population of patients receiving inadequate treatment for intractable pain, new targets need to be considered to better address this largely unmet clinical need for improving their quality of life. A better understanding of the mechanisms that underlie the unique qualities of cancer pain will help to identify novel targets that are able to limit the initiation of pain from a peripheral source–the tumour.

Homogenous and Microbeam X-Ray Radiation Induces Proteomic Changes in the Brains of Irradiated Rats and in the Brains of Nonirradiated Cage Mate Rats:

To evaluate microbeam radiation therapy (MRT), for brain tumor treatment, the bystander effect in nonirradiated companion animals was investigated. Adult rats were irradiated with 35 or 350 Gy at the European Synchrotron Research Facility using homogenous irradiation (HR) or MRT to the right brain hemisphere. The irradiated rats were housed with nonirradiated rats. After 48 hours, all rats were euthanized and the frontal lobe proteome was analyzed using 2-dimensional electrophoresis and mass spectrometry. Proteome changes were determined by analysis of variance (P < .05). Homogenous irradiation increased serum albumin, heat shock protein 71 (HSP-71), triosephosphate isomerase (TPI), fructose bisphosphate aldolase (FBA), and prohibitin and decreased dihydrolipoyl dehydrogenase (DLD) and pyruvate kinase. Microbeam radiation therapy increased HSP-71, FBA, and prohibitin, and decreased aconitase, dihydropyrimidinase, TPI, tubulin DLD, and pyruvate kinase. Cage mates with HR irradiated rats showed increased HSP-71 and FBA and decreased pyruvate kinase, DLD, and aconitase. Cage mates with MRT irradiated rats showed increased HSP-71, prohibitin, and FBA and decreased aconitase and DLD. Homogenous irradiation proteome changes indicated tumorigenesis, while MRT proteome changes indicated an oxidative stress response. The bystander effect of proteome changes appeared antitumorigenic and inducing radioresistance. This investigation also supports the need for research into prohibitin interaction with HSP-70/71 chaperones and cancer therapy.

Identification of capsazepine as a novel inhibitor of system x c − and cancer-induced bone pain

The cystine/glutamate antiporter has been implicated in a variety of cancers as a major mediator of redox homeostasis. The excess glutamate secreted by this transporter in aggressive cancer cells has been associated with cancer-induced bone pain (CIBP) from distal breast cancer metastases. High-throughput screening of small molecule inhibitors of glutamate release from breast cancer cells identified several potential compounds. One such compound, capsazepine (CPZ), was confirmed to inhibit the functional unit of system xc− (xCT) through its ability to block uptake of its radiolabeled substrate, cystine. Blockade of this antiporter induced production of reactive oxygen species (ROS) within 4 hours and induced cell death within 48 hours at concentrations exceeding 25 μM. Furthermore, cell death and ROS production were significantly reduced by co-treatment with N-acetylcysteine, suggesting that CPZ toxicity is associated with ROS-induced cell death. These data suggest that CPZ can modulate system xc− activity in vitro and this translates into antinociception in an in vivo model of CIBP where systemic administration of CPZ successfully delayed the onset and reversed CIBP-induced nociceptive behaviors resulting from intrafemoral MDA-MB-231 tumors.