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-1ß (IL-1ß) 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.

Differences in electrophysiological properties of functionally identified nociceptive sensory neurons in an animal model of cancer-induced bone pain.

BACKGROUND: Bone cancer pain is often severe, yet little is known about mechanisms generating this type of chronic pain. While previous studies have identified functional alterations in peripheral sensory neurons that correlate with bone tumours, none has provided direct evidence correlating behavioural nociceptive responses with properties of sensory neurons in an intact bone cancer model. RESULTS: In a rat model of prostate cancer-induced bone pain, we confirmed tactile hypersensitivity using the von Frey test. Subsequently, we recorded intracellularly from dorsal root ganglion neurons in vivo in anesthetized animals. Neurons remained connected to their peripheral receptive terminals and were classified on the basis of action potential properties, responses to dorsal root stimulation, and to mechanical stimulation of the respective peripheral receptive fields. Neurons included C-, Aδ-, and Aß-fibre nociceptors, identified by their expression of substance P. We suggest that bone tumour may induce phenotypic changes in peripheral nociceptors and that these could contribute to bone cancer pain. CONCLUSIONS: This work represents a significant technical and conceptual advance in the study of peripheral nociceptor functions in the development of cancer-induced bone pain. This is the first study to report that changes in sensitivity and excitability of dorsal root ganglion primary afferents directly correspond to mechanical allodynia and hyperalgesia behaviours following prostate cancer cell injection into the femur of rats. Furthermore, our unique combination of techniques has allowed us to follow, in a single neuron, mechanical pain-related behaviours, electrophysiological changes in action potential properties, and dorsal root substance P expression. These data provide a more complete understanding of this unique pain state at the cellular level that may allow for future development of mechanism-based treatments for cancer-induced bone pain.

Depressive-like behaviours and decreased dendritic branching in the medial prefrontal cortex of mice with tumors: A novel validated model of cancer-induced depression.

Depression is commonly comorbid in cancer patients and has detrimental effects on disease progression. Evidence suggests that biological mechanisms may induce the onset of cancer-induced depression (CID). The present investigation aims to establish a validated preclinical animal model of CID. Female BALB/c mice were allocated to four groups: control (n=12), chronic oral exposure to corticosterone (CORT) (n=12), CORT exposure followed by chronic low dose fluoxetine (FLX) treatment (n=12), and subcutaneous inoculation of 4T1 mammary carcinoma cells (n=13). Anhedonia was evaluated using the sucrose preference test (SPT), and behavioural despair was evaluated using the forced swim test (FST) and tail suspension test (TST). Sholl analyses were used to examine the dendritic morphology of Golgi-Cox impregnated neurons from the medial prefrontal cortex (mPFC). CORT exposure and tumor burden were both associated with decreased sucrose preference, increased FST immobility, and decreased basilar and apical dendritic branching of neurons in the mPFC. CORT-induced behavioural and dendritic morphological changes were reversible by FLX. No differences in TST immobility were observed between groups. On the secondary TST outcome measure, CORT exposure and tumor burden were associated with a trend towards decreased power of movement. CORT exposure induced a positive control model of a depressive-like state, with FLX treatment confirming the predictive validity of the model. This verified the sensitivity of behavioural and histological tests, which were used to assess the CID model. The induction of a depressive-like state in this model represents the first successfully validated animal model of CID.

Inhibition of breast cancer-cell glutamate release with sulfasalazine limits cancer-induced bone pain.

Cancer in bone is frequently a result of metastases from distant sites, particularly from the breast, lung, and prostate. Pain is a common and often severe pathological feature of cancers in bone, and is a significant impediment to the maintenance of quality of life of patients living with bone metastases. Cancer cell lines have been demonstrated to release significant amounts of the neurotransmitter and cell-signalling molecule l-glutamate via the system xC(-) cystine/glutamate antiporter. We have developed a novel mouse model of breast cancer bone metastases to investigate the impact of inhibiting cancer cell glutamate transporters on nociceptive behaviour. Immunodeficient mice were inoculated intrafemorally with the human breast adenocarcinoma cell line MDA-MB-231, then treated 14days later via mini-osmotic pumps inserted intraperitoneally with sulfasalazine, (S)-4-carboxyphenylglycine, or vehicle. Both sulfasalazine and (S)-4-carboxyphenylglycine attenuated in vitro cancer cell glutamate release in a dose-dependent manner via the system xC(-) transporter. Animals treated with sulfasalazine displayed reduced nociceptive behaviours and an extended time until the onset of behavioural evidence of pain. Animals treated with a lower dose of (S)-4-carboxyphenylglycine did not display this reduction in nociceptive behaviour. These results suggest that a reduction in glutamate secretion from cancers in bone with the system xC(-) inhibitor sulfasalazine may provide some benefit for treating the often severe and intractable pain associated with bone metastases.

Oxidative stress and cancer pain.

Breast cancers are the most common source of metastases to bone, of which cancer-induced bone pain is a frequent pathological feature. Cancer-induced bone pain is a unique pain state with multiple determinants that remains to be well understood and managed. Current standard treatments are limited by dose-dependent side effects that can reduce the quality of life of patients. Glutamate is a neurotransmitter and bone cell-signalling molecule that is released via the system x(c)(-) cystine/glutamate antiporter from cancer cell types that frequently metastasize to bone, including breast cancers. In cancer cells, glutamate release is understood to be a side effect of the cellular response to oxidative stress that upregulates the expression and activity of system x(c)(-) to promote the increased import of cystine. Attenuation of glutamate release from cancer cells has been demonstrated to result in reductions in associated cancer-induced bone pain in animal models. This review examines the clinical implications of attenuating cystine uptake and glutamate release in the treatment of cancer-induced bone pain.

Glutamate signaling in healthy and diseased bone.

Bone relies on multiple extracellular signaling systems to maintain homeostasis of its normal structure and functions. The amino acid glutamate is a fundamental extracellular messenger molecule in many tissues, and is used in bone for both neural and non-neural signaling. This review focuses on the non-neural interactions, and examines the evolutionarily ancient glutamate signaling system in the context of its application to normal bone functioning and discusses recent findings on the role of glutamate signaling as they pertain to maintaining healthy bone structure. The underlying mechanisms of glutamate signaling and the many roles glutamate plays in modulating bone physiology are featured, including those involved in osteoclast and osteoblast differentiation and mature cell functions. Moreover, the relevance of glutamate signaling systems in diseases that affect bone, such as cancer and rheumatoid arthritis, is discussed, and will highlight how the glutamate system may be exploited as a viable therapeutic target. We will identify novel areas of research where knowledge of glutamate communication mechanisms may aid in our understanding of the complex nature of bone homeostasis. By uncovering the contributions of glutamate in maintaining healthy bone, the reader will discover how this complex molecular signaling system may advance our capacity to treat bone pathologies.

Extracellular glutamate alters mature osteoclast and osteoblast functions.

Glutamatergic intercellular communication is involved in many aspects of metabolic homeostasis in normal bone. In bone metastasis, the balance between bone formation and degradation is disrupted. Although the responsible mechanisms are not clear, we have previously identified that cancer cell lines used in bone tumour models secrete glutamate, suggesting that tumour-derived glutamate may disrupt sensitive signalling systems in bone. This study examines the role of glutamate in mature osteoclastic bone resorption, osteoblast differentiation, and bone nodule formation. Glutamate was found to have no effect on the survival or activity of mature osteoclasts, although glutamate transporter inhibition and receptor blockade increased the number of bone resorption pits. Furthermore, transporter inhibition increased the area of resorbed bone while significantly decreasing the number of osteoclasts. Alkaline phosphatase activity and extracellular matrix mineralization were used as measurements of osteoblast differentiation. Glutamate significantly increased osteoblast differentiation and mineralization, but transport inhibitors had no effect. These studies support earlier findings suggesting that glutamate may be more important for osteoclastogenesis than for osteoclast proliferation or functions. Since glutamate is capable of changing the differentiation and activities of both osteoclast and osteoblast cell types in bone, it is reasonable to postulate that tumour-derived glutamate may impact bone homeostasis in bone metastasis.

Evidence for the role of matrix metalloproteinase-13 in bone resorption by giant cell tumor of bone.

Giant cell tumor of bone (GCT) is an aggressively osteolytic primary bone tumor that is characterized by the presence of abundant multinucleated osteoclast-like giant cells, hematopoietic monocytes, and a distinct mesenchymal stromal cell component. Previous work in our laboratory has shown that matrix metalloproteinase (MMP)-13 is the principal proteinase expressed by the stromal cells of GCT. The release of cytokines, particularly interleukin-1beta, by the giant cells of GCT acts on stromal cells to stimulate a surge in MMP-13 secretion. The purpose of this study was to determine the bone resorption capabilities of the cellular elements of GCT and the significance of the MMP-13 expression involved in GCT bone resorption. We present a 3-dimensional histomorphometric technique developed to analyze resorption pit depth and yield an accurate measurement of bone resorption with a direct physical view of lacunae on bone slices. In this study, we demonstrate that the mesenchymal stromal cells and the multinucleated giant cells of GCT are independently capable of bone resorption. However, coculture of these 2 cell fractions shows a synergistic increase in bone resorption. In addition, inhibition of MMP-13 reduces resorptive activity of the cells indicating that MMP-13 likely plays an important role in this tumor. This cell-cell cooperation involves giant cell-derived cytokine up-regulation of MMP-13 in the stromal cells, which in turn assists the giant cells in bone resorption. Future research will involve elucidation of the role of cell-cell/matrix communication pathways in bone resorption and tumorigenesis in GCT.

A by-product of glutathione production in cancer cells may cause disruption in bone metabolic processes.

Bone is a frequent site for metastasis of breast and prostate cancers, often resulting in pathologic changes in bone metabolism and severe pain. The mechanisms involved are not well understood, but tumour cells may release factors that interfere with bone homeostasis. Several observations have led us to hypothesize that the functional disruptions in bone metastasis are the result of a biological process common to many cell types. The high metabolic activity characteristic of cancer cells often upregulates oxidative stress protection mechanisms such as the antioxidant molecule glutathione. In maintaining redox balance, this normal metabolic response may result in unintended pathologic effects in certain sensitive organ sites. Malignant glioma cells kill surrounding neurons in the brain specifically by secreting the amino acid glutamate, an obligatory waste product of glutathione synthesis. We suggest that glutamate release is a plausible mechanism that may account for the pathologic changes in bone metastasis, since bone, like brain, is also highly sensitive to glutamatergic disruption. This report reviews the available evidence to draw a mechanistic connection between tumour cell oxidative stress and the pathology seen in patients with bone metastasis.

Cancer cells release glutamate via the cystine/glutamate antiporter.

Although the amino acid glutamate is used as an intercellular signaling molecule for normal bone homeostasis, little is known regarding its possible role in the metabolic disruption characteristic of bone metastasis. We have previously shown in vitro that cancer cell lines relevant to bone metastasis release glutamate into the extracellular environment. This study demonstrates the expression of multiple glutamate transporters in cancer cell lines of non-central nervous system origin. Furthermore, we identify the molecular mechanism responsible for glutamate export and show that this system can be inhibited pharmacologically. By highlighting that glutamate secretion is a common biological feature of cancer cells, this study suggests that tumor-derived glutamate could interfere with glutamate-dependent intercellular signaling in normal bone. Pharmacological interference with cancer cell glutamate release may be a viable option for limiting host bone response to invading tumor cells in bone metastasis.

Luciferase therapeutic microcapsules for gene therapy.

MDCK cells were engineered to express luciferase driven by cytomegalovirus (CMV) or hybrid ubiquitin B (UbB) promoter and encapsulated in alginate-poly-L-lysine-alginate microcapsules. In vitro experiments showed capsules could be monitored individually or in multi-layers quantitatively. When luciferase-expressing and non-luciferase expressing MDCK cells were mixed at different ratios and encapsulated, the signals increased linearly according to the number of capsules, in vitro and in vivo. For CMV-driven luciferase expression, the strongest signal was seen at 4 hours post-implantation, with a subsequent 50% decrease by 24 hours and then declined gradually to 10-20% until day 20. However, retrieved capsules showed good cell viability. When capsules contained plasmid driven by UbB promoter, there was no decline in signal. Our results indicate that luciferase could be used as a marker for microencapsulated cells to monitor the viability and gene expression of the implanted cells.

Cancer cell lines release glutamate into the extracellular environment.

Bone is one of the most frequent sites for metastasis of breast and prostate cancers. Bone metastases are associated with pathologic changes in bone turnover and severe pain. The mechanisms that trigger these effects are not well understood, but it is postulated that tumour cells release factors which interfere with signalling processes critical to bone homeostasis. We have identified that several cancer cell lines known to cause bone disruption in animal models of bone metastasis appear to secrete glutamate into their extracellular environment in vitro. Although these cells also express specific glutamate receptors, the implications of this potentially disruptive chemical signal are discussed in relation to normal glutamate-dependent communication processes in bone and a possible mechanistic connection is made between tumour cell glutamate release and the development of pathological changes in bone turnover.

Cyclooxygenase isoenzymes and patency of ductus arteriosus.

Prenatal patency of the ductus arteriosus is maintained mainly by prostaglandin (PG) E(2). Accordingly, the vessel is endowed in its muscular component with a complete, cyclooxygenase (COX) and PGE synthase (PGES), system for the synthesis of the compound. COX1 is better expressed than COX2, particularly in the premature, but COX2 is more extensively coupled with microsomal PGES (mPGES). No evidence was obtained of either COX being coupled with cytosolic PGES (cPGES). Functionally, these data translate into a differential constrictor response of the ductus to dual, COX1/COX2, vs. COX2-specific inhibitors (indomethacin vs. L-745,337), with the latter being less effective specifically prior to term. This difference, however, subsides upon treatment with endotoxin and the attendant upregulation of COX2 and mPGES. Furthermore, when studied separately, COX1 and COX2 prove to be unevenly responsive to indomethacin, and an immediate and fast developing contraction of the vessel occurs only when COX2 is inhibited. Deletion of either COX gene results into upregulation of NO synthase, and a similar compensatory reaction is expected when enzymes are suppressed pharmacologically. We conclude that PGE(2) and NO can function synergistically in keeping the ductus patent. This arrangement provides a possible explanation for failures of indomethacin or ibuprofen treatment in the management of the prematurely born infant with persistent ductus. Coincidentally, it opens the way to new therapeutic possibilities being based on interference with the NO effector or a more selective disruption, possibly having mPGES as a target, of the PGE(2) synthetic cascade.

Re-expression of TSLC1 in a non-small-cell lung cancer cell line induces apoptosis and inhibits tumor growth.

The TSLC1 tumor-suppressor gene is silenced in a number of human cancer tissues and cell lines, including lung, prostate, liver, stomach, pancreatic, and breast cancers. Expression of TSLC1 in a non-small-cell lung cancer (NSCLC) cell line A549 suppresses tumorigenicity in nude mice. However, the molecular mechanism of TSLC1 action is not yet elucidated. In the present study, we show that the expression of TSLC1 from a recombinant adenovirus vector (Ad-TSLC1) inhibited cell proliferation and induced apoptosis in the NSCLC cell line A549. We also demonstrated that subcutaneous tumor growth in nude mice induced by A549 cells was suppressed to the extent of 70-80% by intratumoral injection of Ad-TSLC1. Re-expression of TSLC1 also resulted in activation of the apoptotic protease caspase-3, accompanied by the cleavage of its substrate poly (ADP-ribose) polymerase (PARP). The antiproliferative and pro-apoptotic activity of TSLC1 required the presence of the FERM-binding and PDZ-interacting motifs located in the cytoplasmic domain. Our results demonstrate the pro-apoptotic and oncosuppressive activity of TSLC1 protein, and suggest the potential of TSLC1 for gene therapy.

Quantification of anti-angiogenesis using the capillaries of the chick chorioallantoic membrane demonstrates that the effect of human angiostatin is age-dependent.

We present a method whereby en face estimation of the chorionic capillary plexus can be generated in the living chick chorioallantoic membrane (CAM) and confirmed by post-fixation cross section analysis. This value does not alter significantly with age and provides a reliable and simple method to evaluate anti-angiogenesis. Anti-angiogenesis may be induced by an intervention, such as a pharmacological agent, applied to the surface of the CAM. We describe the use of silastic rings that are associated with minimal inflammatory reaction, in this process. By estimating changes in the chorionic capillary plexus to quantify anti-angiogenesis, together with silastic rings, we examined the anti-angiogenic effect of human angiostatin and demonstrated that although there is a significant loss of capillaries en face after exposure from days 7 to 9 of incubation, in contrast there is no significant inhibition after exposure to a similar dose of angiostatin from days 11 to 13 of incubation. This not only demonstrates the important effects on neo-angiogenesis compared to mature vessels, but also illustrates the potential of the CAM to readily provide a means for such a comparison.

The cytoplasmic domain is critical to the tumor suppressor activity of TSLC1 in non-small cell lung cancer.

The tumor suppressor gene in lung cancer (TSLC1) encodes a membrane glycoprotein containing extensive homology in the extracellular domain with the immunoglobulin-superfamily cell adhesion molecules. The intracellular cytoplasmic domain (CT) contains a protein 4.1 (FERM) binding motif, and a PDZ-interacting motif. Expression of TSLC1 is silenced in non-small cell lung cancer and in other cancers by promoter hypermethylation. Restoration of TSLC1 expression suppresses tumorigenicity of lung cancer cells. We report here the critical role of the FERM-binding and PDZ- interacting domains of TSLC1 in tumor suppressor activity in non-small cell lung cancer. The entire CT domain [amino acid (aa) 398-442], the FERM binding motif (aa 398-410), or the PDZ-interacting motif (aa 432-442) was deleted to generate mutants CT1, CT3, and CT4, respectively. The lung cancer cell line A549, deficient in TSLC1 expression, was stably transfected with the wild-type TSLC1 or the deletion mutants. The cell lines were then injected into athymic (nu/nu) nude mice, and tumor formation at the sites of injection was monitored. A549 cells stably transfected with the empty vector or mutant TSLC1 constructs induced tumors at the sites of injection within 10 days. In contrast, A549 cells expressing wild-type TSLC1 showed the appearance of tumors after 35 days, and the tumors grew substantially slower. A549 cells expressing wild-type TSLC1 also showed suppression of anchorage-independent colony formation in soft agar and markedly increased cell-cell adhesion activity. These results suggest that the cytoplasmic domain of TSLC1 is important in its tumor suppressor activity, and the tumor suppression activity involve protein(s) interacting with the FERM- and PDZ-interacting regions.

The response of the lamb ductus arteriosus to endothelin: developmental changes and influence of light.

Endothelin-1 (ET-1) is a putative messenger of oxygen in the ductus arteriosus. Since the ability of the vessel to contract to oxygen increases with gestation, we wished to ascertain whether ET-1 action is also developmentally regulated. A corollary objective was to assess whether any gestational variation in the ET-1 contraction is due to a change in the ET(A)-mediated action or to a shift in the balance between opposing, contractile (ET(A) - mediated) and relaxant (ET(B)-mediated), actions. Experiments were performed with isolated ductal strips from preterm (0.7 gestation) and near-term fetal lambs. ET-1 contracted the ductus dose-dependently (10(-10)-10(-7) M) at both ages; however, the peak contraction was about double in magnitude at term. Regardless of age, ET-1 contraction was greater with preparations kept in the dark compared to those exposed to light. This effect of light was not seen after removing the endothelium or when treating the intact tissue with the ET(B) antagonist BQ788 (1 microM). In the dark, however, BQ788 did not modify significantly the ET-1 response at either age. We conclude that ET-1 becomes a stronger ductus constrictor with fetal age, conceivably by acting on ET(A) receptors. Hence, the concept of ET-1 mediating the oxygen contraction is further validated. Peculiarly, the ET-1 contraction is curtailed by light through a hitherto undefined ET(B) receptor-linked process.

Doxycycline decreases tumor burden in a bone metastasis model of human breast cancer.

Bone is one of the most frequent sites for metastasis in breast cancer patients,often resulting in significant clinical morbidity and mortality. Increased matrix metalloproteinase (MMP) activity of tumor cells correlates with a higher invasive and metastatic potential. Members of the tetracycline family of antibiotics, including doxycycline, have potential treatment value for bone metastasis; they inhibit cancer cell proliferation, and they are also potent MMP inhibitors and are highly osteotropic. Doxycycline treatment in an experimental bone metastasis mouse model of human breast cancer MDA-MB-231 cells resulted in a 70% reduction in total tumor burden when compared with placebo control animals. In tumor-bearing animals, the amount of doxycycline incorporated into the radius/ulna as assessed by ELISA was lower than in non-tumor-bearing animals. In doxycycline-treated mice, bone formation was significantly enhanced as determined by increased numbers of osteoblasts, osteoid surface, and volume, whereas a decrease in bone resorption was also observed. Doxycycline treatment may be beneficial for breast cancer patients with or at risk for osteolytic bone metastasis; it greatly reduces tumor burden and could also compensate for the increased bone resorption associated with the disease.