Proteomic analysis of neuroblastoma microenvironment: effect of the host-tumor interaction on disease progression.

BACKGROUND: Children with advanced-stage neuroblastoma (NB) traditionally experience poor outcomes. Because early detection of advanced-stage disease may impact survival, finding new targets for early diagnosis is crucial. Evidence suggests the tumor microenvironment may have profound effects on cancer progression. METHODS: As little is known concerning the NB-host microenvironment, this study applied proteomic techniques, two-dimensional polyacrylamide gel electrophoresis (2D PAGE) combined with matrix assisted laser desorption ionization time-of-flight (MALDI-TOF) mass spectrometry to determine protein differences between cell cultured NB and tumors grown in mice for 2, 4, and 5 wk. RESULTS: We found an increase in proteins in cultured NB compared with implanted mouse tumors during tumor progression. Additionally, analyzing in vivo tumors to cultured NB, we observed less expressed proteins. However, 16 out of 19 proteins were of mouse origin, thus inferring host-derived factors contributing to tumor growth. CONCLUSION: We show that the dynamic relationship between NB and host microenvironment is important for tumor growth and better understanding of this milieu maybe relevant towards finding unique approaches for identifying advanced-stage disease.

Oxidative status in neuroblastoma: a source of stress?

AIM OF STUDY: Reactive oxygen species have been shown to be initiators/promotors of tumorigenesis. Because evidence supports the role of increased oxidative stress in solid tumors, we sought to establish this relationship in neuroblastoma (NB). The aim of the study was to investigate the extent of oxidative DNA damage and antioxidative status in a progressive animal model of human NB. METHODS: Tumors were induced in the left kidneys of nude mice by the injection of cultured human NB cells (10(6)). Blood was collected from tumor-bearing mice and controls at 2, 4, and 6 weeks. Peripheral blood leukocyte oxidative DNA damage was determined using single-cell gel electrophoresis (comet assay), and plasma antioxidant capacity was assessed by the Trolox equivalent antioxidant capacity method. MAIN RESULTS: Levels of oxidative DNA damage in peripheral blood leukocytes of NB-bearing mice were increased by 166%, 110%, and 87% as compared with healthy controls at 2, 4, and 6 weeks, respectively. Plasma total antioxidant values for tumor-bearing mice were not significantly different from control mice. CONCLUSIONS: Our results indicate an increase of oxidative stress in an animal model of human NB, especially in the early stages of growth. Yet, we did not observe an appreciable response in plasma antioxidant activity. Because an altered redox status has been implicated in tumor maintenance and progression, these findings support the notion of a complex oxidant-antioxidant imbalance contributing to NB growth.

Serum protein profiling to identify high-risk neuroblastoma: preclinical relevance of blood-based biomarkers.

INTRODUCTION: Development of early detection assays for advanced stage neuroblastoma (NB) remains elusive. We have previously shown that serum protein profiling technologies can differentiate healthy from NB children. As various sources of patient related bias exist in serum proteins, we hypothesized a well controlled animal model may provide a better method to identify tumor blood-based markers during NB progression. METHODS: Tumors were induced in the left kidneys of nude mice by the injection of cultured human NB cells (10(6)). Sera were collected from control and tumor-bearing mice at 2, 4, and 6 wk. Albumin-depleted sera were subjected to comparative proteomic profiling using 2D gel electrophoresis. Paired samples at each time point were analyzed and differentially expressed serum proteins were identified by mass spectrometry. Additionally, sera proteomic analysis from children with Stage IV NB and healthy controls were performed. RESULTS: Overexpression of five mouse serum proteins [alpha(1)-acid glycoprotein, alpha(1)-antitrypsin, alpha(2)-macroglobulin, serum amyloid P-component, and serum amyloid A) were found only in NB-bearing mice. Changes in protein abundance were found to increase 2.5-fold (P < or = 0.05) between 2-, 4-, and 6-wk old mice. Underexpression of immunoglobulin kappa chain constant region was observed in the sera of tumor bearing mice compared with controls (2.5-fold, P < or = 0.05). Among NB patients, alpha(1)-acid glycoprotein, apolipoprotein A-IV, haptoglobin, and serum amyloid A were found to be up-regulated. CONCLUSIONS: We identified distinct acute phase proteins that show up-regulation in both an animal tumor model and high-risk NB patients. As these serum proteins have been recognized as markers of tumor progression and prognosis in human malignancies, the validation of these polypeptides may enable serum proteomic profiling to become a valuable tool for identifying high-risk NB.

Cadherin-2 participates in the morphogenesis of the zebrafish inner ear.

Molecular mechanisms that control inner ear morphogenesis from the placode to the three-dimensional functional organ are not well understood. We hypothesize that cell-cell adhesion, mediated by cadherin molecules, contributes significantly to various stages of inner ear formation. Cadherin-2 (Cdh2) function during otic vesicle morphogenesis was investigated by examining morpholino antisense oligonucleotide knockdown and glass onion (glo) (Cdh2 mutant) zebrafish embryos. Placode formation, vesicle cavitation and specification occurred normally, but morphogenesis of the otic vesicle was affected by Cdh2 deficiency: semicircular canals were reduced or absent. Phalloidin staining of the hair cell stereocillia demonstrated that cadherin-2 (cdh2) loss-of-function did not affect hair cell number, but acetylated tubulin labeling showed that hair cell kinocilia were shorter and irregularly shaped. Statoacoustic ganglion size was significantly reduced, which suggested that neuron differentiation or maturation was affected. Furthermore, cdh2 loss-of-function did not cause a general developmental delay, since differentiation of other tissues, including eye, proceeded normally. These findings demonstrate that Cdh2 selectively affects epithelial morphogenetic cell movements, particularly semicircular canal formation, during normal ear mophogenesis.

Proteomic analysis of neuroblastoma subtypes in response to mitogen-activated protein kinase inhibition: profiling multiple targets of cancer kinase signaling.

INTRODUCTION: Survival for high-risk neuroblastoma (NB) remains poor despite aggressive therapy. Novel therapies are vital for improving prognosis. We previously showed differential NB subtype sensitivity to p42/44 mitogen-activated protein kinase (ERK/MAPK) pathway inhibition. In this study, we investigated proteomic changes associated with resistance or sensitivity to MAPK kinase (MEK) inhibition in NB subtypes. MATERIALS AND METHODS: SH-SY5Y (N-type), BE(2)-C (I-type), and SK-N-AS (S-type) were treated with MEK inhibitor U0126 (10 microM) for 1 and 24 h. Proteins were extracted from untreated and treated cells and analyzed for differential expression by two-dimensional polyacrylamide gel electrophoresis (2D-PAGE). Selected polypeptides were extracted from the gel and identified by liquid chromatography-linked tandem mass spectrometry (LC-MS/MS). RESULTS: We identified 15 proteins that were decreased by 2.5-fold between untreated and 1 h treated cells and subsequently up-regulated 5-fold after 24 h drug treatment. N-type NB (MEK-resistant) showed the least altered proteomic profile whereas the I-type (MEK-sensitive) and S-type NB (MEK-intermediate) generated significant protein changes. The majority of proteins identified were induced by stress. CONCLUSIONS: Protein differences exist between MEK inhibitor-treated NB subtypes. Identified polypeptides all have roles in mediating cellular stress. These data suggest that inhibition of the ERK/MAPK in NB subtypes leads to an intracellular stress response. The most resistant NB cell line to MEK inhibitor treatment generated the least protective protein profile, whereas the intermediate and most sensitive NB cells produced the most stress response. These findings suggest stress related protein expression may be targeted in assessing a response to ERK/MAPK therapeutics.