Insulin-like growth factor I is the key growth factor in serum that protects neuroblastoma cells from hyperosmotic-induced apoptosis

Neuroblastoma is a childhood tumor of the peripheral nervous system that remains largely uncurable by conventional methods. Mannitol induces apoptosis in neuroblastoma cell types and insulin-like growth factor I (IGF-I) protects these cells from hyperosmotic-induced apoptosis by affecting apoptosis-regulatory proteins. In the current study, we investigate factors that enable SH-SY5Y neuroblastoma cells to survive in the presence of an apoptotic stimulus. When SH-SY5Y cells are exposed to high mannitol concentrations, more than 60% of the cells are apoptotic within 48 h. Normal CS prevents hyperosmotic-induced apoptosis in a dose-dependent manner, with 0.6% CS protecting 50% of the cells, and 3% CS rescuing more than 70% of the cells from apoptosis. Serum also delays the commitment point for SH-SY5Y cells from 9 h to 35 h. A survey of several growth factors, including epidermal growth factor (EGF), platelet-derived growth factor (PDGF), nerve growth factor (NGF), fibroblast growth factor (FGF), and IGF-I reveals that IGF-I is a component of serum necessary for protection of neuroblastoma cells from death. Mitochondrial membrane depolarization occurs in greater than 40% of the cells after mannitol exposure and caspase-3 activation is increased in high mannitol conditions after 9 h. IGF-I blocks both the mitochondrial membrane depolarization and caspase-3 activation normally induced by hyperosmotic treatment in neuroblastoma cells. Our results suggest that (1) IGF-I is a key factor in serum necessary for protection from death and (2) IGF-I acts upstream from the mitochondria and the caspases to prevent apoptosis in human neuroblastoma. J. Cell. Physiol. 182:24–32, 2000. © 2000 Wiley-Liss, Inc.Peer Reviewedhttp://deepblue.lib.umich.edu/bitstream/2027.42/34442/1/3_ftp.pd

Phase II study of the farnesyltransferase inhibitor R115777 in advanced melanoma (CALGB 500104)

BACKGROUND: Multiple farnesylated proteins are involved in signal transduction in cancer. Farnesyltransferase inhibitors (FTIs) have been developed as a strategy to inhibit the function of these proteins. As FTIs inhibit proliferation of melanoma cell lines, we undertook a study to assess the impact of a FTI in advanced melanoma. As farnesylated proteins are also important for T cell activation, measurement of effects on T cell function was also pursued. METHODS: A 3-stage trial design was developed with a maximum of 40 patients and early stopping if there were no responders in the first 14, or fewer than 2 responders in the first 28 patients. Eligibility included performance status of 0–1, no prior chemotherapy, at most 1 prior immunotherapy, no brain metastases, and presence of at least 2 cutaneous lesions amenable to biopsy. R115777 was administered twice per day for 21 days of a 28-day cycle. Patients were evaluated every 2 cycles by RECIST. Blood and tumor were analyzed pre-treatment and during week 7. RESULTS: Fourteen patients were enrolled. Two patients had grade 3 toxicities, which included myelosuppression, nausea/vomiting, elevated BUN, and anorexia. There were no clinical responses. All patients analyzed showed potent inhibition of FT activity (85-98%) in tumor tissue; inhibition of phosphorylated ERK and Akt was also observed. T cells showed evidence of FT inhibition and diminished IFN-γ production. CONCLUSIONS: Despite potent target inhibition, R115777 showed no evidence of clinical activity in this cohort of melanoma patients. Inhibition of T cell function by FTIs has potential clinical implications. Clinicaltrials.gov number NCT0006012

The role of the type I insulin -like growth factor receptor in protection of human neuroblastoma cells from apoptosis.

The Insulin-like growth factor family (IGFs) plays a role in several cellular functions, including mitogenesis, differentiation, and survival from apoptosis. The biological effects of the IGFs are mediated through the type I IGF receptor (IGF-IR) and its downstream signaling pathways. In this series of studies, the role of IGF-IR activation by IGF-I in protecting human neuroblastorna cells from hyperosmotic-induced apoptosis was examined. SH-SY5Y human neuroblastoma cells show the morphological characteristics of apoptosis by 24 h of exposure to high concentrations of mannitol and are protected by calf serum. IGF-I is the principal growth factor in calf serum responsible for protection from apoptosis. Hyperosmotic exposure causes both mitochondrial membrane depolarization and caspase-3 activation, 2 of the early events in the apoptotic cascade, and these 2 events are prevented by IGF-I addition. Since the biological effects of IGF-I occur through IGF-IR activation, SHEP cells, which express very little IGF-IR, and SHEP cells transfected with the IGF-IR (SHEP/IGF-IR cells) were used to investigate the role of IGF-IR activation in protection of neuroblastorna cells from hyperosmotic-induced apoptosis. Serum withdrawal induced apoptosis occurs in SHEP cells and is prevented by overexpression of the IGF-IR. Both SHEP and SHEP/IGF-IR cells undergo apoptosis upon exposure to high concentrations of mannitol, and this is prevented by IGF-I addition. Mitochondrial membrane depolarization and caspase-3 activation are both prevented by IGF-IR activation in SHEP cells. Overexpression of the anti-apoptotic protein Bcl-2 prevents mitochondrial membrane depolarization, caspase-3 activation, and apoptosis in SHEP cells. Therefore, hyperosmotic-induced apoptosis in SHEP cells is a caspase dependent, Bcl-2 mediated process which is prevented by IGF-IR activation. The principal pathway involved in IGF-I mediated rescue of SHEP cells from apoptosis is the PI-3K pathway. PI-3K activates the downstream effector Akt, and the tumor suppressor PTEN inactivates Akt through the dephosphorylation of the lipid phosphatidlinositol 3,4,5-phosphate. Overexpression of wild type PTEN enhances apoptosis, decreases IGF-I mediated protection from apoptosis. PTEN mutants without lipid phosphatase activity have Akt activity and apoptosis levels similar to control cells. Therefore, the PI-3K-Akt pathway is important for IGF-I mediated survival in SHEP cells, and this effect can be modulated by PTEN.Ph.D.Biological SciencesCellular biologyHealth and Environmental SciencesNeurosciencesOncologyUniversity of Michigan, Horace H. Rackham School of Graduate Studieshttp://deepblue.lib.umich.edu/bitstream/2027.42/132263/2/9959877.pd

Integrin Expression Regulates Neuroblastoma Attachment and Migration

AbstractNeuroblastoma (NBL) is the most common malignant disease of infancy, and children with bone metastasis have a mortality rate greater than 90%. Two major classes of proteins, integrins and growth factors, regulate the metastatic process. We have previously shown that tumorigenic NBL cells express higher levels of the type I insulin-like growth factor receptor (IGF-IR) and that β1 integrin expression is inversely proportional to tumorigenic potential in NBL. In the current study, we analyze the effect of β1 integrin and IGF-IR on NBL cell attachment and migration. Nontumorigenic S-cells express high levels of β1 integrin, whereas tumorigenic N-cells express little β1 integrin. Alterations in (3, integrin are due to regulation at the protein level, as translation is decreased in N-type cells. Moreover, inhibition of protein synthesis shows that β1 integrin is degraded more slowly in S-type cells (SHEP) than in N-type cells (SH-SY5Y and IMR32). Inhibition of α5β1 integrin prevents SHEP (but not SH-SY5Y or IMR32) cell attachment to fibronectin and increases SHEP cell migration. Increases in IGF-IR decrease β1 integrin expression, and enhance SHEP cell migration, potentially through increased expression of αvβ3. These data suggest that specific classes of integrins in concert with IGF-IR regulate NBL attachment and migration

Degradation and Dephosphorylation of Focal Adhesion Kinase During Okadaic Acid-Induced Apoptosis in Human Neuroblastoma Cells

Focal adhesion kinase (FAK) prevents apoptosis in many cell types. We have reported that tyrosine residues in FAK are dephosphorylated and FAK is degraded during mannitol-induced apoptosis in human neuroblastoma cells. Several studies suggest that FAK dephosphorylation and degradation are separate events. The current study defines the relationship between FAK dephosphorylation and degradation in neuroblastoma cells using okadaic acid (OA). OA, a serine phosphatase inhibitor, promotes serine/threonine phosphorylation, which in turn blocks tyrosine phosphorylation. OA induced focal adhesion loss, actin cytoskeleton disorganization, and cellular detachment, which corresponded to a loss of FAK Tyr(397) phosphorylation. These changes preceded caspase-3 activation, Akt and MAP kinase activity loss, protein ubiquitination, and cellular apoptosis. Insulin-like growth factor-I prevented mannitol-induced, but not OA-induced, substrate detachment and FAK Tyr(397) dephosphorylation, and the effects of OA on FAK Tyr(397) phosphorylation were irreversible. The proteolytic degradation of FAK is temporally distinct from its tyrosine dephosphorylation, occurring when apoptotic pathways are already initiated and during a generalized destruction of signaling proteins. Therefore, agents resulting in the dephosphorylation of FAK may be beneficial for therapeutic treatment, irrespective of FAK protein levels, as this may result in apoptosis, which cannot be prevented by growth factor signaling

Bcl-2 and N-Myc Coexpression Increases IGF-IR and Features of Malignant Growth in Neuroblastoma Cell Lines

The bcl-2 and c-myc oncogenes cooperate to transform multiple cell types. In the pediatric malignancy NB(2), Bcl-2 is highly expressed. In tumors with a poor prognosis, N-Myc, a protein homologous to c-Myc, is overexpressed as a result of gene amplification. The present study was designed to determine whether Bcl-2 cooperates with N-Myc to bestow a tumorigenic phenotype to neuroblastoma (NB) cells. NB cell lines that at baseline express neither Bcl-2 nor N-Myc were stably transfected to express these gene products. In this model, we found Bcl-2 rescues N-Myc-expressing cells from apoptosis induced by serum withdrawal. Coexpression of Bcl-2 and N-Myc supports growth in low serum conditions and anchorage-independent growth in soft agar. Similarly, in vivo tumorigenic and angiogenic activity was dependent on coexpression. Our data further suggests that the mechanism underlying these changes involves the receptor for insulin growth factor type I (IGF-IR)