p75-Nerve Growth Factor as an Antiapoptotic Complex: Independence versus Cooperativity in Protection from Enediyne Chemotherapeutic Agents

ABSTRACT Growth factors, including nerve growth factor (NGF), have been hypothesized to play a role in resistance to chemotherapeutic agent-induced apoptosis. Induction by NGF of resistance to apoptosis is primarily thought to be the result of its binding to its high-affinity receptor, TrkA. The low-affinity NGF receptor, p75, has long been thought merely to facilitate NGF binding to TrkA. However, we have previously shown that the binding of NGF to its low-affinity receptor, p75, protects neuroblastoma cells that do not express TrkA against apoptosis induced by enediyne chemotherapeutic agents. In cells that express both receptors, it is not clear what determines which receptor is responsible for the protective effect of NGF. We now show that, in enediyne-treated SH-SY5Y neuroblastoma transfectants with native levels of p75 and a low TrkA/p75 ratio (1/100), the anti-apoptotic effect of NGF requires binding to p75. In contrast, in transfectants with native levels of p75 and a high TrkA/p75 ratio (100/100), NGF treatment prevents enediyneinduced apoptosis by a mechanism independent of p75 binding. Treatment of low TrkA/p75 ratio cells with NGF results in activation and nuclear translocation of NF-B and tyrosine phosphorylation of TrkA. Analogous treatment of high TrkA/p75 ratio cells results only in phosphorylation of TrkA even though nuclear factor (NF)-B signaling is not inactive and can be initiated by other ligands. The ratio of TrkA/p75 in cells that express both receptors probably contributes to the determination of which of the two known roles of p75 (i.e., TrkA independent or TrkA facilitatory) are responsible for NGF-mediated protection from enediyne-induced apoptosis

The Role of p75NTR and its signaling pathways in Fenretinide (4-hydroxyphenyl retinamide)-induced apoptosis in neuroblastoma cells

Thesis (Ph.D.)--University of Rochester. School of Medicine & Dentistry. Dept. of Pediatrics, 2013.OBJECTIVE: Neuroblastoma is a common, frequently fatal, neural crest tumor of childhood. Chemotherapy-resistant neuroblastoma cells typically have Schwann cell-like (“S-type”) morphology and express the p75 neurotrophin receptor (p75NTR) and are often refractory to chemotherapy. p75NTR has been previously shown to modulate the redox state of neural crest tumor cells. The retinoic acid analogue, fenretinide(4HPR), has been shown to cause apoptosis by increasing generation of reactive oxygen species in cancer cells. We tested the hypothesis that expression of the p75 neurotrophin receptor (p75NTR), enhances neuroblastoma responsiveness to 4HPR. METHODS: Neuroblastoma cell lines derived from minimal residual disease were used to determine the effect of p75NTR signaling on susceptibility to 4HPR-induced apoptosis. p75NTR signaling was knocked down using transfection and concentration- and time-cell survival studies were performed to determine sensitivity to 4HPR. Changes in oxidative stress as a result of 4HPR treatment were determined using redox-active dye staining and site-specific antioxidants. Western blotting and site-specific stains were used to determine the components of the signaling pathway involved in 4HPR-induced apoptosis. RESULTS: Knockdown of p75NTR attenuated 4HPR-induced accumulation of mitochondrial superoxide and apoptosis. Overexpression of p75NTR had the opposite effects. Treatment of SH-EP1 neuroblastoma cells with 4HPR for 72 hrs revealed no change in production of cellular hydroxyl radical or cytosolic superoxide, but a sharp increase was observed in mitochondrial superoxide production. Pretreatment of cells with 2-thenoyltrifluoroacetone or dehydroascorbic acid uniquely prevented mitochondrial superoxide accumulation and cell death after 4HPR treatment. Mitochondrial complex II is the likely site of 4HPR-induced superoxide generation and p75NTR-induced potentiation of these phenomena. MAPK signaling resulting in JNK phosphorylation is one of the central pathways regulating apoptosis following 4HPR treatment. Akt signaling is a protective signal which is enhanced in p75NTR knockdown neuroblastoma cells CONCLUSION: Modification of expression of p75NTR in a particular neuroblastoma cell line modifies its susceptibility to 4HPR. Mitochondrial oxidative stress, JNK phosphorylation and suppression of Akt signaling are important intermediates for this potentiation. in S-type neuroblastoma tumors with high p75NTR expression when treated with 4HPR are highly sensitive to drug treatment. Phosphorylation of JNK leading to mitochondrial ROS production and cell death are some of the intermediate steps involved in this process. AKT phosphorylation is a protective signal that increases resistance to drug treatment. p75NTR is a biomarker for efficacy of 4HPR against neuroblastoma. Enhancers of p75NTR expression or signaling could be potential drugs for use as adjuncts to chemotherapy of neural tumors