RESUMO
Neuroblastoma (NB) is associated with MYCN oncogene amplification occurring in approximately 30% of NBs and is associated with poor prognosis. MYCN is linked to a number of genes including ornithine decarboxylase (ODC), the rate-limiting enzyme in polyamine biosynthesis. ODC expression is elevated in many forms of cancer including NB. Alpha-difluoromethylornithine (DFMO), an ODC inhibitor, is currently being used in a Phase I clinical trial for treatment of NB. However, cancer cells treated with DFMO may overcome their polyamine depletion by the uptake of polyamines from extracellular sources. A novel polyamine transport inhibitor, AMXT-1501, has not yet been tested in NB. We propose that inhibiting ODC with DFMO, coupled with polyamine transport inhibition by AMXT-1501 will result in enhanced NB growth inhibition. Single and combination drug treatments were conducted on three NB cell lines. DFMO IC50 values ranged from 20.76 to 33.3 mM, and AMXT-1501 IC50 values ranged from 14.13 to 17.72 µM in NB. The combination treatment resulted in hypophosphorylation of retinoblastoma protein (Rb), suggesting growth inhibition via G1 cell cycle arrest. Increased expression of cleaved PARP and cleaved caspase 3 in combination-treated cells starting at 48 hr suggested apoptosis. The combination treatment depleted intracellular polyamine pools and decreased intracellular ATP, further verifying growth inhibition. Given the current lack of effective therapies for patients with relapsed/refractory NB and the preclinical effectiveness of DFMO with AMXT-1501, this combination treatment provides promising preclinical results. DFMO and AMXT-1501 may be a potential new therapy for children with NB.
Assuntos
Poliaminas Biogênicas/metabolismo , Proliferação de Células/efeitos dos fármacos , Eflornitina/farmacologia , Inibidores Enzimáticos/farmacologia , Neuroblastoma/tratamento farmacológico , Inibidores da Ornitina Descarboxilase , Transporte Biológico/efeitos dos fármacos , Caspase 3/fisiologia , Linhagem Celular Tumoral , Sinergismo Farmacológico , Genes myc , Humanos , Neuroblastoma/patologiaRESUMO
OBJECTIVE: Platelet-derived growth factor (PDGF) and its receptor, PDGFR, promote fibrosis in systemic sclerosis (SSc; scleroderma) dermal fibroblasts, and such cells in scleroderma skin lesions produce excessive reactive oxygen species (ROS). PDGFR is phosphorylated upon PDGF stimulation, and is dephosphorylated by protein tyrosine phosphatases (PTPs), including PTP1B. This study was undertaken to determine whether the thiol-sensitive PTP1B is affected by ROS in SSc dermal fibroblasts, thereby enhancing the phosphorylation of PDGFR and synthesis of type I collagen. This study also sought to investigate the effect of a thiol antioxidant, N-acetylcysteine (NAC), in SSc. METHODS: Fibroblasts were isolated from the skin of patients with diffuse SSc and normal healthy donors for cell culture experiments and immunofluorescence analyses. A phosphate release assay was used to determine the activity of PTP1B. RESULTS: Levels of ROS and type I collagen were significantly higher and amounts of free thiol were significantly lower in SSc fibroblasts compared to normal fibroblasts. After stimulation with PDGF, not only were PDGFR and ERK-1/2 phosphorylated to a greater extent, but also the ability to produce PTP1B was hampered in SSc fibroblasts. The activity of PTP1B was significantly inactivated in SSc fibroblasts as a result of cysteine oxidation by the raised levels of ROS, which was confirmed by the oxidation of multiple PTPs, including PTP1B, in SSc fibroblasts. Decreased expression of PTP1B in normal fibroblasts led to increased expression of type I collagen. Treatment of the cells with NAC restored the activity of PTP1B, improved the profile of PDGFR phosphorylation, decreased the numbers of tyrosine-phosphorylated proteins and levels of type I collagen, and scavenged ROS in SSc fibroblasts. CONCLUSION: This study describes a new mechanism by which ROS may promote a profibrotic phenotype in SSc fibroblasts through the oxidative inactivation of PTP1B, leading to pronounced activation of PDGFR. The study also presents a novel molecular mechanism by which NAC may act on ROS and PTP1B to provide therapeutic benefit in SSc.