A Preliminary Study On The Functiona Of Cancer Cachexia And PLK1 Inhibitor RO3280 In Neuroblastoma

Part one.A preliminary study on the cancer cachexia in neuroblastomaObjective:Neuroblastoma is the most common extracranial malignant solid tumor of childhood,accounting for～15%of cancer-related deaths in children.Cancer cachexia is a common multifactor syndrome of tumors,mainly characteristiced with weight loss,skeletal muscle loss.About 50%of cancer deaths is associated with cancer cachexia.The research of mechanisms of cancer cachexia is hot.To explore the mechanisms of cancer cachexia in neuroblastoma we try to establish a cancer cachexia model of neuroblastoma:KP-N-NS cancer cachexia model,trying to explore its mechanism.Method:In vivo:Twelve nude mice were randomly divided into two groups.Six nude mice were injected with KP-N-NS cells subcutaneously.Six nude mice were injected with PBS volume subcutaneously.The weight of nude mice and the food consumption were monitored.Six weeks later,viable nude mice were killed,collecting the skeletal muscle and heart specimens.H&E staining method was used to observe the change of the nude mice in skeletal muscle and myocardial.Immunofluorescence method was uesd to detect Myosin expression level in skeletal muscle.Immunohistochemical method was used to detect protein expression levels related of proliferation,autophagy,ubiquitinin in skeletal muscle.In vitro:The culture medium that was used to train KP-N-NS was named conditioned medium(CM).CM(CM group)or Angiogenin(Angiogenin group)was used to culture C2C12 cells.Twelve hours later,The number of C2C12 cells were counted.C2C12 cells was induced to differentiate for 3 days.Then,CM(CM group)or Angiogenin(Angiogenin group)was used to culture C2C12 cells for 24 hours.Immunofluorescence method was used to observe the diameter of myotube of C2C12 cells.C2C12 cells were treated with CM for 15 minutes,30 minutes,1 hour or 24 hours.Western Blot method was used to detect protein expression levels related of proliferation,autophagy,ubiquitinin in skeletal muscle.The expression of cytokines in CM was detected by protein chip.Results:The weight of KP-N-NS nude mice is lighter.The KP-N-NS nude mice eated less food.The skeletal muscle of KP-N-NS nude mice was less.The expression of Myosin of KP-N-NS nude mice in skeletal muscle decreased.The expression of Fbxo32 of KP-N-NS nude mice in skeletal muscle increased.The KP-N-NS neuroblastoma cancer cachexia model was established successfully.The number of C2C12 cells treated with CM decreased.The diameter of skeletal muscle myotube decreased.The level of Stat3 phosphorylation increased.The the expression of Angiogenin in CM increased significantly.The number of C2C12 cells treated with Angiogenin decreased.The diameter of skeketal muscle myotube decreased.Conclusion:The KP-N-NS cancer cachexia model was builded successfully.The expression level of Myosin,Fbxo32,Stat3,Akt and mTOR were changed.Angiogenin could lead to the changes similar to cancer cachexia in vitro.Part two.The PLK1 Inhibitor RO3280 Induces Cell Death in Neuroblastoma through Apoptosis and AutophagyBackground:Neuroblastoma is the most common extracranial malignant solid tumor of childhood.Polo-like kinase 1(PLK1),a serine/threonine kinase,is overexpressed in many human tumors.The PLK1 inhibitor RO3280 has been shown to suppress cell growth in leukemia cells.However,the role of RO3280 in neuroblastoma has not been investigated.The purpose of this study was to study the therapeutic effects of RO3280 in treating neuroblastoma cells.Methods:The neuroblastoma cell growth was examining using Cell Counting Kit-8(CCK8)assay and colony formation assay.Cell apoptosis and cell cycle distribution were determined using Annexin V/PI staining and flow cytometry.LC3-GFP transfection followed by fluorescence confocal microscopy and transmission electron microscope were performed to assess the cell autophagy.Multiple apoptosis markers and autophagy markers were also assessed.Gene expression profiles of neuroblastoma cells were analyzed with real-time polymerase chain reaction(PCR).Results:RO3280 decreased neuroblastoma cell growth in a concentration-dependent manner.The apoptosis rate and cell percentage at G2/M phase of SH-SY-5Y and NGP cells treated with RO3280 were higher than those of untreated control.After RO3280 treatment,much more LC3-GFP formed fluorescence spots and autophagosomes were found in neuroblastoma cells.Further,cleaved Caspase-3,cleaved PARP and elevated LC3-Ⅱproteins were observed in neuroblastoma cells treated with RO3280.Besides,the expression levels of numerous cell apoptosis-related genes and cell autophagy-related genes were affected by RO3280 in neuroblastoma cells.Conclusion:RO3280 significantly reduced neuroblastoma cell growth through inducing cell apoptosis and cell autophagy,providing the theoretical basis for the clinical use of RO3280 in neuroblastoma treatment.