The Mechanism And Functional Study Of YM155 In Osteosarcoma Cell F5M2

Background:Osteosarcoma is a malignant bone tumor characterized by highly aggressive behavior and mostly occurs around the knee joint in adolescents.The standard treatment for osteosarcoma is the combination of chemotherapy and surgery,which raises the 5 years’ metastasis-free survival rate to 55 to 70 percent in osteosarcoma patients.Many scholars believe that the current treatment and the survival rate of osteosarcoma have entered into a relative stagnation,because 15%-20% of patients have occurred metastasis when firstly diagnosed and about 30% will occurr metastasis within a year after diagnosis.Moreover,the prognosis is not promising as these patients are not sensitive to current chemotherapeutic drugs.Therefore,it is imperative to develop new drugs.YM155 is one of the imidazole derivatives and validated to be a specific inhibitor of survivin.YM155 could bind to survivin promoter and down-regulated its expression,resulting in decresing anti-apoptosis effect in various tumors.Previous studies demonstrated that YM155 could inhibit survivin expression,promote apoptosis and therefore stop tumor for growing in various tumors,including chronic lymphocytic leukemia,prostate cancer,non-small cell lung cancer,glioblastoma,bladder cancer,etc.By far,the application of YM155 to multiple tumors has been tested by human clinical trials.Moreover,YM155 was found to promote autophagy-dependent apoptosis which might be a novel point for researches in tumor apoptosis and new drugs.Survivin has been demonstrated to be highly expressed in osteosarcoma and closely related to its surgical staging in our previous studies.RNA interference vector can decrease survivin expression,resulting in decreasing proliferation capabilities and increasing apoptosis rate of osteosarcoma cells.Such studies provide theoretical support and give a primary basis for the further studies about anticancer drugs.Objective:1.To explore the effect of YM155 on the proliferation,clone formation,invasion,migration,cell cycle and apoptosis of osteosarcoma F5M2 cell;2.To explore the role of YM155 on apoptosis of osteosarcoma F5M2 cell and its associated mechanisms;3.To explore whether osteosarcoma F5M2 cell undergoes autophagic process when treated with YM155;4.To briefly discuss the feasibility of clinical application of YM155 as a new anti-tumor drug.Methods:1.The effect of YM155 on malignant behavior of F5M2 cells was detected by MTT assay,plate clone formation assay,cell scratch assay,Transwell assay and flow cytometry.The effect of YM155 on cell morphology was observed by transmission electron microscopy.2.q RT-PCR and Western-blot experiments were used to investigate the mechanism of YM155 in mediating osteosarcoma cell apoptosis.3.F5M2 cells was transfected by autophagy labeled adenovirus(m RFP-GFP-LC3).Confocal microscopy and MDC staining were used to observe a utophagy after YM155 treatment.4.To investigate the effect of decreasing autophagy activity on apoptosis,flow cytometry was used to detect apoptosis rate of F5M2 cells in three groups:(1)Control;(2)3-MA(Autophagy inhibitor)+YM155 treatment;(3)YM155 treatment.Results:1.Osteosarcoma F5M2 cells were treated with YM155 in different concentrations.Compared with the control group,MTT assay revealed that cell inhibition rates are obviously higher in YM155 group with all concentrations except 1 nmol/L.Plate clone formation assay revealed that the number of cell clone formation was significantly lower in the concentration of 15 and 30 nmol/L than that of the control group,which was in a reverse dose-dependent manner.Cell scratch assay demonstrated that the migration distance decreases with the increase of YM155 concentration of 15 and 30 nmol/L compared with the control group.Transwell cell invasion assay revealed that the number of cells that passed through Transwell membranes significantly decreased in YM155 group with the concentration of 15 and 30 nmol/L compared with the control group.Flow cytometry revealed that among the concentration of 5,15 and 30 nmol/L,there was no significant difference in the percentage of tumor cells in each phases compared with the control group.Flow cytometry revealed that cell apoptosis rate significantly increased in 15 and 30 nmol/L than that of control group.Through Transmission electron microscopy,we observed the poor growing status of F5M2 cells when treated with YM155.The surrounding microvilli almost disappeared which might be associated with cell metastasis.F5M2 cells,when treated with YM155,also displayed cell phenotype changes,including increasing vacuoles and swelling mitochondria with increasing mitochondrial membrane permeability.Chromatin boundary could be observed in the nucleus and led to apoptosis.2.q RT-PCR and Western-blot experiments revealed that YM155 could obviously inhibit survivin m RNA and protein expression,and increase casepase-3 activation in F5M2 cells with the concentration of 15 and 30 nmol/L,compared with the control group.3.Osteosarcoma F5M2 cell was successfully transfected by autophagy labeled adenovirus(m RFP-GFP-LC3).Autophagosome and autolysosome were observed by confocal microscopy in F5M2 cells of all groups while it was higher in YM155 group with the concentration of 10 nmol/L compared with the control group.Autophagosome was observed in each group through MDC staining,the number of which was much higher in YM155 group with the concentration of 10 nmol/L compared with the control group.4.Flow cytometry revealed that the apoptosis rate was lower in 3-MA+YM155 group than in YM155 group.Conclusion:1.YM155 could inhibit proliferation,clone formation,invasion and migration of osteosarcoma F5M2 cells.YM155 could also induce F5M2 cells apoptosis and had no obvious effect on cell cycle,it may exist tissue specificity.2.YM155 induced F5M2 cells apoptosis through inhibiting survivin expression and activating caspase pathway.3.YM155 could induce autophagy and autophagy flux in osteosarcoma F5M2 cells.The inhibition of autophagy was revealed to abolish the pro-apoptosis effect of YM155 but its mechanismwas unclear.4.This study laid the foundation for the application of YM155 in the treatment of osteosarcoma.However,its effectiveness and safety need to be further investigated in future studies.