Histone Deacetylase Inhibitor SAHA Inhibits RD Cells Proliferation And Induces Apoptosis In Vitro

Background and objective:Rhabdomyosarcoma(rhabdomyosarcoma,RMS)is the most common soft tissue sarcoma of the childhood,is considered a malignant tumor derived from skeletal muscle cells(cancer),accounting for 10% of pediatric malignant solid tumors,accounting for 50%~70% of the all childhood soft tissue sarcoma.With the comprehensive treatment applied to clinical,RMS prognosis has been significantly improved,5-year overall survival rate has reached to 33%~50%,but there are still more than 50% of patients died of tumor recurrence or distant metastasis.China has a large population and the number of children with rhabdomyosarcoma is large,we are not satisfied with the overall treatment.Comprehensive treatment,including surgery,chemotherapy and radiation therapy,in order to eliminate residual lesions to reduce the recurrence rate,the current advocate for all patients to be chemotherapy,but the side effects of chemotherapy is limited in the use among children.So the appearance of new drugs is an urgent need for the treatment of rhabdomyosarcoma.It was reported that the histones of tumor cells were often deacetylated,and the imbalance of histone acetylation caused by abnormal HDAC was closely related to the occurrence and development of tumor.HDAC inhibitors reversibly reverse the acetylation of histones,leading to regulatory gene expression,cell cycle arrest,tumor cell apoptosis,anti-angiogenesis,DNA damage and repair,and reduced tumor cell invasion and migration.Therefore,HDAC inhibitors are expected to be a new target drug for the treatment of rhabdomyosarcoma.Therefore,the purpose of this study is to analyze the effects of HDAC inhibitor SAHA on the proliferation,cell cycle distribution,apoptosis,autophagy and migration of RD cells,and to explore the mechanism of its induction of apoptosis.Methods:RD cells were treated with various concentrations of SAHA,the effect of detection on cell proliferation by MTT assay.The effect of cell cycle distribution was analyzed by PI staining.The effect of cell apoptosis was analyzed by Annexin V-FITC/PI,The apoptosis of cells was detected again by DAPI staining.The mechanism of apoptosis induction was determined by the following experiments: the mitochondrial membrane potential was measured by JC-1 probe,the expression of apoptosis-related protein,mitochondrial pathway-related protein and ERK signaling pathway protein were detected by western blot.The cell autophagy was detected by MDC staining and the expression of autophagy-related proteins was detected by western blot.The cell migration was detected by cell scratches.The acetylation level of histone H4 was detected by western blot.Results: 1.SAHA inhibited the proliferation of RD cells,and it was time-dependent and concentration-dependent.The IC50 values of SAHA for 24 h,48 h and 72 h of RD cells were 8.503 μmol /L,3.965 μmol/L,1.921 μmol/L,respectively.The ERK signal pathway changes were that ERK protein phosphorylation levels were decreased,while total ERK protein levels did not change significantly.2.The result of flow cytometry showed that RD cells was arrested in G2 / M by SAHA.3.The results of DAPI staining showed that SAHA could induce the apoptosis of RD cells.DAPI staining showed that the number of apoptotic cells increased with the increasing of SAHA concentration,and the result was the same.The results of JC-1 flow cytometry indicated that the level of mitochondrial membrane potential of RD cells decreased gradually with the increasing of SAHA concentrations.The results of western blot revealed that with the increasing of SAHA concentration,the expressions of caspase-3 and PARP-1 increased gradually,the expression of anti-apoptotic protein Bcl-2 was decreased,the expression of pro-apoptotic protein Bax increased and the expressions of cytochrome C and caspase-9 increased correspondingly.4.The results of MDC staining showed that the number of spots with green fluorescent increased gradually with the increasing of SAHA concentration,indicating that cells autophagy has occurred.Western blot results indicated that the expression of p62 protein decreased gradually with the increasing of SAHA concentration,the expression level of Beclin1 protein increased gradually and the expression of LC3-II protein was also increased.5.The results of cell scratches revealed that the healing rate of scratches was gradually decreased with the increasing of SAHA concentration.6.The results of Western blot showed that the acetylation level of histone H4 increased gradually with the increasing of SAHA concentration.Conclusion:1.HDAC inhibitor SAHA inhibits the proliferation of RD cells,resulting in G2 / M phase arrest in RD cells,which was time and concentration dependent;2.HDAC inhibitor SAHA induces apoptosis of RD cells,and the mechanisms underlying apoptosis may be dependent on the mitochondrial pathway and have caspase-dependent,3.HDAC inhibitor SAHA induces RD cell autophagy;4.HDAC inhibitors SAHA inhibits the migration of RD cells.