Actinomycin D Inhibits Cell Proliferations And Promotes Apoptosis In Osteosarcoma Cells

Background and Purpose Osteosarcoma(OS)is the most common malignant primary bone tumor in children and adolescents.OS has a predilection for the metaphyseal portions of the long bone,with the distal femur and proximal tibia accounting for ~15% of primary bones.OS is highly aggressive and it metastasizes primarily to the lung.Patients with OS are between 15-25 years old.The median age of an OS patient is 16 years old,with a male predominance.The high incidence of OS during the adolescence growth spurt indicates there may be a link between this disease and bone development.Histologically,OS is characterized by a proliferation of malignant spindle cells.Although several histological subtypes may exist,including chondroblastic and fibroblastic OS,once the osteoid directly produced by sarcoma cells is found,OS can be diagnosed.With regard to the clinical features,pain and swelling of the soft tissue are the most common symptoms of OS patients.Prior to the use of adjuvant and neoadjuvant chemotherapy,the long-term survival rate subsequent to surgical resection alone was <20%.Luckily,multi-agent chemotherapy regimens that were pioneered in the 1970 s and early 1980 s have dramatically improved the survival rate by up to ~70%,and the necessity for the chemotherapy used for OS patients has been demonstrated by randomized controlled trials.However,the survival rate has plateaued since themid-1980 s,despite advances in anti-OS therapy.In addition,patients with metastatic or recurrent diseases have a <20% chance of long-term survival despite aggressive therapies.To a certain extent,the reason behind this may be ascribed to the chemoresistance to anti-OS therapy.The development of chemoresistance in malignant tumors limits the effectiveness of cytotoxic drugs,and this is particularly true in OS,which is characterized by the frequent refractoriness to chemotherapy.Therefore,elucidation of the mechanisms of chemoresistance and implementation of strategies to overcome chemoresistance will definitely play a pivotal role in improving the survival rate of OS patients.Actinomycin D(ActD),is an anti-neoplastic agent that inhibits RNA synthesis by binding to guanine residues and inhibiting DNA-dependent RNA polymerase.ActD is a known DNA-interacting transcription blocker with anti-cancer activity,working as a cytotoxic inducer of apoptosis against tumor cells.Moreover,ActD has been reported to be an antineoplastic antibiotic that inhibits cell proliferation,by forming a stable complex with double-stranded DNA,thus inhib?iting DNA-primed RNA synthesis.It also causes single-strand breaks in DNA.Nevertheless,ActD exhibits antitumor activity and induces tumor cell apoptosis.Despite these reports,the effects of ActD on osteosarcoma cells have not been extensively investigated.Therefore,in this study,we char?acterized the actions of ActD on MG63 human osteosarcoma cells.Methods Actinomycin D was purchased from Calbiochem(San Diego,CA,USA).Dulbecco’s Modified Essential Medium(DMEM)and supplemented Fetal Bovine Serum(FBS)were purchased from Gibco Invitrogen Corporation(Carlsbad,CA,USA).The Hoechst kit was from Beyotime Biotechnology Co.(Haimen,Jiangsu,China).Other chemicals were of the highest purity available.Human osteosarcoma cell line MG63 was obtained from Shanghai Institute of Cell Biology(Introduced from American Type Culture Collection).The cells were cultured in DMEM supplemented with 10% FBS and antibiotics in 5% CO2 at 37 °C.For the experiments,MG63 cells were plated in a 6-well plates at 1.0×105 cells/m L for Hoechst staining,and 1.0×106 cells/m L for Western blots and real-time PCR assay.Cell proliferation was measured using the sulphorhodamine B(SRB)colorimetric assay.For the preparation of Hoechst staining,MG63 cells were plated with 1.0×105 cells/m L in 6-well plates.After pharmacological manipulations,cells were directly stained with Hoechst kit from Beyotime.Cell counting was carried out through National Institutes of Health software Image J.As for cell viability assay(MTT assay),1 × 103 cells were seeded in 96-well plate for 24 h.On the next day,cells were incubated with Actinomycin D respectively.Then the viable cells were stained by adding 20 μl MTT solution(5 mg/ml)per 100 μl of growth medium.Moreover,western blots and real-time PCR assays were used to examine the proteien and mRNA alternations of ActD treated osteosarcoma cells.All statistical analysis was performed by Image software.Quantitative data were showed in x ± s using ANOVA tests for comparisons.The value 0.05(*),0.01(**)and 0.001(***)was assumed as the level of significance for the statistic tests carried out.Results We demonstrated that Actinomycin D(ActD)may impair osteosarcomatous proliferations and lead to significant apoptosis in MG63 human osteosarcoma cells.Moreover,the proliferative inhibitory effects of ActD on osteosarcoma cells may be at least partly dependent on cyclin protein expressions,because we noted that ActD treatment would inhibit either cyclin gene transcriptions or pro?tein translations.Thus,the reduced cyclin pro?tein expressions may be responsible for the cell cycle arrest and consequently apoptosisConclusions The present results showed that ActD may impair osteosarcomatous proliferations and lead to significant apoptosis in MG63 human osteosarcoma cells,by inhibiting cyclins expressions and arresting cell-cyle progression.Our work have revealed a novel mechanism by which ActD inhibits osteosarcoma cell proliferations and induces apoptosis,and will provide an useful clue to chemotherapy in future treatment of osteosarcoma.