title: Knockdown of autophagy-related protein 6, Beclin-1, decreases cell growth, invasion, and metastasis and has a positive effect on chemotherapy-induced cytotoxicity in osteosarcoma cells.
abstract: Beclin-1, a well-known key regulator of autophagy, has been implicated in many disorders, including cancer, aging, and degenerative diseases. Previous studies demonstrated that Beclin-1 participated in tumorgenesis and was highly expressed in colorectal cancer cells, primary duodenal adenocarcinoma, and hepatocellular carcinoma cells, and overexpression of Beclin-1 could induce autophagic cell death in leukemia cells. However, the exact effects and molecular mechanisms of Beclin-1-mediated autophagy in osteosarcoma are still unknown up to now. Here, we evaluated the role of Beclin-1 in human osteosarcoma cell lines in vivo and in vitro. In order to characterize the endogenous expression of Beclin-1 in osteosarcoma cell lines, we performed real-time PCR and Western blot analysis. We further analyzed the level of Beclin-1 in osteosarcoma cells after chemotherapy and investigated the impact of autophagy inhibition on chemotherapy-induced cytotoxicity. We used the small interfering RNA (siRNA) directed against Beclin-1 to infect the osteosarcoma cell line with relatively high Belcin-1 expression. Furthermore, we determine the functional relevance of Beclin-1 knockdown to osteosarcoma cell growth, migration, and invasion, and investigate the expression levels of matrix metallopeptidase-2 (MMP-2), MMP-9, phosphoinositide 3-kinase p85α (PI3Kp85α), and phosphorylated AKT (p-AKT). As a result, HOS osteosarcoma cells exhibited higher Beclin-1 expression. Anticancer agents including doxorubicin, cisplatin, and methotrexate each induced Beclin-1 up-regulation in human osteosarcoma cells, and siRNA-mediated knockdown of Beclin-1 suppressed cell proliferation, migration, and invasion indicated by 3-(4,5-dimethylthiazolyl-2)-2,5-diphenylthetrazolium bromide, would healing, and transwell assays. Cell apoptosis induced by anticancer agents was markedly increased. Knockdown of Beclin-1 or inhibition of autophagy by 3-methyladenine (an inhibitor of autophagy and PI3K) rendered them significantly more sensitive to chemotherapy. Addition of the pan-caspase inhibitor ZVAD-FMK partly reversed the cisplatin-induced cell death. When Beclin-1 expression was inhibited, the expression of PI3Kp85α, p-AKT, and MMP-9 was downregulated in HOS cells. In addition, the tumor volumes in subcutaneous nude mouse models in Beclin-1-deleted HOS cells were significantly smaller than those of control group. These results suggested that knockdown of Beclin-1 by siRNA exerts inhibitory effects on growth and migration of osteosarcoma cells possibly via blockade of the PI3K/AKT signaling. Beclin-1 knockdown rendered them significantly more sensitive to chemotherapy through activating apoptosis pathway. The results of this study suggest that Beclin-1 plays an important role in proliferation and tumor progression in osteosarcoma and inhibition autophagy can increase the efficacy of anticancer agent therapy.
authors.affiliation: Department of Orthopaedic Surgery, Renji Hospital, School of Medicine, Shanghai Jiao Tong University, Shanghai, 200127, China, WZhangDoctor@163.com.
authors.forename: Wei
authors.initials: W
authors.lastname: Zhang
authors.forename: Qianyi
authors.initials: Q
authors.lastname: Li
authors.forename: Chao
authors.initials: C
authors.lastname: Song
authors.forename: Lifeng
authors.initials: L
authors.lastname: Lao
mesh_terms: D000818:Animals; D017209:Apoptosis / Q000235:genetics; D051017:Apoptosis Regulatory Proteins / Q000096:biosynthesis / Q000235:genetics*; D000071186:Beclin-1; D045744:Cell Line, Tumor; D049109:Cell Proliferation / Q000235:genetics*; D002945:Cisplatin / Q000008:administration & dosage; D004317:Doxorubicin / Q000008:administration & dosage; D006801:Humans; D008565:Membrane Proteins / Q000096:biosynthesis / Q000235:genetics*; D008727:Methotrexate / Q000008:administration & dosage; D051379:Mice; D012516:Osteosarcoma / Q000188:drug therapy / Q000235:genetics* / Q000473:pathology; D015398:Signal Transduction; D023041:Xenograft Model Antitumor Assays
chemical_list: D051017:Apoptosis Regulatory Proteins; C491997:BECN1 protein, human; D000071186:Beclin-1; D008565:Membrane Proteins; D004317:Doxorubicin; D002945:Cisplatin; D008727:Methotrexate