Tumor Biology

, Volume 36, Issue 4, pp 2531–2539 | Cite as

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

Research Article

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.

Keywords

Autophagy Beclin-1 Proliferation Invasion Chemoresistance 

Notes

Acknowledgments

This work was supported by the grants from the National Natural Science Foundation of Youth Program (No. 81101394), Shanghai Special Fund for Outstanding Young Teachers in Universities (No. JDY10080), Shanghai Renji Hospital Fund for Outstanding Youth (No. RJPY10-010), and Shanghai Fund for Young Physician Development (No. 20141051).

Conflicts of interest

No conflict of interest to disclose.

References

  1. 1.
    Jemal A, Bray F, Center MM, Ferlay J, Ward E, Forman D. Global cancer statistics. CA Cancer J Clin. 2011;61:69–90.CrossRefPubMedGoogle Scholar
  2. 2.
    Choong PF, Teh HX, Teoh HK, Ong HK, Choo KB, Sugii S, et al. Heterogeneity of osteosarcoma cell lines led to variable responses in reprogramming. Int J Med Sci. 2014;11:1154–60.CrossRefPubMedPubMedCentralGoogle Scholar
  3. 3.
    He H, Ni J, Huang J. Molecular mechanisms of chemoresistance in osteosarcoma (review). Oncol Lett. 2014;7:1352–62.PubMedPubMedCentralGoogle Scholar
  4. 4.
    Li Z, Zhu WG. Targeting histone deacetylases for cancer therapy: from molecular mechanisms to clinical implications. Int J Biol Sci. 2014;10:757–70.CrossRefPubMedPubMedCentralGoogle Scholar
  5. 5.
    Ouyang L, Shi Z, Zhao S, Wang FT, Zhou TT, Liu B, et al. Programmed cell death pathways in cancer: a review of apoptosis, autophagy and programmed necrosis. Cell Prolif. 2012;45:487–98.CrossRefPubMedGoogle Scholar
  6. 6.
    Kang R, Zeh HJ, Lotze MT, Tang D. The beclin 1 network regulates autophagy and apoptosis. Cell Death Differ. 2011;18:571–80.CrossRefPubMedPubMedCentralGoogle Scholar
  7. 7.
    Wu XY, Chen J, Cao QH, Dong M, Lin Q, Fan XJ, et al. Beclin 1 activation enhances chemosensitivity and predicts a favorable outcome for primary duodenal adenocarcinoma. Tumour Biol. 2013;34:713–22.CrossRefPubMedGoogle Scholar
  8. 8.
    Tong Y, You L, Liu H, Li L, Meng H, Qian Q, et al. Potent antitumor activity of oncolytic adenovirus expressing beclin-1 via induction of autophagic cell death in leukemia. Oncotarget. 2013;4:860–74.CrossRefPubMedPubMedCentralGoogle Scholar
  9. 9.
    Tai WT, Shiau CW, Chen HL, Liu CY, Lin CS, Cheng AL, et al. Mcl-1-dependent activation of beclin 1 mediates autophagic cell death induced by sorafenib and sc-59 in hepatocellular carcinoma cells. Cell Death Dis. 2013;4:e485.CrossRefPubMedPubMedCentralGoogle Scholar
  10. 10.
    Li H, Wang P, Sun Q, Ding WX, Yin XM, Sobol RW, et al. Following cytochrome c release, autophagy is inhibited during chemotherapy-induced apoptosis by caspase 8-mediated cleavage of beclin 1. Cancer Res. 2011;71:3625–34.CrossRefPubMedPubMedCentralGoogle Scholar
  11. 11.
    Pan B, Chen D, Huang J, Wang R, Feng B, Song H, et al. Hmgb1-mediated autophagy promotes docetaxel resistance in human lung adenocarcinoma. Mol Cancer. 2014;13:165.CrossRefPubMedPubMedCentralGoogle Scholar
  12. 12.
    Bao LJ, Jaramillo MC, Zhang ZB, Zheng YX, Yao M, Zhang DD, et al. Nrf2 induces cisplatin resistance through activation of autophagy in ovarian carcinoma. Int J Clin Exp Pathol. 2014;7:1502–13.PubMedPubMedCentralGoogle Scholar
  13. 13.
    Chen Y, Li X, Wu X, He C, Guo L, Zhang S, et al. Autophagy-related proteins lc3 and beclin-1 impact the efficacy of chemoradiation on esophageal squamous cell carcinoma. Pathol Res Pract. 2013;209:562–7.CrossRefPubMedGoogle Scholar
  14. 14.
    Kim SY, Song X, Zhang L, Bartlett DL, Lee YJ. Role of bcl-xl/beclin-1 in interplay between apoptosis and autophagy in oxaliplatin and bortezomib-induced cell death. Biochem Pharmacol. 2014;88:178–88.CrossRefPubMedPubMedCentralGoogle Scholar
  15. 15.
    Ko H, Kim YJ, Amor EC, Lee JW, Kim HC, Kim HJ, et al. Induction of autophagy by dimethyl cardamonin is associated with proliferative arrest in human colorectal carcinoma hct116 and lovo cells. J Cell Biochem. 2011;112:2471–9.CrossRefPubMedGoogle Scholar
  16. 16.
    Jiang ZF, Shao LJ, Wang WM, Yan XB, Liu RY. Decreased expression of beclin-1 and lc3 in human lung cancer. Mol Biol Rep. 2012;39:259–67.CrossRefPubMedGoogle Scholar
  17. 17.
    Wang L, Zhang H, Qian J, Wang K, Zhu J. Interleukin-10 blocks in vitro replication of human cytomegalovirus by inhibiting the virus-induced autophagy in mrc5 cells. Biochem Biophys Res Commun. 2014;448:448–53.CrossRefPubMedGoogle Scholar
  18. 18.
    Yang YH, Li B, Zheng XF, Chen JW, Chen K, Jiang SD, et al. Oxidative damage to osteoblasts can be alleviated by early autophagy through the endoplasmic reticulum stress pathway—implications for the treatment of osteoporosis. Free Radic Biol Med. 2014;77C:10–20.CrossRefGoogle Scholar
  19. 19.
    Li H, Zhang K, Liu LH, Ouyang Y, Bu J, Guo HB, et al. A systematic review of matrix metalloproteinase 9 as a biomarker of survival in patients with osteosarcoma. Tumour Biol. 2014;35:5487–91.CrossRefPubMedGoogle Scholar
  20. 20.
    Wang J, Shi Q, Yuan TX, Song QL, Zhang Y, Wei Q, et al. Matrix metalloproteinase 9 (mmp-9) in osteosarcoma: review and meta-analysis. Clin Chim Acta. 2014;433:225–31.CrossRefPubMedGoogle Scholar
  21. 21.
    Shang HS, Chang JB, Lin JH, Lin JP, Hsu SC, Liu CM, et al. Deguelin inhibits the migration and invasion of u-2 os human osteosarcoma cells via the inhibition of matrix metalloproteinase-2/-9 in vitro. Molecules. 2014;19:16588–608.CrossRefPubMedGoogle Scholar
  22. 22.
    Zheng Y, Wang X, Wang H, Yan W, Zhang Q, Chang X. Bone morphogenetic protein 2 inhibits hepatocellular carcinoma growth and migration through downregulation of the pi3k/akt pathway. Tumour Biol. 2014;35:5189–98.CrossRefPubMedGoogle Scholar
  23. 23.
    Tonini GP, Pistoia V. Molecularly guided therapy of neuroblastoma: a review of different approaches. Curr Pharm Des. 2006;12:2303–17.CrossRefPubMedGoogle Scholar
  24. 24.
    Mohan N, Chakrabarti M, Banik NL, Ray SK. Combination of LC3 shRNA plasmid transfection and genistein treatment inhibited autophagy and increased apoptosis in malignant neuroblastoma in cell culture and animal models. PLoS One. 2013;8:e78958.CrossRefPubMedPubMedCentralGoogle Scholar
  25. 25.
    Fulda S. Targeting inhibitor of apoptosis proteins for cancer therapy: a double-edge sword? J Clin Oncol. 2014;32:3190–1.CrossRefPubMedGoogle Scholar
  26. 26.
    Monteith GR. Prostate cancer cells alter the nature of their calcium influx to promote growth and acquire apoptotic resistance. Cancer Cell. 2014;26:1–2.CrossRefPubMedGoogle Scholar
  27. 27.
    Lee JH, Jung KH, Lee H, Son MK, Yun SM, Ahn SH, et al. Hs-133, a novel fluorescent phosphatidylinositol 3-kinase inhibitor as a potential imaging and anticancer agent for targeted therapy. Oncotarget. 2014;5:10180–97.Google Scholar
  28. 28.
    Hou YJ, Dong LW, Tan YX, Yang GZ, Pan YF, Li Z, et al. Inhibition of active autophagy induces apoptosis and increases chemosensitivity in cholangiocarcinoma. Lab Investig. 2011;91:1146–57.CrossRefPubMedGoogle Scholar
  29. 29.
    Ren Y, Huang F, Liu Y, Yang Y, Jiang Q, Xu C. Autophagy inhibition through pi3k/akt increases apoptosis by sodium selenite in nb4 cells. BMB Rep. 2009;42:599–604.CrossRefPubMedGoogle Scholar
  30. 30.
    Hsieh MT, Chen HP, Lu CC, Chiang JH, Wu TS, Kuo DH, et al. The novel pterostilbene derivative ank-199 induces autophagic cell death through regulating pi3 kinase class III/beclin 1/atgrelated proteins in cisplatinresistant car human oral cancer cells. Int J Oncol. 2014;45:782–94.PubMedGoogle Scholar
  31. 31.
    McKnight NC, Zhong Y, Wold MS, Gong S, Phillips GR, Dou Z, et al. Beclin 1 is required for neuron viability and regulates endosome pathways via the uvrag-vps34 complex. PLoS Genet. 2014;10:e1004626.CrossRefPubMedPubMedCentralGoogle Scholar

Copyright information

© International Society of Oncology and BioMarkers (ISOBM) 2014

Authors and Affiliations

  1. 1.Department of Orthopaedic SurgeryRenji Hospital, School of Medicine, Shanghai Jiao Tong UniversityShanghaiChina

Personalised recommendations