DOI QR코드

DOI QR Code

Inhibition of c-FLIP by RNAi Enhances Sensitivity of the Human Osteogenic Sarcoma Cell Line U2OS to TRAILInduced Apoptosis

  • Zhang, Ya-Ping (Faculty of Life Science and Technology, Kunming University of Science and Technology) ;
  • Kong, Qing-Hong (Faculty of Life Science and Technology, Kunming University of Science and Technology) ;
  • Huang, Ying (Faculty of Life Science and Technology, Kunming University of Science and Technology) ;
  • Wang, Guan-Lin (Faculty of Life Science and Technology, Kunming University of Science and Technology) ;
  • Chang, Kwen-Jen (Faculty of Life Science and Technology, Kunming University of Science and Technology)
  • Published : 2015.04.03

Abstract

To study effects of cellular FLICE (FADD-like IL-$1{\beta}$-converting enzyme)-inhibitory protein (c-FLIP) inhibition by RNA interference (RNAi) on sensitivity of U2OS cells to tumor necrosis factor (TNF)-related apoptosis-inducing ligand (TRAIL)-induced apoptosis, plasmid pSUPER-c-FLIP-siRNA was constructed and then transfected into U2OS cells. A stable transfection cell clone U2OS/pSUPER-c-FLIP-siRNA was screened from the c-FLIP-siRNA transfected cells. RT-PCR and Western blotting were applied to measure the expression of c-FLIP at the levels of mRNA and protein. The results indicated that the expression of c-FLIP was significantly suppressed by the c-FLIP-siRNA in the cloned U2OS/pSUPER-c-FLIP-siRNA as compared with the control cells of U2OS/pSUPER. The cloned cell line of U2OS/pSUPER-c-FLIP-siRNA was further examined for TRAILinduced cell death and apoptosis in the presence of a pan-antagonist of inhibitor of apoptosis proteins (IAPs) AT406, with or without 4 hrs pretreatment with rocaglamide, an inhibitor of c-FLIP biosynthesis, for 24 hrs. Cell death effects and apoptosis were measured by the methods of MTT assay with 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide and flow cytometry, respectively. The results indicated that TRAIL-induced cell death in U2OS/pSUPER-c-FLIP-siRNA was increased compared with control cells U2OS/pSUPER in the presence or absence of AT406. Flow cytometry indicated that TRAIL-induced cell death effects proceeded through cell apoptosis pathway. However, in the presence of rocaglamide, cell death or apoptotic effects of TRAIL were similar and profound in both cell lines, suggesting that the mechanism of action for both c-FLIP-siRNA and rocaglamide was identical. We conclude that the inhibition of c-FLIP by either c-FLIP-siRNA or rocaglamide can enhance the sensitivity of U2OS to TRAIL-induced apopotosis, suggesting that inhibition of c-FLIP is a good target for anti-cancer therapy.

Acknowledgement

Supported by : National Natural Science Foundation of China

References

  1. Abedini MR, Qiu Q, Yan X, et al (2004). Possible role of FLICK-like inhibitory protein (FLIP) in chemoresistant ovarian cancer cells in vitro. Oncogene, 23, 6997-7004. https://doi.org/10.1038/sj.onc.1207925
  2. Brummelkamp TR, Bernards R, Agami R (2002). A system for stable expression of short interfering RNAs in mammalian cells. Science, 296, 550-3. https://doi.org/10.1126/science.1068999
  3. Carlo-Stella C, Lavazza C, Locatelli A, et al (2007). Targeting TRAIL agonistic receptors for cancer therapy. Clin Cancer Res, 13, 2313-7. https://doi.org/10.1158/1078-0432.CCR-06-2774
  4. Cai Q, Sun H, Peng Y, et al (2011). A potent and orally active antagonist (SM-406/AT-406) of multiple inhibitor of apoptosis proteins (IAPs) in clinical development for cancer treatment. J Med Chem, 54, 2714-26. https://doi.org/10.1021/jm101505d
  5. Djerbi M, Screpanti V, Catrina AI, et al (1999). The inhibitor of death receptor signaling, FLICE-inhibitory protein defines a new class of tumor progression factors. J Exp Med, 190, 1025-32. https://doi.org/10.1084/jem.190.7.1025
  6. Debatin KM, Krammer PH (2004). Death receptors in chemotherapy and cancer. Oncogene, 23, 2950-66. https://doi.org/10.1038/sj.onc.1207558
  7. Elnemr A, Ohta T, Yachie A, et al (2001). Human pancreatic cancer cells disable function of Fas receptors at several levels in fas signal transduction pathway. Int J Oncol, 18, 311-6.
  8. Gao S, Wang H, Lee P, et al (2006). Androgen receptor and prostate apoptosis response factor-4 target the c-FLIP gene to determine survival and apoptosis in the prostate gland. J Mol Endocrinol, 36, 463-83. https://doi.org/10.1677/jme.1.01991
  9. Hernandez A, Wang QD, Schwartz SA, et al (2001). Sensitization of human colon cancer cells to TRAIL-mediated apoptosis. J Gastrointest Surg, 5, 56-65. https://doi.org/10.1016/S1091-255X(01)80014-7
  10. Iannitti T, Morales-Medina JC, Palmieri B (2014). Phosphorothioate oligonucleotides: effectiveness and toxicity. Curr Drug Targets, 15, 663-73. https://doi.org/10.2174/1389450115666140321100304
  11. Li CL, Chang L, Guo L, et al (2014). $\beta$-elemene induces caspase-dependent apoptosis in human glioma cells in vitro through the upregulation of bax and fas/fasl and downregulation of bcl-2. Asian Pac J Cancer Prev, 15, 10407-12.
  12. Micheau O, Thome M, Schneider P, et al (2002). The long form of FLIP is an activator of caspase-8 at the fas death-inducing signaling complex. J Biol Chem, 277, 45162-71. https://doi.org/10.1074/jbc.M206882200
  13. Mezzanzanica D, Balladore E, Turatti F, et al (2004). CD95-mediated apoptosis is impaired at receptor level by cellular FLICE-inhibitory protein (long form) in wild-type p53 human ovarian carcinoma. Clin Cancer Res, 10, 5202-14. https://doi.org/10.1158/1078-0432.CCR-03-0537
  14. Marina N, Gebhardt M, Teot L, et al (2004). Biology and therapeutic advances for pediatric osteosarcoma. Oncologist, 9, 422-4. https://doi.org/10.1634/theoncologist.9-4-422
  15. Mori T, Doi R, Toyoda E, et al (2005). Regulation of the resistance to TRAIL-induced apoptosis as a new strategy for pancreatic cancer. Surgery, 138, 71-7. https://doi.org/10.1016/j.surg.2005.03.001
  16. Mirabello L, Troisi RJ, Savage SA (2009). Osteosarcoma incidence and survival rates from 1973 to 2004: data from the Surveilance, epidemiology, and end results program. Cancer, 115, 1531-43. https://doi.org/10.1002/cncr.24121
  17. Mollaie HR, Monavari SH, Arabzadeh SA, et al (2013). RNAi and miRNA in viral infections and cancers. Asian Pac J Cancer Prev, 14, 7045-56. https://doi.org/10.7314/APJCP.2013.14.12.7045
  18. Marczak A, Denel-Bobrowska M, Rogalska A, et al (2014). Cytotoxicity and induction of apoptosis by formamidinodoxorubicins in comparison to doxorubicin in human ovarian adenocarcinoma cells. Environ Toxicol Pharmacol, 39, 369-83.
  19. Nakajima A, Kojima Y, Nakayama M, et al (2008). Downregulation of c-FLIP promotes caspase-dependent JNK activation and reactive oxygen species accumulation in tumor cells. Oncogene, 27, 76-84. https://doi.org/10.1038/sj.onc.1210624
  20. Okano H, Shiraki K, Inoue H, et al (2003). Cellular FLICK/caspase-8-inhibitory protein as a principal regulator of cell death and survival in human hepatocellular carcinoma. Lab Invest, 83, 1033-43. https://doi.org/10.1097/01.LAB.0000079328.76631.28
  21. Paddison PJ, Caudy AA, Bernstein E, et al (2002). Short hairpin RNAs (shRNAs) induce sepuence-specific silencing in mammalian cells. Genes Dev, 16, 948-58. https://doi.org/10.1101/gad.981002
  22. Rao DD, Wang Z, Senzer N, Nemunaitis J (2013). RNA interference and personalized cancer therapy. Discov Med, 15, 101-10.
  23. Safa AR (2012). c-FLIP, a master anti-apoptotic regulator. Exp Oncol, 34, 176-84.
  24. Soares PI, Dias SJ, Novo CM, et al (2012). Doxorubicin vs. ladirubicin: methods for improving osteosarcoma treatment. Mini Rev Med Chem, 12, 1239-49. https://doi.org/10.2174/138955712802762022
  25. Thorburn A (2004). Death receptor-induced cell killing. Cell Signal, 16, 139-44. https://doi.org/10.1016/j.cellsig.2003.08.007
  26. Ullenhag GJ, Mukherjee A, Watson NF, et al (2007). Overexpression of FLIPL is an independent marker of poor prognosis in colorectal cancer patients. Clin Cancer Res, 13, 5070-5. https://doi.org/10.1158/1078-0432.CCR-06-2547
  27. Valnet-Rabier MB, Challier B, Thiebault S, et al (2005). c-FLIP protein expression in burkitt's lymphomas is associated with a poor clinical outcome [J]. Br J Haematol, 128, 767-73. https://doi.org/10.1111/j.1365-2141.2005.05378.x
  28. Wiley SR, Schooley K, Smolak PJ, et al (1995). Identification and characterization of a new member of the TNF family that induces apoptosis. Immunity, 3, 673-82. https://doi.org/10.1016/1074-7613(95)90057-8
  29. Woo SM, Min KJ, Kwon TK (2012). Calyculin A causes sensitization to tumor necrosis factor-related apoptosis-inducing ligand (TRAIL)-induced apoptosis by ROS-mediated down-regulation of cellular FLICE-inhibiting protein (c-FLIP) and by enhancing death receptor 4 mRNA stabilization. Apoptosis, 17, 1223-34. https://doi.org/10.1007/s10495-012-0753-y
  30. Zhang X, Jin TG, Yang H, et al (2004). Persistent c-FLIP(L) expression is necessary and sufficient to maintain resistance to tumor necrosis factor-related apoptosis-inducing ligand-mediated apoptosis in prostate cancer. Cancer Res, 64, 7086-91. https://doi.org/10.1158/0008-5472.CAN-04-1498
  31. Zhu JY, Giaisi M, Kohler R, et al (2009). Rocaglamide sensitizes leukemic T cells to activation-induced cell death by differential regulation of CD95L and c-FLIP expression. Cell Death Differ, 16, 1289-99. https://doi.org/10.1038/cdd.2009.42

Cited by

  1. Co-Eradication of Breast Cancer Cells and Cancer Stem Cells by Cross-Linked Multilamellar Liposomes Enhances Tumor Treatment vol.12, pp.8, 2015, https://doi.org/10.1021/mp500754r
  2. Facile synthesis of magnetic core–shell nanocomposites for MRI and CT bimodal imaging vol.3, pp.34, 2015, https://doi.org/10.1039/C5TB00775E
  3. Overcoming resistance to TRAIL-induced apoptosis in solid tumor cells by simultaneously targeting death receptors, c-FLIP and IAPs vol.49, pp.1, 2016, https://doi.org/10.3892/ijo.2016.3525
  4. Therapeutic potentials of short interfering RNAs vol.101, pp.19, 2017, https://doi.org/10.1007/s00253-017-8433-z