Effect of Mad2 on paclitaxel-induced cell death in ovarian cancer cells

Abstract

In this article, the status of spindle assembly checkpoint and the alteration of its major component, Mad2 protein level were examined in A2780 and SKOV3 ovarian cancer cell lines. Recombinant eukaryotic expression plasmid pEGFP-Mad2 was transfected into paclitaxel-resistant SKOV3 cells and Mad2 protein was knocked down by Mad2-specific siRNA in paclitaxel-sensitive A2780 cells. Then the expression level of Mad2 gene was detected by Western blotting. Flow cytometry revealed that SKOV3 cells were not fully arrested in G₂/M phase in contrast to A2780 cells in the presence of paclitaxel. However, paclitaxel sensitivity assay showed that sensitivity to paclitaxel was reversed after the transfection in both cell lines in terms of number of cells arrested at G₂/M phase and the expression of Bcl-2 was significantly changed. These results suggest that weakened spindle checkpoint with reduced expression of Mad2 is associated with resistance to paclitaxel in ovarian cells and Bcl-2 may be involved in this process.

Keywords

paclitaxel resistance / ovarian cancer / Mad2 / Bcl-2

References

[1] RowinskyE.K.. The development and clinical utility of the taxane class of antimicrotubule chemotherapy agents. Annu Rev Med, 1997, 48: 353-374

[2] KovárJ., EhrlichováM., SmejkalováB., et al.. Comparison of cell death-inducing effect of novel taxane SB-T-1216 and paclitaxel in breast cancer cells. Anticancer Res, 2009, 29(8): 2951-2960

[3] RowinskyE.K., DonehowerR.C.. Paclitaxel. New Engl J Med, 1995, 332(15): 1004-1014

[4] WineyE., HuneycuttB.J.. Centrosomes and checkpoints: the MPS1 family of kinases. Oncogene, 2002, 21(40): 6161-6169

[5] FiskF.A., WineyM.. Mps1 flies into new areas. Curr Biol, 2004, 14(24): R1058-R1060

[6] ZhouJ., YaoJ., JoshiH.C.. Attachment and tension in the spindle assembly checkpoint. J Cell Sci, 2002, 115(18): 3547-3555

[7] BurkeD.J., StukenbergP.T.. Linking kinetochore-microtubule binding to the spindle checkpoint. Dev Cell, 2008, 14(4): 474-479

[8] BlagosklonnyM.V., FojoT.. Molecular effects of paclitaxel: myths and reality (a critical review). Int J Cancer, 1999, 83(2): 151-156

[9] SinghP., RathinasamyK., MohanR., et al.. Microtubule assembly dynamics: an attractive target for anticancer drugs. IUBMB Life, 2008, 60(6): 368-375

[10] TakahashiT., HarukiN., NomotoS.. Identification of frequent impairment of the mitotic checkpoint and molecular analysis of the mitotic checkpoint genes, hsMAD2 and p55CDC in human lung cancers. Oncogene, 1999, 18(30): 4295-4300

[11] WangX., JinD.Y., WongY.C., et al.. Correlation of defective mitotic checkpoint with aberrantly reduced expression of MAD2 protein in nasopharyngeal carcinoma cells. Carcinogenesis, 2000, 21(12): 2293-2297

[12] WangX., JinD.Y., NgR.W., et al.. Significance of MAD2 expression to mitotic checkpoint control in ovarian cancer cells. Cancer Res, 2002, 62(6): 1662-1668

[13] McGroganB.T., GilmartinB., CarneyD.N., et al.. Taxanes, microtubules and chemoresistant breast cancer. Biochim Biophys Acta, 2008, 1785(2): 96-132

[14] González MartínA.. Anti-angiogenic therapy in ovarian cancer: a great expectation to be confirmed. Clin Transl Oncol, 2009, 11(9): 559-560

[15] JemalA., MurrayT., SamuelsA., et al.. Cancer statistics. CA Cancer J Clin, 2003, 53(1): 5-26

[16] RichardsonA., KayeS.B.. Drug resistance in ovarian cancer: the emerging importance of gene transcription and spatio-temporal regulation of resistance. Drug Resist Updat, 2005, 8(5): 311-321

[17] YamadaH.Y., GorbskyG.J.. Spindle checkpoint function and cellular sensitivity to antimitotic drugs. Mol Cancer Ther, 2006, 5(12): 2963-2969

[18] MalmancheN., MaiaA., ClaudioE.S.. The spindle assembly checkpoint: Preventing chromosome mis-segregation during mitosis and meiosis. FEBS Letters, 2006, 580(12): 2888-2895

[19] ZhouT., BaoY., YeS., et al.. Effect of spindle checkpoint on Akt2-mediated paclitaxel-resistance in A2780 ovarian cancer cells. J Huazhong Univ Sci Technolog [Med Sci], 2010, 30(2): 206-11

[20] GorbskyG.J., ChenR.H., MurrayA.W.. Microinjection of antibody to Mad2 protein into mammalian cells in mitosis induces premature anaphase. J Cell Biol, 1998, 141(5): 1193-1205

[21] SudoT., NittaM., SayaH., et al.. Dependence of paclitaxel sensitivity on a functional spindle assembly checkpoint. Cancer Res, 2004, 64(7): 2502-2508

[22] LeeE.A., KeutmannM.K., DowlingM.L., et al.. Inactivation of the mitotic checkpoint as a determinant of the efficacy of microtubule-targeted drugs in killing human cancer cells. Mol Cancer Ther, 2004, 3(6): 661-669

[23] SrivastavaR.K., MiQ.S., HardwickJ.M., et al.. Deletion of the loop region of Bcl-2 completely blocks paclitaxel-induced apoptosis. Proc Natl Acad Sci USA, 1999, 96(7): 3775-80

[24] WolterK.G., WangS.J., HensonB.S., et al.. (−)-gossypol inhibits growth and promotes apoptosis of human head and neck squamous cell carcinoma in vivo. Neoplasia, 2006, 8(3): 163-172

[25] MacklerN.J., PientaK.J.. Drug insight: Use of docetaxel in prostate and urothelial cancers. Nat Clin Pract Urol, 2005, 2(2): 92-100