Tumor Biology

, Volume 31, Issue 3, pp 225–232 | Cite as

Depression of MAD2 inhibits apoptosis and increases proliferation and multidrug resistance in gastric cancer cells by regulating the activation of phosphorylated survivin

  • Li Wang
  • Fang Yin
  • Yulei Du
  • Bei Chen
  • Shuhui Liang
  • Yongguo Zhang
  • Wenqi Du
  • Kaichun Wu
  • Jie Ding
  • Daiming Fan
Research Article


Mitotic arrest-deficient 2 (MAD2) is one of the essential mitotic spindle checkpoint regulators, and it can protect cells from aberrant chromosome segregation. The Mad2 gene is very rarely mutated in many kinds of human cancer, but aberrantly reduced expression of MAD2 has been correlated with defective mitotic checkpoints in several human cancers. We have previously found that the MAD2 expression level is also shown to be associated with the multidrug resistance of tumour cells. In this study, we constructed a small interfering RNA (siRNA) eukaryotic expression vector of MAD2 and downregulated MAD2 expression in the gastric cancer cell line SGC7901 by transfection of MAD2-siRNA. SGC7901 cells stably transfected with the MAD2-siRNA exhibited significantly increased expression of phosphorylated survivin protein and enhanced drug resistance. Furthermore, MAD2-siRNA suppressed the proliferation of SGC7901 cells and inhibited tumour formation in athymic nude mice. This study clearly reveals that downregulation of MAD2 could regulate the cell cycle, increase proliferation, and improve the drug resistance of gastric cancer cells by regulating the activation of phosphorylated survivin. It also suggests both that MAD2 might play an important role in the development of human gastric cancer and that silencing the MAD2 gene may help to deal with the multidrug resistance of gastric cancer cells.


Gastric cancer Mitotic arrest-deficient 2 Mitotic spindle checkpoint Survivin Drug resistance siRNA Apoptosis 



mitotic arrest-deficient 2










3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide


phosphate-buffered saline


multi-drug resistance




multi-drug resistance-associated protein


propidium iodide


small interfering RNA



We are grateful to Dr. Bin Guo for his proofreading of the manuscript. We thank technician Yunxin Cao for excellent technical assistance. This study was supported in part by grants from the Chinese National Foundation of National Sciences (C03031905, 30973422, 30600551, and 30530780).

Supplementary material

13277_2010_36_MOESM1_ESM.jpg (121 kb)
Supplementary Fig. 1 Effects of MAD2-siRNA on the apoptosis of gastric cancer cells treated with cisplatin for 24 hours. The apoptosis rate is increased in the SGC7901/MAD2-siRNA cell line compared with control and blank vector groups. (JPEG 121 kb)
13277_2010_36_MOESM2_ESM.jpg (201 kb)
Supplementary Fig. 2 Effects of MAD2-siRNA on cellular sensitivity to chemodrugs. The value shown is the mean of three determinations. IC50 values of the cells described as above in vitro. a Cell number was evaluated by the absorbance at 490 nm in an MTT assay at the indicated time. Representative experiment of three, with similar results. b Soft agar clone-forming assay of the cells were performed as described above. The data represent means ± SD of three independent experiments. (JPEG 201 kb)
13277_2010_36_MOESM3_ESM.jpg (118 kb)
Supplementary Fig. 3 Transplanted tumours in the BALB/c nu/nu mice after chemotherapy. a ADR-A; b ADR-B; c ADR-C (ADR-A: the ADR therapy group that was injected into the SGC7901/MAD2-siRNA gastric carcinoma cells subcutaneously; ADR-B: the ADR therapy group that was injected into the SGC7901/psilencer gastric carcinoma cells subcutaneously; ADR-C: the ADR therapy group that was injected into the SGC7901 gastric carcinoma cells subcutaneously). The tumour sizes of the SGC7901/MAD2-siRNA group grew more slowly than the other two groups after drug therapy. (JPEG 118 kb)
13277_2010_36_MOESM4_ESM.jpg (36 kb)
Supplementary Fig. 4 MAD2 protein expression in the transfected tissues of nude mice analysed by Western blot (ADR-A: the ADR therapy group that was subcutaneously injected into SGC7901/MAD2-siRNA gastric carcinoma cells; ADR-B: the ADR therapy group that was subcutaneously injected into SGC7901/psilencer gastric carcinoma cells; ADR-C: the ADR therapy group that was subcutaneously injected into SGC7901 gastric carcinoma cells; VCR-A: the VCR therapy group that was subcutaneously injected into SGC7901/MAD2-siRNA gastric carcinoma cells; VCR-B: the VCR therapy group that was subcutaneously injected into SGC7901/psilencer gastric carcinoma cells; VCR-C: the VCR therapy group that was subcutaneously injected into SGC7901 gastric carcinoma cells). The Western blot shows the levels of MAD2 protein in the transplanted gastric tumour that was subcutaneously injected into SGC7901/MAD2-siRNA carcinoma cells; the levels were very low compared with the other groups. (JPEG 36 kb)
13277_2010_36_MOESM5_ESM.jpg (265 kb)
Supplementary Fig. 5 The effect of the MAD2-siRNA on drug cellular adriamycin accumulation was analysed by using FCM. The drug fluorescence intensity is expressed as the mean of fluorescence that could be calculated from the flow cytometric profiles. After being incubated with 1 or 5 mg/L of adriamycin for 1 hour, the positive fluorescence rates of SGC7901/MAD2-siRNA cells were significantly lower than that of SGC7901 and that of SGC7901/psilencer cells. The adriamycin-releasing ratio of the three groups of cancer cells. A Intracellular accumulation fluorescence intensity of adriamycin in gastric cancer cells; R Intracellular retention fluorescence intensity of adriamycin in gastric cancer cells (*P < 0.05: blank vector vs. SGC7901/MAD2-siRNA). (JPEG 264 kb)
13277_2010_36_MOESM6_ESM.jpg (51 kb)
Supplementary Fig. 6 Western blot analysis of P-gp, MRP, Raf-1, p-cdc2, and cyclin B in three cell lines. β-actin was used as a loading control (lane 1, SGC7901; lane 2, SGC7901/psilencer; lane 3, SGC7901/MAD2-siRNA). It showed that the downregulation of MAD2 could raise the levels of P-gp, Raf-1, and p-cdc2, but it did not influence the level of CyclinB and MRP. (JPEG 51 kb)


  1. 1.
    Fang G, Yu H, Kirschne MW. Control of mitotic transitions by the anaphase-promoting complex phil. Trans R Soc Lond. 1999;B354:1583–90.CrossRefGoogle Scholar
  2. 2.
    Hisaoka M, Matsuyama A, Hashimoto H. Aberrant MAD2 expression in soft-tissue sarcoma. Pathol Int. 2008;58:329–33.CrossRefPubMedGoogle Scholar
  3. 3.
    Dobles M, Liberal V, Scott ML, Benezra R, Sorger PK. Chromosome missegregation and apoptosis in mice lacking the mitotic checkpoint protein Mad2. Cell. 2000;101(6):635–45.CrossRefPubMedGoogle Scholar
  4. 4.
    Michel LS, Liberal V, Chatterjee A, Kirchwegger R, Pasche B, Gerald W, et al. MAD2 haploin sufficiency causes premature anaphase and chromosome instability in mammalian cells. Nature. 2001;409:355–9.CrossRefPubMedGoogle Scholar
  5. 5.
    Pennisi E. Cell division gatekeepers identified. Science. 1998;279:477–8.CrossRefPubMedGoogle Scholar
  6. 6.
    Hernando E, Orlow I, Liberal V, Nohales G, Benezra R, Cordon-Cardo C. Molecular analyses of the mitotic checkpoint components hsMAD2, hBUB1 and hBUB3 in human cancer. Int J Cancer. 2001;95(4):223–7.CrossRefPubMedGoogle Scholar
  7. 7.
    Gemma A, Hosoya Y, Seike M, Uematsu K, Kurimoto F, Hibino S, et al. Genomic structure of the human MAD2 gene and mutation analysis in human lung and breast cancers. Lung Cancer. 2001;32(3):289–95.CrossRefPubMedGoogle Scholar
  8. 8.
    Imai Y, Shiratori Y, Kato N, Inoue T, Omata MJ. Mutational inactivation of mitotic checkpoint genes, hsMAD2 and hBUB1, is rare in sporadic digestive tract cancers. Cancer Res. 1999;90(8):837–40.Google Scholar
  9. 9.
    Percy MJ, Myrie KA, Neeley CK, Azim JN, Ethier SP, Petty EM. Expression and mutational analyses of the human MAD2L1 gene in breast cancer cells. Genes, Chromosomes Cancer. 2000;29(4):356–62.CrossRefPubMedGoogle Scholar
  10. 10.
    Li Y, Benezra R. Identification of a human mitotic checkpoint gene: hsMAD2. Science. 1996;274:246–8.CrossRefPubMedGoogle Scholar
  11. 11.
    Takahashi T, Haruki N, Nomoto S, Masuda A, Saji S, Osada H, et al. 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;18:4295–300.CrossRefPubMedGoogle Scholar
  12. 12.
    Wang X, Jin D-Y, Wong YC, Cheung ALM, Chun ACS, Lo AKF. Correlation of defective mitotic checkpoint with aberrantly reduced expression of MAD2 protein in nasopharyngeal carcinoma cells. Carcinogenesis. 2000;21:2293–7.CrossRefPubMedGoogle Scholar
  13. 13.
    Hisaoka M, Matsuyama A, Hashimoto H. Aberrant MAD2 expression in soft-tissue sarcoma. Pathol Int. 2008;58(6):329–33.CrossRefPubMedGoogle Scholar
  14. 14.
    Ruddy DA, Gorbatcheva B, Yarbrough G, Schlegel R, Monahan JE. No somatic mutations detected in the Mad2 gene in 658 human tumors. Mutat Res. 2008;641(1-2):61–3.PubMedGoogle Scholar
  15. 15.
    Wang X, Jin D-Y, Ng RWM, Feng H, Wong YC, Cheung ALM, et al. Significance of MAD2 expression to mitotic checkpoint control in ovarian cancer cells. Cancer Res. 2002;62:1662–8.PubMedGoogle Scholar
  16. 16.
    Zhao YQ, You H, Liu F, An HZ, Shi YQ, Yu Q et al (2002) Differentially expressed gene profiles between multidrug resistant gastric adenocarcinoma cells and their parental cells. Cancer Lett. 185211–8.Google Scholar
  17. 17.
    Yin F, Hu WH, Qiao TD, Fan DM. Multidrug resistant effect of alternative splicing form of MAD2 gene-MAD2beta on human gastric cancer cell. Chin J Oncol. 2004;26:201–4.Google Scholar
  18. 18.
    Yin F, Du Y, Hu W, Qiao T, Ding J, Wu K, et al. Mad2β, an alternative variant of Mad2 reduce mitotic arrest and apoptosis of gastric cancer cells induced by adriamycin. Life Sci. 2006;78:1277–86.CrossRefPubMedGoogle Scholar
  19. 19.
    Pan Y, Bi F, Liu N, Xue Y, Yao X, Zheng Y, et al. Expression of seven main Rho family members in gastric carcinoma. Biochem Biophys Res Commun. 2004;315(3):686–91.CrossRefPubMedGoogle Scholar
  20. 20.
    Smits VA, Klompmarker R, Arnaudm L, Rijksen G, Nigg EA, Medema RH. Polo-like kinase-1 is a target of the DNA damage checkpoint. Nat. Cell Biol. 2000;2:672–6.Google Scholar
  21. 21.
    Huang T-S, Duyster J, Wang JYJ. Biological response to phorbolester determined by alternative G1 pathway. Proc Natl Acad Sci USA. 1995;92:4793–7.CrossRefPubMedGoogle Scholar
  22. 22.
    Huang T-S, Shu C-H, Yang WK, Whang-Peng J. Activation of CDC 25 phosphatase and CDC 2 kinase involved in GL331 induced apoptosis. Cancer Res. 1997;57:2974–8.PubMedGoogle Scholar
  23. 23.
    Huang T-S, Yang WK, Whang-Peng J. GL331-induced disruption of cyclin B1/CDC2 complex and inhibition of CDC2 kinase activity. Apoptosis. 1996;1:213–7.CrossRefGoogle Scholar
  24. 24.
    Sudo T, Nitta M, Saya H, Ueno NT. Dependence of paclitaxel sensitivity on a functional spindle assembly checkpoint. Cancer Res. 2004;64(7):2502–8.CrossRefPubMedGoogle Scholar
  25. 25.
    Schwartz GK. Development of cell cycle active drugs for the treatment of gastrointestinal cancers: a new approach to cancer therapy. J Clin Oncol. 2005;20:4499–508.CrossRefGoogle Scholar
  26. 26.
    Pawlik TM, Keyomarsi K. Role of cell cycle in mediating sensitivity to radiotherapy. Int J Radiat Oncol Biol Phys. 2004;59(4):928–42.CrossRefPubMedGoogle Scholar
  27. 27.
    Garber PM, Rine J. Overlapping roles of the spindle assembly and DNA damage checkpoints in the cell-cycle response to altered chromosomes in Saccharomyces cerevisiae. Genetics. 2002;161:521–34.PubMedGoogle Scholar
  28. 28.
    Mikhailov A, Cole RW, Rieder CL. DNA damage during mitosis in human cells delays the metaphase/anaphase transition via the spindle-assembly checkpoint. Curr Biol. 2002;12:1797–806.CrossRefPubMedGoogle Scholar
  29. 29.
    Tulub AA, Stefanov VE. Cisplatin stops tubulin assembly into microtubules. A new insight into the mechanism of antitumor activity of platinum complexes. Int J Biol Macromol. 2001;28:191–8.CrossRefPubMedGoogle Scholar
  30. 30.
    Chow JP, Siu WY, Fung TK, Chan WM, Lau A, Arooz T, et al. DNA damage during the spindle-assembly checkpoint degrades CDC25A, inhibits cyclin-CDC2 complexes, and reverses cells to interphase. Mol Biol Cell. 2003;14(10):3989–4002.CrossRefPubMedGoogle Scholar
  31. 31.
    Musacchio, Hardwick KG (2002) The spindle checkpoint: structural insights into dynamic signaling. Nat. Rev. Mol. Cell. Biol. 3731–41.Google Scholar
  32. 32.
    Cleveland DW, Mao Y, Sullivan KF (2003) Centromeres and kinetochores from epigenetics to mitotic checkpoint signaling. Cell 112407–21.Google Scholar
  33. 33.
    Glotzer M. Mitosis: don’t get mad, get even. Curr Biol. 1996;6:1592–4.CrossRefPubMedGoogle Scholar
  34. 34.
    Wang X, Jin D-Y, Wong HL, Feng H, Wong Y-C, Tsao SW. MAD2-induced sensitisation to vincristine is associated with mitotic arrest and Raf/Bcl-2 phosphorylation in nasopharyngeal carcinoma cells. Oncogene. 2003;22:109–16.CrossRefPubMedGoogle Scholar
  35. 35.
    Fung MKL, Han H-Y, Leung SCL, Cheung HW, Cheung ALM, Wong Y-C, et al. MAD2 interacts with DNA repair proteins and negatively regulates DNA damage repair. J Mol Biol. 2008;381(1):24–34.CrossRefPubMedGoogle Scholar
  36. 36.
    Zhang P, Cong B, Yuan H, Chen L, Lv Y, Bai C, et al. Overexpression of spindlin1 induces metaphase arrest and chromosomal instability. J Cell Physiol. 2008;217(2):400–8.CrossRefPubMedGoogle Scholar
  37. 37.
    Du Y, Yin F, Liu C, Hu S, Wang J, Xie H, et al. Depression of MAD2 inhibits apoptosis of gastric cancer cells by upregulating Bcl-2 and interfering mitochondrion pathway. Biochem Biophys Res Commun. 2006;345(3):1092–8.CrossRefPubMedGoogle Scholar
  38. 38.
    Fujita T, Washio K, Takabatake D, Takahashi H, Yoshitomi S, Tsukuda K, et al. Proteasome inhibitors can alter the signaling pathways and attenuate the P-glycoprotein-mediated multidrug resistance. Int J Cancer. 2005;117:670–82.CrossRefPubMedGoogle Scholar
  39. 39.
    Zhao YQ, You H, Liu F, An HZ, Shi YQ, Yu Q, et al. Differentially expressed gene profiles between multidrug resistant gastric adenocarcinoma cells and their parental cells. Cancer Lett. 2002;185:211–8.CrossRefPubMedGoogle Scholar

Copyright information

© International Society of Oncology and BioMarkers (ISOBM) 2010

Authors and Affiliations

  • Li Wang
    • 1
  • Fang Yin
    • 1
  • Yulei Du
    • 1
  • Bei Chen
    • 1
  • Shuhui Liang
    • 1
  • Yongguo Zhang
    • 1
  • Wenqi Du
    • 1
  • Kaichun Wu
    • 1
  • Jie Ding
    • 1
  • Daiming Fan
    • 1
  1. 1.State Key Laboratory of Cancer Biology & Digestive Diseases, of Xijing HospitalFourth Military Medical UniversityXi’anChina

Personalised recommendations