Abstract
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.
Similar content being viewed by others
Abbreviations
- MAD2:
-
mitotic arrest-deficient 2
- VCR:
-
vincristine
- ADR:
-
Adriamycin
- 5-FU:
-
5-fluorouracil
- CDDP:
-
cisplatin
- MTT:
-
3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide
- PBS:
-
phosphate-buffered saline
- MDR:
-
multi-drug resistance
- P-gp:
-
P-glycoprotein
- MRP:
-
multi-drug resistance-associated protein
- PI:
-
propidium iodide
- siRNA:
-
small interfering RNA
References
Fang G, Yu H, Kirschne MW. Control of mitotic transitions by the anaphase-promoting complex phil. Trans R Soc Lond. 1999;B354:1583–90.
Hisaoka M, Matsuyama A, Hashimoto H. Aberrant MAD2 expression in soft-tissue sarcoma. Pathol Int. 2008;58:329–33.
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.
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.
Pennisi E. Cell division gatekeepers identified. Science. 1998;279:477–8.
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.
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.
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.
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.
Li Y, Benezra R. Identification of a human mitotic checkpoint gene: hsMAD2. Science. 1996;274:246–8.
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.
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.
Hisaoka M, Matsuyama A, Hashimoto H. Aberrant MAD2 expression in soft-tissue sarcoma. Pathol Int. 2008;58(6):329–33.
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.
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.
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.
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.
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.
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.
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.
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.
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.
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.
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.
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.
Pawlik TM, Keyomarsi K. Role of cell cycle in mediating sensitivity to radiotherapy. Int J Radiat Oncol Biol Phys. 2004;59(4):928–42.
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.
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.
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.
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.
Musacchio, Hardwick KG (2002) The spindle checkpoint: structural insights into dynamic signaling. Nat. Rev. Mol. Cell. Biol. 3731–41.
Cleveland DW, Mao Y, Sullivan KF (2003) Centromeres and kinetochores from epigenetics to mitotic checkpoint signaling. Cell 112407–21.
Glotzer M. Mitosis: don’t get mad, get even. Curr Biol. 1996;6:1592–4.
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.
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.
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.
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.
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.
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.
Acknowledgments
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).
Author information
Authors and Affiliations
Corresponding author
Additional information
Li Wang, Fang Yin and Yulei Du contributed equally to this paper.
Electronic supplementary material
Below is the link to the electronic supplementary material.
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)
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)
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)
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)
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)
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)
Rights and permissions
About this article
Cite this article
Wang, L., Yin, F., Du, Y. et al. Depression of MAD2 inhibits apoptosis and increases proliferation and multidrug resistance in gastric cancer cells by regulating the activation of phosphorylated survivin. Tumor Biol. 31, 225–232 (2010). https://doi.org/10.1007/s13277-010-0036-6
Received:
Accepted:
Published:
Issue Date:
DOI: https://doi.org/10.1007/s13277-010-0036-6