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

  • Research Article
  • Published:
Tumor Biology

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.

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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

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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).

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Authors and Affiliations

  1. State Key Laboratory of Cancer Biology & Digestive Diseases, of Xijing Hospital, Fourth Military Medical University, Xi’an, 710032, China

    Li Wang, Fang Yin, Yulei Du, Bei Chen, Shuhui Liang, Yongguo Zhang, Wenqi Du, Kaichun Wu, Jie Ding & Daiming Fan

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  1. Li Wang
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  2. Fang Yin
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  3. Yulei Du
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Correspondence to Daiming Fan.

Additional information

Li Wang, Fang Yin and Yulei Du contributed equally to this paper.

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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)

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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

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  • DOI: https://doi.org/10.1007/s13277-010-0036-6

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