Effect of the tumor suppressor gene ING4 on the proliferation of MCF-7 human breast cancer cells

Wei,Qinjun; He,Wei; Lu,Yajie; Yao,Jun; Cao,Xin

doi:10.3892/ol.2012.744

Effect of the tumor suppressor gene ING4 on the proliferation of MCF-7 human breast cancer cells

Authors:
- Qinjun Wei
- Wei He
- Yajie Lu
- Jun Yao
- Xin Cao
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Affiliations: Department of Biotechnology, Nanjing Medical University, Nanjing 210029, P.R. China
Published online on: June 7, 2012 https://doi.org/10.3892/ol.2012.744
Pages: 438-442

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Abstract

Inhibitor of growth 4 (ING4) is a member of the ING family and acts as a tumor suppressor protein. To investigate the impact of ING4 on breast cancer proliferation, the present study examined the antitumor effects caused by upregulation in the expression of ING4 and its possible mechanism of effect in MCF-7 cells. A plasmid-based expression system encoding the ING4 gene was used to construct a stable cell line and overexpress ING4 in MCF-7 cells. Real‑time PCR and western blot analysis were used to detect the mRNA and protein expression levels of ING4, respectively. Cell growth was examined by methylthiazolyltetrazolium (MTT) assay. Cell cycle distribution and cell apoptosis were measured by flow cytometry. The expression of p21, p53 and bax genes were tested by real-time PCR and western blot analysis. The stably transfected cell lines pcDNA3.1(+)/ING4 (with the ING4 gene) and pcDNA3.1(+) (an empty vector) were established. The expression levels of ING4 mRNA and protein in the stable cell line expressing pcDNA3.1(+)/ING4 were significantly higher than those of the two control cell lines. The cell proliferation of stably transfected cells was inhibited, and the inhibitory rate was 62.58±2.93%. Based on the changes in cell cycle distribution in stably transfected cells compared with two control cell lines, a number of cells were blocked in the G0/G1 phase 67.82±3.78% (P<0.05). In addition, the apoptotic rate was significantly higher, at 31.51±3.02% (P<0.05). Real-time PCR revealed that p21 and bax mRNA expression were increased (P<0.01), but the expression of p53 did not change significantly (P>0.05) in the stably transfected cell lines. Western blot analysis results of p21, bax and p53 were in accordance with real-time PCR results. ING4 upregulation may inhibit breast cancer cell proliferation and accelerate the process of apoptosis. It is suggested that ING4 plays a significant role in the suppression of breast cancer progression.

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1	Jemal A, Bray F, Center MM, Ferlay J, Ward E and Forman D: Global cancer statistics. CA Cancer J Clin. 61:69–90. 2011. View Article : Google Scholar
2	Wang Y and Li G: ING3 promotes UV-induced apoptosis via Fas/caspase-8 pathway in melanoma cells. J Biol Chem. 281:11887–11893. 2006. View Article : Google Scholar : PubMed/NCBI
3	Moreno A, Palacios A, Orgaz JL, Jimenez B, Blanco FJ and Palmero I: Functional impact of cancer-associated mutations in the tumor suppressor protein ING4. Carcinogenesis. 31:1932–1938. 2010. View Article : Google Scholar : PubMed/NCBI
4	Li J and Li G: Cell cycle regulator ING4 is a suppressor of melanoma angiogenesis that is regulated by the metastasis suppressor BRMS1. Cancer Res. 70:10445–10453. 2010. View Article : Google Scholar : PubMed/NCBI
5	Xie Y, Sheng W, Miao J, Xiang J and Yang J: Enhanced antitumor activity by combining an adenovirus harboring ING4 with cisplatin for hepatocarcinoma cells. Cancer Gene Ther. 18:176–188. 2011. View Article : Google Scholar : PubMed/NCBI
6	Garkavtsev I and Riabowol K: Extension of the replicative life span of human diploid fibroblasts by inhibition of the p33ING1 candidate tumor suppressor. Mol Cell Biol. 17:2014–2019. 1997.PubMed/NCBI
7	Shiseki M, Nagashima M, Pedeux RM, Kitahama-Shiseki M, Miura K, Okamura S, Onogi H, Higashimoto Y, Appella E, Yokota J and Harris CC: p29ING4 and p28ING5 bind to p53 and p300, and enhance p53 activity. Cancer Res. 63:2373–2378. 2003.PubMed/NCBI
8	Garkavtsev I, Kozin SV, Chernova O, Xu L, Winkler F, Brown E, Barnett GH and Jain RK: The candidate tumour suppressor protein ING4 regulates brain tumour growth and angiogenesis. Nature. 428:328–332. 2004. View Article : Google Scholar : PubMed/NCBI
9	Shi X, Hong T, Walter KL, Ewalt M, Michishita E, Hung T, Carney D, Peña P, Lan F, Kaadige MR, et al: ING2 PHD domain links histone H3 lysine 4 methylation to active gene repression. Nature. 442:96–99. 2006.PubMed/NCBI
10	Cai L, Li X, Zheng S, Wang Y, Wang Y, Li H, Yang J and Sun J: Inhibitor of growth 4 is involved in melanomagenesis and induces growth suppression and apoptosis in melanoma cell line M14. Melanoma Res. 19:1–7. 2009. View Article : Google Scholar : PubMed/NCBI
11	Hassler M, Seidl S, Fazeny-Doerner B, Preusser M, Hainfellner J, Rössler K, Prayer D and Marosi C: Diversity of cytogenetic and pathohistologic profiles in glioblastoma. Cancer Genet Cytogenet. 166:46–55. 2006. View Article : Google Scholar : PubMed/NCBI
12	Li J, Martinka M and Li G: Role of ING4 in human melanoma cell migration, invasion and patient survival. Carcinogenesis. 29:1373–1379. 2008. View Article : Google Scholar : PubMed/NCBI
13	Gunduz M, Nagatsuka H, Demircan K, Gunduz E, Cengiz B, Ouchida M, Tsujigiwa H, Yamachika E, Fukushima K, Beder L, et al: Frequent deletion and down-regulation of ING4, a candidate tumor suppressor gene at 12p13, in head and neck squamous cell carcinomas. Gene. 356:109–117. 2005. View Article : Google Scholar : PubMed/NCBI
14	Zhang X, Xu LS, Wang ZQ, Wang KS, Li N, Cheng ZH, Huang SZ, Wei DZ and Han ZG: ING4 induces G2/M cell cycle arrest and enhances the chemosensitivity to DNA-damage agents in HepG2 cells. FEBS Lett. 570:7–12. 2004. View Article : Google Scholar : PubMed/NCBI
15	Tapia C, Zlobec I, Schneider S, Kilic E, Güth U, Bubendorf L and Kim S: Deletion of the inhibitor of growth 4 (ING4) tumor suppressor gene is prevalent in human epidermal growth factor 2 (HER2)-positive breast cancer. Hum Pathol. 42:983–990. 2011. View Article : Google Scholar : PubMed/NCBI
16	Soliman MA and Riabowol K: After a decade of study-ING, a PHD for a versatile family of proteins. Trends Biochem Sci. 32:509–519. 2007. View Article : Google Scholar : PubMed/NCBI
17	Radhakrishnan SK, Feliciano CS, Najmabadi F, Haegebarth A, Kandel ES, Tyner AL and Gartel AL: Constitutive expression of E2F-1 leads to p21-dependent cell cycle arrest in S phase of the cell cycle. Oncogene. 23:4173–4176. 2004. View Article : Google Scholar : PubMed/NCBI
18	Niculescu AB III, Chen X, Smeets M, Hengst L, Prives C and Reed SI: Effects of p21 (Cip1/Waf1) at both the G1/S and the G2/M cell cycle transitions: pRb is a critical determinant in blocking DNA replication and in preventing endoreduplication. Mol Cell Biol. 18:629–643. 1998.PubMed/NCBI
19	Pierrat B, Simonen M, Cueto M, Mestan J, Ferrigno P and Heim J: SH3GLB, a new endophilin-related protein family featuring an SH3 domain. Genomics. 71:222–234. 2001. View Article : Google Scholar : PubMed/NCBI
20	Weng C, Li Y, Xu D, Shi Y and Tang H: Specific cleavage of Mcl-1 by caspase-3 in tumor necrosis factor-related apoptosis-inducing ligand (TRAIL)-induced apoptosis in Jurkat leukemia T cells. J Biol Chem. 280:10491–10500. 2005. View Article : Google Scholar : PubMed/NCBI
21	Zhang H, Cowan-Jacob SW, Simonen M, Greenhalf W, Heim J and Meyhack B: Structural basis of BFL-1 for its interaction with BAX and its anti-apoptotic action in mammalian and yeast cells. J Biol Chem. 275:11092–11099. 2000. View Article : Google Scholar : PubMed/NCBI
22	Tzouvelekis A, Aidinis V, Harokopos V, Karameris A, Zacharis G, Mikroulis D, Konstantinou F, Steiropoulos P, Sotiriou I, Froudarakis M, et al: Down-regulation of the inhibitor of growth family member 4 (ING4) in different forms of pulmonary fibrosis. Respir Res. 10:142009. View Article : Google Scholar : PubMed/NCBI