Document Type : Original Article(s)
Authors
- Tahereh Kalantari 1, 2
- Bahram Mohseni-Aghdam 1
- Fatemeh Nasri 2
- Gholamhossein Tamaddon 1, 2
- Mohsen Kalantari 3
1 Division of Medical Biotechnology, Department of Medical Laboratory Sciences, School of Paramedical Sciences, Shiraz University of Medical Sciences, Shiraz, Iran
2 Diagnostic Laboratory Sciences and Technologym Research Center, School of Paramedical Sciences, Shiraz University of Medical Sciences, Shiraz, Iran
3 Department of Clinical Sciences, School of Veterinary Medicine, Shiraz University, Shiraz, Iran
Abstract
Background: A high number of human breast cancers overexpress the murine double minute (MDM2) gene which blocks the p53 protein which plays an important role in arresting the cell growth. The present study aimed to investigate the efficacy of siRNA specific MDM2 in knocking down MDM2 and its subsequent effects on p53 to exert antiproliferative effects on Michigan Cancer Foundation-7 (MCF-7) breast cancer cells.
Method: In this in vitro study, we used the specific siRNA of the MDM2 gene to knock down the expression of the MDM2 protein in the MCF-7 cell line. The expression of MDM2, BCL2-associated X (BAX), BH3 interacting-domain death agonist (BID), and B cell lymphoma 2 (BCL2) genes was evaluated using the real-time polymerase chain reaction (PCR) technique. The apoptosis level was also assessed using the flow cytometry technique by the Annexin V test.
Results: The results showed that the entry of MDM2 siRNA into MCF-7 cells significantly reduced the mRNA expression of MDM2 gene (P-value < 0.05). Besides, the expression of the antiapoptotic gene of BCL2 significantly decreased (P-value < 0.05) in transfected MCF-7 cells, while that of BAX and BID genes increased (P-value < 0.05).
Conclusion: Based on the results, MDM2 inhibition is conducive to prevent cancer metastasis by the induction of cancer cell apoptosis. Moreover, it can be considered in cancer therapy along with chemotherapy.
Keywords
How to cite this article:
Kalantari T, Mohseni-Aghdam B, Nasri F, Tamaddon G, Kalantari M. The efficacy of siRNA specific MDM2 in the induction of apoptosis in MCF-7 breast cancer cell line. Middle East J Cancer. 2022;13(4):565-72. doi: 10.30476/mejc. 2021.89554. 1532.
2.Haupt Y, Maya R, Kazaz A, Oren M. MDM2 promotes the rapid degradation of p53. Nature. 1997;387(6630): 296-9. doi: 10.1038/387296a0.
3.Haupt S, Buckley D, Pang JM, Panimaya J, Paul PJ, Gamell C, et al. Targeting Mdmx to treat breast cancers with wild-type p53. Cell Death Dis. 2015;6(7):e1821. doi: 10.1038/cddis.2015.173.
4.Fakharzadeh SS, Trusko SP, George DL. Tumorigenic potential associated with enhanced expression of a gene that is amplified in a mouse tumor cell line. EMBO J. 1991;10(6):1565-9.
5.Momand J, Wu HH, Dasgupta G. MDM2--master regulator of the p53 tumor suppressor protein. Gene. 2000;242(1-2):15-29. doi: 10.1016/s0378-1119(99) 00487-4.
6.Manfredi JJ. The Mdm2-p53 relationship evolves: MDM2 swings both ways as an oncogene and a tumor suppressor. Genes Dev. 2010;24(15):1580-9. doi: 10.1101/gad.1941710. Erratum in: Genes Dev. 2010; 24(18):2105.
7.Jacob AG, Singh RK, Comiskey DF Jr, Rouhier MF, Mohammad F, Bebee TW, et al. Stress-induced alternative splice forms of MDM2 and MDMX modulate the p53-pathway in distinct ways. PLoS One. 2014;9(8):e104444. doi: 10.1371/journal. pone.0104444.
8.Bieging KT, Mello SS, Attardi LD. Unravelling mechanisms of p53-mediated tumour suppression. Nat Rev Cancer. 2014;14(5):359-70. doi: 10.1038/ nrc3711.
9.Phillips A, Teunisse A, Lam S, Lodder K, Darley M, Emaduddin M, et al. HDMX-L is expressed from a functional p53-responsive promoter in the first intron of the HDMX gene and participates in an autoregulatory feedback loop to control p53 activity. J Biol Chem. 2010;285(38):29111-27. doi: 10.1074/jbc. M110.129726.
10.Lenos K, Grawenda AM, Lodder K, Kuijjer ML, Teunisse AF, Repapi E, et al. Alternate splicing of the p53 inhibitor HDMX offers a superior prognostic biomarker than p53 mutation in human cancer. Cancer Res. 2012;72(16):4074-84. doi: 10.1158/0008-5472.CAN-12-0215.
11.Iwakuma T, Lozano G. MDM2, an introduction. Mol Cancer Res. 2003;1(14):993-1000.
12.Momand J, Zambetti GP, Olson DC, George D, Levine AJ. The MDM-2 oncogene product forms a complex with the p53 protein and inhibits p53-mediated transactivation. Cell. 1992;69(7):1237-45. doi: 10.1016/ 0092-8674(92)90644-r.
13.Francoz S, Froment P, Bogaerts S, De Clercq S, Maetens M, Doumont G, et al. MDM4 and MDM2 cooperate to inhibit p53 activity in proliferating and quiescent cells in vivo. Proc Natl Acad Sci U S A. 2006;103(9):3232-7. doi: 10.1073/pnas.0508476103.
14.Wade M, Li YC, Wahl GM. MDM2, MDMX and p53 in oncogenesis and cancer therapy. Nat Rev Cancer. 2013;13(2):83-96. doi: 10.1038/nrc3430.
15.Lundgren K, Montes de Oca Luna R, McNeill YB, Emerick EP, Spencer B, Barfield CR, et al. Targeted expression of MDM2 uncouples S phase from mitosis and inhibits mammary gland development independent of p53. Genes Dev. 1997;11(6):714-25. doi: 10.1101/ gad.11.6.714.
16.Park HS, Park JM, Park S, Cho J, Kim SI, Park BW. Subcellular localization of MDM2 expression and prognosis of breast cancer. Int J Clin Oncol. 2014;19(5):842-51. doi: 10.1007/s10147-013-0639-1.
17.Yu Q, Li Y, Mu K, Li Z, Meng Q, Wu X, et al. Amplification of Mdmx and overexpression of MDM2 contribute to mammary carcinogenesis by substituting for p53 mutations. Diagn Pathol. 2014;9:71. doi: 10.1186/1746-1596-9-71.
18.Ozenne P, Eymin B, Brambilla E, Gazzeri S. The ARF tumor suppressor: structure, functions and status in cancer. Int J Cancer. 2010;127(10):2239-47. doi: 10.1002/ijc.25511.
19.Hu W, Feng Z, Ma L, Wagner J, Rice JJ, Stolovitzky G, et al. A single nucleotide polymorphism in the MDM2 gene disrupts the oscillation of p53 and MDM2 levels in cells. Cancer Res. 2007;67(6):2757-65. doi:10.1158/0008-5472.CAN-06-2656.
20.Li H, Liu Q, Wang Z, Fang R, Shen Y, Cai X, et al. The oncoprotein HBXIP modulates the feedback loop of MDM2/p53 to enhance the growth of breast cancer. J Biol Chem. 2015;290(37):22649-61. doi: 10.1074/jbc. M115.658468.
21.Araki S, Eitel JA, Batuello CN, Bijangi-Vishehsaraei K, Xie XJ, Danielpour D, et al. TGF-beta1-induced expression of human Mdm2 correlates with late-stage metastatic breast cancer. J Clin Invest. 2010;120(1): 290-302. doi: 10.1172/JCI39194.
22.Koom WS, Park SY, Kim W, Kim M, Kim JS, Kim H, et al. Combination of radiotherapy and adenovirus-mediated p53 gene therapy for MDM2-overexpressing hepatocellular carcinoma. J Radiat Res. 2012;53(2): 202-10. doi: 10.1269/jrr.11110.
23.Koom WS, Park SY, Kim W, Kim M, Kim JS, Kim H, et al. Combination of radiotherapy and adenovirus-mediated p53 gene therapy for MDM2-overexpressing hepatocellular carcinoma. J Radiat Res. 2012;53(2): 202-10. doi: 10.1269/jrr.11110.
24.Kondo S, Barnett GH, Hara H, Morimura T, Takeuchi J. MDM2 protein confers the resistance of a human glioblastoma cell line to cisplatin-induced apoptosis. Oncogene. 1995;10(10):2001-6.
25.Hayashi S, Ozaki T, Yoshida K, Hosoda M, Todo S, Akiyama S, et al. p73 and MDM2 confer the resistance of epidermoid carcinoma to cisplatin by blocking p53. Biochem Biophys Res Commun. 2006;347(1):60-6. doi: 10.1016/j.bbrc.2006.06.095.
26.Turbin DA, Cheang MC, Bajdik CD, Gelmon KA, Yorida E, De Luca A, et al. MDM2 protein expression is a negative prognostic marker in breast carcinoma. Mod Pathol. 2006;19(1):69-74. doi: 10.1038/ modpathol.3800484.
27.Karni-Schmidt O, Lokshin M, Prives C. The roles of MDM2 and MDMX in cancer. Annu Rev Pathol. 2016;11:617-44. doi: 10.1146/annurev-pathol-012414-040349.
28.Vousden KH, Lane DP. p53 in health and disease. Nat Rev Mol Cell Biol. 2007;8(4):275-83. doi: 10.1038/ nrm2147.
29.Brady CA, Attardi LD. p53 at a glance. J Cell Sci. 2010;123(Pt 15):2527-32. doi: 10.1242/jcs.064501.
30.Haupt S, Berger M, Goldberg Z, Haupt Y. Apoptosis - the p53 network. J Cell Sci. 2003;116(Pt 20):4077-85. doi: 10.1242/jcs.00739.
31.Oda E, Ohki R, Murasawa H, Nemoto J, Shibue T, Yamashita T, et al. Noxa, a BH3-only member of the Bcl-2 family and candidate mediator of p53-induced apoptosis. Science. 2000;288(5468):1053-8. doi: 10.1126/science.288.5468.1053.
32.Nakano K, Vousden KH. PUMA, a novel proapoptotic gene, is induced by p53. Mol Cell. 2001;7(3):683-94. doi: 10.1016/s1097-2765(01)00214-3.
33.Sax JK, Fei P, Murphy ME, Bernhard E, Korsmeyer SJ, El-Deiry WS. BID regulation by p53 contributes to chemosensitivity. Nat Cell Biol. 2002;4(11):842-9. doi: 10.1038/ncb866.
34.Findley HW, Gu L, Yeager AM, Zhou M. Expression and regulation of BCL-2, BCL-XL, and BAX correlate with p53 status and sensitivity to apoptosis in childhood acute lymphoblastic leukemia. Blood. 1997;89(8):2986-93.
35.Geng QQ, Dong DF, Chen NZ, Wu YY, Li EX, Wang J, et al. Induction of p53 expression and apoptosis by a recombinant dual-target MDM2/MDMX inhibitory protein in wild-type p53 breast cancer cells. Int J Oncol. 2013;43(6):1935-42. doi: 10.3892/ijo. 2013.2138.