Research Article

ZEB2 Knock-down Induces Apoptosis in Human Myeloid Leukemia HL-60 Cells

Author(s): Masoumeh Fardi*, Ali Mohammadi, Behzad Baradaran and Sahar Safaee

Volume 21, Issue 2, 2021

Published on: 20 January, 2021

Page: [149 - 159] Pages: 11

DOI: 10.2174/1566523221999210120210017

Price: $65


Introduction: Acute myeloid leukemia (AML) is the most prevalent type of cancer in the adult hematopoietic system. Conventional therapies are associated with unfavorable side effects in individuals diagnosed with AML. These after-effects with partial remission reflect the urgent need for novel therapeutic approaches for inducing apoptosis, specifically in malignant cells, without affecting other cells. As a transcription factor (TF), ZEB2 (Zinc Finger E-Box Binding Homeobox 2) regulates the expression of specific genes in normal conditions. However, increased expression of ZEB2 is reported in various cancers, especially in AML, which is related to a higher degree of apoptosis inhibition of malignant cells. In this work, the role of ZEB2 in apoptosis inhibition is surveyed through ZEB2 specific knocking-down in human myeloid leukemia HL-60 cells.

Materials and Methods: Transfection of HL-60 cells was conducted using ZEB2-siRNA at concentrations of 20, 40, 60, and 80 pmol within 24, 48, and 72 h. After determining the optimum dose and time, flow cytometry was used to measure the apoptosis rate. The MTT assay was also utilized to evaluate the cytotoxic impact of transfection on the cells. The expression of candidate genes was measured before and after transfection using qRT-PCR.

Results: According to obtained results, suppression of ZEB2 expression through siRNA was associated with the induction of apoptosis, increased pro-apoptotic, and decreased anti-apoptotic gene expression. Transfection of ZEB2-siRNA was also associated with reduced cell proliferation and viability.

Conclusion: Our study results suggest that ZEB2 suppression in myeloid leukemia cells through apoptosis induction could be a proper therapeutic method.

Keywords: Acute myeloid leukemia, ZEB2 (Zinc Finger E-Box Binding Homeobox 2), RNAi, targeted therapy, apoptosis, Leukemia.

Graphical Abstract
Acute Myeloid Leukemia Treatment (PDQ®)–Patient Version. National Cancer Institute. 2018.
Acheampong DO, Adokoh CK, Asante DB, et al. Immunotherapy for acute myeloid leukemia (AML): a potent alternative therapy. Biomed Pharmacother 2018; 97: 225-32.
[] [PMID: 29091870]
Verschueren K, Remacle JE, Collart C, et al. SIP1, a novel zinc finger/homeodomain repressor, interacts with Smad proteins and binds to 5′-CACCT sequences in candidate target genes. J Biol Chem 1999; 274(29): 20489-98.
[] [PMID: 10400677]
Fardi M, Alivand M, Baradaran B, Farshdousti Hagh M, Solali S. The crucial role of ZEB2: From development to epithelial-to-mesenchymal transition and cancer complexity. J Cell Physiol 2019.
[] [PMID: 30773635]
Hegarty SV, Sullivan AM, O’Keeffe GW. Zeb2: A multifunctional regulator of nervous system development. Prog Neurobiol 2015; 132: 81-95.
[] [PMID: 26193487]
Epifanova E, Babaev A, Newman AG, Tarabykin V. Role of Zeb2/Sip1 in neuronal development. Brain Res 2019; 1705: 24-31.
[] [PMID: 30266271]
Yin SY, Peng AP, Huang LT, Wang YT, Lan CW, Yang NS. The Phytochemical Shikonin Stimulates Epithelial-Mesenchymal Transition (EMT) in Skin Wound Healing. Evid Based Complement Alternat Med 2013; 2013: 262796.
[] [PMID: 23861701]
Ye C, Hu Y, Wang J. MicroRNA-377 Targets Zinc Finger E-box-Binding Homeobox 2 to Inhibit Cell Proliferation and Invasion of Cervical Cancer. Oncol Res 2019; 27(2): 183-92.
[] [PMID: 29523224]
Balcik-Ercin P, Cetin M, Yalim-Camci I, et al. Genome-wide analysis of endogenously expressed ZEB2 binding sites reveals inverse correlations between ZEB2 and GalNAc-transferase GALNT3 in human tumors. Cell Oncol (Dordr) 2018; 41(4): 379-93.
[] [PMID: 29516288]
Wang T, Chen X, Qiao W, Kong L, Sun D, Li Z. Transcription factor E2F1 promotes EMT by regulating ZEB2 in small cell lung cancer. BMC Cancer 2017; 17(1): 719.
[] [PMID: 29115924]
Chen P, Liu H, Hou A, et al. Prognostic Significance of Zinc Finger E-Box-Binding Homeobox Family in Glioblastoma. Med Sci Monit 2018; 24: 1145-51.
[] [PMID: 29476046]
Yan Z, Tian X, Wang R, et al. Title Prognosis Significance of ZEB2 and TGF-β1 as well as Other Clinical Characteristics in Epithelial Ovarian Cancer. Int J Gynecol Cancer 2017; 27(7): 1343-9.
[] [PMID: 30814239]
Shibue T, Weinberg RA. EMT, CSCs, and drug resistance: the mechanistic link and clinical implications. Nat Rev Clin Oncol 2017; 14(10): 611-29.
[] [PMID: 28397828]
Santamaria PG, Moreno-Bueno G, Portillo F, Cano A. EMT: Present and future in clinical oncology. Mol Oncol 2017; 11(7): 718-38.
[] [PMID: 28590039]
Song KA, Niederst MJ, Lochmann TL, et al. Epithelial-to-Mesenchymal Transition Antagonizes Response to Targeted Therapies in Lung Cancer by Suppressing BIM. Clin Cancer Res 2018; 24(1): 197-208.
[] [PMID: 29051323]
Zhou P, Wang C, Hu Z, Chen W, Qi W, Li A. Genistein induces apoptosis of colon cancer cells by reversal of epithelial-to-mesenchymal via a Notch1/NF-κB/slug/E-cadherin pathway. BMC Cancer 2017; 17(1): 813.
[] [PMID: 29202800]
Arber DA, Orazi A, Hasserjian R, et al. The 2016 revision to the World Health Organization classification of myeloid neoplasms and acute leukemia. Blood 2016; 127(20): 2391-405.
[] [PMID: 27069254]
Almond LM, Charalampakis M, Ford SJ, Gourevitch D, Desai A. Myeloid Sarcoma: Presentation, Diagnosis, and Treatment. Clin Lymphoma Myeloma Leuk 2017; 17(5): 263-7.
[] [PMID: 28342811]
Wilson CS, Medeiros LJ. Extramedullary Manifestations of Myeloid Neoplasms. Am J Clin Pathol 2015; 144(2): 219-39.
[] [PMID: 26185307]
Sawyers C. Targeted cancer therapy. Nature 2004; 432(7015): 294-7.
[] [PMID: 15549090]
Human Protein Atlas 2019.
Li H, Mar BG, Zhang H, et al. The EMT regulator ZEB2 is a novel dependency of human and murine acute myeloid leukemia. Blood 2017; 129(4): 497-508.
[] [PMID: 27756750]
Hong L, Han K, Wu K, et al. E-cadherin and ZEB2 modulate trophoblast cell differentiation during placental development in pigs. Reproduction 2017; 154(6): 765-75.
[] [PMID: 28912304]
Dai Y-H, Tang YP, Zhu HY, et al. ZEB2 promotes the metastasis of gastric cancer and modulates epithelial mesenchymal transition of gastric cancer cells. Dig Dis Sci 2012; 57(5): 1253-60.
[] [PMID: 22350782]
Vandewalle C, Comijn J, De Craene B, et al. SIP1/ZEB2 induces EMT by repressing genes of different epithelial cell-cell junctions. Nucleic Acids Res 2005; 33(20): 6566-78.
[] [PMID: 16314317]
Kahlert UD, Joseph JV, Kruyt FAE. EMT- and MET-related processes in nonepithelial tumors: importance for disease progression, prognosis, and therapeutic opportunities. Mol Oncol 2017; 11(7): 860-77.
[] [PMID: 28556516]
Wu S, Du Y, Beckford J, Alachkar H. Upregulation of the EMT marker vimentin is associated with poor clinical outcome in acute myeloid leukemia. J Transl Med 2018; 16(1): 170.
[] [PMID: 29925392]
Chen S-C, Liao T-T, Yang M-H. Emerging roles of epithelial-mesenchymal transition in hematological malignancies. J Biomed Sci 2018; 25(1): 37-7.
[] [PMID: 29685144]
Navarro F, Lieberman J. miR-34 and p53: New Insights into a Complex Functional Relationship. PLoS One 2015; 10(7): e0132767.
[] [PMID: 26177460]
Chang CJ, Chao CH, Xia W, et al. p53 regulates epithelial-mesenchymal transition and stem cell properties through modulating miRNAs. Nat Cell Biol 2011; 13(3): 317-23.
[] [PMID: 21336307]
Jing J, Xiong S, Li Z, et al. A feedback regulatory loop involving p53/miR-200 and growth hormone endocrine axis controls embryo size of zebrafish. Sci Rep 2015; 5: 15906.
[] [PMID: 26507500]
Okada N, Lin CP, Ribeiro MC, et al. A positive feedback between p53 and miR-34 miRNAs mediates tumor suppression. Genes Dev 2014; 28(5): 438-50.
[] [PMID: 24532687]
Rajabi H, Alam M, Takahashi H, et al. MUC1-C oncoprotein activates the ZEB1/miR-200c regulatory loop and epithelial-mesenchymal transition. Oncogene 2014; 33(13): 1680-9.
[] [PMID: 23584475]
Brabletz S, Brabletz T. The ZEB/miR-200 feedback loop--a motor of cellular plasticity in development and cancer? EMBO Rep 2010; 11(9): 670-7.
[] [PMID: 20706219]
Hill L, Browne G, Tulchinsky E. ZEB/miR-200 feedback loop: at the crossroads of signal transduction in cancer. Int J Cancer 2013; 132(4): 745-54.
[] [PMID: 22753312]
Gill JG, Langer EM, Lindsley RC, et al. Snail and the microRNA-200 family act in opposition to regulate epithelial-to-mesenchymal transition and germ layer fate restriction in differentiating ESCs. Stem Cells 2011; 29(5): 764-76.
[] [PMID: 21394833]
Siemens H, Jackstadt R, Hünten S, et al. miR-34 and SNAIL form a double-negative feedback loop to regulate epithelial-mesenchymal transitions. Cell Cycle 2011; 10(24): 4256-71.
[] [PMID: 22134354]
Imani S, Wei C, Cheng J, et al. MicroRNA-34a targets epithelial to mesenchymal transition-inducing transcription factors (EMT-TFs) and inhibits breast cancer cell migration and invasion. Oncotarget 2017; 8(13): 21362-79.
[] [PMID: 28423483]
Alves-Fernandes DK, Jasiulionis MG. The Role of SIRT1 on DNA Damage Response and Epigenetic Alterations in Cancer. Int J Mol Sci 2019; 20(13): E3153.
[] [PMID: 31261609]
Donmez G, Outeiro TF. SIRT1 and SIRT2: emerging targets in neurodegeneration. EMBO Mol Med 2013; 5(3): 344-52.
[] [PMID: 23417962]
Zhao LJ, Kuppuswamy M, Vijayalingam S, Chinnadurai G. Interaction of ZEB and histone deacetylase with the PLDLS-binding cleft region of monomeric C-terminal binding protein 2. BMC Mol Biol 2009; 10: 89.
[] [PMID: 19754958]
Nakano K, Vousden KH. PUMA, a novel proapoptotic gene, is induced by p53. Mol Cell 2001; 7(3): 683-94.
[] [PMID: 11463392]
Shibue T, Takeda K, Oda E, et al. Integral role of Noxa in p53-mediated apoptotic response. Genes Dev 2003; 17(18): 2233-8.
[] [PMID: 12952892]
Schuler M, Maurer U, Goldstein JC, et al. p53 triggers apoptosis in oncogene-expressing fibroblasts by the induction of Noxa and mitochondrial Bax translocation. Cell Death Differ 2003; 10(4): 451-60.
[] [PMID: 12719722]
Chipuk JE, Kuwana T, Bouchier-Hayes L, et al. Direct activation of Bax by p53 mediates mitochondrial membrane permeabilization and apoptosis. Science 2004; 303(5660): 1010-4.
[] [PMID: 14963330]
Basu A, Haldar S. The relationship between BcI2, Bax and p53: consequences for cell cycle progression and cell death. Mol Hum Reprod 1998; 4(12): 1099-109.
[] [PMID: 9872359]
Croce CM, Reed JC. Finally, An Apoptosis-Targeting Therapeutic for Cancer. Cancer Res 2016; 76(20): 5914-20.
[] [PMID: 27694602]
Ren T, Zhu L, Cheng M. CXCL10 accelerates EMT and metastasis by MMP-2 in hepatocellular carcinoma. Am J Transl Res 2017; 9(6): 2824-37.
[PMID: 28670372]
Mendez MG, Kojima S, Goldman RD. Vimentin induces changes in cell shape, motility, and adhesion during the epithelial to mesenchymal transition. FASEB J 2010; 24(6): 1838-51.
[] [PMID: 20097873]
Meyer SE. From EMT to HSC to AML: ZEB2 is a cell fate switch. Blood 2017; 129(4): 400-1.
[] [PMID: 28126955]
Chou Y-S, Yang M-H. Epithelial-mesenchymal transition-related factors in solid tumor and hematological malignancy. J Chin Med Assoc 2015; 78(8): 438-45.
[] [PMID: 26078096]
Duan X, Fu Z, Gao L, et al. Direct interaction between miR-203 and ZEB2 suppresses epithelial-mesenchymal transition signaling and reduces lung adenocarcinoma chemoresistance. Acta Biochim Biophys Sin (Shanghai) 2016; 48(11): 1042-9.
[] [PMID: 27733346]
Fang S, Zeng X, Zhu W, Tang R, Chao Y, Guo L. Zinc finger E-box-binding homeobox 2 (ZEB2) regulated by miR-200b contributes to multi-drug resistance of small cell lung cancer. Exp Mol Pathol 2014; 96(3): 438-44.
[] [PMID: 24769353]
Du B, Shim JS. Targeting Epithelial-Mesenchymal Transition (EMT) to Overcome Drug Resistance in Cancer. Molecules 2016; 21(7): E965.
[] [PMID: 27455225]
Zhang Y, Xu L, Li A, Han X. The roles of ZEB1 in tumorigenic progression and epigenetic modifications. Biomed Pharmacother 2019; 110: 400-8.
[] [PMID: 30530042]
Wu W-S, You RI, Cheng CC, Lee MC, Lin TY, Hu CT. Snail collaborates with EGR-1 and SP-1 to directly activate transcription of MMP 9 and ZEB1. Sci Rep 2017; 7(1): 17753.
[] [PMID: 29259250]
Bae G-Y, Choi SJ, Lee JS, et al. Loss of E-cadherin activates EGFR-MEK/ERK signaling, which promotes invasion via the ZEB1/MMP2 axis in non-small cell lung cancer. Oncotarget 2013; 4(12): 2512-22.
[] [PMID: 24318272]
Yokoyama K, Kamata N, Fujimoto R, et al. Increased invasion and matrix metalloproteinase-2 expression by Snail-induced mesenchymal transition in squamous cell carcinomas. Int J Oncol 2003; 22(4): 891-8.
[] [PMID: 12632084]
Miyoshi A, Kitajima Y, Sumi K, et al. Snail and SIP1 increase cancer invasion by upregulating MMP family in hepatocellular carcinoma cells. Br J Cancer 2004; 90(6): 1265-73.
[] [PMID: 15026811]

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