Knockdown of Enhancer of Zeste Homolog 2 Affects mRNA Expression of Genes Involved in the Induction of Resistance to Apoptosis in MOLT-4 Cells

Author(s): Adel Naimi*, Sahar Safaei, Atefeh Entezari, Saeed Solali, Ali Hassanzadeh

Journal Name: Anti-Cancer Agents in Medicinal Chemistry
(Formerly Current Medicinal Chemistry - Anti-Cancer Agents)

Volume 20 , Issue 5 , 2020

Become EABM
Become Reviewer
Call for Editor

Graphical Abstract:


Background: The Enhancer of Zeste Homolog 2 (EZH2) is a subunit of the polycomb repressive complex 2 that silences the gene transcription via H3K27me3. Previous studies have shown that EZH2 has an important role in the induction of the resistance against the Tumor necrosis factor-Related Apoptosis-Inducing Ligand (TRAIL)-Induced Apoptosis (TIA) in some leukemia cells.

Objective: The aim of this study was to determine the effect of silencing EZH2 gene expression using RNA interference on the expression of death receptors 4 and 5 (DR4/5), Preferentially expressed Antigen in Melanoma (PRAME), and TRAIL human lymphoid leukemia MOLT-4 cells.

Methods: Quantitative RT-PCR was used to detect the EZH2 expression and other candidate genes following the siRNA knockdown in MOLT-4 cells. The toxicity of the EZH2 siRNA was evaluated using Annexin V/PI assay following the transfection of the cells by 80 pM EZH2 siRNA at 48 hours.

Results: Based on the flow-cytometry results, the EZH2 siRNA had no toxic effects on MOLT-4 cells. Also, the EZH2 inhibition increased the expression of DR4/5 but reduced the PRAME gene expression at the mRNA levels. Moreover, the EZH2 silencing could not change the TRAIL mRNA in the transfected cells.

Conclusion: Our results revealed that the down-regulation of EZH2 in MOLT-4 cells was able to affect the expression of important genes involved in the induction of resistance against TIA. Hence, we suggest that the silencing of EZH2 using RNA interference can be an effective and safe approach to help defeat the MOLT-4 cell resistance against TIA.

Keywords: Leukemia, EZH2, TRAIL, death receptor, PRAME, knockdown, siRNA.

Whitehead, T.P.; Metayer, C.; Wiemels, J.L.; Singer, A.W.; Miller, M.D. Childhood leukemia and primary prevention. Curr. Probl. Pediatr. Adolesc. Health Care, 2016, 46(10), 317-352.
[] [PMID: 27968954]
Reckel, S.; Hamelin, R.; Georgeon, S.; Armand, F.; Jolliet, Q.; Chiappe, D.; Moniatte, M.; Hantschel, O. Differential signaling networks of Bcr-Abl p210 and p190 kinases in leukemia cells defined by functional proteomics. Leukemia, 2017, 31(7), 1502-1512.
[] [PMID: 28111465]
Short, N.J.; Jabbour, E.; Albitar, M.; de Lima, M.; Gore, L.; Jorgensen, J.; Logan, A.C.; Park, J.; Ravandi, F.; Shah, B.; Radich, J.; Kantarjian, H. Recommendations for the assessment and management of measurable residual disease in adults with acute lymphoblastic leukemia: A consensus of North American experts. Am. J. Hematol., 2019, 94(2), 257-265.
[] [PMID: 30394566]
Giebel, S.; Marks, D.I.; Boissel, N.; Baron, F.; Chiaretti, S.; Ciceri, F.; Cornelissen, J.J.; Doubek, M.; Esteve, J.; Fielding, A.; Foa, R.; Gorin, N.C.; Gokbuget, N.; Hallbook, H.; Hoelzer, D.; Paravichnikova, E.; Ribera, J.M.; Savani, B.; Rijneveld, A.W.; Schmid, C.; Wartiovaara-Kautto, U.; Mohty, M.; Nagler, A.; Dombret, H. Hematopoietic stem cell transplantation for adults with Philadelphia chromosome-negative acute lymphoblastic leukemia in first remission: a position statement of the European Working Group for Adult Acute Lymphoblastic Leukemia (EWALL) and the Acute Leukemia Working Party of the European Society for Blood and Marrow Transplantation (EBMT). Bone Marrow Transplant., 2019, 54(6), 798-809.
[] [PMID: 30385870]
Arora, R.S.; Arora, B. Acute leukemia in children: A review of the current Indian data. South Asian J. Cancer, 2016, 5(3), 155-160.
[] [PMID: 27606304]
Terwilliger, T.; Abdul-Hay, M. Acute lymphoblastic leukemia: a comprehensive review and 2017 update. Blood Cancer J., 2017, 7(6), e577-e577.
[] [PMID: 28665419]
Küley-Bagheri, Y.; Kreuzer, K.A.; Monsef, I.; Lübbert, M.; Skoetz, N. Effects of all-trans retinoic acid (ATRA) in addition to chemotherapy for adults with acute myeloid leukaemia (AML) (non-acute promyelocytic leukaemia (non-APL)). Cochrane Database Syst. Rev., 2018, 8 CD011960
[] [PMID: 30080246]
Mei, L.; Ontiveros, E.P.; Griffiths, E.A.; Thompson, J.E.; Wang, E.S.; Wetzler, M. Pharmacogenetics predictive of response and toxicity in acute lymphoblastic leukemia therapy. Blood Rev., 2015, 29(4), 243-249.
[] [PMID: 25614322]
Cornelissen, J.J.; Blaise, D. Hematopoietic stem cell transplantation for patients with AML in first complete remission. Blood, 2016, 127(1), 62-70.
[] [PMID: 26660427]
Su, M.; Alonso, S.; Jones, J.W.; Yu, J.; Kane, M.A.; Jones, R.J.; Ghiaur, G. All-Trans retinoic acid activity in acute myeloid leukemia: Role of cytochrome P450 enzyme expression by the microenvironment. PLoS One, 2015, 10(6), e0127790-e0127790.
[] [PMID: 26047326]
Cholewa-Waclaw, J.; Bird, A.; von Schimmelmann, M.; Schaefer, A.; Yu, H.; Song, H.; Madabhushi, R.; Tsai, L-H. The role of epigenetic mechanisms in the regulation of gene expression in the nervous system. J. Neurosci., 2016, 36(45), 11427-11434.
[] [PMID: 27911745]
Chen, Z.J. Genetic and epigenetic mechanisms for gene expression and phenotypic variation in plant polyploids. Annu. Rev. Plant Biol., 2007, 58, 377-406.
[] [PMID: 17280525]
Sharma, S.; Kelly, T.K.; Jones, P.A. Epigenetics in cancer. Carcinogenesis, 2010, 31(1), 27-36.
[] [PMID: 19752007]
Ahuja, N.; Sharma, A.R.; Baylin, S.B. Epigenetic therapeutics: A new weapon in the war against cancer. Annu. Rev. Med., 2016, 67, 73-89.
[] [PMID: 26768237]
Shaw, T.; Martin, P. Epigenetic reprogramming during wound healing: loss of polycomb-mediated silencing may enable upregulation of repair genes. EMBO Rep., 2009, 10(8), 881-886.
[] [PMID: 19575012]
Koppens, M.A.; Tanger, E.; Nacerddine, K.; Westerman, B.; Song, J.Y.; van Lohuizen, M. A new transgenic mouse model for conditional overexpression of the Polycomb Group protein EZH2. Transgenic Res., 2017, 26(2), 187-196.
[] [PMID: 27807665]
Abdel-Wahab, O.; Pardanani, A.; Patel, J.; Wadleigh, M.; Lasho, T.; Heguy, A.; Beran, M.; Gilliland, D.G.; Levine, R.L.; Tefferi, A. Concomitant analysis of EZH2 and ASXL1 mutations in myelofibrosis, chronic myelomonocytic leukemia and blast-phase myeloproliferative neoplasms. Leukemia, 2011, 25(7), 1200-1202.
[] [PMID: 21455215]
Cao, R.; Wang, L.; Wang, H.; Xia, L.; Erdjument-Bromage, H.; Tempst, P.; Jones, R.S.; Zhang, Y. Role of histone H3 lysine 27 methylation in Polycomb-group silencing. Science, 2002, 298(5595), 1039-1043.
[] [PMID: 12351676]
Safaei, S.; Baradaran, B.; Hagh, M.F.; Alivand, M.R.; Talebi, M.; Gharibi, T.; Solali, S. Double sword role of EZH2 in leukemia. Biomed. Pharmacother., 2018, 98, 626-635.
[] [PMID: 29289837]
Booth, C.A.G.; Barkas, N.; Neo, W.H.; Boukarabila, H.; Soilleux, E.J.; Giotopoulos, G.; Farnoud, N.; Giustacchini, A.; Ashley, N.; Carrelha, J.; Jamieson, L.; Atkinson, D.; Bouriez-Jones, T.; Prinjha, R.K.; Milne, T.A.; Teachey, D.T.; Papaemmanuil, E.; Huntly, B.J.P.; Jacobsen, S.E.W.; Mead, A.J. Ezh2 and Runx1 mutations collaborate to initiate lympho-myeloid leukemia in early thymic progenitors. Cancer Cell, 2018, 33(2), 274-291.
[] [PMID: 29438697]
Patnaik, M.M.; Vallapureddy, R.; Lasho, T.L.; Hoversten, K.P.; Finke, C.M.; Ketterling, R.; Hanson, C.; Gangat, N.; Tefferi, A. EZH2 mutations in chronic myelomonocytic leukemia cluster with ASXL1 mutations and their co-occurrence is prognostically detrimental. Blood Cancer J., 2018, 8(1), 12.
[] [PMID: 29358618]
Brunetti, G.; Di Benedetto, A.; Posa, F.; Colaianni, G.; Faienza, M.F.; Ballini, A.; Colucci, S.; Passeri, G.; Lo Muzio, L.; Grano, M.; Mori, G. High expression of TRAIL by osteoblastic differentiated dental pulp stem cells affects myeloma cell viability. Oncol. Rep., 2018, 39(4), 2031-2039.
[] [PMID: 29484421]
Naimi, A.; Entezari, A.; Hagh, M.F.; Hassanzadeh, A.; Saraei, R.; Solali, S. Quercetin sensitizes human myeloid leukemia KG-1 cells against TRAIL-induced apoptosis. J. Cell. Physiol., 2019, 234(8), 13233-13241.
[] [PMID: 30589076]
Saraei, R.; Soleimani, M.; Movassaghpour Akbari, A.A.; Farshdousti Hagh, M.; Hassanzadeh, A.; Solali, S. The role of XIAP in resistance to TNF-related apoptosis-inducing ligand (TRAIL) in Leukemia. Biomed. Pharmacother., 2018, 107, 1010-1019.
Mérino, D.; Lalaoui, N.; Morizot, A.; Solary, E.; Micheau, O. TRAIL in cancer therapy: present and future challenges. Expert Opin. Ther. Targets, 2007, 11(10), 1299-1314.
[] [PMID: 17907960]
Hassanzadeh, A.; Farshdousti Hagh, M.; Alivand, M.R.; Akbari, A.A.M.; Shams Asenjan, K.; Saraei, R.; Solali, S. Down-regulation of intracellular anti-apoptotic proteins, particularly c-FLIP by therapeutic agents; the novel view to overcome resistance to TRAIL. J. Cell. Physiol., 2018, 233(10), 6470-6485.
[] [PMID: 29741767]
Kong, X.; Luo, J.; Xu, T.; Zhou, Y.; Pan, Z.; Xie, Y.; Zhao, L.; Lu, Y.; Han, X.; Li, Z.; Liu, L. Plumbagin enhances TRAIL-induced apoptosis of human leukemic Kasumi‑1 cells through upregulation of TRAIL death receptor expression, activation of caspase-8 and inhibition of cFLIP. Oncol. Rep., 2017, 37(6), 3423-3432.
[] [PMID: 28498435]
Naimi, A.; Movassaghpour, A.A.; Hagh, M.F.; Talebi, M.; Entezari, A.; Jadidi-Niaragh, F.; Solali, S. TNF-related apoptosis-inducing ligand (TRAIL) as the potential therapeutic target in hematological malignancies. Biomed. Pharmacother., 2018, 98, 566-576.
Rathore, R.; McCallum, J.E.; Varghese, E.; Florea, A.M.; Busselberg, D. Overcoming chemotherapy drug resistance by targeting inhibitors of apoptosis proteins (IAPs). Apoptosis: Int. J. Program. Cell Death, 2017, 22(7), 898-919.
Haimovici, A.; Humbert, M.; Federzoni, E.A.; Shan-Krauer, D.; Brunner, T.; Frese, S.; Kaufmann, T.; Torbett, B.E.; Tschan, M.P. PU.1 supports TRAIL-induced cell death by inhibiting NF-κB-mediated cell survival and inducing DR5 expression. Cell Death Differ., 2017, 24(5), 866-877.
[] [PMID: 28362429]
von Karstedt, S.; Montinaro, A.; Walczak, H. Exploring the TRAILs less travelled: TRAIL in cancer biology and therapy. Nat. Rev. Cancer, 2017, 17(6), 352-366.
[] [PMID: 28536452]
Jazirehi, A.R.; Arle, D. Epigenetic regulation of the TRAIL/Apo2L apoptotic pathway by histone deacetylase inhibitors: an attractive approach to bypass melanoma immunotherapy resistance. Am. J. Clin. Exp. Immunol., 2013, 2(1), 55-74.
[PMID: 23885325]
Fröhlich, L.F.; Mrakovcic, M.; Smole, C.; Lahiri, P.; Zatloukal, K. Epigenetic silencing of apoptosis-inducing gene expression can be efficiently overcome by combined SAHA and TRAIL treatment in uterine sarcoma cells. PLoS One, 2014, 9(3) e91558
[] [PMID: 24618889]
De Carvalho, D.D.; Binato, R.; Pereira, W.O.; Leroy, J.M.; Colassanti, M.D.; Proto-Siqueira, R.; Bueno-Da-Silva, A.E.; Zago, M.A.; Zanichelli, M.A.; Abdelhay, E.; Castro, F.A.; Jacysyn, J.F.; Amarante-Mendes, G.P. BCR-ABL-mediated upregulation of PRAME is responsible for knocking down TRAIL in CML patients. Oncogene, 2011, 30(2), 223-233.
[] [PMID: 20838376]
Mello, B.P.; de Carvalho, D.D.; Campos, A.H.; Soares, F.A.; Amarante-Mendes, G.P. Regulation of TRAIL expression by PRAME and EZH2 as potential therapeutic target against solid tumors. BMC Proc., 2013, 7(Suppl. 2), 10-P10.
Medinger, M.; Lengerke, C.; Passweg, J. Novel therapeutic options in Acute Myeloid Leukemia. Leuk. Res. Rep., 2016, 6, 39-49.
[] [PMID: 27752467]
Ludwig, H.; Delforge, M.; Facon, T.; Einsele, H.; Gay, F.; Moreau, P.; Avet-Loiseau, H.; Boccadoro, M.; Hajek, R.; Mohty, M.; Cavo, M.; Dimopoulos, M.A.; San-Miguel, J.F.; Terpos, E.; Zweegman, S.; Garderet, L.; Mateos, M-V.; Cook, G.; Leleu, X.; Goldschmidt, H.; Jackson, G.; Kaiser, M.; Weisel, K.; van de Donk, N.W.C.J.; Waage, A.; Beksac, M.; Mellqvist, U.H.; Engelhardt, M.; Caers, J.; Driessen, C.; Bladé, J.; Sonneveld, P. Prevention and management of adverse events of novel agents in multiple myeloma: a consensus of the European Myeloma Network. Leukemia, 2018, 32(7), 1542-1560.
[] [PMID: 29720735]
de Miguel, D.; Lemke, J.; Anel, A.; Walczak, H.; Martinez-Lostao, L. Onto better TRAILs for cancer treatment. Cell Death Differ., 2016, 23(5), 733-747.
[] [PMID: 26943322]
Chamuleau, M.E.; Ossenkoppele, G.J.; van Rhenen, A.; van Dreunen, L.; Jirka, S.M.; Zevenbergen, A.; Schuurhuis, G.J.; van de Loosdrecht, A.A. High TRAIL-R3 expression on leukemic blasts is associated with poor outcome and induces apoptosis-resistance which can be overcome by targeting TRAIL-R2. Leuk. Res., 2011, 35(6), 741-749.
[] [PMID: 21281967]
Zhou, J.; Lu, X.; Tan, T.Z.; Chng, W.J. X-linked inhibitor of apoptosis inhibition sensitizes acute myeloid leukemia cell response to TRAIL and chemotherapy through potentiated induction of proapoptotic machinery. Mol. Oncol., 2018, 12(1), 33-47.
[] [PMID: 29063676]
Hao, X.S.; Hao, J.H.; Liu, F.T.; Newland, A.C.; Jia, L. Potential mechanisms of leukemia cell resistance to TRAIL-induced apopotosis. Apoptosis: Int. J. Program. Cell Death, 2003, 8(6), 601-607.
Lim, E.J.; Yoon, Y.J.; Heo, J.; Lee, T.H.; Kim, Y-H. Ciprofloxacin enhances TRAIL-induced apoptosis in lung cancer cells by upregulating the expression and protein stability of death receptors through CHOP expression. Int. J. Mol. Sci., 2018, 19(10), 3187.
[] [PMID: 30332761]
Park, S.H.; Kim, J.L.; Jeong, S.; Kim, B.R.; Na, Y.J.; Jo, M.J.; Yun, H.K.; Jeong, Y.A.; Kim, D.Y.; Kim, B.G.; You, S.; Oh, S.C.; Lee, D.H. Codium fragile F2 sensitize colorectal cancer cells to TRAIL-induced apoptosis via c-FLIP ubiquitination. Biochem. Biophys. Res. Commun., 2019, 508(1), 1-8.
[PMID: 30409427]
Kang, Y.J.; Kim, I.Y.; Kim, E.H.; Yoon, M.J.; Kim, S.U.; Kwon, T.K.; Choi, K.S. Paxilline enhances TRAIL-mediated apoptosis of glioma cells via modulation of c-FLIP, survivin and DR5. Exp. Mol. Med., 2011, 43(1), 24-34.
[] [PMID: 21150246]
Gibson, S.B.; Oyer, R.; Spalding, A.C.; Anderson, S.M.; Johnson, G.L. Increased expression of death receptors 4 and 5 synergizes the apoptosis response to combined treatment with etoposide and TRAIL. Mol. Cell. Biol., 2000, 20(1), 205-212.
[] [PMID: 10594023]
Zhu, H.; Wang, J.; Yin, J.; Lu, B.; Yang, Q.; Wan, Y.; Jia, C. Downregulation of PRAME suppresses proliferation and promotes apoptosis in hepatocellular carcinoma through the activation of P53 mediated pathway. Cell. Physiol. Biochem.: Int. J. Experim. Cell. Physiol. Biochem., Pharmaco, 2018, 45(3), 1121-1135.
Mathur, R.; Sehgal, L.; Berkova, Z.; Samaniego, F. Histone 3 Methyltransferase (EZH2) inhibition enhances TRAIL-induced apoptosis in mantle cell lymphoma cells by accelerated cFLIP degradation. Blood, 2013, 122(21), 4425-4425.
Braun, F.K.; Mathur, R.; Sehgal, L.; Wilkie-Grantham, R.; Chandra, J.; Berkova, Z.; Samaniego, F. Inhibition of methyltransferases accelerates degradation of cFLIP and sensitizes B-cell lymphoma cells to TRAIL-induced apoptosis. PLoS One, 2015, 10(3), e0117994-e0117994.
[] [PMID: 25738497]
Zhang, Y.; Kinkel, S.; Maksimovic, J.; Bandala-Sanchez, E.; Tanzer, M.C.; Naselli, G.; Zhang, J-G.; Zhan, Y.; Lew, A.M.; Silke, J.; Oshlack, A.; Blewitt, M.E.; Harrison, L.C. The polycomb repressive complex 2 governs life and death of peripheral T cells. Blood, 2014, 124(5), 737-749.
[] [PMID: 24951427]

Rights & PermissionsPrintExport Cite as

Article Details

Year: 2020
Published on: 29 January, 2020
Page: [571 - 579]
Pages: 9
DOI: 10.2174/1871520620666200130091955
Price: $65

Article Metrics

PDF: 21
PRC: 1