Effect of Zebularine on Apoptotic Pathways in Hepatocellular Carcinoma Cell Lines

Masumeh Sanaei, Fraidoon Kavoosi


Background: The alteration of DNA cytosine methylation is one of the most common epigenetic changes that can play a significant role in human cancers. The enzymes involved in DNA methylation of promoter regions of the genes are DNA methyltransferases (DNMTs). The therapeutic activities and apoptotic effects of DNA methyltransferase inhibitors (DNMTIs) have been reported in various cancers. This study was assigned to assess the effect of zebularine on intrinsic and extrinsic pathways, DNAT 1, 3a, and 3b, p21, and p53, viability, and apoptosis in hepatocellular carcinoma (HCC) cell lines. Methods: Hepatocellular carcinoma cell lines (HCCLM3, MHCC97H, and MHCC97L) were purchased from the National Cell Bank of Iran, Pasteur Institute, treated with zebularine, and the MTT assay was performed. Then, flow cytometry assay and real‑time RT‑PCR analysis were performed with zebularine. Statistical comparisons between groups were made using GraphPad Prism software version 8.0. A significant difference was considered as P < 0.05. Results: Zebularine up‑regulated DR4, DR5, FAS, FAS‑L, TRAIL, Bax, Bak, Bim, p21WAF/CIP1 (p21), and p53 and down‑regulated DNMTs (DNAT 1, 3a, and 3b), Bcl‑2, Bcl‑xL, and Mcl‑1, significantly resulting in apoptosis induction in HCC cell lines. Maximal and minimal apoptosis was seen in HCCLM3 and MHCC97L cell lines, respectively. Conclusions: Our findings indicated that DNMTI zebularine can induce apoptosis and inhibit cell growth through both pathways (extrinsic and intrinsic) in HCC cell lines HCCLM3, MHCC97H, and MHCC97L.


Apoptosis; extrinsic; intrinsic; pathway; zebularine

Full Text:



Momparler RL, Bovenzi V. DNA methylation and cancer. J Cell

Physiol 2000;183:145‑54.

Margot JB, Ehrenhofer‑Murray AE, Leonhardt H. Interactions

within the mammalian DNA methyltransferase family. BMC Mol

Biol 2003;4:1‑9.

Haggarty P, Hoad G, Harris SE, Starr JM, Fox HC, Deary IJ, et al.

Human intelligence and polymorphisms in the DNA methyltransferase

genes involved in epigenetic marking. PLoS One 2010;5:11329‑35.

Brown R, Strathdee G. Epigenetic cancer therapies: DNA

methyltransferase inhibitors. Expert Opin Investig Drugs


Flotho C, Claus R, Batz C, Schneider M, Sandrock I, Ihde S, et al.

The DNA methyltransferase inhibitors azacitidine, decitabine and

zebularine exert differential effects on cancer gene expression in

acute myeloid leukemia cells. Leukemia 2009;23:1019‑28.

Matthews GM, Lefebure M, Doyle MA, Shortt J, Ellul J,

Chesi M, et al. Preclinical screening of histone deacetylase

inhibitors combined with ABT‑737, rhTRAIL/MD5‑1 or

‑azacytidine using syngeneic Vk MYC multiple myeloma. Cell

Death Dis 2013;4:798‑806.

Ruiz‐Magaña MJ, Rodríguez‐Vargas JM, Morales JC,

Saldivia MA, Schulze‐Osthoff K, Ruiz‐Ruiz C. The DNA

methyltransferase inhibitors zebularine and decitabine induce

mitochondria‐mediated apoptosis and DNA damage in p53

mutant leukemic T cells. Int J Cancer 2012;130:1195‑207.

Walia YK, Sharma V. Role of HDACs and DNMTs in cancer

therapy: A review. Asian J Adv Basic Sci 2013;1:62‑78.

Pulukuri SMK, Rao JS. Activation of p53/p21Waf1/Cip1 pathway

by 5‑aza‑2’‑deoxycytidine inhibits cell proliferation, induces

pro‑apoptotic genes and mitogen‑activated protein kinases in

human prostate cancer cells. Int J Oncol 2005;26:863‑71.

Hussein MR, Haemel AK, Wood GS. Apoptosis and melanoma:

Molecular mechanisms. J Pathol JPTLAS 2003;199:275‑88.

Yagita H, Takeda K, Hayakawa Y, Smyth MJ, Okumura K.

TRAIL and its receptors as targets for cancer therapy. Cancer Sci


Sanaei M, Kavoosi F, Salehi H. Genistein and trichostatin a

induction of estrogen receptor alpha gene expression, apoptosis

and cell growth inhibition in hepatocellular carcinoma HepG

cells. Asian Pac J Cancer Prev 2017;18:3445‑51.

Cao XX, Mohuiddin I, Chada S, Mhashilkar AM, Ozvaran MK,

McConkey DJ, et al. Adenoviral transfer of mda‑7 leads to

BAX up‑regulation and apoptosis in mesothelioma cells,

and is abrogated by over‑expression of BCL‑XL. Mol Med


Huang Y‑W, Wang L‑S, Chang H‑L, Ye W, Dowd MK,

Wan PJ, et al. Molecular mechanisms of (‑)‑gossypol‑induced

apoptosis in human prostate cancer cells. Anticancer Res


Zhang X‑Q, Dong J‑J, Cai T, Shen X, Zhou X‑J, Liao L.

High glucose induces apoptosis via upregulation of Bim

expression in proximal tubule epithelial cells. Oncotarget


Xu Y, Liu L, Qiu X, Liu Z, Li H, Li Z, et al. CCL21/CCR7

prevents apoptosis via the ERK pathway in human non‑small

cell lung cancer cells. PLoS One 2012;7:33262‑70.

Zhang Y‑L, Pang L‑Q, Wu Y, Wang X‑Y, Wang C‑Q, Fan Y.

Significance of Bcl‑xL in human colon carcinoma. World J

Gastroenterol 2008;14:3069‑76.

Wang B, Ni Z, Dai X, Qin L, Li X, Xu L, et al. The Bcl‑2/

xL inhibitor ABT‑263 increases the stability of Mcl‑1 mRNA

and protein in hepatocellular carcinoma cells. Mol Cancer


Nakamoto N, Higuchi H, Kanamori H, Kurita S, Tada S,

Takaishi H, et al. Cyclooxygenase‑2 inhibitor and interferon‑β

synergistically induce apoptosis in human hepatoma cells in vitro

and in vivo. Int J Oncol 2006;29:625‑35.

Puppo F, Contini P, Ghio M, Brenci S, Scudeletti M, Filaci G,

et al. Soluble human MHC class I molecules induce soluble

fas ligand secretion and trigger apoptosis in activated CD8 +

Fas (CD95)+ T lymphocytes. Int Immunol 2000;12:195‑203.

Herbeuval J‑P, Boasso A, Grivel J‑C, Hardy AW, Anderson SA,

Dolan MJ, et al. TNF‑related apoptosis‑inducing ligand (TRAIL)

in HIV‑1‑infected patients and its in vitro production by

antigen‑presenting cells. Blood 2005;105:2458‑64.

Sanaei M, Kavoosi F, Roustazadeh A, Golestan F. Effect of

genistein in comparison with trichostatin a on reactivation of

DNMTs genes in hepatocellular carcinoma. JCTH 2018;6:141‑8.

Pirouzpanah MB, Sabzichi M, Pirouzpanah S, Chavoshi H,

Samadi N. Silibilin‑induces apoptosis in breast cancer cells by

modulating p53, p21, Bak and Bcl‑XL pathways. Asian Pac J Cancer Prev 2015;16:2087‑92.

Wiklund ED, Bramsen JB, Hulf T, Dyrskjøt L, Ramanathan R,

Hansen TB, et al. Coordinated epigenetic repression of the

miR‐200 family and miR‐205 in invasive bladder cancer. IJC


Gray SG, Baird A‑M, O’Kelly F, Nikolaidis G, Almgren M,

Meunier A, et al. Gemcitabine reactivates epigenetically silenced

genes and functions as a DNA methyltransferase inhibitor. Int J

Mol Med 2012;30:1505‑11.

Soncini M, Santoro F, Gutierrez A, Frigè G, Romanenghi M,

Botrugno OA, et al. The DNA demethylating agent decitabine

activates the TRAIL pathway and induces apoptosis in acute

myeloid leukemia. Biochim Biophys Acta Mol Basis Dis


Jin S, Cojocari D, Purkal JJ, Popovic R, Talaty NN,

Xiao Y, et al. 5‑Azacitidine induces NOXA to prime AML

cells for venetoclax‑mediated apoptosis. Clin Cancer Res


Nakamura K, Aizawa K, Nakabayashi K, Kato N, Yamauchi J,

Hata K, et al. DNA methyltransferase inhibitor zebularine

inhibits human hepatic carcinoma cells proliferation and induces

apoptosis. PloS One 2013;8:54036‑42.

Billam M, Sobolewski MD, Davidson NE. Effects of a novel

DNA methyltransferase inhibitor zebularine on human breast

cancer cells. Breast Cancer Res Treat 2010;120:581‑92.

Sanaei M, Kavoosi F. Investigation of the effect of zebularine

in comparison to and in combination with trichostatin A on

p21Cip1/Waf1/Sdi1, p27Kip1, p57Kip2, DNA methyltransferases

and histone deacetylases in colon cancer LS 180 cell line. Asian

Pac J Cancer Prev 2020;21:1819‑28.

Li Y, Hu W, Shen D‑Y, Kavanagh JJ, Fu S. Azacitidine

enhances sensitivity of platinum‑resistant ovarian cancer

cells to carboplatin through induction of apoptosis. AJOG


Shin DY, Park Y‑S, Yang K, Kim G‑Y, Kim W‑J, Han MH, et al.

Decitabine, a DNA methyltransferase inhibitor, induces apoptosis

in human leukemia cells through intracellular reactive oxygen

species generation. Int J Oncol 2012;41:910‑8.

Najem SA, Khawaja G, Hodroj MH, Rizk S. Synergistic effect of

epigenetic inhibitors decitabine and suberoylanilide hydroxamic

acid on colorectal cancer in vitro. Curr Mol Pharmacol


Zhou P, Lu Y, Sun X‑H. Effects of a novel DNA methyltransferase

inhibitor Zebularine on human lens epithelial cells. Mol Vis


Napso T, Fares F. Zebularine induces prolonged apoptosis effects

via the caspase‑3/PARP pathway in head and neck cancer cells.

Int J Oncol 2014;44:1971‑9.