International Journal of

Molecular Sciences

Review
The Curious Case of the HepG2 Cell Line: 40 Years of Expertise
Viktoriia A. Arzumanian * , Olga I. Kiseleva and Ekaterina V. Poverennaya

Institute of Biomedical Chemistry, 119121 Moscow, Russia; olly.kiseleva@gmail.com (O.I.K.);

k.poverennaya@gmail.com (E.V.P.)
* Correspondence: arzumanian.victoria@gmail.com; Tel.: +7-96-0889-7117

Abstract: Liver cancer is the third leading cause of cancer death worldwide. Representing such
a dramatic impact on our lives, liver cancer is a significant public health concern. Sustainable
and reliable methods for preventing and treating liver cancer require fundamental research on its
molecular mechanisms. Cell lines are treated as in vitro equivalents of tumor tissues, making them a
must-have for basic research on the nature of cancer. According to recent discoveries, certified cell
lines retain most genetic properties of the original tumor and mimic its microenvironment. On the
other hand, modern technologies allowing the deepest level of detail in omics landscapes have shown
significant differences even between samples of the same cell line due to cross- and mycoplasma
infection. This and other observations suggest that, in some cases, cell cultures are not suitable as
cancer models, with limited predictive value for the effectiveness of new treatments. HepG2 is a
popular hepatic cell line. It is used in a wide range of studies, from the oncogenesis to the cytotoxicity
of substances on the liver. In this regard, we set out to collect up-to-date information on the HepG2
cell line to assess whether the level of heterogeneity of the cell line allows in vitro biomedical studies
as a model with guaranteed production and quality.





 Keywords: HepG2 cell line; mutations; hepatocellular carcinoma; hepatoblastoma; hepatocytes
Citation: Arzumanian, V.A.;
Kiseleva, O.I.; Poverennaya, E.V. The
Curious Case of the HepG2 Cell Line:
40 Years of Expertise. Int. J. Mol. Sci. 1. Introduction
2021, 22, 13135. https://doi.org/ Cell lines have revolutionized scientific research because of their similarity to primary
10.3390/ijms222313135
tissues, low cost, and ease of use and culture. In addition, such cells provide an unlimited
supply of biomaterials, and their use in research avoids ethical problems associated with
Academic Editor: Hiroaki Taniguchi
the utilization of animal and human tissues [1]. The widespread use of cell lines has been
found in the production of vaccines [2], cytotoxicity testing [3,4] and the identification
Received: 5 November 2021
of drug metabolic pathways [5,6], the study of gene function [7], the creation of artificial
Accepted: 2 December 2021
Published: 4 December 2021
tissues (for example, artificial skin [8]), and the synthesis of therapeutic proteins [9,10].
The liver is an organ with high regenerative capacity and complex functions [11]. It
maintains various physiological processes [12], predominantly replenishing and storing
Publisher’s Note: MDPI stays neutral
with regard to jurisdictional claims in
rapidly mobilized energy reserves in glycogen, regulating carbohydrate metabolism, and
published maps and institutional affil-
neutralizing and removing metabolic products from the body [13]. In addition, the liver
iations. takes part in the metabolism of nutrients, receiving digestive products in the form of
glucose, amino acids, fatty acids, and glycerol. Moreover, there are many different types
of liver diseases. These can be inherited (Wilson disease, hemochromatosis, and alpha
1-antitrypsin deficiency) [14] or acquired (such as hepatitis and liver cancer) [15,16].
In many studies, immortalized hepatic cell lines are used instead of liver biopsies
Copyright: © 2021 by the authors.
due to their high cost, high invasiveness, and reduced activity of several key enzymes.
Licensee MDPI, Basel, Switzerland.
This article is an open access article
Moreover, there is no technique enabling the maintenance of liver biopsies in culture
distributed under the terms and
for time-consuming studies [17]. At the same time, based on the descriptions in the
conditions of the Creative Commons databases, the hepatic cell lines are described as “epithelial morphology”. Thus, the
Attribution (CC BY) license (https:// human cell lines THLE-2 and THLE-3, considered models of normal hepatocytes, are
creativecommons.org/licenses/by/ controversial. Epithelial cells include hepatocytes and cholangiocytes. These cells perform
4.0/). different functions, with hepatocytes responsible for the formation of bile; the metabolism

Int. J. Mol. Sci. 2021, 22, 13135. https://doi.org/10.3390/ijms222313135 https://www.mdpi.com/journal/ijms

Int. J. Mol. Sci. 2021, 22, x FOR PEER REVIEW 2 of 19

Int. J. Mol. Sci. 2021, 22, 13135 THLE-2 and THLE-3, considered models of normal hepatocytes, are controversial. Epi- 2 of 19

thelial cells include hepatocytes and cholangiocytes. These cells perform different func-
tions, with hepatocytes responsible for the formation of bile; the metabolism of glucose,
amino acids and
of glucose, lipids;
amino acids theanddetoxification of bilirubin and
lipids; the detoxification of ammonia;
bilirubin andandammonia;
the productionand the
of serum proteins [3,18]. Cholangiocytes, in turn, modulate the composition
production of serum proteins [3,18]. Cholangiocytes, in turn, modulate the composition of bile, secrete
chloride
of bile,and carbonate
secrete chloride ions,andandcarbonate
secrete and absorb
ions, andwater
secretewhen
andbile passes
absorb waterthrough
whenthe bile
intrahepatic bile duct [19]; in the event of a malfunction of hepatocytes,
passes through the intrahepatic bile duct [19]; in the event of a malfunction of hepatocytes, cholangiocytes
replace them [20,21].
cholangiocytes However,
replace them the article
[20,21]. describing
However, thethe characteristics
article describing ofthethe THLE-2 and of
characteristics
THLE-3 cell lines
the THLE-2 andmentioned
THLE-3 cell their similarity
lines mentioned with human
their hepatocytes
similarity [22]. Such
with human inaccura-
hepatocytes [22].
cies in wording
Such can affect
inaccuracies the design
in wording can of studies
affect the and the of
design interpretation
studies and of theexperiments
interpretation de- of
signed and performed
experiments designed to and
answer the biomedical
performed to answer question.
the biomedical question.
It should also be noted that, in addition to
It should also be noted that, in addition to epithelial epithelial cells, thethe
cells, liver includes
liver includes Kupffer
Kupffer
cells (~15%
cells (~15%of of
thethetotal
totalliver cellcell
liver population),
population), stellate cells
stellate (~8–10%),
cells (~8–10%), andandliver sinusoidal
liver sinusoidal
endothelial
endothelial cells
cells (~3%)
(~3%) (see(see Figure1).1).This
Figure Thisdiversity
diversitypresents
presentsresearchers
researchers with
with the daunt-
daunting
ingtask
taskofofcreating
creatingaaliver livermodel,
model,both bothin invivo
vivoand
andin invitro,
vitro,that
thatwould
wouldinclude
include all all types
types of
of cells [23].

Figure 1. Types
Figure of human
1. Types liver
of human cells
liver and
cells their
and distribution.
their distribution.

TheTheuseuse
of of
immortalized
immortalized hepatic
hepatic tumor
tumor cellcell
lines hashas
lines become
become a widespread
a widespread practice
practice
notnot
only forfor
only thethe
cancer
cancerresearch,
research, butbut
also forfor
also thethestudy
studyof of
hepatitis
hepatitis B (HBV)
B (HBV) and hepatitis
and hepatitis
DD (HDV)
(HDV) viral
viral infections.
infections. Thus,
Thus, thethe complete
complete cellcell cycle
cycle of ofHDVHDV replication
replication andand expres-
expres-
sion/replication
sion/replication of HBV
of HBV were were found
found in cell
in cell lines
lines HepG2HepG2 andandHuh7Huh7 [24,25].
[24,25]. Additionally,
Additionally,
mechanisms
mechanisms of of
HBV HBV viral
viral entry
entry were
were discovered
discovered in in HepaRG
HepaRG cellcell lines
lines [26].
[26].
Currently, there are about 40 various hepatic tumor cell
Currently, there are about 40 various hepatic tumor cell lines, but the most lines, but the most commonly
commonly
used
used areare HepaRG,Huh7,
HepaRG, Huh7,SK-Hep-1,
SK-Hep-1,Hep3B,Hep3B, and HepG2,HepG2, obtained
obtainedfrom fromvarious
varioustumors
tumors [20]
(Figure
[20] (Figure1).1).Among
Among the the cell
cell culture mentioned
mentioned above, above,the theHepG2
HepG2cell cellline
linehas
has gained
gained
popularity
popularity due dueto toitsits
widewide range
range of of applications
applications in in scientific
scientific research.
research. Therefore,
Therefore, at at
thethe
time of writing, the database PubMed (https://pubmed.ncbi.nlm.nih.gov/, accessed on 23on
time of writing, the database PubMed (https://pubmed.ncbi.nlm.nih.gov/, accessed
23 November
November) 2021) contained
contained a total of
a total of 34,021 34,021available
articles articles available after afor
after a search search for “HepG2”;
“HepG2”; 6455
6455 for the Huh7 cell line; 2994 articles for the Hep3B cell line;
for the Huh7 cell line; 2994 articles for the Hep3B cell line; 876 about HepaRG; and 625 876 about HepaRG; and
625 articles on SK-Hep-1
articles on SK-Hep-1 (Figure 2). (Figure 2).
Int.
Int.J. J.Mol.
Mol.Sci.
Sci.2021,
2021,22,
22,x 13135
FOR PEER REVIEW 3 3ofof1919

Figure 2. Percentage of manuscripts using human hepatic cell lines in accordance with the corre-
Figure 2. Percentage of manuscripts using human hepatic cell lines in accordance with the corre-
spondingsearch
sponding searchterm
termon
onPubMed
PubMed(accessed
(accessedon
on2323November
November2021).
2021).

A meta-analysis of the articles showed that each of the cell types comes from a specific
A meta-analysis of the articles showed that each of the cell types comes from a spe-
type of cancer. For example, the Huh7 cell line has the characteristics of HCC [27], MT-
cific type of cancer. For example, the Huh7 cell line has the characteristics of HCC [27],
CHC01 originates from ICCA cells [28], and HepG2—HB [29]. In Table 1, we summarize
MT-CHC01 originates from ICCA cells [28], and HepG2—HB [29]. In Table 1, we summa-
the characteristics of the top five most studied hepatic tumor cell lines. Characteristics for
rize the characteristics of the top five most studied hepatic tumor cell lines. Characteristics
all hepatic cell lines are presented in Supplementary Material Table S1.
for all hepatic cell lines are presented in Supplementary Material Table S1.
Table 1. Characteristics of the top five most studied tumor hepatic cell lines.
Table 1. Characteristics of the top five most studied tumor hepatic cell lines.
Number of Number of
Cell Type Mutated Genes Number of Number of Arti-Reference
Cell Type Mutated Genes
Chromosomes Articles (PubMed) Reference
Chromosomes cles (PubMed)
HepG2 HB HepG2 CTNNB1
HB 50–60
CTNNB1 50–60 34,021 34,021 [12,30]
[12,30]
PLIN2, ANXA1, PLIN2, ANXA1,
HepaRG HCC HepaRG H2AFY,
HCCSNX1, H2AFY, SNX1,
46 46 880 880 [26,31–33]
[26,31–33]
GCHFR, APO GCHFR, APO
KDR, POLD3, KDR, POLD3,
Huh7 HCCHuh7 HCC 55–63 55–63 6463 6463 [34–37]
[34–37]
TERT, TP53 TERT, TP53
Hep3B HCCHep3B HCC RB1
AXIN1, AXIN1, RB1
≈60 ≈60 2994 2994 [34,38]
[34,38]
SK-Hep-1 Adenocarcinoma CDKN2A, BRAF 56–64 602 [39,40]
SK-Hep-1 Adenocarcinoma CDKN2A, BRAF 56–64 602 [39,40]

This review provides generalized information on the HepG2 cell line, examples of its
This review
use, assessment ofprovides generalized
its characteristics, andinformation on theasHepG2
its applicability modelcell line, examples of its
objects.
use, assessment of its characteristics, and its applicability as model objects.
2. Historical Background
2. Historical Background
Among hepatic cell lines, HepG2 cells were the first to exhibit the key characteristics
Among hepatic cell lines, HepG2 cells were the first to exhibit the key characteristics
of hepatocytes. This line was isolated in 1975 and described as hepatocellular carcinoma
of hepatocytes. This line was isolated in 1975 and described as hepatocellular carcinoma
(or hepatoma, HCC) [41]. On the other hand, the earlier cell line SK-Hep1, created in 1971,
(or hepatoma, HCC) [41]. On the other hand, the earlier cell line SK-Hep1, created in
although considered a model of HCC, does not possess critical markers of liver cells, in-
1971, although considered a model of HCC, does not possess critical markers of liver cells,
cluding the expression of albumin and alpha- and gamma-fibrinogen [42].
including the expression of albumin and alpha- and gamma-fibrinogen [42].
A patent for the HepG2 cell line, “a human hepatoma-derived cell line”, was filed in
A patent for the HepG2 cell line, “a human hepatoma-derived cell line”, was filed
1980 by researchers at the Wistar Institute. Since then, HepG2 cells have been entered into
in 1980 by researchers at the Wistar Institute. Since then, HepG2 cells have been entered
the ATCC (American Type Culture Collection, Rockville, MD, USA) repository as a hu-
into the ATCC (American Type Culture Collection, Rockville, MD, USA) repository as a
man cell line (HB 8065), “derived from the liver tissue of a 15-year-old white male with a
human cell line (HB 8065), “derived from the liver tissue of a 15-year-old white male with
well-differentiated hepatocellular carcinoma” [29].
a well-differentiated hepatocellular carcinoma” [29].
As
Asininseveral
severalcases,
cases,aa classification
classification error crept in,
error crept in, and
andthe
theHepG2
HepG2cell
cellline
linewas
wasmistak-
mis-
takenly labeled
enly labeled as hepatocellular
as hepatocellular carcinoma
carcinoma (HCC)
(HCC) instead
instead of hepatoblastoma
of hepatoblastoma (HB), (HB),
which
which caused considerable confusion for 30 years. This was discovered after
caused considerable confusion for 30 years. This was discovered after an investigation an investiga-
by
tion by Lopez-Terrada
Lopez-Terrada D. et al.D.[29]
et al.
on[29]
the on the nature
nature of HepG2.
of HepG2. In addition
In addition to cytological
to visual visual cytolog-
signs
Int. J. Mol. Sci. 2021, 22, x FOR PEER REVIEW 4 of 19

Int. J. Mol. Sci. 2021, 22, 13135 4 of 19

ical signs of the original cell preparations, from the molecular side, losses in the chromo-
some 4q3 region were found, which are associated with t(1; 4) translocation—a common
occurrence
of the originalwith cell
HB [43], as well asfrom
preparations, withtheother characteristic
molecular HB chromosomal
side, losses abnormal-
in the chromosome 4q3
ities, including
region were found,trisomies
which 2 are
andassociated
20 [29]. Other signs
with t(1; 4) of an HB nature common
translocation—a include the corre-
occurrence
sponding parameters
with HB [43], as well of the Wnt/β-catenin
as with signaling
other characteristic pathway andabnormalities,
HB chromosomal dysregulationincluding
of cell
growth
trisomiesand2survival pathways
and 20 [29]. such as
Other signs of fetal
an HB and embryonal
nature includeHB the[44], a characteristic
corresponding dele-
parameters
tion of the
of the third exon of the
Wnt/β-catenin CTNNB1
signaling gene, which
pathway is identical to that
and dysregulation described
of cell growth inandepithelial
survival
type HB, as such
pathways well as similar
fetal and gene expression
embryonal HBprofiles in HepG2 and
[44], a characteristic tumor of
deletion cells
theinthird
hepato-exon
blastoma [29].
of the CTNNB1 gene, which is identical to that described in epithelial type HB, as well as
Regarding
similar the differences
gene expression profilesbetween
in HepG2 the and
HepG2 tumorcellcells
line in
and normal hepatocytes,
hepatoblastoma [29]. the
crucialRegarding
point is thethe weak or absent
differences expression
between of the cytochrome
the HepG2 cell line and P450
normal(CYP) superfamily,
hepatocytes, the
crucial point
CYP3A4, CYP2C9,is theCYP2C19,
weak or absent
CYP2A6, expression
CYP2D6, ofetc.
the [45],
cytochrome
which are P450 (CYP) superfamily,
involved in phase 1
CYP3A4, oxidation
xenobiotic CYP2C9, CYP2C19,
in the liver.CYP2A6, CYP2D6,
Nevertheless, it isetc. [45], which
believed that theareHepG2
involvedcellinline
phase
has 1
retained most of the metabolic functions of normal hepatocytes; therefore, it is used has
xenobiotic oxidation in the liver. Nevertheless, it is believed that the HepG2 cell line to
retained
study mosteffects
the toxic of theofmetabolic functions
heavy metals, of normaland
nanoparticles, hepatocytes; therefore,
drugs in vitro [46]. it is used to
study the toxic effects of heavy metals, nanoparticles, and drugs in vitro [46].
3. Comparison of Liver Cancer
3. Comparison of Liver Cancer
One of the possible reasons for the popularity of the use of the HepG2 cell line is
relatedOne of the
to the possible
fact that mostreasons
casesforofthe popularity
primary liverofcancer
the useare of the HepG2 cell line
hepatocellular is related
carcinoma
[47]. Liver cancer is the third leading cause of cancer death worldwide [48]. There Liver
to the fact that most cases of primary liver cancer are hepatocellular carcinoma [47]. are
cancer is the third leading cause of cancer death worldwide [48].
several types of primary liver cancer: HCC (80–90% of cases), intrahepatic cholangiocar- There are several types
of primary
cinoma (ICCA, liver cancer:
10–15% of HCC
cases),(80–90%
HB andofangiosarcoma
cases), intrahepatic cholangiocarcinoma
(AS) [49]. It should be noted(ICCA, that
10–15% of cases), HB and angiosarcoma (AS) [49]. It should be noted that hepatoblastoma
hepatoblastoma is the most common malignant liver tumor in children; it accounts for
is the most common malignant liver tumor in children; it accounts for about 70% of cases,
about 70% of cases, followed by HCC, accounting for 27% [50].
followed by HCC, accounting for 27% [50].
Each type of liver cancer and its subtype differs in cytological and molecular charac-
Each type of liver cancer and its subtype differs in cytological and molecular char-
teristics (Figure 3). As mentioned above, one of the hallmarks of HB at the molecular level
acteristics (Figure 3). As mentioned above, one of the hallmarks of HB at the molecular
is damage to the CTNNB1 gene encoding β-catenin in the third exon in the form of dele-
level is damage to the CTNNB1 gene encoding β-catenin in the third exon in the form of
tions or insertions [51], but most often, the loss of serine/threonine residues (in codons
deletions or insertions [51], but most often, the loss of serine/threonine residues (in codons
S33, S37, S45 and T41), as well as the substitution of tyrosine for alanine (T41A) [52]. The
S33, S37, S45 and T41), as well as the substitution of tyrosine for alanine (T41A) [52]. The
frequency of mutations of this gene is observed in 50–90% of patients [51,53]. It should be
frequency of mutations of this gene is observed in 50–90% of patients [51,53]. It should be
noted that in the case of HCC, mutations in CTNNB1 can also be observed, but they are
noted that in the case of HCC, mutations in CTNNB1 can also be observed, but they are
much less common (20–40% of patients [54]) and are represented by changes in codons
much less common (20–40% of patients [54]) and are represented by changes in codons
32,
32,33,
33,38,
38,andand4545[55].
[55].Mutations
Mutationsininβ-catenin
β-cateninleadleadtotoitsitsaccumulation
accumulationinintumor tumorcellscellsdue
due
totothe loss of original function, i.e., impairment of the Wnt/β-catenin
the loss of original function, i.e., impairment of the Wnt/β-catenin signaling pathway, signaling pathway,
which
whichplaysplaysaavital
vitalrole
roleininthe
the development,
development, regeneration,
regeneration, and and metabolic
metaboliczoning
zoningprocess
processof
ofthe
theliver
liver[56].
[56].

Figure 3. Significant liver cancer markers.

Int. J. Mol. Sci. 2021, 22, 13135 5 of 19

The key features of HCC [57,58] and several oncological diseases [59] are a mutation of
the TERT gene promoter, which triggers the activation of telomerase reverse transcriptase,
the process of tumor formation. Promoter mutations have been identified as the most
frequent genetic changes in HCC, with an overall incidence of about 30–60% [60]. The
second most frequent disorder in HCC is mutations in the tumor suppressor gene TP53 [61],
some mutations of which contribute to the emergence of the pro-oncogenic function of the
encoded protein p53—increased cell proliferation, drug resistance, and increased migration
and invasion of cells, as well as the stimulation of neoangiogenesis [62,63]. Such hot-spot
mutations (in particular, R249S and V157F) are associated with a poor prognosis for patients
with HCC [64].
Hepatic AS and ICCA also have a TP53 gene mutation associated with reduced
survival [65–67]. In ICCA, mutations are also often observed in the KRAS and ARID1A
genes [68,69], the proteins involved in the cell signaling pathways that control cell growth,
maturation, and death. Sometimes, mutations occur in genes involved in Wnt signaling,
i.e., CTNNB1, AXIN1, and APC [70,71].
One of the biomarkers of tumor processes in the liver is a change in the level of alpha-
fetoprotein (AFP)—plasma protein produced by the yolk sac and the fetal liver during fetal
development [72,73]. An increase in the AFP level promotes the proliferation of tumor cells
and the formation of blood vessels and enhances the antiapoptotic effect of cancer cells [74].
Changes in the AFP level also predict the course of disease, the therapeutic response, and
the occurrence of relapse [75].
Thus, existing differences and similarities in the molecular characteristics of liver
cancer types raise a logical question about the applicability of the HepG2 cell line as a
model of normal and pathological cellular processes in each case.

4. Comparison of HepG2, Normal Hepatocyte, HB, and HCC

According to their cytological characteristics, HepG2 cells are certainly the most similar
to tumor cells in hepatoblastoma, but at the same time, they retain features characteristic
of normal hepatocytes (see Table 2). Thus, the average diameter of a HepG2 cell is about
10–20 µm, of a hepatocyte, 15 µm, and tumor cells with HB, 10–20 µm. We did not find
exact data for HCC cells, but all tumor cells have a diameter >10 µm.

Table 2. Comparison of HepG2 cells with hepatocytes and cells with HCC and HB.

HepG2 Cells Hepatocyte Cells Cells with HB Cells with HCC

>10 µm,
10–20 µm, round or spindle-shaped and
Cell size and shape 12–19 µm, polygonal 15 µm, cube
angulated show bizarre anaplastic
figures
The numbers of
Large nuclei, 3–7
Two or more nuclei Small, round, mitochondria and ER 3
nucleoli, low
Subcellular occupy 5–7% of the cell inconspicuous nucleoli; is reduced, and have an
mitochondrial content,
components volume; high SER1 and low mitochondrial and abnormal structure,
and poorly developed
mitochondria RER 2 content characteristic of
SER 1
stressful conditions
Number of Aneuploidy, Aneuploidy,
50–60 Polyploidy
chromosomes >46 >46
Genome stability, 7.5 pg genomic DNA, ~6 pg genomic DNA,
Genome unstable Genome unstable
DNA content genome unstable stable genome
1 SER, smooth endoplasmic reticulum; 2 RER, rough endoplasmic reticulum; 3 ER, endoplasmic reticulum.

About 20% of human hepatocytes are binucleated or polyploid [76,77]; often, their
nuclei are anisokaryotic. HepG2 cells contain three to seven nuclei [78], which account for
up to 25% of the total cellular protein, although their size is somewhat more prominent
than in normal hepatocytes, containing up to 10% of the total protein in the cell. In tumor
Int. J. Mol. Sci. 2021, 22, 13135 6 of 19

cells, which are also characterized by an abnormal number of chromosomes, an increase in

the number of nuclei is observed—up to seven per cell.
Hepatocytes are rich in the smooth endoplasmic reticulum (SER) and mitochondria,
reflecting their intense protein synthesis and energy metabolism, respectively [79]. HepG2
SER cells are poorly developed, and the number of mitochondria is half that of hepatocytes.
Ultrastructural analysis of cells in HB revealed well-formed intercellular junctions, numer-
ous intermediate filaments, and rare cytoplasmic organelles: mitochondria and a rough
endoplasmic reticulum [80]. In tumor cells with HCC, the numbers of mitochondria and
endoplasmic reticulum are also significantly reduced, and they have an abnormal structure
characteristic of stressful conditions.
Thus, according to the cytological characteristics, it can be noted that HepG2 cells
occupy an intermediate state between normal hepatocytes and tumor cells, for which
significant changes in the epigenetic regulation of nuclear and mitochondrial genes are
observed [81,82].

4.1. Genome
Numerous studies have shown that this cell line contains fairly stable chromosomal
abnormalities [30,83–86]. HepG2 cells contain translocations between the short arms
of chromosomes 1 and 21 [86], trisomies of chromosomes 2, 16, and 17, and tetrasomy
of chromosome 20 [30,87]. The number of chromosomes varies from 50 to 60 [13,14],
corresponding to the hyperdiploid karyotype [88]. More than 100 of the chromosomes are
observed in some cases, characterized by tetraploid enlargement [86,89]. The HepG2 cell
contains about 7.5 pg of DNA, 15% more than in a normal somatic cell [12,90].
In addition to the marker mutations discussed above (see Figure 3) [30], in an article
by Zhou et al. in which the genome of the HepG2 cell line was studied, 377 SNVs and
255 indels, which are private protein-altering (PPA), were found using whole-genome
sequencing [30]. Some are accounted for among the characteristic mutations by well-
known oncogenes and tumor suppressors, such as NRAS, STK11/LKB1, and PREX2, and
several genes associated with tumor processes, CDK12 IKBKB and RP1L1.
In an article by Tianyou et al. that studied polymorphisms in the NRAS and KRAS
genes in Chinese children, no significant association was found between the risk of de-
veloping hepatoblastoma and these polymorphisms [91]. There are also no data on the
relationship of mutations in the PREX2 gene with hepatoblastoma; however, it has been
shown that in 23.5% of patients with HCC, there is a non-silent somatic mutation of
PREX2 [92]. A similar situation for the RP1L1 gene is defined as a driver in HCC [15,93–95],
but there are no data on the relationship with the development of HB.
The HepG2 cell line carries a mutation in the TERT promoter that is common in
HCC—C228T [96]. In HB, a somatic mutation is also observed in the promoter of this gene—
C250T [51,52,97]. It should be noted that a mutation in the TERT promoter contributes to
immortalization, protecting telomeres in cancer cells.
The TP53 gene can be found in liver cancer in two types: mutant and wild. Wild-type
TP53 [98] is observed in the HepG2 cell line, as in HCC and HB [99]. In HCC, mutant
TP53 tumors have higher malignant potentials than those with wild-type TP53 [100]. The
TP53 gene is critical in suppressing cancer in humans, as it plays a role in cell cycle arrest,
apoptosis, and ageing. Thus, this mutation in the gene can promote cell proliferation.
Thus, it is worth noting that parts of the driver mutations in the genes of the HepG2
cell line coincide with mutations in HCC and HB. Furthermore, a marker deletion in the
reading frame of the CTNNB1 gene for HB also occurs in HCC in 19% of patients [54].

4.2. Transcriptome
A transcript is the next stage after the genome in the transfer of biological information,
at which new characteristics appear, such as changes in the number of transcripts encoded
by one gene and their expression. In total, about 14,000 genes are expressed in HepG2
cells [101]. At the transcriptome level for the HepG2 cell line, it has been shown that
Int. J. Mol. Sci. 2021, 22, 13135 7 of 19

50 genes are upregulated in comparison with normal hepatocytes [102]. Thus, genes
associated with cancer have an increased expression level in the HepG2 cell line, and genes
active in hepatocytes are associated with xenobiotic metabolism.
An analysis comparing results of the transcriptome profiling of hepatic cell lines and
tumor cells in HB and HCC showed significant differences in the level of DLK1 gene
expression in HepG2 cells [102–104], which may be necessary for driving chemoresis-
tance and potentiating malignancy in cancers. A change in the level of DLK1 expression
was also observed in tumor cells, and the gene was more actively expressed in HB cells
than in HCC [44,105]. A similar pattern was found in the case of insulin-like growth
factor 2 (IGF2) [106–109], which is associated with the development of various diseases in
children—for example, Beckwith–Wiedemann syndrome [110–113]. Moreover, increased
IGF2 expression is associated with a poor prognosis of the course of cancer because it
supports both the proliferation and migration of tumor cells [114–116].
In HepG2, there is increased expression of the Wnt signaling pathway antagonist
DKK1 [102], increasing the proliferation, colony-forming ability, and invasion. However, it
is believed that altered DKK1 expression has no effect on tumor development in HB due to
the accumulation of β-catenin [44]. In the case of HCC, overexpression of DKK1 leads to
an increase in cell invasiveness through the MMP-7 signaling pathway, which is associated
with poor prognosis [117].
Another example of a gene with altered expression is the GPC3 gene encoding
glypican-3, which is overexpressed in HepG2 cells and tumor cells in HCC [118,119].
GPC3 is considered a potential biomarker for the early diagnosis of HCC [119,120]. When
studying the functional response to the suppression of the GPC3 gene in HepG2 and Huh7
cells, a more pronounced response was revealed in the first case which, in both cases,
consisted of a decrease in cell profiling [121].
In a study by Tyakht et al. [102], increased expression of genes encoding the insulin-like
growth factor 2 mRNA-binding proteins, IGF2BP1 and IGF2BP3, was shown in HepG2 cells
compared with normal hepatocytes. These genes are also overexpressed in HB [122,123],
but in the case of HCC, a change in expression has only been shown for IGF2BP1, an
increased value of which is associated with a poor prognosis for the course of the dis-
ease [124–126].
Compared with normal hepatocytes, HepG2 cells exhibit a decreased expression level
of CYP genes [102,127,128], which play a decisive role in the metabolism of endogenous
and exogenous molecules [128–130]. Moreover, reduced expression levels of these genes
are also observed in tumor cells in HCC and HB, which, as expected, correlate with a low
survival estimates [131].
Based on the analysis of transcriptome profiling data, common gene expression pat-
terns are observed in HB, HCC, and HepG2 cells. The HepG2 has also been compared
with other commonly used hepatic cell lines at the transcriptomic level. Thus, a study
comparing HepG2 and HepaRG showed that the HB cell line has a greater affinity with
normal hepatocytes [132]. However, the article by Jennen et al. noticed that HepaRG is
more suited in an in vitro liver model for biological interpretations of the effects of expo-
sure to chemicals, whereas HepG2—for classification studies using the toxicogenomics
approach [133]. With a small proportion of genes differing in expression between HepG2
cells and normal hepatocytes, it is necessary to note significant functional differences in
several genes responsible for metabolism and proliferation.

4.3. Proteome
The HepG2 cell contains, on average, 170 pg of total protein, which is almost three
times less than in hepatocytes (600 pg) [12]. The liver is the main source of blood plasma
proteins—fibrinogen, albumin, and globulins. Comparative analysis of the proteomic
profile of hepatocytes and the HepG2 cell line showed that the titers of serum albumin
(ALB), transferrin (AAA), and serpin (SERPINA1) did not differ. At the same time, plasma
Int. J. Mol. Sci. 2021, 22, 13135 8 of 19

proteins such as ceruloplasmin (CP) and hemopexin (HPX) were detected in trace amounts
or were not found at all in HepG2 cells [12,134].
According to the results of transcriptome profiling in HepG2 cells, the cytochrome
P450 superfamily genes are weakly expressed, as expected—at the proteomic level, the
corresponding proteins are either absent or found at very low concentrations [12]. Thus, it
has been shown that CYP3A4, which is one of the key enzymes of drug cleavage [135], is
100–400 times less in HepG2 cells than in hepatocytes [12,136]. Low expression of these
proteins was also observed in patients with HCC and HB [137,138].
A significant difference in the proteomic profiles of hepatocyte and HepG2 cells is also
observed in the expression of phase II drug-metabolizing enzymes. Thus, the expression
of enzymes of the UGT family—UGT1A1, UGT1A4, UGT1A6, UGT2B7, UGT2B15, and
GSTM1, which play a key role in the metabolism of a wide range of anticancer agents—
was observed either at very low levels in the HepG2 cell line or was not detected at all
in comparison with hepatocytes [12,139]. It has been shown that UGT proteins are also
weakly expressed in tumor cells during HCC [140].
For other phase II drug-metabolizing enzymes, SULT1A1 and SULT2A1, responsible
for transferring a sulfate group from 3’-phosphoadenylyl sulfate to the hydroxyl group
of an acceptor, no differences in titers were found between hepatocyte cells and HepG2.
At the same time, it should be noted that in 50% of HCC cases, there is a decrease in the
level of SULT2A1 [141,142]. Conjugates from phase II metabolism are excreted into the bile
and/or the blood by efflux transporters in the canalicular and basolateral membrane of the
hepatocytes, respectively [12]. This process is sometimes referred to as phase III metabolism.
Most of the tubular outflow transporters are present in HepG2 cells at levels close to those
found in hepatocytes. However, it has been shown that the titers of the BSEP protein,
which carry bile salts, are 100 times lower in HepG2 cells than in hepatocytes [12,143],
which is consistent with studies of the mRNA level [144]. In patients with HCC, the BSEP
level is also decreased, which is associated with a poor prognosis of the course of the
disease [145,146]. Of the basolateral efflux transporters MRP3, MRP4, and MRP6, MRP4
was not detected in HepG2 cells [12,147], and MRP3 and MRP6 titers were 4–20 times
lower in HepG2 cells than in hepatocytes.
Thus, at the proteomic level, HepG2 cells retain and multiply changes in systems
associated with the metabolism of endogenous and exogenous substances compared with
normal hepatocytes. At the same time, the observed changes are similar to processes in
hepatoblastoma and hepatocellular carcinoma cells, which justifies the use of the HepG2
cell line to study the metabolism of anticancer drugs and tumor processes.

4.4. Metabolome
Most of the metabolomic studies performed on the HepG2 cell line aim to investigate
the effect of drugs and chemicals on cells. In total, a small part of the work was carried
out on the comparison of HepG2 and other cell lines. In particular, Chen et al. (2018) [148]
compared the hepatocyte cell lines L-02 and HepG2. Their study showed that in the HepG2
cell line, acetate, creatine, isoleucine, leucine, and phenylalanine were increased, which
indicates a more enhanced lipid metabolism and active absorption of nutrients from the
media than in L-02 cells. However, it should be noted that, according to the STR analysis,
L-02 is a HeLa derivative and not a hepatic cell line [149], which casts doubt on the obtained
assessment of the similarity of the HepG2 cell line with hepatocytes.
When comparing HepG2 cells with other tumor cell lines—MCF7 (angiosarcoma of
the breast), PC3 (prostatic adenocarcinoma), 143B (osteosarcoma), and HEK293 (embryonic
kidney)—it was shown that HepG2 cells had a decreased content of amino acids such as
glutamine, proline, asparagine, aspartate, arginine, methionine, alanine, lysine, threonine,
and leucine [150]. Moreover, HepG2 cells also had a high content of glutamate, phenylala-
nine glycine, acetylcarnitine, and methionine and choline derivatives [151]. Changes in
amino acid levels affect protein synthesis, particularly the secretion of total hepatic protein,
which shows a positive relationship between the concentration of several amino acids
Int. J. Mol. Sci. 2021, 22, 13135 9 of 19

and the amount of total protein [152]. A low level of taurine was also observed in HepG2
cells, of which the primary role in the liver is conjugation with bile acids for excretion
into bile [153]. The total amount of fatty acids was increased compared with the cell lines
MCF7, PC3, 143B, and HEK293, and the hydrolysis of phospholipids was low, as assessed
by glycerophosphoethanolamine, which may indicate the causes of degradation of the
nascent very-low-density lipoproteins [154]. Thus, the observed metabolomic profile of
HepG2 is generally characteristic of immortalized cells, but several differences are specific
for the biological processes of hepatocytes.
The meta-analysis of the literature revealed almost no metabolic studies, including
those comparing HCC and HB with hepatocytes and hepatic cell lines. Thus, due to the
lack of relevant studies, we cannot conclude the similarity of HepG2 with other cells for
use as models; however, the opportunity for further research is open.

4.5. Signalome
In the study of tumor processes, special attention is paid to signaling pathways that
are responsible for the aberrant cell growth, survival, genome maintenance, etc. [155].
Signalome study provides another level for deciphering pathological processes and uses it
to inform new treatments for precision medicine in cancer [156]. However, at the moment
many aspects are still unknown, and the current level of information is limited.
The case of liver cancer focuses on the following pathways: the transforming growth
factor β (TGF-β) [157], proto-oncogene Wnt/β-catenin [158], phosphoinositide 3-kinase
(PI3K)/protein kinase B (Akt) [159], c-Jun N-terminal kinase (JNK)/signal transducer
and activator of transcription (STAT) [160], Hedgehog and tumor protein 53 transduction
pathways [161].
As noted above, one of the key features of HB due to the exon 3 mutation CTNNB1
gene is a violation of the Wnt/β-catenin signaling pathway [162]. This pathway is crucial
in controlling hepatic homeostasis and in maintaining adherens junctions and metabolic
zonation and regeneration, suggesting its role in almost every aspect related to liver func-
tioning [162]. The same mutation is characteristic for the HepG2 cell line [29], with suppres-
sion of the Wnt/β-catenin signaling pathway expectedly leading to apoptosis [163]. Other
altered pathways in HB are signal transducer and activator of transcription 3 (STAT3) [164]
and signaling pathways PI3K/Akt, ERK, and p38 [165].
In the case of HCC, the major pathways involved in the oncogenic process, in ad-
dition, to Wnt/b-catenin [54], are Hedgehog, hepatocyte growth factor/c-MET, vascular
endothelial growth factor (VEGF), mitogen-activated protein kinase (MAPK)/ERK (or Ras-
Raf-MEK-ERK), and PI3K/AKT/Mtor [166]. Changes in the HepG2 cell line are observed
in the transforming growth factor-beta (TGF-β) signaling pathway, which accounts for
38% of gene mutations in HCC [167]. Dysregulation of signaling in the TGF-β pathway
plays a central role in inflammation, fibrogenesis, and immunomodulation in the HCC
microenvironment [168].

4.6. HepG2 for Biomedical Research

Based on the meta-analysis of articles, we have summarized the recommendations
for the use of the HepG2 cell line (Table 3). The table is based only on genomic and
transcriptomic data since nucleic acid sequencing is more sensitive than mass spectrometry
of proteins and metabolites [169]. Available experiments do not allow the whole picture of
the proteome and metabolome to be seen [170,171].
Int. J. Mol. Sci. 2021, 22, 13135 10 of 19

Table 3. Suitability of the HepG2 cell line in various spheres of biomedical research.

Type of Research Advantages Disadvantages Recommendations Valid References 1

According to the article (Ren et al.), the HepG2 cell No valid

Toxicity tests
line may not be a suitable model in investigating experiment.
• Including albumin and AFP2 ; metabolism-mediated toxicity without additional
• No hepatitis B viral genome. modification due to it is lack of metabolic No valid
Drug metabolism • Low expression and activity levels of capability [174,175]. For example, the cell line can experiment.
the drug-metabolizing enzymes [45]; be used in studies of CYP inducers [176].
• There are exceptions such as
• CTNNB1 exon 3 mutation [177]; NQO13 , GSTM34 , and MRP15
The correct use of HepG2 is an HB model, as it has
• Gene expression profiling [127,172,173];
many of the characteristics of HB. According to the
demonstrated cell growth and • The minor phase I enzymes: CYP27B1,
article (Lopez-Terrada D et al.), “the correct
survival pathways deregulation, CYP2W1 are expressed at significantly
attribution of the tumor of origin of this cell line is
similar to that of fetal and higher elevated levels than in human
of crucial interest for investigators studying the
HB model embryonal HB [44]; hepatocytes [172]. [178–183]
biology of hepatocellular neoplasms, particularly
• Losses of the chromosome 4q3 those engaged in novel biology-based
region associated with the t(1;4) classifications, clinical stratification, and
translocation—translocation in therapeutic interventions for pediatric and adult
HB [43]. patient” [29].

According to meta-analysis, the cell line has some

similar mutations to HCC. However, there are a
At the genomic and transcriptomic levels
Partially similar genome and large number of cell lines derived from HCC cells,
HCC model has been shown to be similar to Questionable.
transcriptome profiles. such as Huh-7, HepaRG, etc. Additionally, their
hepatoblastoma.
use is relevant, as there are more overlaps in the
genetic and transcriptome profiles.

• A large number of tumor mutations

affect further levels of information
transfer; There are non-tumor cell lines such as THLE-2 and
• The basal gene expression level, No valid
Hepatocyte model No advantages. THLE-3 that have characteristics similar to
HepaRG cells are closer to primary experiment.
hepatocytes [22].
hepatocytes compared with HepG2
cells [133].

1 “Validexperiment” —were chosen based on the recommendations that were found in the articles; 2 AFP —α-fetoprotein; 3 NQO1 —NAD(P)H quinone dehydrogenase 1; 4 GSTM3—glutathione S-transferase
Mu 3; 5 MRP1—multidrug resistance protein 1.
Int. J. Mol. Sci. 2021, 22, 13135 11 of 19

5. Conclusions
Despite the illustrated differences between the HepG2 cell line and normal hepato-
cytes, the toxic effects of heavy metals, nanoparticles, and drugs are frequently studied
in vitro [46,184–187].
The validity of using HepG2 cells as a model of hepatocytes is controversial because
crucial proteins involved in the metabolism of substances—the liver’s primary function—
are poorly expressed. Furthermore, the shortage of uptake transporters and phase I
enzymes is observed in HepG2 cells, which indicates the need for careful use of this cell line
to predict the metabolism and elimination of xenobiotics in hepatocytes. At the same time,
the use of HepG2 cells to study the metabolism of anticancer drugs is acceptable because
there is a similarity in the expression of phase I, II, and III drug metabolism/transport
proteins in cells with HCC and HB. In addition, due to the low basal activity of CYP
proteins (CYP1A2, CYP2B6, and CYP3A4), the cell line can be used in studies of CYP
inducers [176].
Work is under way to modify the HepG2 cell line to increase the expression of cy-
tochromes for the correct use of HepG2 cells as a model of hepatocytes in the study of
drug metabolism [188]. Another approach is to derive three-dimensional spheroid cell cul-
tures [189–191]. The three-dimensional culture method transforms cells into spheroids and
creates a more physiologically relevant system. As a result, metabolic activity, including
cytochromes, is higher in 3D spheroidal HepG2 models than in 2D cells [142], bringing it
closer to normal hepatocytes.
According to molecular profiling data, the HepG2 cell line can serve as a model
for hepatoblastoma. However, the use of HepG2 cells as an HCC model is incorrect,
and as shown in the study by Choi et al. (2015), there is no expression of the marker of
HCC—hGSTP1 [130].
For 40 years, the HepG2 cell line has been widely used as a model for normal hepato-
cytes, hepatocellular carcinoma, and hepatoblastoma cells in various studies. Researchers
are studying its molecular composition at the genomic, transcriptomic, and proteomic
levels, each time confirming that the HepG2 is only partially similar to primary hepatocytes
and cancer cells. However, even after the publication of such articles, researchers continue
to call the genesis of the cell line differently. The “surprising case” of the HepG2 cell line
lies in its erroneous annotation. This is not critical for some work, such as studying the
amount of protein in a cell. However, if the metabolism of drugs or the processes that occur
in a particular type of cancer are being studied, this is important and may call into question
the relevance of the study.
In this manuscript, we wanted to combine the accumulated knowledge about the cell
line and compare it at different molecular levels with normal hepatocytes, HCC and HB.
Hopefully, our review will simplify the scientists’ question: “Should I use HepG2 in my
research?”

Supplementary Materials: All data are available online at https://www.mdpi.com/article/10.339

0/ijms222313135/s1.
Author Contributions: Conceptualization, E.V.P. and O.I.K.; writing—original draft preparation,
V.A.A., O.I.K., and E.V.P.; visualization, V.A.A.; project administration, E.V.P. All authors have read
and agreed to the published version of the manuscript.
Funding: This work was supported by RSF № 20-14-00328.
Acknowledgments: We would like to thank the reviewers of this manuscript for the most construc-
tive and benevolent review process we have experienced to date.
Conflicts of Interest: The authors declare no conflict of interest.
Int. J. Mol. Sci. 2021, 22, 13135 12 of 19

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