Biochemical Engineering Journal 199 (2023) 109040

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Biochemical Engineering Journal

journal homepage: www.elsevier.com/locate/bej

A systematic investigation and network interaction analysis on preventive

treatments against HepG2 cancer cells from a genomic-level perspective
Elham Amjad a, Solmaz Asnaashari a, *, Raffaele Pezzani b, c, Babak Sokouti a, *, 1
a
Biotechnology Research Center, Tabriz University of Medical Sciences, Tabriz, Iran
b
Phytotherapy Lab, Endocrinology Unit, Dept. Medicine (DIMED), University of Padova, via Ospedale 105, Padova 35128, Italy
c
AIROB, Associazione Italiana Per La Ricerca Oncologica Di Base, Padova, Italy

A R T I C L E I N F O A B S T R A C T

Keywords: Worldwide, the fourth most common malignancy leading to death via cancer transformation is hepatocellular
Hepatocellular carcinoma carcinoma (HCC). Notwithstanding various preventive approaches, the incidence and mortality of patients with
Herbal medicine HCC continue to increase. However, a successful early diagnosis of HCC is beneficial given a potentially curative
Signaling pathway
treatment. We systematically searched the publicly available NCBI microarray database to extract the required
Vitamins
Fatty acid
information on different preventive treatments and their affected genes and signaling pathways on HCC.
Furthermore, the significant genes were analyzed through the Kaplan-Meier tool for overall survival, relapse-free
survival, progression-free survival, and disease-specific survival rates. Subsequently, we assessed the protein-
protein network interaction analysis and their corresponding drugs and diseases. The thorough screening of
the literature microarray gene datasets revealed that various compounds could be implicated in HCC prevention.
The results showed that nine genes (i.e., FGA, FGG, TP53, HELLS, CCNA2, MCM10, MCM2, PCTP, and CYP24A1)
modulated the four above-mentioned survival rates. Furthermore, the associations between genes, drugs, and
related diseases discovered 39 out of 61 medications covered potential anticancer agents. Finally, we demon­
strated that numerous signaling pathways were simultaneously modulated in HCC. In conclusion, this work
underlines the importance of the genomic-level perspective in studying HCC treatment and development.

1. Introduction
According to global cancer statistics reports, in 2020, among thirty-
six types of cancers, liver cancer (i.e., hepatocellular carcinoma – HCC)
stood sixth and third at incidence and mortality rates, respectively [1].
The reported studies showed that the disease was geographically
distributed in eleven major countries from Eastern and South-Eastern
Asia and Northern and Western Africa [1]. As the patients with HCC are
mostly diagnosed and treated by surgery, chemo- and radiotherapies at
higher grades of disease resulting in low treatment performance and
suffering side effects, the diagnosis of disease at low tumor grades will be
essential for effective treatment [2]. Clinical speaking, very early and
early HCC are limited in size to a single tumor < 2 cm and < 5 cm,
respectively [3]. Moreover, the absence of clear symptoms at the early
stages of liver cancer is another problem that needs a more specific
therapy concerning genetic and epigenetic alterations of responsible
genes [4]. It is common for patients to visit a doctor only after the tumor
has grown in size or other complications like liver cirrhosis have
emerged, highlighting the importance of the early-stage diagnosis of
HCC. One of the reasons for this is that a patient’s chance of survival
decreases as time passes from diagnosis to therapy [5].
Gene therapy is one of the unique approaches for disease correction
by delivering a gene into an organism or tissues using vectors. It has
been reported that the gene therapy technique can be a novel leading
complement for available therapeutic agents [6]. For instance, in a
recent study on a mouse model, the authors proposed that a
post-transcriptionally regulated RNA replacement strategy is a safe and
effective gene therapy technique for live cancer treatment [7]. There­
fore, for conducting a reliable gene therapy procedure, identify the
genes involved in the progression of advanced stages of liver cancer.
Additionally, the computational approaches are shown for liver regen­
eration procedure, and papillary thyroid cancer development identified
essential genes and signaling pathways (e.g., mTOR and PI3K) that were
clinically validated through literature evidence [8–10].

* Corresponding authors.
E-mail addresses: asnaasharisolmaz@gmail.com (S. Asnaashari), sokoutib@tbzmed.ac.ir, b.sokouti@gmail.com (B. Sokouti).
1
Primary corresponding author.

https://doi.org/10.1016/j.bej.2023.109040
Received 1 March 2023; Accepted 17 July 2023
Available online 20 July 2023
1369-703X/© 2023 Elsevier B.V. All rights reserved.
E. Amjad et al.
The current systematic review and analysis aims to investigate the
impacts of different drugs, whether they are botanicals or synthetic
chemicals, in HepG2 cell lines based on available microarray datasets.
Our results have been proposed regarding differences in gene expression
levels, involved signaling pathways, biological processes, cellular com­
ponents, molecular functions, and possible identification of the rela­
tionship between significant genes and available drugs. To the best of
our knowledge, this is the first time the general information of drugs’
impact on HepG2 cell line has been studied to obtain significant results
to mitigate liver cancer progression at very early stages. Our data sug­
gest the potential use of natural or synthetic compounds in liver cancer
treatment using a genome-wide perspective.
2. Materials and methods
We systematically searched the National Center for Biotechnology
Information (NCBI) microarray database using a Boolean expression
keyword ("HepG2") AND "Homo sapiens"[porgn:__txid9606] for deriving
the complete GEO datasets (CEL files) and expressed by "Expression
profiling by array." The HepG2 cell line is derived from a 15-year-old
adolescent Caucasian male with hepatocellular carcinoma disease
(hepatoblastoma).
Then, we screened the available GEO datasets (beginning with GSE
terms) for the keywords mentioned above for retrieving their corre­
sponding published accessible research articles. Furthermore, we thor­
oughly reviewed the downloaded papers for the inhibitory effects of
target treatments on HepG2 cell line by taking into account which
essential genes (e.g., DEGs: differentially expressed genes along with
their effective fold changes), gene ontology enrichment for possible
biological processes, and Kyoto Encyclopedia of Genes and Genomes
(KEGG) signaling pathways were involved. It is also worth mentioning
that we considered those dosages of treatments that had no evident toxic
effect. Additionally, the cumulative list of genes derived from the
eligible studies was used for protein-protein interaction network anal­
ysis using Cytoscape 3.7.1 [11] by employing STRING database v.11.0b
[12] currently updated on October 17, 2020, including 24,584,628
proteins from 5090 organisms with 3123,056,667 interactions and
ClusterOne 1.0 algorithm [13] for identifying the hub genes and the
genes with the highest connectivity degree. We set the values for pa­
rameters used in the input genes for the STRING database and Cluster­
One 1.0 as default (i.e., confidence score cutoff value = 0.4 and p-value
≤ 0.05 for modules for being significant). Subsequently, the communal
genes among the reported genes in the literature and those significant
ones from protein-protein interaction (PPI) network analysis were the
target input for further studies using Kaplan-Meier plot on liver cancer
through various survival rates OS (n = 364), RFS (n = 316), PFS (n =
370), and DSS (n = 362) [14] (OS: overall survival, RFS: relapse-free
survival, PFS: progression-free survival, and DSS: disease-specific
survival).
Finally, we inspected the gene-drug relationships based on the list of
the genes in Table 1 using the Drug-Gene Interaction Database (DGIdb,
www.dgidb.org) website [15–17] to further screen the obtained drugs
for their corresponding reported literature diseases.
3. Results
The search results revealed that forty nine GEO datasets (i.e.,
GSE163950, GSE163130, GSE138569, GSE138376, GSE132119,
GSE122660, GSE114743, GSE95298, GSE82299, GSE85746, GSE79473,
GSE71547, GSE69850, GSE69851, GSE62813, GSE73396, GSE53216,
GSE74000, GSE71606, GSE45802, GSE45635, GSE58235, GSE47739,
GSE57753, GSE57928, GSE57893, GSE57071, GSE51952, GSE54257,
GSE54255, GSE38122, GSE51143, GSE48157, GSE39291, GSE45945,
GSE36244, GSE36243, GSE40580, GSE28878, GSE22790, GSE28590,
GSE6878, GSE6878, GSE12161, GSE15162, GSE12939, GSE9517,
GSE9166, GSE9166) were available according to the Boolean search
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Biochemical Engineering Journal 199 (2023) 109040
term and selected criteria. We found nine out of forty-nine GEO datasets
to satisfy the current systematic reviews’ criteria with inhibitory effects
on the HepG2 cell line (shown in Table 1). Among the important genes
extracted from nine studies, only ten (i.e., group A: DGAT1, PCTP,
HSDL2, SNAP23, PNPLA3, JAK1, GSTA1, HELLS, CYP24A1, CYP1A1)
were in common. The PPI network analysis showed that four modules
(shown in Fig. 1) were statistically significant. Hence, eight genes (i.e.,
group B: GSTP1, DGAT1, FGA, FGG, CCNA2, MCM10, MCM2, TP53)
showed the highest connectivity degree in the identified modules and
the whole network. However, a single gene DGAT1 was common be­
tween groups A and B.
According to Table 2, nine genes (i. e. FGA, FGG, TP53, HELLS,
CCNA2, MCM10, MCM2, PCTP, and CYP24A1) were found to be sig­
nificant in all survival rates, including OS, RFS, PFS, and DSS (p-values
are in green color). Moreover, three genes (p-values in cyan color) were
responsible for OS and DSS survival rates (GSTA1, HSDL2, and JAK1),
and two genes (p-values in yellow color), SNAP23 and GSTP1, were
accounted for RFS and PFS rates.
The outcomes of gene-drug relationships and their related diseases
are summarized in Fig. 2, where 39 out of 61 drugs obtained from the
associations of genes and drugs correspond to anticancer therapeutic
agents.
4. Discussion
Although clinicians have achieved considerable advancements in
cancer treatment, gene therapy can be an intriguing alternative to
chemotherapy or radiotherapy regimens. Gene therapy techniques as
novel treatment options have had successful results, especially in pre­
clinical settings. Given such premises, its potential efficacy in patients
affected by HCC could be an attractive therapeutic option. However,
safe and efficient HCC gene therapy delivery demands various viral and
anti-viral vectors advances. These include adenoviral, retroviral, and
adeno-associated viral vectors and the use of nanoparticles, i.e., lipo­
somes, exosomes, and virosomes [18,19].
In the current study, we showed in Table 1 that the use of vitamins (e.
g., vitamin D), fatty acids, and botanicals with antioxidant properties
played an inhibitory effect on HepG2 cell lines.
Considering the effect of vitamin D in HCC, various studies demon­
strated the direct relationship between vitamin D deficiency and the risk
of liver cancer [20,21]. In line with the advantages of using vitamin D,
two systematic reviews and meta-analyses showed that low levels of 25
(OH)D3 were more prevalent in patients with HCC than healthy in­
dividuals [20,21]. Moreover, the anti-inflammatory and
immuno-regulatory properties of vitamin D suggested that determina­
tion of vitamin D levels was not only vital in comorbidities, specifically
during the intensive care unit stay, but also in the prevention and
treatment of liver disorders and liver cancer [22–29].
Dietary fat is composed mainly of four kinds of fatty acids: saturated
fatty acid (SFA), monounsaturated fatty acid (MUFA), n6-
polyunsaturated fatty acid (n6PUFA), and n3-polyunsaturated fatty
acid (n3PUFA). Increased SFA, MUFA, and n6PUFA levels have been
implicated in spreading pancreatic cancer cells to the liver, while
increased n3PUFA levels have been shown to reverse the effect [30].
Several different tumor types, including hepatocellular carcinoma,
showed high de novo fatty acid (FA) generation and cholesterol
biosynthesis, suggesting that this pathway could play a role in tumori­
genesis [31]. Indeed, treatment of advanced liver cancer can be done by
omega-3 polyunsaturated fatty acid [32], sorafenib [33], Amaranthus
spinosus plant (Family: Amaranthaceae) [34], and the polyphenol
phloridzin [35]. Their anticancer activities are based on suppressing FA
synthesis through the Wnt/β-catenin signaling pathway. For example,
A. spinosus extracted fatty acids possessed antiproliferative activity, and
fatty esters of phloridzin showed chemotherapeutic effects in the HepG2
cell line [34]. In HCC, the heterogeneity of the disease demonstrates the
need for various therapeutic and preventative strategies to be effective.
E. Amjad et al. Biochemical Engineering Journal 199 (2023) 109040

Table 1
Nine eligible microarray datasets extracted from the publicly available NCBI-GEO database with their corresponding characteristics.
Item Microarray Intervention No. of Total No. Of Important GO enrichment Category Related term Signaling
Dataset Samples DEGs Genes analysis level Pathway

1 GSE138376 Vitamin D 9 samples For FC > 1.5 or MOGAT1, GO:0033993, Biological Lipid metabolism NA
for < 1.5, 298 Genes DGAT1, GO:0008202, Processes
comprative (Upregulated: AGPAT2, GO:0045444,
analysis 229 and LPGAT1, CDS1, GO:0008610,
between Downregulated: SLC27A2 / GO:0030258,
VitD vs. no 69) FATP2, GO:0030258,
VitD SLC6A12, AQP3, GO:0030258,
PCTP, MAT1A GO:0030258,
GO:0030258,
GO:0030258,
GO:0019216,
GO:0019216,
GO:0019216,
GO:0033189,
GO:0006869,
GO:0006869,
GO:0006869,
GO:0050873,
GO:0002933,
GO:0045834,
GO:0008203
2 GSE122660 Fatty Acid 6 samples 571 DEGs HSDL2, SNAP23, NA NA NA NA
for DMSO VAMP3, TUFM,
and 72 h FA PNPLA3, HNF4A,
treatment JAK1, JAK2,
DST, GSTA1,
TIMP1
3 GSE122660 Fatty Acid + 6 samples 278 DEGs PDK4, HSDL2, NA NA NA NA
TNF-alpha for DMSO SNAP23,FLOT1,
and 72 h ANXA 2, SLC2A
FA+T 1, PNPLK 3,
treatment PPARA, JAK1
4 GSE114743 Melicope 4 Samples For FC > 2 or < BBC3, CDKN1A, Apoptosis of Biological n-Hexane extract Cell Cycle
ptelefolia leaf 2, 1290 DEGs CDKN2B, DDIT3, cervical cancer Processes Control of
n-Hexane GABARAPL1, cell lines, Cell Chromosomal
extract GADD45A, JUN, viability of Replication, Role
NDRG1, TP53, cervical cancer of BRCA1 in DNA
CCNA2, HELLS, cell lines, G1- Damage
MCM2, MCM10, phase of tumor Response,
PLK1, RRM2, cell lines, M-phase Hereditary Breast
SKP2 of tumor cell Cancer Signaling,
lines, Checkpoint ATM Signaling,
control, Self- Cell Cycle: G2/M
renewal of tumor DNA Damage
cell lines Checkpoint
Regulation,
Estrogen-
mediated S-phase
Entry, Mitotic
Roles of Polo-Like
Kinase, Unfolded
protein response,
NRF2-mediated
Oxidative Stress
Response,
Mismatch Repair
in Eukaryotes
5 GSE71606 Tamarindus 6 Samples For FC > 1.5 or FGA, SERPINE1, Lipid metabolism, Biological Hematological Coagulation
indica leaf < 1.5 FGG, small molecule Processes system system,
SERPPINC1, biochemistry, development and Superpathway of
KNG1, hematological function, Lipid cholesterol
SERPIND1, disease, metabolic and biosynthesis,
BHMT, PCSK9, Ophthalmic transport Intrinsic
PNPLA3, SCD, Disease, process, prothrombin
CES1, DHCR24, Connective Tissue Carbohydrate activation
EGR1, APOM, Disorders, metabolic pathway,
SLC6A4, Inflammatory process, Immune
SLC23A2, Disease, Digestive Regulation of protection/
DGAT1, PHYH, System hormone, antimicrobial
PCTP, CFB, Development and Inflammatory response,
PLIN2, G6PC, Function, Organ response, Xenobiotic
GK, TCF7L2, Morphology, Defense response metabolism
SHBG, TTR, Developmental to virus/ signaling
(continued on next page)

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Table 1 (continued )
Item Microarray Intervention No. of Total No. Of Important GO enrichment Category Related term Signaling
Dataset Samples DEGs Genes analysis level Pathway

PLA2G12B, Disorder, bacterium,

S100A3, IL18, Carbohydrate Xenobiotic
LEAP2, MX1, Metabolism, metabolic
IFNGR1, ANXA3, Small Molecule process, Immune
EPHX1, Biochemistry, response
ALDH6A1, Free Radical
GSTM4, Scavenging,
ALDH9A1, Hereditary
ADH6, Disorder,
CYP24A1, Neurological
FCGRT Disease,
Organismal Injury
and
Abnormalities
6 GSE51143 RVX-208 8 Samples For FC > 1.5 or HIST2H4A, NA NA Lipid metabolism NA
bromodomain < 1.5 RABL2A,
CYP1A1, KRCC1,
RFC1, DHRS2,
SRP9,
RPL23AP82,
ZNF737, MYBL1,
SLFN5,
HIST1H2BK,
ZNF43, KLRAP1,
STOX1,
HIST1H2AM,
ST3GAL3,
HIST1H2BD,
C6orf203, NPIP,
DNAJC28,
PNPLA8,
LINC00467,
ZNF280C,
SNORD59B,
HIST1H2BC,
TST, ZNF117,
RPS3A, TNS4,
LIF, CACNB4,
LOC151009,
EREG, FLJ11710,
DGKK,
FAM209B,
SH3TC2,
PCDHA13, TBX4,
CYP4Z1,
MAB21L3,
ZNF208, VPS52,
LINC00652
7 GSE40580 Indo-Tibetan 2 Samples For FC > 2.0 or CYP1A1, PTGR1, Drug Metabolism, Biological These genes are Metabolism of
polyherbal < 2.0 SLC7A11, Amino Acid Processes associated with xenobiotics by
preparation AKR1B10, ASNS, Metabolism, signaling cytochrome
Padma 28 CYP24A1, Small Molecule pathways P450,
PMAIP1, NUPR1, Biochemistry, essential for the Glutathione
SCHIP1, GSTA1, Drug Lipid metabolic metabolism,
FECH, CD14, Metabolism, processing of Aminoacyl-tRNA
AKR1C2, Small Molecule drugs, amino biosynthesis,
LOC374443, Biochemistry, acids, lipids, LPS/IL-1
BCAT1, Vitamin and small molecules, mediated
KLHDC9, HELLS, Mineral vitamins and inhibition of RXR
CARS, AKR1C1, Metabolism, Gene minerals. function, Nrf2-
ABCC4, PIR, Expression, mediated
RAB27A, Cancer, oxidative stress
HMOX1, GCLM, Connective Tissue response, Aryl
RBM39, DKK1, Disorders, Hydrocarbon
SCN1A, Molecular Receptor
SLC13A3, Transport, Amino Signaling
MT1M, LGR5, Acid Metabolism,
SLC26A3, Genetic Disorder,
SH3PXD2A, Drug Metabolism,
SUCLG1 Vitamin and
Mineral,
Metabolism,
Molecular
Transport,
Cardiovascular
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Table 1 (continued )
Item Microarray Intervention No. of Total No. Of Important GO enrichment Category Related term Signaling
Dataset Samples DEGs Genes analysis level Pathway

System,
Development and
Function, Cell To-
Cell Signalling
and Interaction,
Inflammatory
Response, Cell-
To-Cell Signaling
and Interaction,
Tissue
Development,
Cell Morphology
8 GSE12939 84 drug 24 Samples ≥ 2 fold Upregulated NA NA Lipid metabolism several
metabolism- Genes: ABCC1, transcriptional
related genes CYP1A1, regulation
and anticancer CYP2B6, ADH4, pathways
drug such as ALDH1A1,
paclitaxel , CHST1, FAAH,
docetaxel, GAD1, GPX1,
Zden and GSTA3, GSTP1,
colchicine LPO, MGST1,
NAT2, NOS3,
PON2, SNN,
ASNA1
9 GSE9517 Cysteine 15 Samples ranged from 1.3 Downregulated Cancer, Cell cycle, Biological A consequence of NA
deprivation to 14.9 Genes: MT2A, DNA replication, Processes sulfur amino acid
CYP2J2, ALAD, recombination, restriction
FBP1, GPX3, and repair, appears tobe the
HSD17B2, Reproductive upregulation of
MGST2 system disease, the cellular
Cell morphology, capacity to cope
Small molecule with oxidative
biochemistry, Cell and chemical
death, Cellular stresses via the
assembly and integrated stress
organization, response.
Cellular growth
and proliferation,
Connective tissue
disease,
Connective tissue
development and
function,
Respiratory
disease, Gene
expression,
Cellular
movement,
Cellular
development,
Lipid metabolism,
Amino acid
metabolism

According to a preclinical work, two fatty acid ethers of isopropylamine
propanol with minimal toxicity constitute novel derivatives that are
helpful in the prevention of liver cancer, according to a preclinical work
[36].
Herbal medicine can be utilized to treat a wide range of illnesses,
including cancer. Natural compounds’ anticancer property is mediated
by their hepatic antioxidant activity, which is particularly effective in
treating liver cancer. Various works investigated the antioxidant activity
of natural compounds through in vivo and in vitro studies. The analyses
and tests on anticancer properties of Azadirachta indica [37], Suaeda
monoica [38], soyasaponins [39], Veronica Ciliata [40,41], Cystoseira
genus macroalgae [42], lithium carbonate nanosized particles [43],
Pleurotus pulmonarius [44], ursolic acid [45], rice bran phytic acid [46],
extracts from various potatoes [47], Chlorella Vulgaris [48], Enicostemma
littorale [49], and TNP-470 [50] revealed the inhibitory effects against
hepatocellular carcinoma models through their antioxidant properties.
The promotion of cancer by food additives, contaminants, nutrient
deficiencies, and their resultant biochemical alterations is a known way
cancer can develop [51]. In contrast to the previous finding, other food
such as coffee, whose increased consumption has been linked to a
decreased risk of liver cancer. These findings indicate that the inverse
relationship between coffee consumption and the incidence of hepato­
cellular carcinoma may be partially explained by inflammation decre­
ment and hepatocellular damage reduction [52]. On the other hand,
several malignancies have been linked with the prevalence and prog­
nosis of systemic inflammatory markers such as C-reactive protein
(CRP). In a clinical trial, patients with elevated baseline CRP levels
showed an increased risk of liver cancer and mortality from chronic liver
disease [53]. Very little epidemiological evidence is available despite
low vitamin D levels seen in patients with chronic liver illness and a
potential connection to liver cancer. People with low vitamin D levels
had a reduced chance of chronic liver disease mortality, while people
with greater calcium levels had a higher risk of developing liver cancer.
It has been indicated that vitamin D may have a preventive effect in

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E. Amjad et al. Biochemical Engineering Journal 199 (2023) 109040

Fig. 1. - (a) Module 1 with 11 nodes, 33 edges and p-value= 7.51E-04, (b) Module 2 with 11 nodes, 29 edges and p-value= 0.005, (c) Module 3 with 13 nodes, 47
edges and p-value= 0.021, and (d) Module 4 with 19 nodes, 22 edges and p-value= 0.022.

hepatocellular carcinoma [54]. Liver tumor development, in which stem
cells or progenitor cells proliferate and convert into cancer stem cells, is
more often now associated with the TLR signaling pathway than before.
Two instances were included: one was caused by long-term feeding of
ethanol and lipopolysaccharide stem cell/progenitor cells. In contrast,
the other was caused by long-term feeding of carcinogenic drugs. These
two instances confirm that inflammation leads to cancer development
and proves that the TLR signaling pathway is needed for stem cell/­
progenitor activation and transformation [55].
At the moment, the idea of a ’cancer stem cell’ may account for a
portion of the process of hepatocellular carcinoma development [56].
One of the primary sources of therapeutic target identification in he­
patocellular carcinoma is signaling pathways. Until now, no other
approach has been so successful at achieving survival improvements

6
E. Amjad et al.
Table 2
Final list of genes used in four different survival rates.
The p-values in green color demonstrate significant genes in all survival rates. The p-values in cyan color are related to the significant genes for OS and DSS survival
rates, and the p-values in yellow color are the genes accounted to RFS and PFS rates. The p-values in red show the non-significant genes for different survival rates.
when using a multikinase inhibitor (e.g., Sorafenib). It reinforces the
significance of increasing our knowledge of how the networks of
signaling pathways in transformed cells interact. Many signaling path­
ways are involved in the process of hepatocarcinogenesis, including ones
that are directly linked to growth factors, cell differentiation, and blood
vessel development are de-regulated in hepatocellular carcinoma [57].
In another study, the impacted HCC signaling pathways include the
tumor growth factor-β (TGF-β), Wnt/B-catenin, Hedgehog, Notch, IGF,
PDGF, FGF EGF, HGF, VEGF, JAK/STAT, Hippo, and HIF. By small
compounds, herbal medicines, and miRNA, the pathways will be sup­
pressed to trigger cell cycle arrest apoptosis and prevent HCC invasion.
Additionally, several signaling pathways may be targeted simulta­
neously, allowing better control over malignant HCC utilizing in vitro
and in vivo experimental models [58]. Overproduction of triacylglycerol
(TAG) in the liver or low-density lipoproteins has detrimental effects on
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Biochemical Engineering Journal 199 (2023) 109040
metabolic health and should be addressed by the liver [59]. The ultimate
phase of the triacylglycerol biosynthetic process in eukaryotic species is
carried out by two enzymes, DGAT1 and DGAT2. There are two diac­
ylglycerol acyltransferases that are found in the same cells, but their
roles are distinct, even though they catalyze the same process [60].
Adipose, small intestinal, and hepatic tissue all produce lipoproteins,
and these two enzymes play a significant role in the lipoprotein synthesis
process [61]. Curcumin and the hydro-alcoholic extract of turmeric have
been shown to drastically reduce the expression of genes involved in
lipid synthesis, such as DGAT1 [62].
The current outcomes show the significant importance of the list of
genes involved in precisely various cancer types. Moreover, it is worth
noting that more than four-fifths of the whole drugs are related to
anticancer, rheumatoid arthritis, and lipid reduction agents. Further­
more, the results suggest a possible role in further clinical and
E. Amjad et al.
Fig. 2. - A pie chart illustration of the gene-drug-disease associations.
experimental investigations to pave the way for an effective new treat­
ment for liver cancer.
5. Conclusion
The suppression of hepatocellular carcinoma development is the
principal target of the world’s cancer prevention communities. This
study focused on investigating the potential effects of various preventive
treatments against HepG2 cell lines at the genomic level using publicly
available microarray datasets. The outcomes indicated that the protec­
tive and nutritional roles of compounds such as vitamins, fatty acids, and
botanicals could decrease the incidence of HCC. Moreover, the role of
involved signaling pathways and their dysregulation in the process of
HCC should be taken into account. On the other hand, studying the
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Biochemical Engineering Journal 199 (2023) 109040
network-based relationships between genes and drugs and their related
diseases proposed that almost two-thirds of the medicines were anti­
cancer therapeutic agents. Finally, it is suggested that gene therapy can
be a suitable technique that can have a remarkable impact on HCC
growth and development.
Ethics approval and consent to participate
This article does not contain any studies with human participants or
animals performed by any authors.
Consent for publication
Not applicable.
E. Amjad et al. Biochemical Engineering Journal 199 (2023) 109040

Funding [20] Z. Yi, L. Wang, X. Tu, Effect of vitamin D deficiency on liver cancer risk: a
systematic review and meta-analysis, Asian Pac. J. Cancer Prev. 22 (2021)
991–997.
Not applicable. [21] Y. Zhang, X. Jiang, X. Li, M.A. Găman, H. Kord-Varkaneh, J. Rahmani, A. Salehi-
Sahlabadi, A.S. Day, Y. Xu, Serum vitamin D levels and risk of liver cancer: a
systematic review and dose-response meta-analysis of cohort studies, Nutr. Cancer
Declaration of Competing Interest 73 (2020) 1–9.
[22] J. Yin, L.Y. Yin, N.D. Freedman, T.Y. Li, S.M. Dawsey, J.F. Cui, P.R. Taylor, B. Liu,
J.H. Fan, W. Chen, C.C. Abnet, Y.L. Qiao, Independent and joint associations
The authors declare that they have no known competing financial between serum calcium, 25-hydroxy Vitamin D, and the risk of primary liver
interests or personal relationships that could have appeared to influence cancer: a prospective nested case–control study, Cancer Epidemiol. Biomark. Prev.
29 (2020) 2057–2064.
the work reported in this paper. [23] Y. Li, Q. Lin, S. Chang, R. Zhang, J. Wang, Vitamin D3 mediates miR-15a-5p
inhibition of liver cancer cell proliferation via targeting E2F3, Oncol. Lett. 20
Data Availability (2020) 292–298.
[24] X.F. Guo, T. Zhao, J.M. Han, S. Li, D. Li, Vitamin D and liver cancer risk: a meta-
analysis of prospective studies, Asia Pac. J. Clin. Nutr. 29 (2020) 175–182.
No data was used for the research described in the article. [25] T.L. Gomes, R.C. Fernandes, L.L. Vieira, R.M. Schincaglia, J.F. Mota, M.S. Nóbrega,
C. Pichard, G.D. Pimentel, Low vitamin D at ICU admission is associated with
cancer, infections, acute respiratory insufficiency, and liver failure, Nutrition 60
Acknowledgements (2019) 235–240.
[26] L. Cai, L. Luo, Z. Tang, X. Meng, Combined antitumor effects of 1,25-dihydroxy
Not applicable. vitamin D3 and Notch inhibitor in liver cancer, Oncol. Rep. 40 (2018) 1515–1524.
[27] S. Khan, A. Ali, S. Khan, A. Bakillah, G. Damanhouri, A. Khan, A. Makki, I. Alansari,
N. Banu, Current therapies in alleviating liver disorders and cancers with a special
References focus on the potential of Vitamin D, Nutr. Metab. 15 (2018) 13.
[28] A. Duran, E.D. Hernandez, M. Reina-Campos, E.A. Castilla, S. Subramaniam,
S. Raghunandan, L.R. Roberts, T. Kisseleva, M. Karin, M.T. Diaz-Meco, J. Moscat,
[1] H. Sung, J. Ferlay, R.L. Siegel, M. Laversanne, I. Soerjomataram, A. Jemal, F. Bray,
p62/SQSTM1 by binding to vitamin D receptor inhibits hepatic stellate cell
Global cancer statistics 2020: GLOBOCAN estimates of incidence and mortality
activity, fibrosis, and liver cancer, Cancer Cell 30 (2016) 595–609.
worldwide for 36 cancers in 185 countries, CA: A Cancer J. Clin. 71 (2021)
[29] J.B. Wang, C.C. Abnet, W. Chen, S.M. Dawsey, J.H. Fan, L.Y. Yin, J. Yin, J.
209–249.
M. Major, P.R. Taylor, Y.L. Qiao, N.D. Freedman, Association between serum 25
[2] K.W. Zhang, D. Wang, H. Cai, M.Q. Cao, Y.Y. Zhang, P.Y. Zhuang, J. Shen, IL-6
(OH) vitamin D, incident liver cancer and chronic liver disease mortality in the
plays a crucial role in epithelial-mesenchymal transition and pro-metastasis
Linxian Nutrition Intervention Trials: a nested case-control study, Br. J. Cancer 109
induced by sorafenib in liver cancer, Oncol. Rep. 45 (2021) 1105–1117.
(2013) 1997–2004.
[3] D.J. Erstad, K.K. Tanabe, Hepatocellular carcinoma: early-stage management
[30] S. Qiu, F. Wang, J. Hu, Y. Yang, D. Li, W. Tian, X. Yuan, Y. Lv, M. Yu, Increased
challenges, J. Hepatocell. Carcinoma 4 (2017) 81–92.
dietary fatty acids determine the fatty-acid profiles of human pancreatic cancer
[4] S. Yang, S. Lin, K. Liu, Y. Liu, P. Xu, Y. Zheng, Y. Deng, D. Zhang, Z. Zhai, N. Li,
cells and their carrier’s plasma, pancreas and liver, Endocr. J. 67 (2020) 387–395.
X. Ren, Z. Dai, H. Kang, Identification of an immune-related RNA-binding protein
[31] L. Che, W. Chi, Y. Qiao, J. Zhang, X. Song, Y. Liu, L. Li, J. Jia, M.G. Pilo, J. Wang,
signature to predict survival and targeted therapy responses in liver cancer,
A. Cigliano, Z. Ma, W. Kuang, Z. Tang, Z. Zhang, G. Shui, S. Ribback,
Genomics 113 (2021) 795–804.
F. Dombrowski, M. Evert, R.M. Pascale, C. Cossu, G.M. Pes, T.F. Osborne, D.
[5] W.C. Tsai, P.T. Kung, Y.H. Wang, W.Y. Kuo, Y.H. Li, Influence of the time interval
F. Calvisi, X. Chen, L. Chen, Cholesterol biosynthesis supports the growth of
from diagnosis to treatment on survival for early-stage liver cancer, PLoS ONE 13
hepatocarcinoma lesions depleted of fatty acid synthase in mice and humans, Gut
(2018), e0199532.
69 (2020) 177–186.
[6] K. Kamimura, T. Yokoo, H. Abe, S. Terai, Gene therapy for liver, Cancer: Curr.
[32] F.Z. Chang, Q. Wang, Q. Zhang, L.L. Chang, W. Li, Omega-3 polyunsaturated fatty
Status Basic Clin. Cancer 11 (2019) 1865.
acid inhibits the malignant progression of hepatocarcinoma by inhibiting the Wnt/
[7] S.R. Han, C.H. Lee, J.Y. Im, J.H. Kim, J.H. Kim, S.J. Kim, Y.W. Cho, E. Kim, Y. Kim,
β-catenin pathway, Eur. Rev. Med. Pharmacol. Sci. 22 (2018) 4500–4508.
J.-H. Ryu, M.H. Ju, J.S. Jeong, S.-W. Lee, Targeted suicide gene therapy for liver
[33] G. Liu, S. Kuang, R. Cao, J. Wang, Q. Peng, C. Sun, Sorafenib kills liver cancer cells
cancer based on ribozyme-mediated RNA replacement through post-transcriptional
by disrupting SCD1-mediated synthesis of monounsaturated fatty acids via the
regulation, Mol. Ther. - Nucleic Acids 23 (2021) 154–168.
ATP-AMPK-mTOR-SREBP1 signaling pathway, FASEB J. 33 (2019) 10089–10103.
[8] E. Amjad, S. Asnaashari, B. Sokouti, Induction of mTOR signaling pathway in the
[34] A. Mondal, T. Guria, T.K. Maity, A. Bishayee, A novel tetraenoic fatty acid isolated
progression of papillary thyroid cancer, Meta Gene 24 (2020), 100704.
from Amaranthus spinosus inhibits proliferation and induces apoptosis of human
[9] E. Amjad, S. Asnaashari, B. Sokouti, The role of PI3K signaling pathway and its
liver cancer cells, Int. J. Mol. Sci. 17 (2016) 1604.
associated genes in papillary thyroid cancer, J. Egypt. Natl. Cancer Inst. 33 (2021)
[35] S.V.G. Nair, H.P. Ziaullah, Vasantha Rupasinghe, Fatty acid esters of phloridzin
11.
induce apoptosis of human liver cancer cells through altered gene expression, PLoS
[10] S. Asnaashari, E. Amjad, B. Sokouti, A comprehensive investigation on liver
One 9 (2014), e107149.
regeneration: a meta-analysis and systems biology approach, Clin. Exp. Hepatol. 7
[36] M. Cao, V. Prima, D. Nelson, S. Svetlov, Composite fatty acid ether amides suppress
(2021) 183–190.
growth of liver cancer cells in vitro and in an in vivo allograft mouse model, Cell.
[11] P. Shannon, A. Markiel, O. Ozier, N.S. Baliga, J.T. Wang, D. Ramage, N. Amin,
Oncol. 36 (2013) 247–257.
B. Schwikowski, T. Ideker, Cytoscape: a software environment for integrated
[37] M. Iman, M. Taheri, Z. Bahari, The anti-cancer properties of neem (Azadirachta
models of biomolecular interaction networks, Genome Res. 13 (2003) 2498–2504.
indica) through its antioxidant activity in the liver; Its pharmaceutics and toxic
[12] D. Szklarczyk, A.L. Gable, D. Lyon, A. Junge, S. Wyder, J. Huerta-Cepas,
dosage forms; A literature review, Journal of Complementary and Integrative
M. Simonovic, N.T. Doncheva, J.H. Morris, P. Bork, L.J. Jensen, C.V. Mering,
Medicine, (2021) Published online May 10, 2021.
STRING v11: protein-protein association networks with increased coverage,
[38] A.A.A. Al-Shawi, M.F. Hameed, N.H. Ali, K.A. Hussein, Investigations of
supporting functional discovery in genome-wide experimental datasets, Nucleic
phytoconstituents, antioxidant and anti-liver cancer activities of Saueda monoica
Acids Res. 47 (2019) D607–d613.
Forssk extracted by microwave-assisted extraction, Asian Pac. J. Cancer Prev. 21
[13] T. Nepusz, H. Yu, A. Paccanaro, Detecting overlapping protein complexes in
(2020) 2349–2355.
protein-protein interaction networks, Nat. Methods 9 (2012) 471–472.
[39] E.S. Hwang, Antioxidant and quinone reductase activity of soyasaponins in
[14] O. Menyhárt, Á. Nagy, B. Győrffy, Determining consistent prognostic biomarkers of
Hepa1c1c7 mouse hepatocarcinoma cells, Prev. Nutr. Food Sci. 22 (2017)
overall survival and vascular invasion in hepatocellular carcinoma, R. Soc. Open
300–305.
Sci. 5 (2018), 181006.
[40] Q. Lu, Y. Sun, Y. Shu, S. Tan, L. Yin, Y. Guo, L. Tang, HSCCC separation of the two
[15] S.L. Freshour, S. Kiwala, K.C. Cotto, A.C. Coffman, J.F. McMichael, J.J. Song,
iridoid glycosides and three phenolic compounds from Veronica ciliata and their in
M. Griffith, O.L. Griffith, A.H. Wagner, Integration of the Drug-Gene Interaction
vitro antioxidant and anti-hepatocarcinoma activities, Molecules 21 (2016) 1234.
Database (DGIdb 4.0) with open crowdsource efforts, Nucleic Acids Res. 49 (2021)
[41] L. Yin, Q. Lu, S. Tan, L. Ding, Y. Guo, F. Chen, L. Tang, Bioactivity-guided isolation
D1144–d1151.
of antioxidant and anti-hepatocarcinoma constituents from Veronica ciliata,
[16] M. Griffith, O.L. Griffith, A.C. Coffman, J.V. Weible, J.F. McMichael, N.C. Spies,
Chemistry Central, Chem. Central J. 10 (2016) 1–8.
J. Koval, I. Das, M.B. Callaway, J.M. Eldred, C.A. Miller, J. Subramanian,
[42] C. Vizetto-Duarte, L. Custódio, G. Acosta, J.H.G. Lago, T.R. Morais, C.B. De Sousa,
R. Govindan, R.D. Kumar, R. Bose, L. Ding, J.R. Walker, D.E. Larson, D.J. Dooling,
K.N. Gangadhar, M.J. Rodrigues, H. Pereira, R.T. Lima, M.H. Vasconcelos,
S.M. Smith, T.J. Ley, E.R. Mardis, R.K. Wilson, DGIdb: mining the druggable
L. Barreira, A.P. Rauter, F. Albericio, J. Varela, Can macroalgae provide promising
genome, Nat. Methods 10 (2013) 1209–1210.
anti-tumoral compounds? A closer look at Cystoseira tamariscifolia as a source for
[17] K.C. Cotto, A.H. Wagner, Y.Y. Feng, S. Kiwala, A.C. Coffman, G. Spies, A. Wollam,
antioxidant and anti-hepatocarcinoma compounds, PeerJ 2016 (2016), e1704.
N.C. Spies, O.L. Griffith, M. Griffith, DGIdb 3.0: a redesign and expansion of the
[43] V.I. Konenkov, Y.I. Borodin, O.P. Makarova, N.P. Bgatova, L.N. Rachkovskaya,
drug-gene interaction database, Nucleic Acids Res. 46 (2018) D1068–d1073.
Effects of lithium carbonate nanosized particles on oxidant-antioxidant status in
[18] B.D. Mckiver, M. Damaj, D. Sarkar, Assessment of current gene therapy practices in
tumor tissue of hepatocarcinoma-29, Patol. Fiziol. i Èksperimental’naia Ter. 59
hepatocellular carcinoma, Gastrointest. Disord. 2 (2020) 469–480.
(2015) 57–64.
[19] K.E.R. Bowman, P. Lu, E.R.V. Mause, C.S. Lim, Advances in delivery vectors for
gene therapy in liver cancer, Ther. Deliv. 11 (2020) 833–850.

9
E. Amjad et al.
[44] W.W. Xu, B. Li, E.T. Lai, L. Chen, J.J. Huang, A.L. Cheung, P.C. Cheung, Water
extract from Pleurotus pulmonarius with antioxidant activity exerts in vivo
chemoprophylaxis and chemosensitization for liver cancer, Nutr. Cancer 66 (2014)
989–998.
[45] L. Liu, J. Wu, J. Zhang, Z. Li, C. Wang, M. Chen, Y. Wang, Y. Sun, L. Wang, C. Luo,
A compatibility assay of ursolic acid and foodborne microbial exopolysaccharides
by antioxidant power and anti-proliferative properties in hepatocarcinoma cells,
J. Food, Agric. Environ. 10 (2012) 111–114.
[46] M.E. Norhaizan, S.K. Ng, M.S. Norashareena, M.A. Abdah, Antioxidant and
cytotoxicity effect of rice bran phytic acid as an anticancer agent on ovarian, breast
and liver cancer cell lines, Malays. J. Nutr. 17 (2011) 367–375.
[47] Q. Wang, Q. Chen, M. He, P. Mir, J. Su, Q. Yang, Inhibitory effect of antioxidant
extracts from various potatoes on the proliferation of human colon and liver cancer
cells, Nutr. Cancer 63 (2011) 1044–1052.
[48] N.A. Mukti, S. Sulaiman, S.M. Saad, J.M.H. Basari, M.A. Rahman, W.Z.W. Ngah, Y.
A.M. Yusof, Chlorella vulgaris exhibited antioxidant and antitumour effects against
liver cancer in in vivo and in vitro studies, Sains Malays. 38 (2009) 773–784.
[49] R. Gopal, R. Udayakumar, Enzymatic and non-enzymatic antioxidant activities of
Enicostemma littorale in p-DAB induced hepatocarcinoma in rats, Int. J.
Pharmacol. 4 (2008) 369–375.
[50] J.L. Mauriz, M.C. Durán, V. Molpeceres, J.P. Barrio, J. Martín-Renedo, J.
M. Culebras, J. González-Gallego, P. González, Changes in the antioxidant system
by TNP-470 in an in vivo model of hepatocarcinoma, Transl. Res. 150 (2007)
189–196.
[51] B. Reddy, L. Cohen, G. Mccoy, P. Hill, J. Weisburger, E. Wynder, Nutrition and its
relationship to cancer, Adv. Cancer Res. 32 (1980) 237–345.
[52] K. Aleksandrova, C. Bamia, D. Drogan, P. Lagiou, A. Trichopoulou, M. Jenab,
V. Fedirko, I. Romieu, H.B. Bueno-de-Mesquita, T. Pischon, K. Tsilidis, K. Overvad,
A. Tjønneland, M.C. Bouton-Ruault, L. Dossus, A. Racine, R. Kaaks, T. Kühn,
C. Tsironis, E.M. Papatesta, G. Saitakis, D. Palli, S. Panico, S. Grioni, R. Tumino,
P. Vineis, P.H. Peeters, E. Weiderpass, M. Lukic, T. Braaten, J.R. Quirós, L. Luján-
Barroso, M.J. Sánchez, M.D. Chilarque, E. Ardanas, M. Dorronsoro, L.M. Nilsson,
M. Sund, P. Wallström, B. Ohlsson, K.E. Bradbury, K.T. Khaw, N. Wareham,
M. Stepien, T. Duarte-Salles, N. Assi, N. Murphy, M.J. Gunter, E. Riboli, H. Boeing,
D. Trichopoulos, The association of coffee intake with liver cancer risk is mediated
by biomarkers of inflammation and hepatocellular injury: data from the European
10
Biochemical Engineering Journal 199 (2023) 109040
Prospective Investigation into Cancer and Nutrition, Am. J. Clin. Nutr. 102 (2015)
1498–1508.
[53] W. Chen, J.B. Wang, C.C. Abnet, S.M. Dawsey, J.H. Fan, L.Y. Yin, J. Yin, P.R.
Taylor, Y.L. Qiao, N.D. Freedman, Association between C-reactive protein, incident
liver cancer, and chronic liver disease mortality in the Linxian Nutrition
Intervention Trials: a nested case-control study, Cancer epidemiology, biomarkers
& prevention: a publication of the American Association for Cancer Research,
cosponsored by the American Society of Preventive Oncology, 24 (2015) 386–392.
[54] J.B. Wang, C.C. Abnet, W. Chen, S.M. Dawsey, J.H. Fan, L.Y. Yin, J. Yin, J.
M. Major, P.R. Taylor, Y.L. Qiao, N.D. Freedman, Association between serum 25
(OH) vitamin D, incident liver cancer and chronic liver disease mortality in the
Linxian Nutrition Intervention Trials: a nested case-control study, Br. J. Cancer 109
(2013) 1997–2004.
[55] S.W. French, J. Oliva, B.A. French, J. Li, F. Bardag-Gorce, Alcohol, nutrition and
liver cancer: role of Toll-like receptor signaling, World J. Gastroenterol. 16 (2010)
1344–1348.
[56] K. Song, J. Wu, C. Jiang, Dysregulation of signaling pathways and putative
biomarkers in liver cancer stem cells (Review), Oncol. Rep. 29 (2013) 3–12.
[57] A. Moeini, H. Cornellà, A. Villanueva, Emerging signaling pathways in
hepatocellular carcinoma, Liver Cancer 1 (2012) 83–93.
[58] Z. Farzaneh, M. Vosough, T. Agarwal, M. Farzaneh, Critical signaling pathways
governing hepatocellular carcinoma behavior; small molecule-based approaches,
Cancer Cell Int. 21 (2021) 208.
[59] H.R. Wurie, L. Buckett, V.A. Zammit, Evidence that diacylglycerol acyltransferase 1
(DGAT1) has dual membrane topology in the endoplasmic reticulum of HepG2
cells, The, J. Biol. Chem. 286 (2011) 36238–36247.
[60] H.R. Wurie, L. Buckett, V.A. Zammit, Diacylglycerol acyltransferase 2 acts
upstream of diacylglycerol acyltransferase 1 and utilizes nascent diglycerides and
de novo synthesized fatty acids in HepG2 cells, FEBS J. 279 (2012) 3033–3047.
[61] P.J. McFie, P. Chumala, G.S. Katselis, S.J. Stone, DGAT2 stability is increased in
response to DGAT1 inhibition in gene edited HepG2 cells, Biochim. Et. Biophys.
Acta - Mol. Cell Biol. Lipids 1866 (2021), 158991.
[62] R. Taebi, M.R. Mirzaiey, M. Mahmoodi, A. Khoshdel, M.A. Fahmidehkar,
M. Mohammad-Sadeghipour, M.R. Hajizadeh, The effect of Curcuma longa extract
and its active component (curcumin) on gene expression profiles of lipid
metabolism pathway in liver cancer cell line (HepG2), Gene Rep. 18 (2020),
100581.