СУЛЬФОРАФАН ПРИГНІЧУЄ ГЕПАТОЦЕЛЮЛЯРНУ КАРЦИНОМУ У МИШЕЙ ШЛЯХОМ АКТИВАЦІЇ P53, ЗМЕНШЕННЯ ОКСИДАТИВНОГО СТРЕСУ ТА ІНГІБУВАННЯ АНГІОГЕНЕЗУ
Background. Hepatocellular carcinoma (HCC) is the most common primary liver malignancy. Sulforaphane (SF) is a natural isothiocyanate that exhibits anticarcinogenic activity against various cancer cells in vivo and in vitro, with no observed side effects. It also has a prophylactic effect against ea...
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| Опубліковано в: | Експериментальна онкологія |
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| Дата: | 2026 |
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| Сторінки: | 120-132 |
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2026
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Experimental Oncology| _version_ | 1874183254774382592 |
|---|---|
| author | A. Omran, Kholoud I. Mahmoud, Yomna A. Mahmoud, Asmaa H. Fares, Nagui |
| author_facet | A. Omran, Kholoud I. Mahmoud, Yomna A. Mahmoud, Asmaa H. Fares, Nagui |
| author_institution_txt_mv | [
{
"author": "Kholoud A. Omran",
"institution": "Department of Zoology, Faculty of Science, Ain Shams University, Cairo, Egypt",
"orcid": ""
},
{
"author": "Yomna I. Mahmoud",
"institution": "Department of Zoology, Faculty of Science, Ain Shams University, Cairo, Egypt",
"orcid": ""
},
{
"author": "Asmaa A. Mahmoud",
"institution": "Department of Zoology, Faculty of Science, Ain Shams University, Cairo, Egypt",
"orcid": ""
},
{
"author": "Nagui H. Fares",
"institution": "Department of Zoology, Faculty of Science, Ain Shams University, Cairo, Egypt",
"orcid": ""
}
] |
| author_sort | A. Omran, Kholoud |
| baseUrl_str | https://exp-oncology.com.ua/index.php/Exp/oai |
| collection | OJS |
| container_end_page | 132 |
| container_issue | 2 |
| container_start_page | 120 |
| container_title | Експериментальна онкологія |
| container_volume | 48 |
| datestamp_date | 2026-08-21T12:36:18Z |
| description | Background. Hepatocellular carcinoma (HCC) is the most common primary liver malignancy. Sulforaphane (SF) is a natural isothiocyanate that exhibits anticarcinogenic activity against various cancer cells in vivo and in vitro, with no observed side effects. It also has a prophylactic effect against early stages of some cancer models, including HCC. The study aimed to assess the therapeutic anticarcinogenic effect of SF in experimental murine HCC in vivo and to estimate possible underlying mechanisms of its effects on cell proliferation, angiogenesis, and antioxidant status. Materials and Methods. HCC was induced in mice by a single intraperitoneal dose of 100 mg/kg of diethylnitrosamine, followed by 22 weekly intraperitoneal doses of 0.5 mg/kg of carbon tetrachloride. After the induction of HCC, SF (2 mg/kg body weight) was given orally from weeks 25 to 28. Then we conducted histopathological examination, biochemical analysis of blood sera, and immunohistochemical analysis of marker expression in liver sections. Results. SF improved liver structure and function in HCC-bearing mice and survival rate, significantly reduced the expression of tumor marker alpha-fetoprotein and the number of hepatic nodules, and downstaged HCC. These changes were accompanied by an increase in total antioxidant capacity, expression of p53 and Bax, as well as a significant decrease in lipid peroxidation and immunoreactivity of PCNA, VEGF, and Bcl-2 in liver tissues. Conclusion. SF decreased the number of tumor nodules and ameliorated HCC by activating p53 expression, inducing apoptosis, and inhibiting proliferation, oxidative stress, and angiogenesis. These findings support further translational evaluation of SF as a potential therapeutic agent for HCC. |
| doi_str_mv | 10.15407/exp-oncology.2026.02.120 |
| first_indexed | 2026-08-22T01:00:34Z |
| format | Article |
| fulltext |
■
120 ISSN 1812-9269. Experimental Oncology 48 (1). 2026
ORIGINAL CONTRIBUTIONS
C i t a t i o n: Omran KA, Mahmoud YI, Mahmoud AA, Fares NH. Sulforaphane suppresses hepatocellular carcinoma in
mice via activating p53, mitigating oxidative stress, and inhibiting angiogenesis. Exp Oncol. 2026; 48(2): 120-132. https://
doi.org/10.15407/exp-oncology.2026.02.120
© PH “Akademperiodyka” of the NAS of Ukraine, 2026. This is an open access article under the CC BY-NC-ND license
(https://creativecommons.org/licenses/by-nc-nd/4.0/)
Hepatocellular carcinoma (HCC) is the most com-
mon primary liver malignancy and accounts for
more than 80% of liver tumors worldwide [1, 2]. It
is the sixth most common cancer and the third
most common cause of cancer-related death world-
wide [2, 3]. Most HCC patients are diagnosed at the
advanced stages of the disease, when surgical op-
tions are no longer feasible [4]. Fewer than one-
third of these patients benefit from chemotherapy
[5], which remains the primary treatment option
https://doi.org/10.15407/exp-oncology.2026.02.120
Kholoud A. Omran *, Yomna I. Mahmoud,
Asmaa A. Mahmoud, Nagui H. Fares
Department of Zoology, Faculty of Science,
Ain Shams University, Cairo, Egypt
* Correspondence: E-mail: kholoudomran_p@sci.asu.edu.eg,
shafaamrschool@hotmail.com
SULFORAPHANE SUPPRESSES
HEPATOCELLULAR CARCINOMA
IN MICE VIA ACTIVATING p53,
MITIGATING OXIDATIVE STRESS,
AND INHIBITING ANGIOGENESIS
Background. Hepatocellular carcinoma (HCC) is the most common primary liver malignancy. Sulforaphane (SF) is a
natural isothiocyanate that exhibits anticarcinogenic activity against various cancer cells in vivo and in vitro, with no
observed side effects. It also has a prophylactic effect against early stages of some cancer models, including HCC. The
study aimed to assess the therapeutic anticarcinogenic effect of SF in experimental murine HCC in vivo and to estimate
possible underlying mechanisms of its effects on cell proliferation, angiogenesis, and antioxidant status. Materials and
Methods. HCC was induced in mice by a single intraperitoneal dose of 100 mg/kg of diethylnitrosamine, followed by
22 weekly intraperitoneal doses of 0.5 mg/kg of carbon tetrachloride. After the induction of HCC, SF (2 mg/kg body
weight) was given orally from weeks 25 to 28. Then we conducted histopathological examination, biochemical analysis
of blood sera, and immunohistochemical analysis of marker expression in liver sections. Results. SF improved liver
structure and function in HCC-bearing mice and survival rate, significantly reduced the expression of tumor marker
alpha-fetoprotein and the number of hepatic nodules, and downstaged HCC. These changes were accompanied by an in-
crease in total antioxidant capacity, expression of p53 and Bax, as well as a significant decrease in lipid peroxidation and
immunoreactivity of PCNA, VEGF, and Bcl-2 in liver tissues. Conclusion. SF decreased the number of tumor nodules
and ameliorated HCC by activating p53 expression, inducing apoptosis, and inhibiting proliferation, oxidative stress,
and angiogenesis. These findings support further translational evaluation of SF as a potential therapeutic agent for HCC.
Keywords: hepatocellular carcinoma, sulforaphane, Bax, Bcl-2, VEGF, PCNA, p53, oxidative stress, mice.
https://doi.org/10.15407/exp-oncology.2026.02.120
https://doi.org/10.15407/exp-oncology.2026.02.120
https://creativecommons.org/licenses/by-nc-nd/4.0/
https://doi.org/10.15407/exp-oncology.2026.02.120
mailto:kholoudomran_p@sci.asu.edu.eg
mailto:shafaamrschool@hotmail.com
ISSN 1812-9269. Experimental Oncology 48 (2). 2026 121
Sulforaphane Suppresses Hepatocellular Carcinoma in Mice via Activating p53, Mitigating Oxidative Stress
for advanced HCC [6]. However, drug resistance
within six months of initiating the regimen [5, 7],
intolerable adverse effects, and high cost remain
the obstacles to effective chemotherapy [8]. These
challenges have encouraged many researchers to
discover and develop natural product-derived sub-
stances for treating cancer with higher therapeutic
efficacy, lower cost, and fewer side effects com-
pared to chemical drugs [9, 10]. Sulforaphane (SF)
is one of the most extensively studied isothiocya-
nates and has been frequently examined for its an-
ticancer effects [11—13]. SF is widely recognized
as a promising natural chemopreventive agent both
in vitro and in vivo against many cancers, including
cervical, breast, bladder, renal cell, lung, prostate,
and colon cancers [14—18], as well as hepatic can-
cer [17, 19]. SF also exhibits apoptotic, antioxidant
[20], and anti-inflammatory effects [21], suppor
ting its potential as a candidate in cancer chemo-
prevention [22, 23]. In addition to its putative be
neficial pharmacological effects and efficacy, SF is
non-toxic and has no side effects [24—26].
Despite the extensive published work on the
beneficial effects of SF, studies carried out on its ef-
fectiveness against HCC are rare. Previous studies
evaluating the anticarcinogenic effect of SF in liver
cells were conducted mostly in vitro [27, 28]. Ho
wever, those carried out in experimental animals
studied the prophylactic effects on the early stages
of HCC [29] or transplanted tumors in nude mice
[30]. Therefore, the present work was designed to
evaluate the anticarcinogenic effect of SF against
advanced HCC in vivo and estimate possible un-
derlying mechanisms of cell proliferation, angio-
genesis, and antioxidant status.
Materials and Methods
Chemicals. Diethylnitrosamine was sourced from
Sigma–Aldrich (USA) at a concentration of 0.95 g / mL
(Sigma N0258-1G). Carbon tetrachloride was ac-
quired from Research Lab (Egypt), identified by se-
rial #02076 and batch #02076170415. DL-SF, with a
purity of ≥90% (HPLC), in liquid form, was pro-
cured from Sigma–Aldrich (USA) under the code
S4441-5MG, with CAS number 4478-93-7. Kits for
assessing serum alanine aminotransferase (ALT;
catalog # 264001), aspartate aminotransferase (AST;
catalog # 260001), albumin (Alb; E-EL-R0362),
and total protein (TP; catalog # MBS9389057) were
obtained from SPINREACT, S.A./S.A.U. (Spain).
The serum AFP ELISA kit was sourced from
CUSABIO (USA). Liver malondialdehyde (MDA;
Cat. # MD2529) and total antioxidant capacity
(TAC; Cat. # LS-F14457) were analyzed by the
manufacturer’s protocols using commercially avail-
able kits. All the other chemicals utilized were of
analytical grade.
Animals. Forty male albino mice, aged six weeks
and weighing 19—25 g, were sourced from the
Biological Unit of Theodore Bilharz Institute in
Giza, Egypt. These mice belonged to the CD1
strain. Before commencing the experiments, a one-
week acclimatization period was allowed in the
laboratory setting. Throughout the study, the mice
had ad libitum access to water and standard meal
pellets. The animal experiments adhered strictly to
the guidelines outlined in the National Institutes of
Health Guide for the care and use of laboratory an-
imals (NIH Publications No. 8023, revised 1985).
Induction of HCC. The method for inducing
HCC followed the protocol described by Uehara
et al. [31]. Initially, a single intraperitoneal injection
of diethylnitrosamine (DEN) at a dose of 100 mg/kg,
freshly prepared and diluted in sterile 0.9% sodium
chloride saline, was administered. Subsequently, to
potentiate the carcinogenic effects of DEN, rats re-
ceived biweekly intraperitoneal injections of carbon
tetrachloride (CCl4) for 22 weeks. CCl4 was admin-
istered at a dose of 0.5 mL/kg, diluted in sterile corn
oil, following the approach outlined by Dapito et al.
[32] and Tolba et al. [33].
Experimental design. The mice were randomly
divided into three groups: a negative control group
receiving the vehicle (n = 10), an HCC control group
(n = 15), and a group treated with SF (2 mg / kg body
weight) (n = 15), which commenced treatment
from week 25 until week 28 following HCC induc-
tion. This dose and schedule were selected based
on preliminary dose-finding studies identifying
2 mg/kg as the minimum effective dose, consistent
with previous literature demonstrating that SF at
this dose exerts anticarcinogenic effects without
observable toxicity in murine models [34]. Treat-
ment initiation after HCC establishment was de-
signed to model a therapeutic, rather than purely
preventive, intervention.
Sample collection. After the 28-week experi-
ment, the mice were weighed before being eutha-
nized via isoflurane inhalation. Following a 12- to
122 ISSN 1812-9269. Experimental Oncology 48 (2). 2026
K.A. Omran, Y.I. Mahmoud, A.A. Mahmoud, N.H. Fares
14-h fasting period, blood samples were collected
from the retroorbital vein of each mouse. These
samples were allowed to clot at room temperature
and then centrifuged at 6000 g for 15 min at 4 °C to
obtain sera, which were subsequently stored at
–20 °C for future biochemical analyses. After blood
sampling, the animals were humanely sacrificed,
and their livers were promptly excised, washed with
saline, and dried with blotting paper. Liver weights
were recorded, and any visible lesions were noted.
Liver sections were preserved in a 10% neutral
buffered formalin solution for histological and im-
munohistochemical examinations.
Biochemical assays. Serum levels of ALT, AST,
total bilirubin, and albumin, as well as MDA and
total antioxidant capacity (TAC), were determined
following the guidelines provided by the respective
kit manufacturers. Quantification of serum alpha-
fetoprotein (AFP), a specific tumor marker for
HCC, was performed using the enzyme-linked im-
munosorbent assay (ELISA) technique.
Histopathological estimation. The left median
lobe liver samples were fixed in 10% neutral buf
fered formalin for 24 h. Subsequently, they under-
went overnight rinsing under running tap water
and were then dried before being embedded in pa
raffin wax. Transverse sections, 5 μm thick, were
prepared and stained using hematoxylin and eosin
according to the method described by Gamble [35].
Immunohistochemistry. Hepatic tissue sections
were processed conventionally for analysis. The fol-
lowing antibodies were employed: monoclonal
mouse p53 antibody (IR616) at a dilution of 1:100
(DO-7, Dako, Denmark), rabbit monoclonal Bcl‑2
antibody at a dilution of 1:250 (E17, ab32124, Abcam,
UK), rabbit at polyclonal Bax a dilution of 1:250 (Sc-
526, Santa Cruz Biotechnolog y, U S A ) ,
rabbit monoclonal PCNA antibody (EPR3821) at
a dilution of 1:100 (Abcam, UK), and mouse
monoclonal VEGF antibody (A17877) at a dilu-
tion of 1:150 (AB clonal, China). Immunohisto-
chemical (IHC) analysis was performed following
the instructions provided with each kit. Staining
Fig. 1. Effect of SF on survival of mice with HCC (Ka-
plan–Meier plots)
Fig. 2. Effect of SF on general appearance of experimental mice. a — normal; b — HCC con-
trol; c — HCC animals treated with SF
ISSN 1812-9269. Experimental Oncology 48 (2). 2026 123
Sulforaphane Suppresses Hepatocellular Carcinoma in Mice via Activating p53, Mitigating Oxidative Stress
intensity was assessed semi-quantitatively using a
scale ranging from 0 to 3: 0 indicating no staining,
1 indicating weak staining, 2 indicating moderate
staining, and 3 indicating strong staining.
Statistical analysis. The mean values and stan-
dard errors of the means were used to represent nu-
merical data. The statistical analyses were conducted
using GraphPad Prism software (version 5.0, Graph-
Pad Software, USA). Data were initially subjected to
one-way analysis of variance (ANOVA) followed by
post hoc multiple comparisons using Tukey’s test to
compare between groups. A significance level of
p < 0.05 was considered statistically significant.
Results
Survival rate. No mortality was observed among
mice in the negative control group throughout the
experiment. In contrast, the HCC control group ex-
hibited a substantial mortality rate, reaching 72.2%
by week 28. However, HCC animals treated with SF
from week 25 to week 28 demonstrated a lower
mortality rate of 50% compared to the HCC control
group (Fig. 1). The Kaplan–Meier survival analysis
showed that SF treatment significantly improved
survival compared to the HCC control group, with
50% of SF-treated mice surviving to week 28 com-
pared to only 27.8% of HCC controls (log-rank test,
p < 0.05). Mortality was primarily attributed to tu-
mor progression based on clinical signs (ascites,
weakness, hair loss, and weight loss), with no tox-
icity-related deaths observed.
Clinical symptoms. Mice in the negative control
group displayed activity and had white, soft fur,
maintaining a steady increase in body weight over
the 28-week experiment period (Fig. 2, a). Con-
versely, the HCC control mice showed signs of
physical inactivity, with light, rough fur that ap-
peared slightly yellowish. Ascites and soft, yello
wish stool were also observed in this group (Fig. 2, b).
Additionally, the weights of mice in the HCC con-
trol group significantly decreased compared to
those in the negative control group (Fig. 3). In con-
trast, SF-treated HCC mice exhibited activity, had
white, soft fur, and showed slight abdominal edema
(Fig. 2, c). Interestingly, the weights of mice in this
group did not significantly decrease compared to the
HCC control group. Fig. 3 illustrates the average
body weight changes observed during the 28‑week
study across the groups.
Gross morphology of the liver. As depicted in
Fig. 4, in the negative control group, the livers dis-
played a normal appearance, size, and color, devoid
of any tumor signs. Conversely, the livers from the
HCC control group exhibited hepatomegaly (as in-
dicated in Table 1), characterized by a light brown
color and rough surface. The liver surfaces exhibi
ted the emergence of multiple nodules of varying
sizes. However, treatment of HCC mice with SF re-
Fig. 3. Effect of SF on body weight change in HCC mice.
The differences are significant compared to the normal
group: † p < 0.05. The differences are significant compared
to the HCC control group: * p < 0.05
Fig. 4. Effect of SF on liver gross morphology in experimental mice: a — negative control group, b — HCC control
group, c — HCC SF-treated group
124 ISSN 1812-9269. Experimental Oncology 48 (2). 2026
K.A. Omran, Y.I. Mahmoud, A.A. Mahmoud, N.H. Fares
sulted in a significant reduction in liver relative
weight. Furthermore, the liver surface of the HCC
SF-treated group displayed a finer granular appea
rance with few small nodules (Fig. 4).
Biochemical parameters. HCC mice exhibited a
notable rise in the serum levels of AST, ALT, total
bilirubin, and AFP compared to the negative con-
trol group (Table 2). Conversely, albumin levels
were significantly reduced in HCC mice compared
to the negative control. Treatment of HCC mice
with SF resulted in a significant elevation in albu-
min levels compared to the HCC control group.
Furthermore, serum levels of ALT, AST, total bili-
rubin, and AFP were significantly reduced in the
SF-treated HCC group compared to the HCC cont
rol group (Table 2).
Total antioxidant capacity and lipid peroxida-
tion. The HCC control group exhibited a notably
significant increase in hepatic MDA levels compared
to the negative control group, while in the SF-treated
group, there was a significant decrease in MDA le
vels compared to the HCC control group (Fig. 5).
Additionally, HCC mice displayed a highly signifi-
cant decrease in liver TAC concentration compared
to the negative control group, whereas treatment
with SF significantly increased liver TAC concentra-
tion compared to the HCC control group (Fig. 6).
Liver histology. The evaluation of hematoxylin and
eosin-stained liver sections from a negative control
mouse revealed normal architecture characterized by
indistinct polygonal lobules (Fig. 7, a). Each lobule
exhibited a central vein and radiating hepatic strands
separated by sinusoids, containing one- to two-cell-
thick hepatocytes. The hepatocytes were polygonal in
shape with granular and eosinophilic cytoplasm, of-
ten with rounded, binucleated nuclei. The sinusoids
were lined with endothelial cells and contained scat-
tered Kupffer cells (Fig. 7, g). Conversely, the liver
architecture in the HCC control group exhibited sig-
nificant alterations, including the emergence of nodu
les and new blood vessels. The hepatic tissue lacked
well-defined lobules and cell plates, being invaded by
Table 1. Effect of SF onrelative liver weight and hepatic
nodules in HCC-bearing mice
Group Liver relative weight
Nodular count
Total nodules < 2 mm 2—5 mm > 5 mm
Normal 3.94 ± 0.09 0 0 0 0
HCC control 8.94 ± 0.47†† 26.44 ± 6.44†† 15.89 ± 4.1†† 9.22 ± 2.62†† 1.22 ± 0.4††
HCC + SF 5.8 ± 0.24** 9.44 ± 0.51* 7.0 ± 0.4 2.67 ± 0.63* 0.33
Notes: The differences are significant compared to the negative control group: †† p < 0.001. The differences are significant
compared to the HCC control group: * p < 0.05, ** p < 0.001.
Table 2. Effect of SF on serum ALT, AST, albumin,
total bilirubin, and AFP in HCC-bearing mice
Group ALT
(IU/mL)
AST
(IU/mL)
Albumin
(μg/mL)
Total bilirubin
(µmol/mL)
AFP
(ng/mL)
Negative control 52.60 ± 6.94 83.8 ± 2.36 152.09 ± 11.61 7.48 ± 0.85 1.9 ± 0.51
HCC control 116.71 ± 9.45† 127.6 ± 13.08† 55.14 ± 7.22†† 39.5 ± 2.91†† 14.17 ± 1.68†
HCC + SF 75.5 ± 4.6* 80.4 ± 6.46* 119.95 ± 8.7** 12.98 ± 2.01** 6.75 ± 0.77*
Notes: The differences are significant compared to the negative control group: † p < 0.05, †† p < 0.001. The differences are
significant compared to the HCC control group: * p < 0.05, ** p < 0.001.
Fig. 5. Effect of SF on MDA in normal and HCC-bearing
mice. The differences are significant compared to the nor-
mal group: †† p < 0.001. The differences are significant com-
pared to the HCC control group: * p < 0.05
ISSN 1812-9269. Experimental Oncology 48 (2). 2026 125
Sulforaphane Suppresses Hepatocellular Carcinoma in Mice via Activating p53, Mitigating Oxidative Stress
unpaired arteries (Fig. 7, b). The hepatic strands ap-
peared disorganized and collapsed, with compressed
sinusoids, portal veins, and inflammatory infiltrates
(Fig. 7, e). Hepatocytes displayed well-differentiated
features with eosinophilic cytoplasm, pleomorphic
nuclei often exhibiting irregular membranes, dividing
nuclei, and prominent nucleoli. Sinusoids appeared
obliterated (Fig. 7, h). In contrast, the liver in the
HCC group treated with SF displayed a partially
restored architecture, with peripheral nodules
Fig. 6. Effect of SF on TAC in normal and HCC-bearing
mice. The differences are significant compared to the nor-
mal group: †† p < 0.001. The differences are significant
compared to the HCC control group: * p < 0.05
Fig. 7. Effect of SF on liver pathology in HCC-bearing mice: a, d, g — normal mice; b, e, h — HCC control; c, f, i — HCC
treated with SF 2 mg/kg
126 ISSN 1812-9269. Experimental Oncology 48 (2). 2026
K.A. Omran, Y.I. Mahmoud, A.A. Mahmoud, N.H. Fares
observed. The hepatic tissue appeared divided into
indistinct lobules, with portal veins exhibiting various
orientations and compressed central veins (Fig. 7, c).
Normal lobules with dilated portal veins and intense
inflammation were observed, while central veins
were challenging to identify (Fig. 7, f). Hepatocytes
exhibited granulated cytoplasm, slightly compressed
sinusoids, inflammatory infiltrate, and occasional
apoptotic hepatocytes (Fig. 7, i).
Immunohistochemical study. IHC studies were
conducted to assess the expression of p53, Bax, Bcl-2,
PCNA, and VEGF in hepatic tissues, aiming to evalu-
ate the effects of SF treatment (Fig. 8, Table 3). In the
negative control group, p53 expression was barely de-
tectable in the nuclei of hepatocytes, while a mild ex-
pression of p53 was observed in hepatocytes of the
HCC-SF-treated group, compared to the dense ex-
pression in the HCC group (Fig. 8, Table 3). The liv-
er sections obtained from the negative control and
HCC mice displayed weak to moderate Bax reac-
tions, respectively, whereas in the HCC-SF-treated
group, the expression of Bax was strong (Fig. 8,
Table 3). Conversely, Bcl-2 expression was weak in the
HCC-SF-treated group and moderate in HCC mice
(Fig. 8, Table 3). Strong positive nuclear expression of
PCNA was observed in the HCC control group, while
the immunoreaction was weak in the negative control
group and mild in the HCC SF-treated group (Fig. 8,
Table 3). VEGF expression was restricted and weak
along the sinusoids and portal vessels in the negative
control group. In contrast, VEGF was diffused in
strong positive immunoreactivity along with the he-
patocytic cytoplasm in the HCC control group. Weak
immunoreactivity was observed in the HCC SF-treat-
ed group along the sinusoids, portal vessels, and some
hepatocytes (Fig. 8, Table 3).
Discussion
The current study investigated the potential anti-
carcinogenic effects of SF against experimental
HCC. The findings revealed that SF enhanced sur-
vival rates, significantly reduced nodular counts
and AFP levels, improved hepatic biomarkers, and
mitigated the histopathological manifestations of
HCC. Although the precise mechanisms under
lying SF’s antitumor effects on HCC remain par-
tially elucidated, previous research suggests that SF
exerts inhibitory effects on cancer cell growth and
metastasis through various pathways. These path-
ways include anti-inflammatory, proapoptotic, and
cell cycle-arresting actions [36—39]. SF has been
shown to induce DNA fragmentation and activate
caspase families, indicating that its antiproliferative
activity primarily involves apoptosis [40]. More-
over, SF has been demonstrated to suppress cell
proliferation and growth by inhibiting transfor
ming growth factor-beta-induced epithelial-mes-
enchymal transition in HCC [19].
SF is well known for its antioxidant activity [39,
41] and ability to safeguard DNA from degradation
caused by highly reactive electrophiles, achieved by
enhancing the antioxidant system’s activity and in-
hibiting inflammation [24, 42]. Its antioxidant
properties are associated with several pathways, in-
cluding attenuation of inflammation by reducing
mRNA expression of interleukin-6 and interleu-
kin-1 in liver tissue [43—46]. This effect is medi-
ated through the inhibition of nuclear factor kappa
B and the upregulation of transcription of Nrf2, a
critical factor in maintaining cellular health and
protection against toxic chemicals and lifestyle-re-
lated factors [36, 42, 47, 48]. SF’s impact on the
Keap1/Nrf2/ARE pathway contributes to its protec-
tive properties in various disease models, including
liver diseases and cancer [24, 49—52].
Beyond Nrf2, a variety of transcriptional factors
are affected by SF through oxidative stress modula-
tion, including NF-κB, activator protein 1, p53, hy-
poxia-inducible factor 1-alpha, peroxisome prolif
erator-activated receptor-γ, and β-catenin/Wnt [53].
As an electrophile, SF can react with protein thiols to
form thionoacyl adducts, affecting cysteine residues
in the Keap1 protein. This interaction disrupts Nrf2-
Keap1 binding and allows nuclear accumulation of
Nrf2 [54]. The resulting Nrf2 activation enhances the
transcription of antioxidant response elements, ex-
plaining the observed reduction in oxidative stress
markers in our study. Concurrently, SF inhibits NF-
κB activation [36, 42] and reduces VEGF expression
[40], contributing to the anti-inflammatory and anti-
angiogenic effects observed in our HCC model.
A balanced interpretation of SF’s antioxidant ef-
fects requires consideration of the dual role of reac-
tive oxygen species (ROS) in cancer biology. SF ex-
hibits a seemingly contradictory dual role: it induces
mitochondrial dysfunction and apoptosis in cancer
cells while protecting normal cell mitochondria
from oxidative damage [55]. This biphasic behavior,
mediated partly through the Nrf2-dependent mito-
ISSN 1812-9269. Experimental Oncology 48 (2). 2026 127
Sulforaphane Suppresses Hepatocellular Carcinoma in Mice via Activating p53, Mitigating Oxidative Stress
Fig. 8. Immunostaining for p53, Bax, Bcl-2, PCNA, and VEGF in the liver of normal and HCC-bearing mice
128 ISSN 1812-9269. Experimental Oncology 48 (2). 2026
K.A. Omran, Y.I. Mahmoud, A.A. Mahmoud, N.H. Fares
chondrial biogenesis and differential ROS thresholds
for cell death [55], suggests that the antitumor effects
observed in our HCC model cannot be attributed
solely to ROS scavenging. Furthermore, SF has been
shown to inhibit HCC cell proliferation and epithe-
lial-mesenchymal transition through ROS-depen-
dent pathways [19], indicating that the therapeutic
window for antioxidant-based strategies requires
careful evaluation in future dose-response studies.
Our findings suggest that treatment of HCC-bearing
mice with SF restored the reduced TAC activity and
prevented lipid peroxidation of hepatocyte cell
membranes, indicating SF’s antioxidant potential
against HCC-related oxidative stress. This aligns
with prior studies by Chi et al. [53] and Kikuchi et
al. [42], which demonstrate SF’s effectiveness as an
antioxidant by reducing MDA levels and inducing
antioxidant enzymes such as glutathione S-transfer-
ase, superoxide dismutase, glutathione, and glutathi-
one peroxidase. The antioxidant effect of SF may
prompt apoptosis in HCC animals [10, 56—58], sug-
gesting a potential link between SF’s anticarcinogen-
ic effect and apoptosis induction. This notion is sup-
ported by increased IHC expression of p53 and Bax,
along with a decrease in Bcl-2 observed in SF-treat-
ed mice in our study, consistent with findings by
Kaboli et al. [59]. Additionally, SF downregulates
PCNA expression in HCC mice, similar to the previ-
ous reports by Kaboli et al. [59] and Rai et al. [60].
Earlier studies have indicated SF’s anti-angiogenic
effect on HCC by reducing VEGF levels [40, 59].
Furthermore, Liu et al. [61] demonstrated that SF
interferes with endothelial cell proliferation, migra-
tion, and tube formation, inhibiting the pro-angio-
genic effect of HepG2 cells both in vitro and in vivo.
SF affects the interaction between HCC cells and en-
dothelial cells by inhibiting signal transducer and
activator of transcription HIF1-alpha/VEGF signal-
ing in cancer cells, thereby suppressing HCC-in-
duced angiogenesis and exerting an anti-tumor ef-
fect. Our study also showed a decrease in the IHC
expression of VEGF in HCC animals treated with SF,
further supporting SF’s anti-angiogenic activity.
In our study, SF demonstrated a hepatoprotec-
tive effect by reducing elevated serum levels of AST,
ALT, and total bilirubin while increasing albumin
levels after 30 days of therapy in mice with induced
HCC. These results align with prior investigations
conducted by Lee et al. [37], Abdelhamid et al. [62],
and Guan et al. [39]. SF also improved liver archi-
tecture and reduced the number of nodules in HCC
mice, aligning with the findings from Abdelhamid
et al. [62], who observed an enhanced liver archi-
tecture following SF treatment in DEN-induced he-
patic damage, attributing it to the suppression of
thiobarbituric acid reactive substances and induc-
tion of hepatic phase 2 antioxidant enzymes, in-
cluding glutathione S-transferase, along with the
modulation of inflammatory pathways.
Although SF-treated mice exhibited lower body
weight compared to the untreated HCC control mice
in our experiment, they were more active. This de-
crease in body weight may be attributed to SF’s up-
regulation of lipid metabolism-related enzymes/pro-
teins, inducing adipocyte lipolysis and inhibiting ad-
ipocyte differentiation, as suggested by Du et al. [63].
Ferramosca et al. [64] and Du et al. [63] further dem-
onstrated SF’s ability to improve lipid profiles and de-
crease body weight, liver weight, and lipid profile pa-
rameters such as total cholesterol, triglycerides, low-
density lipoprotein, and cholesterol in rodents.
One of our study limitations is that the IHC
data are semi-quantitative; quantitative methods
such as Western blotting or qPCR would provide
more precise validation of the observed changes
in p53, Bax, Bcl-2, PCNA, and VEGF expressions.
Additionally, only a single dose of SF (2 mg/kg)
was used. Future studies incorporating multiple
dosing regimens are needed to characterize dose-
response relationships and identify an optimal
therapeutic dose of SF for HCC.
In summary, SF exhibited an anticarcinogenic ef-
fect in the HCC model, characterized by tumor re-
Table 3. Effects of SF on the IHC expression
of marker proteins in hepatic tissues
of experimental animals
Protein
Experimental groups
Negative control HCC HCC + SF
p53 0.48 ± 0.09 2.13 ± 0.15† 0.64 ± 0.13*
VEGF 0.26 ± 0.08 2.09 ± 0.18† 0.81 ± 0.16*
PCNA 0.39 ± 0.09 2.81 ± 0.07† 1.64 ± 0.14†
Bax 0.59 ± 0.11 1.61 ± 0.17† 2.48 ± 0.15†
Bcl-2 0.29 ± 0.08 1.90 ± 0.13† 0.87 ± 0.19†
Notes: Data are expressed as mean ± SEM (n = 6/group).
0 — negative; 1 — weak; 2 — moderate; 3 — strong
reaction. The mean value was significantly different from
that of the negative control group: † p < 0.05, or from the
HCC control group: * p < 0.05.
ISSN 1812-9269. Experimental Oncology 48 (2). 2026 129
Sulforaphane Suppresses Hepatocellular Carcinoma in Mice via Activating p53, Mitigating Oxidative Stress
gression, increased survival rate, suppression of AFP,
improvement of liver biomarkers, and downstaging
of HCC pathology. SF’s anticarcinogenic effect was
mediated by restoring antioxidant capacity, activating
p53 expression, increasing Bax levels, and suppres
sing PCNA, VEGF, Bcl-2, and oxidative stress. SF, as
a natural product with antioxidant and angiogenic
properties, holds promise as a treatment candidate
for HCC. Clinical translation of these findings re-
quires consideration of SF’s limited oral bioavailabi
lity and appropriate human equivalent dosing. How-
ever, further studies are warranted to fully elucidate
the mechanisms underlying the anticarcinogenic and
liver-regenerating effects of SF at the molecular level.
Credit authorship
contribution statement
Kholoud A. Omarn and Yomna I. Mahmoud con-
ceived and designed the study. Kholoud A. Omarn
did the laboratory work and wrote the manuscript.
Yomna I. Mahmoud reviewed the manuscript. As-
maa A. Mahmoud analyzed the data and reviewed
the manuscript. Nagui H. Fares is the main super-
visor and reviewed the manuscript. This study was
performed as part of Kholoud A. Omarn’s disserta-
tion for the degree of PhD in histology. All authors
read and approved the final manuscript.
Ethical approval
Animal handling procedures were conducted fol-
lowing the ethical guidelines set by the Ethical
Committee of Ain Shams University (REC), under
approval number ASU-SCI/ZOOL/2024/5/1.
These procedures were also compliant with the
National Institutes of Health Guide for the Care
and Use of Laboratory Animals (NIH Publications
No. 8023, revised 1978). The research was carried
out at the Faculty of Science, Ain Shams Univer-
sity, Abbassia, Cairo, Egypt.
Funding
This research did not receive any specific grant
from funding agencies in the public, commercial,
or not-for-profit sectors.
Data availability
The datasets used and analyzed during the current
study are available from the corresponding author
upon reasonable request. All data analyzed during
this study are included in this article.
Declaration of competing interests
The authors declare no conflicts of interest.
Acknowledgment
The authors would like to acknowledge the De-
partment of Zoology and the Department of En-
tomology for providing laboratory space and
types of equipment for the achievement of the
current work.
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Submitted: April 22, 2026
Холуд А. Омран, Йомна І. Махмуд,
Асмаа А. Махмуд, Нагуї Х. Фарес
Факультет природничих наук,
Університет Айн Шамс, Каїр, Єгипет
СУЛЬФОРАФАН ПРИГНІЧУЄ ГЕПАТОЦЕЛЮЛЯРНУ
КАРЦИНОМУ У МИШЕЙ ШЛЯХОМ АКТИВАЦІЇ P53,
ЗМЕНШЕННЯ ОКСИДАТИВНОГО СТРЕСУ
ТА ІНГІБУВАННЯ АНГІОГЕНЕЗУ
Стан питання. Гепатоцелюлярна карцинома (ГЦК) є найпоширенішим первинним злоякісним новоутворенням
печінки. Сульфорафан (SF) — це природний ізотіоціанат, який проявляє активність проти різних типів злоякіс-
них клітин in vivo та in vitro без особливих побічних ефектів. Він також має профілактичний ефект стосовно
ранніх стадій розвитку пухлин на деяких моделях раку, включаючи ГЦК. Метою дослідження було оцінити тера-
певтичний протираковий ефект SF на експериментальній моделі індукованої ГЦК у мишей in vivo та дослідити
можливі основні механізми його впливу на клітинну проліферацію, ангіогенез та антиоксидантний статус. Мате-
ріали та методи. ГЦК індукували в мишей одноразовою внутрішньочеревною дозою 100 мг/кг діетилнітрозаміну,
з наступними 22 щотижневими внутрішньочеревними дозами 0,5 мг/кг чотирихлористого вуглецю. Після індук-
ції ГЦК SF (2 мг/кг маси тіла) застосовували перорально з 25-го по 28-й тиждень. Результати. SF дещо нормалі-
зував структуру та функцію печінки в мишей з індукованою ГЦК, покращивши виживаність, значно зменшивши
експресію пухлинного маркера альфа-фетопротеїну та кількість пухлинних фокусів у печінці, при цьому ГЦК
набувала ознак, що відповідали меншому ступеню прогресії. Ці зміни супроводжувалися збільшенням загальної
антиоксидантної здатності, імуноекспресії р53 та Bax, а також значним зниженням перекисного окислення ліпідів
та імунореактивності PCNA, VEGF та Bcl-2 у тканинах печінки. Висновок. SF зменшив кількість пухлинних фо-
кусів у печінці, спричинивши морфологічні зміни, що відповідали меншому ступеню прогресії ГЦК, активуючи
білок-супресор пухлинного росту p53, індукуючи апоптоз та пригнічуючи проліферацію, оксидативний стрес та
ангіогенез. Все це свідчить про необхідність подальшої трансляційної оцінки SF як потенційного терапевтичного
засобу для ГЦК.
Ключові слова: гепатоцелюлярна карцинома, сульфорафан, Bax, Bcl-2, VEGF, PCNA, p53, оксидативний стрес,
миші.
https://doi.org/10.1016/j.eujim.2019.05.002
https://doi.org/10.1016/j.eujim.2019.05.002
https://doi.org/10.1080/13510002.2020.1739870
https://doi.org/10.1016/j.biopha.2019.109635
https://doi.org/10.3390/cancers12051273
https://doi.org/10.1038/s41598-017-12855-w
https://doi.org/10.1038/s41598-017-12855-w
https://doi.org/10.21608/blj.2021.180497
https://doi.org/10.21608/blj.2021.180497
https://doi.org/10.1038/s41598-021-87367-9
https://doi.org/10.3748/wjg.v23.i23.4146
|
| id | oai:ojs2.ex.aqua-time.com.ua:article-644 |
| institution | Experimental Oncology |
| issn | 2312-8852 |
| keywords_txt_mv | |
| language | English |
| last_indexed | 2026-08-22T01:00:34Z |
| publishDate | 2026 |
| publisher | PH Akademperiodyka |
| record_format | ojs |
| resource_txt_mv | exp-oncologycomua/f0/2154bbe98247cbf50f3c57ad0c3088f0.pdf |
| spelling | oai:ojs2.ex.aqua-time.com.ua:article-6442026-08-21T12:36:18Z SULFORAPHANE SUPPRESSES HEPATOCELLULAR CARCINOMA IN MICE VIA ACTIVATING p53, MITIGATING OXIDATIVE STRESS, AND INHIBITING ANGIOGENESIS СУЛЬФОРАФАН ПРИГНІЧУЄ ГЕПАТОЦЕЛЮЛЯРНУ КАРЦИНОМУ У МИШЕЙ ШЛЯХОМ АКТИВАЦІЇ P53, ЗМЕНШЕННЯ ОКСИДАТИВНОГО СТРЕСУ ТА ІНГІБУВАННЯ АНГІОГЕНЕЗУ A. Omran, Kholoud I. Mahmoud, Yomna A. Mahmoud, Asmaa H. Fares, Nagui гепатоцелюлярна карцинома, сульфорафан, Bax, Bcl-2, VEGF, PCNA, p53, оксидативний стрес, миші hepatocellular carcinoma, sulforaphane, Bax, Bcl-2, VEGF, PCNA, p53, oxidative stress, mice Background. Hepatocellular carcinoma (HCC) is the most common primary liver malignancy. Sulforaphane (SF) is a natural isothiocyanate that exhibits anticarcinogenic activity against various cancer cells in vivo and in vitro, with no observed side effects. It also has a prophylactic effect against early stages of some cancer models, including HCC. The study aimed to assess the therapeutic anticarcinogenic effect of SF in experimental murine HCC in vivo and to estimate possible underlying mechanisms of its effects on cell proliferation, angiogenesis, and antioxidant status. Materials and Methods. HCC was induced in mice by a single intraperitoneal dose of 100 mg/kg of diethylnitrosamine, followed by 22 weekly intraperitoneal doses of 0.5 mg/kg of carbon tetrachloride. After the induction of HCC, SF (2 mg/kg body weight) was given orally from weeks 25 to 28. Then we conducted histopathological examination, biochemical analysis of blood sera, and immunohistochemical analysis of marker expression in liver sections. Results. SF improved liver structure and function in HCC-bearing mice and survival rate, significantly reduced the expression of tumor marker alpha-fetoprotein and the number of hepatic nodules, and downstaged HCC. These changes were accompanied by an increase in total antioxidant capacity, expression of p53 and Bax, as well as a significant decrease in lipid peroxidation and immunoreactivity of PCNA, VEGF, and Bcl-2 in liver tissues. Conclusion. SF decreased the number of tumor nodules and ameliorated HCC by activating p53 expression, inducing apoptosis, and inhibiting proliferation, oxidative stress, and angiogenesis. These findings support further translational evaluation of SF as a potential therapeutic agent for HCC. Стан питання. Гепатоцелюлярна карцинома (ГЦК) є найпоширенішим первинним злоякісним новоутворенням печінки. Сульфорафан (SF) — це природний ізотіоціанат, який проявляє активність проти різних типів злоякісних клітин in vivo та in vitro без особливих побічних ефектів. Він також має профілактичний ефект стосовно ранніх стадій розвитку пухлин на деяких моделях раку, включаючи ГЦК. Метою дослідження було оцінити терапевтичний протираковий ефект SF на експериментальній моделі індукованої ГЦК у мишей in vivo та дослідити можливі основні механізми його впливу на клітинну проліферацію, ангіогенез та антиоксидантний статус. Матеріали та методи. ГЦК індукували в мишей одноразовою внутрішньочеревною дозою 100 мг/кг діетилнітрозаміну, з наступними 22 щотижневими внутрішньочеревними дозами 0,5 мг/кг чотирихлористого вуглецю. Після індукції ГЦК SF (2 мг/кг маси тіла) застосовували перорально з 25-го по 28-й тиждень. Результати. SF дещо нормалізував структуру та функцію печінки в мишей з індукованою ГЦК, покращивши виживаність, значно зменшивши експресію пухлинного маркера альфа-фетопротеїну та кількість пухлинних фокусів у печінці, при цьому ГЦК набувала ознак, що відповідали меншому ступеню прогресії. Ці зміни супроводжувалися збільшенням загальної антиоксидантної здатності, імуноекспресії р53 та Bax, а також значним зниженням перекисного окислення ліпідів та імунореактивності PCNA, VEGF та Bcl-2 у тканинах печінки. Висновок. SF зменшив кількість пухлинних фокусів у печінці, спричинивши морфологічні зміни, що відповідали меншому ступеню прогресії ГЦК, активуючи білок-супресор пухлинного росту p53, індукуючи апоптоз та пригнічуючи проліферацію, оксидативний стрес та ангіогенез. Все це свідчить про необхідність подальшої трансляційної оцінки SF як потенційного терапевтичного засобу для ГЦК. PH Akademperiodyka 2026-08-21 Article Article application/pdf https://exp-oncology.com.ua/index.php/Exp/article/view/644 10.15407/exp-oncology.2026.02.120 Experimental Oncology; Vol. 48 No. 2 (2026): Experimental Oncology; 120-132 Експериментальна онкологія; Том 48 № 2 (2026): Експериментальна онкологія; 120-132 2312-8852 1812-9269 10.15407/exp-oncology.2026.02 en https://exp-oncology.com.ua/index.php/Exp/article/view/644/472 Copyright (c) 2026 Experimental Oncology https://creativecommons.org/licenses/by-nc-nd/4.0/ |
| spellingShingle | гепатоцелюлярна карцинома сульфорафан Bax Bcl-2 VEGF PCNA p53 оксидативний стрес миші A. Omran, Kholoud I. Mahmoud, Yomna A. Mahmoud, Asmaa H. Fares, Nagui СУЛЬФОРАФАН ПРИГНІЧУЄ ГЕПАТОЦЕЛЮЛЯРНУ КАРЦИНОМУ У МИШЕЙ ШЛЯХОМ АКТИВАЦІЇ P53, ЗМЕНШЕННЯ ОКСИДАТИВНОГО СТРЕСУ ТА ІНГІБУВАННЯ АНГІОГЕНЕЗУ |
| title | СУЛЬФОРАФАН ПРИГНІЧУЄ ГЕПАТОЦЕЛЮЛЯРНУ КАРЦИНОМУ У МИШЕЙ ШЛЯХОМ АКТИВАЦІЇ P53, ЗМЕНШЕННЯ ОКСИДАТИВНОГО СТРЕСУ ТА ІНГІБУВАННЯ АНГІОГЕНЕЗУ |
| title_alt | SULFORAPHANE SUPPRESSES HEPATOCELLULAR CARCINOMA IN MICE VIA ACTIVATING p53, MITIGATING OXIDATIVE STRESS, AND INHIBITING ANGIOGENESIS |
| title_full | СУЛЬФОРАФАН ПРИГНІЧУЄ ГЕПАТОЦЕЛЮЛЯРНУ КАРЦИНОМУ У МИШЕЙ ШЛЯХОМ АКТИВАЦІЇ P53, ЗМЕНШЕННЯ ОКСИДАТИВНОГО СТРЕСУ ТА ІНГІБУВАННЯ АНГІОГЕНЕЗУ |
| title_fullStr | СУЛЬФОРАФАН ПРИГНІЧУЄ ГЕПАТОЦЕЛЮЛЯРНУ КАРЦИНОМУ У МИШЕЙ ШЛЯХОМ АКТИВАЦІЇ P53, ЗМЕНШЕННЯ ОКСИДАТИВНОГО СТРЕСУ ТА ІНГІБУВАННЯ АНГІОГЕНЕЗУ |
| title_full_unstemmed | СУЛЬФОРАФАН ПРИГНІЧУЄ ГЕПАТОЦЕЛЮЛЯРНУ КАРЦИНОМУ У МИШЕЙ ШЛЯХОМ АКТИВАЦІЇ P53, ЗМЕНШЕННЯ ОКСИДАТИВНОГО СТРЕСУ ТА ІНГІБУВАННЯ АНГІОГЕНЕЗУ |
| title_short | СУЛЬФОРАФАН ПРИГНІЧУЄ ГЕПАТОЦЕЛЮЛЯРНУ КАРЦИНОМУ У МИШЕЙ ШЛЯХОМ АКТИВАЦІЇ P53, ЗМЕНШЕННЯ ОКСИДАТИВНОГО СТРЕСУ ТА ІНГІБУВАННЯ АНГІОГЕНЕЗУ |
| title_sort | сульфорафан пригнічує гепатоцелюлярну карциному у мишей шляхом активації p53, зменшення оксидативного стресу та інгібування ангіогенезу |
| topic | гепатоцелюлярна карцинома сульфорафан Bax Bcl-2 VEGF PCNA p53 оксидативний стрес миші |
| topic_facet | гепатоцелюлярна карцинома сульфорафан Bax Bcl-2 VEGF PCNA p53 оксидативний стрес миші hepatocellular carcinoma sulforaphane Bax Bcl-2 VEGF PCNA p53 oxidative stress mice |
| url | https://exp-oncology.com.ua/index.php/Exp/article/view/644 |
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