Estrogen Attenuates Diethylnitrosamine-Induced Hepatocellular Carcinoma in Female Rats via Modulation of Estrogen Receptor/FASN/CD36/IL-6 Axis

Abstract

This study was designed to evaluate the potential protective impact of estrogen and estrogen receptor against diethylnitrosamine (DEN)-induced hepatocellular carcinoma (HCC) in rats. The levels of liver injury serum biomarkers, liver content of interleukin-6 (IL-6), relative liver weight and distortion of liver histological pictures were significantly increased in ovariectomized (OVX) rats and SHAM rats that received DEN alone and were further exaggerated when DEN was combined with fulvestrant (F) compared to non-DEN treated rats. The OVX rats showed higher insults than SHAM rats. The tapering impact on these parameters was clear in OVX rats that received estradiol benzoate (EB), silymarin (S) or orlistat (ORS). The immunohistochemistry and/or Western blot analysis of liver tissues showed a prominent increase in fatty acid synthase (FASN) and cluster of differentiation 36 (CD36) expressions in OVX and SHAM rats who received DEN and/ or F compared to SHAM rats. In contrast to S, treatment of OVX rats with EB mitigated DEN-induced expression of FASN and CD36 in liver tissue, while ORS improved DEN-induced expression of FASN. In conclusion, the protective effect against HCC was mediated via estrogen receptor alpha (ER-α) which abrogates its downstream genes involved in lipid metabolism namely FASN and CD36 depriving the tumor from survival vital energy source. In addition, ORS induced similar mitigating effect against DEN-induced HCC which could be attributed to FASN inhibition and anti-inflammatory effect. Furthermore, S alleviated DEN-induced HCC, independent of its estrogenic effect.

INTRODUCTION

Hepatocellular carcinoma (HCC) is the fifth most commonly diagnosed cancer and many epidemiological studies have identified obesity as a significant risk factor in its development.¹⁾ Although various mechanisms have been proposed to underlie obesity-mediated hepatocarcinogenesis including elevation of proinflammatory cytokines, adipokines dysregulation, oxidative stress, and altered gut flora, the process remains poorly understood. Metabolic reprogramming of cancer cells is crucial to generate energy and nutrients needed for cancer cell activities.²⁾ It is also believed that de novo lipogenesis contributes to the synthesis of membranes and signaling molecules in proliferating cancer cells.³⁾ Moreover, metabolic reprogramming has been disclosed to be controlled by tumor suppressor genes and proto-oncogenes, proposing its primary role in carcinogenesis.⁴⁾

Hepatic lipogenesis may be implicated in liver cancers, as metformin, an oral hypoglycemic agent which inhibits hepatic lipogenesis and gluconeogenesis, markedly decreased the risk of liver cancers in diabetic patients.⁵⁾ In vivo and in vitro reports also revealed that the metformin-mediated inhibition of HCC could be abolished by the overexpression of sterol regulatory element-binding protein-1c (SREBP-1c), a key transcription factor of lipogenic genes.⁶⁾ These findings also reinforce the role of hepatic lipogenesis in HCC. Interestingly, a fructose rich diet, which induces hepatic lipogenesis produced a greater metabolic effect in male than that in females,^7,8) indicating that hepatic lipogenesis has a vital role in male gender-specific pathway in male HCC patients.

HCC is one of the four major liver diseases related to gender disparity that include steatosis,⁹⁾ hepatitis,¹⁰⁾ cirrhosis,¹¹⁾ and HCC.¹²⁾ A study carried out in the United States by Weston et al.¹³⁾ revealed that the incidence of non-alcoholic fatty liver disease (NAFLD) was 3.5 times higher in men than in women. In addition, men are about three to five times more likely to develop HCC than women.¹⁴⁾ This gender disparity is also observed in rat HCC models.^15,16) Also, the development of chemically (diethylnitrosamine (DEN))-induced HCC was inhibited by administration of estrogens to male rat.¹⁵⁾ Nonetheless, the mechanisms participated in this gender disparity and the antitumor activities of estrogens are still unknown.¹⁷⁾

Moreover, postmenopausal females have a higher HCC prevalence than premenopausal ones.¹⁸⁾ Several studies showed that 17β estradiol (E₂) administration markedly diminished the inflammation and injury associated with a chemical carcinogen of diethylnitrosamine.¹⁶⁾ Administration of E₂ to male mice also inhibited the development of diethylnitrosamine-induced HCC.¹⁹⁾ Yang et al.²⁰⁾ suggested that estrogen-attenuated HCC progression, through inhibiting tumor-associated macrophage alternative activation using orthotropic and ectopic mouse models with mouse HCC cell lines. These findings suggest that estrogens may have a suppressive role in the development of human liver cancer. Liver is a hormone sensitive organ that was found to express estrogen receptors (ER) in both male and female patients.²¹⁾ ERs including ER-α and ER-β classically function as transcription factors, shuttling between the cytoplasm and nucleus for regulating the expression of various genes involved in cell cycle, proliferation, apoptosis, and inflammation.²²⁾

Changes in fatty acid (FA) metabolism in cancer cells have received less attention, but recent studies have shown the importance of lipid metabolic reprogramming in tumorigenesis.¹⁾ FAs function as energy sources, signaling molecules, structural components of the cell membrane and storage compounds, all of which are vital for cancer cell survival and proliferation. Normal cells preferentially use circulating exogenous lipids, whereas cancer cells, including HCC cells, show a high rate of de novo lipid synthesis.¹⁾ Hence, enzymes of FA pathways have attracted attention as a potential therapeutic target. Recently, it has been reported that E₂ treatment decreases gene expression of SREBP-1c and its target genes fatty acid synthase (FASN) and cluster of differentiation 36 (CD36) in liver. It is suggested that E₂ directly regulates SREBP-1c, which have an estrogen response element in its promoter region.^23,24)

Interestingly, orlistat, a pancreatic lipase inhibitor, approved by U.S. Food and Drug Administration (FDA) as an anti-obesity drug inhibits FASN, and shows antitumor effects in several types of cancers, such as ovarian cancer.²⁵⁾ It acts as an irreversible inhibitor that forms a covalent adduct with the active serine of the thioesterase domain of FASN and has been shown to halt cell proliferation in several prostate cancer cell lines in vitro and inhibit prostate tumor growth in murine xenografts. Moreover, it promotes apoptosis and reduces proliferation in HER2-overexpressing ovarian and breast cancers.²⁶⁾

Therefore, this study aimed to evaluate the impact of estrogen/estrogen receptor(s), estrogen receptor antagonist (fulvestrant), orlistat and estrogen receptor-β agonist (silymarin) on DEN-induced HCC through their effect on lipid metabolic enzymes and transporters expression with special emphasis on FASN.

MATERIALS AND METHODS

Ethical Approval and Animal Care

The study was performed under the approval of the Institutional Animal Fare and use committee of Fayoum University, and strictly followed the guidelines of the National Institute of Health Guide for the Care and Use of Laboratory Animals. The ethical approval number is AEC-2101.

Two months old adult Wistar albino female rats, weighing approximately 180–200 g were obtained from the animal facilities, Department of Pharmacology and Toxicology, Faculty of Pharmacy, Al-Azhar University. For acclimatization, rats were housed in polyethylene cages for two weeks in controlled housing conditions (room temperature 25±2 °C, humidity (50–70%), and 12/12 h dark–light cycles) and kept free on a standard diet. Rats were given ad libitum access to food and water.

Chemicals and Reagents

Diethylnitrosamine (DEN) (CAS No: 55-18-5) and orlistat powder (CAS No.: 96829-58-2) were purchased from Sigma-Aldrich Co. (St. Louis, MO, U.S.A.). Fulvestrant (250 mg/5 mL 2 prefilled syringes for injection) was purchased from AstraZeneca (London, U.K.). Silymarin was gained from Medical Union Pharmaceuticals Co. (Cairo, Egypt). Estradiol benzoate was obtained from Misr Co. for Pharmaceutical Industries (Cairo, Egypt). All other chemicals and reagents were of the highest commercially available grade.

Experimental Design and Treatment Protocol

Ovariectomy and SHAM Operation

Wistar albino adult female rats were fasted overnights, then anesthetized using thiopental sodium, the bilateral ovariectomy was performed according to the technique established formerly by Lasota and Danowska-Klonowska.²⁷⁾ Under the same condition of ovariectomy, similar placebo surgery (SHAM) was managed without the elimination of the ovaries. Before starting the specified treatment, rats were allowed to recuperate for four weeks with daily wound disinfection by povidone-iodine (Nile for Pharmaceuticals & Chemical Industries Co., Cairo, Egypt).

Treatment Protocol

Sixty-six female virgin albino rats were allocated into 11 groups (n = 6) as follows:

• •   Group 1 (Control): received water for 16 weeks.
• •   Group 2 (SHAM): received water for 16 weeks.
• •   Group 3 (SHAM + DEN): SHAM rats received DEN with drinking water for 8 weeks at 100 mg/L using the gavage technique followed by drinking water only for the next 8 weeks.²⁸⁾
• •   Group 4 (OVX): received water as DEN solvent for 16 weeks.
• •   Group 5 (OVX + DEN): OVX rats received DEN in drinking water for 8 weeks followed by water only for another 8 weeks.²⁸⁾
• •   Group 6 (SHAM + DEN + F): SHAM rats received DEN in drinking water for 8 weeks, followed by water only for another 8 weeks in addition to weekly injection with F (5 mg/kg) for 16 weeks from the start to the end of experiments.²⁹⁾
• •   Group 7 (OVX + DEN + F): OVX rats received DEN with drinking water for 8 weeks, followed by drinking water for another 8 weeks and weekly injection with F (5 mg/kg) for 16 weeks from the start to the end of experiments.²⁹⁾
• •   Group 8 (OVX + DEN + EB): OVX rats received DEN with drinking water for 8 weeks, followed by drinking water for another 8 weeks and subcutaneous weekly injection of E₂ (30 µg/kg) for 16 weeks from the start to the end of experiments.³⁰⁾
• •   Group 9 (OVX + DEN + ORS): OVX rats received ORS (240 mg/kg/d) intraperitoneally for 16 weeks³¹⁾ and DEN with drinking water for 8 weeks, followed by only drinking water for another 8 weeks.
• •   Group 10 (OVX + S): OVX rats received S (50 mg/kg/d) orally for 16 weeks.³²⁾
• •   Group 11 (OVX + DEN + S): OVX rats received S (50 mg/kg/d) orally for 16 weeks and DEN with drinking water for 8 weeks and only drinking water for another 8 weeks.

The body weight of each rat was estimated at first day of the experiment and then at weekly basis before receiving the treatment items all over the experimental duration.

The appropriate dosing of F, EB, S, and ORS was chosen according to the previous studies and were further confirmed based on our introductory pilot experience. Just before the rat’s euthanizing and after general anaesthesia with 50 mg/kg IP pentobarbital (Nembutal injection ^®50 mg/mL, Akorn Pharmaceuticals, U.S.A.), the blood samples from each rat (2 mL) were withdrawn via retro-orbital plexus bleeding technique using heparinized tubes.³³⁾ Subsequently, the rats were euthanized by cervical dislocation technique according to the standard animal euthanasia method guidelines developed by the Canadian Council on Animal Care (CCAC), in 2010. The rat livers were collected, weighed, and macroscopically examined, and then liver’s left lobe was preserved in a 10% buffered formalin solution and processed for immunohistochemical (IHC) and histopathological assessment.

Blood samples were allowed to clot at room temperature for 15–30 min. The sera were separated by centrifugation at 1500 × g for 10 min in a refrigerated centrifuge. Then, the separated sera were stored at −80 °C until used for biochemical analysis.

Evaluation of Liver Status

Determination of Weight Gain and Relative Liver Weight

At time of euthanasia, animals were weighed and compared to their initial corresponding body weight to calculate weight gain. Thereafter, the livers of rats were rapidly removed and weighed to calculate relative liver weight.

Macroscopical Examination

Liver was excised and washed with saline and macroscopically examined; the DEN-induced liver nodules were counted in each animal. The total number of nodules per group was calculated, the number of animal bearing tumor/each group and relative size of nodules were identified.

Assessment of Serum Biomarkers of Liver Injury

An automated clinical chemistry analyzer Spinlab photometer was used after standardization for the quantitative colorimetric assay for estimating the biochemical serum parameters of liver injury using commercial kits for alanine aminotransferase (ALT) (Cat. No. AL 1031 (45)), aspartate aminotransferase (AST) (Cat. No. AS 10 61 (45)), alkaline phosphatase (ALP) (Cat. No. AP 10 20), total bilirubin (T.Bil) (Cat. No. BR 1111) and direct bilirubin (D.Bil) (Cat. No. BR 1112) according to supplier’s instructions (Bio-Med Company, Cairo, Egypt). Moreover, HCC serum marker AFP was calorimetrically estimated using rat AFP enzyme-linked immunosorbent assay (ELISA) Kit (Cat. No. E-EL-R0153) according to supplier’s instructions (Elabscience Company, TX, U.S.A.).

Histopathological Assessment of Liver Tissues

Formalin preserved liver parts from different treatment groups were dehydrated in ascending grades of ethanol, cleared in xylene, embedded in paraffin, and sectioned at a thickness of 4–5 µm. Sections were stained by a combination of hematoxylin–eosin (H&E) according to the previously described method.³⁴⁾ Histopathological examinations were carried out by well-trained histopathologist who was blind to treatment protocol. The microscopic lesions score was conducted as described by Patial et al.³⁵⁾ Mitotic index was calculated in liver by counting the mitotic bodies per ten microscopic fields per animal and graded with the following scores: scores 0–1: occasional/in few places (mild); score 2: fairly common/multifocal areas (moderate); score 3: diffuse (severe); scores 4–6: very severe.

Evaluation of Interleukin-6 (IL-6) in Liver Tissue

The liver tissue level of the inflammatory cytokine IL-6 was estimated using IL-6 ELISA kit (Rat IL-6 ELISA Kit (ab234570) according to manufacturer’s instructions (Abcam Company, U.K.) and was correlated to the total liver tissue protein using Lowry et al. method.³⁶⁾

Immunohistochemical Assessment of FASN and CD36 Expressions in Liver Tissues

Formalin-fixed, paraffin-embedded liver tissue sections from all treatment groups had been treated by heat-induced epitope retrieval technique (temperature 50 °C for up to 60 min), then, incubated with FASN antibody (sc-48357), and CD36 antibody (sc-7309) obtained from Santa Cruz Biotechnology (TX, U.S.A.) at dilution of 1 : 100. After washing, the sections were incubated with the secondary antibody, goat anti-rat immunoglobulin G (IgG) (HRP; Cat. No. 31471; Thermo Scientific, MA, U.S.A.) at dilution of 1 : 10000. The tissue sections were auto-stained with a high-sensitivity visualization system (Envision™ FLEX, High pH, and Link system) in the autostainer Link 48 in which the software had been pre-programmed according to antibodies manufacturer’s recommendations. The sectioned slides were examined using a light microscope to assess the expression of FASN and CD36 in all groups. Six images for each slide were obtained (400×). Immunoreactivity was evaluated by estimating the area percentage of the positive brown immunostained cells, using computed image analysis (Leica Qwin software 500, Germany).

Western Blotting Technique for ER-α and FASN Enzyme Assessment

For estimating the liver tissue expression of ER-α and FASN enzyme, the Western blotting technique was performed as previously described.³⁷⁾ Briefly, liver tissue was homogenized, total protein was estimated and then an equal amount of protein from each liver homogenate was loaded per lane and separated on a 10 % sodium dodecyl sulfate (SDS)-Tris-glycine polyacrylamide gel electrophoresis (PAGE) gel. Protein bands were transferred to a nitrocellulose membrane using a semi-dry blotter (Bio-Rad). The blot was subsequently blocked with Tris Buffered Saline +Tween 20 (TBS-T) buffer containing 5% skim milk powder for 1 h at room temperature. Then, the blot was probed overnight at 4 °C with the rat monoclonal anti-ER-α (Cat. No. sc-65715, Santa Cruz Biotechnology), FASN (Cat. No. sc-48357, Santa Cruz Biotechnology), or glyceraldehyde-3-phosphate dehydrogenase (GAPDH) (Cat. No. sc-137179, Santa Cruz Biotechnology) antibodies at dilutions of 1 : 1000. Subsequently, incubation with alkaline phosphatase secondary antibody goat anti rat-IgG-AP (Cat. No. A18868, Thermo Fisher Scientific) was performed for 1 h at dilution of 1 : 5000 at room temperature. Blots were finally analyzed using 5-bromo-4-chloro-3-indolyl-phosphate (BCIP) in conjunction with nitro blue tetrazolium (NBT) (BCIP/NBT) for the colorimetric detection of alkaline phosphatase activity.³⁸⁾

RESULTS

Liver Status

Effect of Treatment on Body Weight Gain, and Relative Liver Weight

The weight gain in the OVX group was significantly higher than that in SHAM group. The same trend was observed in SHAM + DEN + F group compared to SHAM + DEN group. There was no statistically significant difference between SHAM + DEN + F and OVX + DEN groups. Also, there was no significant difference in weight gain between SHAM+ DEN group and OVX groups treated with DEN combined with EB, S, or ORS groups. However, all these groups showed statistically significantly lower weight gain compared to SHAM and OVX groups (Table 1).

Table 1. Effect of Estradiol Benzoate, Fulvestrant, Silymarin, and/or Orlistat on Body Weight and Relative Liver Weight in Female Wistar Albino Rats

Group	Body weight gain (g)	Liver weight/body weight ratio (%)
Control	60.0 ± 5.13	2.82 ± 0.40
SHAM	60.2 ± 3.18	2.82 ± 0.33
SHAM + DEN	36.3 ± 4.36 a,b	4.27 ± 0.24 a,b
OVX	72.8 ± 4.83 a,b,c	2.43 ± 0.27 c
OVX + S	72.3 ± 5.20 a,b,c	2.76 ± 0.13 c
OVX + DEN	48.7 ± 8.89 a,b,c,d,e	4.98 ± 0.33 a,b,c,d,e
SHAM + DEN + F	48 ± 6.16 a,b,c,d,e	4.93 ± 0.21 a,b,c,d,e
OVX + DEN+ F	47 ± 7 a,b,c,d,e	5.44 ± 0.18 a,b,c,d,e
OVX + DEN + EB	35.7 ± 4.8 a,b,d,e,f,g,h	4.06 ± 0.25 a,b,d,e,f,g,h
OVX + DEN + S	36 ± 4.24 a,b,d,e,f,g,h	4.22 ± 0.36 a,b,d,e,f,g,h
OVX + DEN + ORS	33.3 ± 3.56 a,b,d,e,f,g,h	4.22 ± 0.17 a,b,d,e,f,g,h

• Data are presented as means ± S.D. (n = 6/group) for each group. • a, b, c, d, e, f, g, or h indicates significant difference from control, SHAM, SHAM + DEN, OVX, OVX + S, OVX + DEN, SHAM + DEN + F or OVX + DEN + F, respectively at p ≤ 0.05 using one-way parametric ANOVA, followed by post hoc Tukey’s test. • SHAM: placebo surgery, OVX: ovariectomized rats, DEN: diethylnitrosamine, F: fulvestrant, EB: estradiol benzoate, S: silymarin, ORS: orlistat. HCC: hepatocellular carcinoma.

The relative liver weight was statistically significantly higher in the SHAM group compared to the OVX one. Data illustrate an absence of statistically significant difference between the SHAM + DEN group and OVX + DEN treated with EB, S, or ORS groups. There was also no statistically significant difference between OVX + DEN group and the SHAM + DEN + F one. Nevertheless, this ratio was statistically significantly higher in OVX + DEN + F group compared to both SHAM + DEN + F and OVX + DEN groups (Table 1).

Effect of Treatment on Macroscopical Examination of Gross Liver

It was performed to identify the amplification or identification of HCC. Normal SHAM and OVX rats who did not receive DEN treatment did not show any hepatic nodules. However, all groups that received DEN treatment showed DEN-induced liver nodules (Fig. 1, Table 2). The tumor incidence was exaggerated in OVX + DEN and SHAM + DEN +F groups showing 83.3% tumor incidence with total number of nodules/groups of 70 and 71, respectively. However, it was highest and overdone in OVX + DEN + F group showing 100% tumor incidence with 93 nodules. The DEN-induced tumor incidence and total nodules number were alleviated in OVX rats assigned in groups co-treated with EB, S, or ORS showing 66.6% tumor incidence and total nodules per each group were 47, 46, and 45, respectively (Table 2). The same trend was observed concerning the relative size of nodules (Table 2).

Fig. 1. Representative Pictures of Gross Livers Collected from Female Wistar Albino Rats Allocated in Different Treatment Groups

Macroscopic observation showed DEN-induced HCC nodules with different sizes and scores among treatment groups. SHAM: placebo surgery; OVX: ovariectomized rats; DEN: diethylnitrosamine; F: fulvestrant; EB: estradiol benzoate; S: silymarin; ORS: orlistat.

Table 2. Macroscopical Examination of the Gross Liver of Female Ovariectomized and SHAM Albino Rats Treated with Diethylnitrosamine, Fulvestrant, Estradiol Benzoate, Silymarin, or Orlistat

Group	Number of rats bearing nodules/total number of rats per group	Incidence of nodules (%)	Total number of nodules/groups	Relative size of nodules (% of number size)			Average number of large nodules/number of tumor per rat
				≤1 mm Small nodules	1–3 mm Moderate nodules	≥3 mm Large nodules
SHAM + DEN	4/6	66.6%	45	27 (60%)	11 (24%)	7 (16%)	0.125 ± 0.05
OVX + DEN	5/6	83.3%	70	37 (53%)	18 (26%)	15 (21%)	0.256 ± 0.02
SHAM + DEN + F	5/6	83.3%	71	37 (52%)	20 (28%)	14 (20%)	0.196 ± 0.02
OVX + DEN+ F	6/6	100%	93	39 (42%)	29 (31%)	25 (27%)	0.328 ± 0.09 a
OVX + DEN + EB	4/6	66.6%	47	28 (59%)	11 (24%)	8 (17%)	0.15 ± 0.04 b
OVX + DEN + S	4/6	66.6%	46	27 (58%)	11 (24%)	8 (18%)	0.15 ± 0.02 b
OVX + DEN + ORS	4/6	66.6%	45	27 (60%)	10 (24%)	7 (16%)	0.15 ± 0.03 b

• Data were presented as means ± S.D. (n = 4–6/group) and statistically analyzed by Kruskal–Wallis test (one-way non-parametric ANOVA test) at p ≤ 0.05, followed by post hoc Dunn’s test. • a and b indicate significant difference from SHAM + DEN and OVX + DEN + F, respectively. • SHAM: placebo surgery, OVX: ovariectomized rats, DEN: diethylnitrosamine, F: fulvestrant, EB: estradiol benzoate, S: silymarin, ORS: orlistat. HCC: hepatocellular carcinoma.

Effect of Treatment on Serum Biomarkers of Liver Injury

Data analysis of the liver injury serum biomarkers (ALT, AST, ALP, AFP, T.Bil, and D.Bil.) pointed to a statistically significant increase in the levels of these biomarkers in all DEN-treated groups compared to non-DEN treated groups. However, OVX rats treated with DEN alone or combined with F illustrated significantly higher serum levels of these biomarkers compared to SHAM rats that received the same treatments (Fig. 2).

Fig. 2. Effect of Treatments on Serum Biomarkers for Liver Injury in Female Wistar Albino Rats

(A) AFP: alpha-fetoprotein, (B) ALP: alkaline phosphatase, (C) AST: aspartate transaminase, (D) ALT: alanine transaminase, (E) T.Bil: total bilirubin, (F) D.Bil: direct bilirubin. Data were presented as means ± S.D. (n = 6/group) and statistically analyzed by one-way parametric ANOVA test, followed by post hoc Tukey’s test. a, b, c, d, e, or f indicates statistically significant difference from SHAM, SHAM + DEN, OVX, OVX + DEN, SHAM + DEN + F, and OVX + DEN + F, respectively at p ≤ 0.05. SHAM: placebo surgery, OVX: ovariectomized rats, DEN: diethylnitrosamine, F: fulvestrant, EB: estradiol benzoate, S: silymarin, ORS: orlistat.

Interestingly, OVX rats treated with DEN in combination with EB, S or ORS exhibited significant lower levels of all these hepatic injury biomarkers specially the liver tumor biomarker AFP compared to OVX rats treated with DEN alone, DEN plus F as well as SHAM rats treated DEN plus F (Fig. 2).

Histopathological Examination of Liver

Microscopical examination of liver tissue section stained with H&E disclosed undoubted liver tissue deterioration combined with a statistically significant increase in the mitotic index in groups that received DEN compared to those that did not receive it. The mitotic index was represented by multiple focal areas of anaplastic hepatocytes detected all over the parenchyma. There were focal areas of necrosis in the parenchyma, and focal anaplastic areas of hepatocytes were defined by fine and thick fibrous tissue (hepatocellular carcinoma). The mitotic index scoring system estimated a remarkable statistically significant increase in these scores in OVX group treated with DEN compared to SHAM treated with DEN (Figs. 3A, B). Treatment of SHAM and OVX rats with a combination of DEN and F resulted in a statistically significant increase in the HCC-mitotic index scores compared to corresponding rats treated with DEN alone. Additionally, there was a remarkable statistically significant increase in these scores in OVX + DEN + F group compared to SHAM + DEN + F group (Figs. 3A, B). The histopathological examination estimated an apparent statistically significant decrease in the DEN-induced HCC markers (mitotic index) in each of the OVX + DEN + EB, OVX + DEN + S, and OVX + DEN + ORS groups compared to OVX + DEN (Figs. 3A, B).

Fig. 3. Histopathological Examination of Liver Tissue Sections Collected from Different Treatment Groups of Female Wistar Albino Rats

A: Representative micrographs for liver tissue section stained with H&E with magnification 40X. SHAM: show no histopathological change; SHAM + DEN: multiple focal areas of anaplastic hepatocytes (HCC) were detected all over the parenchyma (black arrow); OVX: the portal area showed dilatation in the portal vein with few inflammatory cells infiltration in the portal area as well as the formation of new bile ducts (black arrow); OVX + DEN & SHAM + DEN + F: focal anaplastic areas of hepatocytes defined by fine as well as thick fibrous tissue show HCC (black arrows); OVX + DEN + F: diffuse oval cells hyperplasia was observed associated with dilatation of the central veins, the anaplastic activity of the hepatocytes was observed forming HCC with a papillary pattern, as well as a trabecular pattern; OVX + DEN + EB, OVX + DEN + S, and OVX + DEN + ORS: few areas of anaplastic hepatocytes were detected (black arrows). B: Estimating the effect of treatment on mitotic index in liver tissue of female albino rats. Data are expressed as median ± interquartile range (n = 6/group) and statistically analyzed by Kruskal–Wallis test (one-way non-parametric ANOVA test), followed by post hoc Dunn’s test. a, b, c, d, e, or f indicates statistically significant difference from SHAM, SHAM + DEN, OVX, OVX + DEN, SHAM + DEN + F, and OVX + DEN + F, respectively at p ≤ 0.05. SHAM: placebo surgery, OVX: ovariectomized rats, DEN: diethylnitrosamine, F: fulvestrant, EB: estradiol benzoate, S: silymarin, ORS: orlistat, HCC: hepatocellular carcinoma, M: mean.

Effect of Treatment on Proinflammatory Interleukin-6 in Liver Tissue

DEN produced a significant induction of the proinflammatory cytokine IL-6 in liver tissue of SHAM and OVX rats compared to control groups with a paramount increase in OVX rats compared to SHAM rats. Additionally, co-treatment with F and DEN resulted in a significant rise in IL-6 compared to their corresponding rats treated with DEN alone in both SHAM and OVX groups. The expression of IL6 in OVX + DEN + F was statistically significantly higher than its expression in SHAM + DEN + F group (Fig. 4).

Fig. 4. Effect of Diethylnitrosamine, Estradiol Benzoate, Fulvestrant, Silymarin, and Orlistat on Liver Content of Interleukin-6 in Female Wistar Albino Rats

Data were presented as means ± S.D. (n = 6/group) and statistically analyzed by parametric one-way ANOVA, followed by post hoc Tukey’s test. a, b, c, d, e, or f indicates statistically significant difference from SHAM, SHAM + DEN, OVX, OVX + DEN, SHAM + DEN + F, and OVX + DEN + F, respectively at p ≤ 0.05. SHAM: placebo surgery, OVX: ovariectomized rats, DEN: diethylnitrosamine, F: fulvestrant, EB: estradiol benzoate, S: silymarin, ORS: orlistat.

Furthermore, a statistically significant decrease in the liver tissue content of IL-6 level was detected in the OVX rats that received the EB, S, and ORS in combination with DEN compared to those OVX rats treated with DEN only (Fig. 4).

FASN, CD36 and ER-alpha Expressions in Rat Liver Tissue

The expressions of FASN and CD36 in liver tissue collected from all treatment groups were evaluated via immunohistochemical investigation and furthermore the FASN expression was additionally confirmed via Western blotting technique which was also used to evaluate the expression of ER-α in liver tissue of rats.

Immunohistochemical analysis documented a statistically significant increase in the FASN expression in the groups treated with DEN compared to non-DEN-treated groups with exception of OVX + DEN + ORS group which exhibited lower expression compared to OVX control group. The apparent increase was statistically significant in OVX + DEN group compared to the SHAM + DEN group (Figs. 5A, B). Also, SHAM + DEN + F and OVX + DEN + F groups expressed significantly higher expressions levels of FASN compared to their corresponding group treated with DEN. In the same line, OVX + DEN + F expressed higher expression of FASN than SHAM + DEN + F (Figs. 5A, B).

Fig. 5. Immunohistochemical Assessment of Fatty Acid Synthase Expression in Liver Tissue of Female Wistar Albino Rats Post 16 Weeks Treatment with Diethylnitrosamine, Estradiol Benzoate, Fulvestrant, Silymarin, and Orlistat

A: Representative FASN immunohistochemical images from different groups with magnification power 400X. B: FASN reactivity scores. Data were presented as means ± S.D. (n = 6/group) and statistically analyzed by parametric one-way ANOVA test, followed by post hoc Tukey’s test. a, b, c, d, e, f, g, or h indicates statistically significant difference from SHAM, SHAM + DEN, OVX, OVX + DEN, SHAM + DEN + F, OVX + DEN + F, OVX + DEN + EB, and OVX + DEN + S, respectively at p ≤ 0.05. SHAM: placebo surgery, OVX: ovariectomized rats, DEN: diethylnitrosamine, F: fulvestrant, EB: estradiol benzoate, S: silymarin, ORS: orlistat.

Data analysis of the immunohistochemical investigation revealed a statistically significant decrease in FASN enzyme expression in the OVX rats treated with DEN plus EB or ORS compared to those OVX rats that received DEN alone. However, there was no statistically significant difference between OVX rats treated with DEN plus S compared to those OVX rats treated with DEN only (Figs. 5A, B). Furthermore, the Western blot technique showed similar results concerning FASN expression (Fig. 6B).

Fig. 6. The Protein Expression of Fatty Acid Synthase and Estrogen Receptor-Alpha in Liver Tissue of Female Albino Rats in Response to 16 Weeks Treatment with Diethylnitrosamine, Estradiol Benzoate, Fulvestrant, Silymarin, and Orlistat

The expression of estrogen receptor-alpha (A) and fatty acid synthase (B) in liver were analyzed by Western blot technique and normalized against glyceraldehyde-3-phosphate dehydrogenase (GAPDH) as endogenous control. (C, D) Data were presented as means ± S.D. (n = 3/group) and statistically analyzed by parametric one-way ANOVA, followed by post hoc Tukey’s test. a, b, c, d, e, f, g, or h indicates statistically significant difference from: Sh + D, OVX + D, Sh-C, OVX-C, OVX + D + E, OVX + D + S, OVX + D + F, or OVX + D + O, respectively at p ≤ 0.05. Sh-c: placebo surgery, OVX-c: ovariectomized control rats, D: diethylnitrosamine, E: estradiol benzoate, S: silymarin, O: orlistat, F: fulvestrant. FASN: fatty acid synthase enzyme.

In addition, the Western blot technique referred to a remarkable increase in the expression of FASN protein coincided with a decrease in ER-α expression and vice versa in all treatment groups (Figs. 6A, B).

Concerning the effect of treatment on CD36 expression, immunohistochemical investigation estimated a statistically significant increase in CD36 expression in the OVX control group compared to SHAM control one and a remarkable statistically significant increase in all groups that received DEN compared to those that did not receive it. There was a delectable statistically significant increase in CD36 expression in OVX + DEN group compared to the SHAM + DEN group (Figs. 7A, B). The data also referred to a statistically significant increase in CD36 expression in the SHAM + DEN + F group compared to the SHAM + DEN with an absence of a statistical difference between SHAM + DEN + F and OVX + DEN group. However, its expression in the OVX + DEN + F group was statistically significantly higher than both SHAM + DEN + F and OVX + DEN (Figs. 7A, B). The absence of a statistically significant difference in CD36 expression between OVX + DEN + S, OVX + DEN + ORS, SHAM + DEN + F, and OVX + DEN was clear, however, CD36 expression in these four groups was significantly higher than its expression in OVX + DEN + EB group (Figs. 7A, B).

Fig. 7. Immunohistochemical Evaluation of CD36 Expression in Liver Tissue of Female Wistar Albino Rats in Response to 16 Weeks Treatment with Diethylnitrosamine, Estradiol Benzoate, Fulvestrant, Silymarin, and Orlistat

A: Representative CD36 immunohistochemical photomicrograph from different treatment with magnification power 400X. B: CD36 reactivity scores. Data were presented as means ± S.D. (n = 6) and statistically analyzed by parametric one-way ANOVA test, followed by post hoc Tukey’s test. a, b, c, d, e, f, or g indicates statistically significant from SHAM, SHAM + DEN, OVX, OVX + DEN, SHAM + DEN + F, and OVX + DEN + EB, respectively at p ≤ 0.05. SHAM: placebo surgery, OVX: ovariectomized rats, DEN: diethylnitrosamine, F: fulvestrant, EB: estradiol benzoate, S: silymarin, ORS: orlistat.

DISCUSSION

Liver cancer is considered as a universal health challenge and its prevalence is growing globally. A recent study predicted that, by 2025, more than one million people will be influenced by HCC annually.³⁹⁾ Several epidemiological reports showed that, prevalence of HCC in postmenopausal females is increased and estrogen treatment markedly ameliorated it.^40–42) The protective impact of E₂/ERs has been of interest to many researchers; however, previous studies have unresolved issues related to the E₂/ERs protective pathway in HCC malignant liver cells. We aimed in our study to detect the potential protective impact of E₂/ER-α on DEN-induced HCC via modulation of lipid metabolism.

Our designed DEN-treatment protocol had been successful in induction of HCC in rats indicated by the elevated serum levels of AFP, the dependable marker in HCC diagnosis⁴³⁾ and moreover HCC was confirmed by the observed liver nodules in DEN-treated rats upon the macroscopical examination and the detected clear anaplastic activity in DEN-treated liver tissue post-histopathological investigation, in addition to a significant elevation of liver injury serum biomarkers and liver content of IL-6.^44,45) Failure of rats to gain body weight and distortion of relative liver weight were also indicative for severe liver injury.⁴⁶⁾

It was reported that the liver function tests are elevated approximately in 90% of the patients suffered from HCC and considered as a valuable purpose in the diagnosis of HCC.⁴⁵⁾ In the same line, there was a significant association between elevated IL-6 level and the increased risk of HCC. Whereas the IL-6 titers were four-fold higher in HCC patients than in cirrhotic patients and 25-fold higher than in healthy controls. Therefore, IL-6 could be considered a successful tumor marker for HCC, in particular when it is combined with the AFP test.⁴⁴⁾ Based on that, we considered in our study design, the liver injury serum biomarkers, and the liver tissue inflammatory mediator IL-6 levels besides macroscopical and histopathological investigation are dependable HCC progression markers.

In humans, HCC is showing a serious sexual disparity with a significantly increased incidence in men which explains the protective role of estrogen.^15,47) Our results concerning serum biomarkers levels, serum AFP, liver tissue content of IL-6 side by side to macroscopical and histopathological investigation were in line with the previous studies and clinical data confirming the protective and anti-cancer effects of E₂/ER-α against HCC development^48,49) where DEN-induced HCC was lower in SHAM rats compared to OVX rats. The next relevant clinical question was whether the protective effects of sex is due to E₂ as hormone or through its receptor, therefore we decided to use a selective estrogen receptor blocker (F)/agonist (EB). Our results showed that animals that received F increased the severity of DEN-induced HCC, while EB-treated animals improved it as in: 1) SHAM rats compared to SHAM treated with estrogen receptor antagonist (F), 2) OVX rats compared to OVX + F, 3) OVX rats treated with EB compared to OVX rats. This suggests that estrogen hormone provided protection at least in part through its receptor. Where still remain unknown this protective effect was due to ER-α or ER-β, so we used S as selective ER-β agonist, which ameliorated DEN-induced HCC as showed in OVX rats treated with (S) compared to OVX rats.

Lipid metabolism supplies the required building blocks needed to maintain tumor growth and acts as a secondary source for ATP production. FASN serves as a central regulator of lipid metabolism and implicated mainly in the development and progression of tumors. Moreover, inhibition of FASN has an effective and selective antitumor effect on cancer cells.⁵⁰⁾ Interestingly, CD36 expression is increased in hepatocytes of patients suffering from HCC and this elevated expression has been combined with the higher fatty acids uptake providing sustained development for many tumor cells including breast cancer and HCC.^50,51)

The hepatic E₂/ER-α signaling plays a critical role in the maintenance of gluconeogenesis and lipid metabolism in hepatocytes and dysfunction of ER-α is combined with obesity and metabolic dysfunction in humans.⁵²⁾ Hence, ER-α possesses a tumor suppressor effect which abrogates the progression of HCC through lipid metabolism-linked signaling pathways.⁵³⁾ Our data gained from both immunohistochemical, and Western blot techniques analysis referred to the increase the expression of FASN which is coincided with the decrease in ER-α expression and vice versa among treatment groups. These harvested data also pointed to the tumor suppressor impact of EB and ER-α synchronized with a decrease in DEN-induced FASN and CD36 expressions in liver tissue collected from SHAM versus OVX rats and from EB treated OVX rats versus EB-untreated OVX rats. In addition, this particular and fundamental role of ER-α not ER-β as a suppressor for DEN-induced HCC is indicated by the absence of the influence of S on the expressions of FASN and CD36 since the major constituent of silymarin, silybin, binds selectively to the cytosolic ER-β.⁵⁴⁾ However, from our point of view, we suggest that the clear calming impact of silymarin on DEN-induced HCC markers is mainly due to its potent anti-inflammatory and antioxidant effects⁵⁵⁾ and it was documented that the HCC occurs in cirrhotic livers is mediated via chronic inflammation associated with severe oxidative stress and silymarin could protect against these effects.^55,56)

The nuclear ERs bind with their target sites through two definite modes: estrogen response element (ERE)-dependent and ERE-independent pathways. The control of gene expression by the attachment of the E₂/ERs complex to the ERE is alternatively known as “ERE-dependent signaling pathway.” Several DNA binding experiments have mentioned that ERs attach to the ERE half-site 5′-AGGTCA-3′, besides ERE palindromes.⁵⁷⁾ On the other hand, the transcriptional regulation of target genes via binding of E₂/ERs complex with transcription factors specificity protein 1 (SP1), bound to their cognate regulatory elements on DNA is through a consensus sequence known as GC box (GGGCGG) referred to the ERE-independent signaling pathway.⁵⁸⁾

In silico analysis of FASN and SREBP-1c DNA gene promoters sequence demonstrated the presence of the cognate regulatory element of SP1⁵⁹⁾ supporting our data that restoration of E₂/ER-α could regulate FASN and SREBP-1c expression. In line with our data, Liu et al.⁶⁰⁾ detected that the protein or mRNA expression levels of FASN were increased in the OVX rats. Of note, they also detected that treatment with E₂ restored it. In addition, it was reported that E₂ treatment decreases gene expression of SREBP-1c and its target genes FASN and acetyl-CoA carboxylase which catalyze the rate-limiting step of de novo lipogenesis and regulates fatty acid b-oxidation in hepatocytes in the liver.²⁴⁾

The preliminary in silico analysis of CD36-DNA gene promoters sequence elucidated the presence of the cognate regulatory ERE half-site 5′-AGGTCA-3′, referring to the potential tapering impact of E₂ on CD36 expression.⁶¹⁾ Experimentally, it was revealed that hormone-dependent T47-D and MCF-7 cancer cells, down-regulate CD36 in response to E₂, and F blocks this effect, suggesting that the E₂/ERs play a role in down-regulating CD36 expression and F prevents loss of CD36 as a novel mechanism for its anti-estrogen effect in breast cancer cells.⁵¹⁾

Our data gathered post-IHC investigation referred to an increment of FASN and CD36 expression in OVX rats treated with DEN or even in the OVX rats that did not receive DEN. In addition, the SHAM rats in which the ERs were blocked by F displayed the same results. Besides, this increment in FASN and CD36 expression was synchronized to the estimated exaggerated DEN-induced HCC markers in OVX rats that received DEN and SHAM rats that received DEN plus F compared to SHAM rats that received the same DEN treatment alone. It is noteworthy, that EB utilized in our experimental design is considered a prodrug of E₂ to compensate the OVX rats for its depleted E₂. These estimated data proposed the tapering impact of E₂/ER-α on FASN and CD36 in the production of lipids, which are the energy source essential for the survival of liver cancer cells, illustrating the signaling pathway involved in the mitigating impact of E₂/ER-α on DEN-induced HCC. On the other hand, FASN and CD36 expression are not affected by ER-β agonist (S) as showed in OVX + S and OVX + DEN + S compared to OVX + EB and OVX + DEN + EB.

The conceivable antitumor impact of ORS as FASN inhibitor on DEN-induced HCC observed in our study was explained via its well-established halting effect on FASN enzyme which is strongly associated with tumor development.⁶²⁾ This concept is based on results of Lee et al.⁶³⁾ reported the apoptosis inducing effect of amentoflavone (FASN inhibitor) in SK-BR-3 human breast cancer cell line. Furthermore, Knowles et al.⁶⁴⁾ suggested the possible mechanism by which ORS inhibits FASN expression via increase DNA damage-inducible transcript-4 (DDIT4) expression which down-regulates mammalian target of rapamycin (mTOR), a key activator of SREBP-1c, resulting in inhibition of SREBP-1c leading to decreased FASN expression.^65,66) The suppressive effect of ORS on SREBP-1c protein expression has also been documented by Chyau et al.⁶⁷⁾ in high-fat high-fructose diet mice. Data analysis of the present study post-IHC realization revealed the absent impact of ORS on CD36 expression and the clear inhibitory effect on FASN expression which is mainly linked to its alleviating influence on DEN-induced HCC markers away from its effect on CD36 expression.

In conclusion, estrogen protects rats against DEN-induced HCC. This protective effect was mediated via ER-α which abrogates its downstream genes involved in lipid metabolism namely FASN and CD36 depriving the tumor from survival vital energy source and inhibition of proinflammatory IL-6. Orlistat induced similar mitigating effect against DEN-induced HCC which could be attributed to FASN inhibition and anti-inflammatory effect. In addition, silymarin alleviated DEN-induced HCC through its anti-oxidant and anti-inflammatory effects not through its estrogenic effects on FASN and CD36.

Author Contributions

MSA, AMM, MHH, RA, BEME, EME and SAS designed the study, acquired, analyzed, and validated data. MSA provided the necessary tools and reagents and performed all experimental studies under a direct supervision of AMM, MHH, RA and EME. MSA and MHH wrote the first original draft of the manuscript. All authors have revised and approved the manuscript at its final form. EME submitted the manuscript to journal as corresponding author.

Conflict of Interest

The authors declare no conflict of interest.

Data Availability

The datasets supporting the conclusions of this article are included within the article. Any further details about data and materials of this study are transparent and available upon request.

Supplementary Materials

This article contains supplementary materials.

REFERENCES

• 1) Nakagawa H, Hayata Y, Kawamura S, Yamada T, Fujiwara N, Koike K. Lipid metabolic reprogramming in hepatocellular carcinoma. Cancers (Basel), 10, 447 (2018).
• 2) Vander Heiden MG, Cantley LC, Thompson CB. Understanding the Warburg effect: the metabolic requirements of cell proliferation. Science, 324, 1029–1033 (2009).
• 3) Currie E, Schulze A, Zechner R, Walther TC, Farese RV Jr. Cellular fatty acid metabolism and cancer. Cell Metab., 18, 153–161 (2013).
• 4) Navarro C, Ortega Á, Santeliz R, Garrido B, Chacín M, Galban N, Vera I, De Sanctis BJ, Bermúdez V. Metabolic reprogramming in cancer cells: emerging molecular mechanisms and novel therapeutic approaches. Pharmaceutics, 14, 1303 (2022).
• 5) Chen HP, Shieh JJ, Chang CC, Chen TT, Lin JT, Wu MS, Lin JH, Wu CY. Metformin decreases hepatocellular carcinoma risk in a dose-dependent manner: population-based and in vitro studies. Gut, 62, 606–615 (2013).
• 6) Bhalla K, Hwang BJ, Dewi RE, Twaddel W, Goloubeva OG, Wong KK, Saxena NK, Biswal S, Girnun GD. Metformin prevents liver tumorigenesis by inhibiting pathways driving hepatic lipogenesis. Cancer Prev. Res., 5, 544–552 (2012).
• 7) Couchepin C, Lê KA, Bortolotti M, da Encarnaçao JA, Oboni JB, Tran C, Schneiter B, Tappy L. Markedly blunted metabolic effects of fructose in healthy young female subjects compared with male subjects. Diabetes Care, 31, 1254–1256 (2008).
• 8) Samuel VT. Fructose induced lipogenesis: from sugar to fat to insulin resistance. Trends Endocrinol. Metab., 22, 60–65 (2011).
• 9) Yang X, Zhang YK, Esterly N, Klaassen CD, Wan YJ. Gender disparity of hepatic lipid homoeostasis regulated by the circadian clock. J. Biochem., 145, 609–623 (2009).
• 10) Yang F, Yin Y, Wang F, Zhang L, Wang Y, Sun S. An altered pattern of liver apolipoprotein A–I isoforms is implicated in male chronic hepatitis B progression. J. Proteome Res., 9, 134–143 (2010).
• 11) Huang CK, Lee SO, Lai KP, Ma WL, Lin TH, Tsai MY, Luo J, Chang C. Targeting androgen receptor in bone marrow mesenchymal stem cells leads to better transplantation therapy efficacy in liver cirrhosis. Hepatology, 57, 1550–1563 (2013).
• 12) Yeh SH, Chen PJ. Gender disparity of hepatocellular carcinoma: the roles of sex hormones. Oncology, 78 (Suppl. 1), 172–179 (2010).
• 13) Weston SR, Leyden W, Murphy R, Bass NM, Bell BP, Manos MM, Terrault NA. Racial and ethnic distribution of nonalcoholic fatty liver in persons with newly diagnosed chronic liver disease. Hepatology, 41, 372–379 (2005).
• 14) Bosch FX, Ribes J, Díaz M, Cléries R. Primary liver cancer: worldwide incidence and trends. Gastroenterology, 127 (Suppl. 1), S5–S16 (2004).
• 15) Shimizu I, Yasuda M, Mizobuchi Y, Ma YR, Liu F, Shiba M, Horie T, Ito S. Suppressive effect of oestradiol on chemical hepatocarcinogenesis in rats. Gut, 42, 112–119 (1998).
• 16) Naugler WE, Sakurai T, Kim S, Maeda S, Kim K, Elsharkawy AM, Karin M. Gender disparity in liver cancer due to sex differences in MyD88-dependent IL-6 production. Science, 317, 121–124 (2007).
• 17) Maeda S, Kamata H, Luo JL, Leffert H, Karin M. IKKbeta couples hepatocyte death to cytokine-driven compensatory proliferation that promotes chemical hepatocarcinogenesis. Cell, 121, 977–990 (2005).
• 18) Balogh J, Victor D 3rd, Asham EH, Burroughs SG, Boktour M, Saharia A, Li X, Ghobrial RM, Monsour HP Jr. Hepatocellular carcinoma: a review. J. Hepatocell. Carcinoma, 3, 41–53 (2016).
• 19) Nakatani T, Roy G, Fujimoto N, Asahara T, Ito A. Sex hormone dependency of diethylnitrosamine-induced liver tumors in mice and chemoprevention by leuprorelin. Jpn. J. Cancer Res., 92, 249–256 (2001).
• 20) Yang W, Lu Y, Xu Y, Xu L, Zheng W, Wu Y, Li L, Shen P. Estrogen represses hepatocellular carcinoma (HCC) growth via inhibiting alternative activation of tumor-associated macrophages (TAMs). J. Biol. Chem., 287, 40140–40149 (2012).
• 21) Villa E, Colantoni A, Grottola A, Ferretti I, Buttafoco P, Bertani H, De Maria N, Manenti F. Variant estrogen receptors and their role in liver disease. Mol. Cell. Endocrinol., 193, 65–69 (2002).
• 22) Iyer JK, Kalra M, Kaul A, Payton ME, Kaul R. Estrogen receptor expression in chronic hepatitis C and hepatocellular carcinoma pathogenesis. World J. Gastroenterol., 23, 6802–6816 (2017).
• 23) Zeng H, Qin H, Liao M, Zheng E, Luo X, Xiao A, Li Y, Chen L, Wei L, Zhao L, Ruan XZ, Yang P, Chen Y. CD36 promotes de novo lipogenesis in hepatocytes through INSIG2-dependent SREBP1 processing. Mol. Metab., 57, 101428–101439 (2022).
• 24) Bajic VB, Tan SL, Chong A, Tang S, Ström A, Gustafsson JA, Lin CY, Liu ET. Dragon ERE Finder version 2: A tool for accurate detection and analysis of estrogen response elements in vertebrate genomes. Nucleic Acids Res., 31, 3605–3607 (2003).
• 25) Menendez JA, Lupu R. Fatty acid synthase and the lipogenic phenotype in cancer pathogenesis. Nat. Rev. Cancer, 7, 763–777 (2007).
• 26) Menendez P, Wang L, Bhatia M. Genetic manipulation of human embryonic stem cells: a system to study early human development and potential therapeutic applications. Curr. Gene Ther., 5, 375–385 (2005).
• 27) Lasota A, Danowska-Klonowska D. Experimental osteoporosis—different methods of ovariectomy in female white rats. Rocz. Akad. Med. Bialymst., 49 (Suppl. 1), 129–131 (2004).
• 28) Zhang Y, Li X, Li X. Curcumae ameliorates diethylnitrosamine-induced hepatocellular carcinoma via alteration of oxidative stress, inflammation and gut microbiota. J. Inflamm. Res., 14, 5551–5566 (2021).
• 29) Yamamoto S, Yamashita T, Ito M, Caaveiro JMM, Egashira N, Tozaki-Saitoh H, Tsuda M. New pharmacological effect of fulvestrant to prevent oxaliplatin-induced neurodegeneration and mechanical allodynia in rats. Int. J. Cancer, 145, 2107–2113 (2019).
• 30) Eshraghi-Jazi F, Talebi A, Mirsaeedi FS, Ahmadian S, Moslemi F, Nematbakhsh M. Gentamicin induced nephrotoxicity: the role of sex hormones in gonadectomized male and female rats. Scientifica, 2016, 5025097 (2016).
• 31) Cervantes-Madrid D, Dominguez-Gomez G, Gonzalez-Fierro A, Perez-Cardenas E, Taja-Chayeb L, Trejo-Becerril C, Duenas-Gonzalez A. Feasibility and antitumor efficacy in vivo, of simultaneously targeting glycolysis, glutaminolysis and fatty acid synthesis using lonidamine, 6-diazo-5-oxo-L-norleucine and orlistat in colon cancer. Oncol. Lett., 13, 1905–1910 (2017).
• 32) Shaker E, Mahmoud H, Mnaa S. Silymarin, the antioxidant component and Silybum marianum extracts prevent liver damage. Food Chem. Toxicol., 48, 803–806 (2010).
• 33) Sharma A, Fish BL, Moulder JE, Medhora M, Baker JE, Mader M, Cohen EP. Safety and blood sample volume and quality of a refined retro-orbital bleeding technique in rats using a lateral approach. Lab. Anim (NY), 43, 63–66 (2014).
• 34) Carleton HM, Drury RAB, Wallington EA. Carleton’s histological technique. Oxford University Press, New York (1980).
• 35) Patial V, S M, Sharma S, Pratap K, Singh D, Padwad YS. Synergistic effect of curcumin and piperine in suppression of DENA-induced hepatocellular carcinoma in rats. Environ. Toxicol. Pharmacol., 40, 445–452 (2015).
• 36) Lowry OH, Rosebrough NJ, Farr AL, Randall RJ. Protein measurement with the Folin phenol reagent. J. Biol. Chem., 193, 265–275 (1951).
• 37) Mahmood T, Yang PC. Western blot: technique, theory, and trouble shooting. N. Am. J. Med. Sci, 4, 429–434 (2012).
• 38) Ewees MG, Messiha BAS, Abo-Saif AA, Bayoumi AMA, Abdel-Bakky MS. Interference with coagulation cascade as a novel approach to counteract cisplatin-induced acute tubular necrosis; an experimental study in rats. Front. Pharmacol, 9, 1155 (2018).
• 39) Villanueva A. Hepatocellular carcinoma. N. Engl. J. Med., 380, 1450–1462 (2019).
• 40) Villa E. Role of estrogen in liver cancer. Womens Health (Lond.), 4, 41–50 (2008).
• 41) Hsieh CJ, Hong MK, Chen PC, Wang JH, Chu TY. Antiestrogen use reduces risk of cervical neoplasia in breast cancer patients: a population-based study. Oncotarget, 8, 29361–29369 (2017).
• 42) Simin J, Tamimi R, Lagergren J, Adami HO, Brusselaers N. Menopausal hormone therapy and cancer risk: an overestimated risk? Eur. J. Cancer, 84, 60–68 (2017).
• 43) Zhang J, Chen G, Zhang P, Zhang J, Li X, Gan D, Cao X, Han M, Du H, Ye Y. The threshold of alpha-fetoprotein (AFP) for the diagnosis of hepatocellular carcinoma: a systematic review and meta-analysis. PLOS ONE, 15, e0228857 (2020).
• 44) Shakiba E, Ramezani M, Sadeghi M. Evaluation of serum interleukin-6 levels in hepatocellular carcinoma patients: a systematic review and meta-analysis. Clin. Exp. Hepatol., 4, 182–190 (2018).
• 45) Chan AW, Chan SL, Mo FK, Wong GL, Wong VW, Cheung YS, Chan HLY, Yeo W, Lai PBS, To K. Albumin-to-alkaline phosphatase ratio: a novel prognostic index for hepatocellular carcinoma. Dis. Markers, 2015, 564057 (2015).
• 46) Hassan MM, Abdel-Wahab R, Kaseb A, Shalaby A, Phan AT, El-Serag HB, Hawk E, Morris J, Raghav KPS, Lee J, Vauthey JN, Bortus G, Torres HA, Amos CI, Wolff RA, Li D. Obesity early in adulthood increases risk but does not affect outcomes of hepatocellular carcinoma. Gastroenterology, 149, 119–129 (2015).
• 47) Wu Y, Yao N, Feng Y, Tian Z, Yang Y, Zhao Y. Identification and characterization of sexual dimorphism-linked gene expression profile in hepatocellular carcinoma. Oncol. Rep., 42, 937–952 (2019).
• 48) O’Brien MH, Pitot HC, Chung SH, Lambert PF, Drinkwater NR, Bilger A. Estrogen receptor-α suppresses liver carcinogenesis and establishes sex-specific gene expression. Cancers, 13, 2355 (2021).
• 49) Yamamoto R, Tatsuta M, Terada N. Suppression by oestrogen of hepatocellular tumourigenesis induced in mice by 3′-methyl-4-dimethylaminoazobenzene. Br. J. Cancer, 68, 303–307 (1993).
• 50) Fhu CW, Ali A. Fatty acid synthase: an emerging target in cancer. Molecules, 25, 3935 (2020).
• 51) Uray IP, Liang Y, Hyder SM. Estradiol down-regulates CD36 expression in human breast cancer cells. Cancer Lett., 207, 101–107 (2004).
• 52) Qiu S, Vazquez JT, Boulger E, Liu H, Xue P, Hussain MA, Wolfe A. Hepatic estrogen receptor α is critical for regulation of gluconeogenesis and lipid metabolism in males. Sci. Rep., 7, 1661 (2017).
• 53) Meng X, Liu X. Therapeutic value of estrogen receptor α in hepatocellular carcinoma based on molecular mechanisms. J. Clin. Transl. Hepatol, 10, 140–146 (2022).
• 54) Ortona E, Locatelli SL, Pagano MT, Ascione B, Careddu G, Dupuis ML, Marconi M, Carlo-Stella C, Malorni W, Matarrese P, Pierdominici M. The natural estrogen receptor beta agonist silibinin as a promising therapeutic tool in diffuse large B-cell lymphoma. Anticancer Res., 42, 767–779 (2022).
• 55) Mastron JK, Siveen KS, Sethi G, Bishayee A. Silymarin and hepatocellular carcinoma: a systematic, comprehensive, and critical review. Anticancer Drugs, 26, 475–486 (2015).
• 56) Muriel P. Role of free radicals in liver diseases. Hepatol. Int., 3, 526–536 (2009).
• 57) Gruber CJ, Gruber DM, Gruber IM, Wieser F, Huber JC. Anatomy of the estrogen response element. Trends Endocrinol. Metab., 15, 73–78 (2004).
• 58) William SK, Michael RC, Charlotte AS, Michael AP. Concepts of Genetics. 9th edition. Pearson/Benjamin Cummings, San Francisco (2009).
• 59) ‹https://switchdb.switchgeargenomics.com/productinfo/id_708922.html›, accessed 10 March, 2021.
• 60) Liu Q, Li R, Chen G, Wang J, Hu B, Li C, Zhu X, Lu Y. Inhibitory effect of 17β-estradiol on triglyceride synthesis in skeletal muscle cells is dependent on ESR1 and not ESR2. Mol. Med. Rep., 19, 5087–5096 (2019).
• 61) ‹https://switchdb.switchgeargenomics.com/productinfo/id_716068.html›, accessed 10 March, 2021.
• 62) Fako VE, Zhang JT, Liu JY. Mechanism of orlistat hydrolysis by the thioesterase of human fatty acid synthase. ACS Catal., 4, 3444–3453 (2014).
• 63) Lee JS, Lee MS, Oh KW, Sul JY. Fatty acid synthase inhibition by amentoflavone induces apoptosis and antiproliferation in human breast cancer cells. Biol. Pharm. Bull., 32, 1427–1432 (2009).
• 64) Knowles LM, Yang C, Osterman A, Smith JW. Inhibition of fatty-acid synthase induces caspase-8-mediated tumor cell apoptosis by up-regulating DDIT4. J. Biol. Chem., 283, 31378–31384 (2008).
• 65) Xu SM, Xu Y, Cheng XG, Yang LQ. Tilianin protects against nonalcoholic fatty liver disease in early obesity mice. Biol. Pharm. Bull., 46, 419–426 (2023).
• 66) Soliman GA. The integral role of mTOR in lipid metabolism. Cell Cycle, 10, 861–862 (2011).
• 67) Chyau CC, Wang HF, Zhang WH, Chen CC, Huang SH, Chang CC, Peng RY. Antrodan alleviates high-fat and high-fructose diet-induced fatty liver disease in C57BL/6 mice model via AMPK/Sirt1/SREBP-1c/PPAR pathway. Int. J. Mol. Sci., 21, 360 (2020).