Gallic acid against hepatocellular carcinoma: An integrated scheme of the potential mechanisms of action from in vivo study

Abstract

The global burden of hepatocellular carcinoma is increasing; actually, it is estimated as 750,000 new cases annually. This study was initiated to emphasize the possibility that gallic acid could alleviate hepatocarcinogenesis in vivo. In this study, 40 rats were enrolled and distributed as follows; group 1 was set as negative control, while all of groups 2, 3, and 4 were orally received N-nitrosodiethylamine for hepatocellular carcinoma induction. Group 2 was left untreated, whereas groups 3 and 4 were orally treated with gallic acid and doxorubicin, respectively. The current data indicated that gallic acid administration in hepatocellular carcinoma bearing rats yielded significant decline in serum levels of alpha-fetoprotein, glypican-3, and signal transducer and activator of transcription 3 along with significant enhancement in serum suppressors of cytokine signaling 3 level. Also, gallic acid–treated group displayed significant downregulation in the gene expression levels of hepatic gamma glutamyl transferase and heat shock protein gp96. Intriguingly, treatment with gallic acid remarkably ameliorated the destabilization of liver tissue architecture caused by N-nitrosodiethylamine intoxication as evidenced by histopathological investigation. In conclusion, this study demonstrates that the hepatocarcinogenic effect of N-nitrosodiethylamine can be abrogated by gallic acid supplementation owing to its affinity to regulate signal transducer and activator of transcription 3 signaling pathway through its outstanding bioactivities including antioxidant, anti-inflammatory, apoptotic, and antitumor effects.

Keywords

Hepatocellular carcinoma gallic acid signal transducer and activator of transcription 3 signaling pathway tumor markers rats

Introduction

The rate of cancer incidence dramatically increases annually; nearly, 10.9 million of new cancer cases and 67 million of cancer-related deaths are recorded every year. Liver cancer is considered as the most common disease among males worldwide.¹

Hepatocellular carcinoma (HCC) is a life-threatening disease that is regarded as the fifth most frequent cancer whose incidence rate was estimated to be about 626,000 cases every year.² It is a form of aggressive malignancies that is highly associated with viral hepatitis B and C chronic infection, cirrhosis, in addition to other environmental factors such as exposure to aflatoxin B1, and alcohol overconsumption.³ In Egypt, HCC is responsible for about 14.8% of all cancer fatality with a greater prevalence in males (17.3%) over females (11.5%). It is ranked as the second most incident cancer in Egyptian men and the eighth most common among Egyptian women.⁴

Recently, HCC prognosis has been largely improved owing to the promising diagnostic tools and surgical resections. However, the long-term survival rate is not satisfactory due to the high recurrence rate even after hepatic resection (50%–70% at 5 years). Moreover, no effective systemic chemotherapeutic drugs have been established for HCC so far.⁵ Therefore, new therapeutic strategies are required for HCC retraction.

Anticancer agents originated from natural sources represent a promising alternative to the currently standard therapies for HCC and other cancers.⁶ Gallic acid (3,4,5-trihydroxybenzoic acid) is one of the most putative polyphenols existing in pomegranate, grapes, nuts, green tea, oak bark, different berries, mango, as well as in red wines. Also, it is considered as one of the major active compounds in tannins, known as galatotanin. This compound has been shown to exhibit a wide range of pharmacological activities. Away from its medicinal characteristics, it also has a variety of applications as a food preservative in beverages and a chelating agent in cosmetics, dye synthesis, and leather industry.⁷

This study was tailored to assess the anticancer potency of gallic acid against HCC in the experimental model with special concern to the mechanisms of action.

Materials and methods

Extraction, preparation, and isolation of gallic acid from Punica granatum peel

Punica granatum peels were authenticated and extracted according to previously published work.⁸ Then, the extract was filtered, evaporated under reduced pressure, lyophilized, and loaded on a polyamide 6S column chromatography (80 × 3 cm). The column was eluted with H₂O, and then, H₂O-CH₃OH mixtures of decreasing polarity and 10 fractions (1 L, each) were collected. The obtained phenolic fraction was subjected to column chromatography on cellulose and n-butanol (n-BuOH) saturated with H₂O as an eluent to give sub-fraction which was further fractionated on Sephadex LH-20 to yield gallic acid. The chemical structure of gallic acid was identified using proton nuclear magnetic resonance (¹H NMR) spectroscopy and carbon-13 nuclear magnetic resonance (¹³C NMR) spectroscopy.

Chemicals and drugs

N-nitrosodiethylamine (NDEA; CAS no. 55-18-5) was supplied from Sigma-Aldrich Co. (St. Louis, MO, USA), while doxorubicin (Adricin) was purchased from EIMC United Pharmaceuticals, Egypt. All the other chemicals were of high-purity grade in accordance with international standards.

Experimental animals

In total, 40 adult male rats (Wistar strain) weighing 170–200 g were supplied from a breading stock maintained in the Animal House of the National Research Centre, Giza, Egypt. The animal house was ventilated with a 12-h light/dark cycle at the ambient temperature of 25°C–30°C throughout the experimental period with free access to tap water and a standard rodent chow (Wadi El Kabda Co., Cairo, Egypt). Rats were allowed to adapt to their environment for at least 10 days before the initiation of the experiment. Housing and management of animals and experimental protocol followed the guidelines of animal experiments and was approved by the Ethical Committee of the Medical Research of the National Research Centre, Egypt (approval no. 14023).

In vivo study

The rats were randomly categorized into four groups (10 rats/group). (1) Negative control group: the rats in this group were healthy and orally received normal saline (0.9%) five times weekly during the entire experimental period. (2) HCC group: the rats in this group were orally administered NDEA (dissolved in 0.9% normal saline) with a dosage of 20 mg/kg body weight five times weekly for 6 weeks according to the method explained by Darwish and El-Boghdady.⁹ (3) Gallic acid–treated group: in this group, HCC-bearing rats were orally treated with a dosage of 50 mg gallic acid/kg body weight¹⁰ five times weekly for 5 weeks. (4) Doxorubicin-treated group: in this group, HCC-bearing rats were intraperitoneally injected with doxorubicin as a reference drug with a dosage of 0.72 mg/rat corresponding to the human dosage of 40 mg/m² according to Barnes and Paget¹¹ once a week for 5 weeks.

Following the last treatment, the diets were withheld from the experimental rats for 12 h, and then, blood samples were collected, under diethyl ether anesthesia, from the retro-orbital venous plexus in clean centrifuge tubes and allowed to coagulate at room temperature. Serum samples were separated by centrifugation at 1800×g for 15 min at 4°C using cooling centrifuge for biochemical measurements and retained at −20°C till analysis. After collection of blood samples, the rats were sacrificed by exsanguinations, and a middle abdominal incision was performed and the liver of each rat was quickly dissected out and washed in ice-cold saline. Then, a large section from each liver was immediately frozen in liquid nitrogen and kept at −80°C preceding the molecular genetics investigation, while a small section from each liver was placed in formalin saline (10%) to be used in histological slides.

Biochemical assays

Serum alpha-fetoprotein (AFP), glypican-3 (GPC-3), signal transducer and activator of transcription 3 (STAT3), and suppressors of cytokine signaling 3 (SOCS3) levels were assayed by enzyme-linked immunosorbent assay (ELISA) using kits acquired from Glory Science Co., Ltd (USA) according to the manufacturer’s instructions of the kits.

Molecular genetics investigation

Total RNA was isolated from the liver tissues of rats of different groups using TRI reagent (BioShop Canada Inc., Burlington, Canada) according to the manufacturer’s protocol. Then, isolated RNA was reverse transcribed into complementary DNA (cDNA) using RevertAid First-Strand cDNA Synthesis Kit (Fermentas, USA). The subsequent polymerase chain reaction (PCR) was performed using 5 µg of cDNA in a final volume of 20 µL containing 10× PCR buffer, 10 mM deoxynucleotide triphosphates (dNTPs), 5 U/µL of Taq DNA polymerase (Fermentas), and 10 µM of each specific primers. Glyceraldhyde 3-phosphate dehydrogenase (GAPDH) was used as an internal control with the following primer sequences: F: 5′-CAAGGTCATCCATGACAACTTTG-3′ and R: 5′-GTCCACCACCCTGTTGCTGTAG-3′ according to Paul and Kundu’s¹² published sequence. Primer sequences for gamma glutamyl transferase (GGT) were as follows: F: 5′-CTCTGCATCTGGCTACCCAC-3′ and R: 5′-GGATGCTGGGTTGGAAGAGG-3′ according to Carrasco-Legleu et al.’s¹³ published sequence. Primer sequences for heat shock protein glycoprotein 96 (HSPgp96) were as follows: F: 5′-ACACGGCTTGCTAAACTTCT-3′ and R: 5′-ACTACAGTCTGCGGTCCAAA-3′ according to Wu et al.’s¹⁴ published sequence. The PCR cycling was performed using a gradient thermal cycler (Bio-Rad, USA) as follows: initial denaturation at 94°C for 5 min, followed by denaturation at 94°C for 30 s. Amplification was carried out using 35 cycles with annealing temperature at 58°C for GGT and GAPDH and at 53°C for HSPgp96 for 30 s, followed by extension at 72°C for 1 min and a final extension at 72°C for 8 min. The PCR products were subsequently electrophoresed in 2% agarose gel stained with ethidium bromide (Sigma, USA) and visualized and photographed by gel documentation system (Bio-Rad). The amplicon size was determined by comparison with DNA ladder (100 bp; Fermentas). All gene expression levels were semi-quantified using LabImage analysis (LabImage 2.7.0; Kapelan Bio-Imaging GmbH, Leipzig, Germany) software and were normalized against GAPDH gene expression. All primer pairs used were synthesized by Metabion (Germany).

Histological examination

After fixation of the liver specimens in formalin saline (10%) for 24 h, the tissues were washed with a running tap water, followed by ascending grade of ethyl alcohols (30%, 50%, 70%, 90%, and absolute) for dehydration. Specimens were then cleared in xylene and embedded in paraffin in a hot oven of 56°C temperature for 24 h. Paraffin wax tissue blocks were prepared for sectioning into sections of 4 µm thickness using a sledge microtome. The obtained tissue sections were placed over glass slides, deparffinized, and stained by hematoxylin and eosin (H&E) stain for histopathological examination under the electric light microscope.¹⁵

Statistical analysis

The experimental results were represented as arithmetic means with their standard errors (SEs). Data were statistically analyzed by one-way analysis of variance (ANOVA) test using the Statistical Package for the Social Sciences (SPSS) 14 followed by determining the least significant difference (LSD) to evaluate the significance between groups; p < 0.05 was considered significant.

Results

Identification of the isolated compound

The pure compound was isolated as white amorphous powder. Spraying with FeCl₃ gave blue color indicating its phenolic nature. The ultraviolet (UV) spectral data exhibited an absorption band (λ_max 272) characteristic for phenolic acids.¹⁶

¹H NMR spectrum showed singlet signal at δH 6.92 integrated for two aromatic protons assigned for H-2 and H-6. It showed abroad singlet at δH 9.17 integrated for three protons assigned for three hydroxyl groups at positions 3, 4, and 5, while ¹³C NMR spectrum showed carbonyl carbon atom at δc 167.96 and three oxygenated aromatic carbon atoms appearing at δc 145.85 (C-3 and C-5) and 138.44 (C-4). It exhibited three non-oxygenated aromatic carbon atoms at δc 120.91 (C-1) and 109.20 (C-2 and C-6).

From the above-mentioned data and by comparison with previously reported ones^17,18 and co-chromatography with authentic sample, the isolated compound could be identified as 3,4,5-trihydroxy benzoic acid (GA). ¹H NMR at δ9.17 (brs, three OH at positions 3, 4, and 5) and 6.92 (2H, s, H-2, and H-6) and ¹³C NMR at δ167.96 (C = O), 145.85 (C-3 and C-5), 138.44 (C-4), 120.91 (C-1), and 109.20 (C-2 and C-6).

Biochemical markers

The data in Table 1 represent the consequence of the treatment with gallic acid and doxorubicin on serum tumor markers’ level in HCC rat model. HCC group experienced significant augmentation (p < 0.05) in serum AFP, GPC-3, and STAT3 levels associated with significant drop (p < 0.05) in serum SOCS3 level in comparison with the negative control group, whereas the treatment of HCC-bearing rats with gallic acid or doxorubicin yielded significant reduction (p < 0.05) in serum AFP, GPC-3, and STAT3 levels concomitant with significant elevation (p < 0.05) in the serum SOCS3 level relative to the untreated HCC counterparts.

Table 1.

Impact of gallic acid and doxorubicin on serum AFP, GPC-3, STAT3, and SOCS3 levels in HCC rat model.

Groups	Parameters
Groups	AFP (ng/mL)	GPC-3 (pg/mL)	STAT3 (pg/mL)	SOCS3 (pg/mL)
Negative control	18.49 ± 0.29	8.75 ± 0.39	1316.75 ± 81.10	163.62 ± 5.49
HCC	39.14 ± 0.97^a	29.2 ± 0.75^a	2328.50 ± 108.10^a	111.75 ± 3.36^a
Gallic acid–treated	28.57 ± 1.18^b	18.86 ± 0.56^b	1683.20 ± 89.50^b	158.75 ± 4.52^b
Doxorubicin-treated	30.78 ± 1.41^b	19.43 ± 1.26^b	1760.80 ± 106.0^b	150.45 ± 3.47^b

AFP: alpha-fetoprotein; GPC-3: glypican-3; STAT3: signal transducer and activator of transcription 3; SOCS3: serum suppressors of cytokine signaling 3; HCC: hepatocellular carcinoma; SE: standard error.

Data are expressed as mean ± SE of 10 rats/group.

Significant change at p < 0.05 in comparison with the negative control group.

Significant change at p < 0.05 in comparison with the untreated HCC group.

Hepatic GGT and HSPgp96 genes’ expression

Figure 1 and Table 2 illustrate the influence of treatment with gallic acid and doxorubicin on hepatic GGT and HSPgp96 gene expression levels in HCC rat model. The untreated HCC group showed significant amplification (p < 0.05) in the expression level of both genes versus the negative control group, while the treatment of HCC-bearing rats with gallic acid or doxorubicin evoked significant downregulation (p < 0.05) in the expression level of hepatic GGT and HSPgp96 genes as compared to the untreated HCC counterparts. Interestingly, the rats in HCC group treated with gallic acid elicited significant downregulation (p < 0.05) in the expression level of the hepatic HSPgp96 gene when compared with those treated with doxorubicin.

Figure 1.

Agarose gel electrophoresis showing (a) GGT and (b) HSPgp96 mRNA expression in the liver tissue by RT-PCR analysis. GAPDH expression with product size of 496 bp, GGT expression with product size of 418 bp, whereas HSPgp96 expression with product size of 404 bp. Lane (1) represents the negative control group and lane (2) represents HCC group. Lane (3) represents gallic acid–treated group, whereas lane (4) represents doxorubicin-treated group. Lane M represents DNA ladder (100 bp).

Table 2.

Impact of gallic acid and doxorubicin on hepatic GGT and HSPgp96 genes’ expression in HCC rat model.

Groups	Parameters
Groups	GGT expression	HSPgp96 expression
Negative control	No expression	0.29 ± 0.008
HCC	1.42 ± 0.12^a	0.76 ± 0.015^a
Gallic acid–treated	0.43 ± 0.04^b	0.36 ± 0.015^bc
Doxorubicin-treated	0.51 ± 0.04^b	0.54 ± 0.04^b

GGT: glutamyl transferase; HSPgp96: heat shock protein gp96; HCC: hepatocellular carcinoma; SE: standard error.

Values are represented as mean ± SE (six rats/group).

Significant change at p < 0.05 in comparison with the negative control group.

Significant change at p < 0.05 in comparison with the HCC group.

Significant change at p < 0.05 in comparison with the doxorubicin-treated group.

Histological examination

Optical micrograph of the cross-sectioned liver tissue of rat in the negative control group shows a normal histological structure of the central vein and surrounding hepatocytes in the hepatic parenchyma as illustrated in Figure 2, while optical micrograph of the liver tissue section of rat in the untreated HCC group reveals the portal area with fibrosis extended and divided the hepatic parenchyma into lobules of dysplastic with a prominent nucleoli degenerated hepatocytes (Figure 3). On the contrary, optical micrograph of the liver tissue section of rat in HCC group treated with gallic acid shows congestion in the portal vein with inflammatory cell infiltration as well as multiple newly formed bile ductules (Figure 4). Optical micrograph of the cross-sectioned liver tissue of rat in HCC group treated with doxorubicin reveals severe congestion in the portal vein with a focal degeneration of hepatocytes (Figure 5).

Figure 2.

Photomicrograph of liver tissue section of negative control rat shows normal histological structure of central vein (CV) and surrounding hepatocytes (h) in the parenchyma (H&E, 16×).

Figure 3.

Photomicrograph of liver tissue section of untreated HCC-bearing rat shows fibrosis (f) in the portal area between the cystic bile duct (a) which extended and divided the hepatic parenchyma into dysplastic and degenerated hepatocytes (H&E, 16×).

Figure 4.

Photomicrograph of liver tissue section of HCC-bearing rat treated with gallic acid shows congestion in portal vein (PV) with inflammatory cells infiltration (m) in portal area with multiple number of newly formed bile ducts (H&E, 16×).

Figure 5.

Photomicrograph of liver tissue section of HCC-bearing rat treated with doxorubicin shows severe congestion in portal vein (PV) with degeneration in the hepatocytes (d) (H&E, 16×).

Discussion

Natural products have aroused a great attention in the past 30 years owing to its interesting characteristics in tackling cancer. Thus, they represent good candidates for developing new chemopreventive and anticancer agents. The present setup indicated that oral administration of gallic acid could modulate the activities of tumor markers and counteract HCC induced by NDEA in the experimental rats.

The metabolic activation of NDEA in the liver results in the generation of the reactive oxygen species (ROS) responsible for extensive oxidative stress. Such oxidative stress mainly contributes to hepatocarcinogenesis through several mechanisms including DNA adduct formation, lipid and protein damage, and aberration of several signaling pathways especially that implicated in HCC.¹⁹

The current data revealed that administration of NDEA significantly increased serum AFP level. This finding is highly confirmed by the study of Song et al.²⁰ AFP has been long used as a standard diagnostic marker for HCC screening.²¹ AFP is a fetal glycoprotein whose synthesis is restricted only to the fetal liver and the yolk sac during pregnancy. Actually, its serum level shows an extensive drop directly after the birth and its synthesis is ceased in the adult liver.²² Such observed motivation in serum AFP level is most likely due to the upregulation of AFP gene in NDEA-administered rats due to necrosis of hepatocytes.²³ Moreover, the resynthesis of AFP by neoplastic hepatocytes may be attributed to the aberrant upregulation of nuclear factor kappa B (NF-κB) known to be involved in hepatocarcinogenesis.²⁴ NF-κB is a transcription factor regulating the expression of many genes implicated in cell proliferation, inflammation, cell death resistance, and carcinogenesis such as cyclooxygenase-2 (COX-2).²⁵ A former study of Cui et al.²⁶ demonstrated that the inflammation resulted from COX-2 upregulation can enhance AFP production by neoplastic hepatic cells.

GPC-3 is a heparan sulfate proteoglycans that coordinate the signaling pathways regulating cellular growth and differentiation.²⁷ GPC-3 serum level was found to be significantly enhanced in HCC group. This result is in consistent with that of Suzuki et al.²⁸ Chan et al.²⁹ suggested that the GPC-3 gene amplification in HCC could be owed to c-Myc gene overexpression. That is because the transcription factor c-Myc has been found to be the main regulator of GPC-3 gene expression through its direct binding with GPC-3 gene promoter. Moreover, it has been demonstrated that STAT3 indirectly plays a critical role in stimulating GPC-3 expression via stimulating the expression of c-Myc.²⁸ Overall, these data indicate that c-Myc may potentiate the proliferation of neoplastic heaptocytes via upregulating the GPC-3 pathway through increasing its messenger RNA (mRNA) and protein levels.²⁷ Furthermore, Capurro et al.³⁰ concluded that GPC-3 can augment the progression of HCC via activating Wnt signal transduction through mediating its binding with its receptors.

The current findings showed that NDEA administration elicited significant rise in serum STAT3 level. STAT3 is considered as one of the major signaling pathways mediating the injury-inflammation-regeneration cycle associated with chronic liver diseases and HCC development.³¹ High levels of interleukin-6 (IL-6) were found to be closely related with the development of HCC through promoting its hepatic cancer cell survival via activating STAT3.³² STAT3 acts as an intracellular signal transducer and a transcriptional activating factor that is activated in response to proliferative stimuli like many cytokines such as IL-6 or growth factors like epidermal growth factor (EGF). STAT3 motivation involves its phosphorylation at its Tyr-705 residue by different kinases such as Janus kinases (JAK), followed by its homodimerization and nuclear translocation ending up with binding to its DNA regulatory sequences upstream its target genes leading to their transcriptional regulation. STAT3 was found to potentiate proliferation, angiogenesis, and cell survival–related genes such as cyclin D1, c-Myc, and survivin. Moreover, STAT3 activation has been found to be turned off by several proteins such as tyrosine phosphatases (SHP1 and SHP2) and suppressor of cytokine signaling.³³

Tabulated results recorded significant reduction regarding serum SOCS3 level in HCC-bearing rats. This result is in coherence with that of Ji et al.³⁴ SOCS3 has been described as a negative regulator of JAK/STAT pathway.³⁵ It has been found to be epigenetically silenced in many cancer types including HCC.³⁶ Moreover, Ogata et al.³⁷ mentioned significant decline in SOCS3 mRNA levels in cancerous tissues than in non-cancerous ones in more than 90% of HCC patients. Hence, its downregulation may be correlated with HCC progression and poor prognosis. Such reduction in serum SOCS3 levels may be resulted from the aberrant mechanism of IL-6/STAT3 signaling in HCC. Since it has been indicated that activated IL-6/STAT3 signal pathway might induce SOCS3 hypermethylation via increasing the activity of DNA methyltransferase-1 (DNMT1) resulting in the progression of cancer and metastasis.³⁸

On the opposite side, gallic acid administration in HCC group significantly dampened the elevation of the serum AFP level. The proposed mechanism by which gallic acid could recover AFP serum level may stem from the suppression of COX-2 gene expression by gallic acid treatment, which is known to modulate the transcription of AFP. It has been shown that gallic acid inhibits the expression of COX-2 gene via suppressing the activity of NF-κB in the colon tissue of ulcerative colitis-induced mice.³⁹ Moreover, these investigators speculated that gallic acid administration may lead to a decline in the protein synthesis by the hepatic tumor cells owing to its antitumor efficacy which in turn might be responsible for tumor growth shrinkage leading to such regression in AFP synthesis by tumor cells. This suggestion is highly supported by Ji et al.⁴⁰ who demonstrated that gallic acid treatment results in the suppression of lung xenograft tumor growth in a murine model.

In light of our results, treatment of NDEA-challenged rats with gallic acid experienced significant depletion in serum GPC-3 level. The current setting postulated that the observed decline in GPC-3 serum level by gallic acid administration could be attributed to its antitumor activity exerting inhibition of tumor size which consequently results in a suppression of protein synthesis by tumor cells. Such hypothesis is validated by the study of Banerjee et al.⁴¹ which revealed that pomegranate juice, which has been found to be enriched with gallic acid, repressed the number of aberrant colorectal crypt foci in azoxymethane-induced rats owing to its anti-inflammatory and anticancer activity. Moreover, gallic acid could contribute to the attenuation of oxidative stress resulted by NDEA-metabolic products due to its antioxidant properties.⁴² This in turn may prevent hepatic cell injury caused by NDEA and hence hinder the liberation of such tumor markers into the serum.

On the other side, the treatment of HCC group with gallic acid caused significant drop in serum STAT3 level. This effect could be explained by the antioxidant and antiproliferative potentials of gallic acid.⁴³ Moreover, this finding is in respect with Pandurangan et al.³⁹ who showed that gallic acid blocks the activation and nuclear accumulation of phosphorylated STAT3, preventing the degradation of the inhibitory protein IκB and suppressing the nuclear translocation of p65-NF-κB. Furthermore, such study mentioned that gallic acid significantly downregulates the gene expression level of IL-6 which in turn hinders the activation of STAT3. These findings postulated that gallic acid exerts anti-inflammatory effects through the retraction of NF-κB and IL-6/p-STAT3 activation.

In this study, treatment of HCC group with gallic acid significantly amplified serum SOCS3 level. The study of Kuppan et al.⁴⁴ showed parallelism with our study. These authors proved the upregulation of SOCS3 gene expression by gallic acid treatment in high-glucose treated human monocytes (THP-1) through inhibiting NF-κB signaling pathway and modulating the tumor necrosis factor alpha (TNF-α) and IL-6 gene expression levels. Therefore, our study suggests that the observed increase in the serum SOCS3 levels by gallic acid may be ascribed to its anti-inflammatory action via hampering NF-κB and JAK/STAT signaling pathways and subsequent cytokine and chemokine expressions.

GGT is an enzyme responsible for the degradation of extracellular glutathione, supplying the cells with amino acids required for synthesis of new intracellular glutathione compounds.⁴⁵ GGT is mainly secreted in serum by hepatic Kupffer cells and endothelial cells of bile ducts, and its activity was shown to be elevated in patients with HCC and chronic liver disease.⁴⁶ In this study, NDEA administration elicited significant amplification in hepatic GGT levels in HCC group. This result is in harmony with that reported by Carrasco-Legleu et al.¹³ Such elevation in the expression of this gene reflects the toxic effect of NDEA on liver tissue. NDEA has been shown to be metabolized by cytochrome P-450 generating free radicals in the liver which in turn overwhelms the antioxidant status causing progressive oxidative stress that triggers carcinogenesis.⁴⁷ It has been suggested that redox regulation of many genes in response to ROS/electrophiles may result in modulation of GGT expression levels.⁴⁸ Moreover, a study by Jayakumar et al.⁴⁹ documented that HCC-bearing rats experienced significant perturbation in glutathione levels owing to its exhaustion in scavenging free radicals released upon metabolic activation of NDEA. Such reduction in glutathione activity may promote GGT activity in hepatic tissue via stimulating its mRNA transcriptional level.⁵⁰

The current data indicated that NDEA administration resulted in significant overexpression of HSPgp96 level in hepatic tissue. This finding is in consensus with that of Wang et al.⁵¹ who found that HSPgp96 is highly expressed during HCC progression. Carcinogenic agents trigger the expression of HSPgp96 causing a malignant transformation through inhibiting hepatocytes apoptosis.¹⁴ The stimulation of HSPgp96 gene expression by NDEA administration in HCC group could be resulted from the formation of protein adducts through interaction of NDEA metabolites with proteins eliciting HSP activation.⁵² Moreover, such oxidation products induce heat-shock transcription factor (HSF)-1 activity which in turn promotes HSP gene transcription.⁵³

Conversely, treatment of HCC-bearing rats with gallic acid caused significant downregulation in the expression level of hepatic GGT gene. This finding goes hand in hand with that observed by Olayinka et al.⁴² This decrement in GGT expression level could be attributed to the free radicals scavenging potency of gallic acid,⁵⁴ hence preventing hepatic damage caused by NDEA oxidation products and minimizing the consequent oxidative stress. Also, the antioxidant properties of gallic acid may lead to increasing serum level of glutathione which is known to modulate GGT expression levels.

Data in this study indicated that gallic acid–treated group exhibited significant downregulation in the HSPgp96 gene expression level in the hepatic tissue. This effect could be owed to the antioxidant properties of gallic acid⁵⁵ which lead to the suppression of the oxidative stress resulting from free radicals generated during NDEA metabolism. Such modulation of oxidative stress may be responsible for the observed downregulation in HSPgp96 gene expression due to the inactivation of HSF-1 which is known to promote HSPgp96 gene expression.⁵⁶

The histopathological features of liver tissue displayed by the untreated HCC group come in line with those described by the study of Seufi et al.,⁵⁷ whereas those revealed by gallic acid–treated group indicated a remarkable improvement in the liver tissue architecture owing to the reversion of the detrimental effects of NDEA intoxication by gallic acid. Such effect could be attributed to the antioxidant, chemopreventive, anti-inflammatory, and antiproliferative properties of gallic acid.⁵⁸

Our results revealed that treatment of HCC-bearing rats with doxorubicin evoked significant decrease in serum AFP, GPC-3, and STAT3 levels accompanied with significant increase in the serum SOCS3 level. Furthermore, doxorubicin treatment produced significant downregulation in the expression level of hepatic GGT and HSPgp96 genes, while the histological investigation of the liver tissues of HCC-bearing rats treated with doxorubicin showed severe congestion in the portal vein with focal degeneration of hepatocytes. Findings as such might go back to the antiproliferative and apoptotic effects exerted by doxorubicin.⁵⁹ Kusuzaki et al.⁶⁰ reported that doxorubicin treatment results in decreasing the volume of the malignant soft tissue tumors. Moreover, Zanini et al.⁶¹ declared that high doxorubicin concentration causes a progressive downregulation in HSP expression in neuroblastoma and Ewing’s sarcoma cell lines. There are two proposed mechanisms by which doxorubicin could act on cancer cells; intercalation into DNA and disruption of topoisomerase II mediated DNA repair and generation of free radicals with their damaging impact on cellular membranes, DNA, and proteins.⁶² Denard et al.⁶³ reported that doxorubicin suppresses the cellular proliferation via promoting de novo synthesis of ceramide, which in turn activates the transcription factor (CREB3L1). Doxorubicin was shown to stimulate the proteolytic cleavage of CREB3L1 by site-1 protease and site-2 protease, permitting the N-terminus domain to be translocated to the nucleus where it activates the transcription of genes encoding cell cycle inhibitors such as P₂₁.

Conclusion

Generally speaking, gallic acid offers a multitalented therapeutic approach in retracting the aggressiveness of HCC in rat model. We suggest that the regulation of STAT3 signaling pathway via the outstanding bioactivities of gallic acid including antioxidant potential, anti-inflammatory effect, apoptotic action, and antitumor impact may be the probable mechanism by which it can offer its therapeutic action against HCC. These findings could pave the way for the development of functional compound from gallic acid–rich plants for mitigation of hepatic carcinogenesis.

Footnotes

Acknowledgements

The authors express sincere appreciation to Prof. Adel Bakeer Kholoussy, Professor of Pathology, Faculty of Veterinary Medicine, Cairo University, for his kind cooperation in conducting histological examination in this study.

Declaration of conflicting interests

The author(s) declared no potential conflicts of interest with respect to the research, authorship, and/or publication of this article.

Funding

The author(s) disclosed receipt of the following financial support for the research, authorship, and/or publication of this article: This work was supported and funded by research project grant (ID 10010106) from National Research Centre, Giza, Egypt.

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