The Role of Thymoquinone in Mitigating Carbon Tetrachloride-Induced Hepatocellular Carcinoma in Rats: Targeting the CHOP-1/JNK/P38 MAPK, NFĸB/TNF-α/IL-10, and Bax/Bcl-2/Caspase-3 Signalling Pathways

The present study was conducted to evaluate the effect of thymoquinone (TQ) on hepatocellular carcinoma (HCC) in rats. Our study has reported that TQ treatment of experimentally-induced HCC results in the up-regulation of the Jun-N-terminal kinase and p38 mitogen activated protein kinase pathway (JNK/p38 MAPK) and the enhancement of anti-inflammatory, anti-oxidant, and pro-apoptotic machineries. TQ resulted in a significant decrease in the levels of nuclear factor kappa-light-chain-enhancer of activated B-cells (NFêB), tumor necrosis factor-á (TNF-á), and a significant increase in the anti-inflammatory interleukin-10 (IL-10). The pro-apoptotic effect of TQ was demonstrated through stimulating the apoptotic Bcl-2-associated X (Bax) gene and inhibiting the anti-apoptotic B-cell lymphoma 2 (Bcl-2) gene together with increasing the level of caspase 3 and up-regulating the C/EBP homologous protein (CHOP-1) gene expression. TQ treatment also enhanced the activity of the ROS scavenger, superoxide dismutase (SOD), and decreased the level of the lipid peroxidation product malondialdehyde (MDA). TQ-dependent suppression of HCC was associated with the up-regulation of JNK/p38 MAPK, enhanced CHOP-1 expression, and subsequently increased Bax gene expression.

Hepatocellular carcinoma (HCC) accounts for 85-90% of all primary liver cancers and is the third leading cause of cancer-related deaths worldwide (OZAKYOL 2017). Endoplasmic reticulum (ER) stress can lead to ER dysfunction in the terminal phase (BHAT et al. 2013), thus triggering an unfolded protein response (UPR). The UPR has a dual function in cells. One function is to alleviate damage and promote homeostasis in cells, and the other is to induce a pro-apoptotic effect in cells exposed to chronic or overwhelming ER stress (WANG et al. 2014).
The key regulator of ER stress-induced apoptosis is the C/EBP homologous protein (CHOP). Generally, the role of autophagy in liver cancer is dynamic and changeable (CHANG et al. 2012). CHOP can also regulate the Bax and Bcl-2 family. Thus, overexpression of CHOP can induce cell cycle arrest leading to cell apoptosis; even more CHOP-deficient cells were resistant to ER stress-induced apoptosis (HU et al. 2019).
Both c-Jun NH 2 -terminal kinase (JNK) and the p38 mitogen activated protein kinase family (p38 MAPK) are the upstream regulators of CHOP. They cooperate together to regulate apoptosis (PARK et al. 2014). P38 dis-regulation is implicated in the development of solid tumors, such as lung and liver cancers (IYODA et al. 2003). Both in-vivo and in-vitro studies have demonstrated that the initiation of HCC was inhibited upon activation of the reactive oxygen species/p38 MAPK pathway (CHIBA et al. 2014).
The potential anti-neoplastic role of thymoquinone (TQ) is mediated via inhibition of cell proliferation, the activation of cell cycle arrest, and the triggering of anti-inflammatory, pro-apoptotic, and anti-oxidant mechanisms (HARON et al. 2018). The contribution of oxidative stress in promoting hepatocarcingenesis was confirmed. In the ovarian cancer cell line, the anti-cancer effect of TQ involves the activation of apoptosis and cellular oxidative stress. In renal carcinoma, Caki cell lines incubated with TQ (75 µM), the key anti-apoptotic factor, Bcl-2, was suppressed in a dose dependent manner causing apoptosis (PARK et al. 2016).
Thus, the present study was designed to evaluate the efficacy of using thymoquinone in experimentallyinduced HCC and to assess the hepatocellular signalling of JNK/p38 MAPK and the downstream CHOPregulated pathways.

Materials and Methods
This study was performed at the Unit of Molecular Biology at the Medical Biochemistry and Molecular Biology, Physiology, Anatomy and Embryology Departments. The current protocol was revised and approved by the Institutional Animal Care and Use Committee (CU-IACUC), (approval No. CU/III/F/23/18). All procedures and animal experiments were carried out in accordance with the international Helsinki guidelines.

Animals
Twenty four female adult albino rats were included in this study, inbred strain (Cux1: HEL1) and of matched age (6 months -1 year) and weight (200-250 g). All animal experiments were performed according to the Guide for the Care and Use of Laboratory Animals published by the US National Institutes of Health (NIH publication No.85-23, revised 1996), and according to the standard guidelines of the Institutional Animal Care and Use Committee and after Institutional Review Board approval. Animals were maintained in an air-conditioned animal house with specific pathogen free conditions. Animals had free access to standard laboratory food and water ad libitum.

Experimental design
After acclimatization, the animals were divided into 3 groups (8 rats/group). Group I (control) served as a negative control group (normal healthy rats). The remaining sixteen rats were injected with diethylnitrosamine (DEN) and carbon tetrachloride (CCl 4 ) for induction of HCC (RAGHUNANDHAKUMAR et al. 2013). Then, they were subdivided into 2 groups: Group II (HCC): experimental HCC group (pathological control) and Group III (HCC-Thymoquinone): HCC rats received a single intraperitoneal injection of thymoquinone (TQ) (20 mg/kg body weight) (KE et al. 2015) after induction of HCC. The rats were euthanized by cervical decapitation 48 hours after giving TQ.

Induction of hepatocellular carcinoma
Hepatocarcinogenesis was induced chemically by injecting a single intra-peritoneal dose of diethylnitrosamine at a dose of 200 mg/kg body weight, followed by weekly single subcutaneous injections of carbon tetrachloride (CCl 4 ) at a dose of 3 ml/kg body weight for 6 weeks (RAGHUNANDHAKUMAR et al. 2013) (Fig. 1).

Reagents:
Diethylnitrosamine (DEN): purchased from Sigma-Aldrich Egypt, number C1900 in a solution form and was given diluted with castor oil at a ratio 1:1.
Thymoquinone (TQ) (2-isopropyl-5-methyl-1,4benzoquinone): was obtained from (Sigma-Aldrich, Tokyo, Japan) and was prepared as a 100 mM solution and stored at -30°C until use. Olive oil (Ajinomoto, Japan) was used for dissolving the TQ. The TQ solution was prepared by dissolving it in 0.5% dimethyl sulphoxide (DMSO) followed by the addition of olive oil as previously described (ABUKHADER 2012;EL-SAYED et al. 2019).
At the planned time, blood was collected from each rat, for assessment of serum alanine transaminase (ALT) and aspartate transaminase (AST), and alpha fetoprotein (AFP).
The second part was fixed overnight in 40 g/l paraformaldehyde in PBS at 4°C for a histopathological R.E. HUSSEIN et al. examination by hematoxylin and eosin (HE) stained sections.

Estimation of serum ALT and AST levels
Using a spectrophotometer, ALT and AST activity values were detected from the calibration curve and were expressed in U/l.

Estimation of AFP using ELISA (Uscn, Life science Inc, UK)
A microtiter plate coated with an antibody specific to AFP was used. Then according to manufacturer protocol, the substrate reaction was spectrophotometrically measured.

Estimation of SOD and MDA in rat liver tissue using colorimetry
After tissue homogenization, the samples were centrifuged; the supernatant was removed for assay and stored on ice. SOD Activity (U/g tissue) was estimated according to manufacturer instructions and the estimated values of MDA are expressed in mmol/mg and compared to the reference standard according to manufacturer protocol.
Estimation of caspase-3, NFêB, TNF-á and IL-10 levels in rat liver tissue using ELISA (eBioscience, San Diego, CA, USA) The provided microtiter plate was pre-coated with an antibody specific to caspase-3, NFêB, TNF-á, and IL-10. Standards or samples were then added. Streptavidin conjugated to Horseradish Peroxidase (HRP) was added to each microplate well and incubated. After TMB substrate solution was added, only those wells that contained (caspase-3, NFêB, TNF-á, and IL-10) biotin-conjugated antibody, and enzymeconjugated avidin would exhibit a change in color. The enzyme-substrate reaction was terminated by the addition of an acid solution and the color change was measured using spectrophotometry at a wavelength of 450 nm. The concentration of (Caspase-3, NFêB, TNF-á, and IL-10) in the samples was then determined by comparing the O.D. of the samples to the standard curve according to manufacturer's instruction.
qRT-PCR gene expression of CHOP, Bax, and Bcl-2 in rat liver tissues After homogenization, total RNA was extracted from liver. The bound total RNA was further purified by simple washing steps. Finally, the total RNA was eluted from the membrane by the addition of Nuclease-Free Water. The yield of total RNA obtained was determined spectrophotometrically at 260 nm. Total RNA (0.5-2 ìg) was used for cDNA conversion using a high capacity cDNA reverse transcription kit (#K1621, Fermentas, USA). Real-time qPCR amplification and analysis were performed using an Applied Biosystem with software version 3.1 (StepOne™, USA) as previously described (AZIZ et al. 2014). After the RT-PCR run, the data were expressed in Cycle threshold (Ct). The PCR data sheet includes the Ct values of the assessed genes (CHOP, Bax, and Bcl-2) and those related to the reference housekeeping gene (â-actin gene).
Estimation of JNK and p38 MAPK using the Western Blot Technique RIBA lysis buffer PL005 was provided by Bio BASIC INC. (Marhham Ontario L3R 8T4 Canada). The lysis buffer was added to the tissues with an additional protease inhibitor and phosphatase inhibitor buffer to maintain protein integrity and high biological activity. The supernatant was transferred to a new tube for further protein concentration determination analysis. A Bradford Protein Assay Kit (SK3041) for quantitative protein analysis was provided by BIO BASIC INC. Markham Ontario L3R 8T4 Canada. A Bradford assay was performed according to manufacture instructions. Then, the proteins were separated by polyacrylamide gel electrophoresis. The membrane was using tris-buffered saline with Tween 20 (TEST) buffer and 3% bovine serum albumin (BSA) at room temperature for 1 hr. Samples were then incubated overnight in each primary antibody solution, against the blotted target protein (anti JNK antibody and anti p38 MAPK antibody) at 4°C. Incubation was done in a HRP-conjugated secondary antibody (Goat antirabbit IgG-HRP-lmg Goat mab -Novus Biologicals) solution against the blotted target protein for 1 hour at room temperature. Chemiluminescent substrate (Clarity TM Western ECL substrate -BIO-RAD, USA cat#170-5060) was applied to the blot according to the manufacturer's recommendation. The chemiluminescent signals were captured using a CCD camera-based imager. Image analysis software was used to read the band intensity of the target proteins against the control sample after normalization by beta actin on the Chemi Doc MP imager.

Histopathological examination
The liver from all groups was dissected and immediately fixed in 10% formol saline. The specimens were impregnated in paraffin. Serial sections of 5-ìm thickness were cut and subjected to hematoxylin and eosin staining and histo-pathological examination.

Statistical Analysis
Data were coded and analyzed using the Statistical Package for Social Science (SPSS) version 22 (IBM Corp., Armonk, NY, USA) and were tested for normality using the Shapiro-Wilk test and proved to not be deviated from normal distribution. Data were summarized using mean and standard deviation. Statistical significance of the differences between the mean values of the different groups was tested using Twoway analysis of variance (ANOVA) followed by Bonferroni multiple comparison post-hoc testing when comparing more than 2 groups for appraisal of statistical significance. Probability values of less than 0.05 (p < 0.05) were considered statistically significant.

Improved liver function biomarkers and alphafetoprotein (AFP) with thymoquinone
Deterioration of liver function was observed in the HCC group and detected by a significant increase in ALT and AST levels in the HCC group compared to the normal control (p <0.001). Significant increase in AFP level was observed in the HCC group compared to the normal control (p <0.001). However, TQ resulted in a significant decrease in AFP level compared to the HCC group and a significant decrease in ALT and AST levels compared to the HCC group (p <0.001) ( Table 2).

Interference with oxidative stress secondary to thymoquinone intake
Combined DEN and CCL 4 used for induction of HCC could enhance oxidative stress in the HCC group. Data showed a significant increase in MDA and a significant decrease in SOD in the HCC group compared to the normal control (p <0.001), however, the intake of TQ significantly reversed these changes compared to the HCC group (p <0.001) ( Table 2).

Thymoquinone enhances the host defence strategy by regulating inflammatory machinery
Data showed a significant decrease in levels of IL-10 while levels of NFêB and TNF-á were significantly increased in the HCC group compared to the normal control (p <0.001). Intake of TQ resulted in a significant increase in IL-10 and a significant decrease in NFêB and TNF-á as well compared to the HCC group (p <0.001) (Fig. 2).

Table1
Primers Sequence of studied genes CHOP, Bax, Bcl-2, and the housekeeping gene beta actin

Enhanced apoptotic process in the livers of HCC rats after treatment with thymoquinone
The key apoptotic executor, caspase-3 showed a significant increase in the HCC-thymoquinone group compared to the HCC group (p <0.001). A significant increase in Bcl-2 gene expression was also observed in the HCC group compared to the normal control (p <0.001). Whereas, levels of Bax gene expression were significantly increased in the TQtreated groups compared to both the control and HCC groups (p <0.001) ( Table 3). The data also demonstrated a significant decrease in CHOP gene expression in the HCC and HCC+TQ groups compared to the normal control (p <0.001 and p = 0.029, respectively). However, a significant increase in CHOP gene expression was observed in the TQ-treated group compared to HCC group (p <0.001) ( Table 3).

Thymoquinone upgrades the expression of p38 MAPK and JNK in liver tissues of induced HCC
The band intensity of estimated p38 MAPK protein showed a significant increase in the HCC group and TQ-treated group compared to the normal control (p <0.001). Also, the result showed a significant increase in JNK protein expression in the TQ-treated groups compared to the control and HCC group (p <0.001) (Fig. 2).
Thymoquinone improves the hepatic histo-patholoical changes of experimentally-induced HCC Combined DEN and CCL 4 induced pleopathological architectural and nuclear changes including nuclear atypia, pleomorphism, hyperchromatosis, and scattered mitotic figures. Thymoquinone treatment ameliorated those deleterious effects where the hepatic tissue displayed improved structure with only minimal desmoplasia and localized clear cell changes (Fig. 3).

Discussion
Thymoquinone (TQ) is the main constituent in Nigella sativa; it could exert multiple anticancer effects (WOO et al. 2012). The anti-cancer effect of TQ is mediated through induction of apoptosis and cell cycle arrest (MAJDALAWIEH & FAYYAD 2016). In agreement with our results, TQ can ameliorate CCl 4induced hepatotoxicity through suppression of lipid peroxidation, ALT and AST cleavage, and induction of caspases (RAHMANI et al. 2019). TQ treatment has also been reported to up-regulate JNK phosphorylation in human colon cancer cells (EL-NAJJAR et al. 2010) which is also concordant with our results.
Decreased expression of caspase-3 also correlates with a poor prognosis of HCC, which emphasizes the role of caspase-3 in the pathogenesis of the disease. The deficiency of caspase-3 may enhance p38 activa- tion in the liver tissues of mice treated with DEN by increasing the expression of TNFá (SHANG et al. 2018). In this regard, current results reported a significant decrease in caspase-3, and an increase in TNFá levels as well as p38 MAPK in the HCC group compared to the control group. Deregulated MAPK signalling pathways are often implicated in various types of human tumors, as HCC (KIM et al. 2017), p38 MAPK negatively regulates cell cycle progression and oncogene-induced premature cell senescence thus; it acts as a tumor suppressor factor (BULAVIN & FORNACE 2004). ROS could augment and activate p38 MAPK (LIU & CHANG 2009). Moreover, TQ was found to increase p38 protein expression in liver tissues, and suppress the antiapoptotic/pro-survival Bcl-2 gene expression as well. In this context, Torres and colleagues (TORRES et al. 2010) related the enhanced apoptosis in pancreatic cancer cells by TQ to its promotion of p38 MAPK and JNK signalling pathways.
JNK deficiency in liver tissues could increase hepatocyte death leading consequently to increased compensatory proliferation and HCC development (DAS et al. 2011). Notably, in CPT-11-R LoVo colon cancer cells, thymoquinone intake could activate JNK and p38 thereby inducing apoptosis before autophagy-mediated cell death (CHEN et al. 2015).
Excessive endoplasmic (ER) reticulum stress increases the C/EBP homologous protein (CHOP) marker, which is a major pro-apoptotic factor (RON & WALTER 2007). ER stress has crucial roles in mitochondrial dysfunction by augmenting CHOP expression. Incubating bladder cancer cells with TQ has also been reported to induce cytotoxicity and apoptosis, mainly through overexpression of CHOP, increasing Bax/Bcl-2 ratio and augmenting caspases (ZHANG et al. 2018).
Following exposure to cytotoxic agents, Bax undergoes oligmerization in the mitochondria and endoplasmic reticulum membranes, allowing for the permeabilization of these membranes, and then re-leases activators of the final effectors of apoptosis such as cytochrome c and SMAC/Diabalo (SHARPE et al. 2004;ANNIS et al. 2005). Thus, preventing Bax oligomerization abrogates apoptosis. However, decreased Bcl-2 could allow Bax oligomerization to induce apoptosis (DLUGOSZ et al. 2006). Previous studies demonstrated that CHOP promotes apoptosis by decreasing Bcl-2 expression (LIU & CHANG 2009). Our results also document enhanced CHOP expression and decreased Bcl-2. Thus, down-regulating the key factor of cell life and death is a promising target in cancer therapy.
One of the therapeutic potentials of thymoquinone is its immune-regulatory effect against HCC induced by DEN (BIMONTE et al. 2019). In hepatocytes, NF-êB has a contradictory effect being pro-inflammatory and anti-apoptotic, which may exert a negative impact on hepatocytes. In addition, nuclear translocation of NF-êB inhibits JNK activation (LUEDDE & SCHWABE 2011). TQ is able to suppress TNF-á-induced NFkappa B activation (BIMONTE et al. 2019). Activation of NF-êB components is involved in hepatic injury, fibrosis, and subsequent carcinogenesis (LUEDDE & SCHWABE 2011). Even more, JNK activation could promote regression of the NF-êB-induced antiapoptotic factors, thus data obtained indicate that TQ may enhance cancer cell apoptosis which could decrease tumor resistance to chemotherapy and radiotherapy. Taken together, our findings suggest that TQ could be considered as a potential drug for targeting hepatocarcinogenesis via activating JNK, p38 MAPK, and enhancing production of pro-apoptotic factors including the final effector of apoptosis, caspase-3. Table 3 Levels of caspase-3 and the relative gene expression of Bax, Bcl-2, and CHOP-1 in all studied groups. Values presented as mean ±SD (n=8) *: statistically significant compared to corresponding value in group I and #: statistically significant compared to corresponding value in group II Parameter Group 1 (normal control) Group 2 (HCC) Group 3 (HCC-Thymoquinone)