Introduction

Hepatocellular carcinoma (HCC) is a primary malignancy of the liver, occurs predominantly in patients with underlying chronic liver disease, and is the most common cause of death in people with cirrhosis (Satir, 2007). HCC is now the second leading cause of cancer deaths worldwide (Marquardt, Andersen, & Thorgeirsson, 2015). The occurrence of HCC is most elevated in Asia and Africa, where the endemic high commonness of hepatitis B and hepatitis C firmly inclined to the advanced of chronic liver disease and following the development of HCC (Chatterjee & Mitra, 2015).

Angiogenesis (spreading and formation of new blood vessels) plays a critical role in physiological and pathological human conditions (Kim & Byzova, 2014) and results from an imbalance between proangiogenic factors and inhibitors (Mekuria & Abdi, 2017). VEGF activates VEGFR, which will activate downstream pathways lead to stimulates the release of endothelium-derived nitric oxide (NO) from venous endothelial cells, and up-regulates the expression of nitric oxide synthase (NOS), which lead to vasodilation in physiological and pathological angiogenesis (Simons, Gordon, & Claesson-Welsh, 2016).

Vascular endothelial growth factors (VEGF) and the related receptors seem to play a critical role in cancer development and metastasis (Olsson, Dimberg, Kreuger, & Claesson-Welsh, 2006; Sullivan & Brekken, 2010), and they are critical for HCC growth and development (Morse et al., 2019).

In the course of the most recent decade, FDA was approved many anti-angiogenic agents targeting VEGF for the treatment of cancers, as bevacizumab, sorafenib, and pazopanib, etc. (Meadows & Hurwitz, 2012). The most common problems for these approved drugs that it's expensive, often show a poor biodistribution and pharmacokinetic profile, multidrug resistance and having severe side effects (e.g., hypertension, proteinuria, thrombosis, bleeding, etc.) (Ebos & Kerbel, 2011). Moreover, these inhibitors developed multidrug resistance and enhanced invasiveness during treatments that have limited effects on the overall survival, and authenticated prognostic biomarkers are unavailable for observing the response to a treatment (Ellis & Hicklin, 2008). Cancer is a complex disease that involves many pathways and biological mechanisms, in other hand phytochemicals (plant extracts) containing a lot of compounds which act on different biological mechanisms and pathways at the same time which make it suitable field for searching for anticancer treatment (Singh, Sharma, Kanwar, & Kumar, 2016).

Brassicaceae is one amongst the major spermatophyte families with around 338 genera and over 3709 species distributed worldwide (Al-Shehbaz, Beilstein, & Kellogg, 2006). Regarding fifty-three genera and 107 species distributed in Egypt (Boulos, 1999). It is the 4^th of 11 large families that are extensively spread in all phytogeographic regions within the Egyptian flora (Boulos, 1995). Historically, Plants of this family were usually utilized for their antidiabetic, antibacterial, antifungal, antirheumatic, and anticancer action (Rizk, 1986). The genus Erucaria is delineated by four species that grow in Egypt; Erucaria crassifolia, Erucaria hispanica, Erucaria microcarpa, and Erucaria pinnata.

In the present study, we try to evaluate the antitumor activity of methanol extract of Erucaria pinnata plant against HCC with antiangiogenic effect targeting on VEGF/VEGFR-2 in vitro and in vivo studies, our promising result have pushed us to investigate the phytochemical constituent of the extract using HPLC and to try to understand the molecular biology mechanism behind this activity using docking study focusing in the anti-angeogenisis activity of this extract.

Materials and Methods

Chemicals and Kits

Chemicals and reagents were high analytical grade, namely Aldrich-Sigma Chemical (St. Louis MO, USA) & ADWIC, Egypt. The VEGF-R₂/KDR ELISA kit was Biovendor (Brno, ‏Czech Republic) and VEGF-A ELISA Kit EIAab (Shanghai, China).

Plant collection and preparation of the extracts

The fresh aerial parts of Erucaria Pinnata (Viv.) were collected at its growth period of spring season from the North-Western Coastal region in Egypt. The plant was air-dried at lab-temperature till constant weight, then ground to a fine powder and kept to be used for different plant analysis. Two kg of plant powder were extracted by percolation with a minimum amount of 70% methanol and purified according to (Graf, Von, Mabry, Markham, & Thomas, 1970; Harborne, 1984). The slurry was allowed to stand for 24 h with occasional stirring and then filtered off. The residue was repeatedly extracted with an excess volume of 70% methanol. Collected filtrates were evaporated under reduced pressure using rotavapour apparatus at a temperature of 55 ºC. Then divided the residue into two parts the first part (E1), the second part was fractionated successively with petroleum ether (E2), chloroform (E3),ethyl acetate (E4) and methyl alcohol 96% (E5) using percolation, each fraction was evaporated under reduced pressure till dryness at 45°C to get sick mass. All the dried residues were stored in the refrigerator at 5 ºC until the use.

Qualitative and Quantitative Determination of Flavonoids Using HPLC

The determination of flavonoids was done using HPLC (Mattila, Astola, & Kumpulainen, 2000). HPLC Agilent 1100 series with the Quaternary pump was used. The flow rate was set at 1 ml/min. The temperature of the column compartment, degaser, and autosampler was adjusted at 35ᵒC. The detector set at wavelength 330 nm for flavonoid compounds. Hypersil column (5µm, 250x4 mm) was used. The standard which used were purchased from El-Gomhoria-chemical company, Egypt, and the work was done in the Food Technology Research Institute, Agric. Res. Center, Giza, Egypt.

Mammalian cell lines

We used the human hepatocellular cancer cell line (HepG2 cells). These lines were obtained from the Pharmacological Unit in the tumor biology department in the National Cancer Institute, Cairo University, and propagated according to the standard protocol (Hay, 2000).

Cytotoxic activity by colorimetric sulphorhodamine-B (SRB) assay

Cell viability was measured according to the method reported by (Skehan et al., 1990). Cells were seeded in 96 well microtiter plates at a concentration of

1000-2000 cells/well, 100μl/well. After 24 hr. Cells will be incubated for 72 hr. with various concentrations of plant extract (0, 5,12.5,25,50 µg/ml). Following 48 hr. The medium was discarded and fixed with 10% trichloroacetic acid 150 µl/well for 1hr. at 4ºC. Then Wells were stained for 10-30 minutes at room temperature with 0.4% SRB dissolved in 1% acetic acid 70µl/well, washed in acetic acid 1% to remove the unbounded dye. The plates were air-dried before the dye was solubilized with 150µl/well of 10mM Tris base (pH 7.4) for 5minutes on a shaker at 1600 r.p.m. The optical density (OD) of each well was measured spectrophotometrically at 540nm on ELISA microplate reader (ELX 800-BioTek-USA). The IC₅₀ values were calculated using sigmoidal concentration-response curve fitting models (sigmaplot software).

Detection of VEGFR-2 /KDR content in cell culture medium by ELISA

We have our Cells from American Type Culture Collection. Cells were cultured according to the standard protocol in a volume of 100µl complete growth medium + 100 ul of the plant extract per well in a 96-well plate for 18–24 hours, the supernatant was collected. VEGFR-2 in the supernatant was determined quantitatively using a human VEGFR-2 ELISA kit, according to the manufacturer’s instructions.

Animals

The work was done in the Cancer Biology department, National Cancer Institute, Cairo University-Egypt. This study involved a total number of 60 adult male Sprague-Dawely albino rats of 100-130 g, body weight. They were housed in separate screen bottom cages under controlled environmental conditions (20-25 ºC, 55-60 % relative humidity and 12 hours light-dark cycle). Food and water were offered ad-libitum. Animals were kept up on stock diet in the form of pellets having the following composition: protein (18.8 % w/w), barley (37 % w/w), and corn (15 % w/w) were the main constituent (Lewi, 1981). The study was managed after obtaining Institutional Animal Ethical Committee (IAEC) clearance.

Experimental design

Animals were divided randomly into four groups. Each group contained fifteen animals.

Group 1 (Normal control)

This is healthy untreated rats eating standard food and drinking tab water ad-libitum.

Group 2 (diethylnitrosamine and carbon tetrachloride) (DEN+CCl₄)

Rats received a single intraperitoneal dose of DEN (200 mg/kg, body weight) to initiate hepatic carcinogenesis. Two weeks later, animals received subcutaneous injections of CCl₄ once a week in a dose of 3 ml/kg bodyweight for 6 weeks to stimulate liver cell proliferation and regeneration. The experiment continued for 44 weeks.

Group 3 (Plant extract (E1))

Rats treated daily with 70% methanol plant extract of Erucariapinnata alone by oral gavaging at a dose of 1 ml (400 mg/ kg b.wt.) for 44 weeks.

Group 4 (DEN+CCl₄+E1)

Rats treated as group 2 with daily orally administrated by a dose of 1 ml (400 mg/ kg b.wt.) of the 70% methanol extract (E) till the end of experimental.

Blood sampling for biochemical assays

After 44 weeks, the rats were sacrificed. Blood samples were collected, and the serum was then separated and divided into three aliquots and used fresh or kept frozen at -80ºC and thawed only once for demand.

Tissue Preparing

liver tissue was divided into two sections, the first section was kept at 10% neutral-buffered formalin for histopathological investigation, while the second one was homogenized to obtain tissue homogenate supernatant, which are used for estimation of our parameters.

Estimation of liver injury markers in blood serum

Alanine aminotransferase (ALT) and aspartate aminotransferase (AST) activities were measured based on the principles described by (Bergmeyer, Scheibe, & Wahlefeld, 1978), the activity of alkaline phosphatase (ALP) was estimated according to the method described by (King & Armstrong, 1988), γ-Glutamyl transferase (γ-GT) was determined by the method of (Persijn & Slik, 1976), The total bilirubin level (TBL) was determined according to the method of (Walter & Gerade, 1970), Total protein and albumin concentrations were determined colorimetrically by the methods adopted by (Doumas, Watson, & Biggs, 1997; Gornall, Bardawill, & David, 1949) respectively and The serum globulin concentration was calculated by subtracting the value of serum albumin from the total serum protein

(Globulin conc. (g/dl)) = Total protein conc. (g/dl) - albumin conc. (g/dl)).

Liver antioxidant enzymes

The activity of superoxide dismutase (SOD) and Catalase (CAT) in the liver were measured by the procedure described by (Cohen, Dembiec, & Marcus, 1970; Marklund & Marklund, 1974), respectively.

Tumor marker

Alpha-fetoprotein (AFP) measured quantitatively by solid-phase enzyme-linked immunosorbent assay Elisa according to the method of (Belanger, Sylvestre, & Dufour, 1973).

Quantitative Determination of Vascular Epidermal Growth Factor (VEGF-A)

Vascular endothelial growth factor (VEGF) concentration in the serum was depended on binding VEGF antigen to a specific immobilized antibody. The formed immune complex binds to avidin-peroxidase conjugate, and a color developed in proportion to the amount of VEGF bound, which was measured at 450 nm.

Histopathological Assessment

Tissue specimens were dehydrated, cleared, immersed, and sectioned (5 µm) using ethanol series, xylene, Paraplast wax, and microtome, respectively. Liver sections were mounted on a glass slide then the tissue sections were deparaffinized and stained by haematoxylin and eosin.

Molecular docking

To study the possibility of our extract to act as anti-angiogenesis by inhibited the activation of VEGFR-2 kinase, a docking study was performed by maestro 11.4 to determine the ability of flavonoid aglycones with a higher concentration in our extract (HPLC result) to bind with and inhibit VEGFR-2 tyrosine kinase. (PDB entry: 3VID) used for our study. Ligprep have been used to prepare our compounds for docking. Protein preparation have been done using the protein preparation wizard. Validation of our docking study was done by docking of the co-crystallized ligand (4TT), the result indicates that our protocol is valid with rmsd= 0.3561 and the same amino acid interactions were recognized (one hydrogen bond with Cys 919, one hydrogen bond with Glu 917 and one aromatic hydrogen bond with Lys 920). The ligand-binding area was determine using the co-crystallized ligand in 3VID, to avoid any false positive result and to make the docking study highly accurate two type of constraints have been used the first one is the positional constraint where, we define spherical regions of indole ring of the co-crystalized ligand should be occupied by particular ligand atom during docking, the second one is the H-bond constrain where we pick Cys 919, Glu 917 and Lys 920 should participate in hydrogen bond (acceptor or donor) interaction during docking also extra precision (XP) setting have been used during docking.

Statistical analysis

All results were expressed as the mean ± standard errors of mean (SEM). Statistical analysis was performed using statistical packages for the Social Science for Windows (SPSS, Version 11.0, Chicago, IL, USA). The data were analyzed by Student’s t-test. The P-value of less than 0.05 were considered statistically significant.

Results and Discussion

Cytotoxic potential assessment against liver cancer cell (HepG2) cell line

Serial dilution of crude methanol 70% (E1) and its successive extracts (petroleum ether (E2), chloroform (E3), ethyl acetate (E4), 96% methyl alcohol (E5)) of E. Pinnata (0, 5, 12.5, 25 and 50 µg/ml) were examined for their cytotoxicity potency against hepatic carcinoma (HEP-G2) using sulforhodamine B stain (SRB) assay. It was observed from the obtained results in (Table 1) and (Figure 1) that, all extracts of E. Pinnata achieved cytotoxic effect against HEPG2 While the crude methanol 70% recorded the best potent cytotoxicity at low concentration (IC₅₀=13.6 µg∕ ml) followed by the petroleum ether extract (IC₅₀= 15.8 ug∕ml) While the 96% methyl alcohol extract recorded the lowest cytotoxic effect (IC_{50 =}23.3 µg ∕ ml).

Flavonoids Constituent of 70% methanolic extract by HPLC

Quantitative and qualitative estimation for the flavonoid (Table 2) compounds of the 70% methanolic extract of E. Pinnata were achieved by using HPLC, where, each compound was separated. Identification was done using authentic pattern. We found that rutin was the highest concentration of flavonoids compounds by 2412 mg/100mg, and rhamnetin was the lowest concentration of 0.8569 mg/100mg in the plant.

VEGFR-2 /KDR content in cell culture medium

It was clear that in Table 3, the crude methanol 70% inhibited the VEGFR-2 in HepG2 cell line by (72.36%).

Effect of 70% methanolic extract of Erucaria pinnata on liver markers in blood serum

From our study, its clear that Group 2 (DEN+CCl₄) developed hepatocellular carcinoma while in Group 4 (DEN+CCl₄+E1), the carcinogenesis process was prevented or at least showed (hepatoprotective activity) as its clear from the level of hepatic serum parameters. While in Group 3 (E1) recorded no significance change comparing to Group 1 (normal control group) (Table 4).

Antioxidant and oxidative stress determination

Results arranged in Table 5, showed that administration of DEN+CCl₄ in rats produced highly significant decreases in the level of SOD and CAT at (p < 0.001) by (-74.58% & -41.71%) respectively when compared with HCC group, while rats treated with extract alone showed highly significant increase (p < 0.001) in the levels of antioxidants including SOD and Catalase in the liver, when compared to the control group by (54.99% & 17.26%) respectively. Post-treatment with Plant Extract (400 mg/kg body weight daily for 44 weeks) caused significant increases (p < 0.001) in the hepatic levels of SOD and Catalase by (239.63% & 33.42%) respectively as compared to untreated HCC rats.

Effect of 70% methanolic extract of Erucaria pinnata on serum AFP and VEGF-A level

Alpha-fetoprotein (AFP) and Vasculoendothelial growth factor (VEGF) were found to be significantly increased in DEN+CCl₄ treated animals (P<0.001) when compared to control group by (5148.57% & 142.14%) respectively. While rats treated with (E1) alone recorded no significant change comparing with normal control and Post-treatment with 70% methanolic extract of E. Pinnata at the tested dose level significantly reduces the elevated levels of tumor-specific markers AFP and VEGF (P<0.001) when compared to DEN+CCl₄ treated animals group by (-95.65% & -41.54%) respectively, all values are presented in (Table 6).

Histopathological study

Microscopically liver of control untreated rat normal architecture of hepatic strands around the central veins, normal portal tract (PT) with normal portal veins (PV) and normal bile duct (BD), The liver showed intact hepatocytes with normal sinusoids in between. The hepatic cells are polygonal in shape with one or two rounded nuclei (Figure 2 &Figure 3). Meanwhile, the liver of rat treated with (DEN+ CCl₄) showed the clear-cell variant of hepatocellular carcinoma is usually arranged in a trabecular pattern and is characterized by a clear cytoplasm that contains glycogen and a variable amount of fat vesicles as (Figure 4).

Moreover, the examined section revealed that liver tissue showing expanded portal tract and showing dilated portal veins (PV) with bile duct proliferation (blue arrow) and inflammatory infiltrate (black arrow) with complete nodules as (Figure 5). Tumor cells are large, eosinophilic with prominent granular eosinophilic cytoplasm, vesicular nuclei, and conspicuous nucleoli or dense lymphoid infiltrate in a portal tract (nodular cirrhosis) this was obvious in (Figure 6), and hepatocellular carcinoma were identified, with pseudo glandular and infestation of blood cells were observed, tumor cells are surrounded by hyaline fibrous bands pale bodies may be observed in the tumor cells (Figure 7). While HCC was identified with the chronic cholangitis, peircholangiolar fibrosis which indicates the malignancy happened and proliferating bile ducts (black arrow) with an area of bile duct cystification (blue arrow) (Figure 8).

Histopathological liver of rat treated with (E1) revealed normal hepatic architecture (Figure 9 & Figure 10). Histopathological Liver of the DEN+CCl₄-rats treated with (E1) revealed significant improvement compared to DEN+CCl₄ treated group no evidence of HCC and showed normal central vein (blue arrow) surrounded by hepatocytes arranged in single-cell cords (black arrow) with scattered mild steatosis (yellow arrows) (Figure 11), bi-nucleation cell and mild perivascular lymphocytes infiltrate (Figure 12) this mean that the extract of Erucaria pinnata showed a sign of protection.

Docking study

This study revealed that our extract contains flavonoid aglycones (quercetin and luteolin) that could stably bind to the ATP-binding pocket of

VEGFR-2 with almost the same pattern like co-crystallized ligand (Table 7, Figure 13). Previous studies demonstrated that a variety of natural angiogenesis inhibitors, such as ellagic acid prevented VEGFR-2 phosphorylation via competing with cellular ATP binding to the ATP-binding sites.

Natural products which originated from the plant are affluent source for many small molecules which causing tumor cell kill by interacting with a predefined target(s) present on malignant cells and exhibited low toxicity to normal tissues.

In the present study, the cytotoxic activity of different extracts of Erucaria pinnata plant against HEPG2 cell line was evaluated, the 70% methanolic extract (E1) recorded the best potent cytotoxicity at low concentration so we analysis the flavonoids constituent of this extract using HPLC which show that our extract is rich with important flavonoids compounds (rutin, quercetin, leutolin, etc.) so we evaluated it's antitumor activity against hepatocellular carcinoma (HCC) induced by (DEN+CCl₄) in rats and evaluated its anti-angeogenic activity ( in vitro, in vivo and docking study). Our study showed that the prolonged administration of (E1) a no significant change in liver function tests, AFP, and VEGF but was showed an extremely significant increase in SOD and CAT in comparison with a normal control group.

It is known that diethylnitrosamine (DEN) is an effective environment carcinogen used as an initiator for hepatic cancer in an animal model with another factor like CCl₄ as a promoter of carcinogenesis (Subramanian et al., 2007). A reactive ethyl diazonium ion is produced from bioconversion of DEN by cytochrome P450 within the rat liver, and it reacts with DNA creating adducts that's identified as the initial step in DEN-evoked carcinogenesis (Verna, Whysner, & Williams, 1996). Moreover, free radicals were produced due to the metabolization of CCl₄ by liver microsomal cytochrome P450, which exhaust hepatic antioxidants and harm macromolecules, leading to advance tumor formation (Weber, Boll, & Stampfl, 2003).

So, (DEN+CCl₄) stimulate oxidative stress which induces liver tissue injury and caused a significant increase in the activities of liver enzymes, the liver completely lose its functions and significant decrease the antioxidant enzyme activity (SOD and CAT which are involved in scavenging superoxide anion and hence reducing the toxic effect caused by these radicals). In this study, it was observed that the administration of our extract to (DEN+CCl₄) treated rats showed highly significant decrease in the biochemical liver marker enzyme activities and highly significant increase in total protein, albumin, SOD and CAT activities This showed that 70% methanolic extract of Erucaria pinnata can reduce reactive free radicals, thereby reducing oxidative damage to the tissues besides improving the activity of hepatic antioxidant enzymes.

We also interested in our study by another factor which has high specificity for HCC, the diagnosis of tumor response to therapy and is detected with elevated levels is AFP (Afzal et al., 2012). There are many reports which show that AFP plays an important role in the regulation of tumor growth and cell differentiation (Li et al., 2002). In the present study, serum AFP level of DEN+CCl₄ treated rats showed a significant increase in comparison to that of the control group, confirming the occurrence of premalignant liver changes in DEN+ CCl₄ treated rats. Treatment with (E1) significantly reduced serum AFP levels.

This improvement in the biochemical parameters were accompanied with improvement in the histopathological abnormalities. All this finding agrees with the phenolics and flavonoids' rich nature of this extract, which were identified by HPLC and many compounds have been reported to possess antioxidant and free radical scavenging ability, which could possibly be accounted for the antitumor property of this plant (Hay, 2000; Kandaswami et al., 2005).

The Fifth Hallmark of Cancer "Sustained Angiogenesis" is an important target in cancer thereby due to the role of angiogenesis in cancer development and metastasis is critical and the main problem of anti-angeogenisis medication is the development of resistance (most of them is working only in single step in the angeogenic pathway), expensive cost and toxicity (most of them is protein in nature). So, it is important to distinguish new and effective anti-angiogenic molecules. Several studies have indicated that natural plant products can meet these criteria (Lu, Bhat, & Basu, 2016).

In this study, we tried to determine the anti-angeogenic potential of our extract. The extract effectively inhibited VEGFR-2 expression in vitro and significantly inhibited the concentration of VEGF-A in cancer-bearing animals. This antiangiogenic activity of our plant extract may be due to the presence of high contents of flavonoids in the extract. Numerous flavonoids, for example, rutin, genistein, luteolin, kaempferol, quercetin, naringin, and apigenin have shown strong inhibition to cell proliferation and VEGF expression (Luo, Jiang, King, & Chen, 2008; Oak, Bedoui, & Schini-Kerth, 2005).

Our results were confirmed by docking study revealed that our extract contain flavonoid aglycones (Quercetin and Luteolin) that could stably bind to the ATP-binding pocket of VEGFR-2 with almost the same pattern like co-crystallized ligand (which mean the ability of our extract to inhibit the kinase enzyme activity of the receptor domain) also its previously reported that flavonoids can prevent overproduction of NO, all that proves that our extract have anti-angeogenic activity through working in different steps in the angeogenic pathway which will decrease the chance of resistance to its anti-angeogenic activity.

From another view, flavonoids could reduce any side effect associated with angiogenesis target therapy as flavonoids exerts vasodilator, antiplatelet and antiproliferative effects and reduces blood pressure, oxidative status and end-organ damage in hypertensive humans and animal models (Perez et al., 2014).

Conclusions

This is the first study showed that 70% methanolic Erucaria pinnata extract possesses strong anticancer activity against DEN-induced HCC due to its antioxidant activity and antiangeogenic activity through working in different steps in the angeogeniesis pathway in the same time due to its flavonoids phytochemical constituents which give our extract the advantage of being strong antiangeogenesis medication with less toxic effect. These findings provide useful documents for further study on our plant to identify its effect in another hallmarks of cancer.