Pharmaceutical efficacy of harmalol in inhibiting hepatocellular carcinoma

Diethylnitrosamine (DEN) promoted by carbon tetrachloride (CCl4) forms DNA adducts inducing hepatocellular carcinoma (HCC). Plant alkaloid, harmalol, is being used as a therapeutic agent against HCC due to its accessibility and efficacy by apoptosis and inhibiting proliferation of cancer epithelial cells. Seven groups of Swiss albino mice were taken. Different stages of liver tissues and serum from various experimental groups were collected before and after harmalol treatment. The investigation was carried out by enzyme assay, bilirubin level in the blood, DNA, RNA, normal serum protein of liver tissue, and alpha-feto protein estimation of serum. Gross morphological assessment of liver, histological, and different apoptosis markers viz. p53, caspase3, and cytochrome C expression were analyzed by RT-PCR and Western blot. Harmalol (10 mg/kg B.W. per week, I.P.) for 9 weeks showed a significant reduction in hepatocellular foci, nodules, and carcinoma ultimately retaining the normal morphology. It further induces ROS-dependent apoptosis through mitochondrial cytochrome C release that induces p53 by caspase3 activation. The investigation will eventually help to develop more effective chemotherapeutic drugs from the natural source.


Background
The liver is a key metabolic organ that is essential for the production of blood proteins, lipid and sugar metabolism, and detoxification of naturally occurring and foreign harmful chemicals. In Asia and Africa, hepatocarcinogenesis is one of the major causes of cancer deaths [1]. To maintain its mass and many essential functions, the liver possesses a remarkable regenerative capacity, but the latter also renders it highly susceptible to carcinogenesis. In fact, liver cancer often develops in the context of chronic liver injury [2]. The most common form of primary liver cancer, hepatocellular carcinoma (HCC), involves multistep processes which are correlated with different genetic alterations that ultimately lead to malignant transformation of the hepatocytes [3]. Among several liver cancer-inducing agents, diethylnitrosamine (DEN) (Fig. 1a, inset) often induces malignant tumors in the target organ and is speciesspecific. DEN has been reported to cause the generation of ROS, forming DNA-carcinogen adducts in the liver and resulting in oxidative stress and cellular injury [4]. This potent genotoxic compound is widely found in the environment and present in tobacco smoke, varieties of food such as cheese, salted and dried fish, soybean, cured meat, alcoholic beverages, cosmetics, agricultural chemicals, several pharmaceutical agents, and ground water having a high level of nitrates making the human population vulnerable to its exposure and is also synthesized endogenously [5,6]. As preservatives, nitrites and nitrite salts are used in numerous foodstuffs in the food industry since 1920 to preserve the natural color of meat and the typical taste of processed, smoked, and cured meat and meat products. Meat proteins especially amines can react with the nitrites and produce N-nitrosamines [7]. As nitrosamine is found in food preservatives as an environmental dietary carcinogen, an intensive study has been conducted on DEN induced hepatocarcinoma in a mouse model as a potent genotoxic agent [8,9]. Initially, rapid killing of hepatocytes was induced by DEN alone. But DEN by itself requires a long time to induce hepatocellular carcinoma but the assistance of tumor promoters like CCl 4 ensures a shorter time span for the development of liver cancer [10]. Hence, this study concerns the application of DEN associated with CCl 4 (Fig. 1b) to induce HCC in male Swiss albino mice model, which is again another potent hepatotoxin [11,12]. Female mice have been reported to be resistant to HCC; hence, only male mice have been used and analyzed in this study [13]. White albino mice are internationally accepted model which could be extrapolated to human beings. Further, their ease of maintenance, handling, and their high reproduction rate make them particularly suitable models for human-oriented research. Their genome has been sequenced and many genes have human homologs.
The present investigation focused to elucidate the beneficial effect of natural plant-derived β-carboline alkaloid, harmalol, against HCC in the mice model to design a better therapeutic agent worldwide. Earlier, the aqueous and alcoholic extracts of Peganum harmala seeds having βcarboline alkaloid extracts have been reported to show potent antitumor and antiproliferative activity [14]. P. harmala extract caused a significant improvement in liver and kidney function in mice administered methotrexate drug [15]. However, our previous investigations on this naturally occurring indole derivative showed that it induced ROS-mediated, caspase 3-and p53-activated apoptosis against in vitro HepG2 cells [16]. It showed intercalative mode of binding, preferred hetero GC sequence specificity [17], and exhibited RNA binding ability [18]. But, most of the detailed therapeutic indications of harmalol are still a lacuna. Hence, this investigation primarily focused on several liver marker enzymes and gross morphological assessment of liver associated with several histological alterations that lead to HCC has been included in this study. Furthermore, the effective minimum dose of harmalol treatment is also included that finally put a conclusion through mRNA expression by RT-PCR and apoptotic marker proteins by western blot study in addition to the in vivo ROS generation analysis.

Chemicals and reagents
Harmalol and CT DNA were purchased from Sigma-Aldrich (St. Louis, MO, USA). The purity of the sample was confirmed as done earlier [17,19]. Their concentrations were determined using their molar extinction coefficient value of 19,000 and 6600 M −1 cm −1 , respectively, at 371 and 260 nm [17]. DEN (N-nitrosodiethylamine) was also purchased from Sigma-Aldrich (St. Louis, MO, USA) and CCl 4 was purchased from Merck, India. All chemical reagents for phosphate buffer Saline and 10% neutral buffer formalin that were used were of analytical grade.

UV-spectroscopic studies
The absorbance spectral studies between DEN and CT DNA were measured on a Jasco V-630 double beam monochromator spectrophotometer (Jasco International Co. Ltd. Tokyo, Japan) of 1-cm pathlength at 25 ± 0.5°C

Animal care and experiment
Male Swiss albino mice (Mus musculus) weighing 20-22 g (4-6 weeks old) were purchased from a registered breeder of animals. All mice were bred and maintained under constant conditions at a temperature of 24 ± 1°C and humidity of 55 ± 5% with 12-h light and 12-h dark cycles. Water and feed were accessible to the mice ad libitum [20].

Experimental design
The dose of both carcinogen and harmalol were selected as per the LD 50 and acute toxicity of mice maintained under constant laboratory conditions [21]. The calculated LD 50 of DEN, CCl 4, and harmalol are 465 mg/kg, 3 ml/kg, and 190 mg/kg body weight, respectively, through I.P. However, the acute toxicity of DEN, CCl 4 , and harmalol are 310 mg/kg, 2.2 ml/kg, and 120 mg/kg body weight, respectively. Figure S1 represents a schematic illustration of in vivo experimental design of induction towards hepatocellular carcinoma and its treatment with harmalol. Experimental male mice were randomly divided into seven groups, each group consisting of five animals and their periodic induction with carcinogen and treatment with harmalol according to Table 1.
Group I: Controls received the vehicle of normal saline (0.2 ml/kg, B.W. I.P.) and made sacrifice after 21st week.
Group II: Received harmalol (10 mg/kg B.W., I.P. per week) for 9 weeks Group VI (pre-treated): Received aqueous solution of harmalol of (10 mg/kg B.W of I.P.) for first 9 weeks followed by administration of DEN + CCl 4 up to the 21st week (dose vide supra) Group VII (post-treated): Mice post-treated with harmalol (10 mg/kg body weight) for 9 weeks after the administration of DEN for 12 weeks (i.e., after administering DEN alone for 12 weeks, the mice were treated with harmalol along with DEN for another 9 weeks) and continued till the end of the experiment.
The carcasses of the sacrificed mice were disposed of by biomedical waste disposal method.

Gross morphological assessment of liver
The mice were weighed periodically and their body weights were recorded. The livers of the respective groups were removed, weighed, and examined for the presence of grossly visible lesions, and morphological changes were observed.

Transmission electron microscopy
Transmission electron microscopy (TEM) was performed using TECNAI 200KV TEM (Fei Electron Optics), 35 mm Photography System from All India Institute of Medical Science, N. Delhi. HepG2 cells were seeded in 100-mm plates at a density of 2 × 10 8 cell in DMEM cell culture media for overnight and after adherence, cells were treated with harmalol of 7, 14, and 21 μM concentrations (concentrations at GI 25 , GI 50 , and GI 75 , respectively) at 37°C for 48 h. Cells were trypsinized and then the cultured materials were centrifuged at 3000 rpm for 4-5 min to discard the supernatant. The pellet was suspended in 0.1 M phosphate buffer (pH 7.4), dispersed, and centrifuged again. The pellet was resuspended and fixed in a mixture of 2% paraformaldehyde and 2.5% glutaraldehyde in buffer for 3-4 h at 4°C, centrifuged for 10 min at 4°C and the supernatant were discarded. The samples were finally prepared for observation.

Assessment of liver function by enzyme assay
Blood from seven different groups of mice was collected from the tail-vein after a specific time interval. Serum was obtained by centrifugation at 3000 rpm for 10 min at 4°C. Biochemical parameters, i.e., ALP [22], lactate dehydrogenase (LDH) assay [23], total bilirubin, AST, and ALT [24] were analyzed according to the reported methods. The kits for ALP, LDH activity, total bilirubin, AST, and ALT were purchased from Span Diagnostic, India.

Determination of ALP
The response of the liver to any form of damage and obstruction is to synthesize more ALP [25][26][27]. Changes in absorbance are measured kinetically at 405 nm due to yellow color formation and are proportional to ALP activity in the sample [25,26,28].

Determination of activity of LDH
Increased levels of LDH were also found in case of hepatic diseases [23]. Serum sample of each group (untreated and treated groups) was mixed well. The initial absorbance A 0 was measured spectrophotometrically at 340 nm and reading was taken repeatedly after every 1, 2, and 3 min. The mean absorbance change per minute (ΔA/min) was calculated as follows: LDH activity in U/L for 25°C/30°C = ΔA/min × 3333 and for 37°C = ΔA/min × 8095

Determination of total bilirubin
Red-purple-colored azobilirubin is measured spectrophotometrically at 540 nm and it is proportional to the concentration of the appropriate fraction of bilirubin. The concentration of total bilirubin was calculated in mg/100 ml.

Detection of activity of AST
Serum AST level is elevated in case of liver diseases associated with hepatic necrosis. Serum samples of each group were mixed with working AST reagent and absorbances were taken at 340 nm. AST

Detection of activity of ALT
Serum ALT level is also elevated in case of liver diseases associated with hepatic necrosis. ALT is a more liverspecific enzyme. Serum sample of each group was mixed well with working AST reagent and absorbances were taken and reading was taken at 340 nm.

Detection of hepatocarcinoma indicators
The activity of hepatic DNA in the liver tissue was assayed by methods of Giles and Myres [29] and Burton [30]. The hepatic RNA level was estimated by methods of Almog and Shirey [31] and Blobel and Potter [32]. Hepatic protein was estimated by the method of Lowry et al. [33]. The activity of serum alpha-fetoprotein (AFP) level was also assayed.

Histological studies
Mice from each group were taken and sacrificed after a specific time interval. After draining the blood, liver samples were excised, washed with normal saline (0.9% NaCl), and processed separately for histopathological observation according to Alarami [34]. The hematoxylin and eosin (H&E) stained sections were examined under a light microscope, Leitz (Biomed), and histopathological changes were captured by a Nikon Camera, EOS700D, 18-55 lens.

RT-PCR and western blot analysis
Equal amounts of total RNA, extracted with TRIZOL Reagent (Himedia, Mumbai, India), were reverse transcribed and then subjected to PCR with enzymes and reagents of the reverse transcription system using the technique of PCR system as reported earlier by Sarkar et al. [16]. Sequences of primers used in the study are given in Table  S1. G3PDH was used as loading control. PCR kit (Taq Mix Kit), RT-PCR kit, and RNasin were purchased from SRL, Pvt. Ltd. Oligonucleotide primers for caspase3, p53, and G3PDH were purchased from Xcelris Labs Limited. Total protein content from the liver of the control, harmalol post-treated, and DEN-and CCl 4 -induced group of mice were extracted and western blot analysis was performed as described by Lee [35]. Tubulin was used as loading control. Anti-active caspase 3 monoclonal primary antibody (17 Kd, anti-mouse, Santa Cruz Biotechnology), tubulin, and anti p53 monoclonal primary antibody (anti-mouse) were purchased from Santa Cruz Biotechnology, and anti-mouse secondary antibody was purchased from Sigma-Aldrich.

In vivo ROS generation and its quantification by FACS
The liver from different groups was collected and a freshly prepared solution of 0.5 mM EDTA and collagenase type I 0.8 mg/ml in DMEM media was added to the section of the liver sac to release the hepatocytes according to Severgnini et al. [36]. The levels of intracellular ROS were assessed by flow cytometry after incubating with DCFH-DA (20 mM) (dichloro fluorescence diacetate) for 30 min at 37°C.

Statistical analysis
The data obtained were calculated by SPSS 17.0. The values were expressed as mean ± SEM. Statistical analysis was performed by one-way analysis of variance (ANOVA). Values of p < 0.05 were considered significant.

UV-vis spectral changes
DEN (diethylnitrosamine) is a potent hepatocarcinogenic nitrosamine compound, which is present in several sources. It is the nitrosamine that has the carcinogenic effect [37]. Endogenous nitrosamines can be formed in a similar way from their precursors, nitrates, and nitrites. The binding between DEN and CT DNA was studied by UV-vis absorption titration (Fig. 1). The characteristic UV-vis spectrum of DEN exhibited maxima at 338 nm in the range of 300-410 nm. On titration with CT DNA of varied concentration and DEN of fixed concentration (15 μM), the spectra exhibited a characteristic hypochromic effect of 18% and a bathochromic shift of 4 nm until saturation was reached at nucleotide phosphate/DEN molar ratio (P/D) 4.0. The characteristic hypochromic effect and bathochromic shift indicated a strong intermolecular interaction. But throughout the binding, no isosbestic point was observed that signifies that the interaction was not equilibrium based non-covalent binding.
In vitro cytotoxicity studies of harmalol in HepG 2 cell line Apart from the binding study, the biological importance was further highlighted by in vitro cytotoxic assays to emphasize the apoptotic induction ability of harmalol in liver cancer cell line HepG 2 . Previously Sarkar et al. [16] had already revealed that harmalol showed cytotoxicity in different human cancer cell lines and out of all, HepG 2 cell line showed maximum cytotoxicity with a minimum IC 50 value of 14.2 μM. Figure 2a depicts the phase-contrast microscopic images of the control and treated HepG 2 cells with different concentrations of harmalol. Furthermore, ultra-morphological study by TEM (Fig. 2b), which is considered the customary to confirm apoptosis, further discovered some early and late AST

Light microscopic studies
Light microscopic images of the intact liver are shown in Fig. 3a-c. Control (Group I) mice showed normal as a reddish-brown wedge-shaped architecture of the liver (Fig.  3a). Macroscopically the liver extracted from group V showed several lesions as multifocal nodular hyperplasia and the entire structure was complete whitish-pink in color with blood drained structure. Multifocal and infiltrating types of growth patterns were observed in DEN+CCl 4 (Group V)-injected mice liver (Fig. 3b). While harmalol post-treated (group VII) mice liver was also found in nearnormal architecture, reddish-brown color, and normal shape and size (Fig. 3c). The results further showed the disappearance of gray-white nodular patches from the surface of the liver after 9 weeks of post-treatment (total accumulation of 90 mg/kg of harmalol). Light microscopic examinations of the liver of the remaining experimental groups have been shown in Figure S2.

General observation and body and liver weight
The final body weight and absolute liver weights were also collated in Table 2. All groups were subjected to dying sacrifice after 21 weeks. Final body weights were remarkably decreased in group III with a body weight of 17.3 ± 3.2 g and group V with 13.6 ± 1.6 g body weight as compared with harmalol-treated and control group (division of various groups in details have been shown in the "Experimental design" section). But, the absolute liver weights in the mentioned groups (i.e., groups III, IV, and V) were significantly decreased, though the decrease was more pronounced among group V and it was 0.96 ± 0.9 g (Fig. 3b). Decreased liver weight in these groups was attributed to decreased body weight.

Assay of liver marker enzymes
After the study of apparent body and liver weight, blood samples from all seven groups were collected and serum level of alkaline phosphatase (ALP), lactate dehydrogenase (LDH), bilirubin, aspartate aminotransferase (AST), and alanine aminotransferase (ALT) were estimated. Data obtained from the biochemical assays were analyzed and presented in Table 3 which showed the mean serum levels of ALP, LDH, bilirubin, AST, and ALT in all healthy or controlled, harmalol control, different groups of carcinogens induced, and harmalol pre-and post-treated groups of mice, respectively. The parameters were further represented as a bar graph (Fig. 4a-e).
The values of all these enzymes were significantly higher (p < 0.05) in DEN+CCl 4 , i.e., group V treated group with 650.85 ± 20 IU/L, 1801.1 ± 60 IU/L, 2.36 ± 0.098 mg/dL, 495.5 ± 18 IU/L, and 477.36 ± 15 IU/L in ALP, LDH, bilirubin, AST, and ALT, respectively. It showed the highest percentage of marker liver enzymes above the normal level. Furthermore, there was a drop in the hemoglobin level in the hepatocarcinoma-affected mice. Normal hemoglobin was found to be 9.7 mmol/L, whereas in the diseased mice it was 8.4 mmol/L, reflecting acute anemia. In the postharmalol-treated group, it was reported to be 9.3 mmol/L.

Alpha-fetoprotein as hepatocarcinoma indicator
The changes in the level of hepatic DNA, RNA, and normal serum protein level in the liver tissue of control and experimental groups were studied and the quantitative values are presented in Table 4 and Fig. 5a. Mice administered with DEN/CCl 4 showed a significant increase in DNA and RNA levels than the control and post-treated groups while the normal serum protein level was significantly low in the group of mice administered with DEN/ CCl 4 compared to control and post-treated groups. But, the activity of serum alpha-fetoprotein (AFP) level was significantly increased in DEN/CCl 4 when compared to    the normal group (Fig. 5b). A significant reduction in serum AFP level was observed in the treatment group (harmalol post-treated) when compared with DEN/CCl 4 group.

Histological study
Apart from the above study, a histological study was performed under light microscope using hematoxylin and eosin stain. Histology signifies the microscopic examination of tissue in order to study the manifestations of the specific disease. Specifically, in clinical medicine, histology, along with its pathological outcome, refers to the examination of a biopsy or surgical specimen, after the specimen has been processed and histological sections have been placed onto glass slides. In the presence of combined carcinogen, DEN+CCl 4 several characteristics of liver necrosis and HCC were found in experimental system after 12 weeks of injection and were presented in Fig. 6 elaborately. Toxic chemicals like several nitrosamine compounds, used as common food additives, could induce HCC and could cause zonal necrosis of the liver [38,39]. Here, DEN+ CCl 4 induced two types of zonal necrosis. Figure 6a shows centrilobular necrosis involving necrosis in hepatocytes around the central vein, while Fig. 6b shows fibrolamellar carcinoma where oncocytes formed cords (A1) and nests (A2) which were separated by bands of fibrous stroma (A3). Figure 6c (B1, B2) showed peripheral or periportal necrosis involving necrosis in parenchyma closest to the arterial and portal blood supply while (C1) and (C2) showed pyknotic condition in the nuclei of hepatic cells. Figure 6d portrayed trabecular or sinusoidal patterns which were actually made up of 2-8-cell-wide layers of tumor cells separated by vascular spaces or sinusoids which are lined by the endothelium layer. Figure 6e (D1, D2) represents different changes in liver tissues like nodule formation and fibrosis or cirrhosis, respectively. Furthermore, in DEN+ CCl 4 -induced mice, several AHF (altered hepatocellular foci) were also observed and represented in the figures below. Among them, large eosinophilic AHF (Fig. 6f), small basophilic AHF (Fig. 6g), and vacuolated cell AHF (Fig. 6h), in which hepatocellular cytoplasm was found to be vacuolated, have been highlighted. Figure 7 further summarized the results of the histological examinations of untreated, carcinogen-induced, harmalol pre-and post-treated mice liver elaborately. Several characteristics of HCC and necrosis described above were identified in the carcinogen-induced group and HCC was found to be developed in the DEN+CCl 4treated group (group V) after the 12th week. HCC was identified in the CCl 4 -treated group (group IV) also. In all DEN-treated groups (group III), large eosinophilic and small basophilic AHF was identified. AHF condition increased in a dose-dependent manner and was statistically significant as compared with harmalol-treated and controlled values. Eosinophilic AHF was positively correlated to dose, but only those identified in group V were statistically significant. DEN+CCl 4 treatment led to a severe pyknotic condition of hepatic cells nuclei, vesiculation in the cytoplasm of the cells, sign of damage in the wall of central veins, hemorrhage, necrotic foci, degeneration, and hypertrophy in some of the hepatic cells. While the DEN-treated group showed a widening of some of the hepatic sinusoids, histology of CCl 4 -treated groups showed different changes in liver tissues like fibrosis (cirrhosis) and mononuclear polymorphic infiltration besides widening in some of the hepatic sinusoids, pyknotic, and vesiculation of hepatic cells. Thus, it can be summarized from the histopathological examination of mice in group III (DEN-treated mice) and group IV (CCl 4 treated mice) that histopathological deformities were far more prominent in group V, i.e., DEN+CCl 4treated group. While control group animals revealed normal architecture and animals pre-treated and posttreated with harmalol (90 mg/kg B.W.) showed fewer neoplastic cells with the near-normal architecture of a very compact hexagonal or pyramidal structure with a very prominent central vein.

Effect of harmalol on p53 and caspase 3 expressions
Hepatoprotective action of harmalol was further confirmed by RT-PCR data revealing that unlike in the control group of mice, harmalol post-treatment resulted in activation of caspase 3 and increased expression of p53 (Fig. 8a). The expression of caspase-3, p53, and cytochrome C that favored apoptosis were up-regulated in a harmalol post-treated group (Fig. 8b). Figure 8b further exhibited the cleaved form of caspase-3, i.e., 17/19 kd. Together, the present results showed that harmalol induces apoptosis by potentially targeting the cleavage of procaspase-3 (34 kd) into active enzyme and both forms of caspase-3 expression by western blot analysis, which are given with its quantitative presentation. Quantification of each marker is been represented by bar graphs. Evidence suggested that the p53 tumor suppressor gene is involved in cell cycle regulation, DNA repair, and programmed cell death, and it further led to caspase activation [40,41].

In vivo ROS detection
The result was further complemented with in vivo ROSdependent cytotoxicity. ROS generation, induced by various anti-cancer agents [42][43][44], plays a key role in apoptosis. ROS levels were found to be elevated in the harmalol post-treated group. The mean fluorescent intensity changed (Table 5) from 21.3 ± 0.9 (control) to 28.6 ± 0.8, 71.4 ± 0.8, and 85.4 ± 0.7 in harmalol posttreated, DEN, and DEN+CCl 4 -induced group, respectively (Fig. 9). ROS intensity increased nearly 4-folds relative to the control.

Discussion
Ability to induce apoptosis in cancer cells is an established hallmark feature of any drug with a good anti-cancer potential [45]. Hence, the study highlighted the apoptotic induction ability of harmalol in an in vivo model to quantify the efficacy of the drug molecule inhibiting carcinogens inducing hepatocellular carcinoma (HCC). In vivo study was performed in seven groups of male Swiss albino mice administered with both DEN and CCl 4 . DEN by itself requires a long time to induce hepatocarcinoma, but the assistance of tumor promoters like CCl 4 ensures a shorter time span for the development of liver cancer [10]. CCl 4 enhanced hepatocyte proliferation through indirect mechanisms. Essentially, their primary effect is cytotoxicity that eventually triggers compensatory proliferation [10,46]. DEN+CCl 4 application leads to dilatation of central veins and pooling of blood in the sinusoids towards the center of the liver lobule with hemorrhage in hepatic tissues, centrilobular and peripheral necrosis, fibrolamellar carcinoma, pyknotic in the nuclei of hepatic cells, Fig. 8 a RT-PCR analysis conducted with a cytosolic fraction of caspase 3 and p53 activation G3PDH was used as an internal control for normalization of equal loading. b Western blot analysis of caspase 3, p53, and cytochrome c from whole protein extracted from the liver of the control, DEN+CCl 4 -induced, and post-treatment with harmalol of HCC-induced mice. Tubuline was used as loading control. Bar graphs represent the quantification of each marker Simultaneous oxidation of α carbon within microsomes induces its DNA damaging activity [47]. Moreover, CYP2E1 had shown to be the principal catalyst for biotransformation of CCl 4 into trichloromethyl free radical [48]. Biometabolism of both DNA adduct forming DEN and CCl 4 mediated hepatic injury is well documented [49,50]. ROS generation from CYP2E1 activity within the hepatocytes contributed to the development of oxidative stress during DEN+CCl 4 exposure, that eventually released stress-specific enzymes in the serum, which is reflected by the marked elevations of liver functional markers such as ALT, AST, LDH, bilirubin, and ALP. Ultimately, abnormal increase in aminotransferases especially ALT reflected liver cell damage [51,52]. Generally, ALT (SGPT, serum glutamic pyruvic transaminase) and AST (SGOT, serum glutamic oxaloacetic transaminase) are found in the cytoplasm and mitochondria of liver cells in high concentrations but low in blood [53]. However, increased activities of these enzymes in serum are due to increased membrane permeability and leakage into the blood circulation when hepatocytes are injured [54], which is the sign of acute necrosis of liver cells. Increased LDH reflected the formation of tumors in the liver. While ALP, an enzyme, is homogenously distributed mainly in the liver, kidney, and bone tissues, a high level of ALP in serum may be a sign of liver damage. Among all liver function tests, ALP is a helpful screening tool to detect hepatic dysfunction. The mechanism by which alkaline phosphatase reaches the circulation due to the leakage from the bile canaliculi into hepatic sinusoids may result from leaky tight junctions [55]. Elevated serum levels of intestinal alkaline phosphatase have been found in patients with cirrhosis [56]. Thus, ALP has been noted to be an HCC prognostic factor in general. The probable overall clinical steps with the outcome have been postulated schematically in Fig. 10. The activity of hepatic tissue was further studied by estimation of DNA, RNA, normal serum protein, and cancer biomarker alpha-fetoprotein [57]. Earlier, it was proved by investigators that there is a significant elevation of DNA and RNA levels and a decrease in normal serum protein in cancer cells in response to a carcinogen like DEN+CCl 4 [58][59][60]. In the present study, harmaloltreated animals showed a significant decrease in DNA and RNA level and restoration of normal serum protein when compared to DEN+CCl 4 group further validating the anti-cancer property of the alkaloid. This effect was probably accompanied by cell cycle arrest which was previously reported by the authors in in vitro HepG2 cells by harmalol [16]. However, an elevated level of alpha-fetoprotein AFP, which is a common diagnostic marker, in the serum has been well documented in HCC and germ cell cancers [60]. It was revealed that elevated level of AFP is observed in the adult animals which are exposed to hepatocarcinogens [60]. There are several reports which show that AFP plays a key role in the regulation of tumor growth and cell differentiation and can stimulate the proliferation of human hepatoma cells possibly through the AFP receptors [61][62][63]. A significant rise in AFP level observed in DEN+CCl 4 group indicates the presence of HCC in mice model. Harmalol posttreated group significantly reduced the rise in AFP level when compared with the DEN+CCl 4 group. This reduction in the treatment group of AFP to a near-normal level may be due to inhibitory effect which confirms the chemopreventive therapeutic action of the alkaloid.
Furthermore, an increase in ROS generation or oxidative stress is an important marker of cancer, and in this study, the ROS intensity has been found to increase nearly 4-folds relative to control in the DEN+ CCl 4 group. ROS actually creates disruptions in redox balance. Several studies revealed that p53 has an important role in glucose metabolism and redox regulation. Lu et al. had very elaborately showed the mechanistic insights into the relationship between ROS, tumor suppressors, and oncogenes in p53 mouse models. Increased expression of p53 decreased ROS levels by inhibiting glycolysis and promoting NADPH generation [64].
The balance between cell proliferation and apoptosis is essential for the development and structure-function maintenance of normal organs [65]. The occurrence of cancer is supposedly due to the repression of the normal apoptotic process, which caused the imbalance between cell proliferation and apoptosis [66,67]. In fact, apoptosis constitutes an innate tissue defense against carcinogens by preventing the survival of genetically damaged cells. As a result, inhibition of apoptosis has been elucidated as the prevailing mechanism of liver tumor promotion in many rodent studies [68,69]. This study has highlighted that it is through mitochondrial cytochrome c release that induces p53 by caspase 3 activation via the intrinsic apoptotic pathway [68,69] which is considered one of the characteristic features of any anti-cancer drug.

Conclusion
The present findings clearly demonstrate the chemopreventive, dose-dependent activity of natural alkaloid, harmalol, against diethylnitrosamine (DEN) promoted by carbon tetrachloride (CCl 4 )-induced hepatocarcinogenesis (HCC) in mice model based on histological study, enzyme assay, and expression of several molecular markers. It has been found that post-treatment of harmalol (10 mg/kg B.W., I.P., per week) for 9 weeks markedly decreased the number of liver tumor nodules, lowered the formation of peripheral and centrilobular necrosis, and helped to regenerate the parenchymal cells in the liver, protecting membrane integrity and decreasing enzyme leakage thereby maintaining the normal histological architect of the liver. It further induces ROS-dependent apoptosis through mitochondrial cytochrome C release that induces p53 by caspase3 activation. The study also highlighted that action of harmalol is mostly therapeutic rather than prophylactic. So posttreatment with harmalol would be far better than pretreatment.
The ultimate understanding of these investigations will eventually help to develop more effective chemotherapeutic drugs from the natural product with enhanced efficacy and lower toxicity that can be targeted effectively to canceraffected specific organ for better therapeutic applications.
Additional file 1: Figure S1. Diagrammatic representation of in vivo experimental design of hepatocellular carcinoma HCC induction and its treatment with harmalol in Mus musculus. Figure S2