Effect of Standardized Decoction of Nigella sativa Seed, Hemidesmus indicus Root and Smilax glabra Rhizome on the Expression of p53 and p21 Genes in Human Hepatoma Cells (HepG2) and Mouse Liver with Chemically-Induced Hepatocarcinogenesis

Purpose : To evaluate in vitro (using human hepatoma HepG2 cells) and in vivo (using mouse liver with diethlynitrosamine (DEN)-induced hepatocarcinogenesis) effect of a standardized decoction on the expression of p53 (tumour suppressor) and p21 (cyclin kinase inhibitor) genes with the long-term goal of developing the formulation into a globally acceptable therapy for hepatocellular carcinoma (HCC). Methods: The effect of the decoction on (a) mRNA and (b) protein expression of p53 and p21 genes in HepG2 cells and mouse livers with DEN-induced early hepatocarcinogenesis were evaluated by (a) reverse transcription PCR (RT-PCR) and (b) immunohistochemical and Western blot analysis, respectively. Results: The results demonstrated that the decoction significantly ( p < 0.001) enhanced the expression of p53 and p21 genes in a time- and dose-dependent manner in HepG2 cells. A dose of 75 µg/ml significantly increased p53 mRNA at 24 and 48 h and p21 mRNA at 12, 24, 48 h of incubation with the decoction ( p < 0.01). Induction of hepatocarcinogenesis in mice significantly increased hepatic expression of both p53 and p21 compared to distilled water control ( p < 0.001), while treatment with the decoction further enhanced expression of both genes in DEN-induced hepatocarcinogenesis ( p < 0.01). Conclusion: Overall, the findings demonstrate that the decoction may mediate its reported antihepatocarcinogenic effect, at least in part, through the modulating activities of genes involved in tumour suppression and cell cycle arrest.


INTRODUCTION
Hepatocellular carcinoma (HCC) is the most frequent primary liver cancer and the fifth commonest neoplasm in the world.Despite the availability of curative surgical methods, the recurrence rate of HCC is reported to be as high as 50 % at 2 years [1].Similarly, adjunct therapies such as radiotherapy, chemotherapy and hormonotherapy have not reduced the number of deaths from HCC to the expected level [2].Further, these treatments produce some very unpleasant side effects such as toxicity to normal cells, bone marrow depletion, adverse effects on the gastro-intestinal tract etc.Therefore, there is an urgent need to search for better control and preventive methods that would not only reduce mortality of HCC patients, but would also have less side effects.
In many Asian countries, some poly-herbal preparations are considered by traditional medical practitioners to be useful for the treatment of cancer, despite lack of evidence from scientifically controlled studies to validate these claims.One such remedy that has been used for many years by a particular family of Ayurveda physicians in Sri Lanka is a decoction comprised of Nigella sativa seeds, Hemidesmus indicus roots and Smilax glabra rhizomes [3,4].Previous in vivo investigations confirmed that this decoction protects against chemically induced carcinogenesis in rat liver, with no significant toxic side effects, thus providing support for its ethnopharmacological use for cancer therapy [3,4].In the past few years, with the sequencing of the human genome providing us with an enormous number of potential targets associated with cancer, the focus of drug development for cancer therapy has moved from cytotoxic compounds identified by screening to identification of compounds that can act at specific molecular targets associated with carcinogenesis.p53 is a tumour suppressor gene that plays an important role in maintaining genetic stability and cell cycle regulation after induction of DNA damage.It is the most commonly mutated gene in human cancers [5].p21, is a cyclin kinase inhibitor (CKI) involved in cell growth arrest [6].Expression of p21 is controlled at the transcriptional level by both p53 dependent and -independent mechanisms [7].Since the integrity of p53 has been reported to be critical in cancer predisposition, onset and therapeutic response, interventions to restore wild-type p53 is an attractive approach for cancer therapy [8].Therefore, identification of natural agents that can restore wild-type p53 (wt p53) may lead to the development of novel drugs for cancer treatment.
The aim of the present investigation was to evaluate in vitro (using human hepatoma HepG2 cells) and in vivo (using mouse liver with diethyl nitrosamine induced hepatocarcinogenesis), the effect of a standardized decoction comprised of N. sativa seed, H. indicus root and S. glabra rhizome on the expression of p53 (tumour suppressor) and p21 (cyclin kinase inhibitor) genes with the long term goal of developing the above herbal formulation into a form that could be used by itself or as an adjunct to existing therapies for the treatment of HCC.

EXPERIMENTAL Collection of plant material
Plant materials for the preparation of extracts were purchased from a reputed vendor of herbal material used by traditional medical practitioners in Sri Lanka (D.J. Fernando Pvt Ltd, Gabose Lane, Colombo 13).Their identities were confirmed by a taxonomist (Mr.

Preparation of plant decoction
The plant decoction was prepared according to the method recommended traditionally for administration to cancer patients [3,4].Sixty grams (60 g) of plant material (composed of a mixture of 20 g each of N. sativa seeds, H. indicus roots and S. glabra rhizomes) were ground and boiled gently with 1.6 L distilled water for approximately 3 h to reduce the volume to 200 ml.The extract was then filtered through a layer of muslin, the filtrate centrifuged at 3000 g for 15 min to remove any debris, and the supernatant freeze-dried and stored at -20 0 C until required.Prior to use, the freeze-dried extract was reconstituted with the appropriate volume of distilled water containing 1 % DMSO.

HepG2 cell culture and treatment
HepG2 cells (ECACC, Salisbury, UK) were grown in monolayer cultures in Dulbecco's Modified Eagle Medium (DMEM) supplemented with 10% fetal bovine serum, and 50 IU/ml penicillin and 50 µg/ml streptomycin.The cells were maintained at 37 0 C in 95 % air / 5 % CO 2 atmosphere, and 95 % humidity.The media were purchased from GIBCO, Invitrogen Corporation, U.S.A, while other chemicals were obtained from Sigma Aldrich (Gillingham, Dorset, UK).The cells were harvested by trypsinization and 2 x 10 5 cells / flask were seeded into 18 T 25 (25 cm 2 ) flasks.Cell cultures were initially maintained in DMEM medium (5 ml) for 24 h at 37 0 C in 95 % air/5% CO 2 atmosphere, and 95 % humidity.After 24 h, the medium from each flask was replaced with fresh medium and cell cultures were treated with different concentrations of the decoction.The concentrations used were 75, 150, 300, 600, and 1200 µg/ml, and triplicate cell cultures were exposed to each concentration.Control cell cultures received only medium containing 1 % DMSO.Treated and control cell cultures were incubated for a further 12, 24 or 48 h.At the end of the incubation period, the cells were harvested and (a) total RNA and protein were extracted for the reverse transcription PCR (RT-PCR) quantification of p53 wt and p21 genes, and western blot analyses of p53 wt and p21 proteins, respectively, or (b) immunohistochemical focusing of the p53 wt and p21 proteins.
The viability of cells (2 x 10 5 cells/ml) incubated with the decoction for 12, 24 and 48 h with different doses of the decoction were evaluated by the MTT assay [9] as described previously [10].

Experimental animals and treatments
C3H male mice (5-6 weeks old) were purchased from the Medical Research Institute (M.R.I.), Colombo, Sri Lanka.They were housed in a temperature-controlled room (25 ± 2 0 C) and maintained on a standard laboratory diet, prepared by the M.R.I. according to a formula recommended by the WHO [11], and water ad libitum.Ethical clearance for the animal experiments was obtained from the Ethical Review Committee of the M.R.I., Colombo, Sri Lanka.The animals were maintained in accordance with guidelines for ethics review of research proposals involving animals in Sri Lanka [12].
A total of thirty two (n = 32) male C3H mice (5 -6 weeks old) were randomly divided into four equal groups (8 mice/group).The mice in group 1 (distilled water control group) were orally fed with distilled water for a period of four weeks.Mice in group 2 (decoction control group) were orally fed with the decoction at a dose of 10 g/kg body weight/day for four weeks.This dose was based on the decoction dose which demonstrated maximum antihepatocarcinogenic effects against diethylnitrosamine (DEN)-induced HCC in rats [3].Distilled water or decoction was orally administered to mice via a Sondi needle [3].Mice in group 3 (positive control group) and group 4 (test group) were injected with DEN in 0.9 % NaCl at a dose of 20 µg/g body weight as a single intraperitoneal injection on day 1 to initiate hepatocarcinogenesis [13].Throughout the experiment, the animals had free access to standard diet and water.From day 2 onwards, DEN-induced animals in the positive control and test groups were orally administered distilled water and the decoction (at a dose of 10g/kg body weight/day) respectively, for a period of four weeks.Animals in the test and positive control groups were subjected to a second dose of DEN (20 µg/g body weight, i.p.), 30 min.before they were sacrificed [14].At the end of the fourth week, liver specimens from all animals were excised under diethyl ether anaesthesia, immediately washed with RNase-free ice cold phosphate buffered saline (pH 7.4) and used for (a) RT-PCR quantification of p53 wt and p21 genes, (b) western blot analysis of p53 and p21 proteins, and (c) immunohistochemical focusing of the p53 and p21 proteins.

Western blot analysis
For in vitro evaluation of p53 and p21 proteins in HepG2 cells by western blot analysis, cells were harvested after 12, 24 and 48 h incubation by trypsinization, and centrifuged at 1000 g for 10 min at 4 o C. The pelleted cells were solubilized for 15 min at 4 °C in lysis buffer [50 mM Tris-HCl pH 7.4, 150 mM NaCl, 1 mM EDTA, 1 % Nonidet P-40, 0.25 % Na-deoxycholate, 1 mM PMSF (phenylmethylsulfonyl fluoride) and proteinase inhibitor cocktail].Lysates were centrifuged at 12,000 g for 15 min at 4 °C to remove insoluble material.The supernatant was collected and protein concentration was determined using the Bradford's reagent.For Western blot analysis, samples (40 µg each) were separated by electrophoresis on 10 % SDS-polyacrylamide gels and transferred onto nitrocellulose membrane.
For evaluation of p53 and p21 protein expression in mouse liver, frozen mouse liver sections (200 mg of each liver specimen) were completely homogenized in 5 ml of ice cold RIPA buffer (50mM Tris-HCl pH 7.4, 150mM NaCl, 1mM EDTA, 1 % NP40, 0.25 % Na-deoxycholate, 1 mM PMSF [phenylmethylsulfonyl fluoride] supplemented with proteinase inhibitor cocktail containing aprotinin and leupeptin).Homogenate was immediately centrifuged at 12,000 g for 15 min at 4 0 C and the protein content of the supernatant was evaluated by Bradford's reagent using pre-plot standard curve.Equal amount (40µg) of liver protein lysate of each experimental group were separated by 10% SDS-PAGE and transferred overnight on to nitrocellulose paper at 4 0 C.
Nitrocellulose membranes onto which protein extracted from HepG2 cells and mouse livers were transferred were pre-blocked with 5% non fat dry milk in PBS for 30 min.Then the membranes were incubated for 16 h at 4 °C before incubating with specific primary antibodies which detect both human and mouse proteins.Bands were normalized using β-actin antibody (Santa Cruz Biotechnology, Santa Cruz, CA).After three washes of 10 min each with 1x TBST (50mM Tris-HCl, pH7.4,150mM NaCl, 1 % Tween 20), each membrane was incubated with the secondary antibody for 30 min at room temperature.The membrane was then washed three times with TBST (10 min/wash) and bands visualized using western blot detection system (Santa Cruz Biotechnology, Santa Cruz, CA).

HepG2
cells were harvested by trypsinization, and 2 x 10 5 cells/ml were plated on sterile coverslips placed in wells of a 24 well cell culture plate.The cells (on coverslips) were initially maintained in DMEM medium (1 ml/coverslip) for 24 hrs at 37 0 C in 95% air/ 5% CO 2 atmosphere, with 95% humidity.Cultures were then treated with the standardized decoction (600µg/ml or 1200 µg/ml) and incubated for 24 h.The doses of 600 µg/ml and 1200 µg/ml decoction were selected for this experiment based on the doses that produced significant enhancement of p53 and p21 gene expression in the experiments described above.Cells were then washed with phosphate buffer saline (PBS), fixed with cold 1:1 acetone: methanol for 15 min and air-dried.
Sections of mouse liver were immediately fixed in 10% formaldehyde, embedded in paraffin.Subsequently, 4 µm sections were used for staining after de-paraffinization in xylene, and rehydration through graded ethanol (AR, 80 -100 %).p53 and p21 activities were monitored in de-paraffinized tissue sections by exposing each section to heat-induced antigen retrieval in 10 mM citrate buffer (pH 6.0), using a pressure cooker [15].

Immunohistochemical analysis
Primary and secondary antibodies and the ABC staining kit were purchased from Santa Cruz Biotechnology (Santa Cruz, CA).The coverslips with HepG2 cells and slides containing deparaffinized mouse liver sections were quenched with 1% BSA in 0.05 M Tris/HCl (pH 7.6) for 30 min, and then incubated for 60 min at 37 0 C with anti-p53 (sc-6343) and anti-p21 (sc-397) rabbit polyclonal antibodies, diluted 1: 200.After washing with PBS (two times), the cells and tissue sections were incubated for 60 min with goat anti-rabbit IgG-HRP (sc-3837 diluted 1: 1000), washed again with PBS (two times) and visualized with ABC staining kit.Cells / tissue sections were counterstained with hematoxylin and examined under a light microscope.

Statistical analysis
The results of RT-PCR were analyzed using Prism 2.01 (Graph Pad Prism, San Diego, USA).One-way analysis of variance (ANOVA) with Dunnett's post test for multiple comparisons was used to find out the concentrations and time points at which the p53 and p21 expression were significantly increased in comparison to controls.Twoway ANOVA analysis was used to detect the effect of different concentrations of the decoction and duration of the treatment on the mRNA expression of p53 and p21 in HepG2 cells.One-way ANOVA with Bonferroni post test was used to compare all pairs of columns and used to analyze the results of p53 and p21 mRNA expression in mouse liver.

Preparation of plant decoction
The yield of the freeze-dried decoction was 15.2 ± 0.3.%

Effect of decoction on HepG2 cell viability
Alterations in viability of cells incubated for 12, 24 and 48 h with different doses of the decoction are shown in Fig 1 .At all three post-incubation time points, statistically significant differences in viability from zero time were observed only at doses above 1200 µg/ml.Hence, in the experiments to evaluate effects on p53 and p21 expression, doses of decoction at or below 1200 µg/ml were used.

Effect of decoction on p21 gene expression in HepG2 cells
As with p53, p21 gene expression was also normalized to the house keeping GAPDH gene.RT-PCR evaluation (Fig. 4) showed that in HepG2 cells, the standardized decoction induced over expression of p21 mRNA in a significant (p < 0.001) time and dose dependant manner.The lowest dose at which statistically significant (p < 0.01) p21 mRNA up regulation was observed at 12, 24 and 48 h post-incubation was 75 µg/ml.
The results of RT-PCR analysis were supported by findings of the western blot analysis and immunohistochemical analysis of the p21 protein expression in HepG2 cells (Figs 3B and 5A).One of the compounds isolated from N. sativa seeds is an anticancer compound, thymoquinone.
We have previously demonstrated that thymoquinone is not present in the decoction used in the present experiment [10].Therefore, some other water-soluble compound(s) is(are) likely to be responsible for the up regulation of p53 and p21 mRNA, and protein levels observed in the present study.Other investigators have reported that several plant compounds such as resveratrol, curcumin and eurycomanone [23] mediate their anti-proliferative effects trough apoptosis and cell cycle arrest.Decoction comprising of N. sativa seed, H. indicus root, and S. glabra rhizome investigated in the present study also appears to exert anti-proliferative and anti carcinogenic effects via apoptosis and cell cycle arrest through up regulation of p53 and p21 genes.

CONCLUSION
transcription reaction mix containing 5µg M-MLV 5X reaction buffer, 10 mM dNTP mix, 25 units of RNasin and 200 units of M-MLV reverse transcriptase in PCR water adjusted to 25 µl.GAPDH amplification was performed as an internal control.Each PCR was carried out in a master mix containing 1X Green Go Taq Flexi Buffer, 2 mM MgCl 2 , 10 mM dNTPs and 1.25 U GoTaq DNA polymerase (Promega Inc.US) with 0.2 mM of respective forward and reverse primers in 50 µl reaction mix.PCR amplification was carried out in a Mastercycler (Eppendorf, Hamburg, Germany) and the PCR conditions for both human and mouse GAPDH were 30 cycles at 94 o C for 30 s, at 54 o C for 30 s, at 72 o C for 1 min.PCR conditions for both human and mouse p53 were 35 cycles at 94 o C for 1.5 min, at 55 o C for 1 min, at 72 o C for 1 min.PCR conditions for mouse p21 were 30 cycles at 94 o C for 1.5 min, at 62 o C for 1 min, at 72 o C for 1 min and for human p21 were 35 cycles at 94 o C for 1.5 min, at 52 o C for 1 min, at 72 o C for 1 min.Primers were purchased from Integrated DNA Technologies, USA.

Figure 1 :
Figure 1: Effect of aqueous extract on overall cell viability-MTT assay (values are mean ±SEM)

Figure 3 :Figure 4 :
Figure 3: Immunohistochemical analysis of p53 and p21 in HepG2 cells and mouse liver.Expression of p53 (A) and p21 (B) protein in HepG2 cells (shown by arrows) was confirmed by immunohistochemical analysis: [a] control (untreated) [b] treated with the decoction (600 µg/ml), [c] treated with the decoction (1200 µg/ml).Section [d] represents the negative control.Expression of p53 (C) and p21 (D) protein (shown by arrows) in mouse liver [a] neither induced with DEN nor treated with the decoction (normal control), [b] treated with the decoction after induction, [c] induced with DEN, [d] the negative control.Hematoxyline was used for counterstaining

Figure 5 :Figure 6 :
Figure 5: Western blot analysis of effects of the decoction on p53 and p21 expression in HepG2 cells and mouse liver with DEN-induced early hepatocarcinogenesis. [A]-Western blot analysis of p53 and p21 expression in HepG2 cells treated with different concentrations (lane 1-control, lane 2-75 µg/ml, lane 3-150 µg/ml, lane 4-300 µg/ml, lane 5-600 µg/ml, lane 6-1200 µg/ml) of the standardized decoction at (a) 12 h post incubation, (b) 24 h post-incubation and (c) 48 h post incubation was performed as described in the Materials and Methods section.Normalization of bands was performed using β-actin protein.[B] Western blot analysis of p53 and p21 expression in mouse livers with DEN-induced early hepatocarcinogenesis treated with the decoction was performed as described in the Materials and Methods section.Lane 1 -distilled water control, Lane 2 -decoction only, Lane 3-induced with DEN, Lane 4 -treated with the decoction post DEN-induction.