INTRODUCTION

Chemoprevention is a promising strategy to prevent cancer by using natural or synthetic substance. Many studies have successfully shown the effectiveness of diets having chemopreventive effects against the growth of cancer cells. White and green tea ameliorated colon cancer by inducing apoptosis and modulation of certain genes (Santana-Rios et al. 2002, Orner et al. 2003). Curcumin in tumeric induced apoptosis by activating caspases in malignant glioma cells (Gao et al. 2005). Zingiber officinale or commonly called ginger is widely used for medicinal purposes since thousands of years ago in Chinese, Arabs, Romans and Indian medicine (Ross 2002). In our previous study, ginger extract was shown to re­duce lipid peroxidation and tumour bur­den in hepatocarcinogenesis-induced rats. Other studies had reported that gin­ger exerted its anti-tumour properties by inhibiting proliferation and inducing apoptosis in HL-60 leukemia cells (Surh & Lee 1998) and Jurkat human T cell leukemia cells through down-regulation of anti-apoptotic protein Bcl-2 and en­hancement of pro-apoptotic protein Bax
(Miyoshi et al. 2003).

Cancer is caused by an imbalance in the rate of proliferation and apoptosis or cell death. Apoptosis is a form of programmed cell death characterized by morphological changes in cells executed by cysteine-aspartate proteases (cas-pases) and regulated by the Bcl-2 family of proteins involved in the signal trans-duction pathways (Coultas & Strasser 2003, Hanson et al. 2008). It is the preferential way of targetting and removing cancer cells. Dietary com-pounds that can trigger apoptosis would be a potential use in cancer chemo-prevention.

Hepatocellular carcinoma (HCC) is the fifth most common cancer worldwide with nearly half a million of new cases an­nually (Wei 2006). In Malaysia, HCC is the eleventh most common cancer among all malignant diseases (Gerard & Halimah 2003). The attributable risk fac­tors of HCC are chronic hepatitis B and C infections, cirrhosis, exposure to aflatoxin and certain chemicals such as polyamine hydrocarbon (PAH), diethylnitrosamine (DEN) and 2-acetylaminofluoren (AAF) (Schafer & Sorrell 1999). Currently, the treatments of HCC include surgical re­section, liver transplantation, drug and radiation therapy (Forner et al 2006). All these procedures posed a lot of risk and side effects. The search for an alternative and safe compound or chemopreventive agent would benefit patients and reduce the unwanted side effects and risk fac­tors. Ginger is an indispensable ingre­dient of Asian foods and this report con­cerns the chemopreventive efficacy of ginger in an ethionine induced liver can­cer carcinogenesis model by examining the expression of pro- and anti-apoptotic proteins, caspase-8 and Bcl-2 respec­tively.

MATERIALS AND METHODS

Animals, Chemicals and Treatment

Thirty male Wistar albino rats aged be­tween 3-4 months and weighing 200-250 g were supplied by the Animal Care Unit of Universiti Kebangsaan Malaysia (UKM) Medical Center. The study was approved by the Animal Ethics of the Fa­culty of Medicine, UKM. Animals were kept in a polycarbonate cage provided with food and waterad libitum. They were maintained under standard condi­tions of temperature and humidity with an alternating light and dark cycle. Rats were randomized into 5 groups of 6 ani­mals each. The first group and the second group served as the control group and were fed with normal rat chow (Gold Coin, Malaysia) and rat chow plus olive oil respectively. The latter served as a control for gavage method and the deli­very of ginger. Rats in group 3 received ginger extract at 100 mg/kg body weight by gavage method. Ginger extract was prepared by ethanol extraction and kept at 40C. It was dissolved in olive oil and force-fed to the rats. Rats in group 4 were fed with choline deficient diet (ICN Biochemicals, USA) plus 0.1% ethionine (Sigma Chemical Co., USA) in drinking water, known as CDE. This is the model to induce the production of oval cells, which are the precursor cells of liver can­cer(Akhurst et al. 2001). Rats in group 5 received ginger as in group 3 plus CDE diet. All rats were killed at 8 weeks and the liver tissues were excised after perfu­sion and embedded in paraffin blocks for immunohistochemical staining.

Liver perfusion and preparation of paraf­fin blocks

The rats were sacrificed using ether. All equipments for dissection were sterilized using 70% alcohol before use. The rats were anasthesized intraperitoneally with Zoletil 50 (0.1 ml/100g body weight), fol­lowed by heparin (25, 000 U/ml) injection to the inferior vena cava. The portal vein was then canulated using an intravenous catheter, size 16G (2.25 inches) for the perfusion procedure. The liver was then perfused in PBS, pH 7.4 for 1 minute at a 10ml/min flow rate at room temperature, followed by 1:1 ratio of 4% para-formal­dehyde and 0.1% glutaraldehyde for 3 minutes. Then the liver was perfused back in PBS for another 2 minutes. A portion of the perfused liver was then immersed in 10% formalin for fixation before embedding in paraffin.

Preparation of tissue sections

The paraffin-embedded tissues were cut at 3 μm thick with a rotatory microtome(Leica, Germany). The tissue sections were placed on poly-L-Lysine (Sigma-Aldrich Co. USA) treated slides with 1:10 dilution. The slides were then dried overnight and stored at room tempe-rature until used for staining.

Hematoxylin & eosin (H&E) staining

The sections were deparaffinized and hydrated with sequential washes in xy­lene and alcohol. Nuclei were stained by immersing in Mayer’s hematoxylin solu-tion (Lab Vision Corp., UK) for 8 mi­nutes and rinsed under running tap wa­ter. The slides were then dipped in 1% acid alcohol to remove excess hematox­ylin followed by immersion in 2% sodium acetate. Slides were rinsed in running water followed by eosin staining for 5 min to stain the cytoplasm. Finally, slides were dehydrated through a series of graded alcohols and mounted withdibutylpthalate xylene (DPX).

Immunohistochemistry for detection of Bcl-2 and caspase-8

Paraffin sections (3 mm thick) were cut from liver specimens. Sections were de­paraffinized and rehydrated by sequential immersion in xylene, a series of alcohol concentrations (100, 95, 80 and 70%), and in running water. Slides were pre-incubated in 3% hydrogen peroxide for 10 minutes to block endogenous perox­idase activity. Slides were washed in Tris-HCl-buffered saline (TBS) before incubating with bovine serum albumin and biotin as a blocking step to reduce nonspecific staining. For detection of Bcl protein, slides were then immersed in target retrieval solution (pH9.9) (DAKO, U.S.A.) at 98oC in a water bath for 20 minutes. For caspase-8, the target re­triev­al solution (pH9.9) (DAKO, U.S.A.) was heated in microwave oven for 20 minutes at 98oC. Slides were left at room temperature for 20 minutes before washing in Tris-buffered saline (TBS) for 3 changes at 3 minutes each. Tissues were then incubated with the primary an­tibody. For evaluation of Bcl-2 expres­sion, tissues were incubated with mono­clonal mouse anti-human Bcl-2 (DAKO, Denmark) at 1:50 dilution for 30 minutes while for the evaluation of caspase-8, tissues were incubated with rabbit poly­clonal to human Caspase-8 (Abcam, UK) at 1:100 dilution for 1 hour.  After several washings, slides were then incubated with secondary antibody conjugated with biotin and streptavidin labeled with horse­radish peroxidase (LSAB kit, DAKO, Denmark) for 30 minutes. The slides were then treated with diamino-benzi­dine, DAB (DAKO, U.S.A.) for 20 minutes before counterstaining with haematoxylin for visualization of antigen. TBS was used in place of the primary antibody for the negative control. Human tonsil and gastric tissues were used as positive controls for Bcl-2 and caspase-8 respec­tively.

Immunostaining analysis

Immunoreactivity evaluation was based on staining intensity and percentage of positive staining of Bcl-2 and caspase-8. Staining intensity was divided into 3 cate­gories: 3+ indicates strongly positive, 2+ indicates moderately positive, and 1+ indicates slightly positive. The percen­tage of positive staining was determined from 1% to 100% of cells stained posi­tively at 10 different fields observed un­der 400X magnification of light micro­scope. Twelve slides (2 slides from each rat) were prepared from a total of six rats to represent each group.

Statistical analysis

Descriptive analysis was used to com­pare the expression of apoptotic protein in different sample groups.

RESULTS

H&E staining for oval cells expression

The control groups (normal rat chow, normal rat chow + olive oil) and rats treated with ginger showed normal rat liver histology with hexagon shaped he­patocytes, round nucleus and distinct cytoplasm. The sinusoids were clearly visible under 400x magnifications with Kupffer cells located at the sinusoidal wall (Figure 1A). A similar morphology of liver cells was also observed in the gin­ger extract group (Figure 1C) and CDE group treated with ginger extract (Figure 1E). Abnormal liver cell morphology was observed in the liver-cancer induced group with irregular shaped hepatocytes and sinusoids (Figure 1D). Numerous oval-shaped cells with large nucleus and scanty cytoplasm located mainly near the periportal site were seen in the liver-can­cer induced group (CDE).

Table 1 shows increased oval cell ex­pression in CDE group (91.6% of sam­ple) which was abrogated after treatment with ginger extract (100 mg/kg body wt).

Bcl-2 expression

Figure 2 represents patterns of Bcl-2 ex­pression in control groups and in CDE group treated with ginger. Figure 2A shows positive staining for Bcl-2 protein in tonsil tissue.

There were no Bcl-2 expression ob­served in the control (and olive oil group, image not included) and ginger extract groups (Figures 2B and 2C). However, Bcl-2 expression was clearly observed in the CDE group (Figure 2D) with 91.6% of the cells positively stained (Table 2) with the following intensities: 66.6% cells with 1+ intensity and 25% cells with 2+ inten­sity (Table 3). Oval cells that were stained with Bcl-2 showed patchy ex­pression in the cytoplasm with no ex­pression observed in the nucleus (Figure 2D, 400x). Treatment of ginger inhibited the expression of Bcl-2 in the CDE group (Figure 2E).

Caspase-8 expression

Figure 3 represents patterns of caspase-8 expression in control and ginger groups and in CDE group treated with ginger. Figure 3A shows positive staining for caspase-8 in human gastric tissue. The expression of caspase-8 was observed in both CDE and CDE group treated with ginger extract (Figure 3D, 3E). No cas­pase-8 expression was observed in the control and ginger groups (Figures 3B, 3C). For CDE group, 41.7% of cells were stained with caspase-8 (Table 4) with the following intensities: 33.4% cells with 1+ intensity and 8.3% cells with 2+ intensity (Table 5). However, for CDE group treated with ginger extract, 100% of the cells were stained positively for caspase-8 with the following intensities: 50% with 3+ intensity, 25% with 2+ inten­sity and 25% with 1+ intensity (Table 4). Cas­pase-8 was expressed in a patchy pat­tern in the cytoplasm of the oval cells of CDE group (Figure 3D). A homogen­ous staining of caspase 8 was observed in the CDE group treated with ginger ex­tract (Figure 3E).

DISCUSSION

Most of the flavouring ingredients in Asian foods are rich in phytochemicals with medicinal properties which include turmeric, cloves, garlic, aniseed, mus­tard, saffron, cardamom and ginger (Sengupta et al. 2004). Some of these spice condiments have been supported by experimental models to have chemo­preventive properties which have the ability to interfere with carcinogenic process. The purpose of chemopreven­tive agent in cancer prevention is to block or cause delay in onset of cancer, pro­gression from precancerous lesion or recurrence after treatment (Tsuda et al. 2004).  

Ginger has been used in time of im­memorial for food condiments and medi­cinal use especially to aid digestion and treat stomach upset, diarrhoea and nau­sea. It was shown to have strong anti-oxidant properties attributed by the bio-active components such as 6-paradol, shogaol, zingerone and gingerol (Masuda et al. 1995, Aeschbach et al. 1994, Chang et al. 1994, Cao et al. 1993, Reddy & Lokesh 1992).  Antioxidative capacity of ginger has been associated with the ability of ginger to inhibit carci­nogenesis by reducing oxidative stress and inducing apoptosis (Shukla & Singh 2006, Katiyar et al. 1996, Manju & Nalini 2005, Rhode et al. 2007).

Our study clearly showed the efficacy of ginger having antitumour properties by inhibiting the proliferation of oval cells as evidenced by the reduced number of oval cells and the perpetuation of normal his­tological structure of liver tissues in liver cancer induced rats treated with ginger extract. Ovalcell proliferation precedes neoplasia in many rodent modelsof he­patocellular carcinoma and in chronic liver disease of human studies (Ackhurst et al. 2001, Lowes et al. 1999). Preven­tion of this proliferativeresponse can re­duce the risk of subsequent carcinoma. This was supported by our previous findings which showed that ginger re­duced oval cell proliferation and liver tu­mour formation in hepatocarcinogenesis induced rats (Mohd Habib et al. 2008). This property of ginger could be attri­buted to the presence of high phenolic compounds such as [6]-gingerol, and [6]-paradol. [6]-gingerol has been shown to suppress experimental metastases in tumour-bearing mice skin carcinogenesis probably via its anti-angiogenic activity (Kim et al. 2005) and inhibition of COX-2 expression along with suppressed NF-kB DNA binding activity (Kim et al. 2004). Since tumour promotion is closely linked to oxidative stress, a compound that ex­hibits antioxidant properties could act as an anticarcinogenic agent (Shukla & Singh 2006). 

Some compounds present in ginger may exert cancer preventive effects by inducing apoptosis in cancerous or transformed cells. (Shukla & Singh 2006). Several studies have reported that compounds in ginger suppress prolifera­tion of human cancer cells through in­duction of apoptosis accompanied by downregulation of anti-apoptotic protein Bcl-2 and enhancement of pro-apoptotic protein Bax expression (Lee & Surh 1998, Miyoshi et al. 2003).  The dimi­nished expression of anti-apoptotic Bcl-2 protein observed in CDE group treated with ginger correlated with inhibition of oval cell expression. This confirmed that ginger must have bioactive compounds to produce such chemopreventive ef­fects. Our findings are supported by Reed et al. (1994), who have shown that Bcl-2 prevented the death of neoplastic cells via apoptosis. Vaux et al. (1988) reported that expression of Bcl-2 induced the tumour formation in mice. Our study showed patchy staining of Bcl-2 in oval cells and the results are supported by Frommel et al. (2000) who reported that Bcl-2 was only expressed in proliferative cells near the periportal site of liver (oval cells) but not in normal hepatocytes, Kupffer cells and bile duct epithelium.

Caspase-8 is an executor zymogen protein involved in apoptosis signaling pathway. Ishiguro et al (2007) proved that [6]-gingerol facilitated TRAIL-induced apoptosis by activation of caspase-3/7 activity in human gasric cancer cells. We observed caspase-8 staining in both CDE and CDE group treated with ginger, with enhanced staining observed in the latter. This observation confirmed that in cancer cells, apoptosis is a natural way of inhi­biting its growth, and chemopreventive agents, having the ability of inducing apoptosis, enhance further the effect of apoptosis as seen with the effect of gin­ger extract.

In conclusion, our study agrees with other studies which provide substantial evidence that ginger extract are effective inhibitors of carcinogenic process exhi­bited by reduction of oval cells, Bcl-2 protein and induction of caspase-8 ex­pression.