Potential Chemoprevention Activity of Pterostilbene by Enhancing the Detoxifying Enzymes in the HT-29 Cell Line

Cancer is one of the leading causes of death in Malaysia (National Cancer Registry, 2007). Early cancer screening allows it to be diagnosed at an early stage and may provide a good prognosis (Ott et al., 2009). However, prevention remains the best way to deal with cancer (Shukla and Pal, 2004) due the fact that cancer treatments reduce the life quality of the cancer patients, with side effects accompanying as well as it is reported to be costly (Kroenke et al., 2010). Although cancer is often associated with genetic factors, but other risk factors such as diet, lifestyle, exposure to carcinogens and radiation, viral infections and diseases are modifiable (Świątkowska, 2007; Dossus, 2008). For example, there are a lot of studies have shown a correlation between the consumption of a diet rich in fruits and vegetables can prevent the formation of certain types of cancer. Phytochemicals are chemicals produced by plants through a specific response to protect plants from pathogen or environmental exposure, such as excessive ultra violet (UV) radiation. Phenolic compounds are one of the phytochemicals that act as phytoelexin in plants and these compounds also have valuable medicinal value.


Introduction
Cancer is one of the leading causes of death in Malaysia (National Cancer Registry, 2007). Early cancer screening allows it to be diagnosed at an early stage and may provide a good prognosis (Ott et al., 2009). However, prevention remains the best way to deal with cancer (Shukla and Pal, 2004) due the fact that cancer treatments reduce the life quality of the cancer patients, with side effects accompanying as well as it is reported to be costly (Kroenke et al., 2010). Although cancer is often associated with genetic factors, but other risk factors such as diet, lifestyle, exposure to carcinogens and radiation, viral infections and diseases are modifiable (Świątkowska, 2007;Dossus, 2008). For example, there are a lot of studies have shown a correlation between the consumption of a diet rich in fruits and vegetables can prevent the formation of certain types of cancer.
Phytochemicals are chemicals produced by plants through a specific response to protect plants from pathogen or environmental exposure, such as excessive ultra violet (UV) radiation. Phenolic compounds are one of the phytochemicals that act as phytoelexin in plants and these compounds also have valuable medicinal value.

Potential Chemoprevention Activity of Pterostilbene by Enhancing the Detoxifying Enzymes in the HT-29 Cell Line Zaliha Harun, Ahmad Rohi Ghazali*
Resveratrol is a phenolic compound that has been the most studied stilbene for its various biological activities including antioxidant, anti-inflammatory, anticancer and other biological activities (Fremont, 2000). It can be found naturally in grapes, berries and red wine (Bhat and Pezzuto, 2002). Pterostilbene, the natural dimethyl ether analogue of resveratrol has similar biological activity to resveratrol (Remsberg et al., 2008). It is also found in fruits such as grapes and berries. Studies have shown that pterostilbene also has a variety of beneficial biological activities such as antioxidant activity (Pari and Satheesh, 2006) and anti-inflammatory (Paul et al., 2009). In addition, pterostilbene showed anticancer activity against several cancer cells such as breast cancer , lung cancer  and pancreatic cancer (Mannal et al., 2010). Its potential as a cancer preventive agent has also been found through inhibition of drug metabolizing enzyme cytochrome P450 1A1, 1A2 and 1B1 (Mikstacka et al., 2007).
Thus, this study was conducted to determine the effect of pterostilbene treatment on the modulation of detoxifying enzymes in the colon cell line. Detoxifying enzymes involves in the conjugation or reduction of reactive species into more polar compounds that are easily excreted (Sheweita and Tilmisany, 2003). Reactive species has been proved to induce oxidative stress resulting in the cellular damage or it eventually can alter the genetic material of a normal cell become cancerous (Ioannides and Lewis, 2004). Thus induction of detoxifying enzymes will give protection to the cell from the action of the reactive species and this can prevent carsinogenesis. Glutation (GSH), the tripeptide molecule is a substrate that is widely conjugated the electrophilic species intracell. Conjugation reaction of the GSH is catalysed by glutation-S-transferase (GST) enzyme (Sheweita and Tilmisany, 2003;Ionescu and Caira, 2005). In conjuction with GST, the NADP(H):quinone oxidoreductase (NQO1) also functions as protective's enzyme by catalysing the reduction of reactive species such as quinone, quinonamine and nitro compounds (Siegel et al., 2004).

Cell culture
The HT-29 cell line was cultured in 75 cm 2 culture flask using modified MacCoy's5 Agrowth media at 37ºC and 5%CO 2 . The media was supplemented with 1% of penicillin/streptomycin and 10% Fetal Bovine Serum (FBS). Subculture was performed when cells reached confluent and usually it was done twice a week.

Preparation of sample (whole cell lysate)
About 11 ml of cells suspension (1x10 5 cells/ml) were seeded onto the culture dish and allowed to attach overnight. Then, the cells were treated with the compounds for 24 hours before harvesting. Finally, the preparation of the sample was done according to method by Inayat-Hussainet al. (2010) for determination of GSH level and GST activities. While, to study NQO1 activity, the sample preparation employed the method by Yakovlev et al. (2004) with slight modification.

Cell viability assay (MTT assay)
The MTT assay was done according to Mossman (1983). About 200 μl cells were seeded at concentration of 5x10 4 cells/ml per well in 96-well plate and allowed to attach overnight at 37 o C in 5%CO 2 . The cells were then treated with pterostilbene (0-100 μM) for 24 hours. About 20 μl MTT solution (5 mg/ml) was added to each well and the plates were incubated at 37°C in the CO 2 incubator for 4 hours. Then, the culture media was removed and finally dimethylsulphoxide (DMSO) (200 μl) was added to each well to sollubilize the purple formazan formed.
Absorbance was read at 570 nm with ELISA microplate reader (Bio-Rad, USA).

Ellman assay
Determination of glutathione level was done using method by Ellman (1959). DTNB is a soluble material that is used to determine the sulfhydril group quantity in a compound. Compounds containing the substance will produce acid 5-thio-2-nitrobenzoic (TNB), which is colored yellow when acting with sulfhydril. Firstly, the glutathione standard curve was prepare dusing a serial dilution of 0-10 mM glutathione with two times dilutional factor in 96-well plate. Glutathione was diluted in 50 μl reaction buffer (0.1 M Na 2 HPO 4 .7H 2 O and 1 mM EDTA) pH 6.5. The amount of sample used in this assay is about 50 μl. The samples were pipetted into empty well. About 50 μl of reaction buffer (0.1 M Na 2 HPO 4 .7H 2 O and 1 mM EDTA) pH 8.0 was added into well containing glutathione and samples. Finally, 10 μl DTNB was added to all wells and the microplate was read at 415 nm of wavelength using microplate reader (Bio-Rad, USA). Determination of protein concentration of the samples were done using Bradford assay (1976).

Glutatione S-transferase (GST) assay
The GST activity of the samples was determined using GST assay (Habig and Jakoby, 1981). GST activity was defined as the rate formation of product (GS-DNB conjugate). The reaction mixture of the GST assay consist of 840 μl of phosphate buffer (100 mM K 2 HPO 4 and 100 mM KH 2 PO 4 ) pH 6.5, 100 μl GSH (1 mM) and 50 μl of the sample that were added into a 1 ml cuvet. Approximately about 10 μl CDNB (75 mM) was added into the reaction mixture to start the reaction. The rate of the reaction (abs/ min) was red using UV spectrophotometer (Secomam, Perancis) at 340 nm wave length for three minutes. The protein concentration of the samples was measured using Bradford assay (1976). The extinction coefficient of 9.6 mM -1 was used to calculate the GST activity of the samples.

NAD(P)H: quinoneOxidoreductase (NQO1) assay
Method by Siegel (2000) was employed to study the NQO1 activity. In this assay, the rate of 2,6-dichlorophenolindolphenol (DCPIP) reduction over a period of time is the key principle in determining the NQO1 activity. In addition, dicoumarol was used as the chemical that exert the competitive inhibition towards the NADPH binding to NQO1 enzyme thus stop the catalytic reaction of the enzyme. The reaction mixture of this assay consisting of 970 μl reaction buffer (25 mM Tris, 1 mg/ ml BSA and 200 µM NADPH) pH 7.4, 20 μl of DCPIP solution (2 mM) and 10 μl samples that was pipetted into 1 ml cuvet. The absorbance was read over one minute using spectrophotometer (Secomam, France) at 600 nm of wave length. On the other hand, for reactions containing dicumarol, about 960 μl reaction buffer (25 mM Tris, 1 mg/ml BSA and 200 µM NADPH) pH 7.4, 20 μl DCPIP solution (2 mM), 10 μl sample and 10 μl dicumarol (2 mM) were prepared as a mixture of reactions. Total protein concentration of the samples was determined by Bradford assay (1976).

Statistical analysis
Data were analysed statistically using IBM SPSS statistic 20 software (SPSS Inc. Chicago, USA) and were presented as mean±SEM.

Results
Result showed that treatment of the pterostilbene in the range of 0-100 µM did not give the IC 50 value (Figure 1). Small reduction of the cell viability was reported when the cells were treated in this range of treatment dose in which there was only 20.17±0.82 % of cell viability reduction for the highest dose, whereas for 50 µM, 25 µM, 12.5 µM, 6.25 µM and 0 µM of the cell viability reduced about 16.38±4.34%, 14.51±1.20%, 13.06±5.84 and 10.88±2.98% in a dose dependent manner respectively. This showed that treatment of pterostilbene with the chosen range of treatment for 24 hours did not toxic to the cell. Thus the doses were suitable to be used to evaluate the potential of this compound. Hence we have chosen treatment of pterostilbene up to 50 µM because it retained 80% of the cell viability. Figure 2 showed the effect of pterostilbene treatment on GSH level in HT-29 cell line. There was an increased of the GSH level compare to the negative control for all the treatment concentration with a significant of increment reported at 50 µM of pterostilbene treatment with the value of 75.54±5.92mmol/mg protein. This value is closely similar with the significant increment of GSH level with vitamin C that was used as positive control with the value of 75.54±7.05 mmol/mg protein. From the result we also could see a slight increasing of the GSH level was in the dose dependent manner but unfortunately the value of pterostilbene treatment at 12.5 µM and 25 µM is not significant (p<0.05) compare to negative control.
The effect of pterostilbene on the GST activity was summarized in the Figure 3. The result showed that treatment of pterostilbene can also induce the GST activity of the HT-29 cell line. From the result there were significant (p<0.05) increase of GST activity at 12.5 µM pterostilbene treatment with the value of 4277.24±489.32 nmol/min/mg protein and 25 µM pterostilbene treatment with the value of 4617.12±345.95 nmol/min/mg protein compare to negative control.
In addition, pterostilbene treatment also induced the NQO1 activity in the cell line. This was shown from the result (Figure 4) in which treatment of pterostilbene at 12.5 µM significantly (p<0.05) increase the NQO1 activity with the value of 3973.82±177.33 nmol/min/mg protein.
Meanwhile, at 50 µM of pterostilbene, the NQO1 activity was reported as 4219.60±321.18 nmol/min/mg protein which was signicantly increase compare to negative control.

Discussion
Before the potential of pterostilbene was evaluated, the optimum dose of the compound on HT-29 cell line was evaluated. This was to ensure the compound did not causing damage or induce any stress towards the cells. From the result, the treatment of this compound (0-100 µM) for 24 hours did not cause cytotoxicity to the cells. This result differs from findings by Nukula et al. (2011) in which there was an IC 50 reported at 15 µM of pterostilbene treatment in HT-29 cell line. This might due to the long incubation period of pterostilbene treatment in the study which was 48 hours. Study by Mannal et al. (2010) on the effect of pterostilbene on cell viability on two types of pancreatic cells showed a concentration and timedependent manner of pterostilbene treatment. Similarly, pterostilbene was reported to reduce the cell viability of the lung cancer cell  and breast cancer lines  in the concentration and time-dependent manner.
In this study, we found that pterostilbene, has reported the same ability with resveratrol in which it can induce the detoxifiying enzymes. In addition our result was also demonstrated the similar significant effect with regard to the dose with the study by Ghazali et al. (2012). In that study, they used the HepG2 cell line to investigate the effect of pterostilbene on GST activity and GSH level. For the GST activity, the study reported a significant increase in post pterostilbene treatment at 25 µM and 100 µM but not 50 µM. This study also found the same findings in which the significant increase in GST activity was reported at 12.5 µM and 25 µM but not 50 µM of pterostilbene treatment. Ghazali et al. (2012) also showed a significant increase of GSH level at 25 µM and 50 µM that also reported in our study in which at 50 µM the GSH level also increase significantly compare to negative control. This suggests that at 25 µM pterostilbene treatment, GST activity increase thus there were a slight GSH level depleted, masking the induction effect by pterostilbene. But when there was no significant increase of GST activity at 50 µM of pterostilbene treatment, the increase of GSH level are apparent.
In addition, epidemiological studies had correlated the risk of colorectal cancer with the dietary factor (Giovannucci et al., 1994) especially the intake of certain food such as the cooked meet that contain high level of polyarometic hydrocarbon (PAH) (Martorell et al., 2010). Reactive species generated from metabolism of PAH can initiate a cascade of oxidation that produced more reactive oxygen species (ROS) which can lead to oxidative stress. Oxidative stress is one of the pathways that might contribute to carcinogenesis (Goetz and Luch, 2008). Previous study has demonstrated chemoprevention of pterostilbene on colon cancer by suppressing the formation of tumorigenesis, inhibit the cell proliferation and exert the anti-inflammation on the colon cancer cell (Paul et al., 2010). Induction of GST activity and GSH level in this cell line indicate that, colon could also acquire cellular protection to prevent carcinogenesis through the conjugation reaction to eliminate the reactive species.
Moreover, protection from reactive quinone species also can be achieved via the induction of NQO1 activity. Study by Yang and Liu (2009) reported that resveratrol contributed to a high NQO1 activity in the hepatoma cell. Our study also revealed that pterostilbene was able to increase the NQO1 activity significantly in HT-29 cell line. This provides a promising effect towards prevention of cellular damage caused by reactive quinone species.
Metabolism of environment pollutant such as benzene  and penthachlorophenol (PCP) (Vaidyanathan et al., 2007) or endogenous quinones produce reactive quinone species that can induce oxidative stress or forming the DNA adduct. Hence, by induction of NQO1 activity, the quinone reactive species can be reduced into a nontoxic quinone derivatives such as hydroquinone via a two electron reduction, by passing the formation of semiquinone .
Cell protection by means of detoxification always involves the conjugation or reduction of the reactive species to become more polar compound and less reactive. In the basal state, the cell might content low activity of detoxifying enzymes, but the activity will increase when there is an induction. There are several inducers that have been recognized to cause the induction of detoxifying enzymes and among them are phytochemicals such as resveratrol that was reported to activate the antioxidant response element (ARE) via Nrf2-ARE pathway. Nuclear factor erythroid 2-related factor 2 (Nrf2) is a transcription protein that translocates from cytoplasm to nucleus to activated ARE sequences that induce the transcription of the detoxifying enzyme such as GST and NQO1 (Lee and Johnson, 2004;Surh et al., 2008). Beside, resveratrol have been reported to increase the expression of Glutamatecysteine-Ligase (GCL) via the same pathway that leads to increase of the GSH level (Kode et al., 2008). Thus this study postulated that, pterostilbene might also able to activate the Nrf2-ARE pathway, leading to trancrsiption of detoxifying enzymes.
In conclusion, pterostilbene is a potential chemoprevention agent through its modulation of detoxifying enzymes.