Rice bran phytic acid (IP6) induces growth inhibition, cell cycle arrest and apoptosis on human colorectal adenocarcinoma cells

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Journal of Medicinal Plants Research Vol. 4(21), pp. 2283-2289, 4 November, 2010 Available online at http://www.academicjournals.org/JMPR ISSN 1996-0875 ©2010 Academic Journals ... Rice bran phytic acid (IP6) induces growth inhibition, ... Shafie
  See discussions, stats, and author profiles for this publication at: https://www.researchgate.net/publication/215861464 Rice Bran Phytic Acid (IP6) Induces GrowthInhibition, Cell Cycle Arrest and Apoptosis onHuman Colorectal...  Article  · November 2010 CITATIONS 13 READS 218 6 authors , including: Some of the authors of this publication are also working on these related projects: Antidiabetic and anti obesity acitivity of leaves and stems of Mikania micrantha   View projectBioactivity of Novel Metal Complexes View projectNorhaizan MEUniversiti Putra Malaysia 104   PUBLICATIONS   670   CITATIONS   SEE PROFILE Abdah AkimUniversiti Putra Malaysia 50   PUBLICATIONS   239   CITATIONS   SEE PROFILE Norazalina SaadUniversiti Putra Malaysia 11   PUBLICATIONS   153   CITATIONS   SEE PROFILE All content following this page was uploaded by Norhaizan ME on 18 January 2017. The user has requested enhancement of the downloaded file. All in-text references underlined in blue are added to the srcinal documentand are linked to publications on ResearchGate, letting you access and read them immediately.  Journal of Medicinal Plants Research Vol. 4(21), pp. 2283-2289, 4 November, 2010 Available online at http://www.academicjournals.org/JMPR ISSN 1996-0875 ©2010 Academic Journals Full Length Research Paper Rice bran phytic acid (IP 6 ) induces growth inhibition, cell cycle arrest and apoptosis on human colorectal adenocarcinoma cells Shafie Nurul-Husna 1 , Mohd Esa Norhaizan 1,2 *, Ithnin Hairuszah 1,2 , Md Akim Abdah 1 , Saad Norazalina 1 and Ismail Norsharina 1   1 Institute of Bioscience, University Putra Malaysia, 43400 Serdang, Selangor, Malaysia. 2 Faculty of Medicine and Health Sciences, University Putra Malaysia, 43400 Serdang, Selangor, Malaysia. Accepted 9 September, 2010 Phytic acid (inositol hexaphosphate or IP 6 ) is one of the bioactive compound that is present in cereals, nuts and legumes. IP 6  is a naturally occurring polyphosphorylated carbohydrate, recognized to posses various significant health benefits including anticancer effects. Several in vitro   and in vivo   studies provide convincing evidence for the anticarcinogenic properties of commercial rice IP 6  whilst the underlying mechanisms by which IP 6  exerts anti-tumsrcenic effects are still not fully known. The purpose of this present study is to investigate the growth inhibitory effects of IP 6  extracted from rice bran on human colorectal cancer cell line (HT-29). IP 6  extracted from rice bran induced marked growth inhibition in HT-29 with an IC 50  value of 12.0 ± 2  g/ml, in a dose and time dependent manner. Flow cytometry was performed for the analysis of cell cycle and apoptosis. Rice bran IP 6 -extract induced cell cycle arrest in HT-29 cell at G 0 /G 1 phase. Staining with Annexin V-based assay and propidium iodide confirmed that apoptosis occurred early and late in the HT-29. IP 6 is expected to exert anticarcinogenic activity through disruption of cell cycle progression and induction of apoptosis. Our study further supports the function of rice bran IP 6  as a chemopreventive agent for human colorectal cancer. Key words:  Phytic acid (IP 6 ), rice bran, colorectal cancer, cell cycle, apoptosis, chemoprevention. INTRODUCTION Colon cancer is the malignant neoplasm of the colonic epithelium. It is the third most common cancer and the third leading cause of cancer related deaths for both men and women in United States (American Cancer Society, 2008) and becoming increasingly common in Asian countries. Epidemiological studies have shown that high fiber foods, such as fruits, vegetables, whole grains and cereals may be protective against colon cancer (Howe et al., 1992; Potter, 1993). Inositol hexaphosphate (IP 6 ), also known as phytic acid or phytate, is a natural dietary ingredient, which is described as ‘‘natural cancer fighter,’’ being an essential component of nutritional diets. Phytic acid is a major *Corresponding author. E-mail: nhaizan@medic.upm.edu.my. Tel: +603 89472427. Fax: +603 89426769. constituent of all plant seeds, occurring at 0.4 to 6.4% (w/w) of most cereals, legumes, nuts, oil seeds and soybean (Shamsuddin et al., 1997) and naturally accounting for 60-90% of the total phosphorus in discrete regions of the seeds, such as the aleurone layer of wheat and rice (Tanaka et al., 1972) and in the germ of corn (O’dell et al., 1972). Over the years, several studies pioneered by Shamsuddin et al. (1996), and other research groups have shown the potential chemopreventive and anticancer effects of IP 6  in various cancer models (Singh and Agarwal, 2005; Fox and Eberl, 2002). In vitro   studies proved that IP 6  has been shown to inhibit growth of human breast, colon, and liver cancer cells, and  rhabdomyosarcoma and erythroleukemia cells; and cell transformation in mouse epidermal JB6 cells (Shamsuddin et al., 1996; Shamsuddin and Said, 1998; Vucenik et al., 1998; Shamsuddin et al., 1992; Huang,  2284 J. Med. Plant. Res. 1997). With regard to in vivo   anticancer efficacy of IP 6 , it has been shown that 1% (w/v) IP 6  in drinking water 1 week before or 2 weeks after the administration of azoxymethane (AOM) inhibits the development of large intestinal cancer in F344 rats (Shamsuddin et al., 1988). Later, it was reported that in same animal model, treatment with 2% (w/v) IP 6  in drinking water, even after 5 months of carcinogen induction, significantly inhibits both number and size of tumors in large intestines (Shamsuddin and Wah, 1989). Other study by Norazalina et al. (2010), revealed that treatment of 0.2% (w/v) of rice bran IP 6  give the greatest reduction in the formation of aberrant crypt foci (ACF) compared to commercial corn IP 6 . Furthermore, administrations of IP 6  in AOM-induced colon carcinogenesis in rat also reduce the incidence and multiplicity of total tumor formation (Norazalina et al., 2010). Various animal studies reported above, have also shown that IP 6  does not cause any adverse side effects or toxicity even at higher doses which are up to 2% (w/v) or 15 mM in drinking water (Shamsuddin and Wah, 1989; Singh et al., 2004; Vucenik et al., 1995). Because cancer is a major public health issue, the dramatic anticancer effect of IP 6  has resulted in our quest for understanding its mechanism of action. A central pathway of cancer inhibition by IP 6  is via control of cell division; and IP 6  reduces the rate of cellular proliferation both in vivo   and in vitro  . Tian and Song (2006), have demonstrated that IP 6  has potent inhibitory effect on proliferation of human colorectal cancer cell line (HT-29) by modulating proliferating cell nuclear antigen (PCNA) and Cip1/p21 expression. Along with this reduction in cell proliferation, IP 6  can regulate the cell cycle to block uncontrolled cell division and force malignant cells either to differentiate or to go into apoptosis (Matejuk and Shamsuddin, 2010). The laboratory investigation on the antitumor efficacy of IP 6  started in mid 1980s by Shamsuddin et al. (1997), and since then, several studies have shown the anticancer effects of commercial rice and wheat bran IP 6  in various in vitro   as well as in vivo   cancer models. To the best of our knowledge, there is no study showing anticarcinogenic effects of rice bran IP 6  on colorectal cancer cells. The study of phytic acid specific from rice bran as anticancer agent is still scarce. Hence, the main purpose of this study was to determine the anticarcinogenic potentials of IP 6  extracted from rice bran on colorectal cancer cells which may critically contribute to its cancer preventive and therapeutic efficacy. MATERIALS AND METHODS Chemicals and reagents Dulbecco's Modified Eagle Medium (DMEM), fetal bovine serum (FBS) and penicillin–streptomycin were from PAA (Austria). MTT ([4,5-dimethylthiazol-2-yl]-2,5-diphenyltetrazoliumbromide, dimethyl sulfoxide (DMSO) and commercial phytic acid were obtained from Sigma (USA). Annexin V-FITC Apoptosis Detection Kit was purchased from BD Biosciences (USA). HT-29 (human colorectal cancer cell line) and BALB/c 3T3 (mouse fibroblast cell line) were bought from American Type Culture Collection (ATCC) (USA). All other chemicals and reagents used were of the highest purity grade available. Sample preparation Rice bran (BERNAS, Malaysia) was stabilized according to the method of Ramezanzadeh et al. (1999). Stabilization was performed to prevent oxidative rancidity during storage. After the stabilization process, total lipid was extracted from rice bran samples by using hexane regarding to the modified method of Hu et al. (1996). Phytic acid (IP 6 ) was extracted from rice bran regarding to the Fruhbeck et al. (1995), with slight modification. The samples were added to hydrochloric acid, HCl (1 g in 20 ml) in pH 1.0. The extraction was carried out at room temperature with constant shaking in an orbital mixer. The obtained creamy mixture was centrifuged at 17300 g for 30 min at 15°C and the supernatants were collected (Norazalina et al., 2010). The modified method of Camire and Clydesdale (1982), was used to neutralize the phytate extract. The neutralized sample was then concentrated by freeze- drying and kept at -20°C. Growth inhibition assay- 3-(4-5- dimethylthiazol-2-yl]-2,5-diphenyltetrazoliumbromide, MTT) HT-29 and 3T3 cell lines were grown in DMEM supplemented with 10% FBS and 100 IU/ml Penicillin and 100 µg/ml Streptomycin, and incubated at 37 ° C under 5% CO 2  in a humidified atmosphere. To evaluate the effect of IP 6  on the proliferation of HT-29 cells, a colorimetric MTT assay was used according to Shamsuddin et al. (1996), and Vucenik et al. (1998). This assay measures the reduction of tetrazolium salt, MTT to a purple-colored formazan product. HT-29 cells were preincubated at density of 1 x10 5 cells/well on 96-well microtitre plates for 24 h. The old medium was tapped out and IP 6  (diluted in medium) in the concentration range of 0-20  g/ml were added into the plate. The plate was incubated for a further 72 h. Then, 20  l of MTT reagent (5.0 mg/ml) was added into each well and the plate was incubated for four more hours at 37°C. Subsequently, 100  l of solubilisation solution (DMSO) was added into each well and the absorbance was read at 570 nm using the microplate reader (Tecan, Switzerland). In this study, the effect of commercial rice phytic acid on cell proliferation was also determined as a comparison and the toxicology study by using normal cell (3T3 cell line). Therefore, we selected 50% growth inhibition concentration (IC 50 ) for the analysis of cell cycle and apoptosis. Cell cycle distribution analysis HT-29 cells were pre-incubated at a density of 1x10 5 cells   in a culture flask for 24 h. The culture medium was replaced with fresh aliquots containing IP 6  compounds at three different concentrations (9.5, 12 and 14.5  g/ml). After 24, 48 and 72 h exposure, the cells were trypsinized, washed three times with ice-cold phosphate-buffered saline (PBS) (10 mM sodium phosphate pH 7.2, 150 mM sodium chloride), re-suspended in 70% ethanol and further incubated at -20°C for 2 h. Then, the cells were washed with PBS and re-suspended in 50  l of RNase solution (10 mg/ml) and stained with 40  l of propidium iodide (1 mg/ml). The cell cycle was analyzed with flow cytometry (Beckman Coulter, USA). Detection of apoptotic cell death This assay was carried out using Annexin V-FITC Apoptosis  Nurul-Husna et al. 2285 Concentration of IP 6  (µg/ml)          Figure 1. Treatment of rice bran IP 6  extracts and commercial rice IP 6  on HT-29 cells. The cell viability was measured by MTT assay after 72 h exposure. The concentration was expressed as a percentage compared to control cells. Detection Kit I according to manufacturer’s protocols. HT-29 cells at a density of 1x10 5 cells in culture flask were pre-incubated for 24 h. The culture medium was replaced with fresh aliquots containing IP 6  extract at three different concentrations (9.5, 12 and 14.5  g/ml) for 24, 48 and 72 h. Then, the cells were trypsinized, washed twice with ice-cold PBS, and re-suspended in 100  l of 1x binding buffer (0.1 M Hepes/NaOH, pH 7.4 and 1.4 M NaCl, 25 mM CaCl 2 ). The cells were added with 5 µl of Annexin V-FITC and 5  l of propidium iodide for staining and were gently vortexed and incubated for 15 min at room temperature in the dark. Another 400 µl of 1x binding buffer was added and the fluorescence of the cells was immediately analyzed by flow cytometry (Beckman Coulter, USA). Statistical analysis Data were expressed as mean ± standard deviation (SD) and statistically analysed by one-way ANOVA using Turkey’s test and applying a significance level of p <0.05. RESULTS Growth inhibition effect of IP 6  on human colorectal cancer cells Different dose of IP 6  ranging from 0-20  g/ml were applied on HT-29, human colorectal cancer cell line and the effect of their growth was determined by MTT assay. There was a dose-related decrease in cell number upon exposure with IP 6  after 72 h of treatments. From the data, we determined that the IC 50  value of rice bran IP 6  and commercial rice IP 6  were 12.0 ± 2 and 14.2 ± 5.3  g/ml, respectively as shown in Figure 1. The results showed that IP 6  extracted from rice bran   has higher sensitivity towards human colorectal cancer cell line (HT-29) compared with commercial rice IP 6 . Rice bran IP 6  also did not cause any toxicity towards normal cells, 3T3 with <10% of cells were died (Data not shown). Effect of IP 6  on cell cycle kinetics Based on the growth inhibitory response of rice bran IP 6  in HT-29 cells, we next examine its effect on cell cycle progression. After 24, 48, and 72 h exposure with IP 6 , cell cycle kinetics of HT-29 cells were analyzed. As shown in Figure 2, IP 6  increased the G 0  /G 1  phase cells due to the increase in IP 6  dosage (Figure 2a) and IP 6  also increased the G 0  /G 1  phase cells due to the increase in exposure times (Figure 2b). Consistence with its effect on cell growth inhibition, IP 6  induced significantly G 0  /G 1  arrest in HT-29. IP 6  treatment (9.5, 12 and 14.5  g/ml IP 6 ) for 24, 48 and 72 h resulted in accumulation of 63-65% ± 0.6 cells in G 0  /G 1  phase compared to control showing 50% ± 3.5 (p < 0.05). Apoptosis induction analysis of IP 6  treated HT-29 cells The Annexin assay revealed that rice bran IP 6 significantly increased total apoptosis of HT-29 cells. IP 6  also increased the early and late apoptotic HT-29 cells in a dose- and time dependent manner. As shown in Figure 3, the total apoptotic cell death was significantly increased after 24 h of IP 6  treatment (9.5  g/ml) compared to the control (p<0.05). IP 6  significantly increased the number of early (30% ± 1.4) and late apoptotic (41% ± 2.9) HT-29 cells in dose dependent manner (Figure 3a) compared to control only <1% of cell  2286 J. Med. Plant. Res. a   a b (h) Figure 2.  Cell cycle kinetics of rice bran IP 6  treated HT-29 cells in different dosage (a) and different exposure times (b). 1x10 5 cells were seeded in culture flasks. After 24, 48 and 72 h exposure to IP 6 , the cell cycle kinetics was analyzed by flow cytometry. The values are presented as mean ± standard error of mean of three determinations, and, where indicated by *, showed a significant difference (P < 0.05) relative to the respective control. death (p<0.05). Furthermore, IP 6  also significantly increased the number of early apoptotic (29% ± 0.8) HT-29 cells in a time dependent manner compared to control <1% of cell death (p < 0.05) (Figure 3b). DISCUSSION IP 6  has been demonstrated to be instantaneously absorbed by variety of cancer cell lines (Shamsuddin, 1999). The rate and pattern by which IP 6  is metabolized by cancer cells varies depending on the cell type (Shamsuddin, 1999). Cells from different srcin have different sensitivity to IP 6  suggesting that IP 6  may affect different cell types through different mechanisms of action (Vucenik and Shamsuddin, 2003). The major finding of this present study is that rice bran IP 6  strongly induced growth inhibition, disruption of cell
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