Microsoft Word - 216 completed-corrected IIUM Engineering Journal, Vol. 12, No. 4, 2011: Special Issue on Biotechnology Mohd. Yusoff et al. 141 PRELIMINARY INVESTIGATION OF MYO-INOSITOL PHOSPHATES PRODUCED BY ASUIA279PHYTASE ON MCF-7 CANCER CELLS N.MOHD. YUSOFF 1 , T. NUGE 1 , N.H. ZAINAN 1 , Y.Z.H-Y. HASHIM 1 , P. JAMAL 1 , ANIS SHOBIRIN MEOR HUSSIN 2 , ABD-ELAZIEM FAROUK 3 AND H.M. SALLEH 1 1 Bioprocess and Molecular Engineering Research Unit, Department of Biotechnology Engineering, International Islamic University Malaysia, Jalan Gombak, 53100 Kuala Lumpur, Malaysia. 2 Department of Food Technology, Faculty of Science and Food Technology, 43400, University Putra Malaysia, Serdang, Selangor, Malaysia. 3 Department of Biotechnology, Faculty of Science, Taif University, 21974 Taif, Al-Hawiayah, P. O. Box. 888, Saudi Arabia. hamzah@iium.edu.my; yumi@iium.edu.my ABSTRACT:Phytate or myo-inositol hexakisphosphates (IP6) is widely distributed in plants like rice bran. The production of myo-inositol phosphate intermediates has received much attention due to the remarkable potential health benefits offered by the compounds. In this study, the cytotoxicity of the partially purified myo-inositol phosphate fractions and commercial IP1 and IP6 were investigated against MCF-7 breast cancer cell lines. The study showed that the commercial standard IP1 and IP6 showed good inhibition towards the MCF-7 cell line. The MCF-7 cells growth was inhibited in minimum concentration of myo-inositol phosphates (<1000 µ g/ml). However, no inhibition observed on the MCF-7 cell line by the myo-inositol phosphates fractions partially purified from rice bran at concentration <1000 µg/ml. The inhibition of MCF-7 was only observed at concentration more than 30 mg/ml with more than 40% cells were inhibited. This indicates that the partially purified rice bran myo-inositol phosphates degraded by ASUIA279 phytase on MCF-7 breast cancer cells exhibit positive results towards the inhibition of cancer cells growth at relatively high concentration. ABSTRAK: Fitat atau myo-inositol hexakisphosphate (IP6) dikenali umum teragih di dalam tumbuhan seperti dedak padi. Penghasilan perantaraan fosfat myo-inositol mendapat perhatian memandangkan ia berpotensi tinggi dalam kesihatan. Dalam kajian ini, kesitotoksikan sebahagian daripada fosfat myo-inositol separa tulen, IP1 komersil dan IP6 komersil dikaji terhadap produk yang berupa sel kekal (cell lines) kanser payu dara MCF-7. Tumbesaran sel MCF-7 direncatkan dalam pekatan minima fosfat myo- inositol (<1000 µg/ml). Tetapi, tidak ada perencatan dilihat terhadap sel kekal MCF-7 oleh sebahagian fosfat myo-inositol separa tulen daripada dedak padi pada kepekatan <1000 mg/ml. Perencatan MCF-7 hanya dilihat pada kepekatan lebih daripada 30 mg/ml dengan lebih daripada 40% sel terencat. Ini menunjukkan bahawa fosfat myo-inositol daripada dedak padi separa tulen terdegradasi oleh fitat ASUIA279 terhadap sel kanser MCF-7 dimana ia menunjukkan keputusan positif terhadap perencatan tumbesaran sel kanser pada kepekatan tinggi. KEYWORDS:myo-inositol phosphates; phytase; MCF-7cell line; cancer IIUM Engineering Journal, Vol. 12, No. 4, 2011: Special Issue on Biotechnology Mohd. Yusoff et al. 142 1. INTRODUCTION Phytate or myo-inositol hexakisphosphates (IP6) is widely distributed in plants, particularly in cereals and legumes, such as corn, soybean, wheat bran, rice bran, cotton seeds, rape seeds and soybean with a concentration range between 0.4% to 6.4% (w/w) and also in mammalian cells at concentrations of 10 µM to 1mM [1]. Phytase (myo- inositol hexakisphosphate phosphohydrolase) is a phosphomonoesterase that acts on phytate in sequential and stepwise manner, releasing inorganic orthophosphate (Pi) and yielding partially phosphorylated myo-inositol phosphates which may again become substrates for further hydrolysis (Fig. 1) [2]. It catalyzes the degradation of phytic acid into lower myo-inositol phosphates; pentakis- (IP5), tetrakis- (IP4), tris- (IP3), bis- (IP2), and mono phosphate/s (IP1); with the release of inorganic orthophosphate (Pi) in sequential manner [3]. Phytases are found naturally in plants and microorganisms, particularly fungi [4]. Fig. 1: Dephosphorylation of myo-inositol hexakisphosphate (phytate) by phytase. Rice bran, the brown part of rice kernel, is a by-product of rice milling process, composed of seed coat, the major part of the germ, and most of the outer layer of the kernel together with some broken kernels [5]. Due to highly nutritious content of fat, protein and vitamins, it has been used as animal feed in most part of the world. The bran constitutes about 8.5% of the whole grain [6]. Current research interest is focused in utilizing this rice bran in many areas such as food, environmental, health and industrial purposes. In addition, rice bran also has been used as fermentation substrate for the production of enzymes such as lipase by Candida sp. [7], in combination with cassava starch and rice hulls for the production of glucoamylase by Aspergillus sp. [8], with wheat bran for the production of alkaline protease by Trichoderma koningii [9] and also for the production of protease from Rhizopus sp. [10]. The production of myo-inositol phosphate intermediates has received much attention due to the remarkable potential health benefits offered by the compounds [11]. Myo- inositol phosphates have potential applications in many fields including pharmaceutical research and development. Myo-inositol (1,3,4,5,6) pentakisphosphate has an antiangiogenic and antitumour effects which are useful for anticancer therapeutic strategies [12]. Some myo-inositol phosphates, including phytate, are present as intracellular molecules [13]. In addition, the second messenger D-myo-inositol (1,4,5) trisphosphate has a range of cellular functions including secretion, contraction, cell division, cell differentiation and cell death cell via mobilizing intracellular Ca 2+ [13]. Meanwhile, it is believed that D-myo-inositol (1,2,6) trisphosphate can prevent diabetes complications and treat chronic inflammations besides cardiovascular diseases [14]. Furthermore, a study showed that a combination of inositol hexakisphosphate (IP6) and IIUM Engineering Journal, Vol. 12, No. 4, 2011: Special Issue on Biotechnology Mohd. Yusoff et al. 143 inositol gave better inhibition in different cancers such as in soft tissue, colon, metastaticlung, and mammary cancers than was either one alone [15]. In this present work, anion-exchange chromatography was used to partially separate myo-inositol phosphates from the reaction mixturecontaining rice bran and ASUIA279 phytase. The partially purified myo-inositol phosphates were then subjected to a cytotoxicity test on MCF-7 cells. 2. MATERIALS AND METHOD 2.1 Production of ASUIA279 Phytase The cells of ASUIA279 [16] were grown in rice bran with distilled water under aseptic condition (pH 7, 37°C, 250 rpm, 60 hour incubation time). Samples were withdrawn at time intervals, centrifuged at 12,000 rpm for 30 min, 16°C to remove the particulates and the supernatant kept at -20°C for further analysis. Twenty milliliters of aliquot were collected and kept at -20°C for storage. 2.2 Phytase Assay Phytase assay was performed according to [17] with minor modifications to measure the enzyme activity of phytase produced. It was measured in a mixture of 25 µl of 0.1 M sodium acetate buffer, pH 4.5 and 10 µl 1.03 mM sodium phytate in 200 µL microcentrifuge tube. Enzyme (5 µl) was added to the mixtures to initiate reaction. The mixtures of the enzyme and buffer was incubated for 30 min at 50°C in block heater (SHT 100D, Stuart Scientific). The released phosphate was quantified by ammonium molybdate method [18]. Stop solution (150 µl) consists of acetone / 5N sulphuric acid / ammonium molybdate (2:1:1) was added to the assay mixture followed by 10 µl 1.0M citric acid. Any cloudiness was removed by centrifugation (MiniSpin®, Eppendorf) at room temperature, 10, 000 rpm for 10 min prior to spectrophotometric measurement at 355 nm by micro plate reader, Infinite® 200 Pro series (TECAN Group Ltd.). The phytase activity was expressed as 1 micromol of phosphate liberated per min (Equation 3.1). (ε microplate = 0.3094 µ / M. cm; d depth of microplate for 200 µl solution = 0.59 cm; t = incubation time (min); Abs355 = Absorbance at 355 nm; Vtotal = total volume of assay solution; Venzyme = volume of enzyme used). enzyme total Vtd VAbs Activity ××× × = ε 355 (1) 2.3 Extraction of Myo-inositol Phosphates Rice bran (donated by BERNAS Tanjung Karang, Selangor Darul Ehsan) was used as a fermentation media for the production of bacterial phytase from local isolates, ASUIA279. The cell culture broth of ASUIA279 was grown in the presence of 10% w/v rice bran with distilled water under aseptic condition and pH 7, 37°C and 250 rpm. Samples withdrawn from 0 hour until 72 hours were then centrifuged at 12,000 rpm for 30 min to remove particulates and the supernatant kept at -20°C for further analysis. 2.4 Separation and Recovery of myo-inositol Phosphates AG1 X-4 (100-200 mesh) (BioRad Laboratories, USA) anion exchanger resin was packed in a glass column (2.5 x 30 cm) and equilibrated with distilled water. About 15 ml of the supernatant was loaded on to the column. Three concentrations of hydrochloric acid (Merck, Germany) were used to elute the sample in a stepwise gradient; 0.1 M, 0.5 M, 1.0 IIUM Engineering Journal, Vol. 12, No. 4, 2011: Special Issue on Biotechnology Mohd. Yusoff et al. 144 M. All collected samples were kept at 4°C. Following thawing, solvent was removed by a rotary evaporator. The dried samples were diluted with a small amount of distilled water and further concentrated by a spin-concentrator. The completely dried residue (partially purified myo-inositol phosphate) was kept in a desiccator to avoid any reintroduction of moisture. The percentage yield of myo-inositol phosphates and myo-inositol phosphate intermediates eluted by three different concentration of HCl from ASUIA279were determined. ( ) 100 1 12 % × − = w ww yield (2) w1 = weight of centrifuge tube w2 = weight of centrifuge tube and dried sample 2.5 Effects of Myo-inositol Phosphates on MCF-7 Breast Cancer Cell Line 2.5.1 Preparation of Standards and Samples for MCF-7 Tests Six standard samples of myo-inositol phosphates were used as reference during the investigation (Table 1). The standards were diluted in 1ml of 10% DMSO. Table 1: Amount of myo-inositol phosphates standards used. Standard Concentration (Stock solution) (g/ml) Concentration (Dilution) (µµµµg/ml) IP1 0.05 1000 IP2 0.0005 100 IP3 0.0025 500 IP4 5 x 10 -5 10 IP5 2.5 x 10 -4 50 IP6 0.125 1000 A small quantity of partially purified myo-inositol phosphates were dissolved in 1ml of 10% DMSO to a maximum concentration (Table 2). MCF-7 cells were cultured in Dulbecco Modified Eagle Medium (DMEM) supplemented with 10% fetal bovine serum and the cell lines were maintained at 37˚C in a 5% CO2 incubator and the media were changed twice weekly. 2.5.2 Cancer Inhibition Assay: Sulforhodamine B (SRB) Assay The SRB assay was performed to assess growth inhibition based on colorimetric principles. This assay estimates cell number indirectly by staining total cellular protein with SRB dye. Briefly, 190 µ l/well of cell suspensions (seeding density of 2.0 x 10 4 cells per well) and 10 µ l/well myo-inositol phosphates of different concentrations (31.25 µ g/ml to 1000 µg/ml prepared in DMSO, Table 2) were seeded in 96-well microtiter plate. The 96-well plate was then incubated for 72 hours at 37ºC and 5% CO2, to allow for cell attachment. Without removing the cell culture supernatant, the cells were fixed with 100 µl of 10% (w/v) trichloroacetic acid at 4˚C for 1 hour and the supernatant was removed from the 96 well plates. The plate was then washed four times with slow running tap water IIUM Engineering Journal, Vol. 12, No. 4, 2011: Special Issue on Biotechnology Mohd. Yusoff et al. 145 and excess water was removed using a blow dryer or just allowed to stand at room temperature. Each well was stained for 30 min with 100 µl of 0.4% (w/v) SRB dissolved in 1% acetic acid and washed four times with 1% acetic acid. The protein bound dye was solubilized with 200 µ l of 10 mmol/l Tris base, pH 10.5. The absorbance (OD) of each well was read on an ELISA plate reader at 510 nm. Percentage of control cell growth was calculated using the formula: % of cell growth = [mean OD sample/mean OD negative control]. A dose response curve was then plotted. Negative control = 10% DMSO. Table 2: Concentration of myo-inositol phosphates in DMSO. Sample aliquot (hour) Eluent (HCl) concentration (Molar, M) Weight of myo- inositol phosphates (µµµµg) Concentration of myo- inositol phosphates in 1 ml 10% DMSO (µµµµg/ml) Before inoculation 0.1 2500 2500 0.5 4000 4000 1.0 2800 2800 0 0.1 6100 6100 0.5 4600 4600 1.0 2600 2600 16 0.1 5800 5800 0.5 40300 40300 1.0 66800 66800 24 0.1 24400 24400 0.5 7700 7700 1.0 3900 3900 40 0.1 13000 13000 0.5 5700 5700 1.0 5500 5500 48 0.1 18300 18300 0.5 5000 5000 1.0 2300 2300 64 0.1 14300 14300 0.5 1500 1500 1.0 4400 4400 72 0.1 5600 5600 0.5 9400 9400 1.0 2500 2500 IIUM Engineering Journal, Vol. 12, No. 4, 2011: Special Issue on Biotechnology Mohd. Yusoff et al. 146 3. RESULTS AND DISCUSSION 3.1 Analysis of Myo-inositol Phosphate Obtained Three different concentration of eluent: 0.1 M, 0.5 M and 1.0 M HCl (Table 3) were used to elute the myo-inositol phosphates from AG1 X-4 resins. Further, ASUIA279 was incubated and monitored for 72 hours. The highest yield of myo-inositol phosphates obtained is at 16 hours incubation time (Fig.2) and then it started to decrease by time. It is known that during enzymatic phytate degradation, the hydrolysis rate decreased markedly [19]. This might be due to product inhibition by phosphate or a lower hydrolysis rate of the lower phosphate esters of myo-inositol. Another factor is because of the conditions used during the hydrolysis reaction [20]. A very slow degradation also had been observed by Van der Kaay and Van Haastert [21] which is during degradation of myo-inositol trisphosphate intermediate and the missing capability to degrade the myo-inositol bisphosphate intermediate. Table 3: Percentage yield of myo-inositol phosphates and intermediates eluted by three different concentration of HCl from ASUIA279. Sample aliquot (hour) Eluent (HCl in M) Weight of myo-inositol and its intermediates (µµµµg) Yield (%) Before inoculation (control) 0.1 0.0811 7.68 0.5 0.0495 4.69 1.0 0.045 4.26 0 0.1 0.1126 10.67 0.5 0.049 4.64 1.0 0.0516 4.89 16 0.1 0.0788 7.47 0.5 0.2009 19.03 1.0 0.02991 28.34 24 0.1 0.0681 6.45 0.5 0.0188 1.78 1.0 0.0111 1.056 40 0.1 0.1046 9.91 0.5 n.a* n.a* 1.0 0.0521 4.94 48 0.1 0.0785 7.44 0.5 0.0441 4.18 1.0 0.0047 0.45 64 0.1 0.1291 12.23 0.5 0.0435 4.12 1.0 0.0414 3.92 72 0.1 0.0546 5.17 0.5 0.149 14.17 1.0 0.0438 4.15 IIUM Engineering Journal, Vol. 12, No. 4, 2011: Special Issue on Biotechnology Mohd. Yusoff et al. 147 0 5 10 15 20 25 30 yield (% ) before inoculation 16 40 64 incubation time (hour) 0.1M HCl 0.5M HCl 1.0M HCl Fig. 2: Fraction of myo-inositol phosphate intermediates separated by anion exchange chromatography from ASUIA279 by three different concentrations of HCl. Since lower myo-inositol phosphates have less negative charge compared to higher myo-inositol phosphates, the lower myo-inositol phosphates (e.g. IP1, IP2, IP3) will be eluted earlier and easily by low concentration of HCl. Meanwhile, higher myo-inositol phosphates (e.g. IP4, IP5, IP6) will be eluted by higher concentration of HCl. Moreover, the effective charges of myo-inositol phosphates (e.g. IP5 and IP6) will be decreased if the acidity of eluent is increasing, which proves the existence of ion suppression mechanism in eluent [22]. The higher the effective charge of the analyte, the longer the retention time of the IPs, and this is also applicable to the isomers. The fluctuated reading of the yield might be caused by the insufficient elution of the myo-inositol phosphates or weaker binding of the phytate to the anion exchanger resin [23]. 3.2 The Effects of Myo-inositol Phosphates to MCF-7 Breast Cancer Cell Line A novel anticancer function of inositol hexaphosphate (IP6) has been shown both in vivo and in vitro [24]. Commonly, most research about inhibition of cancer cells growth were focused on IP6 and applied in various cell lines including human leukemia cells [25], human colon cancer cells [26], both estrogen receptor-positive and estrogen receptor- negative human breast cancer cells [27], laryngeal carcinoma [28], cervical cancer [29], prostate cancer [30], hepatoma [31], pancreatic [32] and melanoma cell line [33]. Due to a limited amount of standards available, different concentration of sample was used. As can be observed from the experiment, multi well plate that contain IP6 and IP1 immediately change to yellow colour after exposure of cell to the solution containing myo- inositol phosphates. In consequence, highest concentration of IP1 and IP6 showed good inhibition towards the MCF-7 cell line (Fig. 3). This showed that IP6 supported by Vucenik and Shamsuddin [15] , and IP1 efficiently inhibit the cancer cell growth. Based on the observation, minimum concentration of myo-inositol phosphates (<1000 µ g/ml) contributed minimum inhibition against the growth of MCF-7 cell line. IIUM Engineering Journal, Vol. 12, No. 4, 2011: Special Issue on Biotechnology Mohd. Yusoff et al. 148 Fig. 3: Optical density of six standards of myo-inositol phosphates measured at 510 nm. The next step is to study the inhibition of partially purified myo-inositol phosphates extracted from rice bran towards the MCF-7 cell line. Table 2 listed the concentration of myo-inositol phosphates used in this study. Prior to that, a serial dilution of samples ranges from 62.5 µ g/ml to 1000 µg/ml were applied to MCF-7 cell line. Nevertheless, no significant changes had been observed throughout the experiment. Once again, lower concentration (<1000 µg/ml) of partially purified myo-inositol phosphates were not capable to inhibit the growth of MCF-7 cell line. Since rice bran only has 4% of phytate content [34] compared to pure phytate, it is relevant if 1000 µ g/ml was not efficiently inhibit the growth of MCF-7 cells as the standard of IP1 and IP6. Theoretically, in 1ml of sample, about 0.04 fractions were myo-inositol phosphates. The insufficient amount of myo-inositol phosphates used before, leads us to use larger amount of myo-inositol phosphates which diluted in 10% DMSO (Table 2). The SRB assay was then carried out and measured by microplate reader at 510 nm. Figure 4 illustrated the percentage of growth for MCF-7 cells after applied by partially purified myo-inositol phosphates. The minimum amount of observed optical density indicated the inhibition of MCF-7 cells growth in the mixture. Two samples showed positive results supporting this conclusion. A decrement of growth had been observed for samples at 16 hour incubation time for 0.5M and 1.0M elution of HCl respectively. The highest concentration contained in both samples proved that the growth of MCF-7 cells really can inhibit by partially purified myo-inositol phosphates. According to statement by Adachi et al. [34], about 1.612 mg/ml and 2.672 mg/ml of phytates content in both samples, respectively. However, for samples containing less than 30mg/ml of partially purified myo-inositol phosphates showed none or minimum inhibition of MCF-7 cells growth. Moreover, throughout the analysis, more than 40% inhibition of MCF-7 cells had been observed by partially purified myo-inositol phosphates extracted from rice bran. About 44.6% and 46.6% of inhibitions had been calculated for both 0.5M and 1.0M elution of HCl at 16 hour incubation time of ASUIA279. Mostly, myo-inositol phosphates consist of IP3, IP4, IP5 and IP6 were in both samples and able to inhibit the MCF-7 cells growth. IP6 inhibited the growth of cancer cell lines in a dose- and time-dependent manner, irrespective of whether they were epithelial or mesenchymal in origin [35]. Moreover, the anticancer activity of IP6 is a result of its rapid intake by tumor cells was shown when MCF-7 human breast cancer cells were incubated with [ 3 H]-IP6. As early as 1 minute after incubation, 3.1% of IP6-associated radioactivity was taken up by MCF-7 cells IIUM Engineering Journal, Vol. 12, No. 4, 2011: Special Issue on Biotechnology Mohd. Yusoff et al. 149 and 9.5% after 1 hour. Anion-exchange chromatography showed that 58% of the absorbed radioactivity was in IP6 form, indicating that externally applied IP6 enters the cells followed by dephosphorylation. However, IP4 appeared to be a predominant metabolite of IP6, which possibly might have important role in its anticancer activity [35]. Shamsuddin and Vucenik [36] cited that IP6 also was used to enhance the anti-proliferative effects of tamoxifen and adriamycin to MCF-7 cells line. As a whole, IP6 and its metabolites can differentially inhibit the proliferation of cancer cells without affecting the normal cells, inhibit cell proliferation of cancer cells irrespective of their hormonal receptor status and individual IP6 metabolite(s) or combinations could be specifically effective against specific cancers, thereby increasing the chances of successful therapy [34]. Fig. 4: Percentage of MCF-7 cells growth after treated with partially purified myo- inositol phosphates from three different elution concentration of HCl at different incubation time. 4. CONCLUSION As a conclusion, the application of partially purified rice bran myo-inositol phosphates degraded by ASUIA279 to MCF-7 breast cancer cells exhibit positive results towards the inhibition of cancer cells growth with more than 40%. Therefore, through this research findings, a promising value-added product can be obtained from low cost and easily available raw material, rice bran, via hydrolysis reaction by phytase. ACKNOWLEDGEMENTS This research work is funded by Sciencefund research project (SF 0106-03), Ministry of Science, Technology and Innovation (MOSTI), Malaysia. Special thanks to SIRIM Berhad for providing samples as well as to all who had contributed directly or indirectly in this research. REFERENCES [1] A. M. 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