Preliminary investigation of myo-inositol phosphates produced by ASUIA279 phytase on MCF-7 cancer cells.

Phytate or myo-inositol hexakisphosphates (IP6) is widely distributed in plants like rice brans. 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 µg/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 ?g/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

Determination of IGF-1-Producing CHO-K1 Growth Phases Using GCMS-Based Global Metabolite Analysis

Mammalian cell lines, in particular CHO-K1 is vital for the multibillion dollar biotechnology industry. The majority of large scale bioprocessing of commercially valuable protein biopharmaceuticals is produced using this type of cell. An ideal mammalian cell system as host for biologics production should retain efficient use of energy sources in order to boost productivity at minimum cost. Various analyses such as cell counting and monitoring of specific biochemical responses are used to provide data to enable bioprocess control in order to achieve the ideal system. Our study aimed to see whether global metabolite analysis using Gas Chromatography Mass Spectrometry (GCMS) would be a potential alternative approach in providing data for bioprocess control. In this study, we analyzed metabolites of CHO-K1 cells at different growth phases using GCMS. CHO-K1 cells producing insulin like growth factor-I (IGF1) were obtained from ATCC. Cells were grown in T-flask and incubated at 37°C/ 5% CO2 until 70-80% confluent in RPMI 1640 media. Samples (cells and spent/conditioned media) were taken at designated intervals for routine cell counting (Trypan Blue dye exclusion method); glucose, glutamine and lactate determination (YSI 2700); IGF-1 production (ELISA kit R&D Sstems, Inc); and global metabolite analysis (GCMS). Conditioned media from each time point were spun down before subjecting into GCMS. Data from GCMS was then transferred to SIMCA P+12.0 for chemometric evaluation using Principal Component Analysis (PCA). The first component, PC1 results was able to explain 36% of the variation of the data with clear separation between exponential phase and other phases (initial and death phase). This suggests that GCMS-based global metabolite analysis has the ability to capture cell growth behaviour and offered insights of factors that may influence the biological system. ABSTRAK: Produk yang berupa sel kekal mamalia, terutamnya CHO-K1 adalah penting dan menguntungkan industri bioteknologi. Majoritinya pemprosesan protein biofarmaseutikal secara besar-besaran dihasilkan dengan menggunakan sel jenis ini. Sistem sel mamalia yang ideal sebagai hos untuk penghasilan produk ubatan harus mengekalkan penggunaan sumber tenaga secara efisien untuk meningkatkan produktiviti pada kos yang minima. Pelbagai analisa seperti penghitungan sel dan pemerhatian tindak balas biokimia tertentu digunakan untuk memberikan data dan untuk menentukan bioproses terkawal untuk mendapat sistem yang ideal. Kajian ini dijalankan untuk mengkaji sama ada analisa metabolit global menggunakan Spektrometri Jisim Kromatografi Gas (Gas Chromatography Mass Spectrometry (GCMS)) boleh berpotensi sebagai pendekatan alternatif dalam membekalkan data untuk kawalan bioproses. Dalam kajian ini, metabolit sel CHO-K1 dikaji pada peringkat tumbesaran berbeza menggunakan GCMS. Sel CHO-K1 menghasilkan insulin seperti faktor pertumbuhan-I (IGF1) didapati daripada ATCC. Sel dibesarkan dalam kelalang-T dan dieramkan pada 37°C/ 5% CO2 sehingga 70-80% konfluen dalam perantara RPMI 1640. Sampel (sel dan perantara yang digunakan/dilazimkan) diambil kiraan pada selang masa yang telah ditetapkan untuk sel rutin (kaedah eksklusi pencelup Trypan Blue), glukosa, glutamin dan penentuan laktat (YSI 2700); penghasilan IGF -1 (kit ELISA R&D Sstems, Inc); dan analisa global metabolit (GCMS). Perantara yang dilazimkan dari setiap poin masa dikurangkan kelajuannya pada setiap pusingan sebelum dianalisa secara GCMS. Data daripada GCMS kemudiannya dipindahkan ke SIMCA P+12.0 untuk taksiran kemometrik menggunakan Analisis Komponen Utama (Principal Component Analysis (PCA)). Keputusan komponen pertama, PC1 berupaya menjelaskan variasi 36% data dengan pengasingan jelas antara fasa eksponen dan fasa-fasa lain (fasa permulaan dan fasa akhir). Ini menunjukkan bahawa analisa metabolit global berasaskan GCMS mampu menjelaskan tingkah laku pertumbuhan sel dan memberikan pemahaman tentang faktor-faktor yang mempengaruhi sistem biologikal. KEY WORDS: CHO-K1 cells, IGF-1, metabolomics, metabolite profile, PCA, GCMS

Preliminary Investigation of Myo-Inositol Phosphates Produced by ASUIA279 Phytase on MCF-7 Cancer Cells

Phytate or myo-inositol hexakisphosphates (IP6) is widely distributed in plants like rice brans. 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 µg/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 ?g/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.. KEYWORDS: myo-inositol phosphates, phytase, MCF-7,  cancer 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