Penghambatan proliferasi sel kanker kolon HCT-116 oleh produk fermentasi pati resistentipe 3 sagu dan beras
Proliferation inhibition of HCT-116 colon cancer cell byfermentation product of resistant starch type 3 derived from sago and rice
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Date
2012Author
Purwani, Endang Yuli
Suhartono, Maggy T.
Purwadaria, Tresnawati
Iskandriati, Diah
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Resistant starch type 3 (RS3) is retrograded starch which is not digested by human starch degrading enzymes, and will thusundergo bacterial fermentation in the colon. The main fermentation products are the Short Chain Fatty Acid (SCFA), mostly acetate, propionate and butyrate. SCFA, especially butyrate, has been implicated in providing protection against cancer. The control of the cell proliferation and apoptosis present obvious target for preventive in cancer. The objectives of the research were: (1) to prepare RS3 derived from sago or rice starch through enzyme hydrolysis, (2) to study the SCFA profile produced in the in vitro fermentation of the RS3 by colonic butyrate producing bacteria, (3) to investigate the effect of the RS3 fermentation product on proliferation inhibition and apoptosis in HCT-116 colon cancer cell line, and explored its mechanism. Sago or rice starch was retrograded and hydrolyzed with amylase(106 Enzyme hydrolysis of retrograded sago or rice starch was beneficial for concentrating RS. RS3 derived from sago contained higher RS (31-38%) than those derived from rice starch (21-26%). The in vitro fermentation revealed that C. butyricum BCC B2571 produced acetate, propionate and butyrate at concentration of 83.70 mM, 47.57 mM, 46.70 mM when the medium was supplemented with RS3 derived from sago treated with amylase (RSSA) at con-centration 1%. High levels of acetate (95mM), propionate (52 mM) and butyrate (59 mM) was also produced by E.rectale DSM17629 in medium supplemented withRS3 derived from sago treated with pullulanse (RSSP) at concentration 1%. The cell free supernatant, either from C.butyricum BCC B2571 grown in medium supplemented with RSSA or E.rectale 17629 grown in medium supplemented with RSSP was applied to treat HCT-116 cells. It was found that the supernatant inhibited proliferation and induced apoptosis ofHCT-116. This treatmentincreas-ed the expression ratio of Bax/Bcl-2, indicating possibility that apoptosis occurred through mitochondrial pathway which simultaneously increased the caspase-3 concentration. U/g starch), pullulanase (4500 U/g starch) and the combination of amylase and pullulanase. The residue of the enzyme hydrolysis was separated and spray dried to obtained RS3. RS3 was further supplemented as carbon source for the in vitro fermentation study. It was performed by Clostridium butyricum BCC B2571andEubacterium rectaleDSM 17629, which have been regarded as beneficial colonicbacteria. The SCFA was analyzed by gas chromatography. Cell free supernatant was collected and applied to treat human colorectal cancer cell HCT-116. HCT-116 cells was cultured in complete medium and after 50% confluent, the incuba-tion was continued for another 48 hours in the absence or presence ofRS3 fermentation product. Cell number, apoptosis, expression of Bcl-2 and Bax gene, as well as caspase-3 enzymes were measured. Kanker kolon dan rektum (colorectal cancer, CRC), adalah pertumbuhan sel yang tak terkendali (neoplasia) pada daerah usus besar sehingga mengganggu sistem pencernaan secara lokal dan mekanisme fisiologis tubuh secara umum.Ada sekitar 1,2 juta kasus CRC pada tahun 2008 atau mencapai sekitar 9,8% di seluruh dunia.Sekitar 80% dari kasus CRC berkaitan dengan diet, 15% disebabkan oleh faktor keturunan sedangkan sisanya berasal dari faktor lain termasuk lingkungan. Keadaan tersebut menunjukkan bahwa CRC dapat dihin-dari dengan mengonsumsi bahan pangan yang sesuai. Pati resisten tipe 3 (Resis-tant starch type 3, RS3) merupakan salah satu komponen bahan pangan yang mampu memenuhi kebutuhan tersebut. Pati tak tercerna (Resistant Starch, RS) didefinisikan sebagai fraksi pati yang tidak terserap oleh sistem pencernaan pada individu yang sehat. Ada empat kelompok RS, yaitu RS1, RS2, RS3, dan RS4. RS1 adalah pati yang secara fisik terperangkap di dalam matriks sehingga tidak dapat diakses oleh enzim. RS2 adalah granula pati mentah yang didominasi oleh struktur kristalin sehingga sulit dihidrolisis oleh enzim. RS3 adalah pati retrogradasi dan RS4 adalah pati modifikasi secara kimiawi. RS langsung memasuki usus besar (kolon) kemudian difermentasi oleh mikroflora yang ada di dalamnyamenghasilkan produk utama asam lemak rantai pendek (short chain fatty acid, SCFA,), yaitu asam asetat, propionat, dan butirat. SCFA terutama butirat merupakan sumber energi utama bagi sel-sel kolon normal dan mampu mencegah/menghambat kanker. Penghambatan proliferasi dan induksi apoptosis merupakan salah satu sasaran dalam pencegahan penyakit kanker.